ADVANCES I N CANCER RESEARCH VOLUME 1 1
Contributors to This Volume Joseph C. Arcos
Saul Kit
M a r y F. Argus
Sidney S. Mirvish
D. Keast
William Regelson
ADVANCES IN CANCER RESEARCH Edited by
ALEXANDER HADDOW Chester Beatty Research Institute Institute of Cancer Research Royal Cancer Hospital, London, England
SIDNEY WEINHOUSE Fels Research Institute Temple University Medical School Philadelphia, Pennsylvania
Volume 7 7
ACADEMIC PRESS
NEW YORK AND LONDON
COPYRIGHT
@ 1968, BY ACADEMIC PRESS, INC.
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United Kingdom Edition published b v ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London W.l
PRINTED I N T H E UNITED STATES OF AMERICA
CONTRIBUTORS TO VOLUME 1 1 Numbers in parentheses refer to thc pagcs on which tlic authors’ contributions begin.
JOSEPHC . ARCOS,Seamen s Memorial Research Laboratory, U . S. Public Health Service Hospital, N e w Orleans, and the Department of Medicine (Biochemistry), Tulane University School of Medicine, N e w Orleans, Louisiana (305)
MARYF. ARGUS,Seamen s Memorial Research Laboratory, U . S. Public Health Service Hospital, N e w Orleans, and the Department of Medicine (Biochemistry), Tulane University School of Medicine, New Orleans, Louisiana (305) D. KEAST,Department of Microbiology, University of Western Australia, Perth, Western Australia (43) SAULKIT, Division of Biochemical Virology, Baylor University College of Medicine, Houston, Texas (73) SIDNEYS. MIRVISH,Department of Experimental Biology, The Weizmann Institute of Science, Rehovoth, Israel; and the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin ( 1 ) WILLIAMREGELSON, Division of Medical Oncology, Department of Medicine, Medical College of Virginia, Richmond, Virginia (223)
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CONTENTS CONTRlRUTORS To V O L U M E
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CONTENTS OF PREVIOVS VOLIJMl<S
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V
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The Carcinogenic Action and Metabolism of Urethan a n d N-Hydroxyurethan SIDNEY
I . Introduction . . I1. TTrethan . . . I11. N-Hydroxyurrthan IV . Urinary Metabolites V . Conclusions . . Refercnces . .
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Runting Syndromes. Autoimmunity. and Neoplasia
D . KEAST
I . Introduction . . . . . . . . . I1. Homologous Disease and the Clinical Syndromc I11. Situations Which Have Been or Could Be Classified Runting Syndromes . . . . . . . I V . Tlic Important Fcatiirtls of Riinting . . . V . Discussion . . . . . . . . . V I . Comments . . . . . . . . . Rcferenccs . . . . . . . . . . . . . . . . . . Addendum
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Viral-Induced Enzymes and the Problem of Viral Oncogenesis
SAULKIT I . Introduction . . . . . . . . . . . . . I1. Virus-Indiiccd Antigen Syntlicsis . . . . . . . . I11. Viral-Inductd Enzymes of D(.osyl.il~oiiiicIt,ic.Acid M~~t:~l)olisiii . IV Viral-Induced Enzymes That Hydrolyze or Modify . . . . . Deoxyribonucleic and Ribonucleic Acids . V . Viral-Induced Riboniicloic Acid Synt.lirtase (Replicase) . . . \'I . Eff(ds of Virus Infection on Host-Cell Nucleic Acid aiid Protein Syntlirsis . . . . . . . . . . VII . Biorlic.iiiicul Asllihcts of Vir:tl Oiicogcric4s . . . . . .
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74 54 59
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CON TEN 'I'S
Vlll
The Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology
WILLIAMREOELSON I . Introduction . . . . . . . . . . . I1. Biological Evidence . . . . . . . . . I11. Growth Control . . . . . . . . . . IV . Radiation . . . . . . . . . . . . V . Morphological Alteration . . . . . . . . VI . Cell Membrane . . . . . . . . . . VII . Surface Charge . . . . . . . . . . VIII . Adenosine 5'-Triphosphate and Polyanions . . . . I X . Calcium . . . . . . . . . . . . X . Adhesion . . . . . . . . . . . . X I . Polysaccharides . . . . . . . . . . XI1. Colloidal Effects . . . . . . . . . . XI11. Hydrophilic Gels . . . . . . . . . . XIV . Surface and Enzyme Activity . . . . . . . XV . Enzyme Inhibition and Activation . . . . . . XVI . Respiratory Enzymes . . . . . . . . . XVII . Hyaluronidase and Glycosidases . . . . . . XVIII . Ribonuclease . . . . . . . . . . . X I X . Deoxyribonuclease . . . . . . . . . X X . Polyphosphatm . . . . . . . . . . XXI . Lipase and Esterase Activity . . . . . . . XXII . Clinical Antimitotic Side Effects, and Clinical and Experimental Antitumor Activity . . . . . XXIII . Summation . . . . . . . . . . . References . . . . . . . . . . .
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223 226 234 241 242 244 246 247 251 257 259 265 268 271 275 278 279 280 282 283 284 286 287 287
Molecular Geometry and Carcinogenic Activity of Aromatic Compounds N e w Perspectives
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JOSEPH C . ARCOSAND MARYF. ARGUS I . Introduction . . . . . . . . . . . . . I1. Condensed Polycyclic Aromatic Compounds . . . . . I11. Conjugated Arylamincs and Compounds Generating Arylamines . Arylhydroxylamines . . . . . . . . . . . IV . Covalent Binding to Proteins and Nucleic Acids . . . . Rcferences . . . . . . . . . . . . .
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305 308 363 433 454
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CUMULATIVE INDEX .
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AUTHOR INDEX
514
CONTENTS OF PREVIOUS VOLUMES Carcinogenesis and Tumor Pathogenesis I . Berenblum Ionizing Radiations and Cancer Austin M . Brues Survival and Preservation of Tumors in the Frozen State James Craigie Energy and Nitrogcn Metabolism in Cancer Leonard D . Penninger and G. Burroughs Mider Some Aspects of the Clinical Use of Nitrogen Mustards calvin T. ~l~~~ and jeanne c. Bateman Genetic Studies in Experimental Cancer L. W . L~~ The Role of Viruscs in the Production of Cancer C . Oberling and M . Guerin Experimental Canccr Chemothcrnpy C . Chester Slock
Volume 1
Electronic Configuration and Carcinogencsis C . A . Coulson Epidermal Carcinogcnesis E . V . Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice L. Dmochowski Hormonal Aspects of Experimental Tumorigenesis ?'. U. Gardner Properties of the Agent of Rous NO. 1 Sarcoma R. J . C . Harris Applications of Radioisotopes to Studics of Carcinogencsis and Tumor Metabolism Charles Heidelberger The Carcinogenic Aminoazo Dycs James A. Miller and Elizabeth C. Miller The Chemistry of Cytotoxic Alkylating Agents M . C. J . Ross Nutrition in Relation to Cancer Albert Tannenbaum and Herbert Silverstone Plasma Proteins in Cancer Richard J . Winzler AUTHOR INDEX-SUBJECT
AlJTIIOIl INDEX-SUBJECT
INDEX
Volume 3
Etiology of Lung Cancer Richard Doll The Experimental Development and Metabolism of Thyroid Gland Tumors Harold P . Morris Elrctronic Structurc and Carcinogenic Activity and Aromatic Molecules: New Developmcmts A . Pullman and B . Pullman Some Aspects of Cnrcinogcncsis P . Rondoni Pulmonary Tumors in Experimental Animals Michael B . Shimkin
INDEX
Volume 2 The Reactions of Carcinogens with Macromolecules Peter Alexander Chemical Constitution and Carcinogenic Activity G. M . Badger
ix
X
CONTENTS 017 I’REVIOUS VOLUMES
Oxidative Mrlabolism of Nwplastic Tissues Sidney Weinhouse AUTHOR INDEX-SUBJECT
INDEX
Volume 4
Advances in Chcmoth-iapy of Cancer in Man Sidney Farber, Rudolj Toch, Edward Manning Sears, and Donald Pinlcel The Use of Myleran and Similar Agents in Chronic I~enltrmias D . A . G. Galton The Employment of Mctliods of Inhibition Analysis in the Normal and Tumor-Bearing Mammalian Organism Abiaham Goldin Some Recent Work on Tumor Immunity P. A . Gorcr Inductive Tissue Interaction in Development Cliflord Grobstein 1,ipids in Cancer Frances L . IIaven and W . I$. Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Propertics of Angular Brnzacridines A. Lacassagne, N . P. Buu-Hoi, R. Daudel, and F. Zajdela The Hormonal Genesis of Mammary Cancer 0.Miihlbock AUTHOR INDEX-SUBJECT
INDEX
Volume 5
Tumor-Host Relations R. W . Begg Primary Carcinoma of the Liver Charles Bemnan Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal P. N . Campbell
Tlie Ncwcir Concept of Cancer Toxin
War0 Nakahara and Fumiko Fukuoka Chcmically Induced Tumors of Fowls P. R . Peacock Anemia in Cancer Vincent E . Piice aiid Robert E . Greenfield Specific Tumor Antigens L. A. Zilber Clirniistry, Carcinogenicity, and Mctabolism of 2-Fluorenaminc and Related Compounds Elizabeth K . Weisburger and John H . Weiibeiger AUTHOR INDF.X-S~lR~lF~CT INEEX
Volume 6
Blood Enzymes in Cancer and Other Diseases Oscar Bodansky T l i ~Plant Tumor Problem Armin C . Braun and Henry N . Wood Cancer Chcmothrrapy by Perfusion Oscnr Creech, Jr., and E d u m d T . Krementz Viral Etiology of Mouse 1,cukcmia Ludwik Gross Radiation Chimcras P. C . Koller, A . J. S. Dairies, and Sheila M . A . Doak Etiology and Pathogenesis of Mouse Leukemia J. F. A . P. Miller Antagonists of Purine and Pyrimidine Metabolites and of Folic Acid G. iM. Timmis Behavior of Liver Enzymes in Hcpatocarcinogenesis George Weber ATJTIIOR INDEX-SIJBJECT
INDEX
Volume 7
dvian Virus Growths and Their Etiologic Agents J . W . Beard
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CONTENTS O F PREVIOIIS \'OI,CJMES
Mcchanisins of Hcsistanve to Antic*anc.cr Agents R . W . Brockman Cross Resistance and Collateral Sensitivity Studies in Cancer Chemotherapy Dorris J . Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W . M . Court Brown and Ishbel M . Touah Ethionine Carcinogenesis Emmanuel Farber Atmospheric Factors in Pathogenesis of Lung Cancer Paul Kotin and Hails L. Falk Progress with Some Tumor Viruses of Chickens and Mammals: The Problem of Passenger Viruses G. Negroni AUTHOR INDEX-SUBJECT
Volume 9 Urinary Enzymcs and Thrir Diagnostic Valuc in Human Cancer Richard Stambaugh and Sidney Weinhouse The Relation of thc Immune Ilraction to Cancer Louis V . Caso Amino Acid Transport in Tumor Cells R. M . Johnstone and P . G. Scholefield Studies on the Development, Biocheniistry, and Biology of Experimental Hepatomas Harold P. Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes, and Bone Marrow Cells I . F . Seitz AUTHOR INDEX-SUBJECT
INDEX
INDEX
Volume 10
The Structure of Tumor Viruses and Its Bcaring on Their Relation to Viruses in General A . P . Howatson Nuclear Proteins of Neoplastic Cells Harris Busch and William J . Steele Nncleolar Chromosomes: Structures, Interactions, and Perspectives M . J . Kopac and Gladys M . Mateyko Carcinogenesis Related to Foods Contaminated by Processing and Fungal Mctabolites H . F. Kraybill and M . B. Shimkin Experimental Tobacco Carcinogenesis Ernest L. W v n d e r and Dietrich H o f f mann
Carcinogens, Enzyme Induction, and Gene Action H . V . Gelboin I n Vitro Studies on Protcin Synthesis by Malignant Cells A . Clark Grifin The Enzymic Pattern of Neoplastic Tissue W . Eugene K n o x Carcinogenic Nitroso Compounds P . N . Mngee and J. M . Barnes The Snlfhydryl Group and Carcinogenesis J . S. Flarington Thv Treatment of Plasma Ccll Myeloma Damel E . Bergsagel, K . M . Grifith, A . Haut, and W . J . Stuckey, Jr.
AUTHOR INUEX-SUBJECT
AUTHOR INDEX-SUBJECT
Volume 8
INUKX
INDEX
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THE CARCINOGENIC ACTION AND METABOLISM OF URETHAN AND N-HYDROXYURETHAN' Sidney S. Mirvish Deportment of Experimental Biology, The Weizmann Institute of Science, Rehovoth, Israel; and the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin
I. Introduction . . . . . . . . . . . . . 11. Urethan . . . . . . . . . . . . . . A. Carcinogenic Effects . . . . . . . . . . B. Biological Activity of Compounds Related to Urethan . . C. Relationships with Nuclcic Acid Metabolism and Other Effects of Possible Relevance to Carcinogenesis . . . . . D. Metabolism of Urethan . . . . . . . . . 111. N-Hydroxyurethan . . . . . . . . . . . A. Chemistry . . . . . . . . . . . . . B. Effects Other than Carcinogenesis . . . . . . . C. Carcinogenic Effects . . . . . . . . . . D. Metabolites in Blood and Tissucs . . . . . . . IV. Urinary Metabolites . . . . . . . . . . . A. Urethan . . . . . . . . . . . . . B. N-Hydroxyurethan . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . .
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I. Introduction
Urethan (ethyl carbarnate; NHz.COOEt) has long been known t o cxhibit a narcotic action and was used for many years as an anesthetic in man. A related effect is its inhibition of sea urchin egg cleavage, which has also bccn known for many years (Cornman, 1950, 1954). I n 1943, Ncttleship et al., while examining the effects of irradiation under uretlian ancsthesin, discovered that injcctions of urethan rapidly induce lung adenomas in mice. For many years the carcinogenic action of urethan was believed to be confined to the lung. Much attention was meanwhile focused on its cytotoxic :rnd anticancer actions. It was found that urethan produced chromosome damage, especially in the rapidly dividing cells of 'This review was begun while the author was an Eleanor Roosevelt Fellow a t the University of Wisconsin, and finished at the Weizmann Institute of Science with the nid of n grant from the International Agency for Research on Cancer. 1
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SIDNEY S. MIRVISH
thr intehtincs ( I h s t i n , 1947; Boyluncl niitl Kollcr, 1954), induced leukopenia (Skipper ef al., 1949; Moeschliii and Bodiner, 1951) , showed antiIcukeinic activity in rodents (Skippel‘ r t a/., 1949), and antitumor action against the Walkcr carcinoma 256 i n rats (Haddow and Scixton, 1946). Urethan was also tested as an anticancer agcnt in man (reviewed by Haddow, 1963) and has been used in particular to treat multiple myeloma (Loge and Rundles, 1951). Finally, urethan is mutagenic for Drosophila (Vogt, 1948) and Escherichia coli (Bryson, 1949) but not for Neurospora (Rogers, 1955). Some of thcse effects are discussed later in more detail. I n 1953 it was shown that urethan is not uniquely a lung carcinogen, as it initiated skin tumors in mice, as revealed when the skins were subsequently painted with croton oil (Graffi e t al., 1953; Salaman and Roe, 1953). Later developments showed that urethan is a “multipotential” carcinogen (Tannenbaum, 1964) and can induce many types of tumor, notably malignant lymphomas of the thymus, hepatomas, mammary carcinomas, and hemangiomas. These tumors were particularly prominent when repeated doses of urethan were administered, by injection or in the drinking water, and when newborn or very young mice were treated with the carcinogen. Also, most of these tumors were only observed whcn the mice were maintained for a t least 1 year after treatment, whereas lung adenomas mostly develop after 2 to 6 months (malignant lymphomas also show a relatively short latent period of 4 to 12 months). Urethan is structurally one of the simplest carcinogens; it is soluble in both water and lipids, and, in fact, was the first water-soluble carcinogen to be discovered. Urethan readily sublimes even a t room temperature, so that weighing small amounts is not easy. It contains amide and ester groups modified by resonance between each other and is readily hydrolyzed by hot acid and alkali to give NH,, CO,, and ethanol, and also cyanate under alkaline conditions; but it shows few other specific chemical reactions. The chemistry of carbamates was reviewed by Adams and Baron (1965). I n this review the carcinogenic actions of wethan will be discussed separately for each type of tumor, with emphasis on possible mechanisms of action, and then the chemical specificity for some of the varied actions of urethan will be examined. Certain metabolic effects of urethan, and the metabolism of urethan itself, will be discussed in relation t o its carcinogenic action. It was recently proposed that urethan might act after metabolic conversion into N-hydroxyurethan (HONH .COOEt) , and, accordingly, the biological actions of N-hydroxyurethan and its metabolism will be discussed, and reasons will be given for the provi-
URETHAN AND N-HTDROXYURETHAN
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sional rejection of this hypothesis. N-Hydroxyurethan is also of interest as it and related hydroxylamine derivatives show certain biological effects probably depending on interactions with deoxyribonucleic acid (DNA) metabolism. The author is indebted t o reviews by Haddow (1963) on the carcinogenic and other effects of urethan, which may be referred t o for various effects not discussed here, and by Shimkin (1955) on lung tumorigenesis, by Handschumacher and Welch (1960) on nucleic acid metabolism, and by Tannenhaum ( 1964) on the “multipotential” carcinogenic actions of urethan. I I . Urethan
A. CARCINOGENIC EFFECTS 1 . Lung Adenonins
I n the classic investigation I y Nettlesliip ef nl. (1943) it was found tliiit multiplc injections of u r e t h n into C3II mice a t the anesthetic dose of 1 mg./gm. body weight induced a 75% incidence of lung adenomas after 7 months (in most work discussed in this review, a single dose of urethan consisted of 1.0 to 1.5 mg./gm.). Strain A mice were originally bred for spontaneous development of lung adenomas, which occurs in 75% incidence a t an age of 18 months, but multiple injections of urethan induced 100% incidence, with an average of 50 adenomas per mouse appearing after 6 months. Most of these adenomas were 2-3 mm. in diameter a t this time, and situated just beneath the pleura. The spontaneous incidence at the same age was 10%. The early work on lung tumorigenesis by urethan, mostly in A and C3H mice, was summarized by Shimkin (1955). It was shown that there is no sex difference, that urethan is active whether injected or given by mouth, and that the tumor yield is proportional to the number of doses (Henshaw and Meyer, 1944, 1945; Larsen, 1946; for recent dose-response curves, see Kaye and Trainin, 1966; Shimkin e t ul., 1966). Similar tumors had previously been induced by intravenous injections of carcinogenic hydrocarbons (see Shimkin, 1955). Urethan also induces lung adenomas when painted on the skin (Cowen, 1950h; Roe and Salaman, 1954; Berenblum and Haran-Ghera, 1957a) or administered in aerosols (Otto and Plotz, 1966). Anesthetics other than urethan were not carcinogenic (Larsen, 1946), and lysergic acid diethylamide, which prevents urethan anesthesia, did not affect its wrcinogenicity (Berenblum et nl., 195911). Althougli t h v tuiriois arc. o f t c w siiiall nix1 do not inctastasize, so111c~later evolve iiito c:irciiioni:ts (Allen, 195G). Also, tlic :id(wom:is (#:in I)e trans-
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SIDNEY S. MIRVISH
planted and may then develop sarcomatous areas (Klein, 1957). I n large doses urethan also induces a low incidence of lung adenomatoses and lung squamous cell carcinomas (Tannenbaum and Maltoni, 1962; Deringer, 1965). Swiss and BALB/c mice show a fairly high spontaneous incidence of lung tumors and are fairly sensitive to lung tumorigenesis by urethan (Law, 1954; Berenblum et al., 1959a; Trainin et al., 1964), e.g., in adult Swiss mice a dose of 1 mg. urethan/gm. induces about three lung adenomas per mouse after 40 weeks (Berenblum e t al., 1959a). Six mouse strains showed correlations between their susceptibilities t o spontaneous, hydrocarbon-induccd, and urethan-induced lung adenomas, with the order of susceptibility A > Swiss > CBA > DBA (Shapiro and Kirschbaum, 1951). I n an investigation by Cowen (1950a) the order was A > CBA > C57BL (the last strain was originally bred for resistance to spontaneous lung tumorigenesis) . The parallel susceptibilities to spontaneous and urethan-induced lung tumors indicate that these mouse strains differ in some parameter which is not peculiarly related to urethan, e.g., the parameter is unlikely to involve differences in the metabolism of urethan. I n genetic experiments, Cowen (1950a) found that crosses between the resistant C57BL and sensitive A strains showed a susceptibility to urethan lung tumorigenesis rather less than that of A mice, and backcrosses with C57BL mice showed segregation into two groups, as expected from Mendelian genetics. Similar results were obtained by Falconer and Bloom (1962; Bloom, 1964; Bloom and Falconer, 1964), except that the sensitivity of the F, and A mice were found to be almost identical. These results indicate that A mice possess a single dominant gene responsible for most of the strain difference, as was prcviously indicated for spontaneous lung tumorigenesis (Bittner, 1938). A heterogenous mouse strain was selectively bred within nine generations t o give two lines showing large differences in their susceptibility to urethan (Falconer and Bloom, 1964). Urethan is particularly suitable because of its even distribution throughout the body (see later) for use in transplantation experiments. Thus Shapiro and Kirschbaum (1951) implanted mouse lung tissue of the susceptible albino and resistant DBA strains into the ears of F, hybrids, and then injected urethan into the hybrids. They found that 12 of 17 albino grafts developed tumors, but oidy 1 of 17 DRA grafts, showing that susceptibility to urethan is mainly an intrinsic propcrty of the lung tissue and not of the whole animal. Then hlalmgren and Saxen l tissue from strain A mice was (1953) showed that ~ h c nn ~ i * n i n lung transplnntcd subcutaricously into host micc previously treated with
U 1 W ~ ' I I A N AND N-IITDROXYURETHAN
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urethan, adenomas developed in the transplants only when the urethan was injected less than 24 hours beforehand, proving that the active carcinogen must have disappeared after this period. Conversely, when lung tissue was transferred from mice previously treated with urethan to untreated mice, 22% of the transplants developed tumors, confirming that urethan acts directly on the lungs. Transplantation experiments by Rogers (1955)attempting t o demonstrate formation of an active urethan metabolite are discussed later. Histological studies have shown that urethan-induced lung tumors mostly arise from the alveolar epithelium in mice (Mostofi and Larsen, 1951 ; Brachetto-Brian, 1951.; Klarner and Gieseking, 1960; Svoboda, 1962; Driessens e t al., 1963) and rats (Rosin, 1949),unlike human lung cancer which usually arises from the bronchial epithelium. As an example of the observable short-term effects, Brachetto-Brian (1951) showed that in Swiss mice, urethan induced necrotic pycnosis of most of the septa1 alveolar cells within 24 hours, followed after 48 hours by proliferation of these cells, which later showed polymorphism and nuclear atypia. The first adenomas appeared after 24 days. Shimkin and Polissar (1955) described the development in the lungs of urethan-treated A mice of numerous hyperplastic foci after 3 to 5 weeks and of tumors after 3 t o 7 weeks, though it was not clear whether the tumors arose from the hyperplastic foci. I n confirmation of the histological findings, radioautography of the lungs after injection of tritiated thymidine revealed a 70% suppression of mitosis 1 day after urethan treatment, followed by a rise to 3 times the normal value on the fourth to seventh days (Foley e t aE., 1963). I n an important in vitro study, treatment of lung organ cultures with urethan led to a loss of loose connective tissue and a slowing of epithelial budding, and the latter was restored to normal by contact with untreated mesenchyme of lung or submandibular gland (Globerson and Auerbach, 1965). Immunological factors appear to be involved in lung tumorigenesis, as the yield of lung adenomas in Swiss mice after injecting urethan or feeding 7,12-dimethyl-1,2-henzanthracenewas increased by neonatal thymectomy (Trainin e t al., 1967). Contrary to a brief report by Imagawa e t aZ. (1957),Casazza et al. (1965) found that infection with influenza virus a t thc tinie of urethan injection did not enhance lung tumorigenesis, which was consirlercd not surprising since the virus mainly affects the bronchial epithelium, whereas urethan affects the alveolar cpithelium. l'uiiitiiig the mouth with cig:irette tar was stated by Di Paolo an(l Pht~c~lic(19G2) to eiiliance uret1i:iii lung tumorigenesis, but the tar alone w t h tiimoi*igchnic,:Ind a ~yrrc~i~gistic (i.e., inorc than additive) effect was not clcarly tlrmon~trated.
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SIDNEY S. MIRVISH
Several reports have appeared on the effects of varying the oxygen pressure on urethan lung tumorigenesis in strain A mice. Thus tumorigenesis was increased by exposing mice to 70% oxygen for 2 days after injection of the urethan (Di Paolo, 1959), and it was also increased in the progeny of pregnant mothers exposed to 10% or 100% oxygen after injecting the urethan, provided that the urethan was injected less than 24 hours before parturition (Di Paolo, 1962). Also, tumorigenesis by urethan was reported by Mori-Chavez (1962) to be increased a t high altitudes in Peru, but was not affected by exposure of the mice to reduced pressure in decompression chambers (Ellis et al., 1966). Some of these effects could be due to changes in the rate of urethan catabolism (see later). Large doses of X-rays have the effect of suppressing lung tumorigenesis by urethan (Gritsiute, 1961; Duplan et al., 1962; Foley and Cole, 1963). I n the last report, a lethal dose of 880r X-rays was administered 3-24 hours after the injection of urethan into (C57BL x A)F, mice, and this suppressed lung tumorigenesis (expressed as tumors per mouse) to 18% of the value for urethan alone. Syngeneic bone marrow was injected after the irradiation to protect the mice. Some effect was observed when the mice were irradiated a t any time from 8 weeks before to 1 week after the urethan injection, and the irradiation was moderately effective when fractionated into 9 X 100 r, or given as a single dose of 500 to 700 r, without protection by bone marrow (Foley and Cole, 1964, 1966). The effect was prevented by shielding the thorax during the irradiation (Foley and Cole, 1964) and was apparent in only one lung when this lung alone was irradiated (Duplan et al., 1962). Finally, irradiation suppressed the urethan-induced hyperplasia in the lung, as measured by the incorporation of tritiated thymidine (Foley et al., 1963). These results suggest that irradiation inhibits urethan tumorigenesis by a direct action on the lung, probably by prcventing proliferation of the cells sensitive to urethan. Small “subliminal” doses of X-rays followed by urethan produced a synergistic increase in lung tumorigenesis, so that irradiation may have opposite effectsdepending on the dose (Cole and Foley, 1966).
3. S k i n Papillomas I n 1953 it was found that skin papillomas develop on application of urethan to the skin, followed by repeated painting with croton oil (Graffi et al., 1953; Salaman and Roe, 1953; Berenblum and Haran, 1955), so that urethan is here an “initiator” according to the terminology of Berenblum and Shubik, in which the carcinogenic hydrocarbons acted as “initiators” and croton oil as “promoters” of skin tumorigenesis. The same effect was obtained when uretltan was administered by irioutli or Iiy in4jcctioit (1T:tr:tn and Rerenbhittt, 19Fi6; Bet*cnhluniaud €Int~an-Ghcr:t,
URETJ3AN AND N-HYDROXYTJRETI-IAN
7
1957:i; Ilitchie, 1957). LJrrt1i:tn :i])pli(~ilt o tlw ~ l i i i iwas cqiizilly cffectivc i n acetone or Carbowas solution (Roc, n t ~ lSalanim, 1954) : ~ n dis ahsorlwd directly itnd not l)y helug licakctl : i i i t l sw:illowrd, as collars which prevented licking did iiot affect thc turnor yicld (Berenblum and HarariGhera, 1957a). The tumor yield was proportional to the dose of urethan, both when the urethan was applied to the skin, where a total dose of 240 mg. per mouse induced five papillomas per mouse (Roe and Salaman, 1954), and when administered by stomach tube, where the same tumor yield was induced by 64 mg. per mouse (Berenblum and Haran-Ghcra, 1957a). The latter workers found that the mean latent period fell from 16 to 8 weeks as the total oral dose was raised from 1 to 64 mg. per mouse; and when a total oral dose of 64 mg. per mouse was divided into five or twenty subdoses, the tumor yield fell about 50%. The promotion with croton oil may be delayed for 8 weeks with no change in tumor yield (Berenblum and Haran-Ghera, 1957a), but when the promotion was delayed for 24 to 30 weeks the tumor yield fell about 50% (Roe and Salaman, 1954; Pound, 1966). Promotion may also be carried out with the noriionic detergent, Tween 60 (Van Esch et al., 1958). Only a few skin tumors arc induced by urethan acting alone, and even this low incidence may depend on traumatic injury acting as a promoter (Chieco-Bianchi et al., 1964). Urethan is thus virtually a pure initiator for the skin, unlike the hydrocarbon carcinogens which are complete skin carcinogens a t repeated doses. This difference may be associated with the fact that urethan, unlike the hydrocarbons, produces little hyperplasia of the skin (Salaman and Roe, 1953). Thus experiments with tritiated thymidine showed that, in the epidermis, urethan induced a short-term reduction in DNA synthesis, perhaps associated with tumor initiation, whereas 7,12-dimethyl-1,2-benzanthraceneinduced in addition a long-term increase in DNA synthesis, associated with the hyperplasia (Garcia and Leiva, 1966). I n exception to the general rule, painting of tiitthan alone induced epidermoid carcinomas but not papillomas in linirless hr/hr mice, though no effect was found in the hybrid haired Hr/ hr mice (Deringer, 1962). Pound et nl. (Pound and Bell, 1962; Pound and Withers, 1963; Pound, 1966) applied croton oil to the skin of mice, then injected urethan, and finally carried out the usual promotion with croton oil. By this means they obtained a fourfold increase in tumor yield, as compared with controls where the croton oil pretreatment was omitted. Similar results were obtained by preliminary trcatment with acetic acid and other chemicals, or by scarring, all of which lead to inflammation and cellular proliferation. A critical time interv:d of 15 to 18 hours between the croton
8
SIDNEY S. MIRVISH
oil (or acotic acid) and urethan treatments gave the maximum tumor yield, and this corresponds to thc period of maximum DNA replic at'ion in the epidermis, prior to a burst of mitoses about 27 hours after the croton oil treatment. 3. Maliytiurd LympIwiiLus
The most usual type of mouse leukemia induced by urethan is a malignant lymphoma (lymphosarcoma) originating in the thyinus (Pietra e t al., 1961), as with mouse leukemia occurring spontaneously or induced by other agents. The induction of this neoplasm by X-rays was stated by Kaplari (1964) to be mediated by ( 1 ) release of a leukemogenic virus, ( 2 ) injury to the thymus, and (3) injury to the bone marrow, influencing thymic regeneration. Investigations into urethan leukemogenesis have mainly attempted to elucidate similar questions, and, in particular, (I) the interactions between urethan, X-rays, and viruses as leukemogenic agents, ( 2 ) the question whether urcthan acts directly on the thymus or whether other organs, eg., bone marrow, are involved, and (3)the reasons for the greater effect of urcthan in newborn and very young mice. The first demonstration that urethan is a coleukemogenic agent was provided by Kawamoto et al. (1958), who showed that leukemogenesis by X-rays, estrogens, and 3-methylcholanthrene in adult mice was augmented by simultaneous treatment with urethan. Then, in 1961, it was discovered that newborn mice are particularly sensitive to urethan leukemogenesis, as a single injection of urethan into newborn Swiss mice induced a 20-30% incidence of leukemia within 15 to 30 weeks (FioreDonati e t al., 1961, 1962; Pictra e t al., 1961; Della Porta et al., 1963). The leukemia incidence was 80-1000/0, with a latent period of 20 to 30 weeks, when multiple doses of urcthan were administered to mice of the C57BL (Doell and Carnes, 1962), (C57BL X A/J)Fl (Klein, 1962), dd (It0 e t al., 1964, 1965), and (C57BL X C3H)Fl (Vesselinovitch and Mihailovich, 1966) strains, starting a t birth or 1 week later. The incidence was higher in female than male dd mice, though there was no sex difference in the Swiss mice of Della Porta e t al. (1963), or in C57BL mice examined by Della Porta e t al. (1967). I n addition, Liebelt e t al. (1964) reported that urethan injected into newborn female C3Hf mice induced ft 30% yield of reticulum cell sarcomas (Type A) after 1 year. In adult mice, urethan acting alone usually shows little leukemogenic activity (Kawamoto e t al., 1958; Berenblum and Trainin, 1960), but very large doses of urethan in the drinking water induced 10-30% incidences of leukemia in adult Swiss and C3H mice after 30 to 50 weeks (Toth et al., 1 9 6 1 ~ Tanncnhaum ; and Maltoni, 1962; Della Porta e t al., 1963). Following on thc rcport of I<:iwnrnoto et al. (1958), Bcrenblum alltl
UItE'I'IIAN
AND N-IIYI)ROXTCI11E'THAN
9
Trninin ( 1960 ; l3erenblum, 1963) found a synergistic effect when multiple injections of urcthan were given to adult C57BL mice after, but not before, a courhe of 4 x 90 r X-rays. Thus for lcukemogenesis, urethan is a promoter or coleukemogenic agent and not an initiator, in contrast to tlic oppositc finding for skin tumorigenesis. Bercnblum and Trainin (1961 ; Berenblurn, 1963) then showcd that X-ray initiation and urethan promotion of leukemogeriesis could be carried out in different mice, since, when minces of various organs from irradiated mice were injected into mice which were subsequently treated with urethan, significantly more leukemias were obtained than in control groups. The results suggested that irradiation elicited a "precursor virus" and the urethan transformed the precursor virus into an active leukemogenic virus; or that the irradiation liberated an active virus, and the urethan injured the thymus or produced systemic effects in such a fashion that the virus had a greater chance of infecting the thymus. As will be shown, subsequent work has tended to support the second hypothesis. First, the thymus of both newborn and young adult mice is particularly susceptible to injury by urethan and shows a maximum weight loss of 50% a t 3 t o 5 days (depending on the age) after injecting the urethan, accompanied by a loss of lymphoid cells from the thymic cortex (FioreDonati and Kaye, 1964; Haran-Ghera and Kaplan, 1964). These cells were later replaced, often in one lobe only, by immature lymphocytes (lymphoblasts) , which failed to differentiate further into mature small lymphocytes (It0 et al., 1964, 1965). Thymus organ culturcs treated with urethan showed necrosis (Tomatis and Wang, 1964) with considerable loss of lymphoid cells, so that only epithelial cells remained, but contact with bone marrow, even across n Millipore filter, led to lymphoid recovery (Globerson and Auerbach, 1965). Analogous results were obtained with lung organ cultures, and i t seems that urethsn-induced injury to these organs is associated with inductive tissue intcractions. Second, some viruses have been found to induce leukemia rather readily i n uretlian-treated mice. Thus the Graffi leukemia virus, which normally acts only in newborn mice, induced a 55% iiiridcnce of leukemia in adult C57BL micc when thcsc were suhsequciitly tiwitctl with urethan, whcrras the incidence was Qnly 16q3 wlien tlic ordcr of twatments was rcversed (C1iicco-Bi:inchi e t al., 1963). Siniil:u~ly,tlic Ioiikcmoqcnic action of the r:icli:ition 1eulieini:i virus ( l m 7 ) in ~ i c w l ~ o C'5713L ~~ii mice was potcntintetL I)y sul)socliielit, iiwi1i:itioii or t reatinent with ~irctlit~ii, which intlurcd higlicr incideuccs of leukeniia with shoiter latent periods (T,icf)ernian et ul., 1964) . Urethnn also somewhat :iccclerated the onset of leukemia in the high-lculremia AKR and C58 strains (Kawamoto et al., 1961; Fiore-Donati e t al., 1962), presumably by accelerating tlie
10
SIDNEY S. MIRVISH
multiplication of the leukemia viruses carried in these strains. These effects appear to depend mainly on the thymic injury caused by urethan, with subsequent regeneration and repopulation by immature lymphocytes, which seem to be the cells most susceptible to the virus infection. Urethan may also induce a general increase in host susceptibility to the virus (Chieco-Bianchi e t aZ., 1963; Lieberman e t al., 1964), and, in fact, urethan enhances the severity of infection with several noncarcinogenic mouse viruses which do not specifically attack the thymus, namely, the viruses of viral pneumonia, coxsackie, and viral hepatitis (see Haddow, 1963). I n contrast to these results, the induction of leukemia in C3Hf*/Lw mice, in which the Moloney leukeniogenic virus is transmitted through the mother’s milk, was strongly suppressed by four urethan injections into the progeny, starting at birth (Law and Precerutti, 1963). I n this case, urethan possibly acted by injuring the virus itself or the virusinfected cells, but the results have not yet been satisfactorily explained. Kaplan (1964) suggested that a leukemia virus could be released from a dormant site by X-irradiation. I n support of this thesis, HaranGhera (1966) prepared cellfree (thymus bone marrow) extracts of mice which had previously been treated with X-rays (4 X 170 r) together with urethan (4 x 1 mg./gm.). She then injected the extracts into newborn thymuses previously implanted under the kidney capsules of adult thymectomixed irradiated hosts. At 10 days after the end of the irradiation, sufficient active agent was present in the extracts to induce a 7576 leukemia incidence, compared with 15% for a control experiment with X-rays alone. At longer periods after the treatment, little or no active agent was found, until it reappeared much later when the leukemias developed. Thus urethan appears to potentiate the release of active agent which is induced by X-rays. Since bone marrow injections are known to suppress X-ray leukemogenesis, a contributing factor to urethan leukemogenesis could be an indirect action on the thymus mediated by the bone marrow, especially as urethan is known to suppress bone marrow mitoses (Rosin, 1951; Rosin and Goldhaber, 1956). In this connection, bone marrow from 30day-old C57BL mice treated with urethan, but not from similarly treated older mice, was less effective than normal.bone marrow in promoting thymic regeneration nnd suppressing leukemia in irradiated mice (Berenblum c t al., 1964; Haran-Ghem and Kaplan, 1964). However, the injection of normal bone marrow did not affect leukeniogenrsis by the S-ray i i t * t 4 h i t i sydcmi in irrwborn or x h l t mice (Rci*enblum et al., 1961, 1966a) . Evidence that uret11:in acts directly on the thymus and not via other oi’gans was provided by Berenblum et al. (1966b), who showed that
+
urethan leukemogenesis in newborn C57BL mice resembled X-ray leukemogenesis in being prevented by thymectomy, but differed from X-ray leukemogenesis in not reappearing when a newborn thymus was later implanted. When adult C57BL mice received multiple urethan treatments after implantation with five newborn thymuses per mouse, a 41% incidence of lymphomas developed, all from the implanted thymuses, as compared with a 2% incidence in nonimplanted controls (FioreDonati e t al., 1965). This again indicates that the thymus is directly attacked and suggests that the newborn thymus is more sensitive to urethan than the adult thymus. The greater susceptibility of newborn and very young mice to urethan leukemogenesis may be attributed to four factors: ( 1 ) the slower elimination of urethan (discussed later) ; (6)the presence of immature lymphoid cells in the thymus; (S) the more severe bone marrow injury produced by urethan in young mice; and ( 4 ) the immunological incompetence of the newborn animals. 4. Hepatomas I n some mouse strains urethan is a fairly powerful liver carcinogen inducing hepatomus, though with a long latent period of about 1 year. As with many other liver carcinogens, the tumors develop mainly in males. Thus a single dose of urcthan increased the high spontaneous incidence of hepatomas in three substrains of C3H mice (Heston e t al., 1960). Repeated administration of urethan to (C57BL X A/J) F, mice from a n age of 1 week, induced hepatomas in 57% of the males and 13% of the females (Klein, 1962). A single dose of urethan a t an age of 1 week was as effective as multiple doses beginning a t the same age, and the peak sensitivity for both sexes was reported to occur a t 1 week and not when newborn (Klein, 1966), though this disagrees with most other work on urethan. High yields of hepatomas were observed 1 year or more after injecting single doses of urethan into newborn C3H, DBA, and Swiss mice (Dolla Porta et al., 1963; Chieco-Bianchi e t al., 1964; Liebelt e t al., 1964; Trainin et al., 1964). The tumor incidence was higher in the males of all three strains, but was still considerable in C3H females, whereas DBA females were completely resistant. I n three of thc reports, large doses of urethan were given to adult mice and failed to induce hepatomas. The hepatomas were transplantable, and hepatoma-bearing C3H/f mice of both sexes showed signs of androgenic stimulation (Liebelt e t al., 1964). Hepatoma induction by urethan treatment of newborn Swiss mice was reduced after castration of the males and increased after castration of the females (Fiore-Donati et al., 1966).
12
SIDNEY 6 . MIRVISH
5. Liver Ilcittnngiomas
Early changes induced by urethan in the livers of adult mice include injury to the endothelial lining of portal veins and sinusoidal capillaries (Doljanski and Rosin, 1944). With repeated doses of urethan, hemorrhagic cysts develop after about 1 year in the liver and, more rarely, other organs (Roe and Salaman, 1954; Berenblum and Haran, 1955; Tannenbaum and Silverstone, 1958). More recent reports have generally considered a t least some of these cysts to be hemangiomas (hemangioendotheliomas) (Heston et al., 1960; Kawamoto et al., 1961; Toth et al., 1961a; Deringer, 1962, 1965), especially since the tumor was successfully transplanted (Deringer, 1962; Trainin, 1963). The highest incidences (up to 100%) have been observed in DBA, C3H, and C58 mice. The tumor has also been induced by single injections of urethan into newborn mice; in this case female DBA mice developed a higher tumor incidence than males, in contrast to the reverse tendency for hepatomas (Trainin et al., 1964). 6. Mammary Carcinomas The induction of mammary carcinomas by large doses of urethan given t o adult female mice was first reported by Tannenbaum et al. (Tannenbaum and Silverstone, 1958; Tannenbaum, 1961; Tannenbaum and Maltoni, 1962), who reported a 100% incidence of these tumors in female C3H mice, with a latent period of 40 to 50 weeks. Della P o r k et al. (1963) found that urethan increased the high spontaneous incidence of the tumor when administered to adult but, surprisingly, not newborn mice of their “CTM” albino strain. Haran-Ghera (1963) found a synergistic effect when urethan or 3-methylcholanthrene was administered to (C57BL X A) Fl mice concurrently with a hypophyseal transplant beneath the kidney capsule. Thus the transplant alone induced a 17% incidence of mammary adenocarcinomas, whereas after additional treatment with ten urethan injections, a similar (24%) incidence of the same tumor was obtained, and, in addition, 15% of mammary adenocanthomas and 17% of mammary carcinosarcomas. The tumor incidence with urethan alone was 7%. Della Porta et al. (1967) recently reported the induction of mammary carcinomas by urethan in adult female C3H/f and (C57BL x C311)F1 mice. 7. Other Tumors in Mice, and Tumors in Other Species Table I [expanded from a table by Tannenbaum (1964)J shows the many types of tumor induced by repeated doses of urethan in mice, rats,
U R E T H A N AND
13
N-IIYDROXYURETHAN
TABLE I TYPESOF TUMOR INDUCED BY MULTIPLEDOSESOF URETHAN IN MICE, RATS, AND HAMSTERS Type of tumor
A. 2Clicea Malignant mesenchymal tumor of interscapular fat pad Cystadenoma of Harderian gland
Papilloma of forestomach
B. Rats Lung adenoma Mammary tumor Zymbal’s gland carcinoma Malignant lymphoma Kidney tumor Neurilemmoma of ear Melanotic lesion of iris
C. Syrian golden hamster Melanotic skin tumor Papilloma and carcinoma of forestomach Mammary tumor Ovarian tumor Adenomatous polyp of cecum Lung adenomatosis Hepatoma Hemangiosarcoma
Comment Rare Rare
Rare
Variable incidence Common Fairly common Rare Rare Rare Fairly common if treating newborn rats
Itef eren ces Tannenbaum and Silverstone (1958); Tannenbaum (1961) Tannenbaum and Silverstone (1958);Tannenbaum (1961) ; Della Porta el al. (1963); Deringer (1965); Klein (1966) Berenblum and Haran-Ghera (1957b); Klein (1966) Jaffb (1947) ; other references reviewed by Shimkin (1955) Tannenbaum et al. (1962) Tannenbaum et al. (1962) Tannenbaum et al. (1962) Tannenbaum et al. (1962) Tannenbaum et al. (1962) Roe et al. (1963)
Common
Pietra and Shubik (1960); Toth et al. (1961b); Rivikre et al. (1964) Common Pietra and Shubik (1960); Toth et al. (1961b) Fairly common Toth et al. (1961b); Rivihre et al. (1964) Fairly common Rivikre et al. (1964) Rare Toth et al. (1961b) Rare Toth et al. (1961b); Rivikre et al. (1964) Rare Toth et nl. (1961b) Eare Toth et al. (1961b)
Tumors listed exclude those discussed in the text.
and hamsters. Hybrid mice dwelop espccially many types of tumor, presumably becausc they livc longer (Tannenbaum, 1961). Taniieiibauiii (1961, 1964) correlated the tuinors with those occurring spontniieously and c~oncludctlt11:it urethan usually acts by hastening the occurrence of a tumor wliicli would norninlly arise spontnneously Ititer in life. Urethan
14
SIDNEY R . MIRVISH
is particularly suitable for studying this hypothesis, as its even distribution ensures access to most organs (see later). Subsequent urethan treatment augmented the induction of subcutaneous sarcomas in mice by implantation of Teflon sheets (Tomatis and Shubik, 1963) but not of 3,4-benzpyrene (Berenblum and Trainin, 1963). The induction of epidermal skin tumors by carcinogenic hydrocarbons was not affected by the simultaneous administration (by skin painting) of urethan (Cowen, 1950b; Schmahl et al., 1964), nor did the hydrocarbons affect urethan lung tumorigenesis or leukemogenesis (Cowen, 1950b; Berenblum and Trainin, 1963). Repeated injections of urethan into tadpoles of Xenopw Zaevw (the South African clawed toad) raised the incidence of lymphosarcoma from the spontaneous 20% level up to 65% (Balls, 1965). I n rabbits, urethan does not induce lung adenomas (Rogers, 1955), but very large doses initiated skin tumors in this species, when promotion was carried out with croton oil or Tween 60 (Parmeggiani e t aZ., 1957). Urethan was also reported not to induce lung tumors in strain L(P) micc (Bhide and Ranadive, 1966), white-footed deer mice (see Shimltin, 1955), guinea pigs, and chickens (Cowen, 1950a). 8. Tumors Induced in Newborn Mice
Urethan is a far more potent carcinogen in newborn than in adult mice. This is shown most strikingly for leukemogenesis and liver tumorigenesis, as discussed earlier, but also applies to the induction of lung adenomas (De Benedictis et al., 1962; Liebelt et al., 1964; Kaye and Trainin, 1966) and skin tumor initiation (Chieco-Bianchi et al., 1964). With regard to the strain sensitivity of newborn mice to single injections of urethan, leukemia develops particularly readily in Swiss mice, lung tumors in BALB/c mice (Trainin et al., 1964), hepatomas in C3H mice of both sexes and DBA males, and liver hemangiomas in DBA females (see earlier for references). It may be worth stressing that intraperitoneal injections into newborn mice should be made through the leg or gluteal muscles in order t o prevent leakages. When urethan is administered to pregnant mice, the offspring develop lung adenomas, and this is discussed together with the metabolism of urethan. It should be mentioned here that this treatment given rcpeatedly can produce lung tumors in the offspring as early as 3 days after birth (Smith and ROUS,1948) and that lung tumors develop in mice suckled by urethan-treated mothers, due presumably to the presence of urethan in the milk (De Benedictis e t al., 1962; Chieco-Bianchi et nZ., 1964). The time rourse of the decrease in i i r e t h n cnwiringcnicity froin I)ir111 to :uliiltlinnrl is also discussed hter.
URETHAN A N D
N-HYDROXTURETI-IAN
15
Rvcrnt, papers by Vesscliuovitcli c t rrl. (1 967; Vesselinovitcli and hiihailovich, 1966, 1967:1,l)) g i w cletnil(d i~lfortiiationon the carcinogenic :tct,ioii of urcthar~in urwhorii n i x l fctnl iiiicc.
B. BIOLOGICAL Awiviw
OY
COMPOUNDS RELATEDTO UKETEIAN
Some clues to the mode of action of urethan may be derived by studying the action of related carbamates. With respect to their nomenclature (see Table 11), methyl carbamate (I, R = Me) refers to the methyl ester of carbamic acid, and ethyl N-methylcarbamste (“N-methylurethan”; 11, R = Me) refers to the N-methyl derivative of ethyl carbamate. The carcinogenic action of urethan (ethyl carbamate; I, R = Et) shows a high degree of chemical specificity (Tables 11, 111, and IV). Thus i-propyl carbamate (I, R = i-Pr) induced only 5% as many lung adenomas in strain A mice as did urethsn, whereas methyl and n-butyl carbamates were inactive (Larsen, 1947b). The last compound even reduced skin tumor initiation by urethan when the two compounds were TABLE I1 STRIJCTURAL FORMULAS OF CARBAMATES AND
Formula No.
I I1 111 IV
Structural formula NHy COOR RNH.COOEt 1lzN.COOEt RCH(NH.COOEt) 2
V
CIIyXH.COOEt
VI
‘2IIz.NH.COOEt HONH.COOEt
RELATEDCOMPOUNDS Name
Alkyl carbamate Ethyl N-alkylcarbamate Ethyl N,N-dialkylcarbamate Methylene diurethaii (13 = H) Ethylidene diuret,hsri (It = Me) Ethylene diurethaii
I
VII VIII IX X XI XI1 XI11 XIV
xv
XVI XVII XVIII
NH.CO.OCI-Iz.CH2
U
EtO.CO.OEt NHrCO.CHz.CI-Ir.CH3 K Hy CSOE1. N H yCO.SEt HzOJ’.O.COOEt Hz03P.NH.COOEt NHz.CO.O.PO3Hz HOOC.CHR.NH.COOEt CH,N(NO).COOEt HN(C0OEt) z N(C0OEt)j
h‘-Hydroxyurethan, ethyl N-h ydroxycarhamate Oxaxolidone Diethyl carbonate n-B u t ylamide Xanthogenamide Tliiourethan Carbethoxyphosphnte Urethan phosphate Carbamyl phosphate Carbethoxy derivs. of amino acids N-Methyl-N-nit rosourethan Biscarbethoxyamine tris-Carbethoxyamine
TABLE I11 C?LRCIXOGENICCOMPOUNDS RELATED TO URETHAN"
Skin Compound Urethan (ethyl carbamate)
Formula N0.b
I, R = Et
A. Carbamates of other dcohds n-Propyl carbamate
I, R = n-Pr
i-Propyl carbamate Ally1 carbamate B,fl$-Trichlorethyl carbamate
I, R = i-Pr I, R = CH2 :CH.CHy I, R = C13C.CHy
B. N-Substituted ethyl earbamates Ethyl N-methylcarbarnate
11, R
=
Me
Ethyl N-ethylcarbamate Ethyl N-n-propylcarbamate Ethyl N-i-propylcarbamate Ethyl N-phenylcarbamate
11, R = E t 11, R = n-Pr 11, R = i-Pr 11, R = Ph
Ethyl N,N-dimethylcarbamate
111, R = Me
Lug tumorigenesisc
tumor initiationc
+++++ +++++ + ++ + ++ + + +++
+ n.t.
+
n.t.
++ n.t. n.t. n.t.
-
+
+; -
-
References
-
z
s
2
Larsen (194713);Berenblum et d. (1959a) Larsen (1947b) Berenblum et al. (1959a) Larsen (194713) Larsen (1948); Roe and Salaman (1955);Berenblum et a/. (1959a) Larsen (1948) Larsen (1948) Larsen (1948) Hueper (1952) ; Engelhoru (1954) ; Salaman and Roe (1956) Larsen (1948); Berenhlum et al. (1959a)
-4
.
i3
Ethyl -Y,-Y-diethylcarbamate Ethyl S,-h--di*-propylcarbamste Ethyl iV,S-di-i-propylcarbamate Methylene diurethan Ethylidene diurethan Ethylene diurethan N-Hydroqurethan
111, R = Et 111, R = n-Pr 111, R = GPr IV: R = H IV, R = Me
v
VI
C. Other compounds i-Propyl S-phenylcarbamate
+ + + +++ ++++ ++ -t+
-
i-Propyl A~-3-~hlorophenylcarl,amate Oxazolidone Diethylcarhonate
VII VIII
n.t. -
Butylamide Carbet hoxyphosphate
IX XI1
+
-
n.t. n.t. n.t. n.t. n.t. n.t.
+++
+ + + -; + +-
Larsen (1948) Larsen (1948) Larsen (194s) L a w n (1948) Larsen (1948) Larsen (1948) Berenblum et al. (1959a); Miller et al. (1960)
Hueper (1952); Engelhorn (1954) ; Van Esch et ol. (1958) Van Esch et ol. (1958) Berenblum et al. (1959a) Salaman and Roe (1956) ; Berenblum et al. (1959a) Berenblum et al. (19594 Berenblum et nl. (1959a)
All tests were carried out on various strains of mice, except those by Engelhorn (1954),who used rats. See Table 11. c (hrcinogenic potency graded approximately from - (negative) to a maximum of ; n.t.-not tested,
++ + + +
18
SIDNEY S. MIRVISIL
TABLE IV COMPOUNDS RELATEDTO URETHANWHICHARE NONCARCINOGENIC OR OF DOUBTFUL CARCINOGENICITY" Compound
A . Carbarnates of alcohols other than ethanol Methyl carbamate n-Butyl carbamate i-Amy1 carbamate p-Hydroxyethyl carbamateb p-Aminoethyl carbamate 8-Chloroethyl carbamate 2-Methyl-2-n-propyl-I ,&propanediol dicarbamate (meprobamate) 7-Phenylpropyl carbamate (phenprobamate)
References Larsen (1947b) ; Roe and Salaman (1955) Larsen (1947b) Larsen (1947b) Berenblum et al. (1959a) Berenblum et al. (1959a) Larsen (1947a) Berenblum el al. (1959a) Jahn and Adrian (1966)
B. N-Substituted ethyl carbarnales Ethyl N-n-butylcarbamateb Larsen (1945) Roe and Salaman (1955) Ethyl N-octadecylcarbamate Ethyl N,N-di-n-butylcarbamate Larsen (1948) Ethyl N,N-diphenylcarbamab Larsen (1948) Carbethoxy derivs. of 4 amino acids (XV). Berenblum el al. (1959a) Urethan phosphate (X11I)bs~ Berenblum et al. (1959a) C. Other compounds Xanthogenamide (X)bic Thiourethan (X1)b.c Carbamyl phosphate (XLV)bsC Ethyl formate Ethyl glycinate Potassium cyanate" a
Berenblum et ul. (1959a) Berenblum et al. (1959a); Roe and Salaman (1955) Berenblum et al. (19,59a) Roe and Salaman (1955) Roe and Salaman (1955) Larsen (1950); Berenblum el al. (1959a)
All tests were carried out on various slrains of mice. Doubtful carcinogenic activity (the remaining compounds were definitely inactive). See Table 11.
administered together (Garcia, 1963). Ethyl N-i-propylcarbamate (11, R = i-Pr) produced 35% as many adenomas as did urethan, but the activity dropped as the N-alkyl group was lengthened, and ethyl N-nbutylcarbamate was inactive. The activity of the urethan molecule thus decreases mucli more rapidly when the ethyl group is replaced by other nlkyl groups, than when the nitrogen atoll1 is ulkylttted, aiid, :icrorditrgly, Bereiibluni et ul. (1959,) suggc.st(h(l that tlit: ethyl ester portion of the molecule was more likely to be involved in tlie reactions leading to carcinogenesis than was tlie amide portion. Alternatively, Larsen (1948) suggestcd that the N-alkyl derivatives may act via dealkylation
to urethan and also that the r:ithcr :tc.tive nictliyleiie atnd ethylidene diurethans (IV, R = H and Me) may act via hydrolysis to urethan. Ethyl N-acetylcarbamate (acetylurethan) is fairly carcinogenic in mice (Berenblum, Boiato-Chen, Haran-Ghera and Mirvish, unpuhlished work) and this might also act after conversion to urethan. Of special interest among carcinogenic compounds discovered in later work is N-hydroxyurethan (VI) and also N-methyl-N-nitrosourethan (XVI) , which induced squanious carcinomas of the esophagus and stomach, possibly via conversion into diazomethane (Schoental, 1960, 1961 ; Druckery et al., 1961; Schoental and Magee, 1962). I n theoretical calculations, the urethan molecule showed a high superdelocalization a t the carboxyl carbon atom, which may facilitate nucleophilic attack a t this site, whereas related less carcinogenic compounds showed lower values for this parameter, except for other alkyl carbamates (Fukui et al., 1961). Ethyl and i-propyl N-phenylcarbamate and related compounds are used as weed killers, and tests have shown that these conipounds are not complete carcinogens, though they show weak initiating action on the skin (Table 111).Some aromatic carbamates, e.g., l-naphthyl N-methylcarbarnate, are powerful anticholinesterases and important insecticides (Casida, 1964), and certain carbamates are used as hypnotics and tranquilizcrs, c g . , mcprobamatc, ethinamatc, and phenprobamate (y-phenylpropyl carbamate). Of these compounds, meprobamate (Miltown) and phenprobamate have been tested for carcinogenic action, with negative results (Berenblum et al., 1959a; Jahn and Adrian, 1966). Polyurethan foams and sheets, like many other polymeric materials, induced sarcomas when implanted subcutaneously in the rat, and sarcomas and carcinomas when implanted intraperitoneally (Hueper, 1964). The polymers were not carcinogenic when fed, and the action appears to be due to unspecific effects a t the polymer surface, though chemical interactions cannot be excluded. I n contrast to carcinogenesis, the anesthetic effect in mice (Larsen, 1947b, 1948; Skipper e t al., 1949) and the inhibition of sea urchin egg cleavage (Corninan, 1950, 1954) both showed an increase in activity as the alkyl chain was lengthened in alkyl carbamates ( I ) , N-alkylcarbamates (11), and N,N-dialkylcarbamates (111) ; also, the thiourethans (X and XI) were more active than urethan. Antileukemic action in mice, however, was as specific as the carcinogenic effect, e.g., methyl, n-propyl, and n-butyl carbamates were inactive, and ethyl N-alkylcarbamates were less active than urethan (Skipper and Bryan, 1949). Similarly, in tests of activity against Walker carcinoma 256 in rats, only urethan and bis- and tris-carhethoxyamine (XVII and XVIII) out of thirty (un-
20
SIDNEY S. MIRVISH
listed) carbaniates showctl appreciablc activity (Rose e t d.,1950) . Strong activity against chick leukosis and the mouse Rscitcs tumor, but not the rat Yoshida tumor, was shown by y-dimethylamino-n-propyl N-p-nitrophenylcarbamate and related compounds (Stern, 1956). Leukopenic action in mice was rather less specific than antileukemic activity, e.g., ethyl N,N-dialkylcarbamates (111) and ethyl N-methylcarbamate (11,R = Me) were leukopenic but not antileukemic, and this was said to indicate that urethan acts more specifically on differentiation than on growth (Skipper et al., 1949). The teratogenic effects of carbamates in Syrian golden hamsters showed a positive correlation with carcinogenesis in mice, since the carcinogens urethan, n-propyl carbamate (I, R = n-Pr) , ethyl N-methylcarbamate (11,R = Me), N-hydroxyurethan (VI), and diethylcarbonate (VIII) were also teratogenic, whereas n-butyl carbamate (I, R = n-Bu) and ethyl N,N-dimethylcarbamate (111, R = Me) were inactive for both types of effect (Di Paolo and Elis, 1967). However, the order of activity was different from that for carcinogenesis, e.g., urethan was less teratogenic than ethyl N-methylcarbamate, and N-hydroxyurethan was by far the most potent teratogen tested (see also Chaube and Murphy, 1966). Methyl carbamate thus shows none of the effects of urethan, whether carcinogenic, anesthetic, or cytotoxic (Dustin, 1947). The reasons for this behavior remain an enigma, especially as a possible explanation, namely that the compound is very rapidly metabolized or excreted, has been eliminated (Boyland and Papadopoulos, 1952) (see later). The slight carcinogenic activity of the higher alkyl carbamates similarly remains unexplained, though their anesthetic activity a t least confirms that these two properties are not related.
C. RELATIONSHIPS WITH NUCLEIC ACIDMETABOLISM AND OTHEREFFECTS OF POSSIBLE RELEVANCE TO CARCINOGENESIS It was suggested in 1946 that the biological effects of urethan depend on direct inhibition of nucleic acid synthesis, especially of the pyrimidine bases (A. R. Todd, quoted by Haddow and Sexton, 1946), and an apparent antagonism between urethan and the pyrimidines does exist. Thus, the in vivo chroinosome-damaging (Boyland and Koller, 1954; E. Boyland, quoted by Haddow, 1963) and anti-cancer (Elion et al., 1960) actions of urethan were antagonized by thymine, thymidine, glutamine, and (for anticancer action) cytidine, 5-methylcytosine, asparagine, and aspartic acid. The anticancer action was synergistic with that of 6-azauracil. Also, growth inhibition of Escherichia coli by urethan (10 mg./ml. medium) was reversed by phenylalanine and 2,6-diamino-
purine, a d the rcycrsal by 2,6-diainiiiopurine was prevented by adciiiiic (Skipper and Schabel, 1952; Wheeler arid Grammer, 1960). Several workers have examined the effects of nucleotides and related compounds on urethan carcinogenesis. Thus Cowen (1949) found that repeated injections of ribonucleotides a t the time of urethan administration inhibited lung tumorigcnesis, and suggested that the action was correlated with the ability of ribonucleotides to elicit Ieukocytosis, which is opposed t o the leukopenic action of urctiian. Skin tumor initiation by urethan was inhibited by the administration of various purine precursors (Roe, 1955), but the results seem difficult to interpret. Rogers (1957) reported that urethan lung caitinogcnesis was inhibited by injection prior to the urethan of DNA ant1 various pyrimidines and their precursors, and potentiated by the folic acid antagonist, aminoptcrin, but thcw results were not confirmed by Routwell (1964) and Kaye and Traiiiin (1966). Spontaneous lung tuinorigenesis was, however, significantly repressed by feeding thymine in the drinking water (Fink and Fink, 1955; Kaye and Trainin, 1966). Colchicine treatment and fasting, which both lead to a suppression of mitosis, did not affect lung tumorigenesis when carried out at the time of urethan administration (Rogers, 1951; Routwell, 1964; Kaye and Trainin, 1966). Skin tumorigenesis by urethan and by 7,12-dimethylbenzanthracene, with croton oil promotion, was 60% inhibited by topical application of actinomycin irnmcdiately aftcr atlministration of the carcinogen (Gelboin et al., 1965). This suggested that tumor initiation required an active ribonucleic acid (RNA) synthesis, but, alternatively, the high local concentration of actinoniycin may have killed the initiated cclls. Pretreatment with folic acid prevented immediate deaths from LD,, doses of urethan and, surprisingly, inhibited the narcotic action but not the leukopenia produced by urethan (Lee and Shubik, 1965). In other in vivo effects, urethan in common with various other drugs and carcinogens produced an acceleration of hexobarbital metabolism, due to an increase in certain liver microsomal enzymes (Fujimoto et al., 1960). Urethan administration produced a rapid decrease in the RNA ant1 DNA content of various tissues in Swiss mice, though not i n I,(€’) mice which are resistant to urcthan (Bhide and Ranadive, 19GG), hut this finding probably reflects general cell injury. Urethan administration also decrcascd the incorporation of 14C-orotic acid into liver RNA, but n-propyl carbamate and phenobarbital gave similar results, and the effect was presumed to be related to the anesthetic action (Kayc, 1966). I n vitro effects are likely to be relevant only a t the biologically active urethan concentration of 0.01 M or less. Urethan a t these concentr a t’Ions
22
SIDNICY S. MIRVISH
was reported to protect m i I,sii~yl pliosphate from pliosphatascs a d , hence, increase urcidosuccinate synthetase activity (Bresnick, 1960), but Kaye (1968) found no in vitro effect on the enzymes involved in pyrimidine biosynthesis. Also, a t these concentrations of urethan, certain transmethylation reactions were inhibited (McKinney, 1950), and the oxidation of tyrosine by tyrosinase to give 3,4-dihydroxyphenylalanine (dopa) was inhibited, though the final nonenzymic oxidation of dopa to melanin was slightly accelerated (Lea, 1950). Urethan did not affect the transition temperature of DNA exccpt a t very high urethan concentrations (Kaye, 1962; Levcne e t al., 1963), did not inactivate the DNA-transforming principle of Haemophilus influenzae (Zamenhof et al., 1953), and did not affect the sedimentation profile of rat liver RNA differently from methyl carbamate (Brown and Ashton, 1962). The clotting of blood plasma and the action of a-chymotrypsin were inhibited by 0.02 M urethan and n-propyl carbamate, and this effect might be related to the inhibition of sea urchin egg cleavage by urethan (Kaye and Temes, 1963). Recently, Giri and Bhide (1967) reported that aspartic transcarbamylase in mouse lungs was decreased to 15% of the control value six hours after the injection of urethan; this enzyme is required for synthesis of the pyrimidine bases and utilizes carbamyl phosphate (XIV), which may be regarded as an analog of urethan.
D. METABOLISM OF URETHAN 1. Determination Urethan may be determined in body fluids by alkaline hydrolysis and measurement of the liberated ethanol (Archer et al., 1948; Boyland and Rhoden, 1949; Mitchell et al., 1949; Beickert, 1950a) or of the ‘“CO, when 14C-ca&oqurethan is used (alkali-labile 14C02)(Berenblum et al., 1958; Kaye, 1960a,b; Mirvish et al., 1964; Cividalli et al., 1965). Urethan may also be determined, e.g. in urine, by KOH/ethanol decomposition to give potassium cyanate, which is estimated by a color reaction with CoCI, (Deveaux e t al., 1963; Boyland and Nery, 1965). Due to the volatility of urethan, solutions should not be evaporated to dryness during determinations. 2. General Metabolisrn
Investigations on urcthan metabolism have largely been directed a t the questions (I) whether urethan acts directly as carcinogen or is first transformed into an active metabolite and ( 2 ) whether this metabolite is N-hydroxyurethan. In connection with the first question, urethan produces chromosome damage to rat tissues i n vivo (Dustin, 1947; Boyland
UREl'E-IAN AND N-IIYI)ROXYURE'rIIAN
23
arid Koller, 1954) but not to plants, e g . , Viciu fuba root tips (Boyland et al., 1965) or tissue cultures of animal cells (Borenfreund et al., 1964). Also, in vitro transform:ition of embryo hamster cells to neoplastic cells has been achieved using the hydrocarbon carcinogens but not urethan (Berwald and Sachs, 1963) , although urethan is carcinogenic to hamsters (see Table I ) . These results suggest that urethan may be converted into an active metabolite and that this occurs only in the intact animal. Rogers (1955) implanted fctal mouse lungs into newborn or young rabbits, rats, and mice, injected urethan into the hosts, and later transplanted the lungs back into adult mice. Adenomas developed in all groups, and it was concluded that active metabolite formation was carried out by all three host species. However, the implanted mouse lungs themselves could have carried out an activating reaction. Rogers (1955) also reported adenomas after fetal mouse lung hashings were incubated in the plasma of urethan-treated rabbits and then implanted into mice, and he concluded that the plasma contained an active metabolite, but this effect was later attributed to unchanged urethan in the plasma (Berenblum et al., 1960). Chemical methods of analysis showed that urethan is rapidly and evenly distributed throughout the body, including the cerebrospinal fluid and brain, in rats, rabbits, and man (Boyland and Rhoden, 1949; Beickert, 1950b, 1951). It was then shown (Bryan et al., 1949; Skipper et al., 1951) that mice metabolized more than 90% of the label in 14C-carbo~y- and 14C-methyZeneurethan (NH,.14C0 *OCH,CH, and NH2.CO*014CH,-CH3)to expired 14C0, within 24 hours, with 4 to 10% excretion of the label in the urine and less than 1% in the feces. After the same period, 1% of the label in '4C-carboxyurethan and 6% of that in 14C-methyleneurethan were retained in the body, with fairly similar concentrations in various organs, but this was attributed to reincorporation into body constituents of the hydrolysis products WCO, and I4Ccthanol, as these compounds themselves gave similar 24-hour retention figures. Sea cucumber sperm, however, showed more 24-hour fixation of l'C-labeled urethnn than of 14C-labeled ethanol and NaHCO, (Cornman e t al., 1951). Berenblum et nl. (1958) compared total tissue "C analyses with specific isotope dilutioii analysis for urethnn using the uretl~an-sn~itliyd~oI complex, :ti"tci- injcction of 1 4 C - c d m r ~ /and - 14C-)nethyleneu~.ethan. By this nm~iistlicy confi txietl thr c\.cii d i h t idxition or uretlian ant1 showecl that most of thr radioactivity i i i plusiii:~and tissues was due to urethan after 0.8 and 6, Imt not 24, hours. Incoiporntion into livcr proteins after 24 houi~sconstituted 60% of the I'C in the livcr for 14C-carbozyurethan and 26% for 14C-methyleneurethan. After 5 hours, urethan was evenly
24
S I D N E Y S. RIlR\’ISH
distributed between the nuclei, mitochondrial, and supernatant fractions of liver and lung homogenates, except that the lung mitochondria may have concentrated urethan preferentially. The 15Nof 15N-labeled urethan was also evenly distributed (Bailey and Christian, 1952). Paper chromatography of plasma showed only one peak corresponding to urethan, with no sign of metabolites, after injection of 1 4 C - ~ a ~ b o ~ y and 14C-methyleneurethan into mice and rabbits (Kaye, 1960a), and thin-layer chromatography of mouse blood extracts gave similar results (Mirvish, 1964, 1966). 3. Rate of Elimination As urethan is evenly distributed in vivo, its rate of catabolism may be followed simply by measuring the rate of urethan elimination from the blood. The latter shows values in micrograms urethan per milliliter blood per hour of about 60 in adult Swiss mice (Kaye, 1960b; Mirvish et al., 1964), 50 in rats (Boyland and Rhoden, 1949), 25 in rabbits (Beickert, 1951), and 4 in man (Beickert, 1950b). The quoted authors found constant catabolic rates down to low urethan levels, which differs from the exponential curves typical for most drugs, and indicates that the catabolizing enzyme becomes saturated at low levels of urethan (Kaye, 1960b). The rate of urethan catabolism was reduced in tumorbearing and leukemic mice (Mitchell et al., 1949; Boyland and Rhoden, 1949; Bryan et al., 1949; Skipper et al., 1951; Beickert, 1952) but not in human cancer patients (Beickert, 1952). Methyl carbamate was eliminated by rats at half the rate for urethan, and its rate of elimination was still slower in tumor-bearing animals (Boyland and Papadopoulos, 1952). Kaye (1960b) found that the rate of urethan catabolism in 13-dayold Swiss mice was two-thirds of that in adults and suggested that the longer retention of the drug, which would allow more time for it to act, might largely explain its twofold greater carcinogenicity in the young mice (Rogers, 1951). Urethan was catabolized a t normal rates in adult mice 1 and 14 days after X-irradiation with 400 r (Cividalli et al., 1965), after treatment with the drug, SKF-525A (which inhibits drug oxidation reactions), and after four injections of urethan given over a 2-week period (Mirvish, unpublished results). After the drnniatic leukemogeiiic action of urethan in newborn mice was discovered, it w:is foruid l y Mirvish ~f nb. (1964) that, newhorn PWK iiiice eliiiiiii:itc uretliati :it, oiily :tl,out o ~ ~ c s - t c ~ of~ t ht,he rate for wlults. Thus, ~1 close of 0.5 ing,/gni. was coml)lrtely eliminated after 8 hours in adult mice but after 72 hours in newborn mice, with 62% retention a t 48 hours. Newborn mice of five strains showed similar slow
URETTHAN A N D N-HYDROXYORETHAN
25
ratcs of uretlian c1liiiiiii:ition (Cividalli c t nl., 1965). A reduced metabolic rate ill newborn mice I K I ~:{lko reported I)y I)oinsky et nl. (1963) for 7,12diniet~liyl-l,2-bcnz;~iitli1~:1ct~11t~. I\ I i i c ~ h lilic iirrt I i : i n indiires iiiorr Iyn1)hoiii:w : i n c l 11111g: i c I ( ~ i I o n I : i iir ~ l l ~ ~ \ v l ] o r inic.c>. n T h - h l ( i \ v w nict:il)oli,\iii of urethnii in iicsu borii iiiice iii:iy 1:irgcbIy cxl)l:iiii tlic grc1:itcr sciisitivity of newborn mice to urethan carcinogencsis for those organs (luiig and skin) which are also affected in adults. Leukemogenesis and livcr tumorigenesis by urethan are more specific to newborn mice, and here other factors are probably also involved, as discussed earlier. The metabolic results also appear t o explain the phenomenon that, whereas lung tumors did develop in the offspring of pregnant mice injected with urethan several days before delivery (Larsen, 1947a; Smith and ROUS,1948; Klein, 1952, 1954), the number of tumors was up to fivefold greater when the injection was made less than 24 hours before delivery, and was cspecially high when the young were delivered by Caesarian section 1-5 hours after the injection (Larsen, 1947a; Klein, 1952, 1954). I n the first case the urethan would have been metabolized by the mothers a t the usual adult rate, whereas in the other cases the young were presumably born with unmetabolized urethan in the body, which they took several days to metabolize. The metabolic results may also be related to thc longer sleeping time induced by urethan in newborn rats and rabbits, though the depth of anesthesis was less than in adults (Weatherall, 1960). The time required for 50% catabolism of urethan by SWR mice fell steadily for the first 20 days after birth, while the rate of elimination increased most rapidly between the fifteenth and twentieth days (Cividalli et al., 1965). These findings correlate fairly well with observations that 5-day-old Swiss mice were almost as sensitive as newborn mice to leukemogencsis (Fiore-Donati et al., 1962) and liver carcinogcwcsis (Chieco-Bianchi et al., 1964) by single doses of urethan, whereas only a small response occurred a t 20 days, and none in adults. Similarly, newborn Swiss mice were 5 times more sensitive than adults to urethan lung tumorigenesis measurcd as tumors per mouse (Chieco-Bianclii et al., 1964), whereas 14-dny-old Swiss mice were twice as sensitive, and 21day-old mice 60% more sensitive (Rogers, 1951). Leukciiiogcnesis by repeated doses of urethan was about half as effective when the first dose was given to 7-day-old mice as when it was given a t birth, and in both groups linear dose-response curves were obtained (Vesselinovitch and Mihailovich, 1966). The lesser response of the older group could thus be ovcrcome by increasing the dose, which supports the view that the diffcrencc may be due to the varying time for which the drug remains in the body. Fiti:Llly, thc high levcl of mitotic abnormalities induced by urethan
26
SIDNEY S. MIRVISH
in nrwborn mice drol)pec\ to adult lcvels after 12 to 15 (lays (WakonigVaartaja, 1964). 4. The Cntctbolizing ErizyitLc The catabolisni of 14C-carbozyurethan by mouse liver homogenates has been investigated by determining 14C0, production. Initial observations (Kaye, 1960b) were later (Mirvish and Kaye, 1964) shown to be due to a rapid and complete attack by the homogenate on a labilc l'CO,-producing impurity in the sample of 14C-ca~bozyurethan.After rcmoval of thc impurity by recrystallization from toluene, the purified 14C-urethan was hydrolyzed in yields of 0.2 to 0.4% by fresh, concentrated liver homogenates, and this was proved to represent hydrolysis of I4C-urethan itself. Yields of 8% 14C0, were obtained using liver slices. The Michaelis constant was about 2.5 X M urethan (0.22 mg./nil.) for both the homogenatc and the slices; this value is rather higher than expected from the in vivo results indicating saturation of thc catabolizing system a t low urethan levels (Kaye, 1960b). The activity of the homogenatc was unstable, showed a p H optimum of about 8.0, and was inhibited 90% by diisopropylfluorophosphate M ) and 67% by N a F (lod3M ) , indicating that an esterase was involved. The catabolism of urethan was also inhibited, probably competitively, by n-propyl carbarnate, n-butyl carbamate, and N-hydroxyurethan, but not by methyl carbamate. The activity was highest in the microsomal fraction and was liberated from this fraction by bile salts, in accord with the known localization of esterases. It is likely, though not proved, that this system in the liver is mainly responsible for in vivo urethan catabolism. I n line with this view, partial hepatectomy produced a prolongation of uretlian anesthesia (Fischer, 1963), and liver microsoma1 esterases (Okagswa et al., 1966) and urethan catabolism (sec earlier) were both reduced in tumor-bcaring animals. Homogenates of newhorn mouse livcrs showed very low catabolic activity toward urethan (Cividalli e t al., 1965), in agreement with thc in vivo results and the known gcncral drficiency of livcr microsornit1 enzymes i n newborn rodrnts. Ill. N-Hydroxyurethan
The suggestion that N-hydroxyurethan (VI ; NHU) is the proximal carcinogen of urethan was first raised tentatively by Berenblum et al. (1959a) and was more strongly advanced by Miller et al. (1960) and Boyland and Nery (1965) after it was discovered that the aromatic amine carcinogens probably act via the N-hydroxy derivatives. The suggestion seemed attractive as NHU shows carcinogenic activity, is
more activc than urethan in certain chemical and biological respects, and is formed in v i m from urethan. A. CI-IEMISTRY N-Hydroxyurethan is a hydroxamate (compare acethydroxamic acid, CH,.CO.NHOH) and an ethyl ester, may be synthesized by reaction of hydroxylamine with ethyl chloroformatc (Cl*COOEt), and is commercially available. I n alkali, it shows an ultraviolet absorption peak a t 217 my ( E = 3800) (Mirvish, 1965), which can be used for its determination and is absent in N-mcthoxyurethan (MeONH. COOEt) (Mirvish, 1966). The purple color with ferric chloride characteristic of hydroxamates is given by NHU, but with a very low extinction coefficient (Boyland and Nery, 1964; Mirvish, 1965). The NHU may be determined (Mirvish, 1965; Philips et nl., 1967) in blood and tissues (but not urine) by acid hydrolysis of trichloroncetic or pcrchloric acid supernatants to give hydroxylamine. The latter is then oxidized with iodine a t an acidic pH, e.g. pH 3.4, to give nitrous acid, which is determined by diazotization. At acidic pH values, unhydrolyzed NHU and hydroxyurea are not completely oxidized, though hydroxamates are. However, Nery (1966) found that complete oxidation with iodine occurred at pH 8.0, and the hydrolysis step may then be omitted. Alternatively, NHU in water or urine solutions may be determined by treatment with sodium pentacyanoammine ferroate and magnesium chloride (as catalyst) to give a colored complex [Fe(CN),.HONH.COOEtI3- (Boyland and Nery, 1964). The synthesis and some reactions of N-hydroxycarbamates, including oxidation reactions, were recently discussed by Boyland and Nery (1966a,b). Several chemical reactions are known which may provide the basis for some of the biological effects of NHU. Thus Freese and Freese (1965, 1966; Freese, 1965) found that transforming DNA of Bacillus subtilis was inactivated on incubation a t 75°C. with as little as 10-5M NHU, hydroxyurea, and hydroxylamine, but not urethan. The process resembles the reduction in DNA viscosity by low concentrations of the cytotoxic and carcinogenic agent, l-methyl-2-p- (i-propylcarbamoyl)-benzylhydrazine (Natulan) (Berneis e t al., 1963, 1964), in that both are free radical reactions which require oxygen, produce H,O,, and are inhibited by catalase and peroxidase. A reduction in DNA viscosity was also reported for hydroxylamine, hydroxyurea, and NHU, but a different mechanism was proposed involving hyponitrous acid (HON: NOH) and attack on presumed amino acid ester links in the DNA (Bendich et al., 1963). The present author showed tliwt the reduction of DNA viscosity by NHU (10-?-10 M ) rcyuirtvl osygeii (unpul)lished results), so that it may rntlier procccd ns i i i t l i o diciiies p~~oposcdfor liydroxylaminc and
28
SIDNEY S. MIRVISI-I
Natulan, in which oxygen is reduced to H,O, and then to the hydroxylfree radical, and this or some other radical is the active agent attacking DNA (Scheme I ) . 1.
HONHsCOOEt
+
2.
HONHeCOOEt
+
O2
-
[ON*CWEt] +
2&OZ-[ON*COOEt] SCHEME I.
+
2&0
t 2 *OH
Boyland et al. (1963; Boyland and Nery, 1965) showed that NHU reacts in vitro with cysteine and N-acetylcysteine to give S-ethylcysteine (cf. XXIV) possibly by degradation of intermediate S-carbethoxycysteine (cf. XXIII) , since in alkaline solution N-acetyl-S-carbethoxycysteine was slowly degraded to S-ethylcysteine. Schoental and Rive (1963, 1965) had previously identified S-carbethoxycysteine as a product of the reaction between N-nitroso-N-niethylurethan (XVI) and cysteine and had shown that S-carbethoxycysteine readily rearranges above pH 8 to N-carbethoxycysteine (XXII). Similar reactions could also conceivably take place with preformed proteins and nucleic acids in the cell (Schoental, 1966). (For formulas of numbers greater than XVIII, see Scheme 11, which shows the N-acetyl derivatives for XXIII and XXIV.) The N-acyloxy derivatives of the aromatic amine carcinogens were recently shown t o react readily with methionine (Lotlikar et al., 1966) and guanine (Miller e t al., 1966), and these may be the “ultimate” proximal carcinogens of the aromatic amines. The NHU metabolite 0-acetyl-NHU (XXI) (Boyland and Nery, 1965) might similarly react in vivo more readily than NHU itself to produce carbamate ( R N H COOEt) or carbethoxy (R. COOEt) derivatives of proteins or nucleic acids. I n this connection, Rose (1967) suggested that 0-esters or O-glucuronides of NHU might react by decomposing to give the very active nitrene, EtO * CO N : . B. EFFEcrs OTHERTHAN CARCINOGENESIS For many noncarcinogenic effects recently investigated, N H U is far more potent than urethan and closely resembles hydroxyurea, which is not carcinogenic (Yamamoto e t al., 1967). Thus, NHU resembled urethan in producing chromosome damage in vivo to rat tissues, especially those with a rapid cell turnover (Philips et at., 19M) but, unlike urethan, NHU also produced chromosome damage in plants (Vicia faba root tips) (Boyland et al., 1965) and tissue cultures of rodent cells (Borenfrcund et al., 1964). I n addition, NHU was much more active than urethan as a teratogen in the rat fetus (Chaube and Murphy, 1966), an inhibitor of the Shope papilloma virus in tissue culture (dc Sousa e t al., 19651, a i d an antitumor agent in rodents (Adamson, 1965; Tarnowski e t al., 1966; Hahn et al., 1966). For the last action, NHU arid hydroxyurea were equally active, hydroxylamine was
URlC'rHAN AND
N-HYDROXPURETHAN
29
inactive, aiicl cross-resistance was demonstrated betwcen N H U and hydroxynrea. 111 cell cultures, X H U and hydroxyurca specifically inhibited DNA4synthesis (Young and Hodas, 1964), and this action was attributed to an inhibition of cytosine diphosphate reduction to deoxycytosine diphosphate (this is probably the main reaction supplying deoxyribosides for D N A synthesis). I n support of this view, the cytotoxicity of hydroxyurea in Chinese hamster cell cultures was partially reversed on adding thymidine or one of the other pyrimidine deoxyribosides (Mohler, 1964), though contradictory results were obtained by Pollak and Rosenkrantz (1967) using mixtures of several deoxyribosides. Hydroxyurea kills cells specifically while they are in the S (DNA-synthesizing) phase, whereas cells in other phases proceed to the beginning of the S phase and then undergo an unbalanced growth, with synthesis of RNA and protein but not D N A (Sinclair, 1965, 1967). Hydroxyurea can thus be used t o synchronize populations in cell cultures. In vivo, hydroxyurea induces karyorrhexis (nuclear degeneration), megaloblastosis, and cell death in tissues with rapidly dividing cells, especially in the intestinal crypts and bone marrow (Frenkel and Arthur, 1967; Philips et al., 1967). The extensive literature on the subject was reviewed by Philips et al. (1967), who showed that hydroxyurea, N H U and ( a t a higher dose) acethydroxamic acid induced similar biological effects, which were not shown by hydroxy lamine. The N H U also induces methemoglobincmia (Philips e t al., 1964) and higher levels of plasma corticosterone and brain 5-hydroxytryptamine than urethan, whereas urethan induces a greater release of adrenal catecholamines (Spriggs and Stockham, 1966). The pattern of rapidly labeled RNA in the liver and lungs was altered after injection of NHU, probably due to the liberation of ribonuclease from lysozymes (K. Williams, 1967 and private communication). These observations suggest that N H U produces its noncarcinogenic biological effects in a fairly direct fashion, whereas urethan requires conversion into an active metabolite. However, they provide no evidence that the active inetabolitc of urethan is NHU. With regard t o the mechanism of action of NHU, i t seems likely t h a t most of the noncarcinogenic effects of N H U depend on an inhibition of D N A synthesis, although n dircct chemical intcraction with DNA ns discussed earlier remains a possibility.
C. CARCINOGENIC EFFECTS N-Hydroxyurethan is not significantly more carcinogenic than urethan, as would be cxpected if N H U was the proximal carcinogen of urethan. Thus, Berenblum et al. (1959a) first reported t h a t N H U induced lung aclenomas and initiated skin tumors (with croton oil promo-
30
S I U N E T S. MIIiVISII
tioii) iu udult Swiss niicc. A1 doses of 25 nig. lwr mouse, the iiicidence and multiplicity for both types of tumor were similar for NHU and urethan, except that the number of lung tumors per mouse for NHU (0.8) was less than for urethan (2.8-3.4). With 11.8 mg. NHU per mouse, however, the incidence of both types of tumor was only one-third of that for a11 equivalent dose of urethan. Similar results were obtained by Miller e t al. (1960) and Kaye and Trainin (1966). Also, ten injections of 11.2 pmoles/gm. of NHU and urethan into C57BL micc, starting a t birth, induced similar incidences and multiplicities of leukemia and lung adenomas (Boiato et al., 1966). However, when single doses of only 2 pmoles/gm. were injected into newborn SWR mice, NHU induced 0.4 and urethan 2.3 lung adenomas per mouse, and i t was concluded that in both adult and newborn mice the ratio of N H U carcinogenicity to that of urethan drops as the dose is reduced (Kaye and Trainin, 1966).
D. METABOLITES IN BLOOD AND TISSUES The metabolism of NHU in mice as reflected by blood and tissue analyses was studied by the author (Mirvish, 1964, 1966). After injecting 10 pmoles NHU/gm. the blood N H U as estimated by the acid-hydrolysis method reachcd maximum levels within 30 minutes and disappeared after 4 hours, with an initial catabolic rate of 3 times that for urethan. After injection of 14C-carbozy-NHU, however, the blood alkali-labile l4C0, decreased much more slowly than NHU itself. This discrepancy was explained by thin-layer chromatography of ethanolic blood extracts, which revealed three radioactive compounds, identified as NHU, urethan, and NHU-N-glucuronide (XIX). It was shown that urethan reached the same blood level as NHU after 80 minutes and accounted for almost all the I4C after 4 hours. As the maximum blood level of urethan (at 4 hours) was 55% of the theoretical initial level of NHU, and urethan is evenly distributed, conversion to urethan must have accounted for a t least 55% of total N H U metabolism. After allowing for the further degradation of urethan occurring during the first 4 hours, total conversion to urethan was estimated to be 70%. Hydroxyurea is similarly reduced in vim t o urea (Adamson et al., 1965). After injection of I4C-NHU, the level of NHU-N-glucuronide was much higher in the liver and kidneys than in the blood, and higher in the red cells than in the plasma, so that this metabolite is not readily diffusible. The three radioactive compounds were evenly distributed in the subcellular fractions of a liver homogenste. The blood metabolites examined 2 hours after injecting 14C-NHU were not affected by the simultaneous injection of unlabeled urethan or by a prior series of injections of unlabeled NHU, but conversion into urethan was significantly inhibited after injecting SKF-525A. In vitro experiments showed that
URETHAN A N D
N-HTDROXYURETHAN
31
mouse liver homogenates hydrolyzed 14C-carbosyNHU to give 2-3% yields of I4CO,, suggesting that this reaction is a significant catabolic pathway in vivo. Thin-layer chromatography of blood extracts, prepared after injection of 14C-carboxyurethan, showed that a t least 96% of the radioactivity consisted of urethan (Mirvish, 1964, 1966). The known rate of NHU disappearance from the blood does not indicate that the low blood level of NHU after injecting urethan is due to an extremely rapid NHU catabolism. Urethan-treated rats excrete only 0.24% of the injected dose as free and conjugated urinary NHU (Boyland and Nery, 1965). Thus N-hydroxylation appears to be only a minor pathway of urethan metabolism. Since about '70% of NHU metabolism proceeds by conversion t o urethan, and NHU is not more carcinogenic than urethan, it seems probable that NHU acts as carcinogen by conversion into urethan, rather than the converse. Several observations fit in with the hypothesis that NHU carcinogenicity depends on the extent of its conversion into urethan. The relative carcinogenicities of NHU and urethan are similar in newborn and adult mice, and, in accord with the proposed hypothesis, blood analyses in newborn C57BL mice showed similar extents of NHU conversion into urethan and NHU-N-glucuronide as in adults, though the rates of these conversions were about 5 times slower (Boiato et al., 1966). If high doses of NHU are more efficiently converted into urethan than low doses (which appears likely but remains to be demonstrated), the hypothesis would explain why the carcinogenicity of NHU relative to that of urethan dropped a t low doses. I n adult mice the drug SKF-525A inhibits NHU conversion into urethan but not urethan catabolism (see earlier), and in accord with the hypothesis, SKF-525A did not affect urethan lung tumorigenesis in adult SWR mice, but reduced NHU lung tumorigenesis to one-third of the figure for NHU alone (Kaye and Trainin, 1966). Finally, if NHU were the active metabolitc of urethan, the relative noncarcinogenicity of methyl and n-propyl carbamates might be due to a lack of conversion into the corresponding N-hydroxycarbamates, which might themselves be strongly carcinogenic. However, methyl and n-propyl N-hydroxycarbamates were found to be noncarcinogenic in mice (Berenblum, Boiato, Haran-Ghcra, and Mirvish, unpublished work). IV. Urinary Metabolites
(See Tablcl V and Scheme 11)
A. TJRETHAS About 5 to 1 0 7 ~of a dose of urethan is excreted in the urine as measured by 14C or chemical analysis (Boyland and Rhoden, 1949; Beickert,
32
SIDNEY S. MIRVISH
1950b, 1951; Skipper et al., 1951). Urethan-treated rabbits showed a levcl of urethan in the urine which closely followed that in thc blood, and the urine contained an unidentified reducing substance (Beickert, 1951). Sulfates and glucuronides of urethan have not been detected (Tsukamoto e t al., 1963; Boyland and Nery, 1965). Also, 1 4 C - ~ a ~ b ~ ~ y urethan produced urinary W-urea, presumably by reincorporation of 14C0, (Mirvish, unpublished results). Boyland et al. (1963; Boyland and Nery, 1965) investigatcd tlic urinary metabolites of urethan, NHU, and related compounds, mostly in rats (Table V, columns a and b ; Scheme 11))though man and rabbits were shown to give similar results. Small amounts of NHU, N-acetylNHU ( X X ) , and O-acetyl-NHU (XXI) were identified as urethan metabolites. O-Acetyl-NHU was probably derived from N-acetyl-NHU, as this rearrangement proceeded fairly rapidly a t room temperature, and both compounds were readily degraded to N H U by 2N ammonia. The total yield of NHU and its acetyl derivatives was 0.24% of the urethan dose, and the yield of the corresponding compounds from methyl, npropyl, and n-butyl carbamate was even less (<0.04%). The same workers also identified N-scetyl-S-carbethoxycysteine (XXIII) and small amounts of N-acetyl-S-ethylcysteine (ethylmercapturic acid) (XXIV) in the urine, and S-ethylglutathione in the bile. These products were presumed to arise from reactions similar to those of NHU and N methyl-N-nitrosourethan with cysteine, discussed earlier.
B. N-HYDROXYURETHAN The same urinary metabolites were found by Boyland and Nery (1965) for NHU as for urethan, but the amounts of unchanged NHU, acetyl-NHU isomers, and N-acetyl-S-ethylcysteine were larger than observed with urethan (Table V, column b) . S-Ethylglutathione sulfoxide (XXV) was isolated from the bile. The similarity in the amounts of urinary urethan and S-carbethoxycysteine produced from urethan and NHU reflect, in the present author's opinion, the 70% conversion of NHU into urethan, and suggest that S-carbethoxycysteinc was formed more directly from urethan than from NHU. I n contrast, the acetylNHU isomers and N-acetyl-S-ethylcysteine may be formed a t least in part directly from NHU, as more was obtained from N H U than from urethan. The investigation by thc author (R'lirvish, 1966) on tlw urinary metabolites of 14C-curbozy-NHU in the rat (Table V, coluinil c ) sllowe(1 much less unchanged NHU than reported by Boyland and Nery. Sirnilar results were obtained for mice as for rats. A new labeled metabolite was shown to liberate hydroxylamine and glucuronic acid on prolonged acid hydrolysis and was identified as NHU-N-glucuronide (XXII) , possibly
URETHAN AND
33
N-HTDROXYURETHAN
TABLE V ,\~-HYDR~XIURETHAN I N T H E RAT"
1 1 R l N A R S RIhTABOLITES O F LTRE'THAN A N U
Yo rniulit
RIetaholites of uret ha 1i
No.
(4
(b)
(c)
2 2 0 10 0 14
2 5 35 3 9 7
0 8 13 -
18 0 13
1 9 O G -
7 0
Metabolite
I, R VIb
Urethan N-Hydroxyure than N-Acetyl-N-hydroxyureLhari 0-Acetyl-N-hydroxyuret hari N-Acetyl-S-carbethoxycysteine N-Acetyl-S-ethylcysteirie N-Hydroxyurethan-N-glucuroriide
=
XXP Xp
Et
I
XXIIIe XXIV XI*
Pvletaboliteb of A -Iiydroxyureth:t~i
-
-
~~~
a The results were obtained by Boylarid and Nery (1965) (columns a and b) and Mirvish (1966) (column c) and are expressed as percent of the injected doses, which wcre 1.0, 0.4,and 0.53 mg./gm. for columns a, b, and c , respectively. * See Table 11. c See Scheme 11.
NH, * CO * NH,* OH
I
C,H,O,* N. COOEt*
%Ox
+
EtOd
NH. COOEt I HS. CH,. CH. COOH
+
NH,
t
(XM)
OH
(xx)
(VI1
CH,*CO.O.NH.COOEt*
(XXI 1 [ON. COOEt] + %O, + *OH
CO,
+
t NH,OH
I
NH. CO- CH, I EtOOC.S*CH,*CH. COOH*
+
EtOH
*Urinary Metabolite **Biliary Metabolite
(xxrn) NH- CO. CH,
I
EtS- CH,. CH. COOH* (XXIV)
I
0 II
t
y-b
EtS. CH,. CH. COOH**
(as CSH deriv. ) (XXV)
Scheme 11.
mixed with NHU-0-glucuronide. The material was not hydrolyzed by P-glucuronidase and was hydrolyzed only slowly by hot acid, similar to known N-glucuronide metabolites of sulfonamides and the carbamates, meprohnm:ite and ethin:imate (Tsukamoto et al., 1963).
34
SIDNEY S. MIRVISH
Later reports give thc fraction of unchanged NHU excreted in the urine as 13-17oJo of the dose in rats (this figure may include NHU-Nglucuronide) (Philips et al., 1967) and 2-3% in mice (Nery, 1968). I n addition, Nery (1968) reported that the N-hydroxylation of urethan in newborn mice was increased by prior treatment with 3-methylcholanthrene, whereas the effect of SKF-525A depended on the time schedule. Traces of nitrite were found in the tissues after the injection of urethan or NHU, especially aft,er pretreatment with 3-methylcholanthrene. The rate of NHU catabolism (i.e. disappearance) was higher in female than in male mice, and ~light~ly higher in 8-day-old than in newborn mice. The NHU was catabolized by rat liver homogenates and slices, and the catabolism by slices was not oxygen-depcndent and was inhibited by urethan, n-butyl carbarnate, and cyanide. It was suggested that urethan and N H U might act by conversion into free-radical intermediates. I n conclusion, it appears unlikely that NHU is the proximate carcinogen of urethan, though i t remains possible that urethan is converted into an unknown active derivative of NHU, to which NHU itself is not readily converted, or that urethan produces high local concentrations of NHU in specific sites not yet identified. However, it seems more likely that the Scarbethoxy- and S-ethylcysteine derivatives provide models of the presumed reactions with macromolecules leading to carcinogenesis and that these derivatives, in turn, were produced from some as yet unknown active metabolite of urethan.
V.
Conclusions
Since urethan is water-soluble and evenly distributed in the body, the organs affected are likely to be those intrinsically most susceptible to urethan carcinogcnesis and not those where the carcinogen is applied (as with the carcinogenic hydrocarbons applied to the skin) or where the carcinogen is concentrated (as perhaps with acetylaminofluorene and dimethylaminoazobenzene) . The species, strain, and individual organs most susceptible to urctlian carcinogenesis may also be those most susceptible to “spontaneous” carcinogenesis, so that urcthan was said by Tannenbaum (1964) to hasten spontaneous carcinogcnie processes. It has not been clearly established to what extent certain organs and mouse strains are peculiarly sensitive to urethan carcinogenesis, as opposed to carcinogenesis by other water-soluble carcinogens. The considerable difference between C57BL and A mice in susceptibility to urethan lung tumorigenesis is probably controlled by a single gene, but unfortunately its phenotype expression remains unknown. X-irradiation may suppress urethan carcinogcnesis, probably by killing or damaging the initiated cells (in the lung), or may enhance
URETHAN A N D N-I1 Y UHOXYUHETIl AN
35
the process (in the thymus, or in the lung with low doses of X-rays) by itself acting as initiator. Urethan coleukemogenesis seems to be mediated by injury to the thymus, which facilitates virus attack on this organ, and may also involve release of a leukemogenic virus. However, viruses have not been shown to be involved in urethan carcinogenesis elsewhere in the body. Finally, immunological reactions may normally act to suppress urethan carcinogenesis, e.g., in the lung. An important factor, especially in newborn mice, accounting for variations in the susceptibility to urethan carcinogenesis is the time for which the urethan remains in the body, which is inversely related to its rate of elimination. There is fairly good evidence suggesting that uretliaii acts after conversion into an active metabolite, as expected from the lack of known chemical reactions specific to urcthan. This metabolite could act by ethylation or carbethoxylation of cell macromolecules, as suggested by the finding of analogous cysteine derivatives in the urine and bile; but reaction with macromolecules has not yet been demonstrated. The available evidence indicates that the supposed active metabolite is not NHU, which conversely seems to act as carcinogen after conversion into urethan. The considerable chemical specificity for the carcinogenic action of urethan, especially the specific requirement for the ethyl group, has not been explained, though an enzyme with narrow specificity could be required for the transformation into an active metabolite. As a similar specificity also applies to the antileukemic action, these two actions may proceed by similar mechanisms, in contrast t o thc anesthetic effect where the structural specificity is much less. N-Hydroxyurethan shows some interesting cytotoxic and anticancer actions more strongly than does urethan and specifically kills cells in the DNA-synthesizing S phase. These effects appear to depend on a suppression of DNA synthesis, or possibly on known chemical reactions of N-hydroxyurethan with DNA. ACKNOWLEDGMENTS I wish to thank Professors C. Heidelberger, J. A. Miller, and R. K. Boutwell at the McArdle Laboratory for Cancrr Research; and Professor I. Berenblum and my other colleagues Drs. Louisc. Boiato-Chen, Nechama Haran-Ghera, A. M. Kaye, B. Toth, and N. Trainin at the Weizmxnn Institute of Science for their invaluable encouragemrnt and advicci.
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SIDNEY S. MIRVISH
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RUNTING SYNDROMES, AUTOIMMUNITY, AND NEOPLASIA D.
Keast
Deportment of Microbiology, University of Western Australia, Perth, Western Australia
I. Introduction . . . . . . . . . . . . 11. Homologous Disease and the Clinical Syndrome . . . 111. Situations Which Have Been or Could Be Classified as Runting Syndromes . . . . . . . . . . . . . A. Chimcras and Runting . . . . . . . . . B. Thymectomy and Wasting Disease . . . . . . C. Chemical Induction of Runting Syndromc.s . . . . D. Bacterial Infection and Runting . . . . . . . E. Sterile Bacterial Vaccines and Runting . . . . . . I?. Subcellular Fractions and Runting . . . . . . . G. Viruses and Runting . . . . . . . . . . H. Experimrntal Leukopenia . . . . . . . . . I. New Zealand Black Strain of Mice . . . . . . IV. The Important Features of Runting . . . . . . . V. Discussion . . . . . . . . . . . . . VI. Comments . . . . . . . . . . . . . References . . . . . . . . . . . . . Addendum . . . . . . . . . . . . .
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I . Introduction
The original “runt” disease or homologous disease was developed as a laboratory model for the study of immunological mechanisms associated with the grafting of skin and organs and the acquisition of tolerance to histoincompatible antigens. The situation leading to runt disease was originally thought t o be essentially a graft-versus-host reaction (GVHR) . However, host responses almost certainly occur. Simonsen ( 1965) summarizes the present situation regarding the host response, thus, “It is well known that the GVH attack provokes a cellular host response (the nature of which is itself unknown). . . .” The important features underlying both of the above situations are the immunological responses of genetically different tissue. Mature iinmunologically competent cells must be present as one of the systems. These cells are triggered into an immune response by genetic differences expressed as antigens in the second system (Billingham and Brent, 1957; Siskind and Thomas, 1959a; Billingham, 1959; Simonsen, 1962). 43
44
D. KEAST
Billingham et ul. (1956) and Uillingliam arid Brcnt (1957) first reported experiments on the production of immunological runting induced by injection of adult homologous spleen cells into neonates. They outlined the gross pathology and time spans involved in the acute disease. They also noted that runting was not produced by cells other than those immunologically competent, Also in 1957, Simonsen reported on immunological runting produced in chicks by intravenous injection of adult homologous spleen cells into chick embryos. While studying the induction of homologous disease in rats, Billingham et al. (1960) showed that the acuteness of the clinical syndrome could be modified with the source of cell used to induce it. They used spleen, lymph node, and thoracic duct lymphocytes as immunologically competent cells. Gowans (1965), in reviewing the role of lymphocytes in the destruction of homografts, surveys the evidence for the small lymphocytes being responsible for the GVHR in mice. Howard et al. ( 1965) have presented evidence that donor lymphocytes may transform into macrophages and take on a phagocytic activity in the GVHR. Nisbet and Heslop (1962) have recorded the existence of a chronic immunological disease, and Schwartz and Beldotti (1963) suggest thc possibility of a subclinical form. Much of the early work on the GVHR was based on the acute homologous disease (Nisbet and Heslop, 1962) where histopathological damage, although indicative of an autoimmune disease, is not truly of autoimmune origin. However, with the extension of the field to the chronic disease, the possibility of true autoimmune reactions becomes of importance. I n the chronic situation there is thc extreme difficulty of dissociating donor from host icsponses unless complete destruction of one or other of these ccll types can be absolutely verified. Several attempts have been made to do this (Davies and Doak, 1960; Howard e t al., 1961; Hildemann, 1964; Owen et al., 1965; Fox, 1966; Elkins, 1966; Santos and Owens, 1966), but the problem still remains unanswered. A survey of the literature for other systems which may be used to help verify the concept of induction of true autoimmunity in the GVHR revealed an unusual number of situations which have been termed “runting syndromes.” I n view of the association between runting and lymphoma (Schwartz and Beldotti, 1965; Stanley and Walters, 1966; Joske et al., 1966; Stanley et al., 1966b), it is clear that an analysis of runting is an essential preliminary t o a greater understanding of neoplasia. The literature has yielded nine other situations worthy of note. Seven have been classified as runting syndromes and two-experimcn tally induced leukopenia and the 100% development of autoimmune reactions within the New Zealand black (NZB) strain of mice-are closely related.
lZUNTIKG SYNDROMES, AUTOIMMUSITY, AND NEOPLASIA
45
Thc seven classified :is potcntial producers of runting syndroines are: 1. Experimental chimeras. These may be subdivided into (a) parabiotic intoxication (Billingham, 1959; Hasek et nl., 1961), (b) radiation chimeras (Porter and Murray, 1958; Trentin, 1958; Loutit and Micklem, 1962; Loutit, 1965), and (c) the parental F, hybrid situation (Kaplan and Rosston, 1959; Cooper and Howard, 1961; Fiscus e t al., 1962; Hilgard et al., 1965) 2. Thymectomy (Parrott, 1962; Miller and Howard, 1964; Miller and Davies, 1964) 3. Chemical induction (Miller and Davies, 1964; Batchelor and Chapman, 1965; Duhig, 1965; Recd and Jutila, 1965) 4. Bacterial infection (Brooke, 1964) 5. Sterile vaccines (Ekstedt and Nishimura, 1964) 6. Subcellular fractions (Stanley and Keast, 1967) 7. Viruses (Stewart e t al., 1959; Nelson and Tarnowski, 1960; Hotchin, 1962, 1965; Sinkovics, 1962; Stanley and Leak, 1963; Stanley e t nl., 1964)
The two associated situations arc: 8. Experimental leukopenia (Chiba et al., 1965) 9. NZB strain of mice (Bielschowsky and Bielschowsky, 1962; Burnet and Holmes, 1965; Holmes, 1965; Howie and Helger, 1965; Barnes and Tuff rey, 1966)
From these examples, it is still difficult to demonstrate true autoiriimunc responses in any of the situations requiring the introduction of live cells. However, the work of Hilgard et al. (1965) seems to indicate that the immunocyte interaction between host and donor cell is, in fact, the only truly important feature in the GVHR and that other damage is secondary. In the cases where no donor cells are added (e.g., thymectomy, chemical induction, subcellular fractions, viruses, experimental leukopenia, and NZB mice), any immunological damage could be of true autoimmune type. It is these, along with the lcss well-defined GVHR, which may be studied with respect to the development of autoimmunity and the possibility of the subsequent development of neoplasia. Linked very closely to this is the reverse situation, known to occur in both humans and exlwiiiicntal anininls, in which ncoplasia may result in autoimniune rcitctioiis in the host (Kaplail and Sniithers, 1959 ; Dameshek and Schwartz, 1960; Videhaek, 1960; Sinkovics, 1962; Damesliek, 1966a; Aniiat:itions, 1966; K w s t :ind Stnnlcy, 1966). The concepts of nutoim-
46
D. KIGAST
rnunity are now well establishctl, and a coiifcreiicc in 1964 entitled Autoimmunity-Experimental and Clinical Aspects" (1965) under the cochairmanship of Dameshek, Witehsky, and Milgram outlined clearly its vast importance overall. The possibility of autoimmunity escalating into neoplasia, in the form of leukemia and malignant lymphoma, has been accepted for some time and experimental evidence is now mounting that this is, indeed, so (Rosenthal et al., 1955; Kaplan and Smithers, 1959; Dameshek and Schwartz, 1960; Sinkovics, 1962,1966;Bielschowsky and Bielschowsky, 1962; Rask-Nielsen, 1963, 1964; Dameshek, 1964; Burnet and Holmes, 1965; East et al., 1965; Grabar, 1965; Walford and Hildemann, 1965; Schwartz and Beldotti, 1965; Stanley e t al., 1966h; Joske et al., 1966; Stanley and Walters, 1966; Walford, 1966; Dameshek, 196613).Some of the most interesting recent work is experimental evidence that certain viruses, in particular reovirus Type 3, may be able to produce a chronic autoimmune disease which in the appropriate circumstances will escalate into neoplasia in the form of malignant lymphoma (Stanley e t al., 1964, 1966b; Joske et al., 1966; Stanley and Waltcrs, 1966; Stanley and Keast, 1967; Keast and Stanley, 1966; Stanley, 1966; Keast and Papadimitriou, 1966). Much of the experimental evidence on the association of autoimmunity and leukemia is based on the mouse environment. Gross (1951) presented the first evidence that latent leukemia viruses exist in mice. These have now been the focal point of much experimental work (Dmochowski, 1960; Moloney, 1964; Pollard and Matsuzawa, 1964; Gross, 1965; Pollard, 1965; Pollard and Kajima, 1966; Dmochowski e t al., 1966). The problem becomes the exclusion of these when implicating autoimmune reactions, whether cell or virus initiated, in the development of leukemia and lymphoma. There is also some evidence that the leukemia viruses themselves may be able to initiate an autoimmune response (Sinkovics, 1962; Rask-Nielsen, 1963, 1964; Dmochowski e t al., 1966; Sinkovics, 1966). This approach has yet to be explored. The purpose of this article is to select and to examine critically the established autoimmune or pseudoautoimmune situations and their possible roles in the development of neoplasia. It is hoped that the omission of many excellent papers is partially compensated for by the inclusion of review articles, where possible. "
II. Homologous Disease and the Clinical Syndrome
The experimental parameters leading to the production of homologous disease in mice and the clinical and histopathological pictures have been extensively reviewed by Billingham (1959) and Nishet and Heslop (1962). Clinical and I~istopiztliologicnlpictures siinilar to these have been
RUNTING SYNDROMES, AUTOIMMUNITY, AND NEOPLASIA
47
further rtyortc~lfor t , h ~rat, (Rillinghtrti~ t c//., 1960; Stastny and Ziff, 1962; Stastriy ef nl., 1963, 1965b) ; rahhits (Porter, 1960) ; chicks (Simonsen, 1962, 1965; Col)plcmi and Michic., 1965) ; aiid the marsupial quokkas, Setoiiiz bruchyurus (8t:inlcy et al., 1 9 6 6 ~ ) . Hildemann et al. (1964), Stastny et al. (1965b), and Weiss and Aisenberg (1965) report on a wide variety of histopathological damage which can occur as a result of homologous disease, and Stastny et al. (1965a,b) suggest that these are consistent with autoimmune disease in man. Simonsen (1965) reports that i t is becoming evident that immunological reactions resulting from the GVHR are of great variety and that each year more are added t o the list. Thc initial set of signs developing as a result of the GVHR which collectively were considered as the clinical syndrome have, therefore, become somewhat obsolete. However, these are listed as it is these gross macroscopic features which have led to the naming of many other situations as runting syndromes. Classically, homologous disease in rodents is defined as stunted growth, hunched posture, kyphosis, alopecia, diarrhea, ataxia, and early death, early splenomegaly but later splenic atrophy, hepatomegaly followed by necrosis, anemia often associated with a positivc direct Coomb’s test, leukopenia, and atrophic changes in the lymphoid tissues. Even here, all signs are not obligative (Nisbet and Heslop, 1962). Most of the early work associated with homologous disease was carried out on the neonate system. Castermans (1958), Martinez et d . (1961), Olincr et ul. (1961), Stastny and Ziff (1962), and Stastny et al. (1963, 1965a) have shown that homologous disease, or a t least a runting syndrome, can be induced in young adult rats and mice under the appropriate experimental conditions. Castermans (1958) and Oliner et al. (1961) have shown that the onset of an acute phase may be delayed for up to 100 days. JI’alford and Hildcmann (1965) and Walford (1966) present evidence for a possible subclinical situation which leads to early death and a high incidence of lymphoma as compared with untreated control animals. It is possible that thc induction of a latent leukcinis virus has occurred. Nisbct and Heslop (1962) also recorded the possibility of a chronic immunological disease which leads to the definition of an animal suffering such a disease as a chronic runt. Schwartz and Beldotti (1963) have shown that X-irradiation caused reactivation of a subclinical homologous disease. It seems, therefore, that with the appropriate experimental manipulation the homologous disease leading to the immunological runt may be extended throughout most of thc animal’s life. Schwartz and Beldotti (1965) have been able to produce animals suffering a chronic immunological disease which may survive for extended periods of tirnc. Kcast (1965, cited in Stanley, 1966) has also been able
48
D. KEAST
to producc thc chronic lroiuologous diswse, where aiiiinals may survive up to a t leabt 300 days. Tliehc animals are likely to exhibit any one or wvmal of the a r ~ i t efeatures of homologoil., disease a t various ~urddined I,imcs t,hroughoiit tlrc 300 clays. T h t h rriny owur or thc t1isr:is.e may subside to a subcliiiical lcvcl ancl the :iiiiind, apart froiu bciiig pcriiianently emaciated, continues to survive. The work of Howard (1963a,b) illustrates a situation in adult F, hybrids injected with parental immunologically competent cells where the GVHR still functions, but in the absence of the early runting syndrome.
Ill. Situations Which Have Been or Could Be Classified as Runting Syndromes
A. CHIMERAS AND RUNTING 1. Experimental Parabiotic Intoxication The tcchniquc of parabiosis was developed by Saucrbuch and Hcyde (1908). General information here is now widespread and it only seems necessary to record that Billingham (1959) noted a striking similarity between parabiotic intoxication and homologous disease. Eichwald et al. (1960) also noted similarities, Early work has been briefly reviewed by HTisek et al. (1961), and more recently complications of chimerism have been briefly outlined by Loutit (1965).
2. Radiation Chimeras and Secondary Disease With the increasing use of atomic energy and its implications, the study of radiation chimeras and their outcome have become of prime importance. The establishment of chimeras for the treatment of radiation accidents (Loutit, 1965) and following national disastcr in atomic warfare makes their etiology of paramount importance. Porter and Murray (1958) noted rapid weight loss and diarrhea in rabbits surviving primary irradiation and subsequent bone marrow homotransplants. Trentin (1958) linked runting with secondary disease of irradiation chimeras and called them immunological illnesses caused by rcaction of grafted cells against the host. Porter (1960) presented further evidence for this type of illness. Loutit and Micklem (1962) have presented evidence for lymphoid depletion being of major importance in the GVHR leading to the secondary disease, which follows radiation chimeras. Loutit (1965) surveys the situation and accepts the earlier association between homologous disease and secondary disease. He ascribes many of the complications of irradiation chimeras to the GVHR.
49
3. I’nrcnfal Spleen Cell Injection i n t o PI Hybrids and Homologous Diseuse Kaplari arid Rosston (1959) and Fiscus et u1. (1962) have shown that immunologically competent parental cells injected into F, hybrids will produce a runting syndromc identical to homologous disease. This has been shown t o be due to the fact that the F, hybrid is a lymphoid chimera of both parents. The parent cells respond to the cells of the genetic type of the other parent present within the F, hybrid. The F, hybrid is unable to respond due to an immunological tolerance developed during embryonic life. Howard e t al. (1961) and Fox (1962, 1966) used this system in attempts to show donor cell proliferation a s the cause of splenomegaly in homologous disease. Elkins (1966) concluded that there was an interdependence between donor and host cells and that host cells still proliferated in this system. Hilgard et al. (1965) made A strain mice tolerant of F, cells of the A x C57BL/1 type. This was achieved by lethal-dose irradiation. The F, cells could not respond to the A parent, as shown above. If these mice were then injected with A strain, adult spleen cells, the animals exhibited homologous disease. Here, the only cell type t o which the injected A strain cells could mount an immune response was the C57BIJ/1 of the injected F, cells. These cells would not respond because of their immunological tolerance induccd in the embryonic state. Animals made tolerant of the C57RL/l antigens, hy multiple antigen injection on the days immediately following birth, would not exhibit homologous disease when adult syngeneic spleen cells were injected into them. Thus the syngeneic cells cause acute homologous disease in a host where lymphoid cells are unresponsive and of a different antigenic type. This is further supported in experiments where the A cells injected were typed against C57BL/1 cells. The homologous disease produced was more acute and death occurred in eight of ten animals tested. Features in common with the classic homologous disease are that intact lymphoid cells are present and t h a t onc of the pair of the reacting system is precluded from reaction against thc injected cells due to an immunological tolernncc estahlished during fetal life. The results indicate that immunological attack on the lymphoid system alone is enough to inducc homologous disease. Further, it scems that the cells attacked must be established within the host tissue. Tlic authors suggest that there may bc 1oc:ilizetl hypersensitivity re:ictionh occurring which pi*oclucc 1or:il tosin coiic.ciitrntiotib high enough to iiitluce the geiieral niatiifestatioris of homologow disease. Howartl 1961 ) lias used :](hilt hyhrid mice injected with parental spleen cells to study pheiion~ena of the GVIIII. He has shown that there is a transitional increase in the phagocytic Ij’,
50
D. KEAHT
activity of the reticuloendothelial system (RES) as measured by the clearance of colloidal carbon from the blood. The phagocytic activity commences after 7 days and reaches as high as 10 times normal a t 15 days and then falls to normal by 20 to 40 days. The greatest increase in number of active RE cells was found in the liver. Cooper and Howard (1961) found that during the course of the increased phagocytic activity the mortality rate from bacteremic infection of Diplococci pneumoniae was drastically reduced and, although mortality to Salmonella typhimuriurn was not reduced, the survival time slightly increased. Howard (1963a,b) suggested that the phagocytosis was a reaction of the host to cell destruction in the lymphoid tissues attacked by the donor cells. He also pointed out that the activation of the RES following GVHR results in increased susceptibility to the lethal effects of other immunological processes. Howard et al. (1965) presented evidence for the conversion of lymphocytes into macrophages during the GVHR. Using a T6 marker chromosome technique, they showed that there was an increase in mitosis of the Kupffer cells of the liver and that these were, in fact, donor cells. These cells were morphologically similar to macrophages and their precursors and were capable of phagocytosis.
B. THYMECTOMY AND WASTINGDISEASE The immunological function of the thymus has been reviewed by Miller (1961, 1964, 1965a). Parrott (1962) likened the wasting syndrome, developing after thymectomy of the neonate t o that of homologous disease. Miller and Howard (1964) recorded that many of the features expressed in the wasting disease were reminiscent of the GVHR. There was an increase in phagocytic activity as measured by carbon clearance from the blood and there was also an increase in the number of Kupffer cells in the liver. They noted that these features may be either dependent on autoimmune or infective processes. Miller and Davies (1964) summarized the historical development and the histopathology of this field and noted that there is a similarity between homologous disease, secondary disease of radiation chimeras, and the wasting syndrome following thymectomy. McIntire et al. (1964) and Wilson et al. (1964) noted that in axenic (germfree) mice the characteristic lymphopenia occurs but that the wasting disease does not develop. The lattcr workers postulated the wasting as a result of bacterial or viral invasion or both, whicli procceds uncontrolled in the normal state. This was somcwliat substantiated when subsequent contamination of the germfree mice by the ilormal bacterial population led to the appearance of the characteristic wasting. However, the former workers found that runting was induced when adult homologous spleen cells were injected into germfree mice. They suggest that
IWNTISG S Y S I ) R O M E S , AUTOIMRIIJNITY, AND NICOl’LASIA
51
:tiiI~iiniiiiiiiic priiicipI(~ :itx* iioL fiinrt iotiing :IS piini:iiy factow i i i the :ic.iitc stage in convcntioti:il :iiiinials hiit that cell breakdown due to invasion by bacteria or viruses leads to the primary features of the wasting syndrome. They do not exclude the possibility of autoantibody production against cell products released as a result of such a bacterial and viral invasion. Weir (1963, 1964) has shown t h a t autoantibody production is possible as a result of release of abnormal amounts of intracellular antigen into the circulation, and Pinckard and Weir (1966) have shown that the antigen is primarily associated with the mitochondrial fraction as prepared by differential centrifugation methods. Richter et al. (1966) have also presented evidence that immunization of rats with homologous and heterologous liver suspensions can induce the production of autoantibodies. Dalniasso et al. (1964) and Rieke (1966) have shown that thymectomy of mice and rats a t birth causes a decrease in the potential of remaining spleen cells and of thoracic duct lymphocytes to initiate homologous disease in the appropriate environment. This suggests t h a t cells surviving thymectomy are unable to mature to become immunologically competent and initiate the GVHR. Archer et al. (1964) and Sutherland et al. (1964) have shown that the appendix, a t least in the rabbit, may play some part in the development of the immunity system in the absence of the thymus. This was further substantiated by the work of Kellum et al. (1965) where irradiation of thymectomized and thymectomizcd-appendcctomized rabbits could lead to autoantibody production as measured by the direct Coomb’s test. Thus the cells t h a t are “inconipletc” in some way may be able to mount autoimmune responses within the parent situation; this may or may not need to be triggered by availability of antigens suggested above. Miller et al. (1965), Taylor (1965), Metcalf (1965), and Miller (1965b) have shown that thymectomy of adult mice may lead to a decrease in immunological responsiveness. The onset of this decrease is delayed for some 6 to 12 months. This represents a chronic situation of lymphoid depletion and could prove t o be a useful expcrimeatal situation for the study of the development of autoimmunity and its possible escalation into leukemia. Miller (1960a,b) and Harris (1965a) have shown that leukemia virus may be associated with the thymus. Miller (1964), Furth et al. (1964), and Law (1966) have discussed the rolc of the thymus i n lcukemin and possihle antibody implications.
C. CHEMICAL INDUCTION OF’ RUNTING SYNDROMES Miller and Davies (1964) cite the use of 19-nortestosterone to retard the development of the bursa in the chick embryo; this results in a wasting syndrome similar to homologous disease. Batchelor and Chapman
(1965) i q o r t on thymic involution and Iyniphopc~nia dcvclo])ing as :i result of estrildiol implantation in mice. The results of tlw horniotw treatment can be modified to give an acute runting syndrome if immunization against an antigen is commenced shortly aftcr the hormone injection. It is suggested that the artificial immunization increases lymphocyte turnover and that this accelerates the appearance of florid runt disease. The authors record that the acute disease resembles “runt” disease. Balner and Dersjant (1966) have presented cvidence that a sex difference exists as regards the runting produced in thymectoniized mice, and they suggest that the natural hormonal pattern may be of importance. Duhig (1965) reports on the production of a wasting disease induced by hydrocortisone acetate injection of micc 4 days of age; this disease could be classified as a runting syndrome. The author c o n d c r s the results to suggest that iiifectious processes contribute to the poor development and early death of thc niice. Reed and Jutila (1965), using cortisol acetate injection, noted a similar syndrome and there was a marked lowering of mortality when the same treatment was applied to germfree mice. The mortality could subsequently be increased by monocontamination of the germfree mice by Escherichia coli, suggesting that infectious principles were again involved in some way.
D. BACTERIAL INFECTION AND RUNTING I n 1964, Brooke reported on a series of experiments involving homologous diseasc where a runting syndrome could be passaged, and the potentiality to runting apparently incrcascd on passage of spleen cells. Subsequently, Salmonella typhimurium was isolated from the system. I n a second series of experiments, the criteria for definition of a runt were further defined and included the splenomegaly and enlarged liver with areas of necrosis characteristic of homologous disease. Active and passive immunization against S.typhimurium protected animals against runting, whereas injection of adult isologous spleen cells, plus homologous spleens containing S. typhimurium, did not decrease the incidence of runting. These results all suggcst that the S. typhimurium was the cause of runting. Runting could not be induced by 8. typhimurium injection into young adult mice. The author raises the question of whether all thc runting described in the literatui-c is t,he result of infection and not of immunological reactions.
E. STERILEBACTERIAL VACCINES AND RUNTING Ekstedt and Nishimura (1964) present results from experiments on mice of protracted intraperitoneal (IP) injections of sterile bacterial vaccines, prepared from Staphylococcus aureus and Group A (Type 30)
HUNTING STNDROhIL5, hUTOIMMU”ITY,
AND NEOPLASIA
53
streptococci. The initial injection was before the animals were 24 hours old and the subsequent course of IP injections was given every other day for from 3 t o 5 weeks. As boon as the injections werc stopped, animals which had apparently developed as runts gained weight and became indistinguishable from normal animals. Electrophoresis of serum yielded no differences or deficiencie. in serum protein patterns when compared with noriiial controls. There heems to be no immunological damage; the only feature allowing the c1:tssification of these animals as runts was retardation in growth as compared with broth-inoculated control :inimaIs, which had developed normally.
F. SUBCELLULAR FRACTIONS AND RUNTING Classically, homologous disease has been accepted as being associated with cell-bound properties of the inoculated intact cells (Nisbet and Heslop, 1962; Billingharn and Brent, 1957; Simonsen, 1962). Stanley and Keast ( 1967) report on preliminary investigations which suggest that subcellular fractions may be able to induce runting indistinguishable from homologous disease. A light mitochondria fraction prepared from n rcovirus Type 3 induced lymphoma (2731/I,) has produced both lymphomas and a runting syndrome. The fraction is apparently cellfree and the runting potential secms to Iw closely linked with the ribonuclease (RNaxe) sensitivity of thc fraction. Intriguing possibilities exist in that the runting may be associated with ribonucleic acid (RNA) , the mitochondria, the lysosomes, thc mici osomes, and, possibly, with antibody present a s a further contaminant. If the preparation is injected into mice of another strain, these albo develop as runts and muscular dystrophy :kssociated with acute histiocyte infiltration is also present. Willoughby ct (11. (1963) obtaincd an extract, lyrnph node permeability factor (LPF), froin guinea pig lymph notle ccllb which apparently aided the devclopincnt of u dclayecl inflailmatory rcsponse. Willoughby et al. (1964) suggcstctl that the activc constitucnt of the extract was a n RNA. Willoughby nnd Spector (1964) presented furtlicr information on the LPF (LNPF) and suggested that, although its chcniical characteristics had yet to be defined, it seenietl to be nssociatcd with delayed hypersensitivity and autoimmune reactions. Walters and Willoughhy (1965) haye shown that this factor is more witlesprcacl in tissue than was first thought and may be extrnctetl from most nucleatcd cells of the rat. The activity of these extracts varies considerably with ccll type but extracts of lymph nodes, from fetus through to adult, seem to contain the most LNPF. Willoughhy :knd Walters (1965) present further evidence that this factor is closely nssociutcd with RNA, and Boughton (1965) suggests that the LNPF may be a n RNA of mol. wt. > 10,000. T h e is also some evidence that
54
D. KEAST
RNA is associated with the typing of cells to respond to immunological stimuli (Friedman, 1964, 1966; Malpoix, 1964; Cohen e t al., 1965). The lymphoma 2731/L is often associated with splenic and thymic atrophy with host animals developing a runting syndrome (Stanley e t nl., 1966b; Keast and Stanlcy, 1966; Joskc e t nl., 1966). The RNase sensitive factor in the light mitochondria may he an RNA citpahle of typing cells to moiint an immune response which, under the appropriatc conrlitionh, will lead t o runting. Lawrence (1955) and Lawrence et al. (196Oa,l)) have been able to produce delayed &in hypersensitivity reactions hy subcellular fractions and the active component seemed to he an RNA. Wilson and Croshy (1962) have prescnted some evidence that sulm?lluIar fractions may hc ahle to yield cytotoxicity. T h y notc that thc cytotoxic factor was variable, lacked specificity for the initiating system, an(l functioned in the nhscnce of complemcnt and that the mitortioncliki fraction seetnctl to he most active. The work of Pinckard and Wc4i. ( 1966) shows tliat tlie mitjochonclria havc thc potent,ial to initiate auto:mtibody production. However, whcthcr tliis could be of thc arutc tylw required under the cxpcrimental conditions of Keast tind Stanley ( 1966) has yet t o be determined. Holm (1966) has prescntcd cvidence for a cellmediated type of autoirnmunc response which seems t o he similar to that of Moller (1965) and Mollcr ~ i n dMiillcis (1965), :uid this iii:iy follow typing by an RNA.
G. V I R U ~ E AND S RUNTING Stanley e t nl. (1953) ibolated from an aboriginal child, 2Yl years old, a virus which produced a characteristic acutc syndrome when inoculated into neonatal mice. They named this 1iep:ttocnccphalitis virus (HEV), and it was Iatcr identified as a rcovirus Type 3 (Stanlcy, 1961). Stnnley et nl. (1964) reportcd in detail on a chronic diecasr which could develop in survivors from t h r acute pIi:ise (Walters ct al., 1963). The cliarartcristics of this chronic phase W(W those of a runting syndrome, and the clinical findings and Iiistol)athology ~ l i o w c ~a ~marked l sinii1:trity to homologous disease. It mas posttul:itcil that the disease was citlier a chronic virus infection or of an immune or autoimmune type. Furthcr cviclencc associatiiig the chronic phase of the d i w w wit11 immunologicd mcch:inisms was presented by Joskc c t 01. (1966). Onc result was the (levclopment of a lyniplioma (Stanley et ol., 196611). Strwart et al. (1959) rcported in a paper on tlie induction of neoplasms by polyoma virus :I set of signs which might develop after virus infection. These were very similar t o those involved in irnniunological runting. The runted animals often presented finally with a neoplasiii. Hotchin (1962) has also discussed a runting-type syndrome which may develop into clii,onic runting. This
is associated with lympliocytic choriomeningitis (LCM) virus. Nelson and Tarnowski (1960) report on the association of a murine hepatitis virus and a runting syndrome. However, they suggest the virus may have been a reovirus Type 3. Stanley and Leak (1963) brought together these references in a n attempt to draw attention to a possible underlying immunological damage resulting from virus damage t o an immunologically immature system, which might then allow the establishment of a clone of cells which would react against the host. Sinkovics (1962, 1962-1963) has presented evidence which may associate a m u r k hepatitis virus and leukemia viruses with runting syndromes, and RaskNielsen (1964) also put forward a similar hypothesis regarding work with a leukemia virus. Hotchin and Collins (1964) and Hotchin (1965) lisvc presented further cvidcnce from the LCM virus system where the runting produced may he interpreted as the result of an autoimmune response induced hy the virus infection. Cremer e t al. (1966) have shown that rats injected a s neonates with Moloney virus develop impaired antibody response to the subsequent injection of sheep red blood cells. This suggests impairment of the irnniunological system. Mims (1964) has shown that the cells of the RES of an animal are intimately involved in the pathogenesis of some virus discascs. Papsldiniitriou (1966) has shown that reovirus Type 3 can multiply in the cytoplasm of lymphocytes in vivo. Presence of virus and its replication in organs of the body may be the result of virus-white cell intcractions following the initial infection. Such intcractions may also hc responsible for the initiation of the situations outlined above.
H. EXPERIMENTAL LEUKOPENIA Cliib:i c t al. (1965) producccl leukopcnia in dogs by inducing an active state of immunity against their own lymphoid cells. The lymphoid cells were made antigenic by coupling them with liuman y-globulin by diazotization. Humoral and cell-bound antibodies agsinst the animals’ own lymphoid cells were demonstrated, and the peripheral lymphocyte c.ouiit, could he reduced hy 40 to 90%. Splenic. :itrophy and lymph notlc :atrophy were noted, and penicillin G and streptomycin were used as a prophylactic measure against infection. Gross features of general health of the animals were not given ; however, the antibiotics may have prevented this situation from developing some of the more acute signs of a runting syndrome. Although thc ages of the animals were not given, this situation could be classified as one with the potential for producing a lunting syndronie, prohably indistingui~li:~I,Ic from homologous disease. The important feature here appears to be the autoimmune nature of the discasc processes. Snclis et al. (1964) and Nagsya and Sieker (1966)
56
D. KEAST
have produced a similar situation by the injection of antilymphocyte serum. The use of this is probably limited, a s illustrated by Batchelor and Howard (1965). 1. NEW ZEALANDBLACKSTRAINOF MICE In the NZl3 strain of mice as developed by Bielschowsky and Bielsclrowsky (1962), all imirnals, both male and female, develop autoiiiiniune reactions in adulthood. Tliese animals probably present the hest cxperimental niodel of spontaneous development of autoimmunity available a t thc prcsent timc. Bielschowsky and Bielscliowsky (1962) noted a high incidrnce of ni:ilign:int lyinphonia developing along with the autoimmune reactions. Howie and Hclger (1965) have discussetl inany of the :iutoimniune reactions of the NZB strain of mice and show that certain of these reactionb may be transfei-red by hybridization of the NZB to othcr strains of mice. Burnet aiitl Holinee (1965), East e t aZ. (1965), aiid Mellors (1966) have all shown that thcre is close association within the NZB tnice I)ctween autoiiiiiiiunit~yand ntwpl:i&. IV. The Important Features of Runting
The situations discussed above which have been called runting syndromes, or which could bc classified as runting syndromes, indicate that the clioicc of thc word runting was, in fact, an unfortunate one. The results, i n general, point to ttic unspecificity of the macroscopic features and that they may bci the results of many different causes. The retardation of growth, tliarrhca, hunched posture, and ataxia, which Icad to the runted animal, niay t)c due to iincontrolled bacterial ant1 virus infection resulting froin destruction of thc normal immunity mechanisms which are directed against such infections (Brooke, 1964 ; Wilson c t nl., 1964; Parrott, 1962; McIntire e t aZ., 1964; Duhig, 1965; Rccd :ind qJutila,1965). Although rel’orts of work with germfree animals suggest a close :iasociatioii between natural bacterial commensals and runting, thcre are, apart from the work of Brooke (1964), no reports of isolation of bacteria from the tissues of runted animals. The work of Cooper arid Howard (1961) and Howard (196313) suggests that while the phagocytic system a t the critical stages of GVHR has ail increased potential to r c n i o ~ ebacteria, the aniinal as a whole becomes more sensitive t o bacterial endotoxins. Bacterial and metabolic toxins may be the cause of a general toxemia and, although bacterial toxins are excluded in the germfree state, metabolic toxins and those produced as a result of delayed hypersensitivity reactions could collectively be important in causing an animal to develop clinically as a runt. McIntire e t al. (1964)
RUNTING SYNDROMES, AUTOIMhlTTNITY, AND NEOPLASIA
57
have shown that injection of itdult spleen cells into germfree mice does cause the animals to develop as runts. For the most part, the I-tisto~)atliologic~~l 1)icture indicates one of immunological tlmiage and, to tlntc, this tins I ) c w taken to he the result of the GVHR (13illitigh:im : i i i ( I 1 3 i w t , 1959; Siskind and Tliomas, 1959a,1~;Nislwt :tiit1 Heslop, 1962; Sirnoiisen, 1962, 1965; Stastiiy et d, 1963, 19651); Gow~tns,1965). Tlicli*ciis, however, iitcrensiitg evidence that, true :iutoiinmunc tnt~clianisins:IIY also opeiating. It may he that, in the acute ruiiting, the GVHR is of I)rime iinportancc with pscu(1o:iutontitibodies (isoantit)otlics) being itivol\~ed.In the chronic phases of homologous diseasc, true autoinitnuno mechaitisrns could be the important featurcs. Hunior:il aspects of the GVHR certainly exist, and the study of these is :i f i t b l t l i n itself. The pioneer work of Gorer and O’Gorman (1956), O’Gotmiti (1960), and Stetson :itid .Jcnseii (1960) has illustrated tlteir iinlmi-t:inco. In vitro stutlies have intlitxtccl that the antibodies arc tlepmrlctit oil coiiiplrmciit for tlicir cytotosicity (I3oysc et nl., 1962a,b; V:trgues, 1965; \Vigzcll, 1965; I3oros et ~ l . 1964). , However, Caren and Hosenberg (1966) have shown tli:it, homograft rejection is not affected by a genetic I:tck of compleincttt i n the i l l vivo system. Posit)ive direct Coonib’s tests antl histopathological lesions associated with thc spleen and lymphoitl tismcs, as well as with the liver suggest “autoantibody” production. Many of the features are reminiscent of delayed hypersensitivity reactions, and the appnrcnt infiltration of histiocytes a t :ireas of necrosis leads to speculation as to whether these cells are there as :I rehult, of, or are the tlirect cause of, the necrotic areas. The spleiioinegaly associated with :mite runting has been the cause of much controversy. Simonsen ( I 957) rcportctl successful serial transfer of splcnomegaly which was thought to be due to rapidly dividing donor cells. Later, Burnet antl Boyer (1960) :ind Pnpermaster et al. (1962) were unable to maintain the serial propagation of splenomegaly for more t h i one or two passages under their expcl*imental conditions. Simonsen (1965) reviews inore recent work in this fieltl antl comes to the conclusion that the reactions involving thc production of the splenomcgaly, in chicks a t least, arc very complicated. H e draws a general conclusion that the GVHR whirli takes place in thc original host seems to cause ail acceleration of iiiimuno1ogic:tl maturation of the host cells. These cells then protlure :i GVHK of their own in the next host. This inirne(liate1y suggests the clt~vt.lopnient of :iutoimmune mechanisms. HOWCVC~, there seenis t o be :i lower age limit 1)elow which the response of the system becomes varial)le. D:tvics and Doak (1960), using a chromosome marker system of iiiitniinologic:~11ycompetent mouse spleen cells, showed that, a t the height of hplenomcgaly, the spleeiis exhibited a high rate of divi-
58
D. KEAST
sion of cells but that these were host cells and not donor cells. Owen et al. (1965) also using a similar system found that the initial increase in spleen weight was largely of host origin. However, when the maximal weight increase was occurring, the rate of proliferation of donor cells exceeded that of the host. Finally, under their experimental conditions, no proliferation of donor cells occurred within the host spleen. Fox (1966), also using a chromosome marker system for the most part, substantiated these results. However, if the host animals had previously been irradiated, the injected cells continued to colonize the lymphoid tissue of the host animal. Hildemann (1964) has shown that in the C57BL- A/,J neonate system, C57BL cells cannot be detected after 48 hours, although the A/J mice still develop as runts. Nowell and Defendi (1964) studied the development of runt disease in rats from 4 to 21 days of age. Their system was also based on a chromosome marker tcchnique. They found that donor cell proliferation was significant in lymph node enlargement but that the splenic enlargement was caused by the host cells. They further suggested that interaction of the two cell systems is fundamental for the pathogenesis of runt disease, even though the exact mechanism is still unknown. Fox (1962) and Howard et al. (1961), using a parent + F, hybrid system to test the GVHR, found that F, cells still proliferated even though the F,-to-parent response is precluded. Elkins (1966) concluded that in this type of system there is an interdependence 1)etween the donor and host mononuclear cells. The donor cells initiate and maintain the GVHR evcn though host cells proliferatc. Auerbncli and Globerson (1966) using an in vitro system found that splenic fragments undergoing a GVHR exhibited rapid cell proliferation. Here, thymic and hormone effects, as well as the migration of cells to the splenic fragment, are excluded. The “splenomegaly” can also be induced in the absence of entry of any cells into the explanted fragment. Wilson (1963) described work on an in vitro phenomenon between immunologically activated lymphoid cells against homologous target cells which itsults in the death and lysis of both cell types. This is known as “allergic death.” I n his system, however, although the activated cells clustered around target cells, an allergic death did not occur. Sinkovics and Howe (1964) and Hildemann (1964) have described in vivo situations involving runting where allergic death could explain the final tlisappearance of donor cells and the splenic atrophy in the later stages of acute runting. Weiss and Aisenberg (1965) have produced electronmicroscopic evidence for the participation of lymphocytes, plasma cells, and histiocytes in tissues of the thymus, spleen, and lymph nodes of rats suffering from runting. They interpret these results as evidence for im-
RUNTIKG hTNDROhCEd, AUTOIAIMUNITT, AND NEOPLASIA
59
nici(li:ite nricl delayed hypersensitivity rcactions being manifested a t the same time i n tlicse tibsiics. Fi,iihaiii (1966) has shown t h a t endotoxin from Escheticlrctr coZi i h e:i1):d)k of iiitluciitg crytliropoiesis and subsetluent spleiiic ritlargemeiit i n niicc. If inet:il)olic*and bacterial toxins are involved in Iioinologou:, clise:tw :ih t l i m i sl pivviously, then the type of splenic cnIa~y,t~ment,i rsultiiig 1r0111 erytlii,opoiesis, caniiot he exclutled especi:tlly a s one of tlic cltnr:tetrt*istic,s :Lssociatctl with many of tlir niurine lrukcniia v i r u s r ~i h , in f w t , splenomeguly (Gross, 1965). Purtiitwnore, Wootlruff :in(I Syiiies ( 1962) have suggested t h a t splenoincgaly may develop in tiirnoi~-l~caring mice clue to chronic aiitigcnic stimulus by new antigeii associat (lcl with tlic tumor. The existence of tumor-specific :tntigenh Iias I ) c w i cht:il)lihltctl for some neopl:isnis (Gorer c.t al., 1962; Old :incl I3oysr, lY64; Oltl et ( 1 1 , 1964), and u review of i w e n t work i n t l i i h ficltl has bct'it prcwntctl by Haddow (1965). Siskintl :itit1 T2ioin:vi (195Yl)) ant1 Nisbc't :ind l-leslol) (1962) have reviewed the hematology of t h :iciitc homologous tlisrahe. The white cell picture is w r y v:iri:tbIe nntl Itakopcni:i ( l o t + iiot secni to I)e uitiversal. As describetl previously, splciiomcygily c:in lw nssociatcvl with high mitotic rates of lyniphoicl cells. 11I espective of whether thehe are host or donor cells, it may be expected that overflow to the circulation is likely and, hence, thc circulating wliitc cell picture may l)c variable. However, a t the onset of splenic ancl lynil)lioitl ati'opliy oiic might, expect Irukopcnia to tlevelop. Koltay et rrl. (19G5) have shown tlint the sei'iiin proteins may be drastirnlly :iltciwl during the course of the GVHH.
V. Discussion Tlic study of homologous tliscasc expressed primaiaily as the GVHR presents to the experimenter a clinical syndrome which, on analysis, c:in be shown to be the result of many different c:iuscs. Of the scven situations cliscussccl wliich may be classified a5 runting syndromes, only two (Brooke, 19G4; 11:ltstetlt and Nishimura, 1964) present 110 immediate cvidence of immunological involvement. It seems, therefore, that the imniunological status of the host is the critical feature overall. Further :inalysis of the imiaining five runting syndromes indicates that, with the exception of tlic syndrome tlcscribetl hy Ililgard e t al. (1965), the direction of thc permancnt immunological response is against the host aninial. It is tlificult to dccitlr mlicther tlic animals used by Hilgard et al. (1966) h l i ~ u l dbe coiisidwed as A stixin mice or as A x C57BI,/l as i ~ g n r d stlicir txpectcd responwcs to the homologous tlise:ise. I f thcy :ire consitlered :is A X C5713TJ/I mice, then it must also be acceptctl that only the lymphoitl system is really important in the production of IiomoIogous disease. Tlic clclayed liypeisetisitivity reactions to other ce1lul:tr
60
D. KEAST
antigens are secondary effects but these antigens must be present for the disease to run its full course. Establishment of a chronic situation may induce the injected A strain cells to mount an autoimmune response to host tissue and this would further support the concept of homologous disease being capable of initiating true autoimmune responses. I n the case of homologous disease, it was originally thought that the donor cells were directing the sole inimunological response against the host animal. The acute homologous disease has been shown to be dependent on cell numbers (Nisbet and Heslop, 1962). Therefore, it should be possible t o reduce donor cell numbers to give a chronic homologous disease. Nisbet and Heslop (1962), Oliner et al. (1961), Walford and Hildemann (1961i), Schwartz and Beldotti (1965), and Keast (unpublished) have :dl obtained evidence that this does occur. However, the gross clinical picture of the acute phase may not be expected to persist, and animals may appear normal for much of the course of the chronic disease. Similar situations are made use of in the treating of irradiation chimeras (Loutit, 1965). Neonatal thymectomy, chemicals, subcellular fractions of some leukemic cells, and certain viruses induce runting syndromes for the most part indistinguishable from homologous disease. These are produced in the absence of addition of donor cells. If these syndromes involve immunological mechanisms, then they must be true autoimmune responses. The viruses, thymectomy, and chemical treatments can be modified to give chronic immunological situations similar in many respccts to those descrihed for homologous disease (Stanley et al., 1964; Batchelor and Chapman, 1965; Miller e t al., 1965; Taylor, 1965; Metcalf, 1965; Miller, 1965b). There is now ample evidence that in honiologous disease the host makes some cellular response (Davies and Doak, 1960; Howard et al., 1961; Fox, 1962; Simonsen, 1965; Owen et al., 1965; Elkins, 1966). This may be interpreted as n response to repair damage done by donor cells (Howard, 19634. However, if the phenomenon of “allergic death” (Wilson, 1963) does occur in v i m (Sinkovics and Howe, 1964; Hildeinann, 1964), then weight of numbers would yield destruction of donor cells (Hildemann, 1964). In situations where this may occur, animals still die of runting. Thus a situation presents itself which may be interpreted as one of acute autoimmunity leading to death. I n the chronic situation, therefore, a true autoimmune disease may develop which can persist throughout the animal’s life. Nisbet and Heslop (1962) using chronic homologous disease and Stanley et al. (1964) using reovirus Type 3 induced runting have obtained evidence of autoantibody production as expressed by positive direct Coomb’s tests and greatly reduced hemoglobin content of blood.
RUNTING SYNDROMES, AUTOIMMUNITY, AND NEOPLASIA
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The histopathology of the chronic reovirus-induced immunological disease (Stanley e t al., 1964) and homologous disease (Billingham and Brent, 1959; Gowans, 1965; Simonsen, 1962, 1965; Stastny et al., 1963, 1965b) is of iinmediatc and delayed hypersensitivity and, in the case of the reovirus Type 3 induced runts, must be of autoimmune type. The histopathology of the acute runting produced by subce1lul:lr fractions of leukemia cells is also comparable t o that of acute homologous disease (Keast, 1966). The development of the true autoirnniune reactions lias yet tJo be :malyzetl. It may Le that estensive damage to the host’s immunity system both encournges n i i d :~llowsan aberrant clone of cells to develop and mount an immune response directed against the host. I n the case of virus-induced autoimmunity the virus may be a direct cause of the extensive damage, may cause cell transformation t o yield the initial aberrant stem cell, or both. Such a cell type could be one which has lost some antigenic marker. It would riot be recognized by the host as “foreign” but, because of its lack of antigen, it may now consider the host as foreign and mount an immune response :kgainst it. Caso (1965) reviews the work of Green and his hypothesis, whereby the above situation may lead a leukemic cell to produce nutoantibodies. A similar situation might well lead to an autoinirnunc reaction of the delayed hypersensitivity typc. There is now evidence that antigenic differences involved need only be very small (Leskowitz e t nl., 1966). There would be permanent antigenic stimulation present for this type of cell and, as Dameshek and Schwartz (1960) and Danieshek (1966b) have suggested, autoimmunity might eventudly escahte into leukemia. Schwartz and Beldotti (1965), Walford and I-Iildemann ( 3 965), Kenst (1965), and Walford (1966) have all obtained experimental evidence that this may be so. These workers have established chronic homologous disease which eventually escalates into leukemia. There is thc possibility that a latent leukemia virus has been induced by the chronic situation. This is almost certainly the case in the work of Walford and Hildemann (1965) and may also be so in the C:LSC of Scliw:irtz : m l Beldotti (1965). A lymphoma (Keast, 1965) induced by the passage of spleen cells from a chronic immunological runt 273 days old into neonate mice of the same strain is being tested for the presence of leukemia virus. Electron-microscopy studies on the lymphoma cells, spleens of animals bearing thc lymphoma, megakaryocytes, and plasma pellets havc to date not shown the presence of virus particles (Papadimitriou, 1966, personal communication). The results from the inoculation of cellfree extracts of lymphoma cells into neonate mice are as yet unavnilable. Rask-Nielsen (1963, 1964) presentcd evidence for the induction of leukemia which she considered to havc resulted from
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autoimmunity involving virus infection. Unfortunately, she was using a leukemia virus which makes interpretation of underlying principles dificult. However, there is the possibility that leukemia viruses 11i:~ybe :hie to initiate autoimmune responses (Sinkovics, 1962, 19GG; T)nioc.liowski e t al., 1966; Crenrer e t al., 1966). .Joske et ul. (1’366) arid Stmley et (11. (1966b) have presented evidence whcwby virus-inclucctl nutoiniinuiic disease can escalate into leukemia. ‘rhcrc seems to be 110 :Lssuci:itiun with induction of a latcnt leukemia virus (Keast ant1 Strbtilt,y, 1966; Keast and Papadiniitnou, 1966). Interesting particles arc’ lwehrnit i i i the cell but are considered to be cytoplasmic vesicles IPtij)titliniiti.ioii, I966 ; Stanley e t nl., 1966a). The work of Bielschowsky tind Biclwliuwsky ( I 962), B u t w t alicl Holmes (1965), East e t al. (1965), antl Mellors (1966) with n NZIZ strain of mice also illustrates that autoimrnunity and 1cukcini:i may be closely related. Aleutian mink disease (Gorliam e l al., 1965; Lender e t al., 1965; Helmboldt and Keiryon, 1965; Williams, 1965) and its tixiisfer by cellfree extracts (Porter e t ul., 1965; Burger et al., 1965) suggests a further direct relationship between virus, antibodies, :md a leukemic state closely resembling multiple myeloiria (Keiiyoii, 1965 ; Daniwhc~k, 1966b). Mims (1964) has reviewed the role of the cells of thc RES in tlic pathogenesis of certain virus diseases. Presence and multiplication of virus in these cells m:iy rcsult in modification to the coursc of disease and the organs that are infected. There is rilso the possibility that the virus may cause gross niodification of these cells. If such modification takes place in the areas of stem cell tlevelopnient (bone marrow; Tyan and Cole, 1965) or areas responsible for selection of acceptable cells (thymus; Miller, 1965a), then i t may lead to the development antl retention of cells which can mount an autoimmune response of some kind. Tyaii :tnd Cole (1965) have presented evidence that the bone marrow may be the site of synthesis of precursor immunological cells arid t1i:it the thymus provides the critical site for the maturatioli of these precursor cells. The work of I):tlniasho et trl. (1964) and Miller e t wl. (1966) suggests that this may be so. It is j)ossil)lc that the maturation of the cells is mediated by means of a thymic humoral factor (Miller, 1964). Virus invasion of the neonate systeiii may, therefore, result in dairragc to the thymus, to the bone marrow whew the precursor cells are developing, or to both. Subsequently, autoimmunity may develop which coultl eventually escalate into leukemia. Papadirnitriou (1966) has shown that reovirus Type 3 can grow in lymphocytes in vim. The growth of virus, if not lethal to these cells, may trigger them into independent cell divisions which finally present as leukemia.
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Gross (1951) has shown that leukemia viruses are present in a latent form in mice. Since that time, there have been many illustrations of their involvenient in experimental situations (Negroni, 1963; Moloney, 1964; Pollartl and Rlntsuzawa, 1964; Pollnrtl, 1965; Gross, 1965; Dmochowski, 1965c; Harris, l965a; Dmocliowski et al., 1966; Pollard and K:ijilna, 1966). Miller (1960a,b) lias shown that the viruses may be associated with the thymus, and Miller (1964) and Furth et al. (1964) review the field of the thymus in leukemia. 1)mochowski et al. (1966) and Siegler and Rich (1966) have presentctl evidence that not all the leukemia viruses are assoriated with the thyinus. A major esperimental problem is the exclusion of these viruses from the environment when trying to show that nnotlicr virus is the initiator of an autoimmune tlise:rse culniinating in leukemia. In the human situation, Dmochowski (1960), Burkitt (1962a,b), Rosenlof a i d Picltett (1962), Annotations (1963), Proceedings 8 t h Conference Internutional Society Geogmphirrxl Pathology (1964), Bell et al. (1964, 1966), Dmochowski (1965n,b) World Health Organization (1965), and Harris (19651)) all preheat evidence, if somewhat circumstanti:il in some cases, for the :issociation of viruses and certain leukemias. Any evidence of autoimmune associ:ttions has not been discussed. Lewis et al. (1966) have shown t h a t X-irradiation and radiomimetic drugs can cause initiation of autoimmunity when patients are already presenting leukemia. Thew are no indications whether patients suffering from the leukemias were predisposed to develop autoimmunity or whether these treatments can cawe partial regression of the leukemia to an autoimmune disease. Rosenthal et al. (1955), Kitplan and Sinithers (1959), Dameshek and Srhwartz (1960), Videbaek (1960), Camniarata et al. (1961), Dameshek (1964, 1966a,b), Dultsin and Sigidin (1964), Kahn et al. (1964), Stanley et al. (1966b), Stanley and Waltcrs (1966), Sinkovics (1966), and Annotations (1966) all present situations where autoimmunity and resulting leukemia may be correlated. The possibility exists that the leukemias inay still retain some of their ability to react against the host. Preliminary esperiments (Keast and Stanley, 1966) with a lyinphonm (2731/L) resulting from virus-induced autoimmuuity (Stanley et al., 196613; *Joske et al., 1966) produced results indicative of immune responses against the host animal. Subcellular fractions of this lymphon~acan also induce autoimmune responses (Kesst and Stanley, 1966). Kunkel and Tan (1964) survey the role of autoantibodies and disease and the 1964 conference, “Autoimmunity-Experimental and Clinical Aspects,” illustrates the growing amount of attention being given to this type of research. Stanley et nl. (19641, Hotchin and Collins (1964), Hotchin ~
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(1965), Mekler (1965), and Joske et a2. (1966) suggest association between viruses and autoimmune diseases, and Stanley and Walters (19%) have presented a hypothesis of virus-induced autoimmunity. It seems, therefore, that many of the autoimniuiie diseases of man and animal are the sequel of chronic immunological injury (Leading article, 1965a,b ; Mekler, 1965; Lewis et at., 1965; Annotations, 1966) and that this may also lead t o leukemia. The autoimmunity inay develop its a result of viral damage to, and transformation of, cells of tlie imn~unologicalsystem of the host. It would be better if autoiiiiinune situations shown to have developed as a result of extraneous factors were named autologous diseases. A t present, experimental models may exist i t 1 chronic liomologous disease, the F, hybrid system, A1cuti:m mink disease slid, most, certainly, the virus-induced autoimmurie situation. VI. Comments
Analysis of the literature incorporated i n this article shows that there are still many unsolved problems directly associated with the cxperimental models available for the study of autoimmunity and its relationship, if any, to neoplasia. Some of the more important problems arc 1. It has yet to be firmly established whether the chronic homologous disease is, in fact, another manifestation of the GVHR. The most important issues here seem to be whether the acute disease is one involving only pseudoautoimniuriity or whether true autoininiunity plays a significant role; and also whether in the chronic disease it is the persistence of donor cells which gives the “autoimmune” reactions or whether the situation is one of true autoimmunity. It is essential for this to be known if the chronic homologous disease is to be considered as an experimental model for the study of the concept that autoimmunity may be a possible forerunner to neoplasia. 2. Closely allied to this is tlic requimnent for large uunibcrs of cells in the production of homologous disease. If the host is precluded from responding to the injected cells, then why cannot lower numbers of cells produce the disease? It may be interesting to see what continued daily injections of small numbers of immunologically competent homologous cells would produce. 3. Some of the more recent literature suggests that the macrophage is playing some important role in the establishment of the homologous disease. It has yet to be established whether this is a purely passive phagocytic role or whether the macrophage is making some active contribution t o the development of the clinical syndrome. 4. The likely role of the thymus in the course of events leading to
RTJNTING STSDROMES, ATlTOIMMVIiITT, AND NEOPLASI.l
65
autoimmunity is still undefinetl. The study of the course of events fob lowing thymectomy in young adulthood in mice may solve some of the sperulations a s to tlic function of the thymus in the field of autoimmunity. 5 . Tlir possilility of the indiiction of runting by nucleic acids should Iw fully investipte(1 as this coultl ht the kcy to the induction of autoiiiiniiiiie reactions within tho RES. Phagocytized, biologically active nuclcir acids or viral RNA or deoxyrihonucleic acid (DNA) may be able to induce cell transformation. 1Jndcr appropriate conditions, this may cwihlc thc cells to mount an autoiinmnne response which eventually leads to iiroplasia. 6. The fact t h t rpoviriis Type 3 has heen shown to induce autoiininunity which may lead to ncoplasin opens a widc field to experimentation. Thr reovirus Typc 3 system has t o he stutlied in detail and other v i r u m , hoth DNA and RNA types, should be looked a t for the property of ability to inducc immunological damagc leading to autoimmunity. The virus associated with Aleutian mink disease and the unexplored possilility tQat the murinc Ieiikemia viruses themselves may induce :tutoimmunc re:tctions would seem to he good starting points. L4CKNOWLEDGMENTS
I make grateful acknowledgment to Professor N. F. Stanley for encouragement :tnd criticism during the course of preparation of this review. I thank Mrs. M. 1,ahrrschagnr for carrfril prtparat,ion of t,he manilscript and checking of references.
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RTJSTIKG STNI)KOMKS, . ~ l ~ r ~ O I ~ I \ l L K I. TWYD, NEOPLASIA
71
ADDENDUM Since t.his artirlr has bcwi submillrti for puhliration. furl h r r reports on the 110ssible influence of f:tc.tors wsociatrtl wit,h thr gut flora on t.hr riint.ing syndrome have become availahlc, and readers arc refrrrrd to : Mcl3ritle (1966), Ekstrdt, and Hayes (1967), Reed and Jutila (1967), Keast (1968a,h), Krast and Wttltcrs (1968), Keast and Stanley (1968), Walbiirg e l al. (1968). Thcw rv1)ort.s put forward evidenre which suggests that the gut inflwnces in thc runting syndrome of mice .may he far morc. important than has hitherto been accepted. If this is so, then the etiology ascribrtl to some of the so-cnllctl autoirnmune situations rcfcrrrtl to within this cliapt(1r niay \veil have to be reviewed.
REFERENCES Ekstedt, R. D., and Hayes, L. L. (1967). J . I m m u n o l . 98, 110. Keast, D. (1968a). Immunology 15, 237. Keast, D. (196813). Immzinology (in press). Keast, D., and Stanley, N . F. (1968). Pathology 1 (in press). Keast, D., and Walters, M . N-I. (1968). Immunology 15, 247. McBride, R. A. (1966). Cancer Res. 26, 1135. Reed, N. D., and Jutila, J . W. (19f37). J . Zmmiuiol. 99, 238. Walburg, H. E., Jr., Cosgove, G. E., and Upton, A. C. (1968). J. Cancer 3, 150.
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VIRAL-INDUCED ENZYMES AND THE PROBLEM OF VIRAL ONCOGENESIS Saul Kit’ Division of Biochemical Virology, Boylor University College of Medicine, Houston, Texas
I. Introduction . , . . . . . . . . . . . . . A. Tumor-Producing Viruses . . . . . . . . . . . B. General Properties of Animal Viruses . . . . . . . . C . Molecular Weights of Viral Nucleic Acids . . . . . . . D. Infectivity of Papovavirus Deoxyribonucleic Acids . . . . . 11. Virus-Induced Antigen Synthesis . . . . . . . . . . A. Viral Capsid Proteins . . . . . . . . . . . . B. “Early Protein” Synthesis in Virus-Infected Cells . . . . . 111. Viral-Induced Enzymes of Deoxyribonucleic Acid Metabolism . . . A. Enzymes Induced by T-Ercn Bacteriophng~s . . . . . . B. Enzy.mes Induced by Coliphage T 5 . . . . . . . . C. Enzymes Induced by Bacillus subtilis Phages . . . . . . D. Enzymes Elicited by Deoxyribonucleic ilcid-Containing Animal Viruses . . . . . . . . . . . . . . . E. Characteristics of the Induction Systems . . . . . . . F. Mutant Virus Strains Defective in Enzyme-Inducing Activity . . G. Distinctive Properties of Viral-Induced Enzymes . . . . . IV. Viral-Induced Enzymes That Hydrolyze or Modify Dcoxyribonucleic and Ribonucleic Acids . . . . . . . . . . . . A. Deoxyribonuclcases of Noninfectcd Escheiichia co2i . . . . B. Induction of Dcoxyribonuclcase Activities by T-Even and T 5 Bacteriophages . . . . . . . . . . . . . C. Deoxyrihonuclcase Induced by Bacillus subtilis Phage SP3 . . . I). Nuclcase Activity Associated with Induction of Phage h . . . E. Hrrprs Simplex- and Poxvirus-Induced Deoxyribonuclcases . . . F. Phosphorylation of Nucleic Acid by an Enzyme from T 4 Bacteriophage-Infected Escherichia coli . . . . . . . . . G. Induction of Deoxyribonucleic Acid Methylase by Phage-Infected Cells . . . . . . . . . . . . . . . . H. Induction of Glucosyl Transferase Activities by T-Even Phage . . I. Host-Controlled Modification and Virus-Induced Enzymes . . . J. Bacteriophage Genes Controlling Sensitivity to Ultraviolet Light . K. Effects of Virus Infection on Solublc Ribonucleic Acid . . . .
74 74 82 83 84 84 84 87 89 89 92 93 94 100 111 119 126 126 127 129 130 132 134 135 137 140 148 150
‘Aided by grants fram the American Cancer Society (E 291), the National Science Foundation (GB 3126), the Robert A. Welch Foundation (Q 1631, and by U.S. Public Hcaltli Service Grants (CA 06656 and 1-K6-A1 2352). 73
74
SAUL KIT
V. Viral-Induced Ribonnrleic Acid Synthetase (Replicase) . . . . A. Characteristics of the Induction Process . . . . . . . B. Double-Stranded Ribonucleic Acid as a Synthetase Product . . C. Mechanism of Replication of a Riboniicleic Acid Virus . . . . D. Propertics of Highly Purifird Ribonucleic Acid Synthetase . . . E. Mutants of Bacteriophage f2 . . . . . . . . . . VI. Effects of Virus Infection on Host-Cell Nucleic Acid and Protein Syntliesis . . . . . . . , . . . . . . . A. Shutdown of Bacterial Metabolism in Cells Infected with T-Even . . . . . . . . . . . . . and T5 Phages B. Ribonucleic Acid Polymcrase Activity in T-Even Phage-Infected . Cells . . , . . . . . . . . . . . . C. Loss of Polyadenylate Polymerase Activity after Phage Infection . D. T4 Phage-Controlled Breakdown of Bacterial Deoxyribonucleic Acid E. Induction of Deoxycytidine Triphosphatase Activity by T-Even Phage . . . . . . . . . . . . . . . F. Thymidylate Synthetase and Thymidylate Nucleotidase Activities of Phage-Infected Bacillus subtilis . . . . . . . . . G. Shutdown of Biosyntlietic Processes in Virus-Infected Animal Cells . VII. Biochemical Aspects of Viral Oncogenesis . . . . . . . . A. Rcplication Cycles of SV40 and Polyoma Virus . . . . . B. Ribonucleic Acid and Protein Syntheses in Papovavirus-Infected . . . . . . . . . . . . . Cell Cultures C. Temporal Relationships of Protein and Nucleic Acid Syntheses during Papovavirus Development . . . . . . . . . . . D. Induction of Enzymes Functioning in the Terminal Pathway of Thymidine Metabolism . . . . . . . . . . . E. Biochemical Changes in Cell Cultures Abortively Infected with . . . . . . . . . . . . . Papovaviruses F. Other Functions of Papovavirus Genes . . . . . . . . . G. Continued Presence of Viral Genome in Transformed Cells H. Enzymes in Tissues Infected with Rous Sarcoma Virus . . . . I. Final Comments . . . . . . . . . . . . . References . . . . . . . . , . . . . . .
151 151 155 157 157 161 162 162 163 164 165 165 166 167 173 173 174 175 182 194 198 201 203 204
207
I. Introduction
A. TUMOR-PRODUCING VIRUSES Five groups of deoxyribonuclcic acid (DNA) -containing animal viruses arc known (Andrewes, 1962, 1964; Wildy and Watson, 1962; Wilner, 1964). The virus groups and some representative members of each group are shown in Table I. Of these, a t least three, and possibly four contain members that produce tumors. Thus, the ability to produce tumors is not unique to any single virus group (Table 11). At least three poxviruses (Fenner and Burnett, 1957) are tumorigenic. The Yaba monkey poxvirus induces histiocytomas in monkeys and similar lesions in humans (Ambrus et al., 1963; Bearcroft and Jameson, 1958; Knto e t al., 1965; Levinthal and Shein, 1964; Niven
VIRAL-INDUCED ENZYMES A N D VIRAL ONCOGENESIS
75
et al., 1961; Yohn et nl., 1964). Tumors produced in monkeys have been exaniincd by electron microscopy (Noyes, 1965). I n thin section, the cytoplasm of the tumor cells contained particles similar in size and shape to vaccinia-a typical poxvirus. The Shope fibroma virus produces fibromas when inoculated into domestic rabbits. The fibroma cells contain Feulgen-positive “B”-type inclusions resembling those found in the cytoplasm of cells infected with other poxviruses. The cytoplasmic inclusions are rich in viral antigen and are centers of DNA synthesis (Kato et al., 1963a,b; Takahashi et al., 1959). Molluscum contagiosum also resembles vaccinia virus in morphology and in site of replication (Noyes, 1965). Scven of the thirty-one human atienovirus serotypcs induce malignant tumors a t the site of inoculation in newborn hamsters (Brandon and McLean, 1962; Ginsberg, 1962; Huchner et al., 1962, 1965; Larsen et al., 1965; Percira e t al., 1965; Trcntin e t al., 1962). Type 12 adenovirus is also oncogcnic in newborn mice and in Mnstomys (Rattus natalensis) (Rabson et al., 1964a-3;Yabe e t ul., 1964). Seven of seventeen simian ntlenoviruses induce tumors in newhorn hamsters (Hoffert e t al., 1958; Huebner e t al., 1962; Hull et al., 1956, 1965). Strain SA-7, recovered from Cercopithems monkeys, appears to be particularly oncogeiiic ; inoculated hamsters develop tumors within 30 to 40 days. The chick embryo-lethal-orphan (CELO) virus may be responsible for inapparent or mild infections of the respiratory tract of chickens. When inoculated into newborn hamsters, this virus induces tumors a t the site of inoculation within 88 to 199 days (Sarma et al., 1965). The adenoviruses also cause malignantlike transformations in hamster cell cultures. Most viruses of the papovavirus group are tumorigenic (Klug, 1965; Klug and Finch, 1965; Melnick, 1962). The rabbit papilloma virus, the first virus of this group studied intensively, produccs a papillomatosis characterized by proliferation of epithelial cells with a thick accumulation of an amorphous keratinized layer on the surface (Shope, 1933). I n 1953, Gross recovered a filterable agent from AK mice which induced carcinomas of the salivary gland in C3H mice (Parotid Tumor Agent) (Gross, 1961). Eddy, Stewart, and co-workers demonstrated that the parotid tumor agent could bc propagated in mouse embryo cells. The virus produced ncoplasins in hamsters, rats, rabbits, and guinea pigs. Because of thc broad spectrum of histologically different neoplasms which the virus produced, Eddy and Stewart named it polyoma virus (de Estable e t al., 196Fi; Eddy and Stewart, 1959; Eddy e t al., 1958; Stewart e t nl., 1957). Thcl first report$ on tlie oncogenic effects of polyonia virus on murine and 11:iniuter c~in1)ryocells in tissuc c i i l t i i i ~w ( w pul~lislied I)y I~iilbecco
4 Q,
TABLE I SOMEDEOXYRIBONUCLEIC ACID-CONTAINING ANIMALVIRUSESAND PROPERTIES OF VIRALN UCLEIC ACIDS Group and virus I. Pox Vaccinia
Size (mr)
No. of capsomeres
230 X 300
-
DNA mol. wt. yo (Guanine cytosine) (daltons X lo6)
+
39,3i
Double
37 37 37 35
Double Double Double Double
23 -23 -23
5657 5 1-54 48-49
Double Double Double
60-75 70 >32 93 54
73,68 73-74 58 55-57 71
Double Double Double Double Double
160
cowpox Rabbit pox Ectromelia Fowl pox
160 160 160 -200
11. Adeno Human Types 2 , 4 Human Type 7 Human Types 12, 1s
70-90
111. Herpes Herpes simplex Pseudorabies Cytomegalovirus Equine abortion Bovine rhiriotracheitis
110 (200)
DNA strandedness
252
162
il)
s P
zw <
IV. Papova Polyoma SV40 Rabbit papilloma Human papilloma Canine papilloma Bovine papilloma Vacuolating virus (rabbit kidney)
V. Picodna Kilham rat virus (RV) Hamster osteolytic (H-1, H-3) R a t virus (X-14) Minute mouse virus Adenosatellite virus (.iSV), human, type 1 Adenosatellite virus (ASV), simian, type 4 a
Circular.
40-50
72 48 41 48 41
Doublea Doublea Doublea Doublea
3
43 46 43
Doublea Doublea Dolibleu
a
43-45 -
Double Doiible (?)
1 5
48
Double (?) Single
3 6 3 0
54-58 58-62
Double Double
3 3 4 2 4 3 -
18 23 18-24 20-26
22 20
32 32 32
-
; g ,
-
i
x =; 9
3 0 I
zN
+
z
E M
i
TABLE I1 TUMOR-PRODUCING VIRUSES Nucleic acid
Tumors in vivo
Group and virus
Morphology
Site of virus maturation
Essential lipids
I
DNA
in
I. Pox Yaba monkey pox Fibroma-myxoma Molluscum contagiosum Deer fibroma
j;
Monkey Rabbit Man Deer
3 Brick
Cytoplasm
Present
Hamster, rat, mouse
Icosahedral
Nucleus
Absent
Frog
Icosahedral
Nucleus
Present
11. Adeno Human Types 3, 7, 12, 14, 18, 21, 31 Simian Types SV1, SV20, sv33, sv34, sv37, SV38, SA-7 Avian (CELO)
111. Herpes Luck6 renal carcinoma
Y r-3
ns.1
j
IV. Papova Polyoma sv40 I3abbit papilloma Canine papilloma Bovine papilloma Humaii papilloma
1:X.i
1
I
V. Muriiie leukemia VI. Muriiie sarcoma (Bittner) V111. Aviau leucosis complex hl yeloblastosis Erythroblastosis Lymphomatosib I:ous sarcoma
Mouse, hamster, rat, rabbit, guinea pig, ferret Hamster, !\ltastnnz,ys Rabbit
Icosahedral
Nucleus
Absent i :
;j
Dog Cow, h m s t e r
P
7 z
3
I21:tll
5-
d
m u
Mouse, rat, hamster Morise, rat hlouse
m
;2, <
Chicken Chicken Chicken, turkey Chicken, duck, rat, turkey, mouse, hamster, giiine:i pig. inmikey
Pleomorphi c, variable shape aud size
Budding a t cell membrane
Present
z B P
2 tl <
ci
a
z r
80
SAUL KIT
and Vogt (1960), Vogt and Dulbecco (1960), and Sachs and Medina (1961 ) . They found that shortly after infection, the mouse cultures began to synthesize infective progeny virus and exhibited a marked cytopathic effect. After about 4 weeks, the cells began to change morphologically, cell destruction decreased, and a new type of cell began to appear, which by 10 weeks had replaced the normal cells. I n hamster cell cultures, smaller amounts of virus were synthesized, little cell degeneration was noted, and the replacement of normal by "transformed" cells took place more rapidly. Subcutaneous injection of 1 to 4 x 10" transformed hamster cells led to the development of tumors a t the site of injection. Simian papovavirus SV40, originally isolated as a contaminant of poliomyelitis vaccine, multiplies with a cytocidal interaction in monkey kidney cells (Sweet and Hilleman, 1960). This virus was subsequently identified as the oncogenic agent responsible for the induction of fibrosarcomas in hamsters and ependyinomas in hamsters and multiniammate rats (Black and Rowe, 1964; Eddy e t al., 1962; Girardi et al., 1962; Kirschstein and Gerber, 1962; Rabson e t al., 1962). Virus SV40 also produced proliferative responses and transformations in human, hamster, mouse, porcine, bovine, rat, rabbit, and guinea pig cell cultures (Ashkenazi and Melnick, 1963; Black and Rowe, 1963; Diderholm et al., 1965, 1966; Girardi e t nl., 1965; Koprowski et al., 1962; Shein and Enders, 1962). Bovine papilloma virus, which is closely related to SV40 and polyoma virus, induces a transformation in bovine and mouse cell cultures, and when injected into newborn hamsters induces fibromata and fibrosarcomata within an average of 6 months (Boiron e t al., 1965). The viral etiology of renal adenocarcinomas of the leopard frog was proposed by Luckk in 1934, in part because tumor cells contained large, highly acidophilic inclusions in the nucleus and because cellfree extracts induced tumors upon inoculation into frogs (see review by Rafferty, 1964). Thc inclusion bodies were shown to be Feulgen positive and contained viruslike particles, 100 mp in diameter. Mature particles were also found in the cytoplasm and extracellular spaces of renal adenocarcinomas, but were conspicuously absent from normal kidneys. The virus particles have been purified by Lunger (1964) and shown to have a close morphological relationship to the herpes group viruses. The fifth group of DNA-containing animal viruses, the picodna virus group (Table I ) , was discovered only recently (Atchison et al., 1965; Brcese e t al., 1964; Dalton et al., 1963; Greene, 1965; Greene and Karasaki, 1965; Mayor and Melnick, 1966; Mayor e t al., 1965a; Payne et al., 1964; Rose e t al., 1966; Toolaii ct al., 1964). At this writing, there is no positive evidence that any of the picodna viruses are oncogenic.
TAE3LE I11 RIBONUCLEIC ACID-CONTAINING ANIMALVIRUSES RNA Virus Mole %
Group Picorna Pi corna Arbovirus Reovirus Myxovirus
Example EMC Polio Sindbis Types 1, 3 Influenza A Newcastle disease
Size (mp)
Stranded
Mol. wt. (daltons X 106)
27 28 40-48 76 80-120 100-200
Single Single Single Double Single Single
3 1.8 8.8 8-10 2.8 7.5
Avian leucoses
Rous sarcoma (Bryan)*
65-90
Single
9.6
80-110 102 100 100
Single Single Single Single
9.8
Murine tumor Murine tumor
;\I yeloblastosis Eryt,hroblastosis Mammary tumor factor Iiauscher leukemia
12 13
A
TI
G
C
Ref.0
27.3 28.5 29.6 29.7 22.0 23.8 26.1 29.9 25.1 22.8 20.9 19.3 24.4
25.6 25.2 19.7 30.5 35.7 29.4 22.0 19.2 22.4 22.5 20.1 28.9 23.7
23.6 24.0 25.8 19.3 19.8 23.8 24.9 20.5 28.3 31.1 36.4 30.2 26.3
23.5 2'2.0 24.9 20.5 22.6 23.0 27.0 30.4 24.2 23.6 22.6 21.6 25.6
a b, c
d e-h i j
k I m II
n o, p q. r
a Key to references: (a) Faulkner et al., 1961; (b) Maassab, 1963; (c) Schaffer et ol., 1960; (d) Pfefferkom and Hunter, 1963; (e) Gomatos arid Tamm, 196313; (f) Gomatos and Stoeckenius, 1964; (g) Mayor et al., 1965b; (h) Gomatos et al., 1962; (i) Agrawal and Bruening, 1966; (j) Duesberg and Robinson, 1965; (k) Scholtissek and Rott, 1964; (1) Crawford and Crawford, 1961; (m) Robinson et ol., 1965; (n) Bonar et al., 1963a,b; (0) Lyons and Moore, 1965; (p) Duesberg and Blair, 1966; (9) Mora et al.. 1966; (r) Galibert ct al., 1966. * Rous sarcoma virus stocks contain avian leucosis virus (RAV).
9
2
82
SAUL K I T
Besides the DNA-containing viruscs, many tunior-l,rotiuciiig, ribonucleic acid (RNA)-containing Tiruses are known (Gross, 1961). The avian leucoses complex, the mammary tumor virus, and the murine leukemia viruses all contain RNA (Table 111). The oncogcnic RNA-eontaining viruses have a complex structure that also characterizes myxoviruses that do not produce tumors (Beard et al., 1963; Dmochowski e t al., 1963; Lyons and Moore, 1965). Quantitative techniques developed for assaying transformation in vitm of chick fibroblast cells to ROUS sarcoma cells greatly stimulated and influenced all subsequent investigations in viral oncogcncsis (Temin and Rubin, 1958). B. GENERALPROPERTIES OF ANIMALVIRUSES Tumor-producing viruses differ greatly in morphology, architecture, nucleic acid content, and nucleotide composition (Tables I to 111).The poxviruses are brick shaped, whereas, the adeno-, papova-, and herpes viruses have icosahedral symmetry. The poxviruses (Nagington and Horne, 1962) and the leukemia viruses (de Th6 and O’Connor, 1966) possess an internal helical nucleoprotciii structure similar in some aspects to the internal nucleoprotein of typical myxoviruses. The RNAcontaining tumor viruses, myxoviruses, herpes viruses, and poxviruses all contain lipid material in their outer membrane. The poxviruses, unlike other DNA-containing animal viruses, replicate in the cell cytoplasm. Poxvirus DNA’s contain relatively low amounts of guanine and cytosine (G C ) (,Joklik, 1962; Pfau and McCrea, 1962; Randall e t al., 1962, 1964; Szybalski et al., 1963). I n contrast the mole percent (G C ) of herpes virus DNA’s is unusually high (55-74 mole 7.)(Ben-Porat and Kaplan, 1962; Crawford and Lee, 1964; Kaplan and Ben-Porat, 1964; Russell and Crawford, 1963, 1964; Soehner et al., 1965; Wildy et al., 1960). Some human adenovirus DNA’s are relatively rich in (G C ) , but the oneogenic human adenoviruses have DNA base ratios more nearly resembling those of vertebrate tissues (Green and Pifia, 1963a,b, 1964; Kit, 1961, 1962; Pifia and Green, 1965). The DNA’s of papovaviruses are circular and also resemble vertebrate DNA’s in base composition (Crawford, 1963, 1964a,b, 1965; Crawford and Black, 1964; Crawford and Crawford, 1963; Dulbecco and Vogt, 1963; Kleinschmidt et al., 1965; Vinograd et al., 1965; Watson and Littlefield, 1960; Weil and Vinograd, 1963). The picodna viruses resemble bacteriophage (9x174 in size and buoyant density. Some viruses of this group appear to contain single-stranded DNA (Cheong et al., 1965; Crawford, 1966; Jamison and Mayor, 1965; Mayor and Melnick, 1966). The DNA of one murine picodna virus has a buoyant density in CsCl of 1.722 gm./cm.?, corresponding to n (G C) content of 48% for
+
+
+
+
VIRAL-INDUCED ENZYMES .4ND VIRAL ONCOGENESIS
83
single-stranded DNA. However, other picodna viruses (the human adenosatellite virus, serotype 1 simian adeno-satellite virus type 4, and the kilham rat virus) all contain doublc-stranded D N A (Rose e t al., 1966). The (G C) content of the D N A of the various adeno-satellite viruses range from 43-62% (Table I ) . Base compositions of some RNA-containing animal viruses are shown in Table 111.
+
C. MOLECULAR WEIGHTSOF VIRALNUCLEIC ACIDS Viral nucleic acids differ greatly in molecular weights and, hence, in polynucleotide chain lengths (Tables I and 111). The animal viruses with the largest nucleic acids are the poxviruses. The DNA’s of these viruses are comparable in size (160 x lo6 daltons) to those of the T-even bacteriophages (130 x lo6 daltons) . Poxvirus D N A molecules consist of about 250,000 nucleotidc base pairs. Assuming that the coding ratio is three nucleotide base pairs per amino acid, this number of nucleotide base pairs would specify about 83,000 amino acids, or about 415 polypeptide chains of 200 amino acids each. B y a similar calculation, it can be estimated that thc adenovirus and herpes virus DNA’s, respectively, contain genetic information for specifying about 60 and 240 polypeptide chains of 200 amino acids. The papovaviruses DNA’s consist of about 5000 nucleotide base pairs, or enough genetic information to code for about 8 or 9 polypeptide chains. This is also the approximate information content of the minute mouse virus and the human adenosatellite virus (Crawford, 1966; Rose e t al., 1966), the picornaviruses, many plant viruses, and influenza virus (Agrawal and Bruening, 1966; Cheng, 1959; Fraenkel-Conrat, 1962; Frisch-Niggcmeycr, 1956). The DNA of the kilham rat virus, however, consists of only 3000 nucleotide I m c pairs; it should be capable of coding only 3 t o 5 proteins a t the most. The RNA-molecular weights of avian tumor viruses are about 10 million daltons, so there is enough genetic information to code for about 50 polypeptide chains. Newcastle disease virus and Sindbis virus also contain about this amount of genetic information, and reovirus has about half this amount (Table 111). Of the known tumor-producing viruses, the papovaviruses contain the least genetic information. Moreover, not all of the eight or nine papovavirus genes are critical for tumor induction. This conclusion follows from reccnt experiments by Benjamin (1965) in which the rate of inuctivnt ion of Iw1yoni:i virus infectivity :ind transforming ability were c~on1l):ircdusing ultr:tviolet (IJV) light, S-irradiation, nitrous acid treatment, or H,”’PO, dccny to inactivatc the virus. Tlic ratc of inactivation
84
SAUL KIT
of transforming ability was 55-65% of that of reproductive ability. These results were interpreted to mean that transformation requires the participation of only about 55 to 65% as much of the viral DNA as does plaque formation. From experiments on the inactivation of polyoma virus by cobalt-60 irradiation, Basilico and D i Mayorca (1965) concluded that the radiation target of the transforming ability was about half the size of the target of the lytic activity. If the functions of papovavirus genes were elucidated, a major step would have been taken in solving the problem of viral oncogenesis. OF PAPOVAVIRUS DEOXYRIBONUCLEIC ACIDS D. INFECTIVITY
It is worth emphasizing that the DNA’s of the papovaviruses are infectious as well as tumorigenic. D i Mayorca and co-workers (1959) first described the isolation from mouse embryo tissue cultures of a nucleic acid, resistant to ribonuclease, but susceptible to destruction by deoxyribonuclease. The isolated material produced cytopathic effects in mouse embryo cultures and tumors in hamsters. Weil (1961) and Orth et al. (1964) confirmed the findings of Di Mayorca et al. (1959), and Weil (1961) developed a plaque-assay method for quantitatively studying the infectivity. Subsequently, DNA-containing extracts endowed with tumorproducing activity were obtained from papilloma and carcinoma tissues of rabbits and from purified rabbit papilloma virus (Ito, 1962). Polyoma virus DNA transformed hamster cells in culture (Crawford et al., 1964) and bovine papilloma virus DNA induced transformations of bovine skin cells in vitro and fibromata and fibrosarcomata when inoculated into newborn hamsters (Boiron et al., 1965). The SV40 DNA produced cytopathic effects in monkey kidney cell monolayers identical to those caused by intact virus and proliferative changes in bovine, hamster, and mouse cell cultures (Black and Rowe, 1965a; Diderholm et al., 1965; Gerber, 1962). The SV40 DNA also induced the formation of the SV40 T-antigen in hamster and monkey kidney cells (Black and Rowe, 1965b). These findings leave little doubt that the biological activity of the oncogenic papovaviruses resides in the nucleic acid moiety of the particles. If. Virus-Induced Antigen Synthesis
A. VIRALCAPSIDPROTEINS The most obvious function of viral genes is that of specifying the amino acid sequencc of capsid proteins. Data tire now available for some viral capsid proteins and will be discussed next. It appears that the capsids of polyonlit :ind ral,I,it papilloma viruses coutain only one type of
VIRAL-INDUCED E N Z Y M E S A N D VIRAL ONCOGENESIS
85
structural polypcptide (Thornc. and W:ir(Ien, 1967). However, 3 different polypeptide chains with an average molccular weight of 16,350 have been identified in the SV40 particle. Two of these, chains A and B, are present in equimolar amounts and account for about 91% of the total virus protein. One A and one B polypeptide chain form a structural subunit, which, in turn, builds the isometric particle shell. Chain C is a basic polypeptide and seems to br localized inside the virus particle (Anderer et al., 1968). The capsid of tobacco mosaic virus (TMV) consists of about 2130 identical protein subunits, each with a molecular weight of 17,500. There are 158 amino acids per subunit. Thus, 474 of the approximately 5500 nucleotides of TMV RNA, or less than 10% of the total, are needed to specify the amino acid sequciice of the capsid subunit (Knight, 1960). The capsid of bacteriophage R17, a small RNA-containing virus, is constructed from identical polypeptide chains having molecular weights of 14,200 (Enger and Kaesberg, 1965). The RNA of the virus has a molecular weight of about 1 million so that about 12% of the genctic information is needed to specify the capsid protein. The DNA’s of phages 9x174 and S13 have about the same information content as T M V RNA. The average molecular weight of yX174 capsid proteins is 25,000 (Carusi and Sinsheimer, 1963). Genetic studies have revealed, however, that two phage genes determine the structures of the phage-coat proteins. Electrophoretic and immunological analyses also lead to the conclusion that two distinct subunits are present in the coat of 9x174 (Dann-Markert e t nl., 1966; Rolfe and Sinsheimer, 1965; Teesman and Tessman, 1966). In this case, 1380 of the 5500 nucleotides of 9x174 and S13 are needed to code for the subunits of the coat proteins. Poliovirus, mouse encephalitis (ME) virus, and encephalomyocarditis (EMC) virus are all niemhers of the picornavirus group. The information content in the RNA’s of these viruses is also of the same order of magnitude as that in T M V RNA. It appears that two or more polypeptides are present in the capeids of the picornaviruses. Poliovirus-coat proteins have been resolved into four components by electrophoretic methods (Maizel, 1963). The coat protein of E M C can be separated into two proteins and that of M E virus into two major and one minor component (Rueckert and Duesherg, 1966; Work, 1964). The M E virus proteins differ in sniino acid content and in tryptic mapping patterns. Maizel (1963) has estimated that the poliovirus-capsid proteins have an average , (19661, who disrupted I>oliomolecular weight of 27,000. I I o w c v ( ~BoryE virus by :i difftli.(lnt ~nrtliotlfroill t h t used by Rlaizel (1963), found the poliovirus protcin niolecular w i g h t to be 42,000. The two rnajor componenth of ITF ~ ~ i r i icapsi(I3 s Ir:td c.htiniatct1 nm1rcuI:ir wlights of 15 t,o
86
SAUL KIT
22,000 and 29,000, respectively. It would appear that onequarter or more of the picornavirus genetic information is utilized for the determination of capsid proteins. Myxovirus particles are complex in structure ; many different proteins are utilized to construct their capsids. Electron-microscopic studies of negatively stained myxovirus particles reveal that the particles consist of two main components, an outer envelope studded with periodic projections and an inner helical structure (Schafer, 1963). Virions of orthomyxoviruses (i,e., influenza and fowl plaque virus) can be broken down by ether treatment, with the release of several constituents. TWOtypes of subunits can be isolated from the outer envelope, a hemagglutinin which has the appearance of star-shaped “rosettes” with spikes on the surface, and the rest of the envelope, which consists of protein and carbohydrate and contains neuraminidase activity. The hemagglutinin carries the strain-specific virus antigen. The RNA is located in the inner helical component, or soluble (S) -antigen, and is identical with the group complcment-fixing antigen of the virus. The protein portion of the helical component probably consists of protein subunits assembled in a manner similar to TMV virus. The proteins obtained by disrupting influenza A (strain BEL) particles have been fractionated by electrophoresis and studied by chemical methods (Laver, 1963a,b, 1964). One fraction, consisting of 37% of thc viral protein, exhibited hemagglutinating activity and possessed a free N-terminal aspartic acid residue together with smaller amounts of N-terminal glycine. The molecular weights of the one or more protein chains in this fraction were estimated to be about 60,000. The second fraction was lacking in N-terminal asparate residues but also appeared to consist of more than one component. The protein in the third fraction was identified as the S-antigen and the molecular weight of this protein, estimated from the basic amino acids of peptide digests, was about 40,000. If the assumption is correct that influenza and other orthoniyxoviruses contain RNA particles of 2.8 X loGdaltons (Agrawal and Bruening, 1966; Schafer, 1963), i t would seem that a large fraction of the genetic information of these viruses is used for specifying viral coat proteins. The information content of adenovirus DNA’s (23 X 10e daltons/ particle) appears to be greatly in excess of that required for synthesis of adenovirus capsid proteins. By the combined use of electron-microscopic and imniunological methods, the relationship between three adenovirus antigens, antigens A, B, and C, and the subunits (capsomeres) of the virion has been elucidated. The adenovirus group-specific nntigcvi [:mt~igc~nA (1,)1 forii~st l i c 240 ( ‘ ; q ~ h o ~ n ( 011 ~ e sthe sut-f:lce of
VIRAL-INDUCED
ENZYMES A N D vILiLiI, ONCOGKNESIS
87
the virus particle which havc six nearest ncighbors. Antigen A (L) particles are round objects 8 nip i n diameter. Antigen B has heen identified with the rcniaining twelvr capomeres at, the vertices of the virus particle which have five ncarcst ncigbbors. Antigen €3 has a round head 8 n ~ in p diameter attached to a 20-1np long and 2-mp wide tail, and a 4-mp knob a t the end. The tail on each antigen B extends outward from the virus surface. Trypsin digests the head of antigen B and converts antigen B to antigen C (E) (Valentine and Pereira, 1965; Wilcox et al., 1963). Antigens B and C are responsible for the hemagglutinating activity of adenovirus subgroups. B y utilizing polyacrylamide gel electrophoresis to fractionate adenovirus proteins, Maize1 (1966) has demonstrated the presence of ten different kinds of structural proteins. Chemical and immunological studies of proteins obtained by electrophoretic fractionation suggest even greater imniunological complexity. At least twenty separate antigens capable of reacting with rabbit pox antiserum have been detected in soluble extracts from rabbit poxvirus-infected HeLa cells (Appleyard and Westwood, 1964).
B. “EARLY PROTEIN” SYNTHESIS IN VIRUS-INFECTED CELLS 1. Td Phage-Infected Bacteria
From the preceding discussion, the conclusion may be drawn t h a t most viral genes do not function in eapsid-protein synthesis. Direct evidence was obtained by Watannbe (1957) that noncapsid, virusspecific proteins were Synthesized in T 2 bacteriophage-infected Escherichiu coli cells. Subsequently, noncapsid, virus-specific antigens have been demonstrated in a variety of virus cell systems. Watariabe (1957) perfornicd the following experiment. Escherichiu coli cells were infected with T 2 phage and incubated with radioactive amino acids to label the newly synthesized proteins. Antiserum prepared against the phage was then used to precipitate the radioactive-coat proteins of the virus. Watanabe observed t h a t a class of virus-specific proteins (“early proteins”) was synthesized which did not precipitate with phage antiserum. The synthesis of the early proteins began within minutes of phage infection, whereas phage-specific coat-protein synthesis commenced about 10 minutes after infection. The early proteins were neither precursors of phage-coat proteins nor were they bacterial proteins. Watanabe speculated that thew early proteins played an important, but yet unknown, role in producing phage-specific coat proteins and phage DNA.
88
SAUL KIT
2. Poliovims-Infected Cells Pioneering experiinent,~were also performed by Watanabe and COworkers (1962) on poliovirus-infected HeLa cells. More recently, two developments made it possible to examine more definitely the total virusdirected protein synthesis after poliovirus infection (Summers et al., 1965) : ( 1 ) conditions were established for complete suppression of hostprotein synthesis prior to any viral-RNA replication, and ( 2 ) improved techniques for sampling proteins separated by high-resolution polyacrylamide gel electrophoresis permitted the identification of radioactive virusspecific proteins in crude extracts from infected cells which had been “pulse”-labeled with radioactive amino acids. It was shown that twelve to fourteen electrophoretically different, virus-specific proteins were formed in poliovirus-infected cells and that many, if not all of these chains were synthesized during most of the virus cycle, although the rates of syntheses of certain proteins varied during infection. Only four of these proteins corresponded to the proteins resolved after disruption of purified poliovirus particles. The others were poliovirus-induced noncapsid proteins. The total number of protein chains resolved corresponds roughly to the number anticipated on the basis of the genetic information contained in the poliovirus RNA.
3. Pseudorabies Virus-Infected Cells Serologically distinct proteins which bear no precursor relationship to the proteins found in noninfected rabbit kidney cells are formed during the early stages of pseudorabies virus replication (Hamada and Kaplan, 1965). 4. Neoantigens Induced by Oncogenic V i w e s
Hamsters bearing tumors induced by SV40 develop antibodies to new antigens (T-antigens) present in both hamster and human cells transformed by the virus. These T-antigens can be detected by complement fixation or by immunofluorescence techniques (Black et al., 1963; Pope and Rowe, 1964; Rapp et al., 1964a). They are also produced in cell cultures of other species in which SV40 induces transformations (Black e t al., 1963; Black and Rowe, 1963; Diderholm et al., 1966) and in monkey kidney cells acutely infected with SV40 (Hoggan et al., 1965; Rapp et al., 1964b; Sabin and Koch, 1964). The SV40-capsid antigens differ from the T-antigens in immunological and physical properties and in time of appearance. The viralcapsid antigens are synthesized later than the T-antigens, they are more stable to heating, and arc coniplctely seditnented with the virus
particles, whcrcas tlie T-antigcns are completely destroyed by heating a t 56°C. for 30 niinutes, and, after centrifugation, they remain in the virus-free supcrnatant fluid (Gilden et nl., 1965; Sabin and Koch, 1964). Neoantigens (T-antigens) have been dctected in ( I ) tumor cells induced by adenoviruses and cell lines transformed by adenoviruses in vitro (Huchner et nl., 1963, 1965; Percira e t nl., 1965), (2) “virus-free” polyoma tumors and mouse and hamster cell cultures transformed by polyoma virus (Habcl, 1965), and ( 3 ) tumors induced in hamsters by the Scliniidt-Ruppin strain of Rous sarcoma virus (Berman and Sarma, 1965) . The adcnovirus-specific T-antigens arc fornied in KB, human amnion, and hamster kidney cell cultures inoculated with Types 12 and 18 adenoviruses (Berman and Rowe, 1965; Gilend and Ginsberg, 1965; Riggs et nl., 1966). The neoantigens can be detccted 3 to 4 hours after infection of K B cells with these adenoviruses, whereas viral-capsid antigens are synthesized 17 hours after infection. As in the case of the SV40 T-antigens, the adenovirus Type 12 T-antigens are more heat labile and thcy are smaller than the viral-structural antigens so t h a t they can bc separated from them by centrifugation in a linear sucrose gradient (Gilead and Ginsberg, 1965). The Schmidt-Ruppin neoantigens are found in chicken tells infected with any member of the avian tumor virus group (Sarma and Huebner, 1965). B y using hamster sera derived from animals carrying tumors induced with adenoviruses Types 3 and 7, i t was shown t h a t there is no crossing with the T-antigens shared by adenovirus Types 12 and 18, or with the T-antigens induced by polyoma virus, SV40, Schmidt-Ruppin strain Rous sarcoma, or with other nonspecific rcacting hainster tissue antigens (Hucbner e t nl., 1965). The SV40 T-antigens react with sera from hamsters bearing SV40 tumors but not with hainster sera from animals bearing adenovirus-induced tumors. Sera from hamsters bearing the Schmidt-Ruppin virus-induced tumors react with preparations from Rous sarcoma, avian leucoses, or RIF (resistance-inducing factor) infected chick cells, hut not with normal chick antigcns, unrelated avian viruses, and nonaviaii tumor virus prcparatioiis (Bcrman and Sarma, 1965). The polyoma T-antigens arc not found in normal animal tissues nor in tumors induced by SV40, adeiioviruses, or methyleholanthrene (Habel, 1965) . Ill. Viral-Induced Enzymes of Deoxyribonucleic Acid Metabolism
A. ENZYMES INDUCED BY T-EVEN BACTERIOPHAGES
It has becii kiiowii since tlie first studies on polymer synthesis in b:ictcrioi,littgr-infcctcd E scherichia coli that the protein produced early in infection is essential to the production of viral DNA. Nevertheless,
90
SAUL KIT
most of this early protein cloes not appear in the bacteriophage itself; indeed, the bulk of the viral-structural proteins are elaborated subsequent to the inception of DNA synthesis. The nature of the early proteins cssential to viral production began to bc clarified after the discovery by Wyatt and Cohen (1953) of a unique pyrimidine, B-hydroxymethylcytosine (HMC) , in the DNA of the T-even bacteriophages. Studics by Cohcn and collaborators also revealed that virus infection induced the ability to syntliesizc the normal pyrimidine, thymine, in an E . coli strain deficient in the ability to makc this substance (Barner and Cohen, 1954). Flaks and Cohen (1957, 1959a) found an enzyme, deoxycytidylate (dCMP) liydroxymethylssc, that catalyzes the synthesis of the unique viral pyrimidine, HMC, from formaldehyde and dCMP. The enzyme was not present in noninfected bacteria but could be detected 2 to 3 minutes after infection. Infection of E. coli cells with disrupted T2 bacteriophage preparations, or with bacteriophages TI or T5, which do not contain HMC, did not lead to the appearance of the enzyme, nor was there any evidence for the presence of the enzyme in an inhibited state in uninfectcd cells (Flaks e t nl., 1959). An enzyme, thymidylatc (dTMP) synthetase, which catalyzed the synthesis of d T M P from dcoxyuridylatc (dUMP) and formaldehyde, was also induced a t about 4 minutes after infection (Flaks and Cohen,
t
HCHO, THFA
0
I
d x P
w
0
t T H F A -DHFA
-
1
J ti -Y TMP
H20
O
H
FIG. 1. Enzymic reactions eatalyrcd by deosycytidylate (dCMP) hydroxyrnethylase, dCMP deaminase, dihydrofolate reductase, and thymidylate synthetase.
VIHAL-INDUCED E N Z Y M E S AKI> V I R A L ONCO(iENES1S
91
1959b). I3y u4ng tl~yniitli~ic-tl~~fic.icllt E . rdi stixiils (15T :ind &), Barrier and Cohen (1959) ~lcnionstrated that both d T M P synthetase and dCMP liydroxyi~~etliylaseincreased by a t least 1000-fold after T-even phage infection. Subsequently, it was found t h a t dihydrofolate (F’H,) reductase, the enzyme which catalyzes the formation of the tctrahydrofolic acid (THFA) coenzyme required in the d T M P syntlietase and dCMP hydroxymethylase reactions, was also induced early after T-even phage infection (Mathews and Cohen, 196313). Noninfected E . coli cells apparently contain little d C M P deaminase activity. However, on infection with T 2 bacteriophage, there was a marked increase in the activity of this enzyme (Keck e t al., 1960). I n contrast to the dCMP deaminase from :mima1 tissues, the T 2 phageinduced deaminaee does not demninate 5-hydroxymethyldeoxycytidylatc. The deplction of this T-even phage DNA precursor is thus prevented (Naley and Maley, 1966). Figure 1 illustrates the reactions catalyzed by the aforementioncd phage-induced enzymes. I n addition to the enzymes shown in Fig. 1, extracts of T2-infected E . coli cells were shown to contain an enzyme that catalyzed the phospliorylation hy adenosine triphosphate (ATP) of hydroxymethylcytosine 5’-niono1,hosphate (({HILIP): dHMP
+ ATP
+ dHUP
+ A I ) P ATI’ dHTP + A I I P
(1 1
Increases after T 2 infection in the levels of thymine and guanine deoxyribonucleoti~le phosphol*ylntlng cnzylnch (by 20- to 45-fold) also occurred bringing the activities of the latter enzymes up t o the level of the adenine deoxyribonucleotide phosphorylating enzyme, which was unchanged in activity (Bello and Bessman, 1963a; Bcllo e t al., 1961a; Bessman and Bello, 1961; Bessman and Van Bibber, 1959; Kornberg e t al., 1959; Sonierville e t al., 1959) :
+ ATP d G N P + A’I’P dAhIP + A’I‘P dTMP
-+ --t
-+
+ dTDPATE’ dTTP + ADP ATP AIIP + dGIlP dGTP + ADP .ITP ATP + dAIIP dA1’P + ADP
ADP
+
-
(2)
(3) (4)
The level of the cytosine deoxyribonuclcotide phosphorylating enzyme remained at the low level a t which i t was present in noninfected cells. The phosphorylation of dcoxythymidine 5’-diphosphatc ( d T D P ) and hydroxymethyl-deoxycytidinc-5’-dipliosphate ( d H D P ) into the triphosphates by extracts from noninfectetl and T2-phage infected cells was studied by Bello and Bessman, 1963b). The enzymes that catalyzc these reactions were already very active in noriinfected cclls and did not increase
92
ShUL KIT
slftcr phage infcction. Tliymit1yl:ttc (tlTMP) kinase, a t its highest lcvel aftcr induction of T2 phage, was still only onc-thirtieth as active as d T D P kinase, and d H M P kinabc cshibited only one-sixtieth the activity of d H D P kinase. The new enzymes induced by T-even phage account for the availability of deoxythymidine 5’-triphosphate (dTTP) , deoxyguanosine 5’triphosphate (dGTP), dATP and of the triphosphate of H M C , for the synthesis of T 2 DNA. Kornberg and co-workers (1959) showed further that after T 2 infection, enzymes were induced which catalyzed the polymerization of deoxyribonucleotide triphosphates into DNA (DNA polymerase) and which transferred glucose froln uridine diphosphate glucose (UDPG) directly to the H M C of DNA (glucosyl transferases) :
+ DNA
--t
DNA(dAhlP, dHMP, dGMP, dTMP)
+
(5)
4 pyrophosphate
HMC-DNA
+ UDPG -+
gl1lcos+HMC-DNA
+ UDP
(6)
Thus, seven or more enzymes catalyzing steps in the terminal pathway of deoxyribonucleotide metabolism and D N A biosynthesis were induced by T-even phage infection (Cohen, 1961). An increase in the availability of the deoxyribose moiety of tlic nucleotides is essential for optiinal rates of DNA biosynthesis. Consistent with this requirement, it was found that enzyme systems catalyzing the reduction of cytidylic acid t o d C M P increased 10 to 27 times by 20 minutes after phage T6r’ infection (Cohen and Barner, 1962; Cohen e t d., 1961). As mentioned previously, enzymes not induced, either because they were already present in excess or because they were not involved in the synthesis of T-even phage DNA wcre dAMP kinase, d C M P kinase, and the deoxyribonucleotide diphosphate kinases. Following T2 infection of Escherichia coli B, there were also no changes in the following enzyme activities: ( 1 ) amino acid “activation” levels for valine, isoleucine, mcthionine, leucine, phenylalanine, tryptophan, and tyrosine ; ( 2 ) P R P P (5-phosphoribose-l-pyrophosphate) synthetase; ( 3 ) orotate plus PRPP conversion to uridylate ; ( 4 ) inorganic pyropliosphatase ; ( 5 ) adenine plus PRPP conversion to adenylate; and (6) adenylate kinase (Kornberg e t al., 1959).
B. ENZYMES INDUCED BY COLIPHAGE T5 Coliphage T5 contains the normal pyrimidine, cytosine, in its DNA. Hence, dCMP hydroxymethylasc and the H M C glycosylating enzymes
arc not, iiitlnced iii Imtcria iiifcctctl with this virus. However, several enzymes wliicli function in D N A biosynthesis arc induced. Barner and Cohcn (1959) demonstrated that after infection of Escherichia coli strains 15T- or B,- by bacteriophage T5, tliyiiiidylate synthetase was produced concomitant with the acquisition of the ability of the infected cells to make viral DNA. Dihydrofolate (FHJ reductase increased about tenfold (illathews and Cohen, 19631)), and the levels of enzymes phosphorylating dTMP, deoxyguanosine 5'-monophosphate (dGMP) , and d C M P increased hy tcn- to fortyfold, bringing their activities up to the level of the dARfP phoaphorylat iiig enzyme, which increased about twofold (Kornberg et al., 1959). Dcoxyri1)onuclcic acid polyiiierase was also induced by phage T5 (dc Wttartl et al., 1965; Orr et nl., 1965). After phage T1 infection, no increases occurred in d T M P synthetase, d C M P hydroxymethylase (Barner ant1 Cohcn, 1959; Flaks et al., 1959), and FH, reductase (Mathews and Cohcn, 19631)). Deoxycytidylate kinase did not increase in E . coli infected with T3 or T 7 phages (Cohen, 1961). Escherichia coli K12 ( h ) cells which wcre lysogenically induced to form phage h were examined to see if a new DNA polymerase was synthesized (Pricer and Weissbach, 1964) . Lysogcriic induction with mitomycin C caused a small twofold rise in the activity of this enzyme. However, after 7300-fold purification of the noninduced and the induced polymcrases, these enzymes wcre indistinguishable in their kinetic properties. It, therefore, seeiiis that after lysogenic induction, a host-cell D N A polymerase is utilized for the replication of h DNA.
C. ENZYMES INDUCED BY Bncilliis siibfilis PHAGES Adenine, guanine, and cytosine are present in the D N A of Bacillus subtilis phages cpe and SP8, but thymine is completely replaced by hydroxymethyluracil ( H M U ) (Kallen et al., 1962 ; Roscoe and Tucker, 1964, 1966). Glucose is bound to the DNL4 of phage SP8 in the amount of 0.98 mole per gram atom of phosphorus. A temperature-sensitive (ts) mutant containing DNA-bound mannose (0.81 mole per gram atom P) has also been isolated (Roscnberg, 1965). Glucose has not, however, been detected in the DNA of phage y e . The 5-hydroxyn~ethyldeoxyuridylicacid (HMdUMP) required for DNA synthesis by these phages is generated froiii tlUMP by a hydroxymethylation reaction analogous to the hydroxymethyldeoxycytidylatesynthesizing mechanisms found in T-even phage-infected Escherichia di : dUMP
+ 5,1O-nietliylerietetr~hydrofolate
--f
H-1IdU.I.ZP
+ 'I'HFAI
(7)
An enzyme catalyziiig this reaction hn. not l ~ c c i iobhervetl in cstracts of 1964) T, . iioiiiiifcctctl bactci3a (1low)c :ml T I I C ~ C
94
SAUL KIT
The cnzyme, dCMP dearninasc also incrcascd ninefold after +e or SP8 infection, but d T M P synthetase activity decreased, suggesting that HMdUMP was synthesized by the following pathway: dCMP deaminaae
dCMP - dUMP
h ydroxymeth ylase
HMdUMP HCHO, THFA
.--)
1
dTMPsynthetase
!
-+
dTMP
(8)
bloiked
Some Bacillus subtilis phages havc dUMP in place of d T M P in the phage DNA (Takahashi and Marmur, 1963a,b). After B. subtilis infection by one of these phages, SP2, high levels of dUMP kinase were detected, although the enzyme was virtually absent from noninfected cells (Kahan, 1963). This enzyinc accounts for the availability of the dUTP required for phage DNA synthesis. Also induced in infected cells was a d T M P phosphatasc which degraded dTMP to thymidine (dT) and orthophosphate, thus reducing the availability of the d T M P needed for B. subtilis DNA synthesis. dUMP
+ ATP
-
ATP. d U D P kiriaso
dUMP kinase
dUDP
dTMP phosphatase
dTMP-
dT
dUTP
+ orthophosphate
(9) (10)
D. ENZYMES ELICITED BY DEOXYRIBONUCLEIC ACID-CONTAINING ANIMALVIRUSES 1. One-Step Growth Curves of Sortie DNA-Containing Animal Viruses
I n T-even bacteriophage-infected Escherichia coli cells, early enzymes are synthesized 2 to 15 minutes after infection. Infectious phage formation begins a t about 20 minutes and lysis occurs a t about 35 minutes. The latent periods for T3 and T 5 are 15 and 35 minutes, respectively, and that of h virulcnt is 45 minutes. The time scale of events is quitc different for animal viruses; hours rather than minutes arc required for virus replication. Moreover, the timing of virus replication varies considerably from one virus group to another, and for a given virus, the growth cycle depcnds on the host. Let us first consider the replication cycles of typical adeno-, pox-, and herpes viruses. Thc cytolytic iiitcixctioii o f tht. pilmvnviruscs with ruscrptiblc cells will hc disciisscrl later.
VIRAL-IKDUCED ENZYMES A N D VIRAL ONCOGENESIS
95
a. Poxviruses. Tlie eclipse 1)eriocl of vaccinia, a typical poxvirus, lasts about 6 hours in 1,RI nio11se fibroblast, cells, t,he virus titer then rapidly incrrascs until ahout, 15 hours. Most of the infect,ious virus remains cellassociated up to 24 hours aftcr infcction. Tlio vaccinia replication cycle in HcLa cells is similar to that in iiiousc fibroblast cells (Dales and Siminovitch, 1961 ; Hanafusa, 1961 ; Kit and Dubbs, 1962a; Salzman, 1960). h. Herpes Viruses. The replication of herpes simplex virus has been studied in human, hamster, murine, and canine cell cultures (Aurelian and Roizman, 1964; Gold et al., 1963; Nii and Kamahora, 1961; Rapp and Hsu, 1965; Roizman, 1963; Russell et aZ., 1964; Siminoff, 1964; Smith, 1963). The replication of pseudorabies, a related virus, has been determined in cultures of rabbit kidney and dog kidney cells (Aurelian and Roizman, 1964; Kaplan and Ben-Porat, 1963). Although herpes simplex virus replicates in cell nuclei, and vaccinia
0
8
16
24
32
40
48
Hours postinoculation of SV15
Fro. 2. Growth of simian adenovirus SV15 in cultures of monkey kidney (CV-1) cells.
in the cell rytoplaxni, the time required for virus growtll is about the same for these two yiruses. An increase in titer of herpes siinplex is genrved after a 4-6 horir eclipse period, and maximal titers are gcqlrt.:~llyattaiticcl Ily 12 to 16 hoiirs. I)iploi(l ~ ‘ l i i i i ( wI i x i n s t c ~cc.lls arc less suscrptiblc to 1 i q ) e s sitiiplex t 11:~n :irv IicLa cell lincs ; it1 Chinese hamstcr cells, the virus lias a latent period of about 12 hours. Virus titers subsequcntly iricrcase until about 24 hours after infection (Rapp and Hsu, 1965). I n clog kidney cells, herpes simplex virus infection is abortive; the virus fails to multiply or to form plaques in these cclls although certain herpes siniplex functions, such as viral-antigen and viral-DNA syntheses are expressed (Aurelian and Roizman, 1964). c. Adenoviruses. The latent period for adenoviruses is considerably longer than for pox- and herpes viruses. The growth of simian adenovirus SV15 in monkey kidney (CV-1) cells is illustrated in Fig. 2. The eclipse period is of about 16 hours duration. Virus titers then increase rapidly to a maximum a t about 36 hours after infection. The growth cycle of human adenovirus Types 2, 5, and 12 in KB and in HeLa cells is similar to that of SV15 in CV-1 cells (Green and Daesch, 1961; Kjellen, 1962; Smith, 1965; Wilcox and Ginsberg, 1963). 2. Biosynthesis of Viral DNA
a. Vaccinia Virus. Since vaccinia virus replicates in the cell cytoplasm, radioautographic techniques have been used to establish the time of formation of viral DNA. After noninfected cells were pulse-labeled with SH-dT, only the nuclei of the cells were radioactive. However, in cells infected with vaccinia and other poxviruses, Feulgcn-positive, radioactive foci were found in the cytoplasm a t the sites of virus-antigen formation. These 3H-dT-labelcd cytoplasmic foci were first detected about 1 to 2 hours after vaccinia infection. The percent of cells exhibiting cytoplasmic labeling increased rapidly so that by 5 to 6 hours, about 90% of the cells were labcled (Cairns, 1960; Kit and Dubbs, 1962a; Kit et nl., 1963a; Shcck and Magec, 1961). A second nicthod for studying viral-DNA synthesis, applicable to viruses otlicr than poxviruses, involves the use of fluorodeoxyuridine (dFU) (Salznian, 1960). In IlcLn cells, dFU causes a complete and immediate suppression of DNA synthesis owing to the inhibition of dTMP synthetase activity by the fluorodeoxyuridylate (FdUMP) generated from dFU. If dFU is added to cclls a t various times after vaccinia infection, any virus formed must contain DNA synthesized prior to the time the inhibitor was added. By comparing the amount of virus formed in 22 hours in cultures inhibited by dFU with that observed in noninhibited controls, one olkains :i nw:isui*rof the fr:wtion of viral DNA syntlicsizcd
1)rior to the tiriic of dFIJ a(1tlition. In tliih way, it was found tli:tt h i i f ficicnt vaccinia D N A was ~yntliesizcclby 6.5 hours to pcrniit niaxinial titers of infcrtious virus. 0. Herpes 17truses.Because of their high (G C) contcnts (Table I ) , the DNA’< of herpes simplex and pseudorabies v i r u m can be separated from host-cell D N A by equilibrium centrifugation in CsCI density gradicnts. By following the appearance in infccted cells of “heavy” DNA, it w:is shown that herpes simplcx and pseudorabies DNA’s ai e synthesized 2 to 8 1ioui.s after infection (Aurclian and Roizman, 1964; Bcn-Porat aiitl Kaplnn, 1963; Kaplan and Ben-Porat, 1963; Russell et nl., 1964). In agreement, with these rcsults, it was found that, in stationary-phase cultures, the incorporation of “-dT into DNA was greatly accelerated and the percent of ccll nuclei labeled with ?H-rlT increased sh:trply a t 2 to 8 hours aftcr infection by these viruses (Bcn-Porat and Kaplan, 1963 ; BenPomt et al., 1961; Dubbs and Kit, 1965; Kaplan and Ben-Porat, 1963; Nii and Kamwhora, 1961 ; Nii et al., 1961). Broniotleoxyuridine (dBU) and iododeoxyuridine (tlIU) inhibit the production of infectious herpes simplex virus. Expcriments analogous to thosc of Salzniari (1960) on the suppression by dFU of vaccinia virus production were carried out to determine the time relationship between the onset of inecnsitivity to dBIJ or dIU and synthesis of progeny virus. Whcn added 1 to 3 hours after infection, dBU suppressed infectious virus formation. If adde(1 :it later tiines, dBU was less effective, and if added 8 hours aftor infection, nornial yields of infectious virus wcrc made a t 24 hours (Roiznian e t al., 1963; Sirriinoff, 1964). Infectious virus forniation did not occur if d I U wits added during the first 4 hours after infection. I n infected cells treated 6 to 12 hours after infection, multiplication tended to level off and thc final viral yiclds diminished. Viral yields a t 17 hours werc almost riormal if dIU was added a t 10 hours after infcction (Roizman et al., 19631. These cxperinients also show that viralDNA synthesis was initiated a t about 2 hours and continuctl for about 6 to 8 hours. c. Adenoviixscs. The time course of adenovirus DNA synthesis has I m n cxaniincd by adding dFU to cell culturcs a t various tiiiws nfter infection and deterniining the final virus yields a t about 35 to 40 hours postinfection (PI). When tlic inhibitor was added up to 7 hours aftcr infmtion, no virus W:L$ subsequently dctvctetl. Howelw, w l r c ~tlFU ~ was nrlded 8 to 21 hour$ nfter infection, iiiriniAng :mioutits of viiaiis n - c ~ ~ Foimcd. ‘I’liyiiii~lii~~ II~ITINYI t l i v iiihiI)itoiy d t c c t ~of tlE’I’. \ Y I N ~ I I(11‘ ws : i t l ( l t s t l within 11it. firht 7 I i o u i . ~ aftt’r itiftiction to ciiltuiw trcated with tlFU :it the tiiiic of iiifcrtion, the growth cui ol)t:Liiiecl n.eiy1 iclc.litic:il to those of noninhibitctl cultures; wlicn clT was added a t 8 to 15 Iioiirs,
+
98
SAUL KIT
virus niaturation in cach cas(' bcg:tn aboiit 7 Iioiirs h t e r and proceeded a t an accelerated rate, confirming that viral-DNA synthesis hegan about 7 hours after infection and that about, 10 hours of viral-DNA synthesis were required to obtain normal virus yields (Green, 1962; Kjellen, 1962; Polasa and Green, 1965; Wilcox and Ginsberg, 1963). In confluent monolayer cultures of monkey kidney cells, viral DNA is made 10 to 18 hours after infection. A marked stimulation of "H-dT incorporation into DNA occurred starting a t 10 hours PI and continued 500
400
a
5
-
-
300-
9
k
6 200._
E
\
E 100 -
I
0
I
4
I
1
8
I
I
I
12
I
16
I
I
20
I
I
24
Hours postinoculation
FIU.3. Incorporation of 3H-thymidine ('H-dT) into DNA of SV15-infected confluent monolayer cultures of CV-1 cells. Infected and noninfected cultures were pulse-labeled 2 hours with 'H-dT beginning at the times indicated on the figure.
until about 18 hours after infection (Fig. 3 ) . Concomitant radioautographic studies indicated that the percent of cells incorporating 3H-dT into nuclear DNA increased a t 8 to 10 hours PI. B y 10 t o 12 hours, 74% of the cells in the infected cultures synthesized DNA compared with 13% of the cells in nonirifected cultures. At 14 t o 18 hours, approximately 90% of the cells of infected cultures were synthesizing
DNA. 3. Eiiz!lmcs Elicited I)?/ POT-, A t d c ~ ? onnd - , l l ~ r p c sV i m s e s
IIannfusa (19ti1) first sliowetl tlint extracts from \ineciniii-iiifected, strain L, mouse fibroblast cells catalyzed "-dT incorporation into DNA more rapidly than did extracts from noninfected cells. Stiniulated en-
VIRAL-INDUCED E N Z Y M E S AND VIRAL ONCOCENESIS
99
zytllic 3H-dT incoi*poration itito I)NA w:i+ :dso fouiitl a f t u vaccinia illfection of IIB cells (Green and Pifia, 1962) and after pseudorabies infection of rabbit kidney cells (Nohara and Kaplan, 1963). Four enzymes function in the metabolic pathway of "-dT incorporation into DNA, namely, d T kinase, d T M P kinase, d T D P kinase, and DNA polymerase. A pronounced enhancement of dT kinase activity was found after vaccinia infection of I,M and KB cells (Green e t al., 1964; Kit e t al., 1962, 1963c), and after cowpox, ectromelia, or rabbit pox infection of HeLa cells (McAuslan and Joklik, 1962). Thymidine kinase activity was also stimulated in several tissue culture lines infected with herpes simplex or pseudorabies viruses (Hamada e t al., 1966; Kaniiya et al., 1965; Kit arid Dubbs, 1963a,b, 1965) and in green monkey kidney ccll cultures infected with sitiiiari ntlenoviruscs SV15 and SV32 (Kit ef al., 1965). Mutant strains of mouse fibroblast cells [ L M ( T K - ) ] and of HeLa cells (HcLa BU-100) have been isolated; these mutant lines exhibit about 0.5 and 3% of the d T kinase activities, respectively, of parental ccll lines. Within 1 to 2.5 hours of infection of the iiiutant cell lines with vaccinia or herpes simplex viruses, thc induction of dT kinase could be demonstrated (Kit e t al., 1963c,d, 1 9 6 6 ~ )The . enzyme continued to increase in activity until 4 to 6 hours after infection a t which time thc activities attained were about 400 to 500 times greater than that of the mutant cell lines and about 3 times greater than that of parental cell lines (Kit and Dubbs, 1963b; Munyoii and Kit, 1965). Stimulations of dTMP kinase activity after poxvirus or pseudorabies virus infections have been reported by Hamada e t al. (1966) and Magee (1962), respectively, but were not confirmed by Grcen e t al. (1964) and McAuslan and Joklik (1962). Thyrnidylate kinase is very unstable ; however, the enzyme can he stabilized and activated by its substrate, dTbIP. Unless proper precautions are taken during extraction and assay, the activity of d T M P kinnse may be underestimated. It has been observed in the author's laboratory that extracts from noninfected cells prepared in buffer containing 0.1 niiM d T M P exhibited 2-3 times the activity of cxtracts prepared in buffer lacking dTMP. A n apparent enhancement of d T M P kinasc activity w n s observed af tcr either vaccinia or herpes simplex infections provitlccl that ccll extracts were prepared without dTMP. However, if the extracts were prepared with buffer containing 0.1 iiiM dTMP, the extracts from noninfcctcd and virus-infected cells had about equal activities. Presumably, vaccinia and herpes simplex infections partially stabiliaed the enzyme in vim. The virus-induced d T M P kinase increases are qualitatively different from those of d T kinase. The d T kinase was in-
I)y 1 ~ s -i ~ n dIicrpcs vii.iixcis iiwslwctivc of whctllcr cell extracts Rcsults similar to werc prepnrctl with or without t l T liiriaw hLal)ilizc~l*a. those described above h a w I m n obtaincd by Coto et al. (1966) in a study of the effects of pscudorabies infection on the d T M P kinase of rabbit kidney cell cultures. Pronounced increases in DNA polymcrase activity occur in cell cultures infected with vaccinia, pscudorabies, or herpes simplex viruses (Green et al., 1964; I-Iamada et al., 1966; Jungwirtli and Joklik, 1965; Kaniiya et al., 1965; Magee, 1962; Russell et al., 1964). A transient two- to threefold increase has also been found 16-20 hours after simian adenovirus SV15 infection of Grccn monkey kidney (GMK) monolayers (Kit et al., 1 9 6 7 ~ ) . I n poxvirus- or licrpes simplex virus-infcctctl cultures the activities of the following enzymes did not increase appreciably ; dAMP, dGMP, dCMP, and d T D P kinases (Green et al., 1964; Hamada et al., 1966; McAuslan and ,Joklik, 1962; Magee, 1962), FH, reductase and dTMP synthetase (Frearson et al., 1965, 1966), dCMP dearninase and uridine phosphorylase (Kit et al., 1964, and 1967a). The activity of uridine kinase decreased in vaccinia virus- and herpes simplex virus-infected cells (Dubbs arid Kit, 1964c; Kit et al., 1964). tliivcd
E. CHARACTERISTICS
O F THE INDUCTION SYSTEMS
1. Requirement for Protein Synthesis
Drugs that inhibit de novo protein synthesis block the increases in d T kinase and DNA polymerase normally observed after virus infections. Puroniycin or DL-p-fluorophenylalanine (FPA) prevent the enzyme increases when added to cultures at the inception of vaccinia or herpes si~nplcx infections. These drugs arrest the enzyme increases when added after thc enzyme induction has commenced. If the infected cultures are subsequently waslicd so as to remove the inhibitors, protein synthesis resumes and d T kinase and DNA polymerase activities increase (Hamada et al., 1966; Jungwirth and ,Joklik, 1965; Kamiya et al., 1965; Kit and Dubbs, 1963b; Kit et nl., 1963b,c; McAuslan, 1963b; McAuslan and Joklik, 1962). 2. Requirement for RiVA Synthesis
Actinomycin D inhibits DNA-dependent RNA synthesis in bacterial and animal cell systems. To learn whether RNA synthesis was required for enzyme induction, poxvirus- and herpes simplex virus-infected cells were treated with actinoniycin D either a t the time of virus infection or a t various tiiiies thereafter. When added just prior to virus infection,
actinomyc.iii 1) iiiliihits tlic iii(1iirtioii of (IT l
s simplex virus was extrtmely sensitive to actinoniyciii D inhibition; at a drug concentration of 0.5 pg./ml., d T kinasc induction was inhibited by 9776 (Table I V ) . At actinoniycin D concentrations of 1 to 2 pg./nil., induction of the enzyme was virtually undctectable. However, much higher concentrations of actinomycin D wcrc TABLE I V ACTINOMYCIN
D
INHIBITlON OF
THYMIDINE ICINASE
INDUCTlON I N
LM(T1I-) CELLSBY VACCINIAAND HERPESSIMPLEX VIRUSES~ Thymidine kiriase activity (prmoles dTMPb formed per pg DNA in 10 minutes a t 38°C.) Herpes simpleuinfected Actinomyciii D cells Concentration (P&h1.) Exp. a 0 0.5 1 2 4 0 8
356 9
Vacciniainfected cells Exp. b
Exp. c
Exp. d
16.1
-
1 0
0
-
12
12
24
4
Noninfected Lhl(TIC-) (.ells exhibited 110 delecta1)le enzyme activity. Experiment a-actinomycin D was added to the cultures 30 minutes prior to herpes simplex iiifec6oii arid d T kiiiase was assayed 5 hours after infection. Experiments b and c-actinomyciii D was added 1 hour prior to vaccinia infection arid dT kiriase was assayed a t 4 hours after infection. Experiment d-actinomycin D was added 1 hour prior to vaccinia infect l o l l niitl d T kinuse was assayed 5 holm after infection. * Thyinidylate.
rcquired to inhibit tlT kinase formation after vaccinia infcction. Altliougli 2.4 pg./nil. actinomycin D suppressed the incorporation of radioactive uridinc into crllular RNA by 95% or more within 1 hour of drug treatment, 4 pg./nil. :ictinomycin D permitted 18-31 '/. of thc normal amounts of dT kinasc to he induced by vaccinia, and cvcn a t nctinomycin concentrations of 8 pg./ml., significant amounts of dT kinase were made. Actinomycin D bincls t o gu:inine groups of DNA. The :mount of actinoniycin D bound by any single D N A is a function of its guanine
102
SAUL KIT
content. It may be recalled that lierpcs simplex 1)NA cout:lins about 37% guanine, whereas vaccinia DNA has a guanine content of only 18%. Thus, the fact that lower actinomycin D concentrations were required to inhibit d T kinase induction by herpes simplex than by vaccinia may be understood in terms of the differences in their DNA guanine content. The experiments imply that the messenger RNA’s functioning in the induction of d T kinase are different in vaccinia and herpes simplexinfected cells (Kit and Dubbs, 1963b; Kit et al., 19G3d). McAuslan (1963b) has investigated the time of synthesis and the comparative stability of the d T kinase messenger RNA. He observed that if the addition of actinomycin D to cowpox virus-infected HeLa cell cultures was delayed until 2 to 4 hours after virus infection (but not earlier), a maximal rate of d T kinase synthesis took place. Therefore, the synthesis of messenger RNA for d T kinase must have been completed within 2 hours after infection. Not only was d T kinase induced in infected cells to which actinomycin D was added 2 to 4 hours after infection, but the synthesis of the enzyme was not shut off a t 5 to G hours as was normally the case. Instead, enzyme synthesis continued for 18 hours, a result indicating that the messenger RNA for d T kinase was very stable. These observations have been confirmed by Jungwirth and Joklik (1965). I n addition, Jungwirth and Joklik (1965) have shown that the messenger RNA for DNA polymerase is synthesized by 2 hours after vaccinia infection and that the DNA polymerase messenger RNA is also functionally active for relatively long periods of time.
3. RNA Polymerase Activity in Cells Infected with DNA-Containing Viruses The way in which DNA-containing viruses initiate either new RNA or protein synthesis is perplexing since, in order for the virus to begin RNA synthesis, an RNA polymerase must be available, and before a new protein can be made, an RNA messenger must be made to code for the new protein. There are four ways in which viral RNA synthesis can be initiated: ( 1 ) by using a pre-existing host-cell RNA polymerase; ( 2 ) by bringing into the cell an RNA polymerase as part of the virus particle; (3) by using a new induced host-cell RNA polymerase; and ( 4 ) by bringing an RNA into the cell with the virus particle that would serve as a messenger for the synthesis of an RNA polymerase. Mechanisms (S) and ( 4 ) require that a new RNA polymerase be formed in infected cells-a process requiring protein synthesis-whereas mechanisms ( 1 ) and ( 2 ) permit the synthesis of RNA without prior protein synthesis. It is known that RNA polymerase activity is not increased in T-even
phage-infected cells; indeed, the activity of this enzyme is reduced after phage infection (Skold and Buchanan, 1964). Moreover, Jungwirth and Joklik (1965) failed to detect a new DNA-dependent RNA polymerase in the cytoplasm of vaccinia-infected HeLa cells. Using a polyauxotrophic mutant of Escherichiu coli deficient in the ability to synthesize thymine, uracil, and histidine, Sekiguchi and Cohen (1964) studied the synthesis of phage-specific RNA after T6r+ infection. Ribonucleic acid was made in the TGr+-infected E . coli cells in the absence of the required amino acid and also in the presence of chloramphenicol. The newly synthesized R N A had the characteristic base ratio and electrophoretic mobility of nornial phage-induced R N A and it was mainly associated with ribosomes. Incubation of the isolated RNAcharged ribosomes with inorganic phosphorus resulted in the selective degradation of the phage-induced RNA. Furthermore, after charging ribosomes with phage-induced RNA in the absence of protein synthesis, it was found t h a t the addition of the essential aniino acid to infected cells produced a stimulated rate of synthesis of two early enzymes, d C M P hydroxymethylase and d T M P synthetase. However, such prior synthesis of RNA did not significantly affect the rate of synthesis nor even the time of appearance of a late enzyme, lysozyme. The formation of T 4 phage-messenger RNA has been demonstrated in cells pretreated with chloromycetin (Nomura e t nl., 1960, 1962; Okamoto et al., 1962) ; therefore, mechanisms ( 3 ) and ( 4 ) can be ruled out for phages T 4 and T6 since the latter mechanisms presuppose protein synthesis prior to phagemessenger RNA synthesis. I n designing experiments on whether protein synthesis is required for the induction of RNA synthesis by vaccinia virus, Munyon and Kit (1966) made use of the following observations. First, Salzinan et nl. (1964) and Becker and Joklik (1964) had shown that, in noninfected cells, RNA, synthesized after short pulses with radioactive precursors, was localized primarily in the nucleus, hut after vaccinia infection, “pulse”-labeled RNA was found in the cytoplasm. This newly synthesized cytoplasmic RNA had thc base composition of vaccinia D N A and formed specific hybrids with vncciiiia DNA. Second, the induction of d T kinase by vwcini:i w t s highly resistant to :~ctinomycin D inhibition (Tahlc 1 1 7 ) . R/Iouw fil)roliIa>t c c ~ l ln~. c i ~f i~r h t 1)rxd r w t c v l with liigli conccntratiotis of actiiioiiiycin 1) to iiiliibit thc. iiicoi,l)ol.iition of ‘13-iiridiiic into hostcell RNA. B y drastically reduciirg the base line of host-cell RNA syntliesis, it was possible to reveal :i \.accinia-clepcndent stimulation of ?ITuridine 1ncorpor:ition into cytoplaemic RNA. Having established that cytoplasmic RNA synthesis was stimulated
104
SAIII, K I T
in vaccinia-ixifectcd cells pretreated with actirioinycin D, it then bccamc possible to test whether protein synthesis was required for this stirnulation. Cells were incubated with both cyclohexiniide and actinomycin D, washed, then infected with vaccinia in the presence of cycloheximide, and further incubated. The presence of cycloheximide before and during infection prevented 3H-leucine incorporation into LM cell proteins or the induction by vaccinia of d T kinase. However, cycloheximide treatment did not inhibit the vaccinia-dependcnt synthesis of cytoplasmic RNA. Similar results were obtained with cells pretreated with puromycin or p-fluorophenylalanine. It would appear that, as in the case of T-even phage-induced RNA synthesis, vaccinia-stimulated cytoplasmic R N A synthesis did not require prior protein synthesis. Thus, the RNA polymerase which catalyzed the formation of the virus-induced RNA was probably made before infection. It was either a preexisting host-cell enzyme or was brought into the cell as part of the infecting virus particle. Joklik (1964a,b) has shown t h a t FPA and puromycin prevent the disassembly of infecting-vaccinia virus so t h a t the virus D N A is not degraded by DNase digestion. Corroborative work by Dales (1965) has been reported showing that infecting-vaccinia particles do not release their D N A into the cytoplasm of the cell in the presence of Streptovitacin A, another inhibitor of protein synthesis. The results of Munyon and Kit (1966) in conjunction with the conclusions of Joklik (1964a,b) and Dales (1965) suggest that vaccinia infection can initiate messenger RNA synthesis without the disassembly of the virus to the point that viral DNA is degradable by DNase and before the DNA is relcascd into the cytoplasm of the cells. 4. Inhibitors of D N A Synthesis itnd Enzyme Induction Although RNA and protein syntheses are required for the iriductioii of early enzymes by phage and animal viruses, D N A synthesis is not needed. Normal levels of d C M P hydroxymcthylase and FH, reductase were made by dFU-treated Bsclieiichia coli cells after T6rC infection, I n addition, a t least one “late” protein, namely, lysozyme was formed (Sckiguchi and Cohen, 1964). T o learn whether DNA synthesis was required for d T kinase induction by wccinia, mouse fibroblast cells were treated with dFU, niitoniyciii C, or l-~-~-:ii~:il~ii~oFUl‘:111OSY]CYtohil~ (:iix-(’) (Kit Pt ( ~ l . ,1963t1, niitl u i 1 ~ ~ u l ~ l i u 4 l1)dw r ~ ~ : ~ t i. oNoilc* n ~ ) of Ilicsc. (Irugs appreciably rcducc~l (IT kinase foimttiou ~llthougli :ill three drugs stlinost coinl)lctcly inhibited D N A biosynthesis. Experiments with vaccinia-infected HeLa cells and with adenovirus
VIRAL-INDUCED E N Z Y M E S A N D VIRAI, ONCOGENESIS
105
FV15-infected inoiiliey kidney cell+ also showed tlint tlFU :ml ara-C, rvbpectively, inhiliited ncitlier d T kinn>e nor DNL1polymerase formatioiih (Jiingwirth :ind Jolilili, 1964; Kit c t ( I / . , 1!167r).
5 . h irz!/me Indiwtion b y U V LicJiit-Ir,atliatcd Virus Particles
It is generally assumed that DNA lesions account for loss of infcctirity of viruses irradiated with UV light. A comparison has been made of the effects of UV radiation on viral infectivity and on enzymc-indiicing c:tpacity (Dirksen et al., 1960; Flaks et aZ., 1959; Keck e t al., 1960; McAuslan, 1963a). \Vhen bacterial cells were infected with phage inactivated by UV light, the DNA content of cells remained constant, whereas in normal infection, it increased rapidly after an initial lag. Early synthesis of RNA was more resistant to damage caused by UV light than was subsequent synthesis of viral DNA, although both processes were progrcssively impaired (Minagawa et al., 1964; Sekiguchi and Colien, 1964). The induction of dCMP deaminase by phage T2 was also more resistant to UV radiation than was phage infectivity. When tlie irradiation reduced infectivity to 0.21 and 0.78% of normal, respectively, enzynieinducing activity was reduced to only 18 and 47% of normal (Kcck et al., 1960). Heavily-irradiated, T-even phage retained partial ability to induce the formation of d T M P and dGMP kinases, dCTPase, d T h l P synthetase, dCMP hydroxymethylase, and FH, reductasc (Dirksen et al., 1960; Flaks et al., 1959; Kozinski and Bessman, 1961 ; Sekiguchi and Cohen, 1964) . Phage of which the DNA had been heavily labeled with 3zPlost their ahility to form plaques a t a rate proportional to the specific activity of the isotope and tlie amount of DNA in the phage. All available cvidencc suggests that the primary lethal action of tlie 32Pdecay is the destruction of genetic material, presumably by scission of the DNA doublc helix. The capacity of cells infected with the 32P-labelcd phage to foriii early proteins was strongly affected by decay of the incorporated isotopc. However, the rate of decay of the initiation of synthesis of dCMP hydroxymethylase, d H M P kinasc, and dCMP clcaniinase was oiily 40 to 50% of that of tlie rate of loss of infective centers (Ebisuzaki, 1962). Also dGMP kinase-inducing activity was more resistant to 32P decay than was loss of infectivity (Kozinski and Bessman, 1961). As first shown by McAuslaii (1963a), the capacity of poxviruses to induce d T kinase was progressively impaired by irradiating virus particles with increasing doses of UV light. Figure 4 illustrates the finding that the ability to induce dT kinase was considerably more resistant to UT’ radiation than was infectivity. Whereas the synthcsis of d T kinase
106
SAUL KIT
0.01 0
30
60
90 120 UV- irradiation (seconds)
150
180
FIG.4. Ultraviolet (UV) light inactivation of vaccinia infcctivity and ability to induce thymidine kinase in LM(TK-) cells (Munyon and Kit, unpublished experiments). A preparation of vaccinia having an initial titer of 2.3 X 10’ PFU/ml., suspended in media containing 0.1% bovine serum albumin, but no calf serum, was irradiated at 4°C. with a 25-watt General Electric germicidal lamp. At 30-second int,ervals, samples were withdrawn and assayed for infcctivit,y and for thymidine kinase-inducing activity (3 hours after infection). (dT = thymicline.)
was induced by vaccinia virus inactivated with UV radiation, syntheses of DNA polymerase and DNase were not. When the proportion of survivors was d T kinase was still induced but even a t a survival of to very little induction of DNA polymerase and DNase occurred (Jungwirth and Joklik, 1965). Vaccinia virus irradiated with UV light is either not “uncoated” a t all or to a very small extent. The results, therefore, suggest that normally “uncoated” viral genomes cause induc-
VIRAL-ISDUCED ENZYMES AND VIRAL ONCOGENESIS
107
tion of the synthesis of DNA polymerase and DNase, whereas only limited ‘(uncoating” is needed for the induction of d T kinase. Although several early enzymes were induced by UV-irradiated bacteriophage, the formation of coat proteins and of lysozyme was almost completely prevented. Lysozynie is normally made a t about 15 minutes after infection with nonirradiated phage; however, a t 69 minutes after infection, the activity of lysozyme in irradiated phage-infected cells was only 10% of the normal value, i n marked contrast t o dCMP hydroxymethylase in the same extract, which was almost twice as high as that in normal infection. Since early enzymes increased during the entire period of incubation of cells infected with irradiated phage, the failure of late proteins to increase in these cells cannot be ascribed to the overall cessation of protein synthesis a t the later period of incubation (Sekiguchi and Cohen, 1964). 6. Extended E n z y m e Synthesis i n Cells Infected with U V -Irradia t ed Virzises
The pattern of enzyme induction ascribed to the phenomenon of “extended enzyme synthesis” is as follows: Early in the infectious cycle, irradiated virus induces enzyme a t the saiiic rate or nearly the same rate as nonirradiated virus. At later times, the amount of enzyme in cells infected with nonirradiated virus levels off or decreases, whereas the enzyme levels in cells inoculated with irradiated virus continue to increase, frequently surpassing the maximum levels obtained in cells inoculated with nonirradiated virus. Extended synthesis of dCMP hydroxyinethylase was observed by Dirksen et al. (1960) when infection of Escherichia coli was produced by W-irradiated T2 phage. Normally, the synthesis of this enzyme was shut off a t about 12 minutes after infection. With phage irradiated with UV light to to survivors, dCMP hydroxymethylase synthesis continued for about 60 minutes. Ultraviolet irradiation had caused a lesion in some system responsible for the cessation of enzyme formation during normal infection. Formation of dCTPase, dTRlP kinase, and dTMP synthetase also continued beyond the normal period whenever infection was produced by irradiated phage. With these three enzymes, however, the initial rate was significantly decreased a t a radiation level that did not affect the initial rate of liydroxymetliylase formation (Dirkscn et al., 1960; Sekiguchi and Cohen, 1964). It seems that tlie factors responsible for the initial formation of these enzymes were more sensitive to W light t t i i t i i tlie oiie fur tlic furniatioii of liyclroxyinethyl:~~~. T h c initial rate of induction of H I 2 rcrluctase activity Ijy UV-irrxli:ttcd phagc was : h o
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SAUL
Iirr
UV exposure
0.5 min I .5min
Control (no UV exp.)
2 min
2.5 min
Hours after infection
FIG.5 . Extcndrd thymidine kinase synthesis after inoculation of LM(TK-) cell cultures with vaccinia virus irradiated with increasing doscs of ultraviolet light (Munyon and Kit, unpublished experiments). (dUMP = deoxyuridylate.)
slower, and FH, reductase increased to only one-half the normal level a t 30 minutes after infection. Extended synthesis of dT kinase was observcd by McAuslan (1963a) after HeLa cell infection with UV-irradiated poxviruses. At 24 hours after infection, the d T kinase induced by irradiated virus reached values up to 38 times that of noninfected cells. Experiments illustrating extended synthesis of d T kinase in LM(TK-) cells infected with UVirradiated vaccinia are shown in Fig. 5 . After 1 minute of irradiation (1.13% survival of infectivity), the initial rate of d T kinase induction was almost normal. However, the lcvels of d T kinase reached a t 10 hours were o w r twice thobc attaincd by nonirradiatc(1 virus. With more heavily irradiated vaccinia, both the initial rates and the 10-hour levels of d T kinase artivity w r c subnormal. McAuslan ( 1 963:~) t1rir~onstr:~ted t h t (17’ liinase induction by UV-
iiw(liattv1 cowl)os vii,iis c*onltl Iw sliiit off by s.ul)ei.iiifcctioii witli :I i~cllatetl livr virus. The :iiwlal)ility of niritant inoiisc fil)rol)la~tcells [ LRI (TI< ) 1 , deficient in (IT kinaw :trtivity, an[I of :L iriutant vwcinia strain (Vtk ), deficient in enzyme-inducing activity, 1)erriiittcd a confirmation of McAuslan’s findings in :t system where noninfected and Vtk--infected cells exhibited essentially no d T kinase activity (Fig. 6 ) . Since nonirradiated virus prevented the rxtendcd d T kinaw synthcsi:, by irradiated virus whcn cells were simultaneously infectcd with both viruses, the shutoff mechanism of enzyme synthesis must be cytoplasmicnlly mediated. Irradiation damages some property of poxvirus DNA wliicli affects the repression of enzyme induction hoiiie time after infection. Repression dominates over induction by irradiated virus. also occurs in poxThe observation t h a t extended enzyme synthc
‘oool
Vtk+(UV) infection
-,P I
//
;
i I
I I
In
Vlk’
I
400-
I
infection I
3 200 -
a‘/
I0
3
Vtk’ (UV) and Vtk- infection (sirnu;toneous)
6
9
12
Hours after infection
FIG.6. Inhibition of extendcd enzyme synthesis in LM(TK-) cells simultaneously infected with UV-irradiated parental vaccinia (Vtk’) and a nonirradiated mutant vaccinia (Vtk-), deficient in thymidine kinase inducing activity (Munyon and Kit, unpublished experiments). (dUMP = deoxyuridylatc.)
110
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virus-infected cclls treated zlt 2 to 4 hours after infection with actinonlycin D suggested an experimental approach toward defining the chemistry of the cytoplasmic repression phenomenon (McAuslan, 1963b). AS actinomycin D blocks the shutoff mechanism, the function of shutoff appears t o be mediated either by some species of RNA or by the product of such an RNA. If thc repressor were RNA, its synthesis should take place even though protein synthesis were inhibited by puromycin ; in this case, removal of puroinycin would not lead to the resumption of enzyme synthesis. If the repressor were protein, it would not accumulate in the presence of puroinycin. I n this case, removal of purornycin would permit enzyme synthesis to continue until the repressor was synthesized. T o test these alternatives, HeLa cells were infected with C O W ~ O X virus; 2 hours later, when synthesis of dT kinase iiiessenger RNA was completed, puroinycin was added. Five and one-half hours after infection, the puroiiiycin block was removed, whereupon enzyme synthesis was resumed a t the preinhibition rate. The repression of enzyme synthcsis did not occur until 4 hours after the release of protein synthesis. Thus, the shutoff mechanism could not be established in the presence of puroinycin, indicating that thc repressor was not simply a species of RNA. Whereas addition of actinomycin D a t 2 hours prevented the shutoff, addition a t the end of the 3.5-hour period of puromycin inhibition failed to prcvent repression, which occurred 4 hours after enzyme synthesis WRS resumed. Therefore, the actinomycin-sensitive step for the establishment of the shutoff must have occurred prior to the addition of actinomycin D, i.e., during the period of puromycin inhibition. Thus, the establishment of the shutoff required the syntheses of both RNA and protein. 7. Extended Enz yine Formation in Cell Cultures Treated with Aminopterin or dFU
It is known that viral D N A is not made by cells infected with UVirradiated viruses. I n order t o inhibit D N A synthesis without affecting the D N A molecule itself, aminopterin or dFU have been applied to infected cells. I n the case of poxvirus-infected HeLa cells, aminopterin treatment resulted in a n impairment of the dT kinase shutoff mechanism, and extended synthesis of d T kinase took place. However, if the infected cultures were incubated with both d T and aminopterin, the inhibition of DNA synthesis was reversed and, concomitantly, d T kinase formation was repressed a t the normal time (McAuslan, 1963a). The addition of dFU to vaccinia-infected HeLa cell cultures also resulted in extended syntheses of d T kinase and of D N A polymerase (Jungwirth and Joklik, 1965). When the dFU-containing media was supplemented
VIRAL-INDUCED ENZYMES A N D \‘IRAL ONCO(;ENE‘IS
11 1
with thymidine, the d F U block of D N A synthesis was overcome and the switchoff of enzyme synthesis took place in the normal manner. Fluorodeoxyuridine inhibits the incorporation of l*C-uracil into DNA of T6r’-infected bacterial cells ; however, the inhibition is not complete. There is no net increase in DNA, but a sniall and significant amount of DNA formation does take place. Possibly, this is because small amounts of d T are generated through the breakdown of bacterial DNA. I n dFUtreated bacterial cells, dCMP hydroxynicthylase and FH, reductase activities increase in a manner similar to normal infection and enzyme synthesis is shut off a t the normal time. There is also a considerable increase in lysozyme, in contrast to the behavior of cells infected with irradiated phage in which the increase in lysozyme activity is inhibited (Sekiguchi and Cohen, 1964). Moreover, phage-structural proteins are made in dFU-treated culturcs a t 33 to 50% of the rate of controls (Ebisuzaki, 1963). Oric can imagine that the transcription of the genes coding for lysozyme actually occurs on thc few replicas of the initial infecting DNA which are formed in the presence of dFU. These results lead t o the conclusion that the cessation of synthesis of early enzymes and inception of synthesis of late proteins require D N A synthesis.
F. MUTANTVIRUS STRAINSDEFECTIVE I N ENZYME-INDUCING ACTIVITY 1. Amber Mutants of Bacteriophage T4
Amber mutants of T 4 are conditional lethal mutants capable of growing on some strains of Escherichia coli K12 (i.e., CR63, a permissive host), but not on E . coli B (a nonpermissive host). Benzer and Champe (1962) have described a similar relationship between certain strains of E . coli and some rII mutants of T4. They suggested that these rII mutants contain a codon which is nonsense in the nonperniissive host, whereas the permissivc host is able to supprcss the mutation making the mutated codon LLiiiissen+e” rather than nonsense. The results obtained by Sarabhai et a l . (1964), using T4 mutants containing amber mutations in genes corresponding to the head protein, suggest that this explanation may apply to :tnibcr mutations, since chain termination occurs during synthesis of the head proteins of E . coli B infected with these mutants. Genetic studics by Epstcin et al. (1963) with ambcr mutants have lr11 to the construction of :t genetic niap for T4 coiitainiiig approximatcly scventy genes oi’ which twenty are believed to function in D N A biosynthesis. Some of the aiiiber mutants (i.e., am N82 and am N122) are completely unable t o reproduce in E . coli B and produce practically no DNA (DO mutants; T:il)le V ) . Other mutants (i.e., a m N81, am N116, and am N130) multiply to a limited extent and phage production is
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TABLE V AMBER MUTANTS O F BACTERIOPHAGE T4 Mutant gene No.
Amber mutant
Mutant type"
Enzyme controlled by gene
1 39 56 41 42 43 43 44 46 47 52 30
am B24 am N l l 6 am E56 am N81 am N122 am B22 am NlOl am N82 am N130 am A456 X 5 am H17 am H39X
DO DD DS DS
Deoxyiiucleoside nioiiophosphute kinase dCTPaseh dCMPc hydroxymethylase DNA polymerase DNA polymerase Unable to cause breakdown of host DNA Unable to cause breakdown of host DNA Polynucleotide ligase
DO DO
DO DO DA DA DD DS
0 DO-no DNA synthesis in Escherichia coli B ; DD-delayed DA-arrested DNA synthesis; DS-some DNA synthesis. Deoxycytidine triphosphatase. c Deoxycytidylate.
DNA synthesis;
roughly related t o the level of DNA production in infected cells. With am N81, DNA synthesis is negligible during the first 20 minutes, but later there is a slow but significant rate of synthesis (DS mutant). With am N116 (DD mutant), DNA synthesis is delayed until approximately 20 minutes and then commences a t a rate comparable t o that observed with cells infected with normal T4 phage (Wiberg e t al., 1962). In the case of am N130, DNA synthesis starts a t a normal rate but stops abruptly a t about 15 minutes after infection (DA mutant). A number of amber mutants have been examined for their ability to induce in E. coli B (the nonpermissive host) the formation of enzymes related to the synthesis of DNA (Warner and Barnes, 1966; Warner and Lewis, 1966; Wihcrg et al., 1962). Amber mutants of T 4 defective in genes 1, 42, 43, and 56, respectively, fail to produce deoxyribonucleoside monophosphate kinase, dCMP hydroxymethylase, DNA polymerase, and deoxycytidine triphosphatase (dCTPase) . Studies with the amber mutants support the hypothesis that phagc DNA synthesis is closely related to the regulation of the synthesis of early enzymes. Iiifcction of E. coli B with DO or DD amber mutants (Table V) generally results in extended synthesis of phage-induced early enzymes. Thus, although mutant an] N122 does not induce thc formation of dCMP hydroxyrnethylasc i n E . coli B, dTMP synthetase, dcoxyribonucleoside inonophosphate kinase, DNA polymerase, dCTPase, H M C
VIRAIA-INDUCED ENZYMES AND VIRAL ONCOGENESIS
113
0-glucohyl t ransferase, antl dCRlP deaniiiiase attain activities two- to fi\clfoltl greater than with wild-type phage, and enzyme synthesis continut~sfor almit 60 minutes after infection. With am N82, all of thew cnzyni~santl :dso dCMP hydroxymethylase are synthesized for an cxtended period of time. With amber mutants am N81 arid am N116, the enzymes are all induced a t higher levels than normal ; however, enzyme synthesis is repressed a t about 20 to 30 minutes after infection when DNA synthcsis commences. Amber mutants am 90 and am H17 induce phage DNA synthesis in E . coli B, although only limited phage production takes place. These mutants are dtfectivc in essential steps other than DNA replication. In the case of am H17 and am 90 and also mutant aiii N130, normal enzyme levels arc inducctl and enzyme synthesis is shut off a t the usual time. Although FH, rcductase activity is induced by a t least t n ~ n t yamber mutants, seldom do the FH, reductase levels induced by the mutants exceed that induced by the wild-type phage, and, in contrast to the other enzymes, extended synthesis is not observed (Warner and Lewis, 1966). These observations suggest that the increase in FH, reductase activity occurring in T4-infected cells may not be subject to the same control as other phage-induced early enzymes. The control of the synthesis of other enzymes is lost if DNA synthesis is not initiated on tiiiit, whereas the control of FH, reductase is unaffected. 2. Temperature-Sensitive Mutants
Temperature-~ensitive(ts) mutants are a second type of conditional lethal mutant. Thc ts mutants generally replicate poorly a t about 40°C. hit do grow a t about 30°C. Among the ts mutants of bacteriophage T4 are two, ts L13 and t s G25W, that contain sites of mutation located in the same gene as that of aniber mutant aiii N122 (gene 42). In infections carried out a t 425°C. with mutant t:: L13, no DNA was made and no dCMP hydroxymethylase WRP detectable. Either the enzyme was not formed a t 42.5"C. or it wa6 rapidly and irrcvcrsihly inactivated a t that temperature. At 30"C., however, mutant ts L13 did induce dCMP hydroxymcthylase activity. With wild-type phage, neither DNA synthesis nor dCMP hydroxymethylase production were impaired a t 42.5"C. (Wiberg and Buchanan, 1964). With mutant ts G25W, just as much dCMP hydroxymcthylase activity was detected with high-temperature infections as with low-temperature infections but very little DNA was synthesized a t 425°C. Although dCMP hydroxymethylase was made a t 42.5"C., the enzyme was very unstable and exhibited other abnormal properties. Probably, mutant ts G25W failed to make DNA a t 425°C.
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because the dCMP hydroxymethylase induced by this mutant functioned poorly. Temperature-sensitive mutants defective in gene 43 have been tested for their ability to induce T4 DNA polymcrasc (de Waard et al., 1965). Of the ts mutants, t s L97 and ts 1,107 induced the synthesis of H M C ,8-glucosyl transferase a t 37"C., thus indicating that infection proceeded successfully. Deoxyribonucleic acid polymerase was not produced by cells infected a t 37"C., but the enzyme was formed a t 25°C. The enzyme induced a t 25°C. showed a significantly higher in vitro activity a t 37°C. than a t 25°C. This suggests that there was a thermolabile step in the formation of the tertiary structure of the protein so that a functional protein was formed a t 25°C. but not a t 37°C. With a third mutant, ts L91, equivalent levels of enzyme were produced a t 37" and 25°C. However, activity measured a t 25°C. was nearly twice as great as that measured a t 37°C. The ratio of activity a t 37°C. to that a t 25°C. The QiiZ for this DNA polymerase was 0.5. On the other hand, the DNA polymerases purified from Escherichia co2i B cells after either wildtype virus or mutant am N82 infection (gene 44) showed a &,;:' of 2.8. Moreover, the ts L91-induced enzyme was considerably more labile than the wild-type enzyme a t 37°C. A DNA polymerase from a bacteriophage T 5 mutant, ts 53 has also been purified. Infection a t either 30" or 43°C. resulted in the induction of polymerase activity. The activity of the T 5 ts 53-induced DNA polymerase, in contrast to the enzyme induced by the wild-type phage, was less active when assayed a t 43°C. than a t 25°C. The Q!gX of the sixtyfold purified polymerase from ts 53 was 0.04 compared t o a value of 1.4 for the wild-type T5-induced enzyme. 3. Mutant Phage Defective in Thymidylate Synthetase-Inducing Activity Using Escherichia coli strain B3, which requires thymine for growth, Wulff and Metzger (1963) have isolated mutant strains of T4 which require exogenous thymine for growth. Similar T4 mutants (td mutants) have been isolated by Simon and Tessman (1963). The mutants map close to amber mutant am 134, in the tail fiber region, a t a position far removed from the region controlling other functions that are expressed early in phage development. I n E . coli B3 infected with td mutants, DNA synthesis was absolutely dependent upon the presence of thymine in the medium. However, aftcr infection of E . coli B, which contains dTMP synthetase activity, DNA was synthesized a t about two-thirds the rate in cells infected by mutant td8 as those infected with td'. I n dilute cultures of E . coli B,
VIRAL-INDUCED ENZ’I-MES ANI) \‘IRAL ONCOGENESIC;
115
the eventual phage yield and the phage growth rate were two- to threefold lower with the mutant than in cultures infected with the wild-type strain. Addition of thymine not only increased DNA synthesis but also mutant-phage production. The ability to induce d T M P synthetase formation in infected cells probably confers a selective advantage on the wild-type strain (Mathews, 1965). Enzyme studies have shown directly t h a t little or no d T M P synthetase is induced in E . coli infccted with three of the T 4 td mutants and a fourth mutant was “leaky” (synthetase induction 19% of normal). However, inductions of d C M P hydroxymethylase, dCTPase, and FH, rcductase were normal with all of the T 4 td mutants (Shapiro et al., 1965). 4. T 4 Miitants Unable to Induce dCilIP Deaminase Activity
Hall and Tessman (1966) have isolated mutants (cd-) of bacteriophage T 4 that were unable to induce d C M P deaminase activity. Extracts of Escherichia coli B cells infected by these mutants showed less than 1% of the activity of extracts from cd+-infected cells. However, the induction by the cd mutants of d T M P synthctabe and d C M P hydroxymethylase activities were normal, suggesting that the mutation was specific for the deaminase. The lack of activity in extracts from cd mutant-infected cells was not due to an excess of inhibitors of d C M P deaniinasc. This was shown by experiments in which extracts from cd- and cd+-infected cells were mixed; the cd- extract did not inhibit the activity of the cd’ extract. Fleming and Bessman (1965) have found that the d C M P dearninase induced by phage T 6 is subject to feedback control, being activated by d C T P and inhibited by dTTP. The deaminase activity of extracts of T 4 cd+-infected E . coli B was increased sixfold by the addition of d C T P to the reaction mixture, suggesting that the activity of the T4-induced deaminase was also regulated by feedback inhibitors and activators. Nevertheless, extracts from cells infected with cd- mutants showed no significant activity even in the presence of dCTP. The isolation of the cd- mutants demonstrates that a t least one T 4 cistron controls the production of dCMP deaminase activity. However, the cd+ function does not appear to be essential for T 4 growth in E . coli B ; the burst size of the cd- mutants was a t Icast one-half that of the wild-type virus. 5 . M u t a n t Virus Strains Defective in H M C cu-Glucosyl Transferuse-Ind ir ciny A ctiv it 1~ From T 2 : t t i ( I T6 hnctt~riopliagcstocks which l i d I)crn treated with Iiydroxyl:itiiiii(i +oluCion, Rc\.cbl :uuI (w-woikrrs (1965) liave isol;itetl
I IG
SAUL KIT
rllutant virus strains defective in H M C a-glucosyl transferase-inducing activity. Pliage were selected which grew on Shigella dysenteriae Sh (permissive host) but not on Escherichia coli B (restrictive host). The mutants were designated T2gt and T6gt, respectively. Upon infection of Shigella dysenteriu Sh, the mutants failed to initiate the synthesis of HMC a-glucosyl transferase, whereas dCMP hydroxymethylase and dTMP kinase were made in normal amounts. I n its reactions with the restrictive host (Escherichia coli B) , T2gt-1 behaved similarly to host-modified T2 (T"2) (see Section IV,I). Adsorption to E . coli B was normal, but thc killing efficiency per particle was only one-half to onc-third. Small amounts of dCMP hydroxymethylase and dTMP kinase were formed. I n mixed infection on E. coli B the mutant T2gt-1 complemented amber mutants of cistron 42 (dCMP hydroxymethylase defective), but not amber mutants of cistron 44 (function unknown). As with T"2, multiplicity activation occurred a t high multiplicities of infection. The DNA of T2gt-1 was as good a substrate for HMC CY-glucosyltransfcrwsc of T2 as was T"2 DNA, indicating the DNA of tliesc phages contained little or no glucose. 6. Vaccinia and Herpes Simplex Mutants Using dBU as a selective agent, a mutant mouse fibroblast line [strain LM (TK-) 1 with the following properties was isolated: 1. The LM(TK-) cells were highly resistant to growth inhibition by dBU, dIU, or excess dT. 2. They were more resistant to (1FU inhibition than parental LM cells. 3. They lacked dT or dU phosphorylating activities although the cells were capable of phosphorylating deoxycytidine, uridinc, or dTMP, and the cells contained normal levels of FH, rerluctase, dTMP synthetaw, and DNA polymerase activities. 4. They failed to incorporate "-dT into DNA but did incorporate 3H-deoxycytidine into DNA (Dubbs and Kit, 1964a; Kit et al., 1 9 6 3 ~ ) . Following infection of LM(TK-) cells by either normal vaccinia or herpes simplex viruses, d T kinase activity was induced, and the capacity of the cells to incorporate 3H-dT into DNA was restored. By infecting LM(TK-) cells with vaccinia or herpes simplex viruses at low input multiplicities in the presence of dBU, mutant virus strains were isolated deficient in d T kinase-inducing activity (Dubbs and Kit, 19641),r, 1965; Kit and Dubbs, 1963b). The vaccinia (Vtk-) and 1i~rl)eshiinplt~s (Htk ) mutants proved to be relatively stable, since thry could be p least 5 times in either IJn4 01' LM(TK-) cells in tllc :il)spllccl of clT3lJ without gross revcrsion.
Thc wrccinin mutants (Vtk-) not only failcd to induce d T kinase activity in IAI (TI<-) cclls, but unlike the 1)arental Vtk' virus, the Vtkvirus did not redore thc capacity of L J I ( T K - ) cells to incorporate "H-dT into DNA. The failure of Vtk--infected LM (TK-) cells to incorporate 3H-dT into D N A was not attributable to the abscncc of DNA synthesis in infected cells. The LM (TK-) cells infected with either Vtk+ or Vtk- viruses incorporated 3H-deoxycytidine into D N A although slightly less than did noninfected LM(TK-) cells. The nuclei of about 36 to 40% of infected T , M (TK-) cells were labeled when incubated with 3H-dcoxycytidine a t 3 to 6 hours aftcr infection, while the nuclei of 42% of the noninfected I,M(TK-) cells were labeled. Foci of radioactivity were found in the cytoplasm of about half of the cells infected with Vtk- virus, indicating the vaccinia D N A was made in the infected cells. The Vtk- mutants probably all hat1 lesions in the same cistron. Scrccning cxperiments wcrc performctl in which LM (TK-) cells were niixcdly infected with pairs of Vtk- virus mutants and challenged with "H-dT a t 2 to 6 hours aftcr infection. Mixed infections with Vtkmutants did not result in 3H-dT incorporation into DNA, as would be expected if complementation had occurred. The Vtk- mutants differed from Vtk+ in several additional properties. Although Vtk' rcplication in LM (TK-) cells was inhibited by 25 pg./ml. dBU or dIU, growth of Vtk- mutants was not inhibited by 200 pg./ml. of the drugs. Significantly, growth of eithcr Vtk or Vtk' was inhibited by dBU in the parental L M cells which have the capacity to phosphorylatc dBTJ. However, even with IJM cells, higher concentrations of dBU were required to inhibit growth of Vtk- than Vtk+ virus. The Vtk- mutants were not only incapable of inducing d T kinase iii I A l ( T K - ) cells, but in simultaneous infections with both Vtk- and Vtk' viruses, the Vtk- mutants inhibitcd the induction of d T kinase by Vtk' virus (Munyon and Kit, 1965). The inhibition of Vtk'-induced dT kinase formation was a function of thc ratio of the input niultiplicities of the Vtk' and Vtk- strains and the inhibitory effect of Vtk- could be potentiated by infecting L h i ( T K - ) cells with Vtk- before infection with Vtk' virus. It is unlikely that the inhibition of d T kinase induction by mixed infection of cells with Vtk' and Vtk- viruses was attributable either t o the formation of inhibitors of dT kinitsc in the Vtk--infcctcd rells or t o t h i s f o 1 ~ l l ~ i ~ tofi ~ Il 'ic ~ ) I ' P s N > ~ ~of. s i l l ' kiitaa~induction. Exl)eriiiicints in which thxtt-iicts f r o i i i Yth'- ~ t r i d from \'tk -iiifecte(l cells were niixetl in vitro h l i o w c v l tli:it thc c1str:icth froit] T'tk -infected cells (lit1 not inliiliit the cwzyiiit* f i ' o t i i T't li '-i ti fwtc(l ccl Is. A ko, :t repressor- type i n h i hi tion would
118
SAUL KIT
be expected to be related to the absolute multiplicity of Vtk- infection since such a mechanism would be expected to be effective regardless of how many superinfecting Vtk+ particles were present. Two hypotheses consistent with the experimental findings are that (1) d T kinase is composed of subunits which polymerize from a pool of protein monomers. If the monomer pool were generated solely by Vtk' infection, an active enzyme would result; whereas in a mixed infection, the monomer pool would be generated by both Vtk+- and Vtk--controlled synthesis. I n this case, hybrid enzymes with low activities would be formed. (2) The Vtk' and Vtk- viruses compete for some limiting cell structure early in the course of infection. If such sites were occupied by a product of the Vtk+ infection, such as messenger RNA bound to ribosomes, an active d T kinase would be formed, The binding of ribosomes by the messenger RNA of Vtk- virus would not result in active enzyme synthesis, and these ribosomes would be unavailable to the messenger RNA of Vtk' virus. This type of inhibition is compatible with the observation that inhibition is related to the ratio of the multiplicities of the two infectiving viruses. Three classes of dBU-resistant herpes simplex mutants (Htk-) have been isolated from LM (TK-) -infected cells (Dubbs and Kit, 1964c; Kit and Dubbs, 196313): (1) Htk- virus similar to the Vtk- strains in that they induced either very low levels of d T kinase activity or none a t all and failed to induce 3H-dT incorporation into DNA; (2) Htk- strains that induced approximately normal levels of 3H-dT uptake into the nuclei of 4 to 14% of the LM(TK-) cells; and (3) mutant Htk- strains that induced only very light 3H-dT labeling in the nuclei of 22 and 63% of the LM (TK-) cells. As in the case of the Vtk- mutants, growth of class I Htk- mutants was not inhibited in LM(TK-) cells even in the presence of 500 pg./ml. dBU although 25 pg./ml. dBU inhibited Htk' replication. Also inhibition of Htk- replication in L M cells required higher concentrations of dBU than inhibition of Htk' replication. Complementation between class I Htk- mutants did not occur in tests of the effects of mixed infection on the 3H-dT uptake into DNA of LM (TK-) cells. A study of enzyme induction in LM(TK-) cells by class I11 Htkmutants (B2010 and B2015) revealed that the induction of d T kinase was a temperature-dependent process. When LM (TK-) cells were infected with mutants B2010 and B2015 a t 37"C., then ( I ) essentially no d T kinase activity was detected in extracts of infected cells when assayed a t 38' or 30°C., (2) approximately 15% of the cells a t 2 to 10 hours after infection displayed more than 40 graiii counts of SH-dT inco 1'1 )o1'21 tion I K T 11 uc~cus , :L i i d ( 3 ) iScpI ic:i t io I I W ~ L H I iot signi fi cttn t 1y in-
VIRAI,-IKDUCED
IINZl'hfES .4ND VIRAL ONCO(;ENESIS
119
hil)ited by 100 ,~g./niI. dBU. Howevcr, w l i t ~ i i LRI (TI<-) wlls were infected a t 31"C., then ( 1 ) approxiinately one-tenth the level of d T 50 to kinase obtained with parental virus strains W R S induced-about 757% of the cells had more than 40 grains per nucleus when the infected cells were incubated witli jH-dT-and (2) replication of mutant B2010 wits partly inhibited and that of mutant B2015 was coiiipletely inhibited by dBU. Once induced a t 31°C. by mutants B2010 and B2015, d T kinase could be assayed a t either 38" or 30°C. I n contrast, class I Htk- mutants and also Vtk- mutants failed to induce d T kinase a t either 37" or 31°C. arid the replication of these mutants a t the higher or tlie lower tempernture was resistant to high levels of dBU (Dubbs and Kit, 1965).
PROPERTIES OF VIRAL-INDUCED ENZYMES G. DISTINCTIVE Viral-induced enzymes generally differ from enzymes present in uninfected cells in kinetic, chromatographic, and immunological properties. Frequently, these altered characteristics are observed even after extensive enzyme purification. I n some instances, geneiic and radiological data also show that enzyme synthesis in virus-infected cells is controlled by viral genes. In cases in which evidence has been obtained by all of these methods, it is difficult to avoid the conclusion that the induced enzymes are new and distinctive viral-specific proteins and that the primary structures of the enzymes are determined by viral genes. In actuality, only a few of the viral-induced enzymes discussed in this chapter have been thoroughly studied. The available evidence for some of the enzymes will now be considered. 1. Phage-Induced dCMP Hydroxymethylase
Extracts from noninfected Eschem’chia co2i cells do not catalyze the dCMP hydroxymethylase reaction nor is the product of this reaction, dHMP, a normal constituent of E . coli cells. Hydroxymethylase activity is induced after infection of E . coli by T-even bacteriophages. From Sepliadex G200 gel filtration and from sedimentation arid diffusion data, the molecular weight of a highly purified dCMP hydroxymethylase was estimated to be about 68,000 (Mathews et al., 1964). Using this value for tlie molecular weight, it was shown that there was less than one active hydroxymethylase molecule per noninfected cell. The question may be asked, ((1s dCMP hydroxymethylase activity produced by the addition or deletion of a peptide fragment from a preformed polypeptide chain?" Perhaps the modified protein would have an altered catalytic function permitting i t to hydroxymethylate dCMP? To answer this question, Mathews and co-worker8 ( 1964) prelabeled cells The cells were then by growth in the presence of ''C-mctl~yl-iiictl~io~iii~e.
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SAUL KIT
infected with phage TGr+in nonr;~tlio:ictivc~iiiediiiiii, a1111tlCAIP hythoxymethylase was extcnsively I)urifid. The piirified (ICRZP hydroxynlethylase was virtually nonrar1io:wtivc. From this c y t ~ r i i i i c ~ n titj , rail 1x2 conwits in;ttlc clc nova :iftc'r infection. cluded that dCMP 1ly~lroxymcthyl:~sc Thus, the experiment contradicts the hypothesis that preexisting cell protein was used to make the viral-induced enzyme. It is known that T4 amber mutants defective in gene 42 failcd to induce the synthesis of dCMP hydroxymethylase in E. coli B cells, the nonpermissive host. However, in E. coli CR63 cells, the perniissive host, these mutants do induce dCMP hydroxymethylase activity, but the enzyme induced in the permissive host has different properties from the enzyme induced by wild-type virus. Extracts prepared from wild-type virus-infected E. coli B or CR63 cells are more active a t 37°C. than at 25°C. The Qlo is 1.6 to 2.0. The dCMP hydroxymethylase formed by the mutants in E. coli CR63 is inactive when assayed a t p H 8 and 37°C. However, when assayed a t 25°C. and pII 7.4, the enzyme does show activity. Thus, after infection of permissive hosts with cistron 42 mutants unstable enzymes with altered pH optima are produced (Dirksen et nl., 1963.) Several properties of the hydroxymethylases induced by two t s mutants of phage T4 and by the wild-type phage hare been compared. Two properties of the wild-type enzyme are ( 1 ) a marked protective effect of both substrate and product, dCMP and dHMP, respectively, against heat inactivation a t 40"C., and ( 2 ) the ability of the enzyme to undergo extensive reactivation after exposure of the unprotected enzyme to heat a t 40°C. Relative to the wild-type enzyme, the ts G25W enzyme had a lower pH optimum and a much smaller temperature coefficient between 0" and 30°C. (for the ts G25W enzyme the ratios of activities a t 30" and 0°C. were 2.9; for the ts+ enzyme the ratio was 11.3). The mutant enzyme was also inactivated far more rapidly a t 40°C. in thc presence of substrate, dCMP, and was quite unstable a t p H 8.3 even :it 0°C. in t h r ahscnce of dCMP. The ts L13 enzyme was similar to the wild-type enzyme in all respects except that i t could not be reactivated after inactivation a t 40°C. in the absence of dCMP. Thus i t is likely that, in oioo, the ts L13 mutant failed to make DNA a t 42.5"C. hecause active enzyme was either not made or was inactivated rapidly and irreversibly, whereas the ts G25W mutant failed to make such DNA at 42.5"C. simply because the enzyme functioned poorly. This demonstration of two different types of alterations in the properties of the dCMP hydroxymethylase induced by two phage mutants, which map genetically a t two different loci within the same gene, eonstitutes strong evidence that the structural gene for this enzyme residcs in the phage rather than in tlic bacterial h t .
2. Phage-Induced d T M P Synthetuse
The Michaelis constant for d U M P for the d T M P synthetase induced by T 2 phage in Escherichia coli B is identical to that induced in E. coli 15T- (Mathews and Cohen, 1963a). However, this constant differs from the coustant of the enzyme induced in either E . coli B or 15T- by T 6 phage. Moreover, the inhibitor constant of FdURiIP of thc d T M P synthetase induced by T6 is over twice as great as the inhibitor constant for the d T M P synthetase induced by T2. These observations show that distinctive enzymes are produced by two related phages. The Michaelis constant for tetrahydrofolate for the dTMP synthetase purified from T2-infected cells differed considerably from that of the enzyme from noninfected cells (Grcenl)crg et al., 1962). The two enzymes also differed in their chromatographic properties. After dicthylaminocthyl (DEAE)-cellulose chromatography, noninfected E . coli cell extracts showcd only oric peak of d T M P synthetase activity, whereas TP-infected cells had two peak$, one corrchponding to the normal host enzyme and a second, new peak. Extracts from infcctcd E . coli 15Tcells, which lacked d T M P synthetase activity, exhibited only one peak, that corresponding to the position on the DEAE-cellulose column of the phage-initiated enzyme. 3. Phage-Indurerl F H 2 Rerlurtuse
Although extended synthesis of FH, reductnse w:is not observed after infection of bacteria with T4 amber mutants or UV-irradiated phage (Sekiguchi and Cohen, 1964; Warner and Tmvis, 1966), the FHr rcductase extensively purified from T6-infected Escheiichia coli differed in so many important respects from the E . coli enzyme that it is probably a. different protein (Mathews and Suthcrland, 1965). The E . coli enzynic exhibited no p H optimum with either reduced nirotinamide adenine dinucleotide (NADH) or reduced nicotinaniide adenine dinucleotide phosphate (NADPH) ; rather it showed a gradual decrease in activity as the pH was raised from 4 to 8. With NADH, the activity fell to zero above pH 7. The T6-induced enzyme, on the other hand, had R definite optimum with each substrate a t about pH 7.0 and was partly active with NADH. Thc E . roli FH, reductase precipitated only in the presence of rrlativcly liigli ( N I I , ) ,PO, concentrations (6&80% h:itur:ition), whcrcah tlie I)h:igc.-iiiducctl c~iizynic~ w u s cwnpletely prc’cipitated a t 50% saturation. Tiic pli;igc.-iiitluced c.lizgiric w : i b uiihtahle, losing all of i t h wtivity during 2 iiiinutch of henting :it 50°C.; the host-cell enzyme retained morc than 60% of its initial activity during 15 minutes a t the same temperature. The E . coli enzyme was slightly activated by 4 M urea, whereas
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SAUL KIT
the T 6 enzyme was almost completely inactivated under the same conditions. The host-cell FH, reductase was much less sensitive to inhibition by aminopterin and its dihydro and tetrahydro derivatives than the enzyme from T6-infected cells. The sedimentation coefficients in sucrose density gradients of the two enzymes differed. The estimated molecular weight of the host-cell enzyme was 22,000, whereas that of the viral enzyme was 31,000.
4. Phage-Induced Deoxyrabonucleoside Monophosphate Kinase The dTMP and the dGMP kinases of normal Escherichia coli cells are easily separated individual enzymes and neither enzyme catalyzes the phoephorylation of d H M P (Bessman and Bello, 1961). After infection by T 2 phage, a kinase was induced which could be separated by DEAE-ccllulose chromatography from the d T M P and dGMP kinases of noninfected cells. The phage-induced enzyme catalyzed the phosphorylation by either ATP or dATP, of dGMP, dTMP, and also d H M P (Bello and Bessman, 1963a; Bello et al., 1961b). This T2-induced kinase has been purified 200-fold, and it appears that a single enzyme utilizes all three nucleotides as phosphate acceptors. The evidence is as follows: ( 1 ) all three activities fractionated similarly; ( 2 ) all three substrates were competitive inhibitors of one another; and ( 3 ) the three activities were inactivated at about the same rate by incubation a t low p H and by tryptic digestion. The dGMP ltinase of normal cells was stimulated five- to tenfold by K', Rb', and NHt; ions; these same salts all inhibited the corresponding enzyme from T2-infected cells (Bello et al., 1961a; Bessman and Van Bibber, 1959).
5. Phage-Induced D N A Polymerases After 600-fold purification, the DNA polymerase induced by T2 infection differed from the normal Escherichia coli polymerase in the following properties (Aposhian and Kornberg, 1962) : ( 1 ) rabbit antisera prepared against purified E . coli polymerase completely inhibited that enzyme but did not affect the T2 polymerase-T2 polymerase antiserum inhibited T2 polymcrase but not E . coli polymerase; (2) a t levels of p-chloroniercuribenzoatc that inhibitcd T2 polymerase almost completely, the E . coli polynicrase rettiincd 73% of its activity; ( 3 ) heatccl DNA w t ~ s10 tiii1t.s more cffective than unheated DNA as a primer for T 2 polyinerase but was not a good primcr for the normal E . coli polymerase. The T2-intluced DNA polymerase catalyzed the replication of approxiniatcly 14 to 35% of the primer DNA; howlcver, the E . coli polymerase, gave a 10- to 20-fold increase in DNA synthesized with
a n:ttive DNA primer. ( 4 ) A niixture of T2-iutlucctt and norinal E’. coli polymerases could be sharply and quantitatively separated by chromatography on a phosphocellulose column. A DNA polymerase has also been purificd 400-fold from T5-infected bacteria (Orr et al., 1965). The T5-inducetl enzyme was similar to the T2-induced enzyme in its requirement for driiatured DNA as primer and it catalyzed no more than a doubling of the DNA added as primer. The T5-induced polymerase was activated about fourfold by 0.2 M salt, whereas the E . coli polymerase was inhibited by salt. The altered properties of DNA polymerases induced by ts mutants of T 4 and T5 are described in Section III,F,2.
6. Vaccinia- and Herpes Simplex-Induced D N A Polymerases The DNA polyinerase induccd in HcLa cells by vaccinia virus had a greater affinity for DNA primer than thr I-IeLa cell enzyme. Both enzymes had a broad pH optimum between pH 7 and 8. However, the ratio of the activity a t pH 9.0 to that a t pH 7.8 was ahout 0.75 in extracts of infected cells and 0.14 for noninfected cells (Jungwirth and Joklik, 1965). Neutralization tests with specific antibody against DNA polynierases from noninfccted and vaccinia-infected cells have shown that a new antigenically distinct enzynie was formed after vaccinia infcction (Magee and Miller, 1966). Evidence that the DNA polymerase activity of herpes simplexinfected cells was not identical with that froin noninfected cells derives from differential behavior of the enzymes with respect to heat inactivation, primer, and uni- and bivalent cation requirements (Keir et al., 1966a). Furthermore, control and herpes siniplex-induced DNA polymerases behaved differently after preincubation with an antiserum against herpes simplex virus-specified proteins. The activity of the infected cell polymerase was stimulated after preincubation with increasing amounts of preimmune serum but was drastically reduced after preincubation with the antiserum to extracts from herpes virus-infected cells. On the other hand, the enzyme preparation from the control cells was stimulated by both preimmune and immune sera (Keir et al., 1966b).
7. Thymidine Kinase Induced by DNA-Containing Animal Viruses Thymidine kinase has been partially purified from noninfected L M mouse fibroblast cells and from vaccinia-infected LM or LM (TK-) cells (Kit and Dubbs, 1965). To learn whether these d T kinase preparations were subject to feedback inhibition by dTTP, the enzymes were incubated with d T T P concentrations ranging from 0.01 to 0.1 m M and with
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SAUL KI T
ATP c.oticcntr:~tions\ytt.yiiig f r o t l l 2.4 to 16.8 null. ' r l ~ ccll'cvt:: of t m " ' thc) i~l~osplioi~ylntioii of 'II-dT : ~ t r t l H-tlU wcre btii(licv1. rTti,ler all the cxperinlciltal collditiotis tcstcd, the cnzyiiics froiii noninfccted and infected cells were about equally susceptible to dTTP inhibition. Pronounced differences were, however, observed with respect to the thernial inactivation of thc enzymes. Whether prepared from LM cells in the logarithmic phase of growth, in the phase of negativc-growth acceleration, or in the stationary phase, the enzymc from noninfected cells was inactivated after preincubation a t 38°C. with a half-life of about 29 to 43 minutes. I n contrast, the enzyme purified from vaccinia-infectcd LM(TK-) cells was considerably more stable; the half-life of the vaccinia-induced enzyme exceeded 300 minutes. Partially purified d T kinase preparations from vaccinia-infected LM cells should represent a mixture of vaccinia-induced and pre-existing host-cell enzymes. Consistent with this concept, it was found that the enzyme from vaccinia-infected LM cells was more stable than the enzyme from noninfccted LM cells but less stable than the enzyme from vaccinia-infected LM (TK-) cells. Similarly, McAuslan (1963b) has observed that the d T kinase activity induced by cowpox virus in HeLa cells was more resistant to heat inactivation than the HeLa cell enzyme. However, the d T kinase induced in LM(TK-)cells by another DNA-containing animal virus, hcrpcs simplex, was unstable. The halflife of the herpes simplcx-inducc(1 enzyme a t 38°C. was about 45 minutes (Kit and Dubbs, 1965). The LM cell (IT kinxse could be protected against thermal inactivation in vitro by the addition of the feedback inhibitor, dTTP (0.01 niM), or the substrates dT (0.1 mM) or ATP (15 m M ) , T o test the possibility that the stability of the vaccinia-induced dT kinase was due to enzymebound nucleosidcs or nucleotides, the enzyme was filtered through Sephadex gels and treated with Norite A (charcoal). This treatment did not cause an inhibition of thc vaccinia-induced dT kinase nor did this treatment diminish the thermal stability of that enzyme. The immunological properties of the vsccinia-induced d T kinasc differed from thosc of the LM cell cnzynic. Antisera prepared by inoculating rabbits with partially purified prcpai-utions of the vacciniainduced d T kinase neutralized the activity of that enzyme in vitro although preirnmune sera or sera from rabbits inoculated with extracts from noninfected LM cells did not (Kit and Dubbs, 1965). The vaccinia-dT kinase antiscra did not, however, inhibit thc in vitro activity of the LM cell enzyme. yGlobulins prepared from the scra of roosters immunized with enzyme prcparations from noninfected and pseudorabies-infected rabbit 011
kitlncy cells huvc~ :~lso beeii tested for inhibitory activity against the I’seudorabies-induced and the rabbit kidney d T kinases. These cxperiinents have ~ h o w nthat the d T kinas? present in infected cells was unrelated antigenically to the protein performing the same function in noninfected cells (Hamada et al., 1966). The d T kinase induced by poxvirus infection had an altered Michaelis constant (IC,) value from that of the enzyme from noninfected cells. Different I<, values were demonstrated with either d T or dU as nucleoside acceptors (Kit and Dubbs, 1965; McAuslan, 1963b). Table VI illusTABLE VI (K,) FOR D E O X Y U R I D I N E FOR I’ARTIALLY PURIFIED TFIYMIDINE KINASEPREPARATIONS FROM NONINFECTED CELLSAND FROM VIRUS-~NFE(’TED CELLS COMPARISON O F h ~ l C l l A E L I SC O N S T A N T S
Cell liiie Mouse fibroblast, (LM)
**
Noiie Noiie None None
3 . 2 0 . 1 (8) 2 . 8 0 . 2 (3) 2 . 8 k 0 . 2 (4) 2 . 8 k 0 . 4 (3)
Mouse kidney (primary)
Vaccinia-infected, 5 hr. Vaccinia-infected, 7 hr. Vaccinia-infected, 7 hr.
7 . 2 0 . 5 (14) 9 . 0 f 0 . 7 (4) 8 . 8 1 . 2 (3)
Mouse fibroblast [LM(TK-)]
Herpes simplex-infected, 7 hr.
1 . 9 f 0 . 2 (7)
CV-1
Adeiiovirus SV15-infected, 20 hr.
5 . 2 f 0. 1 (4)
GXIK (priniary)
cv-1
Mouse kidney (pri1niu-y) Mouse fibroblast) [LM(TK-)I
GMK (primary)
* *
Vnlues given are the means k standard erros of the means. The numbers in parentheses indicate the number of determinations.
trates data on the I<, values for dU of d T kinase preparations from several sources. It may be seen that the dT kinase induced by vaccinia in LM(TK-), GMK, or mouse kidney cells had a K,,, value that was 2 to 3 times greater than that of the corresponding enzyme from noninfected cells. The K , value of the adenovirus SV15-induced enzyme was also elevated. On the otlier hand, the K,,, value of the d T kinase induced by herpes simplex virus was significantly lower than that of all of the other enzymes. Trifluoromethyl-2’-deoxyuridine (F,dT) competitively inhibits the plios~~liorylationof ’H-dU. The ?H-dU phospliorylation reaction catalyzed by the dT kinase from herpes simplex virus-infected cells was conaiderably nwrc sensitive to inhibition by F,dT tlitin either the v w (3ini:i-in(liicedor t l l c not~nlnlT,RI (TI<) ccll (~Ilzytiws.
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SAUL K I T
IV. Viral-Induced Enzymes That Hydrolyze or Modify Deoxyribonucleic and Ribonucleic Acids
A. DEOXYRIRONUCLEASES OF NONINFECTED Escherichia coli Extracts of E. coli can hydrolyze DNA to its constituent nucleoside 5’nionophosphates. The deoxyribonuclease activity is accounted for by at least five physically and catalytically distinguishable enzymes (Lehman, 1963; Oleson and Koerner, 1964; Short and Koerner, 1965). Four of these enzymes are exonucleases, catalyzing the successive removal of mononucleotides from the 3’-hydroxyl termini of polydeoxyribonucleotides. The fifth nuclcase, cndonuclease I, catalyzes the hydrolysis of phosphodiester bonds a t many sites within the polymer and is less fastidious with regard to the secondary structure of the DNA substrate. Endonuclease I attacks both native and denatured DNA’s ; however, denatured DNA is degraded a t about one-fifth the rate of native DNA. The enzyme also hydrolyzes native DNA’s from the T-even phage which contain glucosylated hydroxymethyldeoxycytidylate a t about the same rate as the DNA’s not containing glucose or hydroxymethylcytosine (Richardson, 1966). All of the E. coli nucleases are activatcd by divalent cations and exhibit optimal activity in the pH range of 8 to 10. Although endonuclease I activity is inhibited by RNA, that of the exonucleases of E . coli is not. The presence of endonuclease activity in autolysates of purified ribosomal particles and also in the soluble fraction of the cell not sedimcntable in 90 minutes a t 105,OOOg suggests that more than one endonuclease may actually be present in E . coli cells. Moreover, Weissbach and Korn (1962) have observed two peaks of endonuclease activity in DEAE-cellulose chromatograms of E . coli extracts. Exonuclease I utilizes denatured, single-stranded DNA as substrate ; exonuclease 11, which is physically associated with highly purified DNA polymerase, cleaves either native or denatured DNA. Denatured DNA is hydrolyzed more rapidly than is native DNA. Exoriuclease I11 requires double-stranded DNA. This enzymc (DNA phosphatase-exonuclcase) releases 5’-mononucleotides from DNA and also cleaves 3’-phosphomonoester groups terminating polydeoxyribonucleotides. Exonuclease I V (oligotidase) catalyzes the release of acid-soluble products from partially degraded DNA 20 times more rapidly than from either native or denatured DNA. The influence of glucosylatiori of DNA on the hydrolysis by E . coli exonucleases has been studied by Richardson (1966). Exonuclease I, like endonucleasc I, hydrolyzes T-even phage DNA at only slightly diminished rtitrs conipared to tliosc obscrvucl with T7 DNA. H O W C V C ~ ,
VIRAL-IKDUCED ENZYMES AND VIRAT, ONCOGICNESIS
127
exoiiiicIpzisc TI (tlic nucI(.sse associated with DNA polymerase) and rxonucleaee I11 ( DNL4phoephatase-cxoiiiiclease) hydrolyze native DNA froni T-cvm 1)arteriophxge at, lcss thaii 5% the rate of T7 DNA. The rcduccd activity of moniirIcvisv III is iittributtihle to tlic presence of glucose in the DNA; if modified T2 or T 6 DNA (DNA-containing HMC in place of cytosine but no glucose) is used as substrate, the modified T 2 and T 6 DNA’s are degraded by exonuclease I11 at rates approximately twentyfold greater than those found for T2 and T 6 DNA’s. The phosphatase activity of exonuclease I11 is not significantly influenced by glucosylation of the DNA substrates (Richardson, 1966). OF DEOXYRIBONUCLEASE ACTIVITIESBY T-EVENAND T5 B. INDUCTION BACTERIOPHAGES
Pardee and Williams (1952) first demonstrated an increase in deoxyribonuclease activity in extracts of phage T2-infected Escherichiu coli. A similar increase was found after T5 phage infection (Crawford, 1959) or after infection with phage T2 that had received a dose of UV radiation sufficiently high to prevent phage multiplication (Kunkee and Pardee, 1956). It was postulated by Kozloff (1953) that the apparent increases in the levels of DNase activities were actually due to the partial destruction of an RNA fraction which was a DNase inhibitor and that no change in DNase activity occurred. However, Stone and Burton (1962) demonstrated that when the extracts were incubated with pancreatic ribonuclease to degrade endogenous RNA, the T-even and T5 phageinduced DNase activities still were three- to fourfold greater than the DNase activities of noninfected cells. The T2 phage-induced DNase increase occurred 2-15 minutes after infection, whereas the T5 phageinduced D N a w increase took place between 10 and 25 minutes after infection. The increases were greatest a t about pH 9 and with heated DNA as a substrate and the nucleasc activity was inhibited by addition of exogenous RNA. Addition of chloramphenicol immediately after virus infection prevented the increase in DNase activity suggesting that the increase required concoinitant synthesis of protein. The DNase which appeared after T2 infection was more sensitive to inhibition by p chloromel.curiberizoate than were the activities in noninfected or T5 phage-infected bacteria, suggesting that the nucleases were distinct proteins. In agreement with the results of Stone and Burton (1962), Oleson and Koerner (1964) found that crude extracts of T2- and T6-infected cells cleaved heat-denatured DNA more rapidly than native DNA. Kinetic studies of the degradation of different substrates showed that the enzyme in extracts of infected cells which attacked heat-denatured DNA
128
SAUL KIT
was not, I~owever,thc ciizynic directly responsible for forniation of most of the acid-soluble nucleotides. The initial rate of prodnction of acidsoluble prodiirt,s from Iirat-drnatiirid DNA was only 30% of tJhe rate observed when tlw onzymr attacking rlcnaturctl DNA I d gcricrated sufficient oligonuclcotides to provide substrate for the DNase which rapidly formed acid-soluble products. If partially degraded DNA was furnished as substrate for the latter enzyme, the rate of degradation of this substrate was almost independent of the time of incubation. This enzyme appeared in the fraction of soluble extract precipitated with 45 to 69% saturation of ammonium sulfate. Enzymes in crude extracts could produce acid-soluble products rapidly because of the synergism of several enzymes. Oleson and Koerner (1964) purified the oligonucleotidase from extracts of T2 phage-infected Escherichia coli. This purified enzyme failed to attack native or heat-denatured DNA or RNA a t an appreciable rate, was not inhibited by RNA, required magnesium ions for activity, and was maximally active a t pH 9. Additional DNase activities appeared after infection of E . coli with T-even phage. One of these newly formed enzymes was an endonucleasc which preferentially utilized denatured DNA as substrate (Lehman, 1963). The second was an exonuclease associated with the DNA polymerase induced in E . coli by T-even phage (Short and Koerner, 1965; Weissbach and Korn, 1964). This latter enzyme attacked oligonucleotides prepared by the partial degradation of DNA much more rapidly than it attacked heat-denatured DNA, but differed from the oligonucleotidase first studied by Oleson and Koerner (1964). The T2-induced exonuclease of Oleson and Koerner (1964) was separated froin the T2 phage-induced DNA polymerase-exonuclease either by fractionation with protamine or by chromatography on DEAE-cellulose (Short and Koerner, 1965). Both of the T2 phage-induced oligonucleotidases could be distinguished from all DNases found in noninfccted E . coli. Tlic oligonucleotidases differed from E . coli cndonuclcase I and cxonuclease I1 in that the latter enzyme did not attack partly degraded DNA and thc endonuclease was inhibited by RNA, whereas the oligonucleotidascs were not. The oligonucleotidases could be distinguished from E . coli exonuclease IV by chromatography and from E. coli exonuclease I by their low activity on denatured DNA. The T2 phage-induced oligonuoleotidases could also be separated chromatographically from E . coli exonuclease 11. The oligonucleotidases differed from E . coli exonuclease I11 since the latter enzyme utilized nativc DNA and had low activity on partially degraded DNA.
1. Function of Phrige-Induced Nficlenses
The T-even and T5 colipliages induce the breakdown of liost DNA. It was logical, therefore, to suppose t h a t the induced DNases function in this breakdown. This does not appcar to be tlie case. The T5 phageinduced Dh'ase does not appear until 12 minutes after infection (Stonc arid Burton, 1962). By this time, two-thirds of the bacterial DNA is rendered acid-soluhle. Furthermore, Crawford ( 1959) has shown that the rate of degradation of bacterial DNA is not affected by addition of sufficient chloraniphenicol to prevent any synthesis of protein after phage infection. Thus it appears t h a t in the T5 system (which is closely related to the T-even phage system) the breakdown of host D N A is performed by a nuclease present in the noninfected organism or by a n as yet unidentified virus-induced nuclease. Also, the DNase induced after induction of lysogeiiic phage in E . coli K12 (A) increases greatly but host-cell D N A does not break down. The concept t h a t the induced DNases function to destroy the DNA of superinfecting phage has been considered but evidencc that this is a major function is lacking. There has been conjecture that the induced nucleases play a role in genetic recombination or in DNA replication, but evidence for these functions is also lacking.
C . DEOXYRIBOXTJCLEASE INDUCED BY Bacillus subtilis PHAGE SP3 Following infection of B. subtilts with phage SP3, there was a fiftyfold increase in the activity of a nuclease which degraded heat-denatured DNA to acid-soluble fragments (Trilling and Aposhian, 1965). The incre:tsc in DNase activity was first observed a t about 14 minutes after infection, and maximal activity WRS attained a t about 40 minutes. T h a t protein synthesis was required for thc increase in DNase activity was shown by experiments in which chloraniphenicol addition 5 minutrs after infection prevented the enzyme increase. It is known that phage SP3 infection of B. subtilis causes the induction of defective B. subtilis phages. T o sliow that a defective phage did not cause the DNasc increase, B. szibtilis cells were treated with mitomycin C to induce defective phages. No Dh'ase increases were found after this latter treatment. Chromatography on DEAE-cellulose columns of extracts from noninfected or p1i:ige SP3-infcctctl cc~llsrcvcalecl the presencc of a distinct pc:tk of DNasc activity i n tlie txtractb froin infected cclls. No sucli I)l\'asc activity pcuk w i i h prtwwt in the prcparatioii II om noninfectetl cclls. The pliage SP3-inducecl nuclense required MgCI, for activity and
130
SAUL KIT
was not active when the MgCl, was replaced by CaCI,. I n contrast, the nuclease from noninfected cells showed a 130-fold stimulation of activity when MgCl, was replaced by CaC1,. These results suggest that a new DNase is induced after SP3 infection. The role of the SP3 phage-induced DNase is not known.
D. NUCLEASEACTIVITYASSOCIATED WITH INDUCTION OF PHAGE A Escherichia coli K12 (A) (lysogenic for phage A) can be induced to form vegetative phage by mitomycin C treatment or thymine deprivation. Chromatography on DEAE-cellulose of streptomycin-treated extracts obtained from the bacteria yield four peaks of deoxyribonuclease activity. The relative amounts of DNase activity in the first three peaks did not change after the lysogenic bacteria were induced to form vegetative phage. However, the activity in peak IV, increased thirty- to fiftyfold (Korn and Weissbach, 1963). Experiments with E . coli K12 ( S ) (nonlysogenic) showed that mitomycin C treatment caused no significant increase in nuclease activity in the peak IV region, However, during lethal infection of E . coli K12(S) with A, there was also a marked rise in the phage-related nuclease. Furthermore, a nuclease similar to the A nuclease was formed after mitomycin C induction of E . coli K12 lysogenic for phage 434 (Korn and Weissbach, 1964b). During lethal infection of sensitive cells with A or after mitomycin C induction of E . coli K12(A), the synthesis of the A-associated DNasc began after a 20-minute latent period and continued until phage maturation was complete a t about 80 to 100 minutes (Korn and Weissbach, 1964s). The kinetics of DNasc formation were somewhat different in the case of thyniineless induction. I n this instance, enzyme activity began to increase during the induction period and continued to rise as long as the thymineless state was maintained or until spontaneous lysis of the cultures occurred. However, as soon a s thymine was added to the cultures, thereby allowing phage-DNA synthesis to begin, there was an abrupt cessation of nuclease synthesis. The level a t which DNase synthesis stopped was variable and depended only on the duration of the thymineless state suggesting that an early step in the induction process allowed the expression of the transcription functions of the h prophage independently of its replication and that control of the cessation of virus-directed enzyme formation was in some way related to viral-DNA synthesis. The peak IV enzymes from noninduced cells and the enzyme formed after induction of E . coli K12(h) have been highly purified and appear to be exonuclcases. The h nuclease represent a type of DNase
wliicli difl'crs i n ;it I e a b ~stkvcr:~I features froin the exonurlease fou~ul in the same peak in noninduccd K12(h) or K12(S) cells. The phage exonuclcasc was distinctive in having a more alkaline pH optimum and it showed a inarkcd preference for double-stranded DNA. The corresponding peak I V enzymes from noninduccd E . coli K12(h) or from sensitive E . coli K12(S) cells, or, indeed, from T 4 phage-infected cclls all attacked heated DNA 10 times faster than native DNA (Korn and Wcissbiich, 1963, 1964b; Weissbach and Korn, 1963, 1964). The exonuclease fornied after induction of E. coli K12 (434) was identical in most of its properties to the nuclease associated with the induction of phage h. The 434-associated exonuclease had a pH optimum of 10, exhibited a marked prefercnce for native DNA as opposed to heat-denatured DNA, and was not sensitive to ribonuclease. However, i t differed slightly from the h-associated exonuclease in its behavior on columns of hydroxylapatite and in its more rapid sedimentation in sucrose gradients. That the synthesis of the peak I V exonuclease is controlled by phage h has been established by a study of defective lysogens (Radding, 1964a). Defective lysogens are bacterial cells bearing mutant-defective prophage; they offer an opportunity to study lethal niutations of phage the gcnoines of which can be propagated as prophage along with the Iiost genome, but are unable to produce mature phage particles. Defective lysogens of h fall into two classes: ( 1 ) early mutants which are blocked prior to wgctative replication of tlie phage genome, and ( 2 ) late mutants which are blocked in some subsequent maturative function. Among the flrst class of defectivc lysogens are those with mutations in the N cistron of A. A number of suppressor sensitive ( s u s ) mutants of h in cistron N have been studied. I n a permissive host (i.e., E. coli CR63), these hsus mutants in cistron N undergo replication after treatment by inducers, and exoiiiicleasc activity increases. The sus mutants of h cannot grow in nonperniissive hosts although the latter can be lysogenized by hszis, thereby forrning defective lysogens which cannot be induced to yicld phage. After iiiduction of nonpermissivc hosts carrying hsus mutants in cistroris other than the N cistron, a rapid increase in nuclease activity was observed similar to that found with wild-type A. However, three independently isolated hsus mutants in cistron N failed to show the increase in nuclease activity shown by wild-type h or other sus mutants on treatment with inducing agents. It was a t first supposed that the N cistron was the structural gene for h exonuclease. This conclusion has, however, been questioned (Protass and Korn, 1966). Mutation in the N cistron results in a failure to produce h cxonuclease. However, the production of h endolysin (cistron R ) is also defective in the same mutants. Moreover, the N mutants of h
132
RATTL KIT
fail to hyi1tltty&(>X DNA 01’ X n i c ~ s c ~ ~ liNA, i g ~ ~ rattclhting to tlio drficiciicay of other X-associated functions. The inability of N mutants to syrithesize
exonuclease and endolysin is not due simply to in:tbility to produce h DNA. Mutants in cistrons, N , 0, and P all fail to synthesize X DNA, but the mutants in the 0 and P cistrons do produce both enzymes. It, therefore, appears that N may be a regulatory cistro11, the protein product of which “turns on” functions by allowing the initiation of transcription of early h cistrons. Although defective lysogens carrying mutations in the N cistron of h phage produced little or no A-exonuclease activity upon induction, defective lysogens in cistron TI1 showed a fivefold greater rate of increase in exonuclease activity and 10 to 30 times greater yields of activity than wild-type (Radding, 1964b). When the X exonuclease was highly purified after induction of lysogcn, XTI1, it was found that the hTll-associated exonuclease and the wild-type exonuclease were similar in the following properties: ( 1 ) strong specificity for native DNA; ( 2 ) p H optimum; ( 3 ) divalent cation reyuirement; ( 4 ) K,n value; and ( 5 ) inhibition by sodium chloride. Inhibition and competition experiments with antiserum prepared against partially purified hTll exonuclease demonstrated immunological similarity with the wild-type enzyme. These data indicate that the active site of XTll exonuclease had not been mutationally altered. Presumably, the T11 mutation indirectly enhances the synthesis of X nuclease (Radding, 1966; Radding and Shreffler, 1966).
E. HERPES SIMPLEX-A N D POXVIRUS-INDUCED DEOXYRIBONUCLEASES Pronounced increases in deoxyribonuclease activities occur in cultures infected with poxviruses and with herpes simplex virus (Hanafusa, 1961; Jungwirth and Joklik, 1965; Keir arid Gold, 1963; McAuslan, 1965; Russell et al., 1964). The increases begin a t about 2 hours after infection and closely follow those for virus-induced d T kinase. Further, as in the case of dT kinase, puromycin addition a t the time of infection prevents the increases in DNase activities, and addition of puromycin to infected cultures after thc DNase increases have begun halts further enzyme inductions (.Jungwirth and Joklik, 1965; McAuslan et al., 1965). It appears that a t least three deoxyribonuclease activities are involved in these increases. The three nucleases can be conveniently described by their substrate preference and pH of maximal activity. One DNase (alkaline SS-DNA) was maximally active for heat-denatured DNA at about p H 7.8 to 8.5 (Eron and McAuslan, 1966; Jungwirth and Joklik, 1965; Keir and Gold, 1963). The second nuclease (alkaline DS-DNA) hydrolyzed native D N A a t p H 9.2 and was induced in chick embryo fibroblasts or HeLa cells by cowpox, rabbit pox, and vaccinia viruses
: L i d i l l I-IeLa cell nioiioluycrs I)y Iicrpes siiiil)lox virus. Tlic tliircl iiiicleasc (acid IINase) degracletl thermally denatwed D N A at p H 6 . This latter nucleasc increased five- to tenfold in activity after poxvirus infection of HeLa cell cultures. The alkaline DS-deoxyribonuclease and the acid DNase have been tentatively characterized as exonucleases. NO increase in any DNase, (single- or double-stranded substrate a t alkaline, neutral, or acid pH) was denionstratcd after adenovirus Type 2 infection of KB cells. I n contrast to the puromycin inhibition of the synthesis of poxvirusinduced d T kinase, the puromycin inhibition of the increase in acid DNase was not reversible, suggesting that the mcseenger RNA for acid DNase was comparatively unstable. I n support of this suggestion it was found that R rapid inhibition of the increase in acid 1)Nase followed addition of actinomycin D. Similar results have been obtained with alkaline DS DNase. As previously discussed, UV-irradiated poxviruses induced d T kinase a t the same rate as live virus, but with W-irradiated virus, the normal shutoff of kinase synthesis was impaired. Induction of alkaline D S DNase was found to be more sensitive to UV irradiation than was induction of d T kinase. However, repression of both d T kinase and alkaline DS DNase was abolished by the same UV dose (McAuslan and Kates, 1966). I n contrast to this, when HeLa cells were infected with vaccinia virus irradiatcd with graded doses of UV light (survival lo-* to very little induction of alkaline SS DNase was observed (Jungwirtli and Joklik, 1965). Further evidence that somewhat different control mechanisnis exist for different poxvirus-induced enzymes has been obtained through studies of the effects of inhibitors of DNA synthesis on the regulation of enzyme inductions. I n vaccinia virus-infected HeLa cells, induction of alkaline SS DNase occurred at the same time whether viral DNA was allowed to replicate or not. The enzyme increased rapidly in the presence of dFU or aminopterin, and this increase continued beyond the time when synthesis of the enzyme was switched off under conditions permitting viral-DNA replication. Thus, similar switch-off mechanisms were observed for d T kinase, DNA polymcrase, and alkaline SS DNase in vaccinia-infectetl IleLa cells (Jungwirtli and Joklik, 1965). Similarly, in cowpox- or rabbit pox-infected HeLa cells, dFU addition prior to about 2.5 hours PI prevented the termination of the increase in induced alkaline D S DNase (hl cAuslan and Kates, 1966). In striking contrast to the effect on the regulation of the two alkaline DNases, the addition of dFU to HeLa cells a t the start of infection completely prevented thc normal increase in cowpox virus-induced acid
134
SAUL KIT
DNase activity. Tlic b:t111[7 resultb twre o1)t;tiiicd uhing :iiiiiii~i)t~~riil ill place of dFU. Furthermore, after dFU was removed from cowpox virusinfected cells and dT was added to insure prompt reversal of viral-DNA synthesis, acid DNase activity rapidly increased. This indicates that a mechanism for terminating the increase in acid DNase activity was not established during the delay in initiating DNA synthesis. In a study of pseudorabies virus-infected rabbit kidney cells, Kamiyn et nl. (1964) found that dBU prcvented infectious virus formation although dBU-labeled DNA was produced. Thymidine kinase and the enzymes for incorporating 3H-dCMP into DNA were also induced in the dBU-treated cells. Normally, enzyme synthesis stopped at about 6 hours but the substitution of dBU for d T in the progeny DNA allowed the enzyme increases to continue up to 14 hours after infection. If dBU was added to HeLa cell cultures before 2.5 hours after poxvirus infection, the drug was also effective in preventing thc onset of d T kinase repression (McAuslan and Kates, 1966). Similarly, dBU treatment prevented the termination of alkaline DS DNase formation in poxvirus-infected HeLa cells. I n dBU-treated cultures, an increase of acid DNase activity did take place, but the rate was somewhat lower than i t was in poxvirusinfected cells not treated with dBU. Thus, induction of acid DNasc differed from that of the other DNases in that the control of the increase in its activity was dependent on viral-DNA synthesis.
F.
PHOSPHORYLATION O F
NUCLEICA C I D
BY AN ENZYME: FROM
T4
BACTERIOPHAGE-INFECTED Escherichin coli Bacteriophage T 4 infection of E. coli leads to the appearance of an enzyme, polynucleotide kinase, which catalyzes the transfer of orthophosphate from ATP to the 5’-hydroxyl termini of a wide variety of nucleic acid compounds (Richardson, 1965). The specificity of the enzyme permits the phosphorylation of DNA, RNA, small oligonucleotides, and even nucleoside 3’-monophosphates. Novogrodsky and Hurwitz (1965) have described an enzyme with similar properties from T2 phageinfected bacteria. The T4-induced polynucleotide kinase could he detected 5 minutes after infection and increased to a maximum a t about 20 minutes. The enzyme has been purified and requires divalent cations, 5’-hydroxyl-terminated DNA, and the addition of mercaptoethanol or reduced glutathione for niaxiiuum activity. The Mn++ions can partially replace the Mg++ions. The pH optirnurn for enzyme activity was about 7.4 to 8.0 in tris-fIC1 buffer. The function of polyriucleotide kiiiase is not known. Perhaps the presence of 5’-phosphoryl termini may serve to prevent initiation of hydrolysis by exonucleases. The presence of 3’-phosphoryl end groups
VIRAL-ISDUCED ENZYMES AND L‘IHAL ONCOGENES18
135
in D N A has been shown to prevent exonuclease attack in addition to eliminating their template activity for D N A polymerase. Another possibility is that polynucleotide kinase, together with other enzymes, might lead to the synthesis of a polynucleotide chain bcaring an activated 5’-terminus. This, in turn, could result in the condensation of performed polynucleotide chains.
G. INDUCTION O F I)EOXYRIRONUCLElC ACIDi\/lETHYLASE INFECTED CELLS
BY PHAGE-
Infection of Escherichia coli B with bacteriophages T1, T2, or T 4 results in a pronounced increase in the activity of DNA methyla~e.The largest increase was observed after infection with T 2 phage; T 4 and T1 phages produced smaller increases in t h a t order of decreasing magnitude. After infection with T3, T5, or T 6 phages, a decrease in activity was observed; T 7 and h phages had no effect (Gold e t al., 1964; Hausniann and Gold, 1966). The kinetics of the increase in DNA methylase activity after T 2 phage infection was simihr t o t h a t found with other “early enzymes” induced by T-even phages. The increase was first detected a t 2 minutes after infection and rcaclied inaxinial levels a t about 10 to 15 minutes after infection when phage-DNA synthesis occurred. Mixing extracts from noninfected and from T 2 phage-infected cells resulted in additive activity, suggesting t h a t T 2 phage infection did not inactivate an inhibitor present in excess in normal cells. T h a t de nova protein syntlic.sis was esscntial for the increased DNA methylase activity was shown i n two ways. First, when chloramphenicol was added a t the time of infection, the incrcasc in DNA methylase activity was not observed. Second, a methionine-requiring strain of bacteria was infected in the absence of the necessary amino acid, and enzyme activity followed. I n the presence of mcthionine, D N A methylase activity increased. However, in the absence of methionine, no significant change in enzyme activity took place after infection. When niethionine was added to the culture a t 25 minutes after infection, the rise in D N A methylase activity began without an appreciable lag. The ability of T 2 phage to induce nn increase in D N A methylase activity could be innctiv:ited by preirradiation of the phage with UV light. l3otli the nictliyl:isc~-iiiclucing cap:icity :ind the infectivity of the phage were inactivated oxponentially with single-hit kinetics when bacteria were infected a t a ~nultiplicity of 0.3 particle per bacterium. However, the rate of inactivation of inethylase-inducing activity was about 30% of that of phage-producing activity. With a UV dose of survival, both the rate of increase and the
maximum level of rnetliylasc activity attained were lower than with nonirradiated phage. Even though the increase in activity continued past the normal shutoff time of 10 to 15 minutes, the results were in marked contrast to those found with other early enzymes such as dGMP kinase, where enzyme synthesis continued for relatively long periods of time. Higher doses of UV radiation severely depressed both the rate of increase and the final levels of DNA methylase activity. The UV light experiments tend to discount the possibility that it is a nongenetic factor associated with phage infection that brings about the changes observed. A study of mutant strains of phage T2 has yielded information concerning the regulation of DNA methylase synthesis. All amber mutants tested in permissive hosts had the ability to induce an increase in DNA methylase activity, and the enzyme levels attained were quite similar to those obtained with wild-type phage. In the nonpermissive hobt, however, amber mutants blocked in DNA synthesis induced increased levels of enzyme activity and DNA methylase synthesis continued for a considerable length of time beyond the normal “shutoff” time. Thus, although the regulatory mechanism for shutting off DNA methylase synthesis was relatively resistant to UV light, it was impaired in amber mutants defective in DNA synthesis. The rII mutants of T-even phage are unable to grow normally in Escherichia coli K12 (A) although DNA and protein syntheses proceed until about 10 minutes after infection and then decline abruptly. The total DNA made corresponds to about 10 phage units per cell. The DNA synthesized in E . co2i K12(X) after abortive infection with rII mutants of phage T4 contains hydroxymethylcytosinc, so that the defect associated with the r I I mutation is not a failure to form dCMP hydroxymethylase (Nomura, 1961). When DNA methylase was measured in E . coli K12(A) after infection with TBrII, the increase in x t i v i t y was similar to that obtained with wild-type phage in initial rate, duration, and total amount of enzyme induced (Hausmann arid Gold, 1966). Deoxyribonucleic acid methylase has been highly purified from extracts of phage T2-infected cells. The properties of the induced enzyme differed from those of the host-cell enzyme suggesting that the phage and the host-cell enzymes were distinct proteins. The phage-induced enzyme was more stable than the host enzyme a t all stages of purification. Reagents such as Twccn greatly stimulated the purified phage enzyme but they were without effect on tlie host-cell enzyme. Unlike tlie niethylases of E . coli W or K12 strains, that of E . coZi U catalyzed the transfer of a methyl group only to adenine residues producing 6-methylaminopurine. In strains W and K12, 5-nietliylcytosine was also formed. The methylase purified from phage T2-infected cells, however, catalyzed only
thc form:ition of 6-nid Ii~laniiiioliuriiie,n-lirtlier the hobt w a b the B 01’ the K12 strain of E . c d i . Ah htnted v:irlior, D N A nict I i y l : t ~ (i~i o t uiily w a s not induced in h r toria infcotctl witlr I)li:igc T3 but the wtivity of the pre-existing hostcell enzyme rapidly tlcclinetl. 1l:xtr:tcts of T3-infected cells, when mixed with extracts from either normal cells or T2-infected cells, markedly inhibited the activity of the latter (Gold e t al., 1964). These inhibitions were caused by the induction by T3 phage of an enzyme catalyzing the hydrolysis of S-adeiiosylmethioiiine, the methyl donor rcquired for the DNA methylation reaction (Gefter et nl., 1966) : 8-iidenosylmethionine
---f
thioniethyladenosine
+ homoserine
(11)
This enzyme appeared only after T3 infection. Cell-free extracts from noninfected bactcria or from bactcria infectcd with T1, T5, T7, T-even, or phagea contained no significant liyrlrolytic activity. Enzyme formation required de novo protein synthesis, appeared 2 minutes aftcr infection, reached a maximum a t about 8 niinutcs and then dcclined. The enzyme was isolated from normal T3-infected E . coli and from E . coli infected with UV-irradiated T3. The activity of cell-free extracts induced by UV-irradiated T3 was sevcral times liiglicr than extracts obtained from normal T3-infectctl cclls, and “cxteridcd” enzyme synthesis was observed. The function of the niethylatcd bases in DNA is not unknown. A possible role of inethylation in host-induced modifiration has been suggcsted mid will bc discussed in Section IV,I,5.
H. INDUCTION OF GLUCOSYL TRANSPERASE ACTIVITIESBY T-EVENPHAGE The T-even phage differ from Escherichiu coli in containing H M C in place of cytosine in their DNA’s. I n addition, the DNA’s of the Tcven phages contain glucose linked to the hydroxymethyl group of H M C in characteristic ratios. I n T 2 phage DNA, 25% of the H M C is nonglucosylated, 70% is monoglucosylated, and 5% is diglucosylated. The binding of glucose to the hydroxymethyl group of H M C is by means of an a-glucoside linkage. 111 T 4 phage DNA, all the H M C is in the monoglucosylated form but 70% has the glucose attached in the LYconfiguration and 30% has a /3-linkage. I n T 6 DNA, 25% of the H M C is nonglucosylated, 3% is monoglucosylated with the linkages the same a s in T2, and 72% is diglucosylated. The diglucosyl-HMC is a disaccharide in which the 2 glucose residues are linked t o each other in ;t p linkage and the hydroxymcthyl group of H M C in an N linkage (Kuno and Lehman, 1962; Lichtenstein and Cohen, 1960; Lehman and Pratt, 1960). Enzymes that transfer glucose from UDPG to DNA-containing H M C
138
SAUL KIT
:ire iiitluccd i n Ixicteriii infcctctl with the T-even pl~agosbut not by
phage T5. Enzyme activity is not detectable in lioninfected bacteria (Kornberg e t nl., 1959). The increases in the DNA-glucosylating enzymes (glucosyl transfernscs) commence a t about, 7 minutrs and are normally shut off :it :ibout, 20 niinlitcs aftcr infe(%ion. The fitil11l.c of certain amber inutants of T4 to turn off glucosyl transferase synthesis a t the normal time has been discussed in Section III,F,l, and the isolation of mutant virus strains defective in glucosyI transferase-inducing activity has been described in Section 111,F,5. From the evidence cited, i t is apparent that the induction of glucosyl transferasc activities is a function mediated by T-even phage genes. The glucosyl transferases have been highly purified from T-even phageinfected cells and are conveniently described by their substrate specificities aild the nature of the glucoside linkage formed (Kornberg e t al., 1961). All of the T-even phages induce HMC a-glucosyl transferases. In addition, phages T 4 and T 6 induce H M C p-glucosyl transferascs (Table VII). All three a-glucosyl transferases add glucose to cnzymically TABLE VII EXTENT OF GLUCOSYLATION OF DEOXYRIBONUCLEIC ACID ACCEPTORS BY GLUCOSYL TRANSFERASES~ ~
~
hccept,or DNA:
Trarisferases
Enzymically synthesized HMC-DNA T 2 D N A
T4DNA
T6DNA
(% of total HMCb residues glucosylat,ed) T2 HMC L Y - ~ ~ U C O S Y ~ T4 HMC Oc-glucosyl T6 HMC Cu-glucosyl T4 HMC j3-glUcosyl T6 glucosyl-HMC ~-glllCoSyl
50-58 66-75 50-7 1 70-78 <1
7 7 28 70
<1 <1
<1 <1 <1 25
70
" From Kornberg et al., 1961. HMC-5-hydroxymet hylcytosine.
synthesized, HMC-containing DNA. The T 4 DNA, with no unsubstituted H M C groups, does not serve as acceptor with a-glucosyl transferases. The a-glucosyl transferases of T 4 and T 6 were distinguishable from the enzyme of phage T 2 by the extent to which they added glucose to T 2 DNA. Although there was no detectable transfer to T2 DNA by the enzyine from T2-infected cells, there was a small addition of glucose to T2 DNA by the a-glucosyl transferase from T4- and T6-infected cells. The amount of glucose transferred to the DNA of T2 and T 6 phages
by the j?-gIucosyl transferasc of T4-infected cells was close to the amount of unglucosylated H M C in these DNA’s. It did not react a t all with T 4 DNA, as expected from the absence of unglucosylated H M C residues, but reacted extensively with enzymically synthesized H M C DNA. The TG glucosyl-HMC /I-glucosyl transferase did not bring about any detectable transfer of glucose to enzymically synthesized H M C DNA nor to T 6 DNA; however, it did transfer glucose to T2 and T 4 DNA’s so that the diglucosyl content of these DNA’s was increased to the extent found in T 6 DNA. Although the three a-glucosyl transferases differed in their ability to attach additional glucosyl residues to T2 DNA, the purified enzymes were similar in the following properties. These three enzymes were not readily precipitated from extracts of infected cells by streptomycin sulfate, they were quantitatively held by DEAE-cellulose, and they were eluted from this adsorbent in similar yields a t similar salt concentrations. The a-glucosyl transferases wcre alike in their requirement for a protective sulfhytlryl reagent, their inhibition by phosphate buffer and by MgCI?, and their insensitivity to ethylenediaminetetraacetate (EDTA) . However, the T 4 a-glucosyl traiisferasc differed from the T 2 and T6 enzymes in its K,,, value for UDPG (Josse and Kornberg, 1962; Zimmerman e t al., 1962). Fractionation of crude extracts containing the T 4 HMC ,8-glucosyl transferase showed that this enzyme was more readily precipitated by streptomycin sulfate and was separated from the a-glucosyl transferase early in the purification procedure. The T 6 glucosyl-HMC /I-glucosyl transferase was largely precipitated from extracts with streptomycin sulfate and was weakly held by DEAE-cellulose and was, therefore, readily separated from the T 6 H M C a-glucosyl transferase. The T 6 pglucosyl transferase, like the a-glucosyl transferase, required a sulfhydryl reagent, whcrcas the T 4 p-glucosyl transfcrase could be fractionated and assayed in the absence of a sulfhydryl reagent. Both /I-glucosyl timsferases required MgCI, for maximal activity. A inajoi. question to be nnswercd is how a given glucobylating enzyme rcachcs a fixed limit short of the total number of groups available for glucosylation of the DNA acceptor provided. In relation to this qucstion, Lunt and Burton (1962) and Lunt et al. (1964) have studied dinucleotide and trinucleotide sequence patterns in T2 phage DNA. It was found that the a-glucosyl residues were not distributed a t random among the HMC residues. Nonglucosylated HMC residues in T2 DNA were found predoininantly i n two kinds of sites; those in which a H M C residue was linkctl thrnougli it. 5’-positjon to mothein IlhlC iiuclrotjdr find thoso in w l r i c ~ lit~ 1i~tli~o~yiiic~tlryldcosycyti~liii~ w:th linkctl tlirough its 3’-positioii
140
HAUL
mr
to a purine nucleotide. The simplest interpretation of thc different degrees of glucosylation is that there are two independent factors which may restrict glucosylation of a H M C residue and which presumably reflect the specificity requirements of the T 2 a-glucosyl transferase. Glucosylation seems to be restricted in all sequences containing adjacent H M C nucleotides or when a single H M C nucleotide is linked through its 3'-position to a purine nucleotide. Taking these facts into account, the further glucosylation of T2 DNA by T 4 and T 6 H M C a-glucosyl transferases in vitro has been used to investigate possible differences between the substrate specificities of these enzymes arid T 2 a-glucosyl transferase (dc Waard, 1964a,b). In the in vitro reaction, the T 6 and T 4 a-glucosyl transferases introduccd a further 1.3-6.3 moles of radioactive glucose per 100 gm. atoms of phosphorous in T 2 DNA. The T 4 and T 6 H M C a-glucosyl transferases were able to glucosylate H M C nucleotides situated between two purine nucleotides in T 2 DNA and in certain other sequences where H M C nuclcotides were linked through their 3'-positions to purine nuclcotidcs; the T 2 enzyme was unable to do this.
I. HOST-CONTROLLED MODIFICATION AND VIRUS-INDUCED ENZYMES 1. Host-Controlled Modification of T-Even Phage
Host-controlled modification of viruses is a general term applied to those cases in which passage through certain host strains imparts one or more new, noninheritable properties to a virus without altering its genetic information content. The first well-documented case of hostcontrolled modification concerned the multiplication of phage T2 in certain strains of Escherichia coli B that cannot adsorb phages T3, T4, and T7 (B/3,4,7) (Luria and Human, 1952). Host-controlled modification has since been observed with the other T-even phages and with E . coli phages T1, T3, T7, P2, and A, and with Salmonella and Psezcdomonas phages (Arber, 1965a; Uetake et al., 1964; Holloway, 1965). It will be convenient in the discussion which follows to employ certain abbreviations. Host-modified phages will be designated by asterisks, e.g., T"2, T"4, and T"6 signify host-modified T2, T4, and T 6 phages, respectively. The notation, E . coli K12(X) or E . coli K12(P1) signifies, that E. coli strain K12 is lysogenic for phage X or phage P1. The notation, E . coli B/3,4,7means that this E . coli B strain fails to adsorb phages T3, T4, or T7 and, hence, is resistaiit to thein. Finally, X'B :tnd T4'W3110 denotes that the last host in which h and T4 phages, respectively, have been passed are E . roli drains B and TT'3110. Normal T2 :ind TA 1)li:tgc pnrticlcs pro(lucc plaques with a high
efficiency wlicn plated with E . coli 13 or SliirJella dysenteriae bacteria, but produce no plaques when plated in modcratc amounts with E . coli strain B/3,4,'1.The B/%,,,7 cells adsorb T2 and T6 phages and proceed to carry out one cycle of phage growth yielding a burst of progeny phage particles which are still able to produce plaques when plated on Shigella (perniissive host), but which now possess the novel character of being unable to produce plaques when plated with E . coli B. This character is not inherited, since one further growth cycle in Shigella restores to all progeny phage the capacity to produce plaques in E . coli B. A study of the metabolism of E . coli B/3,4,7revealed that, in contrast to E . coli B, the mutant strain was unable to ferment galactose. In fact, this strain was galactose-sensitive in that growth was stopped in minimal medium by the addition of 0.01 Af galactose, which indicates that the inability to ferment galactose was not duc t o the lack of galactokinase: galactokinase
galactose
+ ATP -
--t
galactose-1-phosphate
+ ADP
Further analysis revealed that galactose-1 -phosphate-uridyl transferase and epimerase activities were present in extracts of E . coli B/3,4,7cells but that the extracts were deficient in UDPG synthctase (pyrophosphorylase) activity:
+
gal~ctose-1-phosptiate VDPG
trantfcmse 7 -
UDP-galwtose
+ glucos~-l-phosphate (13)
1':pirnernue
IJDPG
UDP-galartose
UTP
IT111'0 syrithetrtve
+ ~l~icose-1-phosphate
* UIjPG
(14)
+ pyrophosphate
(15)
The deficiency in UDPG resulting from the mutation in UDPG syiithetase formation has a number of consequences. The UDP-galactose, normally generated in the uridyl transferase reaction, cannot be made. The UDPG and UDP-galactose are necessary for the incorporation of glucose into a polysaccharidc layer of cell walls; the absence of these sugars alters the phage receptor sites in this structure. Moreover, UDPG is utilized for the addition of glucose to the HMC found in the DNA of T-even phages. Therefore, the phage DNA synthesized in E . c d i B/,$,,,, are deficient in glucose. For example, the DNA of T2 pliage particles released by a single cycle of growth in E . coli straiir 13/q,4,7 contained less than 5% of the glucose of an equivalent amount of normal T 2 DNA (Symonds e t al., 1963). I n addition to strain B/3,4,7,coli strains K12-W4597 and HfrC-Ug5 are galactose negative. These bacteria are defective in UDPG synthetase activity and yield modified phage (T") when infected with T-even
I'll:igtb. cell nl:llls of strains ~ 4 5 9 7a i i ~ l~ 9 coii~:iin 5 110 gtllactosc :111(1 tllc3T* I311;lgCS l'roduced in these strains cont:tin little or 110 glucose in thcir DNA (Erikson and Szybalski, 1964; Fukasawa and Saito, 1965; Hattman and Fukasawa, 1963; Shedlovsky and Brenner, 1963; Sundarajan e t al., 1962). The T" phages lack the ability to grow in E . coli B but multiply in strains of Shigella dysenteriae giving rise in a single growth cycle to normal phage. Revertants of E. coli B/3,4,7capable of utilizing galactose have been isolated. These revertants had lost their resistance to phages T3, T4, and T 7 as well as their ability to modify T 2 and T6 to the T" forms. Thcy also h a t 1 rccovered normal UDPG synthetase activity. A study of the biochemistry of permissive bacteria (8. dysenterzhe) and restrictive bacteria ( E . coli K12, strain W3110) following infection by host-modified T N 4has revealed significant data relevant to the failure of T"4 to form plaques on the restrictive host. It was found that the nonglucosylated DNA of T"4 was partially broken down to acid-soluble fragments in strain W3110 but not in S. dysenteriae. This breakdown began immediately after infection and was complete within several iiiinutes, although 50-60% of thc adsorbed phage DNA remained insoluble in acid (Fukasawa, 1964). Similarly, the DNAs of phages T"2 and T"6 were extensively degraded to acid-soluble fragments following infection of restricting E . coli cclls, whereas infection of E . coli with wild-type phage or of Shigella with T" phages c:uised very little degradation of phage DNA (Hattman, 1964b). It would appear that 8. dysenten’ae, the permissive host, does not differentiate between the glucosylated and nonglucosylated DNA's of T4 and T"4 phages, but that restrictive hosts such as E . coli K12-W3110 do so. One may imagine that a restricting factor, presumably a DNase, is localizcd outside the membrane of the restricting host. Thus, the nonglucosylated DNA of an abnormal T"4 phage would be broken down before passing through the protoplast membrane. Support for this view is provided by the finding that DNA-splitting nucleases are released by bacteria upon removal of the cell wall and that the nucleases of restricting strains act preferentially on the DNA of T"4 phage (Fukasawa, 1964). Also, Molholt and Fraser (1965) have found that conversion of restricting bacterial cells to spheroplasts, which results in quantitative loss of periplasmic nucleases, also reverses restriction for nonglucosylated T"2 and T"4 DNA. Richardson (1966) has shown that E . coli exonuclcascs I1 and I11 exhibit greatly reduced rates of hydrolysis of glucosylated DNA in vitro but has pointed out that this result should not be interpreted as suggesting that these two enzymes are responsible for the restriction of T"-even phage. All of the E . coli nucleases are present
VIRAL-INDUCED ENZYMES AND VIRAL ONCOGENESIS
143
i n similar ainourits in extracts of S. dysenteriae which does not restrict the T* phage. Host-modified T* phage are unable to initiate replication of their DNA's in restricting hosts (Fukasawa, 1964; Hattman, 196413). Nevertheless, certain phage functions can be performed. The enzymes, dCMP hydroxymethylase, and d H M P kinase are synthesized by T"4-infected W3110 (restrictive) cells in which replication of viral DNA does not take place. The kinetics of the formation of both enzymes are quite different from those in normal infection with phage T4: ( 1 ) both enzymes are synthesized a t slower rates than normal and are greatly dependent on the multiplicity of infection; and ( 2 ) both enzymes are formed for a longer period before synthesis ceases. I n T"6-infected E . coli B, the initial rates of synthesis of dCMP hydroxymethylase and cu-glucosyl transferase are lower than in wild-type phage infection. A plateau is observed a t 10 to 15 minutes, as in the case of normal T6; however, a t 35 to 40 minutes after T"6 infection, there is a renewal of enzyine formation, possibly due to secondary infection by nonrestricted phage progeny. The enzyme, d T M P kinase, is also made in T"6-infected E . coli B. The induction of DNA methylase activity in E . coli €3 by hostmodified T"2 phage is multiplicity dependent. At an input multiplicity of 50 phages per bacterium, a fortyfold increase was observed. Nevertheless, this was only 30% of the maximum level induced by normal T 2 phage. In the permissive host, S. dysenteriae, infection with either T2 or T"2 resulted in an increase in DNA methylase activity which was independent of the multiplicity of infection (Hausmann and Gold, 1966) . 2. Host-Controlled Modification of Phage h
Host-controlled modification of phage X occurs if Escherichia cola strain C is used as host instead of the usual E . coli K12. Phage A, adapted to E . coli C, grows on E . coli K12 with a probability of only about Other systems producing host-controlled modification of phage h are illustrated in Table VIII (Arber and Dussoix, 1962; Arber, 1965a; Lederberg, 1965) . It can be seen tlii~tX-C (phage grown on strain C) plates with a rehtivc rficiency of 1 on fit. coli C, :is do A*K, h ' K ( P l ) , and X'B, but that X'C has a low plating efficiency 011 h'. c o l i strains K12, K12(P1), and B. Also, X'K has :L plating efficiency of 1 on E . coli strains K12 or C but a. low efficiency of plating on strains B or K12(P1). T w o wstricting actionh exerted in the same cell result in a restricting action which is to some extent additive. The restricting action of prophage
144
H A U L KI'I'
P1 is superimposed on that of the host cell itself. Thus, the efficiency of plating of A.B is 4 lo-* on strain K12 and 10-6-10-7 on strain K12(P1). Escherichiu coli strains lysogenic for prophage P1 not only restrict h phage that has last been propagated on a nonlysogenic strain but also phages T1 and P2 (Lederberg and Meselson, 1964). As in the case of ?**-even phage infection of restrictive cells, the D N A of phage A is rapidly degraded to acid-solublc fragments in nonaccepting cells (Arber and Dussoix, 1962; Dussoix and Arber, 1962; Lederberg and
x
TABLE VISI EFFICIENCY O F PLATING OF PHAGE A VARIANTSIN DIFFERENT Hosr STRAINS" Host strains
c,
Phage variants
K12
A.K, A.Kr-m+ A.K(P 1) k B , bBr-m+ b C , kKr-m-, kBr-m-
1 1 4 x 10-4 4 x 10-4
K12 (P1)
SWW, Br-
B ~
2
x
10-6 1 10-6-10-7 10-6- 10-7
10-4 10-4
~~
1 1 1 1
1 10-4
0 The values are approximate relative titers of host-modified phage on different host strains. The generalized scheme holds in toto for phage X and in part for phages T1 and P2. The K12 strains include, K12, K10, AB259, C600, W3110, W1895, and U'4032. C-type strains include C, mutants of IC12-type strains, and mutants of B (Arber, 1965a; Lederberg, 1965).
Meselson, 1964). This breakdown bcgins shortly after phage attachment and successful D N A infection. Genetic markers for A*K may be rescued from nonaccepting K12 (Pl) cells, however, if the cells are simultaneously infected with both restricted A'K and unrestricted A'K(P1). Since DNA breakdown competes in time with rescue, the probability of marker rescue is high if the nonrestricted phage infects first, but low if the restricted phage infects first. Only closely linked markers have a good chance to be rescued together (Dussoix and Arber, 1962). Degradation of infecting phage D N A by nonaccepting cells has been found not only for T-even E . coli phages and phage A, but also for E. coli phage T1 and for Sal?noneZln phage E .
3. The Functions of Restriction ( r + ) arid Motltficiifiori
(In')
The reproduction of phage A in a new sensitive host strain depends upon two successive host-control mechanisms : ( 1 ) the infecting phage
DNA is either recognized a. iiicompatible with the host and degraded, or is accepted; ( 2 ) if fully accepted, it multiplies and its DNA replicas receive host specificity, i.e., the particular nonheritable stamp given by the host. The two separate functions of restriction and modification are also exercised by prophage P1. Escherichia coli lysogenic for P1 restricts h phage previously propagated in nonlysogenic strains. However, prophage PI also induces modifications of phage A. This modifying action occurs not only when h phage multiplies in cells lysogenic for P1, but also following superinfection of nonlysogenic cells in which X is multiplying (Arbcr and Dussoix, 1962). Glover et al. (1963) have isolated mutants of phage P1 which no longer restrict nonadapted phage A. Some of the mutants still carry out modification, whereas others were also defective in this function. If normal P1 phage is arbitrarily given the genetic structure r+m+,then these two types of mutants can be designated r-m' and r-m-. Thus, the control over the process of restriction and modification induced by P1 are each determined by a different gene. Tests with phages T1 and P2 1i:tr.e shown that r-m- strains of P1 that failed to restrict and niodify X phage were also permissive and nonmodifying for the other phages (Hattman, 1964a). A considerable number of r- mutants of E . coli R l 2 and B lacking the ability to restrict X phage of foreign host specificity have been isolated by Wood (1966). About half of the mutants appeared to be r-m+; the niaj ority of the remainder were r-m-. Mapping experiments using conjugation and phage-mediated transduction have shown that the r character is linked to the threonine locus, on the side opposite to the lcucine markers (Colson et al., 1965). Furthermore, the r characters of K12 were allelic with those of B, since no recombinants were obtained which gave Br and Kl2-type restrictions simultaneously. It might be predicted that bacterial DNA may be restricted and modified by the same factors that act upon the DNA of phage A. Tliib t*e+triction might be responsible for the abnormally low frequency of recombinant formation observed in K12 B crosses. The DNA of strain K12 would thus carry the K12 hoht specificity; DNA from K 1 2 ( P l ) would carry both the K12 and P1 hobt spccificitics, and so on. Good cvidence supporting this prediction has come from bactcrial-conjugation and phage-mediated transduction experiments. For example, the cross of Hfr K12 F-KlB(P1) is 100- to 1000-fold less fertile than the three other combinations, Hfr K12 x F-K12, Hfr K12(P1) x F-(K12), and H f r K12(P1) X F-K12(P1). Furthermore, zygotic induction of prophage A, carried out by the donor strain K12, is reduced in F K12(P1) by the same factor of I00 to 1000. The propcrties of the r~~odification-rcstrictiori
x
x
146
SAUL
Ixr
syst,c111of E. coli wggcst that it scrves :IS a defciise rnccliw~~isirn against inronlil~glliiclcic acid, prcvcnting the expression or integration of fol*eign T)NA without hindering genetic exchange among cells of the fiame strain (Colson et al., 1965; Wood, 1966). : ~ t c.hlot~arnpliciiicol l Mutations to incrp:tsed rcsistaiice l o stre~~toroycin affect the multiplication of host-modified phage B3 in Pseudornonas aeruginosa strains. Mutations to streptomycin and chloramphenicol resistance enable the bacteria to support multipIication of the phage (Holloway and Rolfe, 1964). 4. Heat Sensitivity of the Restricting Function Restriction has becn visualized as either a screening of new DNA by a DNA-site specific degrading activity-successful passage permitting the DNA to operate in that cell-or a scavenging of a new DNA by a nonspecific degrading activity when such DNA fails to be complexed with a site-specific protecting agent (Lederberg, 1965). Different bacterial strains might have different arrays of specific screening enzymes for specific sites. As a counterpart to this activity, the cell might have specific protecting enzymes which could modify otherwise sensitive sites to make these sites resistant to degradation. The breakdown of h phage DNA occurs in restrictive cells infected in the presence of chloramphenicol. Thus, the restricting factor is present prior to h phage infection (Arber, 1965a). The screening function (restriction) of bacteria may be inactivated by a brief preheating period. Cells of Eschem’chia coli B heated a t 51°C. for 10 minutes lost their restriction toward phage h'C600. Heated cells of strain 0 0 behaved in a similar fashion toward phage X'B (Lederberg, 1965). Cells of strain CSOO(P1) lost the P1-dependent restriction and the C600-controlled restriction somewhat independently. I n these heat treatments, the host-specific modification, as measured by the host range of the phage produced, was not noticeably impaired. Heat sensitivity of the screening activity against host-specific phage has been reported for Salmonella phage (Uetake et al., 1964) and for Pseudomonas phage (Holloway, 1965). I n the case of Salmonella, preheating nonpermissive cells a t 49°C. for 3 minutes not only permitted a n increased efficiency of plating of restricted phage but also reduced the breakdown of restricted phage DNA to acid-soluble form. It might be expected that cells could recover from the heat effect by regenerating phage growth restricting factor as time elapsed after heating. I n order to study the mechanism of this recovery, the effects of inhibitors of protein and of nucleic acid synthescs have becn
\ I I L ~ I ~ ~ I ~ D L J CENZYRIES L'D A N D VIRAL ONCOGENEhIS
147
examined. I n cultures treated with chloramplicnicol and heat and infected with restricted phage, the efficiency of plating of the phage remained elevated in contrast to the reduction observed without chloramphenicol. However, the efficiency of plating in cultures treated with mitomycin C and heat decreased a t a rate similar to that of cultures heat treated without mitomycin C. These findings give strong support to the hypothesis that protein synthesis is needed for the regeneration of the restriction factor. 5. D N A Methylation and the Modification Factor
If phage DNA is :tccepted in a host bacterium, the phage multiplies and progeny phages are produced. Those progeny to which the parentalphage DNA molecule is transferred in either conserved or semiconserved form receive the parental-phage host spccificity. All progeny containing only newly synthesized D N A receive only the specificity of the new Iiacterial host. Thus, host specificity is carried on the bacteriophage DNA. The basis for the niodificatioii of the DNA is not known. One attractive possibility is that host specificity is conferred by the alkylation of specific sites on the phage DNA. This could occur in enzymic reactions in which methionine is a specific donor (DNA methylase). To investigate this possibility, phage h has been grown in auxotrophic met-, pro-, or arg- strains of Escherichia coli K12 in the presence of the required amino acids. The phage progeny showed an efficiency of plating of approximately 1 on E . coli K12 or on E . coli C [the general acceptor strain for phage h (Table VIII)]. Howeyer, if met- cells were deprived of mcthionine during a portion of the latent period, the efficiency of plating of the progeny phage was lower on E . coli K12 than on strain C. In similar experiments, a methionine effect has been obtained for all the host specificities investigated: those of K12, of B, and/or prophage P1. Such an effect was not observed following a similar starvation for proline or arginine. These results suggest that the phage DNA is only incompletely supplicd with host specificity in the abscncc of methionine (Arber, 1965b). Coliphage T3 induces an enzyme which catalyzses the hydrolysis of S-adenosylmethionine, the substrate required for the DNA methylase reaction. I n order to test the influence of the T3 coinfection on T1 methylation, a lysate was made from niixedly infected E . coli cells. After reiiioval of phage T3, the T I phage was purified and analyzed for ntlcniiie niul G-iucthylarlcninc. 1t contained less tliaii half the 6-nit.tliyl:d(mirlc f o r l l d i r i T I g ~ ~ o wwithoiitj n T3. To test wllct,JtcI- t!hc mod-
148
SAUL KIT
ification of T1 particles could bc impaired by the encynle induced h' phage T3, strain Bc ( P I ) cells wcre simultaneously infected with the two viruses and tested on Shigella ( P l ) . Only 570 of the cells yielding T1 phage after infection with T1 T3 produced modified phage (Klein and Sauerbier, 1965). These findings support the concept that host-controlled modification of T1 DNA by lysogenic host bacteria involves methylation of the DNA which can bc suppressed by simultaneous infection with phage T3.
+
J. BACTERIOPHAGE GENESCONTROLLING SENSITIVITY TO ULTRAVIOLET LIGHT The UV sensitivity of T2 coliphages is about twice as great as that of T4 coliphages. The level of this sensitivity is controlled by the Vt gene and behaves as a unit character. Mutant T 4 strains (T4v) have also been isolated with increased UV sensitivity similar to that of phage T2 (Harm, 1963). The v+ gene apparently controls reactions with the colicoIiphage system which repair the UV damage in the DNA of the coliphage. There is considerable evidence that thymine dimers are major lethal photoproducts in double-stranded DNA. This damage is subject to different types of reactivation. Bacteria possess two known mechanisms whereby thymine dimers are eliminated from DNA. One of these is photoreactivation (Phr) , whereby thymine dimers are reconverted to thymine in situ in the presence of light and an enzyme fraction present in Escherichia coli and in yeast. I n addition, there is also a dark reactivation process which involves the excision of thymine dimers from the DNA (Setlow and Carrier, 1964; Shuster and Boyce, 1964). Cells able t o undergo the dark reactivation process exhibit postirradiation degradation of DNA manifested as a release of nucleotides into the acidsoluble fraction of the cell and into the surrounding medium. The latter process occurs by a sequence of a t least three major steps. First, a section of the polynucleotide chain containing the thymine dimers is excised. Thymine dimers, as such, are not removed from DNA, since free dimers are not among the products detected in nonhydrolyzed acid-soluble fractions. This indicates that the N-glycosidic bonds are probably not cleaved, but rather that the phosphodiester backbone of the DNA strand containing the dimer is broken. Removal of the DNA fragment containing the thymine dimer is presumably due to the action of an endonuclease, specific for certain defects in the DNA. Second, nucleotides are inserted in the excised region of a single DNA strand by complementary pairing with the intart opposite strand. Third, the broken ~)hospllotlicstcrImkbonc is uijoiiiecl ( h y r e and hlownrd-Flnn(lci.s, 1964;
Setlow and Carrier, 1964) .z A similar clurk repair mechanism operates in the repair of DNL4 damaged by tlic bifunctional alkylating agents, sulfur mustard or nitrogen mustard (Papirnicister and Davison, 1964). Escherichia coli B mutants have been isolated which do not photoI-cactivate UV damage (Phr-) and in which phages are not subject to photoreactivation. It is known that T4v+ gene action does not require visible light, although the damage reparable by the T4v+ gene can also be photoreactivated. It can he shown that the T4v+ gene repair action is not due to the creation by the T4v+ gene of conditions in which the bacterial photoreactivating enzyme can act without light. A comparison of the UV sensitivities of T4v+ and T4v in E . coli B and in the Phr- mutant revealed that the diffcrence between the survival curves for T4v+ and T4v were the same in both these bacterial strains. Thus, the v+ gene controls n dark-acting enzymc of specificity similar to the bacterial-photoreactivating enzyme (Sauerbier, 1964). Bacterial mutants have been iPolated which are deficient in the excision of thymine dimers and their ability to repair UV-irradiated DNA in the dark. These mutants are also unablr to repair UV damage suffered by irradiated phage T 1 DNA (Boyce and Howard-Flanders, 1964; Emmerson and Howard-Flanders, 1965; Shuster and Boyce, 1964). T o learn whether the T4v+ gene acts indirectly through the already existing excision-repair system of the bacterium or whether i t is responsible for the de novo synthesis of a T 4 dimer excision enzyme, the postirradiation survival curves were determined for T 4 grown on a UVresistant and on a UV-sensitive E . coli strain (Haber, 1966). If the phage were responsible for the synthesis of its own excision enzyme system, then there should be no differencc in the survival curves of T 4 grown on the two bacterial strains. If, howevcr, the phage utilized the bacterialexcision enzyme mechanism, there should be decreased T 4 survival in the E . coli-coliphage system in which the host lacks the excision system. Contrary to the second hypothesis, it was found that there was no detectable difference in the survival curves of T4 in the wild-type bacterial strain, which contained its own dirner excision enzyme, and in the mutant E. coli strain which ditl not. It may be concluded that thc v+ locus of T 4 is responsible for the de novo production of components of a reactivation system for UV-induced damage in phage DNA.
'Uninfected and phage T4-infected E . coli contain enzymes which catalyzr the covalcnt joining of polydeoxyribonurleotidcs. Polvnucleotide ligase (sc-nlase) is onr such enzyme which catalyzes the following reactions indicative of joining : (1) tlic conversion of internal 5'-phosphate cnds of DNA to a form resistant to itlhalinr~ phosphatase; ( 2 ) the closure of hydrogen-bonded circular lamhda DNA4 t o covalentlv r l o s d circlcs; and ( 3 ) thr, wpair of sin& ncl I J ,ik< ~ 111 I~ihrlic~tl DNA rriolrvilrs (Becker t l ( I / , 1967 ; \\'cbiss niul I
150
SAUL KIT
K. EFFECTS OF VIRUS INFECTION ON SOLUBLE RIBONUCLEIC ACID Herpes simplex virus contains double-stranded DNA of about 68 mole.
% (G + C) content. Replication of the virus takes place in the nuclei of many mammalian cells, the DNA of which is 40-44 mole % (G C) .
+
The large difference in mole percent ( G + C) content between virus and host DNA suggests the following consideration. Unless there is extensive redundancy, the population of sRNA molecules normally present in the host cell must be optimally adjusted to the translation requirements of messenger RNA transcribed from 40 to 44 mole % (G C) DNA and cannot also be optimally adjusted to the translation requirements of herpes virus messengers transcribed from 68 mole % (G C)
+ +
DNA. In animal cells, ncarest-neighbor analyses of DNA have shown that the doublet CpG occurs with a particularly low frequency. Doublet frequencies based on published nearest-neighbor analyses of human and mouse cell DNA’s have been compared with the doublet frequencies calculated by assuming randomness for the herpes simplex DNA of 68 mole ”/. (G C) content. It was found that the observed frequencies of the four doublets, GpC, CpG, GpG, and CpC, found in mammalian DNA represents 36, 9, 44, and 41% of the respective frequencies calculated for herpes DNA. This suggests that host-cell sRNA would be poorly represented a t the time of extensive synthesis of virus proteins. The eight triplets containing the CpG doublet in particular appear to be in short supply. However, if it is assumed that the invading virus genome itself contains genetic information for certain sRNA’s (and their activating enzymes), then after infection, the sRNA population would change in a direction favoring the requirements for virus-protein synthesis. On the basis of these considerations, hybridization experiments were undertaken to look for homology between labeled sRNA fractionated from herpes simplex and noninfected BHK21 cells and viral and cell DNA (Subak-Sharpe and Hay, 1965). It was shown that virus DNA hybridized specifically with the sRNA of infected cells but not with the sRNA of noninfected cells. The cellular DNA specifically hybridized with both sRNA from infected and noninfected cells. Thus, host-specific sRNA synthesis was not eliminated on infection with herpes simplex virus, but synthesis of virus-specific sRNA was initiated (Hay et al., 1966). About 1.2% of thc virus DNA specifically hybridized with the infected cell sRNA. This represents a length of DNA sufficient to code for 10 to 20 molecular species of sRNA. Further indio:xtiuri> th:it, virus infectioit : t l t c m the sRNA of iilfccte(1 cclls 1i:ive I m n obtninctl by Sucoko :~nd Kano-Sucoka (1964). Tllcse
+
VIRAL-INDUCED ENZYMES AND V I R A L ONCOUENESIS
151
investigators observed that upon phage T 2 infection of Escherichin coli R , L: spccific structural niodific*ation of lcuryl sRNA occurred which roultl he cdctwterl by fractionating the aminoacyl sRNA on methylated albuniin colunins. The modification startcd :it thc tliircl minute after infectioii : u i d w:is cwniplrte by tlic eighth minute. 1)e novo protein synthesis was required for thc modification. When the sRNA’s of noninfected and T2-infected cells were charged with radioactive amino acids and chromatographed on methylatcd albumin-kieselguhr (MAK) columns, only one amino acid showed a clear difference in the profile from the infected cells and from the noninfected cells. This was in the leucyl sRNA peak. The lcucyl sRNA of E . coli showed two major peaks on MAK columns (leuc I and leuc 11),leuc I being the major component. The leucyl sRNA from 3-minute infected cells showed a new component in front of the leucine peak which was not seen in the RNA from cells infected for 8 minutes. At 5 minutes PI, the new component increased, whereas the leuc I peak decreased considerably. After 8 minutes of infection, the new, front component, leucyl sRNA disappeared. PresumabIy, some component of leuc I sRNA is modified first to t,he front leucine peak and then further modified to the one in the leuc I1 region. The alterations in the sRNA could affect the translation of genetic information in infected cells due to the change in the adaptor. If a codon recognition of a particular adaptor out of a set of degenerate adaptors for an amino acid is changed by a structural modification, the messenger RNA of the genes that accommodate the codon corresponding to the modified adaptor should not be translated properly, whereas messenger RNA of the other genes that do not accommodate the codon should be translated normally. This means that by modifying a specific sRNA molecule, the function of some of thc genes which are transcribed can be shut off and the rest of the genes kept functional a t the translation ie~ei. V. Viral-Induced Ribonucleic Acid Synthetase I Replicase)
A. CHARACTERISTICS OF THE INDUCTION PROCESS The replication of RNA viruses logically suggests the existence of a n enzymic mechanism utilizing RNA as a template for the synthesis of RNA containing each of the four ribonucleotides. Until recently, such a n enzymic mechanism was unknown. Ribonucleic acid-dependent incorporation had been restricted to unique sequences of ribonucleotides, such as polyriboadenylic acid, or t o a limited addition of ribocytidylate or riboadenylate to the ends of pre-existing polynucleotide chains. Polynucleotide phosphorylase, although catalyzing the synthesis of polyribo-
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nucleotides, does not provide a mechanism for the synthesis of a specific sequence of ribonucleotides. Furthermore, although the enzyme, RNA polymerase (transcriptase) will utilize, in vitro, RNA as well as DNA as a template, there is no evidence that RNA synthesis by this enzyme, in vivo, is dirccted by RNA. Deoxyribonucleic acid is apparently utilized as a template for all bacterial and animal cell RNA syntheses. Ribonucleic acid-directed RNA synthesis is a prerequisite, however, for multiplication of the RNA of RNA-containing viruses. I n 1962, Baltimore and Franklin (1962a) demonstrated the existence of a particulate system in cytoplasmic extracts of mengovirus-infected L cells which polymerized all four ribonucleoside triphosphates into an acidinsoluble form. Similar RNA synthetase (replicase) enzyme systems were subsequently found in poliovirus-infected HeLa cells (Baltimore et al., 1963a), encephalomyocarditis virus-infected Krebs I1 cells (Horton et al., 1964), vesicular stomatitis virus-infected chick embryo cells (Wilson and Bader, 1965), and in influenza virus-infected chorioallantoic membranes of embryonated eggs (Ho and Walters, 1966). It was also shown that RNA synthetases were induced in Escherichia coli strains infected with f2, MS2, and Qp phages (August et al., 1965; Haruna and Spiegelman, 1965a,b; Weissmann et al., 1963) and in Chinese cabbage cells infected with turnip yellow mosaic virus (Astier-Manifacier and Cornuet, 1965). Ribonucleic acid synthetase activity was not detected, however, in chick embryo cells infected with Rous-associated virus (Wilson and Bader, 1965). The cytoplasmic localization of viral-induced RNA synthetase is one property that distinguishes this enzyme from the cellular RNA polymerase (transcriptase) ; the latter enzyme is localized in the nucleus. I n IleLa cells, a lipid-containing structure is developed in the cytoplasm after poliovirus infection; this structure appears to contain all the necessary machinery for virus production and maturation. Ribonucleic acid synthetase, virus protein, and a substantial portion of the newly forniecl viral RNA rapidly sediment with this structure (Becker et ul., 1963). The lcinetics of RNA synthetase formation in animal cells may be illustrated by events taking place after poliovirus or mengovirus infections. Ribonucleic acid synthetase activity was detected about 2 hours after infection, rose to a maximum a t about 5 hours, and then declined. When puromycin was present from the beginning of the infection, RNA synthetase activity did not appear. Moreover, if puromycin was added 3 hours after infection, the activity of the enzyme declined to negligible levels during the subsequent 2 hours while the activity of the enzyme in nontreated cells rose almost twofold. The fact that the enzyme activity
fell in the absence of continued protein synthesis indicates that RNA synthetasc in its functional form is unstable (Baltimore and Franklin, 1963a,b; Baltimore e t al., 1963a; Eggers e t al., 1963). Ribonucleic acid synthetase is very likely one of the essential proteins previously shown to be needed for viral RNA formation. It had been demonstrated t h a t inhibition of protein synthesis with puromycin a t nny time before the onset of poliovirus-RNA synthesis resulted in a coniplcte inhibition of the forniation of viral RNA. On thc other hand, when puromycin was added a t some time after the initiation of viral RNA synthesis, the formation of viral RNA continued, though a t a decrensiiig rate for approximately 1 hour, after which it came to a complete stop (Levintow et al., 1962; Wecker, 1963). Experiments with guanidine and 2- (a-hydroxybenzyl)benziinidazole (HBB) have provided further evidence for the instability of picornavirus-RNA synthetase. Both these drugs specifically inhibit the multiplication of poliovirus and other viruses of the picornavirus group. Guanidine and HBB also prevent the syntheses of virus RNA and protein. Addition of either of these two drugs to poliovirus-infected cultures a t the time of infection prevented the formation of RNA synthetase. If the compounds were added after enzyme formation had started, these compounds halted any further increase in enzyme, and, i n fact, a marked decrease occurred 4.25 hours after infection (Baltimore et al., 1963a). Replication of picornaviruses is refractory to actinomycin D. Infected cultures treated with this compound induce picornavirus-RNA synthctase and virus RNA. Additioii of actinoiiiycin D to extracts from picornavirus-infected cells does not inhihit ItNA synthetase activity in vitro nor is the cnzynie activity inhibited by DNase treatment. The lack of inhibition by actinomycin D and DNase p i w h d e s D N A as n template for this enzyme and distinguished picornavirus-induced RNA synthetase from host-cell RNA polymerase. A further distinction may be mentioned. The picornavirus-induced RNA synthetase requires Mg++ for activity, and the enzyme is inhibited by A h + + ion$:.This is in contrast to the host-cell RNA polymerase which can utilizc &In++in place of Mg++ (Baltimore and Franklin, 1963a; Horton e t al., 1964). The process of RNA synthetase induction by influenza virus differs in some respects from that by picoriiaviiuscs. Influenza. virus is one of the few RNA viruses inhibited by actinoniycin D. T h e drug inhibits an essential step in the early part of the replication cycle. I n contrast to the lack of inhibition by actinornycin D of R N A synthetasc induction in picornavirus-infeckd ('ells, actinoriiycin D addetl at the time of infection does inhibit the formation of influenza virus RNA synthetase.
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Furthermore, guanidine, which inhibits picornavirus replication and picornavirus RNA synthetase induction, has no effect on influenza virus replication or appearance of RNA synthetase activity in influenza virusinfected cells. In vitro, however, the RNA synthetase induced by influenza virus resembles the enzyme formcd in picornavirus-infected cells. It is refractory to actinomycin D, DNase treatment, or guanidine treatment, requires Mg++ions, is inhibited by RNase, and has a pH optimum of about 8.5, as do the picornavirus-RNA synthetases (Ho and Walters, 1966). Induction of RNA synthetase in bacteria infected with MS2 or f2 phages is first detectable from 6 to 10 minutes after infection. The enzyme increases for about 20 to 25 minutes and then remains constant until the cells lyse. The increase in RNA synthetase activity precedes by about 9 minutes the formation of infectious phage. The time that RNA synthetase attains maximal activity (about 20 minutes after infection) is 15-30 minutes before half the viral RNA and 20-30 minutes before half the progeny phages are made (Lodish and Zinder, 1966a; Ochoa e t al., 1964; Zinder, 1965). As in the case of the enzyme induced by picornaviruses, the f2- and the MS2-induced RNA synthetases polymerize ribonucleoside triphosphates into trichloroacetic acid-insoluble material; the activity is sensitive to RNase, yet insensitive to DNase and actinomycin D and magnesium ions are required for maximum activity. The RNA synthetase of phage f2 is induced in normal amounts even if RNA synthesis is inhibited markedly by uracil or adenine starvation of host bacteria prior to infection. This finding suggests that only input parental RNA molecules are needed for the initial formation of the enzyme. The production of the viral enzyme appears to be mandatory for the synthesis of phage RNA. Chloramphenicol added a t the moment of infection inhibits the induction of RNA synthetase and the production of viral RNA. If added within 10 to 15 minutes, when partial formation of the synthetase has taken place, chloramphenicol does allow normal synthesis of viral RNA even though further protein synthesis is inhibited (Cooper and Zinder, 1963; Paranchych and Ellis, 1964). Indirect evidence implicating the RNA syiithetase in phage f2 replication has been obtained using 1):wtwiaI nuxotrophs requiring histidine 2nd methionine. Thc protein rnpsid of pliagti f2 docs not contain histidine. It can be show11 that the synthesis of f2 becomes independent of the presence of histidine halfway through the latent period but remains dependent on the continued presence of methionine. Thus, an essential protein, other than capsid protein is synthesized after phage f2 infection (Cooper and Zindcr, 1963).
VIRAL-ISDL'CED ENZYMES AND VIRAL ONCOGICNESIS
H. 1)oumE-Smis [)ED R I ~ o N ~ . ( T , Edl(m I ( ~ ih
4
155
SINTHETASE PRODTJC~
Most of the infective RNA isolated from Krebs I1 cells after E M C infection has a size corresponding to the whole RNA contcnt of one virus particle as well as properties indicating t h a t i t exists in solution as single-stranded chains. However, an unusual kind of infective RNA w a s found by Montagnier and Sanders (1963). This new RNA first appcar~(1 a t the time that EMC RNA replication comn~enc~ctl antl increased with time, ebpecially a t 6 hours after infection. The new RNA fraction had the following properties: (I) in a sucrose gradient, it sedimented with a sedimentation coefficient of about 20 S, whereas infectious viral RNA had a sedimentation coefficient of about 3 7 s ; ( 2 ) the 20s RNA was relatively resistant to RNase, whereas RNase treatment caused a dramatic disappearance of the 3 7 s R N A ; (3) the infectivity of the 20 S RNA was not destroyed by RNasc treatment whereas t h a t of the 37 S RNA was; ( 4 ) the 20 S component r forinaldehyde treatment, whereas the 37 S form reacted with formaldehyde; ( 5 ) in (CS)~SO, density gradients, DNA had a buoyant density of 1.42 gm./cm.3 and ribosomal or E M C RNA's had densities of 1.63 gm./cm.?. The 20 S fraction had a buoyant density of 1.57 gni./cm.'; ( 6 ) as in the case of DNA i t had a sharp thermal transition upon melting, whereas single-stranded RNA had a broad transition. The T,n value (midpoint of the thermal transition) WRS 96°C. in 0.15M NaCl, 0.015 M sodium citrate antl 84°C. in 0.015M NuC1, 0.0015 M sodium citrate, values considerahly higher than t h a t of single-stranded RNA ; and (7) the 2 0 s RNA was synthesized from r:idioactive precursors in EMC-infected cells pretreated with actinomycin D, thcrehy demonstrating that it was not synthesized from host D N A templates. Ribonucleic acid fractions sedimenting a t about 18s and with properties similar to 2 0 s RNA of EMC-infected cells were later found in the cytoplasm of poliovirus-infected HeLa cells (Baltimore et nl., 1964) ; M E virus-infected L cells (Hausen, 1965) ; Semliki Forest virus-infected chick embryo fibroblasts (Sonnabend et al., 1964) ; TMV- and turnip yellow mosaic virus-infected plant tissues (Burdon et al., 1964; Mandel et al., 1964; Ralph e t al., 1965; Shipp arid Haselkorn, 1964), and from f2-, MS2-, and R17 phage-infected bacteria (Fenwick e t al., 1964; Kelly anti Sinsheimer, 1964; Lotlidi and Zinder, 1966a). All the properties of the 18 to 2 0 s RNA suggest a double-stranded helical structure. This conclusion was confirmed by X-ray diffraction studies of purified 18 S RNA from MS2-infected bacteria (Langridge et al., 1964). By labeling parental MS2 RNA prior to infection, i t could be shown that part of the parental RNA was converted to an infective,
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cloul)lc-sti.an(lccl forin. A(i(Ii1ioii:il ~ l o i i l ~ l ~ ~ - ~ t i ~ liNA : i i i t l w:is ~ ~ ~ l syiithesizcd cluri~lgMS2 replic:ltioil. T l l i i h , 0110 h t i*antl of the double-btrandcd RNA was derived froin viral RNA (Kelly and Sinsheimcr, 1964). A series of melting and annealing experiments further demonstrated that the second strand was a cornplcment of the first strand (Ochoa et al., 1964). Annealing and nucleotide lmse composition studies also showed t h a t one of the strands of the newly synthesized, double-stranded RNA made in TMVinfected plant leaves contained sequences complementary to T M V RNA (Shipp and Haselkorn, 1964; Weissinann et al., 1965). The double-stranded RNA was not produced when protein synthesis was prevented by addition of chloramphenicol to N1S2-infected bacteria 10 minutes prior to infection. Chloramphenicol added 15 minutes after infection had no effect on the synthesis of this material, indicating that a new phage-induced enzyme was needed for the synthesis of the doublestranded MS2 R N A and that this enzyme had been made by 15 minutes after infection (Kelly and Sinsheinier, 1964). The behavior of double-stranded RNA during a pulse-chase experiment was studied by Fenwick and co-workers (1964). Escherichiu coli cells were first irradiated with UV light to stop ribosonial RNA synthesis and then infected with phage R17. It was shown that the R N A most rapidly labeled after short 3H-uridine pulses sedimented in a broad band in a 16s region of sucrose gradients and had the properties of a doublestranded structure. The effect of RNase on this material and its behavior during a “chase” period in nonradioactive medium suggested that it consisted of a core of double-stranded RNA, one strand of which-the viral strand-was continuously displaced by a growing strand forming single-stranded tails and ultimately free 27S, R17 phage RNA (Fenwick et al., 1964). Pulse-labeling and chase experiments also showed that part of the label within double-stranded ME virus RNA could be replaced by newly synthesized material and t h a t the displaced strand had the propei-ties of ME virus RNA (Hausen, 1965). The double-stranded RNA was not only synthesized in infected cells but also in vitro in the reaction catalyzed by RNA synthetase. The enzymic syntheses of RNase resistant, 18-20 S RNA fractions were demonstrated with RNA synthetase preparations from EMC and poliovirus arid from MS2 and f2 phage-infected cells (August et al., 1965; Baltimore, 1964; Hortoii et al., 1964; Oclioa et al., 1964; Weissmann et al., 1963). Thus, molecules with the properties of double-stranded RNA have been detected in mammalian, bacterial, and plant cells infected with RNA-containing viruses, a s well a s in the product in vitro of virusinduced RNA synthetases from both bacterial and mammalian cells. That the double-stranded RNA is probably relevant to virus growth is
implied by the o1)scrvations that it is infective, tlint sonie of the RNA of the originally infecting virus is transformed t o such a form, and that one of the strands from the double-stranded forni can be displaced to give viral RNA.
C. MECHANISM OF REPLICATION OF A RIBONUCLICIC ACIDVIRUS On the basis of the experiments cited, the following model for the replication of an RNA virus has heen proposed (Ochoa et d.,1964; Fenwick et al., 1964; Lodisli and Zindcr, 19GGa). The parental-virus RNA molecule, immediately upon entering the cell, becomes attached to ribosomes and directs the form:ttion of a11 RNA synthetasc. The synthetase (enzyme I) now u p c ~the parental RNA as template for the synthesis of the complementary RNA stran(1, forming a double-helical RNA structure. This double-stranded RNA (replicative forni) then becomes n templatc for the synthesis of single-stranded viral RNA molerules, which is perhaps accomplished hy a wcond enzyme. A sinall portion of the newly made single-stranded RNA is converted by enzyme I into double strands that form new templates for the second step. This cyclic process continues throughout infection and the number of doublestranded RNA molecules increases in parallel with the number of viral single strands. Some of the single-stranded RNA’s may be taken up by ribosomes and participate in polysonie formation. These RNA strands are transcribed yielding virus-specific proteins such as the subunits of the capsid. Single strands formed later are no longer taken by ribosomes, but by competing subunits of capsid proteins which become abundant. The formation of single-RNA strands from an active double-stranded replicativc form proceeds hy means of an asytnmetrir, semiconservative mechanism. Nascent parental (“plus”) strands ai’e initially found in the duplex, presumably hydrogen bonded to complementary (“minus”) strmds. Synthesis of new “plus” strands proceeds by the displacement of the parental plus strand yielding a dou1)le-stranded core to which is attached parts of one or more, viral plus strands. Finally the parental plus strand is completely displaced.
D. PROPERTIES OF HIGHLYPURIFIED RIBOWJCLEIC ACID SYNTHETASE
As indicated in the previous section, it has been proposed that an early step in the replication of an RNA virus is the conversion of the incoming RNA into an RNase-rcbistnnt, tlouhle-stranded stt ucture whicli tlieii serves as u “replicativo f o n ~ ~for ” 1 1 1 gcnc~i~atiori ~ of \i~igle-str:iii(letI copies of progeny viral l i N A . Xccotdiiig to this view, re1)lication of takes placc hy iiiuch the wine mec11:iiiiiiiis as the replication of 9x1 74 DNA. Spiepc1in:iti nncl 1 J : ~ i ~ u n(19GG) :~ 1i:ive called :ittention
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to the following disturbing properties of the RNase-resistant structure produced in infected cells: ( 1 ) only a small percentage of the infecting viral RNA strands is found in this structure; ( 2 ) the RNase-resistant material tends to accumulate late in infection, long after many mature single strands have been made, a feature not easily reconcilable with the double-stranded structure being a mandatory initial intermediate. These findings are in striking contrast to the +X174 situation, where all infecting strands are converted into a replicative form and where the replicative form of DNA appears in the first stages of infection, long before appearance of single-stranded viral DNA. I n order that the mechanism of viral RNA rcplication might be clarified, RNA synthetase has been purified by several laboratories and the products of the in vitro reaction have been studied. August and co-workers (1965) purified an RNA synthetase 200-fold from extracts of a supressor negative (su-) Escherichia coli strain after infection with a mutant strain of phage f2. This f2 mutant, sus-11, caused the formation in the infected host of excess amounts of “minus” strands (in the double-stranded form) and of several times the normal amount of the enzyme. I n the reaction catalyzed by the purified enzyme, Mg++ions and each of the four ribonucleoside triphosphates of adenine, uracil, guanine, and cytosine were required. The addition of an RNA primer was also needed, but the enzyme was not fastidious as to the source of the RNA. Bacterial-ribosomal and soluble RNA’s as well as f2 or TMV RNA’s all satisfied the requirement for RNA. The enzyme not only catalyzed the polymerization of nucleoside triphosphates into RNA but also the exchange between inorganic phosphate and ribonucleoside triphosphates. The RNA product of the synthetic reaction had a polymer length of a t least 300 to 400 ribonucleotide units. The base composition of the product closely resembled that of the complement of the RNA primer. The product was partly resistant to RNase and possessed a high degree of secondary structure as evidenced by denaturation and renaturation studies (Shapiro and August, 1965). Possibly, this enzyme is the enzyme I referred to in the previous section. Weissmann and co-workers (1963) have obtained a fifty-fold purification of an RNA synthetase isolated from MS2-infected E . coli cells. The MS2 phage-induced enzyme was not inhibited by actinomycin D and it was essentially free of DNA-dependent RNA polymerase, but contained polynucleotide phosphorylase activity. The purified enzyme contained large amounts of RNA; hence, a dependence on exogenous RNA could not 1)c dcnionstrated. Part of the RNA syiithesizcd by the purified RNA synthetase was
VIRAL-ISI)UCED ENZYMES AN11 VIRAL ONCOGENESIS
159
ant to liNasc. Thc RNasc-rcsibtant product obtained l q RNase digestion of tlic reaction niixtiire had a lowrr Iiuoyant density in Cs,SO, than single-stranded RNA ant1 it liatl a +harp thermal transitiori a t 102" to 104"C., indicative of R highly ortlcrcd sccondary structure (Weissrnann and Borht, 1963). In order to determine whether the double-stranded product of the phage MS2 RNA synthetase reaction contained parental-typeJ MS2 RNA in one of its complementary strands, a specific-dilution test was devised based upon thermal denaturation and reannealing of the labeled duplex in the prcsence of nonlabeled MS2 RNA. The results showed t h a t oiily MS2 RNA, but not ribosomal, soluble, or TMV RNA displaced radioactivity froni the double-stranded synthetase product. The amount of labeled-plus strands displaced a t infinite concentrations of cold MS2 RNA could be estimated by extrapolation to be 85% of the radioactivity of the double-stranded fraction of the synthetase product. Presumably, the remainder of the label was in the complementary MS2 RNA-minus strand. It would appear t h a t the RNA synthetase purified by Weissmann and co-workers (1964) and Ochoa and co-workers (1964) was a holoenzyme in association with its natural template. Haruna and Spiegclinan (1965a,k) have purified an RNA synthetasc from bacteria infected with phage MS2 or with another unrelated RNA phage, QP. The use of Escherichia coli strain Hfr (Q-13) as host greatly facilitated the purification of tlie viral-induced enzyme. Escherichia coli Hfr (Q-13) lacks ribonuclease I and polynucleotide phosphorylase activities. The latter enzyme had been particularly difficult to eliminate during the purification of RNA synthetase, and the RNasc I introduced additional complications in the early stages of fractionation. The RNA synthetasc finally purified by Haruna and Spicgelman (1965a,b) was devoid of DNA-dependent RNA polyrnerasc, polyadenylate polymerase, :tnd 1)otassiuin-stimulatcd RNase 11. The purified enzyme from either MS2- or QP-infected cells showed a mandatory requirement for added RNA and a unique preference for homologous RNA. Ribosomal a i d solul,le RNA of the host could not substitute as templates and ncither of thew cellulur types showed any nhility to interfcrc with the tcmp1:ttc functioii of viral RNA. Comparison of the MS2 and QP enzymes isolated from tlic same host showed that each replicase could recognize the RNA genoirie of its origin and required it as a template for normal activity, but neither replicase could function with the other's RNA. The only heterologous RNA showing detectable activity was that of turnip yellow mossaic virus; it supported a synthesis corresponding to 676 of that observed with QP RNA and the Q p RNA syiithetase. The requii-cment for homologous RNA is significant and
1 60
SAUL KIT
provides :I solution for tlic cruciul prol)lcin of ail IlNA virus attcmpting to direct) its own duplication in a n environrnent replete with other RNA niolrculcs. Ry produring :t polyri~crnscthat, ignores the mass of preexistent ccllt~ltirRNA, :I gu:ir:iiitcc is 1)rovitled t11:it rcplicstion is focuscd on a single strand of incoming viral RNA, the ultimate origin of progeny. Added RNase inhibited the synthetase reaction but DNase did not, and an ATP-generating system was not needed. The synthetase had an absolute requirement for divalent ions, magnesium being the preferred ion with homologous RNA. Manganese substituted partially (10%) but induced changes in the nature of the reaction. The enzyme purified by Haruna and Spiegelman (1965a,b) was fully saturated with 0.025 pg. RNA/pg. protein and was competent for prolonged (more than 5 hours) synthesis of RNA. The phage Qp-induced enzyme generated a polynwleotide of the same molecular weight as viral RNA and a t the end of an experiment, the RNA synthesized corresponded to many times the amount of RNA put in a t the beginning. The RNA produced synthetically by the replicase was fully competent to program the production of complete virus particles. Thus, the enzyme was faithfully copying viral RNA (Spiegelman et al., 1965). Using the purified RNA synthetase, Haruna and Spiegelman (1966) have studied the mechanism of viral RNA replication. The data obtained did not confirm the model of viral RNA replication in which a doublestranded template, consisting of a viral-plus strand and its complementary strand, were obligatory intermediates in replication. If purified Q/3 replicase was presented with fragments of &p RNA a slow reaction (10% of normal) of abbreviated duration (30 minutes) was observed. The product was small, biologically inactive, and much of it was complexed in a heat-denaturable RNase-resistant structure. In contrast, when activated by intact templates, &p replicase produccd virtually unlimited amounts of complete and biologically competent replicas of Qp RNA but no heat-sensitive, RNase-resistant material was detected. Neither the base composition nor annealability to plusstrands revealed any compelling evidence for an initial appearance of complete complementary strands. I-Iaruna and Spiegelinan (1966) suggested that the results could be readily described by a nicchanism which involves the production of replicas without the intervention of an initially formed replicating duplex. The base composition, partial RNase resistance, and the limited compIementarity of the early products could be explainable in terms of partial copies of an RNA strand possessing a beginning sequence rich in adeninc and a complemcntary sequence rich in uracil near the end. Contrary to tlie rcsults of Httrun:i and Spicgelnmn (1966), however,
Weiasmarin and Feix (1966) found that whcn incubatcd with nuclcositlc triphosphates and Qp RNA as template, the Qp replicase3 synthesized predominantly, if not exclusively, “minus” strands in the early phase of incubation, and later on mainly “plus” strands. Presumably, further studies will elucidate the reasons for this dibcrepancy.
E. MUTANTS OF BACTERIOPHAGE f2 The study of mutant strains of phage f2 has provided another approach to the problem of the mechanism of replication of viral RNA. A temperature-sensitive f2 mutant (ts-6) g r o w in Escherichia coli strains a t 34°C. but not a t 43°C. Analysis of some of the physiological abnormalities of mutant ts-6 leads to the postulate that it contained a mutation in a gene specifying viral RNA synthetaw (1,odish and Zinder, 1966a). At high temperatures, mutant ts-6 did not initiate any of the events that normally ensue after RNA-phage infection. I n particular, the parental RNA was not converted to a double strand. When cells grown a t low temperatures were shifted t o high temperatiucs, synthesis of doublestranded R N A ceased. Single-stranded viral RNA continued t o be synthesized normally for some time after the shift. Thus, there was an enzyme (enzyme I) t>hatconverted single-stranded viral R N A to double strands, and it was this enzyme which was presumably thermolabile. Since the synthesis of single-stranded R N A proceeded normally for a t least 10 minutes after the shift up, it appears that a second enzyme (enzyme 11) was responsible for that synthe,qis. The sccond enzyme could he either the normal cellular RNA polynierase or a second phage-induced enzyme. The fact that, in time after the increase in temperature, the synthesis of single-stranded RNA was also inhihited implies t h a t this synthesis is coupled to the synthesis of double-stranded RNA. Further genetic evidence for the existence of an f2 phage-induced enzyme which converts single strands to double-stranded RNA has been obtained by using host-dependent, amber mutants (Lodish et al., 1964; Lodish and Zinder, 19661)). Amber mutations in the coat-protein cistron o f phage f2 nltcr the n o m : ~ lprocess of control of synthesis of both viral RNA and of RNA synthetnee. These effects depend on the position of the amber mutation and on the suppressor properties of the host bacterium. Phage mutant sus-3 carrics mi nmbcr codon near the N-terminus of the virus-coat protcin cisti~on.Whcn thi.: mutant is i ~ tod infect Su- h.%cri:i, wliic~li lack :L supi)ttssor gtwc’, inifi:illp little sitigle- or ~ l o i i l ~ l t ~ - ~ t i ~ :\,it ~ n:ilt l ~KN.1 tl 01’ \.it :iI-RNA bytitlieta~c:ti iuadr, althoi~glt t l i c i ~ ~ : i t x ~ n t :plitigv 11 liN.1 i h c o i i \ ~ c . t t t ~ till l t o :L ~ ~ ~ L I I ~ ~ f0t.m ~ ~ nl- ~ ~ I ~ A ~ (1
‘The
QP
Spiegelman.
reglicasr einplovrd in tlic study nns n gift from Drs. Haiunn and
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most as efficiently a s is RNA from wild-type phage. The functional lesion of sus-3 infected Su- bacteria appears to be in the synthesis of single-strandcd viral RNA. Infection with sus-3 of bacterial hosts such as Su 11, which are iionpermissive but partially suppress the amber mutation, results in normal synthesis of single-stranded and 14-18 S doublestranded viral RNA. As in f2 wild-type cells, all enzymes needed for this synthesis are made very early in infection. However, late in infection, all available single-stranded RNA (including RNA from superinfecting wild-type phngc) are converted to small (7 S) double-stranded RNA; this transformation is not merely the consequence of lack of viral-coat protein to encapsulate the RNA, but is dependent on protein synthesis late in infection. Furthermore, the amount of virus-induced RNA synthetase in extracts late in infection by sus-3 is 5-10 times that of extracts from wild-type cells. The excess mutant-induced enzyme has properties expected of a phage-induced enzyme (enzyme I) which converts singlestranded viral RNA into a double-stranded form. An amber mutant (sus-11) carrying a mutation near the middle of the virus-coat protein cistron makes, on both hosts (Su I1 and Su-), normal amounts of single-stranded RNA, but excess amounts of RNA synthetase (enzyme I) and 7 5 double-stranded RNA. Thus, when single-stranded RNA is made normally, but sufficient coat protein is not produced, early cessation of enzyme synthesis does not occur. The cell synthesizes large excess amounts of enzyme I. Acting directly or indirectly, virus-coat protein is the repressor of phage-enzyme synthesis. These results were interpreted in terms of a model coupling, genetic translation and transcription. Viral RNA is translated in two distinct states; as free (parental) RNA, which makes RNA synthetase (s), and as a nascent messenger still bound to its double-stranded RNA template, which makes coat and other phage proteins synthesized late in infection. The translation into protein of the N-terminal region of the coat cistron is necessary for continued synthesis of the nascent messenger RNA. When RNA is synthesized under coiiditions when sufficient coat protein is not made, as in mutant-infected cells, single-st,randed progeny RNA molecules appear analogous to parental RNA molecules and synthesize enzyme I. VI. Effects of Virus Infection on Host-Cell Nucleic Acid and Protein Synthesis
A. SHUTDOWN OF BACTERIAL N~ETABOI~ISM I N CEILSINFECTED WITH T-EVEN AND T5 PHAGES The T-even and T5 coliphages, which are known to destroy the nuclear structurc of Escherichiu coli, arc also thosc phagcs for whicli radiatior~
VIRAL-INDUCED ENZTBlES AND V I R A L ONCOGENESIS
163
experiments inclicate that tlicir iiiultiplication is relatively indepenrlent of the integrity of the host cell. Within minutes of infection with Teven phage, the nuclei of E . coli are disrupted into small blocks of chromatin material, which collect a t the periphery of the ccll. After T5 infection, there is a rapid and progressive deformation of the nucleoids :ind they disappear. This involves the chemical degradation of host DNA and, hence, the destruction of the gcnctic infornintioii of the lioht (Kcllenberger, 1961). After T-even phage infection of growing bacteria, the syntheses of bacterial DNA and all classes of bacterial RNA stop (Volkin and Astrachan, 1956). Protein formation continues but the majority of the proteins synthesized appear to be phage proteins or enzymes associated with phage formation (Cohen, 1948; Nomura et al., 1962). The synthesis of bacterial enzymes comes to a sudden and complete halt and remains a t the preinfectiori level. The bacteria can 110 longer be induced or dercpressed to form P-galactosidase, lysine decarboxylase, aspartic transcarbamylase, dihydroorotic decarboxylase, or alkaline phosphatase (Benzer, 1953; Levin and Burton, 1961 ; Pardee and Kunkee, 1952). During T-even phage growth, the major part of the host DNA is degraded to substrates of low molecular weight. With phage T5, the total DNA decreases very rapidly to reach about one-third the initial level 20 minutes after infection. The profound inhibitions of host-cell metabolic processes and breakdown of host-cell structures are in marked contrast to effects produced by other bacteriophage strains. With phages T1, T7, A, and P1, nuclear breakdown, if it occurs a t all, takes place so late that these phages can use home of the genetic information of the bacteria for their own syntheses. The syntheses of bacterial enzymes and other proteins may also continue during the first part of the infectious cycle. With phage f2, all bacterial metabolic processes continue normally until late in infection, and there is no inhibition of cellular DNA or RNA syntheses. Sucrose-gradient fractionation of RNA labeled after infection reveals csseritially the same pattern as docs RNA from noniiifected cells, and enzymes such as P-galactosidase and alkaline phosphatase can be induced in a normal manner (Zinder, 1965).
B. RIBONUCLE~C ACID POLYMERASE ACTIVITYIN T-EVENPHAGEINFECTED CELLS
It appears tliat a Itlwtively heat-stable substance is produced after T 4 phage iiifection that can effectively inhibit purified DNA-dcpentlent RNA polynierase of noninfected cells (Skold and Buchanan, 1964). The production of this inhibitor can account for the decrease in turnover of messenger RNA and the eventual cessation of bacterial protein synthesis. Ribonucleic acid polymerase activity decreases to 15 to 20% of
164
SATJL K I T
I)I*cinf(!ctioi~ ;icLtivity tluriirg tIro f i r h t 10 to 12 niiiiutes alter '1'4 iiifccation. The decline i i i IiNA polymerase activity is preventcd by the addition of chloraniphcnico1 to cells shortly after their infection, suggesting that polymeruse inhibition may be mediated by :L protcin. Sephadex G25 experiments indicate that the inhibitor has a minimum molecular wcight of about 5000. A similar inhibitor of RNA polymerase is apparently elaborated aftcr T2 phage infection (Khesin e t al., 1962). tllr
C. Loss OF POLYADENYLATE POLYMERASE ACTIVITYAFTER PHAGE INFECTION The abrupt inhibition of R N A polymerase activity in cells infected with T 2 phage was not confirmed by Oritz and co-workers (1965). Although a gradual and variable decrease in thc activity of this enzyme was sonictimes found in cells infected for 20 minutes, Ortiz et al. (1965) never observed a significant loss of RNA polymerase activity with bacteria infected for less than 4 minutes. Ribonucleic acid polymerase activity was also examined in cxtracts of bacteria infected with phages T1 and T7 with similar results to those seen with phage T2. I n contrast, a rapid loss of polyadenylate polymerase activity was observed; a decrease of about 50% occurred within 1 minute of T 2 infection and less than 10% of the normal activity remained after 8 minutes. Inhibition of 32P-orthophosphate incorporation into RNA occurred within 2 minutes after phage addition. Thus, the inhibition of polyadenylate polymerase and cessation of RNA synthesis took place within 1 minute of each ot,lier. Moreover, decreased levels of polyadenylate polymerase were also found in all cases in which cessation of net RNA synthesis was a consequence of phage infection, i.e., with Teven and T5 phages. Convcrscly, following infection with f2, T3, or induction of h phage, when RNA synthesis did not decrease, normal polyadenylate polymerase activity was observed. The inhibition of polyadenylate polymerase appeared t o be specific. Neither the ribonucleotide kinases, polynucleotide phosphorylase, nor RNA adenylatc (cytidylate) pyrophosphorylase showed a Significant change after T 2 phage infection. Of the possible causes for the loss of enzyme activity there was evidence only for induction or releasc by bacteriophage infection of a n inhibitor of the polyadenylate polymerasc reaction. The loss did not occur when cells were infected in the presence of chloramphenicol or a s a rcsult of destruction of Escherichia coli D N A in bacteria grown in the presence of mitomycin. Inhibition was not produced in vitro by components of phage T2, i.e., phage T2 DNA, internal protein, spermidine, or putrescine, but inhibition in vitro did occur when extracts of normal and
VIIIAL-ISI)UCED ENZYMES AND VIRAL ONCO(;ENESIS
165
infected cells wcre incubated together. The iiihibitor was prcsent i n the supernatant fluid of infected cells after high-speed centrifugation, whereas, polyadenylate polymerase itself, was sedimented with ribosomes. It must be granted that the role of polyadenylate polymerase in bacterial metabolism is still unclear; nevertheless, the intriguing finding that a specific inhibitor of this enzyme is elaborated soon after infection by the same phages that shut off bacterial RNA synthesis docs not appear to be coincidental and merits further study. D. T4 PHAGE-CONTROLLED BREAKLXIWN OF BACTERIAL
DEOXYRIBONUCLEIC ACID When one measures the total DNA as a function of time after infection by wild-type phage T4, the breakdown of Escherichia coli DNA, which takes place in these cultures, is obscured because of the concomitant synthesis of phage DNA. However, if infection is carried out with a mutant of T4 that is unable t o induce synthesis of any phage DNA, one observes clearly and directly the conversion of a large portion of the bacterial DNA from an acid-insoluble to an acid-soluble form. sl prior), one might suppose that this conversion involves a DNase. However, although the induction of three new DNases in T-even phage-infected cells has been reported, evidence is lacking that any of them has a direct role in host-cell DNA breakdown (Oleson and Koerner, 1964; Short and Koerner, 1965; Stone and Burton, 1962). I n order t o test the hypothesis that bacterial DNA breakdown was controlled by coliphage genes, Wiberg (1966) tested a series of T 4 phagc mutants known to be defective in DNA synthesis. It was found that mutants in genes 46 and 47 were unable to cause host-cell DNA breakdown (Table V). These results imply that genes 46 and 47 control one or more DNases and that these DNases preferentially attack DNAcontaining cytosine rather than hydroxymethycytosine. It is not known, however, whether genes 46 and 47 are structural genes for a DNase or, instead, control an inducer or activator of a DNase. I n addition to host-cell DNA breakdown, other functions occurring after phage infection that have been suspected of involving DNase activity are (1) genetic exlusion of related superinfecting phage and ( 2 ) genetic recombination. These functions were virtually unimpaired in the T 4 mutants defective in genes 46 and 47.
E. INDUCTION OF DEOXYCYTIDINE TRIPHOSPHATASE ACTIVITYBY T-EVENPHAGE Following T-even phage infection, a new enzyme, dCTPase, is induced which specifically catalyzes the hydrolysis of dCTP and deoxycytidine 5’-
166
SAUL KIT
cliphosphilte (clCDl-’) (Iiocriici. ct d., 3959; Iioi*~il)rrg ct d., J 959; Zinimerman and Kornberg, 1961). ( M g + + )dCTPase
dCTP dCDP
( M g t f ) dCTPase
dCMP
+ pyrophosphate
dCMP + orthophosphate
(17)
This enzyme has a dual function in the promotion of the synthesis of phage DNA. First, it degrades dCTP so that i t cannot be utilized l ~ y DNA polymerase to make Escherichia coli DNA and, second, it generates dCMP which is the precursor of dHMP. Extracts of noninfccted cells have 0.1% or less of the dCTPasc activity observed in extracts of T 2 phage-infectcd cells. The enzyme is not induced by coliphagc T5, which contains cytosine rather than hydroxymethylcytosine in its DNA. Studies with UV-irradiated phage and with phage mutants have provided additional evidence that this enzyme is controlled by T-even phage genes. Normally, the enzyme is detected in about 4 minutes and increases to a plateau at about 15 minutes after infection. With UV-irradiated T 4 phage, the initial rate of enzyme synthesis is decreased; however, dCTPase synthesis is not shut off a t 15 minutes and extended enzyme synthesis is observed (Dirksen e t al., 1960). Infection of E . coli B with amber mutants defective in T 4 phage genes 41, 42, and 44 (Table V) also leads to extcnded synthesis of dCTPase (Wiberg e t al., 1962). Gene 56 appears to control the formation of dCTPase. Amber mutants defective in gene 56 fail completely to induce dCTPase activity (Wiberg, 1966).
F. THYMIDYLATE SYNTHETASE AND THYMIDYLATE NUCLEOTIDASE OF PHAGE-INFECTED Bacillus subtilis ACTIVITIES
The B. subtilis phages, SP8, +e, and SP5C, contain hydroxymcthyldeoxyuridylate in place of d T M P in their DNA’s. I n another B. subtilis phage, SP2, deoxyuridylate rcplaced dTMP in the DNA. The metabolism of dTMP is abnormal in bacteria infected with these phages. Phages SPSC, SP8, and SP2 all induce in infected cells increases in the activity of n 5’-nuclcotidase, specific for rlTMP (Aposhian, 1965; K n h n , 1963) : dTMP
dTMP nucleotidase
dT
+ orthophosphate
The appearaiice of this activity is inhibited by chloramphenicol, indicating that de TLOVO protein synthesis is needed for enzyme formation. The enzyme from phage SP5C-infected bacteria has been purified, and i t
rat;llyzch thc lly~lroly of dThIP, but iiot that of t1coxp:itlciiosiiic 5’iiionol)lio~pliatr (dAMP) , dChIP, ant1 cl(:nlP (Aposhi:in, 1965). The increase in dTAlP nucleotidase is not relxtcd to tlic induction of defective phages known to bc present in Iz. subtilis, since treatnictit of noninfected cells with niitoniycin C, an inducer of thew prophages, does not produce c1TMP nucleotidase activity. The dTh1P nucleotidase of phage ye-infected l m t c r i a has not been stutlictl a t this writing. However, it has been shown that in hactcria infcctcd with this phage, the dTlClP synthetase activity of the host cell declines t o very low levels. This rcduction in dTMP synthctase activity 5eciiis t o bc caused hy the presence of an inhibitor in the infected cell extracts (perhaps d T M P nucleotidase) . If extracts from infected and noninfected bacteria are mixed, the synthetase activity of the latter decreased (Roscoe and Tucker, 1964). As a consequence of thc increased d T M P nucleotidase activity and the rapid inhibition of dTMP synthetase activity, formation of thyminecontaining DNA is prevented. Concurrently, the inductions of d C M P deaminasr, d U M P hydroxyrnethylase, and deoxyuridylate aiid hydroxy~~icthylclcoxyuridylate kinnse activities facilitate phage-DNA synthesis.
G.
SIIUT1)UU‘N OF
BIOSTNTHETIC PROCE5SES
IN
VIRUh-INFECTED
ANIMAL CELLS
1. R N A - Containing Animccl Viruses
The 1-eplication of many RNA-containing animal viruses is relatively independent of host-cell functions. Thus, the picornavii~uses,Newcastle disease virus (NDV) , am1 vesicular stoinatitis virus (VSV) grow normally despite the addition to infected cultu,cs of actinoniycin D, a drug which prevents host-cell RNA polymcrasc function. The replication of thwc viruses is aleo uiiitnpaired by drugs t h a t inhibit DN.4 biosynthesis. In ccIl culture,. infected hy mengovirus, poliovirus, ME virus, NDV, niitl VSV, there ia an early inhibition of cellular RNA and protein syntheses followed by niorpliologiral c l i m g ~ awhich culniiiiatc in the rounding and rlisruption of cell:, (I3al~laniane t nl., 19651; Baltimore and Franklin, 19621); Baltimore et al., 19631); Fenwick, 1963; Hausen and Vcnvoerd, 1963 ; Holl:ind, 1962 ; I-Iomma, ancl Graham, 1963; Martin and Work, 1961 ; Vcnvoeid and Hausen, 1963; Wlicelork aiid Tamni, 1961 ; Zimmermnii c’f rrl , 1963). Ti1 iiiciigo\.ii i i h - i i i f c i ( + d I, c ~ l l s ,DN-4 *yritlicsis is r 1 o r i l l : k I for more t h i 3 h u i 3 a t t e r i n f t d i o i i and t h greatly decieabt~.s.Thii5, the rapid : i r i t l c:115ly iiili~l~itioii of Iio-t-cell RNA syiithcsis is not due to a generalized inhibition of all DNA-dependent processes (Baltimore and Franklin,
168
SAIJL K I T
1962b). A progrcssivc decrease of DNA biohynthesis also occurs in NDV-infcctcd HeLa cells. This decrease coincides in time with a rapid increase in virus antigen and infective particles, and synthesis of DNA stops as the amounts of virus materials produced reach maximal levels (Wheelock and Tanim, 1961). Both puromycin and FPA suppress the inhibition of host-cell RNA and protein syntheses when added early in the infectious cycle (Bablanian et al., 1965a,b; Baltimore et al., 1963b; Bolognesi and Wilson, 1966; Huang and Wagner, 1965; Verwoerd and Hausen, 1963; Wagner and Huang, 1966). These rcsults could meal1 that the inhibitions are caused by proteins that are synthesized under the control of the viral RNA. A t the same time that nuclear RNA synthesis is depressed in mengovirus-infected cells, a pronounced inhibition of RNA polymerase is observed. Within 1 hour of virus infection, RNA synthesis falls to less than 10% of normal and RNA polymerase to less than 50% of normal (Balandin and Franklin, 1964). These inhibitions appear to be mediated by an inhibitor made in the cytoplasm of the infected cells. If control nuclei and cytoplasm from infected cells are mixed, RNA polymerase activity of control cells is inhibited by 61%. However, mixing control nuclei with nuclei of infected cells has no effect on the enzyme activity of the control nuclei and mixing control cytoplasm with nuclei from infected cells does not restore the polymerase activity of the nuclei from infected cells. The cytoplasmic inhibitor of RNA polymerase is present in the postmitochondrial fraction; it is inactivated by trypsin but not by RNase. I n a study of poliovirus-infected HeLa cells, Holland (1962) confirmed the finding of Balandin and Franklin (1964) that RNA polymerase activity is rapidly inhibited after virus infection. This inhibition is not due to the breakdown of HeLa DNA nor to permanent alterations preventing DNA priming nor even to firm masking of DNA by protein in some manner to prevent contact with polymerase. Holland (1962), however, was unable to detcct the presence of a protein inhibitor in the cytoplasm of infected HeLa cells. The inhibition of protein synthesis noted prcviously could be an indirect result arising from virus-induced suppression of messenger RNA synthesis. However, this does not appear to be the primary reason for the arrest of host-protcin bynthesis. Protein synthesis is inhibited by poliovirus infec4ioii tnur(~ntpidly thttii Ly :~c.tinornycinD (Holland, 1962)-a result coinpatible with the hypothesis that virus infection interferes directly with protein-synthesizing mechanisms in intact cells. The release of ribosomes from complexes with cellular messenger RNA might well be one reason for the inhibition of protein synthesis. Consistent
VIRAL-INDUCED ENZYMES AND VIRAI, ONCOGENESIS
169
with this concept, it his heeii found that polyril,osomes are degraded after poliovirus infection of HeLa cells (Penman ef nl., 1963; Rich et nl., 1963). T h e 1.att: of polyrihn,iornal clisriiption increabecl linearly with thc t inic of iiifrvtion. R v l ( ~ i , s c of ~ Iio~t-iiiC~~sc~ligc~i~ R N A fro111polyrihosomcs was not mercly a coriscqucnce of co~npctitionfor ribosomes with virus RNA. Polyribosome breakdown and inhibition of host-cell protein synthesis took place even when viral RNA replication was prevented by guanidine (Bablanian et al., 1965a,b; Penman and Summers, 1965). However, polyribosome breakdown was manifested only after a period of protein synthesis following virus adsorption. This disruption thus may be due to a product of the viral genome which is stable for a t least 1 hour in the absence of protein synthesis and seems to be specific for the host-cell messenger RNA (Penman and Summers, 1965; Willems and Penman, 1966). The inhibitory factor probably acts by affecting messenger RNA on the polyribosomes of the host cell so that it is rendered incapable of attaching ribosomes. The irihibition of protein synthesis by poliovirus does not appear to result froin an effect on the cellular ribosomes. The protein-synthesizing system of the cell is capable of functioning with viral messenger later in infection t o produce viral-specific proteins. Moreover, the host-cell ribosomes are not simply inhibited from functioning with host-cell messenger since newly synthesized messenger RNA, produced in cells infected in the absence of actinomycin D, apparently functions with the existing ribosomes (Willems and Penman, 1966).
2. Metabolism of Reovirus-Infected Cells Reovirus differs from most of the RNA-containing animal viruses in that mature reovirus particles contain double-stranded RNA. Moreover, reovirus replication is inhibited by actinomycin D, whereas that of the picornaviruses, NDV, and VSV is not. Reovirus is a comparatively slowly replicating virus. In L cells, thc latent period for reovirus Type 3 was 8 hours, and maximal yields were not reached until about 17 hours (Gomatos and Tanim, 1963a). Virusspecific RNA formation commenced a t about 6 hours and continued through most of t.he infectious cycle. In contrast, formation of poliovirus particles in HeLa cells increased exponentially beginning a t about 3 hours after infection, and maximal titers were obtained by about 6 hours. With NDV, newly made virus antigens and the first infective virus particles appeared a t 2.5 hours, and infective virus reached a peak a t about 5 hours after infection (Wheelock and Tamm, 1961). Capsid antigen of influenza A virus was detected in infected HeLa cells a t 3 to 4 hours and hecamc promincnt a t 6 to 7 hours (Whcclock and Tamm,
170
SATTI, TiIT
1959) . In I, cclls infectecl with ni~ngovirus,formation of infectious RNA began a t about 4 hourh and ivah completed by 7 hours after infcction; at 6.5 I I O U ~ a11cl ~ t . 1 1 d ~ dby 0 110~1’sPI VIIWS I i i w t i i r a t i o i i i’oiii~ii(~ii(~d (llolllnls :uld G r : h t l l l , 1963). The syntliescs of protein : ~ n dL)NA wcrc not iiiliibited during tlic first 8 hours of infection of L cells with reovirus Type 3 (Gomatos and Tamni, 1963a; Kudo and Graliam, 1965). However, late in infection when infectious reovirus increased exponentially, a pronounced inhibition of DNA synthesis and a moderate inhibition of protein synthesis were observed. There was very little inhibition of either ribosomal RNA or nuclear RNA syntheses up to 12 hours after reovirus infection. 3. Metabolism of Cell Cultures Infected with DNA-Contairwig Aninial Viruses
A severe inhihition of 3H-dT incorporation into host-cell DNA ensues early after infection of cell cultures with vaccinia, cowpox, herpes simplex, and pseudorabies viruses (Ben-Porat and Kaplan, 1963; Dubbs and Kit, 1964b; Hanafusa, 1960; Kaplan and Ben-Porat, 1963; Kato et nl., 1964; Kit and Dubbs, 1963c; Kit et nl., 1963a; Roizman and Roane, 1964). Although host DNA synthesis was inhibited, viral DNA was made. I n cultures infected with poxviruses, 3H-dT labeling of nuclear structures decreased while foci of cytoplasniic labeling developed. In either exponentially growing or stationary phase cells infected with herpes viruses, formation of “light,” cellular DNA synthesis was arrested early in the infectious cycle while the formation of “heavy” viral DNA was accelerated. The inhibition of cellular DNA synthesis was not due to the degradation of that DNA (Kaplan and Ben-Porat, 1963; Kit and Dubbs, 1962b). In pseudorabies virus-infected cells i t was also not attributable to greater affinity of the DNA polymerase for viral DNA than for cellular DNA, nor to the successful competition of viral DNA with cellular DNA to act as a template for DNA replication. Thc inhibitory process was arrestcd by the addition of puromycin to pscudorabies virus-infected cells a t 1 to 2 hours after infection suggesting that a protein was responsihle for the inhibition of the syntliesis of cellular DNA (Bcn-Porat and Kaplan, 1963, 1965). Inhibitions of host R N A and protein syntheses were also observed after poxvirus or herpes virus infections. The capacity of infected LM cells to incorporate 3H-uridine into RNA was reduced within 1 hour after vaccinia infection, and by 6 hours declined to 2570 the rate of noninfected cells. The incorporation of 2-I4C-alanine into protein was inliil)itctl 25% at I hour : ~ n r l 56% hy 7 hours after infection (Kit : L I I ~
I h b b s , 1962a). During the first 4 hours of vaccinia infection of HeLa cells, about 30 to 40% of tlic proteins made were viral proteins; after 4 hours, net ccllular protein synthesis diminished considerably and mostly viral proteins were made (Shatkin, 1963). Vaccinia RNA was detected in the cytoplasm of HeLa cells as early as 0.5 hour after infection. By 1.5 to 2.5 hours, about 50% of the newly formed RNA had the properties of vaccinia-messenger RNA and a t 2.5 to 3.5 hours, 80% was viral-specific RNA. New formation of ribosomal RNA did not occur in the cytoplasm of cultures 3-4 hours after infection (Becker and Joklik, 1964; Stllzman et al., 1964). Between 1 to 3 hours after infection of cell cultures with herpes simplex virus, 3H-uridine incorporation into RNA was reduced by half, relative to that of noninfected cells. The rate of synthesis of ribosomal RNA declined to 39% of the control levcl in the period 3.5-6.0 hours after herpes simplex virus infection. This decline paralleled the time course of inhibitions of soluble RNA (4 S) and host-cell messenger RNA. By 90 minutes after infection, a new RNA species exhibiting a sedimentation coefficient of 2 0 s and capable of hybridizing with herpes simplex DNA was observed. Although host-cell RNA synthesis decreased the formation of herpes-specific RNA increased. A 4 5 fraction capable of hybridizing with herpes simplex DNA was also made (Hay e t al., 1966; Roizman et al., 1965). During the first 3 hours after infection, protein synthesis, as measured by the incorporation of radioactive amino acids into protein, decreased to about 70% of the control rate. Between 3 to 6 hours, this incorporation was generally stimulated but 6 to 10 hours after infection, protein synthesis declined to approximately 60% of that of noninfected cells. Adenovirus Type 2 infection inhibited multiplication of KB cells, but overall nucleic acid and protein syntheses continued throughout much of the infectious cycle a t essentially unchanged rates (Green, 1959; Green and Daesch, 1961; Polasa and Green, 1965). Noninfected cells multiplied three- to four-fold during a 48-hour experimental period. In contrast, multiplication of cells in infected cultures was limited to the first 12 hours and was never more than 1.2- to 1.3-fold. Infected cells did not lyse but increased in size. Protein, DNA, and RNA accumulated continuously in infected KB cells starting within 12 hours after infection. At 36 hours, the content per cell of these polymers was double that of noninfected cells. cell cultures includcs soliiblc The RNA fomed in :Idcnovirus-iiifc~t(~(l RNA, rilmsotiiiil HN-4, :itiil RNA4c.oiiipIrtiirntaiy to hoth liust ~ ( viral 1 DNA's (Rose ct nl., 1965). The presence of viriil-conip1einental.y RNA was detected ns e:trly ns 9 Iiours after infection. A t 28 liours, ahout 36%
172
SAUL K I T
of the newly synthesized RNA formed duplexes with adenovirus Type 2 DNA. I n contrast to viral-complementary RNA, KB cell-complementary RNA remained an almost constant fraction of newly synthesized RNA after adenovirus infection. Overall RNA and protein syntheses were not inhibited in adenovirus Type 5-infected HeLa cells. In fact, an increase in the overall rate of RNA synthesis (presumably viral-messenger RNA) was noted a t 8 to 9 hours and was still evident 24 hours after infection. A significant increase in the rate of protein synthesis occurred a t 14 to 17 hours and a markedly accelerated rate continued until approximately 20 hours after infection (Flanagan and Ginsberg, 1964; Wilcox and Ginsberg, 1963). 4. Decrease in Cellular Enzyme Activities after Virus Infection The uridine kinase activity of LM mouse fibroblast cclls varies with the metabolic condition of the cell. The enzyme exhibits highest activity during exponential growth and declines to about one-third the peak level during the stationary phase. Infection of growing LM cell cultures with either vaccinia or herpes simplex viruses resulted in a rapid decline in uridine kinase activity. Within 5 hours after infection, the enzyme activity fell to less than half the value of noninfected cells. However, uridine phosphorylase activity was not significantly changed in the infected cultures (Dubbs and Kit, 1964b,c; Kit et al., 1964). Extracts from vaccinia-infected cells, when added to those from noninfected cells, did not reduce the in vitro activity of the latter extracts. Thus, the decline in uridine kinase activity was probably not caused by free inhibitors in the infected cell extracts. It is more probable that the uridine kinase decrease was due to a greater turnover of the enzyme after virus infection. As discussed previously, the formation of messenger RNA was inhibited in vaccinia- and herpes simplex-infected cells. This inhibition of messenger RNA synthesis would be expected to contribute t o a decline in the activity of enzymes with a high turnover rate. The inhibition of uridine kinase, in turn, would reduce the capacity of infected cells to incorporate "-uridine into RNA. The d T kinase activity of LM cells was enhanced about threefold after infection by vaccinia or herpes simplex viruses, Since formation of new virus-specific enzymes was taking place, changes in the activities of pre-existing host-cell d T kinase were obscured. However, by infecting LM cells with mutant viral strains deficient in d T kinase-inducing activity, it was possible to follow the effects of virus infection on cellular dT kinase activity (Dubbs and Kit, 1964b,c). Within 5 hours after infection of LM cells with mutant vaccinia (Vtk-) or herpes simplex (Htk-) strains, LM cell clT ltinnse activity declined to about half the
TI
1 ~ ~ 1IS , -D u CED EK z Y M ES A N D
v IHAL
ONCOG ICNESIS
173
iioriiial ~ : 1 1 1 1 1 ~This . t1ccre:~sew:ih not duo to frcv inhibitors in tlie cxtracts from infcctctl cells. Thc results suggest that vaccinia and herpes simplex viruses also enhanced the turnovcr of cellular d T kinase in infected LM ccll Cultures. VII. Biochemical Aspects of Viral Oncogenesis
A. REPLICATIOK CYCLE? OF SV40 AND POLYOMA VIRUS
I n previous sections, the general aspects of viral gene functions havc been considered. Against this background we can now discuss the gene functions of tumor-producing viruses. Considerable progress has recently hccn made in studies of polyoma virus and simian papovavirus SV40, and most of our discussion will be concentrated on thesc two viruses. The SV40 and polyomn viruses have dual capacities; they can actively niultiply in certain types of cells, which arc then killcd, or they can transform other cells. Let us firbt consitlcr the events taking place during active replication of these viruses. Virus SV40 replicates in primary cultures of green monkey kidney (GMK) cells and in CV-1 cells-an established line of GMK cells. The duration of the growth cycle is long compared with other types of animal viruses. Also, the growth of SV40 depends t o some extent on the metabolic condition of the host cells. I n confluent monolaycr cultures of GMK or CV-1 cells, the eclipse period lasted for about 20 to 24 hours (Fig. 7 ) . Intracellular infectious virus, then, increased to a maximum a t about 48 to 55 hours; total infectious virus (intracellular and extracellular) increased until about 72 hours after infection. Vacuolation was first detected about 60 hours after infection, and, by 72 hours, virtually all the cells in the culture displayed typical cytoplasmic vacuolation. At an input multiplicity of 5 to 10 PFU (plaque forming units)/ceIl, 3% of the cells were positive for the SV40 T-antigen at 18 hours, and this percentage increased so that a t 48 hours after infection, approximately 80% of the cells were positive. About 80% of the cells were also positive for the SV40-capsid (V)antigen. At SV40 input multiplicities of about 25 PFU/cell or more, 6080% of the cells were capable of forming infectious centers (Carp and Gilden, 1966; Kit et al., 1966e; Mayor et al., 1962). The rcsults were quite diffeient if rapidly growing cells were infected with SV40. Whereas the yield of SV40 was about 100 PFU/cell in stationary-phase cultures, this yield was less than 2 PFIJ/cell in replicating-phase cultures. The SV40 T-antigen was detected in only 1 to 10% of the rcplicating-phase cells and few cells showed cytopathic changes. Cells infected while rapidly growing continued to multiply despite in-
174
SAUL KIT I o8
x
10'
/?
-E a b v) ._ c t
P
.-
E
b
L
g3 10'
I
I 16
1
I 32
I
I 40
I
I 64
I
1 80
Hours postinoculation
Fm. 7. Growth of SV40 in CV-1 cells (established line of Green monkey kidncy cells).
fection and have been subcultured serially for at least fourteen passages (Carp and Gilden, 1966). Polyoma virus replicates actively in mouse kidney or mouse embryo cells. In confluent monolayer cultures of mouse kidney cells, the eclipse period lasted about 21 hours after which virus titers increased until about 60 hours after infection. At an input niultiplicity of about 100 to 200 PFU/cell, 40-600/0 of the cells were productively infected by polyoma virus (Dullmco et al., 1965; Kit e t al., 196Gd).
B. RIBONUCLEIC ACID AND PROTEIN SYNTHESES IN PAPOVAVIRUS-INFECTED CELL CULTURES The overall rate of 'H-uridine incorporation into RNA was not significantly changed during the first 24 hours after acute infection of
175
VIR.iI,-ISUUC'EU ENZYMES A N D I'INAL ONCOGENEhlS
$ 1 c
c
0
loot
-u-LuJ 0
4
8
12
16
20
24
Hours oostinoculotion
FIG.8. Incorporation of 3H-uridine into RNA of noninfected or SV40-infected CV-1 cells. Seven-day-old cultures (5.4 X loG cclls/culture) were inoculated with 180 PFU/cell of SV40 at t h e zero. At the times indicated in the figure, 3H-uridinc was added and the cultures were incubatcd for an additional 30 minutes.
CV-1 cells by SV40 (Fig. 8 ) . Moreover, protein synthesis, as measured by the 3H-leucine incorporation into protein continued a t an unaltered rate (Carp and Gilden, 1966). Similai-ly,there werc no large differences in the rates of incorporation of "-uridine into total cell RNA or of 'Hleucine into total cell protein during the first 30 hours after polyoma virus infection of confluent mouse kidney cell cultures (Dulbecco et al., 1965). These findings are in marked contrast to the early inhibitions of cellular nucleic acid and protein syntheses observed with most other viruses (Section VI 1 and :ittest to the fact that papovavirus infcctions are moderate.
C. TEMPORAL R E L A T I O N ~ HOFI PPROTEIN ~ A K D SUCLEIC ACIDSYNTHESES DURING PAPOVAVIRLS DEVELOPMENT 1. Virus-Specific RNA
Virus-specific RNA can be detected by pulse-labeling infected cells with RNA prwur>orb :tnd studying the calmcity of the labcled RNA to hybridize with virus DNA. During the first 16 hours aftcr polyoma infection of mouse kidney cells, a .~iiiallbut iiiri.cnsing :mount of hybritlizable RNA was detected. From 16 to 32 hours aftvr infection, the
176
SAUL KIT
relative amount of virus-specific RNA was much greater. However, even during the period of maximum synthesis of polyoma virus RNA (2428 hours), not more than 1% of the labeled RNA could hybridize to polyoma DNA. These results support the conclusion that polyoma infection does not block the synthesis of cellular RNA. I n fact, a comparison of the capacity of RNA from noninfected and from infected cells t o hybridize with cellular DNA suggests that celluIar RNA synthesis may be stimulated by polyoma virus infection (Benjamin, 1966). 2. Virus-Specific Proteins Drug-inhibitor and immunologica1 studies have shown that synthesis of early proteins essential for polyoma virus DNA formation starts a t about 9 hours after infection and that synthesis of virus-capsid proteins commences several hours later. Infectious DNA formation was prevented if puromycin was added to infected cultures a t any time during the first 9 hours after infection. Polyoma virus production was completely blocked by drug addition from 0 to 14 hours after infection. When applied after 14 hours, virus formation was only partly blocked. If puromycin was present for 16 hours and then removed, a normal yield of polyoma virus was obtained at 48 hours (Gershon and Sachs, 1964). Exposure of polyoma virus-infected cells to FPA from 4 t o 15 hours after infection delayed virus maturation by 19 hours, but a t 106 hours, virus yields from FPA-treated and infected control cultures were equal (Munyon e t al., 1964). During lytic infection of mouse embryo cells, T-antigen appeared about 12 hours after polyoma virus infection, reached a peak a t 24 to 48 hours, and then decreased (Habel et al., 1966). Twenty-four hours after infection, over 50% of the cells showed nuclear fluorescence similar to that described for SV40 (Takemoto et al., 1966). As measured by immunofluorescence, polyoma capsid-protein formation was first detected about 14 hours after infection. B y 16 t o 18 hours, most of the cells were producing virus protein (Sheinin, 1964; Sheinin and Quinn, 1965). The kinetics of SV40 tumor (T)-antigen and of viral-capsid antigen formation in CV-1 cells are shown in Fig. 9. An increase in T-antigen was detected a t 10 hours and attained a maximum a t about 30 hours. Formation of viral-rapsid antigen began several hours after that of Tantigen (Kit ef nl., 19GGg). Synthesis of SV40 T-antigen in primary cultures of GMK C C ~ was S abolished by cycloheximide addition froni 0 to 10 hours after infection (Gilden and Carp, 1966). However, inhibitors of DNA synthrsis (i.e., fluorouracil, dFU, ara-C, or dIU) neither reduced nor delayed SV40 T-antigen production (Butel and Rapp, 1965; Gilden and Carp, 1966;
177
VIRAL-INDUCED ENZYMES AND VIRAIJ ONCO(iENES1S
Cril(lcn et (11.. 19G.5; Rlclnick : i i d R:ipp, 1965; R:ipp et al., 1965a). Viral DNA ~ynthc+is also 1va3 not, required for polyoma virus T-antigen protluctioii siuw CF (coinl)lmc~nt3 fixing) titers rrlaclied those of infected control cclls whrn viral rq)lic.:ttioii wis in1iil)itccl by tiFU or ara-C (Habel et ul., 1966). I n contrast to the lack of inhibition of T-antigen formation, the inhibitors of D N A synthesis greatly reduced viral capsid-antigen production (Melnick and Rapp, 1965; Muriyon et al., 1964; Rapp et al., 1965a; Sheinin, 1964). Addition of ara-C a t 8 to 12 hours after infection T - anttqen ( C V - I cells) 256[
-1
80
Hours postinoculation S V 4 0
FIG.9. Kinetics of T-antigen and viral-capsid antigen formations in CV-1 and in mouse kidney cell cultures inoculated with SV40. (CF = complement fixing.)
inhibited viral capsid-antigen formation, but addition of the inhibitor 16 hours after infection or later failed to do so. These results suggest t h a t viral capsid-antigen formation may be dependent on D N A replication. Inhibition of RNA synthesis by actinomycin D treatment prevented either SV40 T-antigen or viral capsid-antigen production.
3. Metabolic Antagomsts and Viral D N A Synthesis From the following experiments, it appears t h a t synthesis of polyonia virus D N A began a t approximately 14 to 1.5 hours PI, and that of SV40 DNA a t about 18 hours PI. When dBU, dIU, or mitomycin C were added to mouse embryo cultures earlier than 14 hours after polyoma virus infection, virus formation was cornpletely blocked. The addition of these drugs a t progressively later intervals of time resulted in the formation of correspondingly greater yields of infectious virus. No inliibition of infectious virus formation wiis obscrved if dIU was added
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a t a time later ttiun 40 hours after infection (Gershon and Saclis, 1964; Rlunyon c f nl., 1964). Tllt~(.ff(\cthof (IFTI and ;ira-C, rcqwctivcly, on SV40 replication have been htucliul hy Uutel and Rnpp (1965) and by Gilden and Carp (1966). No infectious SV40 was detected when dFU or ara-C were added as late as 18 to 22 hours after infection. Addition of the inhibitors a t later times permitted increasing amounts of infectious SV40 formation. The inhibitory effects of DNA antagonists on virus production were reversible (Butel and Rapp, 1965; Gilden and Carp, 1966; Sheinin, 1964). The presence of dFU in cultures of mouse embryo cells infected with polyoma virus for up to 17.5 hours had no effect on the subsequent time course of virus formation when the inhibitor was removed. Also removal of ara-C after 40 hours of treatment of infected GMK cells permitted normal yields of infectious SV40 24 hours later.
4. Stimulation of $H-dT Incorporation into D N A of Znfected Cultures Radioautographic and radioisotope-incorporation studies have provided additional data concerning the timing of DNA synthesis in papovavirus-infected cell cultures. Studies by several laboratories have shown that when confluent monolayer cultures were infected with these viruses the overall ratc of DNA biosynthesis was stimulated (Dulbecco et al., 19fj5; Kit et al., 1966d,e; Minowada, 1964; Minowada and Moore, 1963; Molteni et al., 1966; Weil e t al., 1965; Winocour et al., 1965). Figure 10b illustrates pulse-labeling experiments on the incorporation of 3H-dT into DNA of SV40-infected GMK cell cultures. Similar experiments have been performed with 3H-deoxyadenosine as DNA precursor with csseiitially the same rcsults. A stimulation of DNA biosynthesis was first detected about 16 hours after SV40 infection. A t 30 to 32 hours, the rate of 3H-dT incorporation into DNA of infected cultures was over 3 times that of noninfected cultures. This high rate of DNA biosynthesis continued until about 50 hours after infection. Radioautographic experiments on the same cell populations are shown in Fig. 10a. Initially, less than 10% of the nuclei of noninfected GMK cells were labeled after a 2-hour ‘H-dT pulse. This value increased to about 20% a t about 16 hours after i‘rnock-infection” and then declined. I n SV40-infected cultures, the percentage of cells with 3H-labeled nuclei increased sharply at about 16 hours and exceeded that of noninfected cultures. By 30 to 32 hours after infection, 7 0 4 0 % of the cells exhibited nuclear labeling. The radioautographic experiments demonstrate that many cells not synthesizing DNA initiated DNA synthesis a t about 16 hours after SV40 infection. Colorimetric assays of the total DNA synthesized in infected cultures
VIRAL-INDUCED ENZYMES A N D VIRAL ONCOGENESIS
179
I00
a
SV40 - infected
ao 70 60 -
.-m
50 -
c
40 -
0
8.
30 20 10 I
0 800-
I
I
I
I
b.
700
600
% 500
,E
400
300
'""I I00
u
0
Noninfected
-
L
10
l
~
20 30 Hours postinoculation
-
40
50
FIG. 10. Fh3dioautogral)hic (a) and biochemical (b) studies on the uptake of 'H-thymidinc ('H-dT) by SV40-infected GMK cells. Replicate 9-day-old cultures (14.7 X lo6 cells/culture) were inoculated with approximately 10 PFU/cell of SV40. A t the indicated times, 'H-tlT was nddcd and the cultures wcre furthcr incubated for 2 hours at 37" C.
confirmed that DNA synthesis was enhanced. The total D N A of SV40iiifccted cultures exceeded that of noninfected cultures a t about 30 hours, and a t 48 hours after infection was 43-9076 greatcr than that of noninfected cultures (Kit e t al., 1966e). The 3H-dT incorporation into DNA of confluent monolqcr cultures of inouse kidney cells was significantly increased a t 16 t,o 24 hours after polyoma virus infection; a t 30 Iioiirs, incorpoixtion into 1)NA was 5 times grcltt,ei*i i i infrctctl t l i a i i i i i noninfcxrttvl rult ~ i i ' t + (Kit, rl u L . ) 1966~1). Polyoliia virus infection :tlso incre:wctl ty sovei*alfold the ii~coi~~~orat.iona of J"'-oi.thopliosl,Ii:ltc, "C-Toi.iii:itc~, : i i i ( l ' i ~ ~ I ~ ~ : - L - i i i c t l l i ointo i i i l ~ DNA c
180
SAUL KIT
of mouse kidney cultures. The total DNA syr~thcsized by infected cultures a t 36 hours was 21-63oJo grcater than that of noninfected cultures (Dulbecco et al., 1965; Winocour et al., 1965). The nuclei of about 5% of noninfected mouse kidney cells were labeled after a 2-hour 3H-dT pulse. However, a t 24 to 26 hours after polyoma virus infection, this number increased to 34% and, by 48 t o 50 hours, 65% of the nuclei were labeled (Kit et al., 1966d). 5. Viral-Induced Synthesis of Host D N A
I n confluent monolayer cultures productively infected with papovaviruses, some of the newly synthesized DNA must be viral DNA. HOWever, the total amount of DNA synthesized appears to be far in excess of that needed for infectious virus formation alone, and this suggests that induction of cellular DNA synthesis had also taken place. Experiments from several laboratories strongly support this inference. The evidence is as follows: 1. The rate of DNA biosynthesis a t 16 to 30 hours after infection of stationary-phase, mouse kidney cell cultures with polyoma virus was 10 times greater than that of mock-infected cultures. Chromatography on methylatcd albumin columns revealed that about two-thirds of the newly synthesized DNA was cellular DNA. Therefore, ccllular DNA synthesis in infected cultures exceeded that in rioninfected cultures by a factor of over 6 (Dulbecco et al., 1965). 2. Polyoma DNA contains 0.9 residue of 5-methylcytosine per 1000 DNA nucleotides, whereas mouse kidney DNA has 9-10 residues per lo00 nucleotides of DNA (Winocour et al., 1965). The methyl group of 14CH,-methionine is a precursor of 5-methylrytosine and of thymine, guanine, and adenine. During the period of stimulated DNA synthesis, the pattern of incorporation of radioactivity from 14CH,-methionine into the DNA bases of polyoma virus-infected cultures was similar to that of noninfected cultures but differed from that of purified, polyoma virus DNA. Thus, a substantial portion of the newly made DNA must have been cellular DNA. 3. Noninfected mouse kidncy monolayers incorporate little 14C-dBU into DNA. Following CsCl density gradient centrifugation of this DNA, two bands are observed-a large band corresponding to normal mouse DNA and a small, labeled band corresponding to “hybrid” DNA (onc strand containing 14C-dBU and the other only dT). The uptake of I4CdBU into DNA is markedly stimulated a t 24 to 50 hours after polyoina. virus infection of mouse kidney monolayers. Aiialysis of the DNA of thc infected cultures revealed that most of tllc DNA h : ~ lshiftetl fi-onl tlle positioll of normnl tlcnsity to :i position coriwponding to hybrid DNA.
VIRAL-1NDUCED ENZYMES AND VIRAL ONCOGENESIS
181
Moreover, the hybrid DNA was radioactive proving conclusively that cellular DNA synthesis had been induced by polyoma virus infection (Weil et al., 1965). I n a study of crowded cultures of GMK cells, Hatanaka and Dulbecco (1966) have shown that cellular DNA synthesis is also induced by SV40. The induction of cellular DNA synthesis was initiated about 24 hours after infection and reached a maximum at 36 hours. At about 48 hours, the rate of cellular DNA synthesis decreased rapidly whereas SV40 DNA synthesis increased. I n the experiments so far described, crowded monoIayer cultures were crnployed and about half of the cclls were productively infected. Sheinin and Quinn (1965) have investigated DN-4 synthesis in rapidly growing cultures of mouse embryo cells infccted with approximately 5000 PFU/ cell of polyoma virus. Under the conditions of their experiments, practically all the cells werc productively infected. In contrast to the results obthined with crowded monolayer cultures, Sheinin and Quinn (1965) found that in the rapidly growing cultures, synthesis of host-cell DNA was inhibited by polyoma virus. During the eclipse period, practically all of the DNA made was cellular DNA. Thereafter, cellular DNA synthesis decreased rapidly to very low levels. Polyoma virus DNA synthesis began a t the end of the eclipse period and increased progressively until all the DNA synthesized in the infected cultures was viral DNA (Sheinin, 1966b). I n view of the results of Sheinin (196613) and Sheinin and Quinn (1965), the question arises as t o whether induction of celluIar DNA synthesis occurred only in cells that were not productively infected, i.e., cither in abortively infected cells or, as an indirect phenomenon, in noninfccted cclls. In order to clarify this question, Vogt and co-workers (1966) carried out a combined radioautographic and irnmunofluorescence study. Polyoma-infected confluent monolayer cultures of mouse kidney cells were pulse-labeled with 3H-dT at a time when a high proportion of the DNA synthesized was cellular. The cells were fixed after an appropriate time of incubation to allow for the synthesis of the viral-capsid protein. At least 90% of thc cells synthesizing DNA a t the time of the pulse also made viral-capsid antigen a t the time of fixation. These rcsults showed that the induction of cellular DNA synthesis took place in productively infected cells. The following interpretation of the preceding experiments may be suggested. Iiicluction of cellular DNA synthesis by papovaviruses depends 011 tlic physiologicd conditioii of the cclls. When few cells are in the ‘W-period of t h r initotic cycle, DNA syntliesis in:iy l)c initiiited in ~ n a n y of tlic rcmaining cclls of tlic cultiirc. T-Towever,if the cultures are actively
182
SAUL KIT
growing so that a large proportion of the cells already are synthesizing cellular DNA a t a rapid rate, cellular DNA will not be stimulated. Indeed, late in the infection as viral-DNA synthesis increases and infectious virus particles are formed, cellular DNA synthesis will be progressively inhibited.
D. INDUCTION OF ENZYMES FUNCTIONING IN THE TERMINAL PATHWAY OF THYMIDINE METABOLISM 1. Kinetics of Enzyme Formation
Since DNA synthesis was grossly stimulated in cells infected with papovaviruses, it seemed likely that some of the enzymes of DNA metabolism would also be increased. Seven enzymes catalyze reactions in the terminal pathway of d T metabolism leading to DNA synthesis. This pathway and the pertinent enzymes are schematically depicted in Fig. 11. Six of these enzymes (i.e., dCMP deaminase, d T kinasc, d T M P
4,
(Dihydrofolate reductase)
(dCMP Deaminase)
1,
AT P, Mg++
TPNH
(TMP synthetase)
(TDP kinase)
ATP Mg*
(dTMP kinase)
... ........( Thymidine . . . . .... ATP, kin& Mgt+
synthetasc, dihydrofolate (FH,) reductase, dTMP kinase, and DNA polymerase) increased in cell cultures infected with papovaviruses (Kit e t al., 1965, 1966d,e). The seventh enzyme, d T D P kinase, is present in great excess, and it seems unlikely that this enzyme would be induced by virus infection. Deoxyribonucleic acid polymerasr mid tlT kitlahe activities we^^ tn:irkrtlly stfiiiiul:itorl i n c w r i f l u n i t tiionolayc~r cultuws of citlier nio~i>e
183
\'I RA L- 1N DT 1C ED EX Z Y M ES A N D 1'1R A L ON CO( iEN ESIS
liitliicy oi* iiioiiw t.iiibryo w l l h iiifrcleil wit11 polyoilia v i r w and in either (iAIK or ( X - 1 cells infwtfvl n i t h PV40 (Diilhwco clt (7/., 196.5; F r e a r w i c f ( I / . , 1W5; Iiit r / ( I / . , 1965, 19(if%I,o,g;Sliviniii, 19(i(k). Tho 1iiiicBtic.s of tlic rneyiiic IIICTC:LMX i l l SV40-iiitc~tctlCV-1 cells :ire sliown i n Fig. 12. The d T kiiiase increascs began a t about 12 to 16 hours after SV40 infection, and at 28 to 48 hours tho onzyine activity 1ws 4-15 times greatcr
l2
t
Infected kinose-
/'
\ \
/
0 I
/
/
\
\
\ \
I
\
I
I
\
Infected polymerase
I
\ \
I
I I
I
-a0
10
20
30
40
50
60
70
80
Hours postinoculation SV40
FIQ.12. Kinetics of thymidine kinasc and DNA polymerase formation in confluent monolayer cultures of CV-1 cells inoculated with SV40 at an input multiplicity of 140 PFlJ/cell. Thyinidinr kinase nct,ivity : micromicromoles deoxyuridylatc fornied per microgram protein in 10 niinritcs at 38°C. Dcoxyribonucleic acid polymerase activity : micromicromoles 'H-deoxythyniidinc triphosyhatr incorporated into DNL4per microgram protein in 30 minutcs at 38°C.
than that of noninfected cells. The increase in DNA polymerase activity was less pronounced than t ha t of d T kinase but paralleled that of the latter enzyme. I n polyoma-infected mouse kidney monolayers, the kinetics of d T kinsse and DNA polymerase inductions were about the same as that in SV40-infected monkey kidney cell cultures (Dulbecco e t al., 1965; Hartwell et al., 1965; Hatanaka and Dulbecco, 1966). Two- t o threefold increases in d T M P synthetasc :tncl FH, reductase activit,ics were found following SV40 infection of CV-1 cells or polyoina
SAUL KIT 60
-
1
l2
50 -
40 -
w
3 s
--
30-
0
0
s
2
R 20G
P
10-
5\, Noninfected ( dT kinase) \ a . V-
/'
-
A /
'4 0
I
I
I
10
20
30
'Q-,
I
40 Hours postinoculation of SV40
c
--7
I
50
0
Fro. 13. Kinetics of dihydrofolate (FH,) rednctase and thymidine kinasc induction following infection of 7-day-old CV-1 cells (4.7 X 10' cclls/culture) with 314 PFU/cell of SV40. Dihydrofolate reductase activity : micromicromoles FH, reduced per microgram protein in 10 minutes at 23°C. Thymidine kinase activity: micromicromoles deoxyuridylate formed per microgram protein in 10 minutes at 38°C. (TdR = thymidine.)
virus infection of mouse kidney cells (Frearson e t al., 1965, 1966; Kit e t al., 1966e). Figure 13 illustrates experiments with SV40-infected CV-1
cells in which dihydrofolate (FH,) reductase and d T kinase activities were measured on the same cell extracts. The percentage increase of FH, reductase activity also was not as great as that of d T kinase but occurred over the same time interval. The dCMP deaminase activity of mammalian cell culturcs is very unstable, but the enzyme can be stabilized during extraction and activated during assay by dCTP. As mentioned previously, d T M P kinasc can be stabilized and activated by its substrate, dTMP. Even when extracts were prepared with buffer containing dCTP or dTMP, the activities of dCMP deaminase and d T M P kinase were very low in confluent mouse kidney cell monolayers. The activities of both enzymes were increased severalfold after polyoma virus infection. The time a t which 50% of the increase was achieved was almost the same as for d T kinasc (Dulbccco et al., 1965; Hartwcll e t al., 1965; Kit et al., 1966d).
I n contrast, tlic, :wt ivity of t1CMP dr:uninabe was uiiusually high in GMK or CV-1 cells (about 80 to 100 times greater than in mouse kidney cells). Moderately high levels of d T M P kiiiasc are also found in monkey kidney cells. Thebe two enzymes did not increasc in activity in GMK cells after SV40 infection. Scvrral additional enzymes have h e n stutlictl in papovavirus-infected cell cultures. Uridine kinase, d T M P phosphatase, dAMP kinase, and dCMP kinase did not change appreciably in activity after papovavirus infections (Dulbecco et al., 1965; Kit et al., 1966d,e). Experiments with puromycin and cycloheximide have shown that the papovavirus-induced cnzyrne increases did not occur in the absence of protein synthesis. Addition of puromycin during the first 12 hours after polyoma virus infection prevented the increases in d T kinase, dCMP deaminase, and DNA polymerase in murine cell cultures. Cycloheximide inhibited the stimulation of d T kiiiase and DNA polymerase in SV40infected monkey kidney cell cultures. If the inhibitors of protein synthesis were added to cultures after the enzyme increases had begun, further enzyme induction was curtailed. Moreover, removal of the drugs permitted a renewal of enzyme synthesis after a lag period (Hartwell et al., 1965; Kit et al., 1966d,e; Sheinin, 1966:~). Experiments in which mixtwes of enzymes from both infected and noninfected cells were assayed tlcmonstrated that the increased enzyme activities in infected cells were not attributable to activators in the infected cell extracts. Similarly, the low activities in extracts from noninfected cells were not due to an excess of free inhibitors in the extracts from noninfected cells. 2. Effect of Actinomycin Il
OIL
SV4O-Iduced Enzyme Synthesis
I n order to learn whether RNA synthesis was required for the induction of d T kinase by SV40, CV-1 cells were treated with 1 to 5 pg./ml. actinomycin D a t 2 hours after SV40 infection. Actinomycin D completely inhibited the SV40-induced incrcnw nornially observed a t 26 hours after infection (Kit et nl., 1965, 19GGf). If actinomycin D addition was delayed until 10 to 14 hours after infection, a partial induction of d T kinase took place. If the actinoniycin D was added at 17 to 21 hours after infection, almost normal levels of d T kinase were induced. These results siiggcst that most of thc nicssengvr RNA rrquirctl for (IT kinase formation was niacle by I7 hours aitrr SV40 infcctiun. similarly, nctinoniycin D (1&2.5 pg./'ml.) iitldetl at 2 hours after SV40 iiifection of CV-1 cells completely inliihitctl the increase in FH, rccluctase activity noimally observed 41 hourh after infection. If actiaomycin I)was added a t 12 hours, the SV40-infected cells showed a signifi-
1%
SbtJL KIT
cant increase in FH, reductasc activity a t 41 hours, but still 1 ~ : than s in infected but nontreated cells. Addition of actinomycin D a t 19 hours had little or no effect on the induction of FH, reductase (Frearson et al., 1966).
3. Effect of dBU on Papovavirus-Induced dT Kinases Halogen-containing analogs of d T are known to inhibit cell growth and t o prevent the development of infectious SV40 and other DNAcontaining animal virubes. However, both T-antigen and virus-capsid antigen are synthesized in infected cell cultures treated with the d T analogs. The addition of dBU to SV40-infected cell cultures did not appreciahly affect the ratc of dT kinase induction (Kit et al., 1966e). I n infected cultures not treated with dBU, d T kinase activity was elevated for about 48 hours and then declined sharply a t the time that cytopathology became pronounced. This decline was delayed in SV4O-infected cultures treated with dBU. At 96 hours after infection of dBU-treated cultures, the d T kinase activity was still sixfold greater than the activity of noninfected cultures. Treatment with dBU also did not inhibit the induction of d T kinase by polyoma virus (Kit et al., 1966d) nor the induction of FH, reductase by either SV40 or polyoma virus (Frearson et al., 1966). 4. E f f e c t of Mitomycin C on SV4O-Induced dT Kinase Formation Mitomycin C is a potent inhibitor of cell growth and DNA synthesis. Treatment of bacterial or mammalian cell cultures with this drug leads to an induction of DNase activity and the breakdown of DNA. At concentrations which inhibited DNA synthesis, mitomycin C addition to cultures a t 2 hours did not block the increase in d T kinase observed at 30 hours after SV40 infection. I n some experiments, GMK cell cultures were pretreated with mitomycin C for 16 hours prior to SV40 infection. The infected cultures were then further incubated in the absence of mitomycin C. Despite the pretreatment of GMK cell cultures with mitomycin C, d T kinase activity was 24 times higher in SV40-infected than in control cultures a t 30 hours after infection. Moreover, in cultures in which mitomycin C was present both in the preinfection and postinfection periods, the dT kinase activity of infected cultures was about 10 times greater than that of noninfected cultures. It is probable that prolonged treatment with mitomycin C not only inhibited DNA synthesis hut caused damagc, t o the host-cell DNA. The failure of mitoniyciii C to prevent the increase in d T kinase therefore suggests that nornial undamaged GMK cell DNA w i s not recluirccl for SV40-induced d T kinase synthesis.
5 . Effect of Ara-C on Papovavirus-Induced Enzyme Synthesis l-P-D-Arabinofuranosylcytosine is a potent inhibitor of cell growth, DNA synthesis, and the replication of DNA-containing animal viruses. Tlie mcchilnisin of action of ara-C is different froin that of either initomycin C or dBU. It is thought that ara-C curtails d C T P synthesis by preventing the reduction of ribonucleotides to deoxyribonucleotides. The addition of ara-C (10 pg./inl.) a t 2 hours after SV40 infection of CV-I cells or polyoiiia virus infection of iiiouse kidney cells had no inhibitory effect on the inductions of d T kinase or FH, reductase by these viruses (Frearson et al., 1966; Kit et al., 1966d,e). This concenh t i o n of ara-C did suppress "H-(IT iticorporation into the DNA of the cells. Ara-C treatment of CV-1 cells :ilso fniled to inhibit the induction by SV40 of DNA polymerase nctivity. It may be concluded froin the experiments with dBU, mitomycin C, anti ara-C that DXA synthesis, cell growth, and infectious-virus forinatioii were not required for the inductions of d T kinase, FH2reductase, and DNA polymerase by the papovaviruses.
6. Effect of Ultraviolet Light on the Infectivity and dT Kinuse-Inducing Activity of SV4O Ultraviolet irradiation of SV40 inactivates both infectivity and Tantigen-inducing activity. Loss of T-antigen-inducing activity occurs a t a slower rate than loss of infectivity (Carp and Gilden, 1965). Ultraviolet radiation also reduced the clT kinase-inducing capacity of SV40 (Carp et al., 1966). The loss of virus infectivity occurred 2-6 times faster than enzynie-inducing capacity. It is probable that virus DNA is the primary target of UV radiation. Since increasing doses of UV radiation progressively inactivated the d T kinase-inducing capacity of SV40, it appears that d T kinase synthesis in infected cells is controlled by the virus DNA. However, the UV irradiation experiments do not elucidate the mechanism by which the SV40 nucleic acid controls the forination of d T kinase. This could be due either to effects that act directly on the coding properties of the virus genome or to effects on the DNA of tlic virus which then arc translatrd to t,he control mcclianisnis of tlic cell. 7. Stiniulation by Ara-C and Illitomycin C of the dT Kinase Activity of Noninfected Monkey Kidney Cell Cultures I n connection with the problem of the mechanism of viral-induced enzyme synthesis, it would be useful t o have a method for inducing enzymes in noninfected cell cultures. Comparisons could then be made
188
SAUL KIT
of the enzyme induced in noninfected cells with those induced by virus infection. During study of the effects of drug treatment on the iiiduction of d T kinase in papovavirus-infected cells, a simple procedure was discovered for increasing the d T kinase activity of noninfected cells hy a factor of 3 to 10. It was found that the addition of ara-C (10 to 20 pg./ ml.) to noninfected GMK or CV-1 cells causecl pronounced increases in the activity of d T kinase starting a t about 12 hours after drug treatment (Fig. 14). The ara-C-induced stimulation of dT kinase activity occurred
O6
I
I
I
I
10
20
30
40
Hours after Ara-C addition
FIG.14. Kinctics of ara-C-induced stimulation of dT kinase nciivity of 9-dny-old G M K cell cultures. Arn-C concentration : 10 pg./nil.
in HeLa cell cultures as well a s monkey kidney cell cultures but did not occur after ara-C treatment of LM and LM(TK-) mouse fibroblast cells, primary mouse kidney cells, or HeLa (BU-100) cells (Kit e t al., 1966a,b). I n noninfected CV-1 cell cultures treated with ara-C, a n 84% increase of FH? reductase and two- to threefold increases of dCMP deaminase and DNA polymcrase activities were observed. However, dTMP kinase activity was not significantly changed. The ara-C-induced incrcascs could be reversed by deoxycytidine (dC) . It was also found that an appreciable increase in d T kinase activity occurred after DNA synthesis was inhihited by mitomycin C or amethopterin. The amethopterin inhibition of DNA synthesis could he reversed
189
\ l I t A I ~ - I N L ) U C E D ENZYMES AND \‘1RAL ONCOGENEHIS
by d T ; after d T reversal of the inhibition, the activity of d T kinase rapidly declined. It is attractive to suppose that cell cultures were pseudosynchronized by drug treatment. Cells not in the G, phase of the mitotic cycle entered this phase but were prevented by the inhibitors from entering the “S” phase of DNA synthesis. An accumulation of enzymes related to DNA biosynthesis would appear to be charactcristic of late G1-phase cells. TABLE IX AND ARA-C OR BROMODEOXYURIDINE TREATMENT EFFECTSOF SV40 INFECTION ON THE THYMIDINE KINASEACTIVITY OF GMK CELLS dT kinase activity (ppmoles dUMPn formed per fig. protein in 10 min. at 38°C.) (:onr?mt,m.- - .- -.... I
tion Inhibitor
(pg./ml.)
None Ara-C dBU
None 20 25
a
48 hr. PI 28 hr. P I Nonirifected SV40-infected Noriinfected SV40-infected 1.3 9.9 1.8
6.4 14.7 7.8
0.8 4.8 0.9
5.9 10.9 9.2
Deoxyuridylate.
Table IX shows the dT kinase activity of GMK cclls infected with SV40 and also treated with ara-C or dBU for 28 and 48 hours. Either ara-C treatment or SV40 infection increased the d T kinase of GMK cells, but dBU treatment did not. The effects of ara-C and SV40 infections on d T kinase induction were additivc. Additive effects of ara-C and SV40 infections on the induction of DNA polymerase were also observed. 8. Properties of Partially Purified dT Kinnse
Using a relatively simple procedure consisting of ammonium sulfate fractionation and negative phosphate gel adsorption, d T kinase has been purified approximately twenty- to fortyfold compared with crude-cell cxtracts (Kit et al., 1966d,e). To learn whether SV40 infection produced a d T kinase with altered properties, the I(, of the SV40-induced eiizyme was determined and a comparison was m : d ~with the I<, value of the enzyme from noninfected GMK or CV-I cells. Table X shows that the SV40-induced enzyme had a RnLvalue for d T J about 3 times greater than that of the enzyme from noniiiicctctf cells. As previously stated the d T kinabe activity of noninfected monkey kidney cells wits invl.caits(%(l ~ ( ~ \ . ~ ~ 1 . i t l f o1)s l t l tiwitinl?; vultures L\.itli a i ~ t - Cur
190
SAUL HIT
TABLE! X MICHAELIS CONSTANTS (Km)WITH 3H-DEOXYURIDINE AS NUCLEOSIDE ACCEPTOR AND 5-TRIFLUOROMETHYL-2’-DEOXYURIDlNE INHIBITOR CONSTANTS (ZCJ OF THYMIDINE KINASEPARTIALLY PURIFIED FROM NONINFECTED OR SV40-INFECTED MONKEY KIDNEYCELLS Source of enzyme
Cell cultures incubated in medium containing the compounds listed for the times indicated
K, ( X 10-6 AT)
‘H-dUa
Ki ( X 10-6M) FadTe
None dTb (10 pg./ml.) d o (10 pg./ml.), 16 hr. d T (100 pg./ml.), 41 hr. Ara-Cd (10 pg./ml.), 41 hr. Mitomycin C (20rg./ml.), 16 hr.
2 . 8 k 0 . 2 (3) 2 . 8 k 0 . 1 (2)
4 . 3 f 0 . 7 (3) 2.9 f0.5 (2)
3 . 3 k 0 . 1 (2) 3 . 2 f 0 . 3 (2) 3 . 9 f 0 . 3 (2)
4.0 f 0 . 6 (6) 2.8 k 0.3 (5) 5.1 k 0.4 (5)
Noninfected CV-1 cells
No treatment Ara-C (10 pg./ml.), 28 hr.
2 . 8 f 0 . 2 (4) 2 . 8 f 0.1 (2)
-
SV40-infectled GMK cells for 41 hr.
None d T (100 pg./ml.), 41 hr.
SV4O-infected CV-1 cells for 41 hr.
None
Noninfected GMK cells
+
-
8 . 4 f 0 . 9 (8) 11.0 (1)
15.0 f 2 . 9 (7) -
8 . 1 f 0 . 6 (4)
-
Values shown are the mean k standard error of the mean; the numbers in parentheses indicate the number of determinations. b Thymidine. c Deoxycytidine. 1-8-D-Arabinofurariosylcytosiii~.
mitomycin C. It was, therefore, of interest to learn whether ara-C or mitoinycin C would also change the K , value of the enzyme. It was anticipated that ara-C and mitomycin C would alter the intracellular nucleotide pools and that this might perhaps affect the I<, value. Table X shows that this was not the case. The K , values of the enzymes from ara-C- or mitomycin C-treated cells were similar to those of the enzymes from noninfected cells and differed from the K , value of the enzyme from SV40-infected cells. In an effort t o manipulate the intracellular nucleotide pools, noninfected GMK rcllls were incuhatcd with high ronrcntrations of d T (100 pg.,/nd.) or will) l o w c ~rotic.(iti1i.:itionh of cL1’ I ) l u h cIC (10 pg./llll.). T h e reitmii for : i ( l l l i i i g Iiigli rcrr~c:c~~iti~:il~ori~ o f (IT IWS to r:tiise, i f I)usbil)le,
a n :cc~ci~niul:itio~i of tlTTP, the fecdlmk inhihitor of clT kii1a.w The d T plui. (I(: was adilwl to fwcilitatr i ~ lgrowth l Nrithrr addition of e y c w (IT i ~ o r: i ( l i l i t i o i i of (IT p l l i h d ( ’ :il)prvr.iahly : i l t c w t l thr lim\-aluc of thc 1):ti.ti:LIly p u r i l i t ~ t lc~iizyi~ic~ f i ~ ~ ii i~i o i ~ i i i f t ~r.c,ll,~. t t ~ l The F,dT inhibition constants ( K L )of t l T kiriase are also sliowii in Table X. The K , values of the crizyines prepared from SV40-infected G M K cells were greater than those from noninfected cells. The preceding experiments demonstrate that a d T kinase with altered properties is induced in monkey kidney cells after SV40 infection (Kit e t al., 1966e). I n the author’s laboratory, a similar change in I<, value for dU has not been observed for the enzyme purified from polyoma virus-infected murine cell cultures (Kit et al., 19GGd). However, with dT as nucleoside acceptor, Sheinin (1966a) found three differences between the kinases from normal and polyoma-infected cultures: (1) the K , value of dT kinasc from polyoma virus-infected cells was less than that of normal mouse embryo cells; ( 2 ) in the presence of excess substrate, the phosphorylation of d T was inhibited-a lower dT concentration was required to produce this inhibition with the polyomn-induced enzyme than with thc normal cellular d T kinnse; and ( 3 ) the infected and the noninfccted cell enzymes differed in thermal stability. A number of hypotheses may be considered in accounting for the altered properties of d T kinase in SV40-infectcd cell cultures: (1) d T kinase may be constructed from subunits which either dissociate or polymerize after virus infection; ( 2 ) virus infection might cause a gross change in dT kinase conforniation; ( 3 ) unknown products of SV40 infcction could combine with the cellular clT kinasc, altering the affinity of the enzyrnc for dU and F,dT without grossly changing the molecular weight or conforniation of the enzynic; ant1 ( 4 ) a new crizyme might IJC induced after SV40 infection. T o learn whether there were gross differenccs in the molecular weights of tlT liinnsc from SV40-infected and tioninfected monkey kidney cells, Sephatlex G200, gcl filtration studies wcre carried out. The molecular weight of the SV40-induced d T kinase was ehtirnated to be about 80,000 to 100,000 (Table XI). Thymidine kinnse prepai-ittions from non-infected and from ara-C-trcatcd CV-1 cells were also studicd. Their molecular weights were also :ibout 80,000 to 100,000. It would appear, therefore, that SV40 infection did not inducc x gross change in the molecular weight of monkey kidney d T kinnse. If, as :i result of SV40 infection, a cellular enzyme were either to unfold or to coil to a more compact configuration, the sensitivity of the ciizyrric in the new configuration might he xltered wit11 respect to sulf-
192
SAUL KIT
hydryl reagents, heavy metals, or to reagents that denature the protein. Also thermal stability and sensitivity t o dTTP, the feedback inhibitor, might hc changed. To investigate these poesibilities partially purified dT kiriase 1)rcp:irntions wcre assayrtl in tlic prrsciiw of various concentrations of p-chlol.omercuriberizo:~tc, p-liydroxyrricrcuribcnzoate, sodium dodecylsulfate, cobalt chloride, or dTTP. All these compounds inhibited the activity of d T kinase. However, the inhibitor dose-response curves were about the same for the enzyme from either infected or noninfected cells. The kinetics of thermal inactivation of d T kinase preparations was studied a t 65", 70°, and 75°C. At each of the temperatures, the enzymes from noninfected and from SV4O-infected cells were inactivated at about the same rate. The results do not support the first two hypotheses. The observations could however, be explained either on the bases of the hypothesis that a new virus-specific enzyme is induced or that SV40 infection causes, by unknown mechanisms, smaller changes in either molecular weight or conformation. 9. Properties of Partially Purified DNA Polymerase Deoxyribonucleic acid polymerase preparations from noninfected or SV40-infected CV-1 cells have been purified six- to sixteenfold compared with crude-cell extracts. The purified DNA polyriierasc enzymes required denatured DNA for activity, had the same p H optima, and were drastically inhibited by low concentrations of ammonium sulfate. The enzymes from infected and noninfected cells were very unstable; they were totally inactivated by 5 to 10 minutes of heating a t 50°C. and lost about half of the initial activities in 3 hours a t 38°C. The DNA saturation curves were very similar for the two enzymes but they did differ with respect to the K , value for dTTP. The K , value of the enzyme from SV40-infected cells was about half that of the enzyme from noninfected CV-1 cells. 10. Relationship between SV40-Induced Enzymes and T-Antigens
Enzymes of DNA metabolism and thc SV40 T-antigen wrrc induced in monkey kidney cell cultures a t about the same time and were readily distinguished from the virus-capsid antigen on thc basis of their time of appcarancc and physical properties. The enzymrs and the T-antigens were synthesized in thc presence of ara-C and halogenated pyrimidine deoxyribonucleosides but not in cell cultures treated with actinomycin D or cycloheximide. The SV40 T-antigen has the properties of a protein since it is inactivated by 10 minutes of heating a t 60°C. or by trypsin hut not by RNase or DNase (Gilden and Carp, 1966; Gilden et al., 1965; Hoggan et al., 1965; Rapp e t nl., 1964b, 1965a; Sabin and Koch,
1964). The possibility has h e m considered that SV40 T-antigens might be early enzymes. Sonic of the propertics of the T-antigens arid early enzyiiics are shown in Table XI. Over 90% of the SV40 T-antigen and d T kinase activity were precipitated from centrifuged extracts of infected cells by 20 to 32% saturated ainmoiiium sulfate. Most of the DNA polymerase activity was also precipitated by this concentration of ammonium sulfate, but higher salt concentrations were needed to precipitate dCMP deaminase, FH, reductase, and dTMP kinase. These results demonstrate that the last three enzymes can be physically separated from the T-antigen. Table XI shows that the molecular weights of FH, reductase, d T M P TABLE XI s v 4 0 T-ANTIGEN AND O F ENZYNES FUNCTIONING DEOXYRIBONUCLEIC ACID B~OSYNTHESIS
S O M E P R O P E R T I E S OF IN
Enzyme or antigen SV40-specific T-ant,igen Thymidine kinase Deoxycytidylate deaminase Dihydrofolate reductase Thymidylate kinase D N A polymerase Thymidylats synthetase Q
Saturation with ammonium sulfate required for precipitation (%)
Estimated mol. wt. (Sephadex GI50 chromatography)
20-32 20-32 3.5-60 55-83 31-60 20-40
> 200,000 80-100,000 120,000 20,000 52,000 >200,000
-
.58,0000
M. Y. Lorensen, G. F. Maley, and 1'. Maley (1967).
kinase (and perhaps, dTMP synthetase) are significantly lower than that of the T-antigen. The molecular weight of DNA polymerase is uncertain since i t was estimated under conditions where the enzyme was aggregated. The T-antigen was eluted from Scphadex G200 columns slightly ahead of d T kinase; it differed from (IT lriiiaee in two properties: ( I ) the SV40 Tantigen was :iInio*t coniplctcly iiiactivatccl l)y 10 minutes of hcating at 6OoC., but about 50 to 75% of the SV40-intluced d T kinase activity survived this heat trezitmciit ; : t n t l ( 2 ) tlie T-antigen was firmly absorbed I)y calcium phosphate gels under condition* where little d T kinase w:is hound. Tlicw i)l)hcrwtioiis suggc.d. t l i : i t t l i c SV40 T-:iiitig(m iii:iy not I)c icleiltic.sl with any of the early c-nzyines. ,\lternative possibilities are that the T-antigen is: (1) ;t regulatory poteiii, (2) a nonerlzymatic protein functioning in DNA rcplicntioii ; or (3) :in iiitcrnnl coiiipoiient of the virion.
194
SAUL K I T
E. BIOCHEMICAL CHANCXS IN CELLCULTURES ABORTIVELY INIT CTED WIT11 PAPOVAVIRUSES 4
1. Abortive SV4O Infection of Mouse Kidney Cells Papovavirus SV40 is adsorbed by mouse kidney cell cultures, though more slowly than by monkey kidney cells. The virus enters an eclipse phase in tlie mouse kidney cells which lasts about 24 to 32 hours. Thereafter, virus titers increase until about 40 hours and then decline. Even when high input multiplicities are used, no cytopathic changes are ohscrved although the virus persists for a t least 7 days (Dl1l)l)s et al., 1966; Kit et al., 1966g). I n most of the mouse kidney cells, SV40 undergoes an abortive infection. When infected cultures were trypsinized and individual cclls plated on noninfected CV-1 monolayers, less than 1% of the mouse kidney cells initiated infectious-center formation as compared with 60 to 80% of SV40-infected CV-1 cells. At 36 to 54 hours, the mouse kidney cultures yielded about 1 t o 6 P F U of SV40 per cell, whereas CV-1 cultures yielded about 100 to 300 P F U per cell. However, the yield of SV40 per infectious center was of the same order of magnitude as from CV-1 cells. The number of SV40 particles found in supernatant fluids used for analysis of complement-fixing (CF) antigens was about lo8 per milliliter for the mouse cells, and 1O1O per milliliter for the monkey cells. If one assumes that the virus particles were coming from cells that plated a s infectious centers, then again thcre was no difference in yield between a n infected monkey kidney cell a i d a competently infected mouse cell. The results suggest t h a t infectious SV40 is replicated in only a small percentage of mouse kidney cells. Four of the enzymes of DNA metabolism have been studied in mouhe kidney cell cultures inoculated with SV40 (Kit et al., 1966g). All four ciizymcs, namely, d C M P dr:tniinasc, dT kinase, d T M P kinnse, and DNA polymerase increased considerahly. The kinetics of the increases of d T kinase and DNA polymerase arc illustrated in Fig. 15. Increases in d T M P kinase and dCMP deaminase activities took place a t about the same time as the increases in DNA polymerase and d T kinase. Addition of puromycin a t any time prior to 16 hours after infection prevented the enzyme increases normally ohscrved a t 30 to 32 hours after SV40 infection. The effect of ara-C on enzyme induction has also been studied. The aru-C trcutment ditl iiot in1iil)it ciizyiiic forination in SV40-infected mouse kidney monolayers (Kit, 1988; Kit e l ul., 196Gg). The kinetics of T-antigen form:ition were similar in mouse kidney and CV-1 cell cultin*es, but the titers were about one-tenth as high in
- 0.24
- 0.20
-
.-.-> ~
- 0.16 -
E, -
- 0.12
2
-
c
0
01
a 0
n
- 0.08 -
- 0.04 -
I
0
I
I
10
20
I
I
40
I
50 Hours postinoculation SV40 30
I
I
60
70
J
00
FIG.15. Kinetics of thymidinc kinasrl and DNA polymerase formation in confluent monohyer cultnrcs of mousc ladncp c ~ l l sinoculated w t h sV40 a t an input multiplicity of 170 PFU/ccll. Thymidinc klnase activity : pupmoles dUMP formed prr pg protein in 10 minutes nt 38°C. DNA polymcrase activity: ppmolrs ’HtlTTP incorpoiatrtl into DNA pcr pg protcin in 30 minutes a t 38°C.
mouse kidney as in CV-1 cells (Fig. 9 ) . At 40 hours, SV40 capsid-antigen titers in mouse kitliicy cell cultures were less than I % of those in CV-1 cell cultures. Pulse-labeling experiments demonstrated that the incorporation of ’II-dT into D N A was stimulated two- to tlireefold from 16 to 48 hours aftcr sV40 infection. I n noninfectetl mouse kidney cell cultures, only 2-5% of the cells exhibited lahcled nuclei after a jH-dT pulse. After SV40 infection, approximately 20-2570 of the nuclei were labeled. At 96 hours after infection, over 2070 of the nuclei of the SV40-infected inurine cell cultiirCk wcw %tillIalbeled. E~miti:illyall of the DNA made in abortively infrctcd i i ~ o u ~l i ei t l i i c y cultuiw w:is cellulai~,not viral, DNA (Kit ct u1., 19671)).
2. Enz~j111e.4ctivitics and T-Antigen Titers illouse Kidney Cells
01 Transforwwd
At nhout 2 to 3 weeks aftcr SV40 infection, colonies of transformed mouse kidney cells W‘CIY~ noticc:thlc (Black arid ROUT, 1963; Black
196
SAUL KIT
ct ul.,1963; Kit et d., 19GGg). Hoinc. of tlic “t8ransforni~d” cells have bccn subcultured for 117 passages a t this writing. Primary monolayer cultures of mouse kidney cells showed contact inhibition and attained a population density of approximately 3 to 5 million/55 cm.’ in 5 to 7 days. The cell population then gradually declined, and vcry little cell growth occurred after subculture. The transformed cultures, however, grew to populations of 10 to 15 million/ 55 cm.’ and could be subcultured a t 3- to 4-day intervals. The SV40-transformed cell lines exhibited high levels of d T kinase, dCMP deaminase, dTMP kinase, and DNA polymerase activities. The enzyme levels observed were typical of other established cell lines. The transformed cell lines all contained titers of SV40 T-antigen comparable to those of SV40-transformed hamster tumor cells (H-50) . However, the transforincd mouse cells did not contain detectable SV40capsid antigen or SV40 particles. Attempts to extract infectious virus or infectious DNA from the transformed cells have heen unsuccessful.
3. Michaelis Constants (K,) of dT Kinase and D N A Polymerase fyom SV40-Infected Mouse Kidney Cells and Transformed Im e s ‘
The d T kinase and the DNA polymerase induced in monkey kidney cells by SV40 have altered K , valucs. I n contrast, the enzymes from SV40-infected mouse kidney cells or from transformed cells had the same K , values as the enzymes from noninfected cells, suggesting that the latter enzymes may be derepressed cellular cnzymes, rather than new virus-specific enzymes (Kit, 1966). The implication is that an additional SV40 function is that of dereprrssing cellular cnzyme and DNA syntheses. 4. D N A Synthesis in Polyoma Virus-Infected Rat Embryo Cells R a t embryo cells adsorb polyoma virus but less than 0.1% replicate the virus and no more than 5 4 % undergo morphological transformation. The induction of cellular DNA synthesis was stimulated in rat embryo cells abortively infected with polyoma virus (Sheinin, 1 9 6 6 ~ ) .An induction of cellular DNA synthesis has also been observed in X-irradiated rat and mouse embryo cell cultures after polyoma infection (Gershon et al., 1965). Cultures were irradiated with X-rays prior t o infection to reduce the normal level of cellular DNA synthesis. Combined radioautographic and chemical studies showed that every DNAsynthesizing cell approximately doubled its DNA content. The induction of cellular DNA synthesis in the rat cells was not accompanied by the detectable synthesis of infectious virions, viral antigen, or viral DNA.
Nitrous :tcaitl I r c : t t i i i c I n t of tlic viix+ ii~ac-livalclcl1)NA-iiitluc.iiig (2:ipacity : ~ n dccll-tranbfoi,miiiff cap:tcsity :It :ibout tliv s t m e ratc. This ratc w i only about 2070 of that for inactivation of plaquc-forming ability. The nitrous acid inactivation experiments show the induction of cellular DNA synthesis is a function of the polyoma virm gcnome. 5 . Infection of dT K znase-Deficient Baby Hamster Kidney ( B N K 21) ( ells with Polyoina I-irus
The BHK 21 cells, a stable line of hamster kidney fibroblasts, are cuploid cells which exhibit a generation time of about 12 hours, grow in oriented parallel bundles, and form sheaflike colonies. Transplantation of these cells into hamstcrs gave rise to tunioi*h, but m o i ~t h i n lo6 celh were required for tumor formation. When BHK 21 cells were exposed to polyoma virus, there were no cytopathic effects or consihtent fall in plating efficiency. Most of the cells formed colonies indistinguishable from those of control cells which had not been exposed to the virus. A small portion of the cells with different morphology wcre found to be coniposed of “transformed” cells. Such colonies wei-c round a n d the cells wcre oriented a t random to one another. No plaque-forming viruh could be detected by immunofluorescence in the nuclei of harnster cells infected with polyoma virus. I n the BHK 21 cells, about 5% of the number originally plated show polyoma-nuclear antigen 4 days after infection a t a ratio of lo3 PFU per cell. Under these conditions about 3% of the cells plated were transformed. The cells with fluorescent nuclci were pi obably vegetatively infected and were responsible for the small aniouiits of newly synthesized virus which were detected in the cultures (Frascr et ul., 1966; Stoker and Abel, 1962). Variant cells deficient in d T kinase activity were isolated from populations of BHK 21 cells. Infection of d T kinase-deficient cells with polyoma virus did not cause a n induction of this enzyme, as occurs during the lytic cycle in mouse embryo or mouse kidney cell cultures. However, the enzyme-deficient cells were transformed by polyoma virus. Thymidine infected or kinase was not permanently acquired by even a rare transformed cell (Littlefield and Basilico, 1966). The results indicate that the polyoma virus function of inducing d T kiriasc is not expressed during abortive infection of BHK2l cells. lloreover, the “transformation,” :is judged by altered colonial morphology, loss of contact inhibition, and increased transplantability in hamsters, docs not necessitate functional d T kinase activity. T h e data do not exclude the possihility that d T kinase does play a role in transformation processes when contact-inhibited primary mouse kidney cultures are converted to established cell lines.
~
198
SAUL K I T
1 . l rurul,latitcitioti A ntigetis
Polyoma-induced tumors of hamsters and mice and SV40-transfoi~ned hamster cells contain new foreign antigens. The new antigens can be demonstrated by transplantation-rej ection procedures and by indirect iinniunofluorcscence tests, using hamstcr serum from animals rendered resistant to transplantation by vaccination with the viruses (Habel, 1965; Khera et al., 1963). The transplantation antigens arc distinct from either viral-capsid antigens or T-antigens ant1 they are virus-specific. Hamsters inoculated with SV40 were resistant to challenge with cells transformed i n vivo by SV40, but inoculation of polyorna virus did not confer resistance to challenge with SV40-transformed cells although it did protect against polyoma virus-traiisforrned cells. The SV40-transplantation antigen appcars to be localized a t the surface of cells transformed by that virus (Tevethia and Rapp, 1965). 2. Potentiation by SV4O of Human Adenovirus Growth in G M K Cells
Replication of human adenoviruscs is abortive in GMK cells. Human adenovirus strains adsorb to and penctrate GMK cells and induce adenovirus tuinor antigens, but not adenovirus capsid antigens. .Joint infection of GMK cells with adenovirus and with SV40, however, results in the syntheses of both SV40 and adenovirus tumor and viral antigens, the development of both types of virions in the same cell, and an increase in the infectious titer of both viruses (Feldnian et nl., 1965; Malmgren et nl., 1966; O’Conor e t al., 1963; Rabson et al., 1964b). I n some way SV40 gene products enable human adenovirus strains to ovcrcomc blocks in thc replication cycle which otherwise occur in GMK cells. Thc genetic function supplied by SV40 is not that of providing enzymes for adenovirus DNA replication. Thc rate of synthesis of adenovirus DNA was about the same in GMK cells infected only with adcnovirus (nonproductive cycle) and in KB cells (productivc cycle) or with adenovirus and SV40 (adenoproductive cycle). The failure t o detect adenovirus capsid proteins in GMK cells infected with adenovirus only might be clue to defective production of adcnovirus messenger RNA or to faulty translation so that aberrant protcins antigcnically diffcrcnt from normal capsid protcins arc made (Rap11 et nl., 1966; Reich et nl., 1966). The SV40 gcnetic function rcquired for productive adenovirus infection of GMK cells can be supplied by defcctive particles in SV40adenovirus “hybrid” populations. Thc latter virus preparations consist of two types of particles: one is a n ordinary adenovirion and, the
s~~con(1, a dcfcctive SV40 particle (PARA) cnc:iscd in :HI ndcnovirus capsid (Boeyk et ul., 1965; Melnick et ul., 1965; Rapp et ul., 1964c, 1965b; Rowc, 1965; Rowc and Buum, 1964, 1965; Rowe et al., 1965). The defective PARA particle can induce the syntlicsis of SV40 tumor antigens but not its capsid proteins. I n cells doubly infected with hunian aclcnovirus and PARA particles, the adenovirus supplies the necessary complcmerit of adenovirus genes for encapsidation of the defective SV40 genome and the PARA particle supplies an SV40 function that facilitates adenovirus growth. The adenovirus acts as a “helper” virus for defective PARA particles, and the PARA particles perform as “helper” viruses for adenovirus replication. Adenovirus Type 7-SV40 hybrid populations produced tumors in hamsters having the antigenic characteristics of SV40 virus and produced in vitro transformation of primary hamster kidney cells and diploid human skin fibroblasts (Black and Todaro, 1965; Huebner et al., 1964). With both cell systems, the SV40 tumor antigen could be demonstrated in practically every transformed cell. 3. Induction of Arginase by R a b b i t Papilloma Virus
It has been well established by Rogers (1959) and Rogers and Moore (1963) that tumors provoked by the Shope rabbit papilloma virus on the skin of the cottontail and the doniestic rabbit contain high levels of argiiiasc, although normal skin derived from eithcr adult or embryo rabbits exhibits little or no arginase activity. These observations suggested that a new arginase might be induced by rabbit papilloma virus and motivated Rogers and Bloorc (1963) to undertake an extensive purification of the papilloma enzyme :tnd a comparison of this enzyme with arginasc purificd from rabbit liver and kitlncy ant1 from a tar-intlucetl tumor of thc rabbit. Striking differenrcs we're obscrvctl Iwtwccri tlic physicochemical properties of the papilloma arginnhe utid the other arginases of domestic and cottontail rabbits. The papilloma arginase could be separated from liver : q i n a s e by sedimentation in a sucrose gradient; it had an estimated molecular weiglit of 42,000 compared with 37,000 for liver arginase. The papilloma arginnsc did not cotitiiin Iiiiinganese and was active even after EDTA treatment to eliminate traces of divaleiit ions. However, all other arginases studied required manganese or cobalt ions for activation and liver nrgiti:ihc rontnincd iiinngaiicsc. The p ~ p t i d cpatterlib o f p:ipilloma argiiiasc W C I I ~ w r y tlifi’t~rt~ii t from liver 1irgiiimL. Aniilials rurrylilg viibii+ itiitibo~licsagaitiht the purtfitd papillomn inducetl ~ ~ p i l l o i i i aclcveloped arginnse. Tlie virus itself IWS iiiiuiuiiologicully distinct from the purified arginase and liad no argiuase :ictivity. Rogers and Moore (1963), thcre-
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fore, concluded that the genetic information of the papilloma arginase does not belong to the rabbit chromosomes but is carried by the rabbit papilloma virus. A cogent hypothesis elucidating the function of the papilloma arginase was also presented by Rogers and Moore (1963). In contrast to normal or hyperplastic squamous epithelium, the Shope rabbit papilloma has an extreme paucity of arginine-rich nuclear histones. It was, therefore, plausible to suppose that in the papilloma, the new arginase acted to deplete cellular arginine, which in turn inhibited nuclear histone synthesis. Assuming that arginine-rich histones are “repressors,” their depletion might possibly free the cells for abnormally rapid growth and papilloma formation. The ohservation by Passen and Schultz (1965) that arginase activity increases in papilloma virus-infected cultures of cottontail rabbit skin has further stimulated interest in this experimental system. Unfortunately, however, recent reports from two laboratories cast doubt on some of the original observations. Orth and co-workers (1967) reinvestigated the properties of the arginases from rabbit liver and papilloma. Contrary to Rogers and Moore (1963), they found that both liver and papilloma arginabes previously treated with EDTA were activated t o nearly the same extent by MnC1,; furthermore, both preparations were stabilized by Mn++ions against thermal inactivation. I n addition, liver and papilloma arginases exhibited thc same Michaelis constant for arginine and the same dissociation constant for the competitive inhibitor, L-oriiithine. Liver and papilloma arginases were found to have the same p H optimum and serum of sheep immunized against purified hepatic rabbit arginase precipitated the arginase extracted from the rabbit papillomas. Also, contrary to Rogers and Moore (1963), antibodies directed against the papilloma enzyme were not found in the serum of papilloma-bearing rabbits. Finally, appreciable arginase activity was dctected in extracts of rabbit ear epidermis (though less than in papilloma extracts), and in tumors induced by repeated applications of dimethylbenzanthracene on the ear skin of the domebtic rabbit. Confirmation of some of the findings of Orth et al. (1967) have been reported by Sntoh and co-workers (1967). The latter investigators also found arginase activity to be present in normal rabbit skin and dimethylbenzanthracene-induced papillomas. They found that both hepatic and papilloma arginases required Mn++ for maximum activity and had the same Michaelis constant for arginine. Orth and co-workers (1967) have therefore suggested two alternatives to the hypothesis that a structural gene for arginase belongs to the virus. They suggested that either the virus “induces” the expression of genetic
information cLtrriec1 hy t l i c rahhit. or that tlie \.irii> ~IircIcbtlie pi.efer~ntial growth of particulnr strainh of epidermal cells with a higli arginase con tell 1- . I t is, thcrcfore, unc-lc:Li. :it this writing whether rabbit papilloma virus docs or does not induce a new arginase. It should be pointed out, however, t h a t the suggestion of Rogers and Moore (1963) concerning the biological significance of the enzyme remains of interest whether the enzyme is coded by the virus genome or represents a “derepressed” cellular cnzymc.
G. CONTINUED PRESENCE OF VIRALGEXOMEI N TRANSFORMED CELL? Although SV40 and polyoilia virus :ire dircctly responsible for the induction of tumors in animals an(l for thc transformation of cells in culture, a significant proportion of virus-incluced tumors and transformed cells are free of virus. Tumors originally containing demonstrable virus frequently lose it on subsequent transplantation. The tumor and transformed cells do not spontaneously produce virus nor can they he induced to do so by such treatments as X-irr:idiation, UV-irradiation, nutritional deficiencies, or superinfection with cytolytic or other oncogenic viruses. Nevertheless, there is good evidence t h a t a t least part of the viral genome is integrated in virus-induced tumors and transformed cells. First, i t has been shown t h a t virus-free tumor cells contain specific complementfixing T-antigens and transplantation antigens (Black et al., 1963; Habel, 1965; Pope and Rowe, 1964; Rapp e t al., 19648). Second, nucleic acid hybridization experiments have revealed t h a t virus-specific messcnger RNA’s are produced in transformed cells. For example, Benjamin ( 1966) has shown that a small fraction of the pulse-labeled RNA from virus-free polyoma-transformed cells was capable of hybridizing with polyoma DNA. Similarly, pulse-labeled RNA from SV40-transformed cells hybridized with SV40 DNA. No hybridization was observed between RNA of cells transformed by one of these viruses and DNA of the other virus, or between viral DNA :tiid RNA from normal or spontaneous malignant cells. Virus-free tumors and tiasue culture cells transformed by human adenovirus type 12 also contain new viral-specific tumor antigens (Huebner et al., 1963, 1965). Moreover, a large proportion of the rapidly labeled messenger-RNA fraction in the polyribosomes of the tumor and transformed cells was complementary to adenovirus type 12 DNA (Fujinaga and Green, 1966). I n the case of SV4O-transformed cells, conclusive evidence has been obtained that the entire viral genome is “integrated” during transforniation. I n 1962, Gerher and Kirchstcin reported t h a t SV40 could be recov-
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ercd by seeding SV40 tumor cells onto sellsitive indicator cells. Studies by Sabin and Koch (1964), which showed t h a t minute anlounts of virus roiild he r e c o w r d from tumors indiired by inoculation of 2 to 10 tumor c-clls i n t o : i ~ I i i I i h n i s t w s , I)rovitlc(l strong rvirlcnw that, W 4 O tlinlor c ~ > l l * were carrying thc cntirc SV40 gcnoioe. hIorc conclusive cvideiice was obtained by the studies of Black (1966), who isolated single-cell clones of SV40-transformed cells in the presence of antiserum and showed that these clones yielded infectious SV40. Studies by Tournier et al. (1967) showed by neutralization and immunofluorescence with antiviral sera that SV40 recovered from a clonal strain of SV40-transformed hamster tumor cells was similar t o the parental SV40. The virus recovered from transformed cells was also oncogenic for Syrian hamsters and transformed hamster cells in vitro. I n this author's laboratory, SV40-transformed mouse kidney cells were grown in the presence of susceptible monkey kidney cells (Dubbs et al., 1967). SV40 was readily recovered from: (1) 15 transformed cell lines ; (2) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and dBU; (3) 45 of 46 clonal lines isolated in the presence of antiviral serum; (4) 19 of 19 secondary clones isolated from two-clonal lines; and (5) dRU-resistant transformed cell lines. SV40 recovered from tramformed cells was shown to express in monkey kidney cells a t least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T-antigen, synthesis of viral DNA, induction of d T kinase, induction of D N A polymerase, and induction of host cell D N A synthesis. I n addition, sV40 recovered from the transformed cells induccd T-antigen, d T kinase, d C M P deaminase, d T M P kinase, and D N A polymerase in abortively infected mouse-kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells. Finally, band centrifugation and nitrocellulose chromatography experiments have dcinonstrated t h a t the molecular size and conformation of the DNA obtained from the SV40 strains recovered from transformed cells were indistinguibhable from that of parental SV40 DNA. A clue to the possible mechanism of the activation of SV40 in transformed cells was providcd by Gerber (1966) who observed that more virus is produced if UV-inactivated Sendai virus is added t o mixtures of transformed and susceptible cells. The inactivated Sendai virus promotes cell fusion; it is possible, therefore, that SV40 virus is produced by heterokaryons resulting from fusion of transformed cells with virussusceptible cells. Further evidence in support of this mechanism was obtained by Koprowski et al. (1967) who showed t h a t freezing and thawing
of SV40-tt~;itt~forttt~~c1 hrinian or ittonkcy cells or co-cultivation of vial)le cells of thcse culturcs with suuceptiblc primary cultures of green monkey kidney cells did not yield infectious SV40. Howevcr, when heterokaryons were produccd by treating transformed cells and primary GMK cells with UV-irradiated Sendai virus, infectious SV40 could readily be isolated. The fact that co-cultivation of the transformed and normal cells in absence of Seitdti virus did not result in the production of infectious SV40 is not surprising in view of thc extremely low proportion of heterokaryons formed after mixing these cells. Finally, Watkins and Dulbecco (1967) have demonstr:itecl that the reactiv:ition of SV40 virus in mixtures of tr;iitsformctl mouse (3T3) and susccptible monkey (BSC) cells trcatcd with TJV-inactivatcd Sendai virus occurrcd only in lieterokaryons. Probably, thc susccptiblc monkey cclls contribute to the heterokaryons a, factor lacking in transformed cclls. Thc factor could affect the translation of viral genes. This factor could be, for instance, a t R N A which recognizes a rate triplet; and thc SV40 DNA could bc viewed as carrying a mutation sensitive to a supprcssor prcsent in monkey kidney but not in mousc, human, or hamster cells. I n contrast to the results with SV40-transformcd cells, attempts to reactivate polyoma virus from polyoma-transformed cells have so far heen unsuccessful W a t k i n s and Dulbecco, 1967).
14. ENZYMES IN TISSUES INFECTED WITH Rous SARCOMA VIRUS Infections of chick embryo fibroblasts by avian sarcoma viruses lead to changes in cell shapc and increased capacity to make acid mucopolysaccharides (Erichsen ~t nl., 1961; Grossfeld, 1962; Temin, 1965). Howcver, infection did not alter the capacity of the cells to multiply, to niakc collagen or DNA, RNA, and protein. Thus, the incrcase in the rate of acid mucopolysaccharide production was not necessarily associated with a n altered growth rate. The acid mucopolysaccharide isolated from supernatants of infected and noninfected cells behaved, on electrophoresis and chromatography, in a manner similar to hyaluronic acid. This suggested t h a t enzymes functioning in hyaluronic acid formation might be enhanced by avian sarcoma viruses. I t was found that, beginning a t about 36 hours after infection, hyaluronic acid synthetase activity increased. At 5 to 6 days after infection, the enzyiiic activity reached a maximum about 8 times greater than t h a t of noninfected cclls. I n order to approach the question of wlic~thcr tlic iilclrchasc in Iiyuluronatc~syttthetiisc~rcwiltrcI f r m i productioti uf tl ilew enzyn~eui i i 1 c r e a 4 m ~ u u u tUI activatiuil uf a host cneyme, sevcrnl propertie4 of the rnzyme bysteins froni nonitifectetl and itifcctctl cell. n’ct’c htutlie(1. T l i c two cnzytiich hncl tlic s:tmc pTI optinturn
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and virtually identical mctal ion activation. No significant diffcrences were scen in thermal stability or in K , values for UDP-GlcNAc or UDP-GlcUA (Ishimoto e t al., 1966). The data, therefore, suggest that enhanced activity of a cellular hyaluronate synthetase occurs after infection with avian sarcoma viruses. It is not known whether the increased hyaluronate synthetase represents a secondary change due to alterations in the cell surface or whether the enzyme plays a direct role in changing the morphology and surface properties of the cell. It is of interest that transformation of 3T3 mouse fibroblast cells by SV40 or polyoma viruses and of human diploid fibroblasts by SV40 lead in each case t o a marked reduction in the rate of hyaluronic acid synthesis but not in collagen production (Hamerman e t al., 1965). It remains to be seen whether alterations in formation of hyaluronic acid and other mucopolysaccharides are related to dcvelopment of new transplantation antigens, loss of contact inhibition, or invasive properties of malignant cells. I n addition to hyaluronate synthetase, an increase in the activity of uridine kinase has been described 4-7 days after infection of chick embryo fibroblasts or the chorioallantoic membrane by avian sarcoma viruses. The increase in uridine kinase was not a result of general metabolic changes since control and infected cultures had similar levels of uridine phosphorylase, uracil ribonucleotide phosphatase, an d the kinases for adenosine, guanosine, dT, dA, dU, and dC (Gelbard et al., 1966; Rada and Gregusova, 1964).
I. FINAL COMMENTS The replication cycles of SV40 and polyoma virus are slow compared to most other animal viruses. Even during productive infection, the syntheses of cellular nucleic acids and proteins are not inhibited until late in the infectious cycle. Time is, therefore, available to initiate events which lead to transformation of a small percentage of the cultured cells. I n contact-inhibited cells productively infected with papoviruses, virus DNA is made, but in addition, there is an induction of cellular DNA synthesis. Preceding the syntheses of viral and cellular DNA’s, marked increases occur in six enzymes of the terminal pathway of thymidine metabolism. Virus-specific neoantigens are also developed. These include the T-antigens and a transplantation antigen. Later in the lytic cycle, after progeny viral DNA replicas appear, viral-capsid proteins are made. Also, rabbit papilloma virus induces a new arginase, and SV40 and polyoma virus modify acid mucopolysaccharide metabolism of infected cells. The papovavirus DNA contains genetic information for only about nine :ivcr:igc.-siarcl proteins. Two of tlw protcins migllt \.cry tvc.11 1)e
polypeptide subunits of the viral capsids. Yet, mwi enzymes and at least two neoantigens are induced. It appears that the information content of all of these proteins exceeds that of the papovavirus DNA’s. Thus, i t is unlikely that all of these proteins are direct products of viral gene action. It is more probable that one or more viral products derepress cellular genes so that some of the observed metabolic effects are indirect consequences of papovavirus infections. Aside from the capsid proteins, d T kinase and the DNA polymerase induced during Iytic infection may, perhaps, be direct products of viral gene action. There is stronger evidcnce that the T-antigens are coded by viral genes; these antigens might be regulatory proteins acting a t various levels of gene functions. Papovavirus infection is abortive in some tissue culture lines. Infectious virus, virus DNA’s, and capsid proteins are not made during abortive infections. BHK 21 cell culturcs also do not produce d T kinasc after abortive infection by polyoma virus. Mouse kidney monolayers abortively infected with SV40 do not synthesize a d T kinase or a DNA polymerase with altered properties. However, T-antigens and enzymes associated with DNA metabolism are induced and DNA synthesis is initiated. Synthesis of DNA is also initiated in rat embryo culturcs after polyoma virus infection. Some of the abortively infected cells are transformed. The transformation may be manifested by karyotypic changes, capacity of cells to grow in culture as established lines, loss of contact inhibition, alterations in colonial morphology, and increase in potential to grow as tumors in animals. The transformed cells are positive for the virus-specific Tantigens and have elevated levels of anabolic cnzymcs. It can be shown by nucleic acid hybridization experiments that virus-specific messenger RNA’s are made in transformed cells. Furthermore, in the case of SV40, production of infectious virus can be activated by fusing transformed cells with susceptible monkey kidney cells. These observations indicate that the entire SV40 genome is integrated in a t least some lines of transformed cells. Integration results in the repression of a number of viral genes essential for SV40 replication, but not of the genes controlling the formation of the T-antigen, the transplantation antigen, and the derepression of cellular enzymes functioning in DNA biosynthcsis. The molecular mechanisms underlying integration arc not known. It is by no means certain that thcrc is a linear insertion of the viral genome into the cellular chromosome, as in the case of the integration of phage lambda. Perhaps integration involves a semistable attachment of the viral DNA to a nuclear structure. This process would presumably change the conformation of the SV40 DNA to a noninfectious form. I n those instances where virus cannot be rescued from transformed
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(~clls,iiitcgratioir of niiitailt vii-ii* gonoinc~ (“rlcfcrtive Iysog~llh”) III:iy I)c suspcctcd. This I:ittor Iiyl)otIitLsih i h s i i I ) i r t h t to ~ ~ s ~ ) ( ~ i ~ i ~ i\ i~[t ~, ~i fi it ~~. i: lt tioil sincc: (1) the ii:iturr of the ~~ostulatctI gcnctic tlefccts caii bc investigated by studying. SV40-specific products after activation of virus replication in heterokaryons ; and (2) complementation techniques can perhaps be used to rescue “defective lysogens” after multicellular fusions. Not known a t this writing are the biochemical mecllanisms which lead to derepression of cellular gencs, initiation of cellular DNA synthesis, and integration of viral DNA. The regulatory processes whereby virus development is blocked are also not understood. Perhaps highly specific nucleases play a central role in the diverse events leading to integration, the initiation of cellular DNA replication, chromosome breakage, and karyotypic change. I n abortive infections, specific nucleases might also “restrict” papovaviruses in a manner analogous to thnt observed with restrictive bacterial cells. If there are DNA modification processes akin to those observed with bacteria, additional mechanisms of regulation would be expected. A highly appealing hypothesis which might explain some instances of abortive infection is that codons in the viral DNA’s make “sense” in cells where there is a productive infection but are “nonsense” in nonpermissive hosts. This hypothesis could apply in the case of abortive infections of SV40 in mouse kidney or WI-38 cells, polyoma virus in BHK 21 or rat embryo cells, and human adenovirus strains in GMK cells. The hypothesis focuses attention on the translation mechanisms in protcin synthesis and the possibility that there are specific adaptor RNA patterns in the various cell lines. Furthermore, the adaptor RNA dictionary of cells infected with oncogenic viruses might be modified in a manner analogous t o that observed after herpes simplex virus infection. Modifications occurring after either abortive or productive infections could indirectly affect cellular as well as viral protein syntheses. It is probably safe t o predict that the genetics of oncogenic viruses will be investigated with increasing vigor in the next few years. Studies of SV40-adenovirus “hybrid” populations have heightened interest in defective viruses and helper virus interrelationships. That this phenomenon is significant in viral oncogencsis is apparent from the fact that the Bryan strain of Rous sarcoma virus is also known to be defective and to require helper viruses for the formation of essential capsid components (Hanafusa, 1965; Hanafusa e t al., 1963, 1964s)~).It is also significant that mammalian cell lines can be transformed by certain strains of Rous sarcoma virus. Although these latter Rous sarcoma virus transformed cells do not spontaneously produce virus, they can be activated to do so by treating mixtures of transformed cells and susceptible chick embryo
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cdls with TTV-irradiated Sendai virus. Thus, rebcue of Rous sarcoma virus can bc obtained by niethods analogous t o those which are successful with SV40 virus (Vigier and Montagnier, 1966). Studies of conditional lethal mutants of T4, f2, and 4x174 phages have contributed substantially to the understanding of the regulation of gene function in phage systems. No doubt, the investigation of conditional lethal mutants of oncogenic viruscs will be iiicrcasingly employed in thc clucidation of the incclianisnis of turnor formation.
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Wildy, P., Russcll, W. C., and Horne, R. W. (1960). Virology 12, 204-222. Willems, M., and Penman, S. (1966). Virology 30, 355-367. Wilner, B. I. (IW) “A . Classification of the Major Groups of Human and Lowrr Animal Viruses,” 2nd Ed. Cutter Laboratories, Berkelcy, California. Wilson, R. G., and Badcr, J. P. (1965). Biochim. Biophys. Acta 103, 549-557. Winocow, E., Kaye, A. M.. and Stollar, V. (1965). Virology 27, 156-160. Wood, W. 13. (1966). J . Mol. Biol. 16, 118-133. Work, T. S. (1964). J . Afol. Biol. 10, 544-554. Wulff, D. I,., and Mrtzgrr, K. (1963). Virolngu 21, 400-500. Wyatt, G. R., and Cohrn, S. S. (1053). Biochem. J. 55, 774-782. Yahe. Y., Samper, L., Bryan, E., Taylor, G., and Trentin, J. J. (l(964). Sciencc 143, 4647.
Yohn, D. S., Gracr, .J. T., nntl H;wndig:.c~s, V. A . (1964). Nature 202, 88-883. Zimmc~rm:m,E. F., Hcrter, M., and D:irn(>ll,J. E. (1963). Virology 19, 400-408. Zirnmc~rman,S. B., and Kornhrrg, A . (11961). J . Biol. Chem. 236, 1480-1486. Zimmcrmnn, S. B., Kornhcrg, S. R., and Kornherg, A . (1962). J . B i d . Chem. 237, 512-5 18.
Zinder, N. D. (1965).Ann. lieu. Microbial. 19, 455-472.
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THE GROWTH-REGULATING ACTIVITY OF POLYANIONS: A THEORETICAL DISCUSSION OF THEIR PLACE IN THE INTERCELLULAR ENVIRONMENT AND THEIR ROLE IN CELL PHYSIOLOGY William Regelsonl Division of Medico1 Oncology. Department of Medicine. Medical College of Virginia. Richmond. Virginia
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I Introduction . . . . . . . . I1. Biological Evidence . . . . . . Embryogenesis . . . . . . . I11 Growth Control . . . . . . . IV . Radiation . . . . . . . . . V . Morphological Alteration . . . . . VI Cell Membrane . . . . . . . VII Surface Charge . . . . . . . VIII Adenosine 5'-Triphosphate and Polyanions . I X . Calcium . . . . . . . . . X Adhesion . . . . . . . . X I . Polpsacchnrides . . . . . . . XI1. Colloidal Effctcts . . . . . . . XI11. Hydrophilic Gels . . . . . . . XIV . Surface and Enzyme Activity . . . . XV . Enzyme Inhibition and Activation . . . XVI . Respiratory Enzymes . . . . . . XVII . Hyaluronidasc and G1ycosirl:isi~s . . . XVIII . Ribonuclease . . . . . . . . X I X . Deoxyribonucleasc . . . . . . X X . Polyphosphates . . . . . . . X X I . Lipase and Estcrasr Activity . . . . XXII . Clinical Antimitotic Sidr Effccts, and Clinical Antitumor Activity . . . . . . XXIII . Summation . . . . . . . . References . . . . . . . .
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I Introduction
Natural and synthetic polyanions inhibit tumor growth. and this inhibition is a reflection of a fundamental place for these charged inacroFormerly at the Dcpartmmt of Medicine A. Roswell Park Memorial Institutr. BiifTalo. New York . 223
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WILI,IAM REGELSON
inoleciiles in tlie control of norilia] and iibiioriiial cell divisioli. Thc purpose of this review, and another published previously (Regelson, 1968a) , is to disciiss the theoretical role of polyanions as growth regulators. Historically, thc :inticoagiilant, rffert of the native polyanion, hcparin, and its possible relation to calcium binding was valiicd for impeding the growth potential of tumors. Furthermore, heparin’s ability to alter the isoelectric point of protein provoked speculation (Fisher, 1930, 1936) that it interfered with the nutritional role of protein. Heilbrunn and co-workers (1948, 1952, 1955, 1956) likened changes seen in protoplasm to those seen in blood clotting. The antimitotic actioii of the anticoagulant heparin and related heparinoids and the alteration of the sulfomucopolysaccharides of eggs during fertilization (Runnstrom ct nl., 1949, 1950, 1956, 1957) lent weight t o these assumptions. The mitotic inhibition of Cimetopterus and sea urchin cggs by heparin and related compounds implied a physiological growth-controlling function for polyanions. The systematic study of anionic dyes beginning with Paul Ehrlich and his co-workers led to the development of trypanocidal dyes, such as isainine blue, which may havc shown clinical antitumor activity. Lallier (1956, 1957a,b,c, 1958, 1959, 1964) has shown that these dyes and related compounds produce animalization of sea urchin eggs, indicating a direct effect of these agents on cellular differentiation. In recent years, despite the loss of clinical interest in dye derivatives, attempts to develop synthetic or naturally derived substitutes for heparin (heparinoids) have produced a number of polyanions which possess antimitotic activity (Regelson, 1968a). Clinically and experimentally, these compounds produced alopecia, ulceration of the gastrointestinal (GI) tract, and osteoporosis leading to pathological fractures. This is not surprising as these side effects may reflect inhibition of proliferating tissue. Recent observations relate to the future pharmacological role of polyanions as other than agents of anticoagulant or lipolytic usefulness. Of importance is the observation that pretreatment of mice with these compounds prior to virus inoculation prevents the development of the infection by inhibiting virus replication through interferon induction (Merigan and Kleinschmidt, 1965; Kleinschmidt, 1964; Regelson and Foltyn, 1966; Regelson, 1967; Regelson and Merigan, 1967). This may be of clinical value, as recent evidence indicates that pyran copolymer, a polycarboxylic polyanion, induces interferon in man (Merigan and Regelson, 1967). In recent work, Lampson e t al. (1967) and Tytell et al. (1967) have similarly shown that the double-stranded ribonucleic acid (RNA), obtained from helenine, and Type 3 reovirus induce interfcrori and increase resistance t o in vitro and in vivo virus infection.
Similar activity has been shown for syrlt,hetic multistranded polynucleotide complexes (Field et al., 1967). A variety of homopolymers and COpolymers tested singly and in combination were inactive. Thus, one can consider polynucleotides as having similar activity to polycarboxylic or related polyanions. This work is also of interest in the area of immunology, as synthetic (Braun and Firshein, 1967; Braun, 1967) and naturally derived oligodeoxyribonucleotides enhance antibody response in rodents (Braun and Nakano, 1966; Braun and Firshein, 1967). Thcy can increase host resistance to a variety of grani-negative organisms (Braun, 1965), and in this manner resemble statolon, the interferon-inducing factor derived from Penicilliwm stoloniferu??z (Kleinschmidt et al., 1964 ; Kleinschmidt and Probst, 1962), which may be a phagclikc viral product infecting Pe,zicillium. I n addition, these polynucleotides have direct effects in culture where they modify bacterial and tissue growth (Braun, 1967). Most recently, it has been found by Braun (1967) that the synthetic polyanion, pyran copolymer, possesses not only interferon-inducing capacity (Regelson, 1966; Merigan and Regelson, 1967) but acts similarly to polynucleotides in its action in enhancing in vivo immunological responsiveness in mice. The mechanism for this action is unknown but may be pertinent to the action of these agents in controlling tumor growth. Stimulation of phagocytosis may be the prime mechanism through which both immunological and interferon polyanion effects are manifested, as we have observed that polynucleotides and synthetic polyanions stimulate phagocytosis (Munson et al., 1967). I n view of the recent emphasis on the role of macrophages in stimulating antibody production, the common pathway for stimulation of both interferon and antibody production by polynucleotides and synthetic polyanions may be through their enhancement of phagocytosis. Testing of heparinoid polyanions for antitumor activity led to systematic clinical trail of two agents: polyethylene sulfonate and a polycarboxylic divinyl ether maleic anhydride (pyran copolymer, NSC 46015). In addition, there has been related but independent research on the inhibitory effect of heparin and other anticoagulants on the iniplantation of tumor emboli and the growth of metastatic lesions (Regelson, 1968a). .4p:ut froai rlft.ct 011 cells, heparin :lilt1 rclatcd conipounds can I)c used in the cytstnllization and isoliltion of tobacco mosaic viruses (Cohen, 1942) , and, sut)mluently, it nunher of polyanioirs have been shown to inhibit viruses i n vitro am1 in vico. Tliere is much in the literature rcgarding the inhibition or enhancement of the virulence of viruses by
226
WILLIAM REGELSON
polyanions sonic of which, a s indicated 1)rcvioosly, may be host related, hit there is also a direct action of thesc agents on the virus (Vaheri, 1964). Polyanions also inhibit hormonal action, activate or inhibit enzyme action, pos~css cell surface effects, and interact with nucleohistone. Whatever range of activity and nicclianisni of action polyaiiions possess, this review seeks to present the biological basis for the place of these ncgatively charged niacromolcculcs in rclation to control of cellular proliferation. II. Biological Evidence
EMBRYOGENESIS Thc function of the sperm is not only essential for transfer of genetic information but may also be involved in egg surface and cytoplasmic transformation essential for cell division (Runnstrom and Immers, 1956). Development in the sea urchin egg begins with changes in the cell surface (Loeb et al., 1913). For example, the cortical granules of sea urchin eggs contain acid mucopolysaccharides with sulfuric acid residues (Monn4 and Slautterback, 1950; MomG and Horde, 1951) and are rich in sulfhydryl groups (Afzelius, 1956). At fertilization extrusion and breakdown of cortical granules can contribute to increased permeability to water (Ishikawa, 1954) and to the elevation of the fertilization membrane (Runnstrom, 1949). Similar findings have been found in frogs and fish (Raven, 1961; Aketa, 1954). Cortical granules are also found in thc sea urchin embryo, and these may be involved in the formation of ccllcementing substances (Afzelius, 1956). T h a t there is mctachromatic material in the egg substance of lowcr animals further indicates thc importance of polyanions when egg devclopment begins. Kelly (1953), Mulnard (1958), Messina (1954), Ilalcq (1960), Yamamoto (1961), Vasseur (1948, 1952), and Immcrs (1949, 1950) have shown that the jelly coat that surrounds the egg is a mucopolysaccharide esterified with sulfate with anticoagulant properties similar to heparin (Immers, 1949; Vasseur and Immers, 1949). A release of sulfate occurs on fertilization (Aketa, 1963), and jelly coat substance can inhibit fertilization (Runnstrom, 1950, 1952; Wicklund, 1954) in similar fashion to t h a t obtained with related polyanions: heparin (Gagnon, 1950; Hnrding, 1949, 1950, 1961 ; 1'i;irding and Harding, 1952; Hagstroiii, 195(iL), chititi clisulf:itr (Wicklund, 39543, clioiidroitin sulfate (Esping, l956), dextran sulfate (Hsgstroni, 1956a), polyanethol sulfoliate (liquoid) (Lallier, 1957a) , the polysaccharidcs of human blood group H substance (Harding, 1950; Wickluntl, 1954), and fucoidin
(H:iriIiiig, 1950; R i i i i n ~ t r o i i i :tiid Jlagstruiii, 1955; I
228
WILLIAM REGELSON
bearing Ehrlich ascites tiimor (Heilbrunn el uZ., 1957a,b) on direct intraperitoneal injection; these findings were duplicated by Lippmaii (1965). I n an extensive evaluation of Heilbrunn’s concept regarding the correlation between anticoagulation and antimitotic effects, Deysson and Longevialle (1962) studied the antimitotic action of a variety of anticoagulants on pea seedlings and garlic bulbs. Heparin and polyanetholsulfonate correlated well with their antimitotic effects, which were independent of calcium binding. I n further support for an antimitotic role for heparin and related compounds, tlie coagulation effect of cytolyzed cellular material on jelly coat substance of sea urchin eggs was prevented by heparin (Runnstrom and Monroy, 1950). Heparin prevented the restoration of the fertiliznbility of aged sea urchin eggs by adenosine 5’-triphosphate (ATP) or crystalline serum albumin (Runnstrom and Wicklund, 1949), and samples of heparin, fucoidin, and H substance algo prevented fertilization membrane formation (Harding, 1951). There were differences between sources of heparin, and heating the heparin to 119°C. for 10 to 30 minutes increased its activity. Pcriotlate oxidation completely destroyed antimitotic activity for heparin and for all the polysaccharides studied. The above observations are not surprising in t h a t Fisher and Schmitz (1933) showed that A T P could also counteract the anticoagulant effect of heparin, and McIntyrc and Braverman (1947) showed that heparin inhibited the contraction of actomyosin threads induced by ATP. I n support of this, using Amoeba discoides, Goldacre and Lorch (1950) following microinjection of A T P showed that heparin counteracted effects of A T P on cytoplasmic streaming. Heparin prevented protoplasmic gclntion in the ameba, Chaos chaos, which was reversed by the presence of zinc free insulin (Heilbrunn et al., 1958). As evidence supports a contractile role for mitotic spindle protein, i t is not surprising t h a t metaphase arrest follows exposure of ascites cells or plant cells to heparin (Lippman, 1957; DiMarco et al., 1958; Csaba e t al., 1961 ; Deysson and Longevialle, 1962). As mentioned earlier, it has been the contention of Runnstrom (1949, 1952; Runnstrom and Immers, 1956) that the cortical layer of the unfertilized sea urchin egg contains acid mucopolysaccharides which act as enzyme inliibitors and which control cell division. Similar polysaccharides are involved in the formation of the fertilization membrane in fish eggs (Dettlaff, 1957; Raven, 1961). Also the unfertilized rabbit ovum takes up little sulfate but there is good uptake in the surrounding
granular cells (Otlebiiltl, 1952) , a d tlierc iiiay bc siiiii1:tr iiiatcrid deposited on the egg surface. I n addition to proteolytic enzymes which activate sea urchin eggs, sperm lipases and lysolecithin have been invoked as activators of mitosis following fertilization (Monroy, 1965). The polyanionic jelly coat may have enzyme inhibitory properties which inactivatc or can sequentially activate these enzymes (see Section XV) . Supporting an inhibitory role for the polysaccharide surface layer of the unfertilized egg is the presence in sea urchin sperm of certain enzymes capable of dissolving the jelly coat substance (Lundblad and Monroy, 1950; Numanoi, 1953; Maggio and Monroy, 1956; Hathaway et al., 1960). In particular, polysaccharidases and hyaluronidases in sea urchin sperm (1,undblad and Monroy, 1951 ; Hultin, 1950a,b) dissolve thc jelly coat. For example, Arbacia sperm splits a sulfatc-rich fraction from egg fertilizin (Hathaway, 1959; Hathaway and Warren, 1961) which links dispersal of the jelly coat, the release of 36Sderived from sulfate groups, and an increase in acid formation. This increased acid formation may relate to the splitting off of sulfate groups from the mucopolysaccharides of the jelly and cortical layers by the sulfatases found in sperm (Numanoi, 1953; Runnstrom and Immers, 1956; Aketa, 1963). In addition, following fertilization, sulfated mucopolysaccharides are found in the perivitelline space as determined by staining and "SO0, incorporation (Inimers, 1960) , and it may he that similar polysaccharides are involved in movements governing the infolding of blastulae. A number of agents derivcd from sperm can precipitate the jelly coat substance and so start fertilization membrane formation. These include proteins extracted from sea urchin sperm (Metz, 1949). However, anionic compounds such as cellulose trisulfuric acid, sialic acid, and the anionic dye, suramin, which also precipitate jelly coat substance can behave similarly. These, however, also inhibit fertilization a t nonlethal concentrations (Metz, 1961). Labeled sulfate 35S is incorporated into the egg nucleus as well as the jelly coat. Histochemically, the red fluorescence of Acridine Orange is associated with the presence of RNA and mucopolysaccharides in the cytoplasm. The red fluorescence of the nucleus and nucleolus may be related to mucopolysaccharides (Austin and Bishop, 1959). This is supported by the work of Immers (1956, 1961a,b) who showed a relationship between the appearance of sulfated acid polysaccharitles and amino acid incorporation. Furthermore, metachromatic material is present not only a t the surface of the sea urchin or clam egg but within the cell in the form
of alpha ttnd bcta grwilules (Pasteels and Illulnard, 1957). The beta granules are localized in the vicinity of the nucleus during the resting stage, but during mitosis they can be found bound to the outer fibers of the spindle (Pasteels, 1958) or in association with the aster (Dalcq e t al., 1956; Mulnard e t al., 1959; Mulnard, 1958; Rebhun, 1958, 1959; Kojima, 1959a,b). I n Arbacia, a new set of beta granules is formed a t each mitotic cycle (Mulnard et al., 1959) and synthesis of nuclear material occurs simultaneously with synthesis of metachromatic granules (Dalcq, 1959). On release of cells from high hydrostatic pressure, Marsland (1958) has shown that cell division which accompanies pressure release is linked with simultaneous dissolution of the beta granules and the nuclear membrane. Pressure-induced furrowing in fertilized eggs can be induced independently of the stage of cell division, provided the nuclear membrane and beta-metachromatic granules are disrupted (Zimmerman and Marsland, 1960). Both the metachromatic alpha and beta granulcs of ascidian, annelid, and bivalve eggs are rich in dephosphorylating enzymes that split adenosine 5’-diphosphate (ADP) and ATP (Dalcq and Pasteels, 1963), and ATPase has been found in polar areas of the mitotic spindle of tumor cells (J. F. Hartmann, 1964). Then, too, Golgi elements have a histochemistry resembling the beta granules (Dalcq, 1959; Mulnard e t al., 1959) and, in view of the position of these granules in association with cell division (Dalcq and Pasteels, 1963), i t may be that this common metachromatic material plays a vital role in mitosis. This has been suggested by Kojima (1959a,b) who found that cleavage initiation was associated with these particles. Of additional interest, Kojima (1959h) found that metachromatic granules are associated with the formation of cilia and thus play an animslizing role in egg development. Important t o the initiation of ccll division, sperm contributes material necessary for aster formation (Hiramoto, 1963) and, in view of the possible role of beta-metachromatic granules in spindle formation, an analogous situation to aster formation seems to arise. This is the development of the acrosome filament extruded from the sea urchin sperm as soon as the arrosome touches the outer layer of the jelly coat (Dan, 1960). Of importance to changes in thc surface of the egg, gonadotropin results in the loss of the mammalian zona pellucida around the ovum (Katabcrg and Ilendricks, 1966). Equally interesting, hormonal control of ovulation may be preceded by the formation of depolymerizing enzymes which increase the solubility of ground substance leading to
follicular rupture (Gcrsli and Catchpole, 1949). Similarly, with fertilization, proteolytic enzymes are activated (Lundblad, 1954) and fucoidin, heparin, and other polyanions inhibit proteolytic enzyme activity. This enzyme inhibition by polyanions might be vital to the beginning of cell division in the egg, and, along this line, Monroy et ul. (1965) have shown that activation of ribosomes in unfertilized sea urchin eggs could be induced by careful tryptic digestion in similar fashion to fertilization itself. Fucoidin is also thought to inhibit mitosis by increasing the cross-linkage in the jelly coat substance and the cortex of the Unfertilized egg (Runnstrom and Hagstrom, 1955). Adhesiveness of the cell membrane can play an important part in morphogenesis (Holtfreter, 1944). An example is the production of primary mucopolysaccharides by developing mesenchyme and ectoderm in the sea urchin blastula (Immers, 1960; Motomura, 1960). These mucopolysaccharides may be critical to the phenomenon of gastrulation as there is evidence of increased adhesiveness, produced by these mucopolysaccharides, which governs the growth pattern of ectoderm (Okazaki et nl., 1962). I n the development of the embryo, Yamada (1961) and Tmmers (1960, 1961a,b) have presented evidence for the role of acid mucopolysaccharide secretion in blastopore invagination and the formation of the neural tube. I n further indirect support for the role of polysulfonated polyanions is the necessity of sulfate ions for the development of the sea urchin egg and larva. This is particularly true in the early blastula ,itage but it also occurs during gastrulation (Immers, 1961a,b). Iminers postulates that the regional uptake of sulfate, which correlates with amino acid uptake, may indicate a related role for sulfate incorporation in protein synthesis (Immers, 1961b). More specifically, polysulfates may play a morphogenetic role related to cell adhesion and organization in that sea urchin larvae raised in sulfate free sea water become radializcd or animalized with subwqii(wt failure of development of the blastula. Eggs reared in the cnvironnient of sulfate dcficiency show a decline ill respiration that is thought to be due to a rclcase of harmful substances from the vegetal region which may be detoxified by sulfate. Lack of sulfate brings about R decrease in protein synthesis in the :inimal portion of the embryo without, however, affcctitig 1)rotcin synthesis in the wgrt:il half (Ruiinhfroln e t ul., 1964). Tlicw workcrs poztulatc. that the al)wiiw of 1)rotcili I)ul\.hdfonzltes i t 1 tlw .nlfatr-tleticient enibryo may t e d t i t t ail iilcrtwt. 111 protcwlytic. e ~ ~ z y nactivity ~c’ Iwcauw of tlic f:xilurc of polyui~iousto in:ictivate these enzynicb I Lundblticl, 1954) which would lead to animaliza-
232
WILLIAM REGELSON
tion. Lack of sulfatc also results in abolishment of the capacity of mesenchyme in the vegetal half to show pseudopodal activity with distortion of morphogenetic activity. Support for a nutritional rolc for jelly coat polysaccharides may be found in transfer of oviduct polysaccharides into the egg. The nutritional role of acid polysaccharides on egg development has been more recently discussed by Humphries (1966) who confirmed the presence of acidic polysaccharides in the various layers of jelly coat in the urodele egg. Apart from studies regarding fertilization and morphogenesis ; in isolated cell systems, chondrocytes grown in monolayers transform into stellate cells, stop producing chondroitin sulfate, but begin synthesizing deoxyribonucleic acid (DNA) preparatory to cell division (Holtaer, 1964). Thus, chondroitin sulfate could be a growth regulator as well as a by-product. I n Protozoa, Amoeba transformation to a flagellate form is governed by the presence of cations which could be affected by the cation-binding capacity of polyanions (Willmer, 1961). Heparin can alter the antigenic surface characteristics of paramecia (Austin, 1959), and the animalizing effects of polyanions on sea urchin embryonal development are well known (Lallier, 1966) and discussed elsewhere. These and other observations may be pertinent to the presentation of McLoughlin (1963) , who has stressed the influence of mesenchyme on epithelial differentiation. I n this regard, in embryogenesis the importance of intercellular material, the bulk of which are acidic polysaccharides, is seen in the participation of surface coats of eggs and embryos in early morphogenetic movements, directed movement, contact inhibition, selective reaggregation, and the ability of injected cells to settle in appropriate host tissue. As indicated, the best-known intercellular materials are acid polysaccharides which are secreted in abundance in tissue culture (Grossfeld et al., 1957). A periodic acid-Schiff (PAS) -positive “collagenlike” material may be responsible for dermal induction of epidermis of chick embryo skin (Wessels, 1962). This material can pass through a Millipore filter, and McIloughliii (1963) suggests that the accumulation of acid mucopolysaccharides in the dermis may have cpithelial inductivc properties to give rise to hair, feathers, or other cutaneous structures. Supporting this, in vitro studies suggest that cclls may have nonspecific requirements for macromolcculcs which can lie sntisficcl by polyvinyl pyrrolitlonc or methylcellulosr. As :iii v x i i i r p l c of the clepcndcticc1 of cc4s on iirtcrccllular matrix, hlcloughliii (1963I presents the evidence of Oazello e t ul. (1960) of a fibroblast-free strain of mammary carcinoma cells that can survive and grow in vitro if supported by the addition of chondroitin sulfate. Wilde (1961) found that cxtracellular material obtained from
CROW‘fH-REGUI,ATINC, ACTIVITY 01’ POIAYANIONR
233
disilggregated pigment cells hen added to washed isolated undifferentiated cells accelerated their differentiation, and it is RlcLoughlin’s conclusion t h a t connectivc tis5iie mucopolyPacrharides h a w a maint c w \ i i c c :intl/or organiz:it ioiial cliff(wnti:it ing c , f t v t on rpitliclial cells. Thc role of oriciited surfaces guitliiig tlic locoiiiotioii of cells and nerve fibers (“contact guidance”) (Weiss, 1945) beems to throw light on differentiation and morphogenesis. This concept has been strengthened by the realization that cellular exudates influence the orientation and migration of their originators or that of their neighbors. Proteolytic activity which can destroy these guiding exudates modifies the response of cells to their guiding influence. This concept has been amply discussed by Weiss (1945, 1961) wherein he describes colloidal exudates which orient the growth of Scliwann cells and nerve fibers in tissue culture. The endogenous production of mucopolysaccharides by cells or its exogenous introduction may provide the colloidal exudates responsible for the above phenomena. In malignant growth, tumois invasiveness may similarly be supported by the orientation of connectivc tissue stroma relative to the tumor cells (Lcighton et nl., 1959). This has also been suggested hy the work of Balazs (1961), who, using the vitreous humor as a model system, described the importance of the molecular integrity of hyaluronic acid in maintaining the cellfree character of the vitreous body. In addition to excluding cells and acting as a molecular sieve for the passage of cellular materials, the character of the vitreous polyanionic gel influences the physical characteristics of thc cells migrating within or embedded in it (Balazs, 1960). Cations or radiation can dramatically alter the physical characteristics of these stabilizing hyaluronate gels. Similarly, exposing salamander limb buds to the basic dye, Toluidine Blue, results in altered patterns of rcgeneration (Csaba et al., 1962a, 1964a,b). Invertebrates respond similarly to charged macromolecules, e.g., following exposure of Plannria to graded concentration of heparin, increased cephaliaation (supcrnumerary eyes) occurred with alteration in regenerative capacity (Csaha et al., 1962a,h, 1964a,b). Based on the demonstration in tissue culture that acid mucopolysaccharides may be necessary for cell growth, it is Csaba’s concept that an increase of partial binding of these polyanions can lead to ahherant growth. It is also his thesis that epithelial cells and lymphocytes of the thymus are capable of taking on the characteristics of mast cells (Csaba et al., 1960a,c,d). Intact thymic tissue, following the in vivo administration of heat-treated heparin produces a continuous migration of metachromatic cells up to 20 days later (Csaba e t al., 1960b). This group believes that hcparin precursors or related heparinoids can he picked up by thymic cells after which they
234
WILLTAM REGELSON
are btored aiid syiitlicsize(l into inetachrorn:itic rnatcrial (Csaba et (fl., 1960c,d). They feel that this ib of importance t o ernhryonic devclopment in that inetachromatically staining embryonic tis5ue was found also to take up heparin. With maturity they claim only thc lymphatic organs and thymus function to pick up heparin in this rnaiiner (Csaha et al., 1960a,c,d). Thus it can be seen that polyanions are found in a changing but important relationship to cell division and the development of organizetl structure. In fact, one does not have to separate their controlling role in either area, but ratlicr cell division and morphogenesis are linked by polyanions. Ill. Growth Control
Polyanions are found in association with cellular proliferation in processes other than embryogenesis. Although increases in polyphosphates have not been associated with mitosis in mammals, in bacteria, the appearance of high levels of metachromatic polyphosphates is synchronized with logarithmic growth. Polyphosphates in bacteria may provide phosphate for synthetic purposes required for cell division (Sall et al., 1958). Also, in higher plants, changes in pectic polysaccharides are associated with germination and the appearance of acid pectic components as growth occurs (Gould e t al., 1965). In this regard, native heparin in minute concentrations has been found to stimulate the growth of Lupinus albus seedlings whereas the heparinoid, polyanethol sulfonate, was found to be a growth inhibitor (Macht, 1943). I n animals, tumor growth is associated with increase and/or changes in serum polysaccharides that are structurally important to native polyanions. This may be independent of inflammatory response (Almquist and Lausing, 1957; Tunis and Weinfield, 1962). For examplc, homogenates of Walker 256 tumor produce hexosamines from hexose-6phosphate and glutamine (Kizer and McCoy, 1959), and tumors arc capablc of i n vitro synthesis of hyaluronic acid chains from uridinc diphospho-N-acetylglucosamine and uridine diphosphoglucuronic acid (Glaser and Brown, 1955). In clinical tumor effusions, acid mucopolysaccharides are elevated regardless of the tumor (Castor and Naylor, 1965). This may be dependent on the anatomic locus of the tumor as evidenced by /3-glucuronidase and p-N-acetylglucosaminidase increasing up to tenfold in Ehrlich T2146 and 536 tumors grown subcutaneously as compared to growth in the ascites from (Carr, 1965). Increases in pglucuronidase have been found in regenerating tissue (Kerr and Levvy, 1951) and Carr feels that the invasiveness of these subcutaneous tumor
implants is related to the increase in glycosidases which break down connective tissue mucopolysaccharides. The observation of a contrasting slow growth of subcutaneous Ehrlich tumor in Strong A mice as compared to CAF, mice led t o extraction from the slow-growing Ehrlich tumor of a polysaccharide t h a t reduced the cytoplasmic viscosity of Ehrlich tumor cells and produced mitotic arrest (Nishimura et al., 1958). The effect on cytoplasmic viscosity produced by this polysaccharide was similar to what had been observed earlier on exposure of cells t o ethylenediaminetetraacetate (EDTA) or colchicine (Nishimura et al., 1955). In normal growth, ail increase in the uptake of Toluidine Blue in granulation tissue of rats up to the ninth day of wound healing was found by Balazs and Holmgren (1950), and early aqueous extracts of granulation tissue have growth-promoting effects on fibroblasts in vitro. This has also bccii seen for hyaluronic acid (Balazs et al., 1951). However, granulation tissue extracts more than 5 days old showed the strongest metachromatic dye binding and could inhibit cell growth in tissue culture. A polysaccharide sulfuric acid ester fraction increased both the number of normal and abnormal mitoses of fibroblasts in tissue culture (Peters and Matis, 1961). Similar findings have been obtained by Csaba et al. (1964) for normal thymus, liver, kidney, and heart cells in tissue culture. In this regard, tlie situation is confused a s hyaluronic acid has been found by Csermley and Curri (1958) to block wound healing, but systemic pretreatment of rats with hyaluronic acid increased the granulation reaction (Curri and Tischendorf, 1959). Cutaneous wound healiiig improved following local application of a polyphosphate aiitienzyme and anti-inflammatory agent ( Wechselberger, 1960). Of related interest is the observation that wound healing was blocked by local injection of uncouplers of oxidative phosphorylation, with a rise in hexosamines. This inhibition was reversed with local administration of N-acetyl-D-glucosamine which encouraged the formation of collagen and sulfate incorporation into collagen (Roden, 1956). N-Acetyl-Dglucosamine-trented animals also showed an increase in the number of mast cells within thc wound (Rcynolds et nl., 1959). As indicated earlier, depending on tlie pcriod of extraction following wound healing, metachromatic material derived from wounds can have both growth-promoting and inhibiting effects. This finding is pertinent t o the observation that single injections of 100 pg. of heparin can result in a n increase in hepatic mitotic activity and D N A synthesis in rats killed 2-2 lrorirs l'ollo~ving Iiq)arin iiij(bctioii (Ziiniiirrni:tri : i 1 1 ( 1 Celaezi, 1961).
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Heparitin sulfate and p-heparin showed less activity, whereas chondroitin sulfate, chitin sulfate, and polystyrene sulfonate had no effect. Effects varied with the brand of heparin used (Zimmerman, 19641, and no effect on the regenerating rat liver other than an increase in fat content has been found (Bianchini and Gilberti, 1953). Urodele limb bud regeneration is associated with polysaccharide synthesis which may provide structural support for cell migration (Schmidt, 1962), and Csaba e t al. (1964a,b) has found that the basic dye, Toluidine Blue, which combines with acid polysaccharides, distorts limb bud regeneration in salamanders. Using a breast tissue epithelial tumor, Ozzello e t al. (1960) found that higher-molecular-weight hyaluronic acid or chondroitin sulfate promoted proliferation in tissue culture. Lower-molecularweight fractions were without effect, and the anionic character of these supporting macromolecules was an important factor as growth was not as well maintained by higher-molecular-weight polyvinyl pyrrolidone. Impairment of the tensile strength of wounds by heparin was not found by Bcndix and Nechcles (1949). However, the direct application of heparin interfered with superficial wound healing in the guinea pig (Klingenberg, 1952). A similar finding was obtained by Isidor (1954) who found that direct application of heparin to superficial wounds blocked the utilization of glycogen and interfered with mitosis and resultant fibrous tissue formation. This action of heparin was prevented and reversed by methionine. Of related interest, methionine (Dubin, 1959) and other sulfur-containing compounds are needed for the synthesis of polyamines in bacteria. If this were true, then in wound healing, the polyamincs synthesiecd could block the inhibiting action of heparin. Similar observational support for the block to scar formation in guinea pigs by heparin is that it may be similar to that produced by ascorbic acid deficiency (Ohlwiler e t al., 1959). Heparin given to vitamin C-deficient guinea pigs also hastens the onset of scurvy and shortens survival (Ohlwiler e t al., 1959). In these animals, there was a decrease in the tcrisile strength of wounds and fibrous tissue formation around plastic implants. Heparin also decreases fracture healing in normal animals and these authors postulate that this action of heparin is due to an ascorbic acid antagonism. I n contrast, wc arc confused by thc observations of McCleary e t al. (1949) that heparin in the dog increased the rate of wound healing. This was confirmed hy Fentori and West (1963) who observed that heparin, and particularly histamine preceding heparin, increased the rate of wound healing. Wound healing and tensile strength were impaired with prior administration of the polycationic heparin antagonists, 48/80 and polymixin R. Similar ol)scrv:itiotis wcre also made following reserpine
GROWTH -REGIJIAATING ACTIVITT 01’ I’OLYAN 10sS
237
and serotonin administratioii, and they postulated that the pronlotiotl of wound healing by heparin may be mediated by blocking the antigranulation action of corticosteroids. This may be pertinent to the observation that the exogenous introduction of nicotinamide adenine dinucleotide (NAD) decreased the size and number of granulomata formed by subcutaneous inoculation of carrageenin in rats. The NAT) also reversed the inhibitory effect of cortisone on the respiration of rheumatoid synovial tissue formed by carrageenin-induced proliferation in the knee joint of the rabbit (Luscombe, 1963). Carrageenin stimulates granulation response (Stoloff, 1959), and the answer to these contradictions may be found in studies such as have been done by McCandless (1965) of the structural character of polysaccharides that promote connective tissue growth in rats. She found that A-carrageenin has a potent stimulating effecct on the production of mammalian connective tissue. Similar effects were seen for agar, agarose, furcelleran, mucilage, and polygalacturonic acid. No effects were seen for chondroitin sulfate, fucoidin, pectin, amylopectin, sulfated dextran, laminarin, and other polysaccharides. Sulfation was unnecessary and all compounds that stimulated connective tissue response were essentially linear polymers of galactose. Unfortunately, no studies of the possible enhancing or blocking action of heparin or heparinoids were made. Similar studies have found that systemic high-molecular-weight levan and dextran polymers interfere with wound healing and inflammatory response by blocking granulation tissue through interference with the growth of capillaries and fibroblasts. Low-molecular-weight polymers did not behave this way, so that polysaccharide polymers can inhibit as well as produce local and systemic inflammatory response (Wolman and Wolman, 1956; Shile et al., 1956). The tolerance of patients to heparin is increased during injury and repair secondary to burn trauma in man (Robinson and Hamilton, 1953). This heparin tolerance remains elevated during keloid formation, and the authors postulate that heparin is a controlling factor in fibroblastic proliferation and scarring via its release from the mast cell. In view of the importance of hormones as growth regulators, the following observations are of interest. Following thymectomy, cortisone increased fornialin-induced arthritic reactions in rats (Csaba e t al., 1962). Becausc of the presence of mast cells in the thymus which could give rise to heparin or related polysaecharides, thymectomy might modify inflammatory reactions independent of immunological phenomena via loss of mast cells or mast cell precursors. Cortisone inhibits the mitotic activity in mouse epidermis possibly
bccausc of interference with carbohydrate metabolism (Ghadially and Green, 1954) and decreases and delays skin carcinogenesis (Engelbreth-Holm and Asboe-Hansen, 1953) with degranulation of mast cells. Hyaluronic acid and chondroitin sulfate content of the skin of diabetic rats decreased, while heparin content remained high and fornlalin restorecl the mucopolysaccharide synthetic activity of the diabetic animals (Schiller and Dorfman, 1963). Pertinent to this, Heilbrunn et al. (1958) found heparin to antagonize insulin effects. Heparinlike polysaccharides (Szirtnai, 1962 ; Homburger et al., 1963) are found in menstrual blood, but their irnmediate role other than anticoagulation has not been ascertained, although Szirmai (1964) suggests a heparin-related control of menstruation. Estradiol increased the in vitro incorporation of gluc~samine-l-'~C into acid mucopolysaccharides of normal and neoplastic tissue. Increased incorporation into hyaluronate and chondroitin sulfate was found but not into heparin, and the responsiveness of breast cancer to estradiol was related to increased glucosa~nine-l-'~Cuptake (Sinohara and Skypeck, 1964). Endometrial stroma, oviduct, cervix, and vagina contain metachroniatic substances that bind radioactive sulfate and which are stimulated by estrogen (Zachariae, 1958). Chondroitin sulfate and hyaluronic acid have been isolated from bovine Graafian follicles, and the molecular weight of these mucopolysaecharides decreases with follicular maturation. Prior t o rupture, a substance appears which can produce further decrease in molecular weight (Jensen and Zachariae, 1958). This may relate to the disappearance of the zona pellucida about the ovum of baboons under the influence of gonadotropin (Katzberg and Hendricks, 1966). Hypothyroidism increases the concentration of hyaluronic acid in skin and decreases that of chondroitin sulfate-a situation reversed by thyroid hormone (Schiller e t al., 1962). Cortisone depresses synthesis of chondroitin sulfate and hyaluronic acid (Dziewiatkowski, 1964 ; Schiller and Dorfman, 1957). Parathyroid hormone (Guri and Bernstein, 1964), insulin, and growth hormone (Bernstein et al., 1961) increase the uptake of 3BS and glucose-"C into rachitic rat cartilage mucopolysaccharide in uitro, and the effect of hormones on the turnover of polysaccharides in connective tissue depends on the hormone used and end-organ assayed (Dziewiatkowxki, 1964). All this is pertinent to an early paper by Gersh and Catchpole ( 1949) who discussed the hormonal relationships of ground substances in normal and tumor growth. They claim that ground substances in and around rapidly growing tumors become water-soluble and the hyperplastic fibroblasts are rich in secretory granules. In slow-growing tumors,
the ground substance is iiot water-soluble and the above is not seen. This was associated with collagenase activity in human and rat tumors. They postulated that the growth and spread of neoplasms “may be related to enzymes which depolymerize the ground substance a t the site of invasion.” I n this regard, Walker carcinoma (Grossfield, 1961) and Rous sarcoma give rise to large quantities of hyaluronic acid (Pirie, 1942) and heparin (Harris e t al., 1954) which Sylven (1945) felt may provide a nutritive source for tumor growth. I n support of this, tumor invasiveness of cervical cancer was thought to correlate with increased sti-omal metachromasia. In addition, depolymerization of hyaluronic acid by hyaluronidasc correlated with increased growth activity for Walker c:trcinoina ( G ~ ~ o s ~ f i c1961 l d , ). There is an increase in acid nmcopolysarrliaride production in mammary gland tumors as conqmrcd to normal breast (Gabuniia, 1964), and Csaba et al. (1961) felt that mast cell reactions to tumor growth reflect their ability to pick up polysaccharidcs necessary for tumor proliferation. Support for this may be provided by the observation of Sylven (1941), Laskina (1961), and Ksmei and Nagoya (1964) that the invasiveness of cancer cell lines into normal Fponge matrix explants was governed by the character of the connective tissue in the matrix relative to the tumor cells (Leighton e t al., 1959). In further support for a growth-controlling role of matrix precursors, wglucosamine prevents neural retinal cells from reaggregating following tryptic separation (Garber, 1962), arid mice bearing Sarcoma 37 treated with D-glucosamine showed decrease in the growth rate of the tumor (Quastel and Cantero, 1953). There was a tumoricidal action of Dglucosamine within 2 hours of drug injection. Their concept of inhibition of tumor by D-glucosamine related to a postulated competitive decrease in the availability of ATP for phosphorylation with sugar (Harpur and Quastel, 1949). Glucosamine inhibited tumor cell glycolysis and increased survival of tumor-bearing mice was obtained by Lindner (1961) following injection of glucosamine and N-acetylglucosamine in mice bearing Ehrlich and Yoshida ascites tumor. Lesser effects were obtained with hyaluronic acid, heparin, arid chondroitin sulfate. Mucopolysaccharides obtained from bovine liver inhibited the development of intraperitoneiil chloroleukemia in rats following intraperitone:il injection. Cliondi~oitin sulfate was inactive whereas glucosaniinr iiict’cn~ccl sui,viv:il (\Basustjrriiu et ol., 19641. In contrast, adniini.;tratioii of gluc.utmic acid and glucosarnine to mire bearing sutwutimcoub 13hi~Iicll :isrites tuinor I w u l t t y l i n stiortenetl survival (Csaba e t nl., 1960n, 1961). No measurements of tumor growth are reported, but this is consistent with the concept that connectiw tissue
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WIL1,IAM REGELSON
polyanions or related compounds may control cell growth. Of pertinence, as mentioned previously, Csaba e t al. (1964a,b) feels that heparin is essential for cellular proliferation and that its removal by cationic binding results in inhibition of cell division, whereas its presence in abnormal relationship leads to mitotic abnormalities in tissue culture. I n further support for the role of polyanions in cellular proliferation, leukocytes are aggregated by heparin (Wilander, 1938), and, as discussed earlier (Lindner, 1961), glucosamine and galactosamine inhibited anaerobic glycolysis and oxygen uptake in guinea pig and rat polymorphonuclear leukocytes (Alonso, 1959). Despite this, or because of it, intravenous heparin, chondroitin sulfate, and other sulfated polysacchrides inhibited the mononuclear cell exudation that follows subcutaneous injections of albumin into rabbits. There was no correlation with anticoagulant effect or protamine reversal (Miller and Page, 1963). This may relate to the action of cationic proteins which are present in leukocytes and which produce inflammatory reactions (Janoff and Zweifach, 1964) ; and rabbit polymorphonuclear leukocytes contain acid mucopolysaccharides within cytoplasmic granules (Fedorko and Morse, 1965). The effect of polyanions on patterns of normal cellular growth may be seen in that native heparin produces a marked lymphocytosis as well as neutrophilic leukocytosis following its intravenous administration (Cronkite e t al., 1962; Jansen e t al., 1962). Lymphocytosis occurs via an increased input of lymphocytes into the blood from the thoracic duct. Interesting in this regard is the breakdown of endothelial integrity following excessive heparinization (Yoshimura and Djerassi, 1962). There may be a decrease in the mucopolysaccharides of leukemic cells (Notario and Nespoli, 19611, but a panleukocytosis in rats is caused by heparin, and heparin antogonizes the lymphopenic action of hydrocortisone (Paluska and Hamilton, 1963). Also pertinent is the observation that the synthetic polyanion, ammoniated ethylene maleic anhydride (E/MA\ of mol. wt. 20,000-30,000, causes a marked normocytic anemia [30-600/0 reduction in red blood cells (RBC) ] with a significant increase of circulating nucleated red cells [up to 21 per 100 white blood cells (WBC) ] without a corresponding increase of reticulocytes. The immediate effect of this polymer was to block rcticuloendothelial system (RES) carbon clearance (Old, 1959), but the peripheral wliite 1)lood count i l l t l i c w anininls increased u p to 140,000 per cubic millimeter (iininature and atypical cells up to 20%) in dogs and rats (Mihich et al., 19601, along with an increase in blood viscosity. This was not surprising because macromolecules have been shown to induce lcukocytosis or leukopenia dependent on the branched
OROWT€I-RE(:TILATIN(;
ACTIVITY O F P O I J Y A N I O N S
241
or noribranched polar or nonpolar character of the polymer studied (Weiderheim et nl., 19531 ; and Math6 e t nl. (1963) found that a polysaccharide from visrIiiii alhiiun stiniulatep timt rophilia in chronic lymphocytic Icukrmia. Sitnil:ir 1c.ukocytir-stimu1:tting wtion has h e n seen for the anionic dye, trypan blue, which in similar fashion to EJMA COpolymers induces anemia and changes in blood proteins. On prolonged administration, trypan blue induces sarcomas (Gilman, 1956), a property which is thought to relate to its prolonged storage and stimulation of the RES. Heparin (Monkhouse, 1954) is cleared by the RES and vinyl ether and styrene maleic anhydride, antitumor and antiviral agents produce morphological stimulation of the RES along with evidence of storage within these cells (Regelson, 1966, 1967; Merigan and Regelson, 1967). IV. Radiation
Despite heparinoid-induced loss of hair on prolonged administration, there is metachromatic material which may be a sulfated mucopolysaccharide present in hair follicles during periods of intense proliferation (Sylven, 1950), and repeated local injection of sulfated mucopolysaccharides such as chondroitin sulfate B and heparitin sulfate results in an increased stimulus to pigmented hair growth in the shaved rabbit (Meyer et al., 1961). Complexing with protamine to produce a depot preparation did not prevent this effect. Based on this, the intracutaneous administration of synovial mucopolysaccharides and sulfate-enriched gelatins prevented radiation-induced epilation following 550 r whole body radiation in mice (Bacq, 1962). Heparin alone was inactive, but the blood of heparinized dogs was highly active in accelerating the regeneration of hair following radiation. This is of interest in view of the effects of radiation involution on glucuronide excretion (Chiriboga, 1963) and the block by radiation to the replacement of mucopolysaccharidcs (Gerber et al., 1962). In view of local radiation protection afforded by sulfated polysacrharides, the observations that minute concentrations of heparin in spring water can prolong the life-span of the arthopod, Daphnia magna (Schecter, 1950) and that the longest viability of marine eggs is associated with greatest electronegativity (Dan, 1947a,b) are of interest, as radiation affects the overall process of aging. The role of heparin release following radiation-induced mast cell breakdown is controversial (Allen and Jacobsen, 1947; Laurence e t al., 1948; Engelberg, 1963) and further study is necessary. However, heparin breaks down vascular barriers and red cells appear in lymphatics in similar fashion to lethal radiation effccts (Yoshimura and Djerassi, 1962).
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WILLIAM REGELRON
Of greatrr pertinence KIM^ he the obbervation regarding the loss of viscosity of bovine vitreous humor (high in hyaluronic acid and collagen) followiilg roentgen radiation, which may imply radiation effects in carcinogericsis or cell death independent of that usually ascribed to effects on nucleic acid (Balazs, 1960) . Similarly, ultraviolet radiation depolymerizes hyaluronic acid and alters the biological properties of heparin by decreasing its anticoagulant properties while increasing its reducing power (Balazs e t al., 1959). Exogenous copper and/or ascorbic acid may behave similarly (Balazs, 1960) although the pertinence of this to radiation has not been established (Pirie, 1942; Balazs, 1960). As indicated, radiation with ultraviolet light, electrons (Balazs e t al., 1959), or X-rays (Ragan et al., 1947) produccs alterations in the physical properties of hyaluronic acid, hyaluron sulfuric acid, and heparin. This is manifested by reduction of molecular weight, changes in cationic dye binding, and anticoagulant activity which can occur as a result of depolymerization of the large-molecular-weight polysaccharide molecules into lower-molecular-weight dialyzable compounds. I n view of the sensitivity of this stromal material to irradiation and the heparin-containing, mast cell, cutaneous reactions that accompany radiation and carcinogenesis, investigation of the changed physical state of the ground substance should be looked for i n association with radiation or chemicalinduced carcinogenesis. I n recent studies, pretreatment of mice with a polycarboxylic vinyl ether maleic anhydride (NSC 46015) copolymer did not protect mice from whole body radiation a t the LD,, dose level despite splenomegaly and morphological RES stimulation induced by the drug. Radiation effects in relation to prior stimulation of the RES by these polyanions is still under study (Regclson, 1968a). V. Morphological Alteration
Alterations in the surface of tumor cells have been suggested to be responsible for the developmcnt of malignant change (Pardee, 1964; Kalckar, 1964a,b). Tumor cell membranes possess quantities of carboxylic sialic acid which has been though to be associated with the greater electronegativity of the tumor cell as compared to the normal. Sialic acid may also be responsible for K+ ion transport in cell membranes (Click and Githens, 1965). Neuraminidase decreases the net negative charge on tumor cells and allows for easier deformation of the cell surface (L. Weiss, 1965a). I n view of the possible importance of negatively charged polyanions on the surface of the cell, a study of their action on the developing egg may give insight into their mechanism of action. Lallier (1956, 1957c, 1958, 1966) has studied the animalizing or radi-
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alieing effects on the sea urchin embryo (the formation of a motile hyperciliated surface) of agents having in common sulfonic (Evans Blue, chlorozol sky blue, Niagara Blue) or polycarboxylic (uranine, chrome violet, rose Bengal) acid groups, Animalizing agents interfere with fertilization and embryonic development. This morphogenic activity is independent of the number but is related to the position of acid groups on the molecule (Lallier, 1958). There is a correIation between the animalieing effects of dyes; e.g., Evans and Niagara blues, with their affinity for albumin. Related henzene polyanionic compounds such a s p-toluenesulfonic acid were inactim, hut the napthalene derivative (LalIier, 1958), suramin (Lallier, 1956, 1957a, 1958), lignin sulfonates ( Lallier, 1958), and polyanethanol sulfonate (Lallier, 1957b) wcrc iimong the most active animalizing agents studied. Lallier postulates that the most active animalizing agents have an affinity for the -NH2 groupings in protein. The absence of reactivity bctween the -SH groups and the sulfonatcd aniline dyes (Wilson and Wormall, 1949) suggests that the availability of sulfonic or carhoxyl groups for combination with -NH, and sulfhydryl groups is the factor that leads to aninialization of the embryo. It is possible that ectoplasmic granules in sea urchins, because they are structurally involved with cilia formation (Kojima, 1959b), are also involved in animalization as metachromasia present in these granules may reflect the presence of acidic (heparinlike) polyanions. The anionic dyes differ from the action of fueoidin on the unfertilized sea urchin eggs in that animalizing agents diminish the viscosity of fibrillar protein extracts tlerived from the embryo, whereas vegetalizing agents increase the viscosity of these extracts (IAIiw, 1964). This is directly applicable to Heilbrunn’s concept of sol-gel alteration of protoplasm resulting in inhibition or activation of mitosis. I n this regard, the action of carcinogens in decreasing the surface rigidity of Chaetopterus protoplasm has been used by Heilbrunn (1956a,b) to explain Haddow’s paradoxical observation of the carcinogenic and careinolytic properties of radiation or chemical agents. Heparin and related polyanions can inhibit mitosis in animal eggs or tumors, but evidcnce that the paradox is applicable to polyanions is as follows: the promotion of Rous sarcoma growth by the anionic dye, Aliznriiie sulfate, while virus tumors are inhibited by other polyanions ; the growth-inhibiting versus the animalizing influence of polyanionic dyes and heparinoids on the developing sea urchin embryo (Lallier, 1956, 1957a,b,c, 1958) ; the growth-promoting as well as inhibiting action of nictachi-omatic polysaccharides on wound healing (Balazs rind Holmgrcn, 1949, 1950) ; a n d the ehnnge of eniI)ryonic cliontlrocytrs from rlion(lroitin >ulfntr pro~lucci~sto stellate
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WILLIAM REGELSON
dividing cells with little capacity to synthesize collagen when removed from their cartilaginous matrix (Holtzer, 1964). Raven (1963) suggests that the character of the egg cortex can be altered by follicle cells which support the oocyte against the wall of the gonad. This effect imparts a morphogenetic control to areas of the egg surface, In addition, there is the influence of the products of cells on their own development or on the development of neighboring cells. This is seen in the influence of extracellular material on the differentiation of embryonic cells. For example, various workers (Holtfreter, 1946; Curtis, 1958) have observed that, on disaggregation, gastrula cells give off a material which is referred to as extracellular matrix (ECM) or cement (Moscona, 1960). One of its constituents is ribonucleoprotein. Wilde (1961) who has collected matrix in fairly concentrated form from calcium-deficient disaggregating cells, returned an appropriate quantity of calcium and observed the difference in behavior between washed embryonic cells cultured with and without matrix added to the medium. Cells in small groups differentiated more frequently when suspended in matrix. The association of polyphenols with sulfomucopolysaccharides may also be pertinent to changes in cell surfaces because of the tanning action of polyphenols on the cell surface (MonnB, 1961). Surface-active agents have the opposite effect and can promote dye penetration without lysis of the cell (Hodes e t al., 1960). VI. Cell Membrane
Although direct injection of polyanions into ascitic fluid was without effect on survival of mice bearing ascites tumor, reduction in the quantity of cells present (Regclson, 1968a; Lippman, 1957, 1965) and alteration in cell morphology leading to cell death have been seen (Belkin, 1963). These alterations produced by polystyrene sulfonate are similar to what has been observed for the direct intraperitoneal administration of higher plant polysaccharides into mice bearing Yoshida and Sarcoma 37 ascites cells (Belkin et al., 1959; Belkin, 1963). This was associated with marked swelling of Sarcoma 37 cells and vacuole formation, and, depending on the molecular weight of the polymer used, is a hostmediated reversible rcnction rcquiring I to several hours to develop. The sensitivity of tumor cell lines differed in that, in contrast to Sarcoma 37, L-1210 was unaffected. Similar patterns of vacuoliaation of ascites tumor cells associated with blebbing of the cell surface have been seen with in vitro administration of autonomic blocking agents (Belkin et al., 1962) and compounds which i m c t with pi’otciii-sulfhytlryl groups by alkylntion, oxida-
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tion, or nicrcaptidc formation (Belkin aiid Hardy, 1961). I n contrast to ascites tumor cells, isolated normal cells fail to show bleb formation on exposure to compounds reacting with -SH groups, indicating a possible difference between normal and malignant cell lines. Vacuolization of human aortic intimal and HeLa cells has also been produced by polyvalent cobalt salts secondary to complexing with RNA or mucopolysaccharides within the cell (Lazzarini and Weismann, 1960). Liquoid (polyanetholsulfonic acid) or sodium salt of polyvinyl alcohol polysulfuric acid ester (PVAS) induces phagocytosis of normal platelets. The reaction of these polyanions in normal serum induces morphological alteration in leukocytes indistinguishable from those seen in the lupus cell phenomenon. The fraction of serum responsible for “lupus” cell formation is a 7-glohulin, which is distinct from the fraction responsible for phagocytic promotion. This lupus protein induces marked vacuolization of the cell sufficient to displace the nucleus (Inderbitzin, 1963). Here changes resemble those induced by polyethylene or polystyrene sulfonate on ascites tumor cells. Acid mucopolysaccharides are found within polymorphonuclear leukocyte granules (Fedorko and Morse, 1965), and it has been postulated by these authors that hyaluronate in leukocyte granules may control the availability of lysozomal enzymes t)y affecting lysozomal integrity. Nutritionally, heparin can block the uptake of lipoprotein from isolated human arterial intimal cells (Lazzarini-Robertson, 1961). If specific lipoproteins are needed for cell division, this could result in inhibition. Pertinent to the above, trace metals, particularly the cupric ion, degrade serum lipoproteins (Ray e t al., 1954). This may relate to the formation of lipoperoxides, but regardless of mechanisms, the presence of Cut+ degrades lipoproteins. Chelators, or proteins that bind cupric ions protect, and, since polyanions possess an affinity for Cu++(Wall and Gill, 1954; Smets, 1962), this role a t the cell surface may be a protective one against the oxidizing denaturing effect of metallic ions on the lipoproteins of the cell membrane. Conversely, the entrance of copper-carrying polyanionic complexes within the cell could be damaging to cellular integrity. The anionic detergents protect serum albumin from denaturation (Epstein and Possick, 1961 ; Kondo, 1962; Markus e t al., 1964; Markus, 1965). This protection is afforded only in the presence of positively charged, lysine rcsidues, and the protective effect may originate in crosslinkage of the protein by the detergent molecule (Markus et al., 1964). Similar activity of polynnioiis coulcl 1)rotect against the denaturillg actiorl of c.llzpnlt’a :\a 11;is I w n sugg(’stl’(11,y thc n w k of All(lt with fatty acids.
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WILLIAM REGELSON
VII. Surface Charge
The isolated tumor cell surface posscsses a nct negative charge as determined by electrophoresis (Ambrose et al., 1956, 1958; Purdom e t aZ., 1958; Straumfjord and Hummel, 1959; Mayhew and O’Grady, 1965) which increases during mitosis (Mayhew, 1966). I n the presence of added polyxenylphosphate (PXP), the electrophoretic mobility of 5180 cells was greatly increased. From pH mobility relationships, PXPtreated cells were found to assume some of the anionic properties of PXP. Cyanide and fluoride did not affect the mobility of the cells in the presence of PXP. From combined electrophetic and 32P-uptake measurements with 32P-labeled PXP, surface binding accounts for only a fraction of the total PXP uptake by Sl80 cells, supporting the affinity of tumor cells for polyanions and their associated alteration of physical properties (Straumfjord and Hummel, 1959). Unlike anionic detergents (e.g., sodium lauryl sulfate) (Hodes et al., 1960), cationic detergents do not lyse cell and nuclear membranes (Palmer et al., 1961). Tween 80 can scparate hepatoma ascites islands depending on the strain of tumor. The effect is associated with an increase in the negative electrical charge of the cell surface. The greater the negative electrical charges a t the cellular surface, the more easily does Tween 80 disperse hepatoma islands, and tumor cells with high negative electrical charges may be more loosely bonded to each other (Yamada, 1962). Tissue culture cells are induced to spread on glass in the presence of the basic proteins such as salmine (Lieberman and O m , 1958; Taylor, 1961), and tumor cells clump dramatically in the presence of polycations (Ambrose et aZ., 1958) which supports the ohservations regarding negative surface charge on tumor cells. Evidence for this using a titration technique has been obtained by Terayama (1962) for estimating the surface charge of ascites tumor strains. Differences are observed among various strains of tumor cells which indicates there is a greater net negative charge for all cell strains which grow as single cell suspensions, although some island-forming strains have high net negative charge as well. Of interest is his observation that a high negative charge parallels nitromin sensitivity of the cell system. This might relate to increasing negativity with increased mitotic rate (Terayama, 1962). The author feels that the binding of cells to each other is a function of a lipoprotein surface interaction hecause of the disaggregative effect of deoxycholate. He does not feel mucopolysaccharides are involved because hyaluronidase and rhondroitin sulfate do not affect binding. Similarly, L. Weiss (1963) Iraa foulid tliitt I i y ~ 1 ~ r 0 1 1 i dditl i t ~not ~ affcrt the 1,inding o i iso-
later1 cells to glaw although trypsin and nc~uramini~lase weaken glass adhesion. In contrast,, cationic polymers siich as Polybrene increase cell attachnient, (Nordling et nl., 1965:t,h). Altrrntitivrly, maintenance of the abilities of cells to remain nttaclicd to glahb (r.g., by hydrocortisone) may be related to preVcntion of cell d:tmage with subsequent leak of lysozonial enzymeb from the cell surface which L. Weiss (1965b) feels may facilitate a decrease in cell adhesioii related to surface alteration. The negative charge on the surface of tumor cells is dispersed by neuraminidase with a11 increase in cellular surface deformability (L. Weiss, 1 9 6 5 ~ )and an increase i n the phagocytic ability of monocytes. In this regard, the effect of polyanions on surface deformation of cells will be of interest and, based on previous work, probably would not support ail increase in surface rigidity such as has been postulated for the surface-supporting action of native carboxyl groups provided by neuraminic acid. VIII. Adenosine 5'-Triphosphate and Polyonions
The action of mitotic poisons has been related to effects on A T P metabolism (Lettre, 1952), and ATP may be critical to cell form and motion. Surface and cytoplasmic membranes possess ATPase activity (Wallach and Ullrey, 1962), and protoplasmic streaming involves ATP (Ts'o e t al., 1956). Collagen also possesses ATPase activity (Krane and Glimcher, 1962), but so do basic polypeptides such as polylysine and protamine which increase microsomal ATPase activity in similar fashion to Na+ ions. This ATPase activity was inhibited by 2,4,6-trinitrobenzenesulfamate and by suramin (Schwartz e t al., 1962). However, in similar fashion to collagen, metachromatic alpha and beta granules in invertebrate eggs possess ATPase activity and may play a role in spindle and furrow formation (Dalcq and Pasteels, 1963). Adenosine triphosphate has been localized a t a polar location in the mitotic apparatus of HeLa and Sarcoma 180 cells, and the SH-dependent ATPase activity in these cells was associated with cleavage activity (Hartniann, 1964). I n regard to microsomal polyanions, Schwartz e t al. (1962) have postulated that if the site for Na+ ion affinity in the microsoine includes an acidic group, then i t is understandable why protamine might behave in similar fashion to Na+ ions. This activation of ATPase by Na+ ions or basic polypeptides could be inhibited by polyanions and thus affect the availability of ATP governing ion transport and the physical properties of cellular membranes. These membranes, ns has been seen in the case of a protein-phospholipid complex from beef mitochondria, possess striking affinities for polyphosphates, which can be modified by the tri-
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WI1,LIAM REGELSON
carboxylie citric acitl ion (Hultin :ind I
rodent eggs depeiitling 011 the position of the spindlc :wl furrow forniation (Dalcq, 1959). I n addition to effects on embryos, it is of interest that platelet aggregation or “viscous metamorphosis,” a specific physical alteration in the nonnucleated blood platelet resulting in increased adhesiveness, can be induced by adcnosirie diphosphate (ADP) and prevcntcd by the monophosphate. Heparin inhibits adenylic deamiiiztse and may affect the level of available ATP for this phenomenon (Iliainond, 1955). Collagen (Zucker and Borelli, 1962) and the polyanion, polyanethol sulfonate (liquoid) , also produce platelet aggregation (Bettex-Galland e t al., 1963). Whether this has any bearing on cell division or embryogenesis remains t o be seen, but the action of polyphosphates on embryogenesis is of interest in that there is a fundamcntal similarity between muscle contraction (Grette, 1962) and acrosornal discharge and cell movement which may relate to spindle formation and the phenomenon of cell division (Runnstrom and Gustafson, 1951 ; Lettre, 1952; Weber, 1955). Polyphosphates may affect cell sol-gel alterations and associated cytoplasmic viscosity. This is important, since an increase in cell viscosity of canccr cclls over normal cells, 011 studying nuclear displacement on high-speed centrifugation, was found by Guyer and Claus (1939). This was coi~firmedfor rnethyleholaiithrcnc-induced hyperplastic versus norm:tl skin by Cowdry a n d Paletta (1941), and support for this was found in the morc recent work of Landau and Mc1,car (1961) wlierein micromorphology of F1 and primary human amnion cells differed. Marked cell contraction and bleb formation on pressure release for pressure-contracted F1 cells differed from that seen in human cells, as ATP strengthens the resistance of the cell t o high hydrostatic pressure. Adenosine triphosphate has been implicated in surface gel formation by Landau e t al. (1955). Cytoplasmic blebbing which resembles that occurring a t metaphase or following ATP administration is associated with an increase in ATP following release of cells from high hydrostatic pressure at low tempcraturc (TJandau and AIcLear, 1961 ; 1,andau and Pcabocly, 1963). Also, ATP added t o iinfcrtilizcd sen urchin eggs decrwises tlicir cytoplasniic viscosity a n d effects their fcrti1iz:ibility (Runnstrom and Kriszat, 1950a,l)). Cell contr:tctile proteiti l~chavcs~iiiiilnrlyto actinomyosin threads so th:zt sul)strnte iiiliil)itioii i> rcl:ito(l t o thc ronrcntratioii of ATP or Ca++. H(:paritt ciiii iriliihit coiittxctioii, :ib ciin tltc aiiioiiir (lye, sur:iniin, or sulfliydryl inhil)itors (IIoffrii:iti-Bcrlit~~, 1954u,l); Wcber, 1955) . Tlie latter ties in with I ~ i l l i ~ rtrhwtvttionb ’s regartling animalizntioil of the sea urchin enil)ryo. Fiirtli~~rmore, in motlcl systems, polyortliopliospliate has a dual action in similar fashion to A T P or inosine 5’-triphosphate (ITP),
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WILLIAM REGELSON
which, when broken down, can induce contraction, but when unchanged, has a plasticizing effect and renders the muscle protein structure extensible. Elongation of the cell or spindle protein of cells in the glycerinized model is brought about in the presence of ATP or inorganic polyphosphates (Hoffman-Berling, 1954a,b ; Weber, 1955) , and the viscosity of plasmodia1 proteins of slime mold can show a decrease in viscosity to ATP or an increased viscosity to adenosine 5'-monophosphate (AMP) which governs protoplasmic streaming (Ts'o et al., 1956) and is associated with cell division. Further support for the role of ATP in cell division is the presence of a nucleotide in the isolated mitotic spindle which may be A T P (Mazzia, 1957). This is not surprising as Bell (1958) has found acid-soluble nucleotides diffusing from thc vicinity of chromosomes in the Walker carcinoma. Also, it is well known that low concentrations of A T P increase the furrowing potency of egg systems (Zinimerman et al., 1957; Landau et al., 1955) under a variety of temperature and pressure conditions and can alter the streaming characteristics of protoplasm. Of importance to our discussion is that the action of heparin reverses this effect and, conversely, heparin activity is inhibited by A T P in this and other systems. For example, A T P counteracted the effect of heparin on clotting (Fisher and Schmitz, 1933), and heparin inhibits the contraction of actomyosin threads which normally occurs when ATP is added. Adenosine triphosphate added to aging sea urchin eggs can maintain their capacity for fertilization (Runnstrom and Wicklund, 1949) and antagonizes the inhibiting effects of heparin (Runnstrom and Kriszat, 1950a,b). Microinjection of ATP into Amoeba discoides causes the cytoplasm to flow away from the injection site, whereas heparin induces motion towards i t (Goldacre and Lorch, 1950; Bell and Jeon, 1963). Heparin stimulates pseudopod formation and cell locomotion in Amoeba proteus (Bingley et al., 1962), which Bingley et al. felt was due to depolarization of the cell surface. Pseudopod action may be similar to what occurs immediately after cell cleavage and was associated with increased stickiness of the cell surface (Bell, 1961) and pseudopod fusion (Bell, 1962a,b). Depending on the character of the polyanion, inhibition of locomotion and alteration of cell division can be obtained. The anionic dye, aliztirin sulfonntr, has heen found by Pollack (1927) and Chalkley ( 1935, Chiilklcy and D:tniel, 1934) to iiiliihit locomotion of A ? w P I ~protrus. This will inliibit clcnvage i f administered tluring 1)roph~s'c without affectirig initohis, wit11 subbequent giant cell forination. The reactioii of :i particulnr contractile aystem to polyanions may relate to whether ATP is involved in a process of shortening or elonga-
tion; ATP-induced motility of ciliary models is inhibited (Child, 1961). Swimming activity in the sea anemone is induced by an aminopolysaccharide obtained from dermal spherules of the starfish (Deimosterias imbricata) (Ward, 1965), and dilute solutions of sea urchin jelly coat substance or heparin can increase the motility of sperm, produce acrosoma1 discharge (Dan, 1960), and enhance rather than inhibit fertilization of jellyfree eggs (Hagstrom, 1956b). The importance of acidic proteins obtained from muscle which possess metachromatic qualities has not been determined. This protein forms gels and is precipitated by calcium and basic dyes. Heilbrunn noted the action of calcium in causing muscle contraction and claimed that muscle produced a metachromatic material that could inhibit cell division. The inhibition of contractile protein by polyanions may be of importance as it relates t o inhihition of oiicogenic viral infections. This has been shown in that injection of virus genetic material into bacteria depends on contraction of the viral envelope, which rcscmhles actoniyosin. The effect of polyanions on cell surface is also seen through polyglutamic acid-inhibiting phagocytosis. Here, inclusion of lysine to produce a copolymer with a 7 : 3 ratio has the opposite effect (Blout et d., 1962). Morphological alteration of a variety of cells has been induced by ATP (Hoffman-Berling, 1954a, 195.5). The action of surface polysaccliarides or intercellular cement could he one of control of the level of ATP a t the surface by direct or indirect action. This could, in turn, affect cell morphology, function, and/or the surviving integrity of the cell surface. IX. Calcium
Calcium-binding capacity for heparin has been correlated with anticoagulant activity (Ascoli and Botre, 1962). The ligand structure of acid mucopolysaccharides and the high concentration of chondroitin sulfate and calcium are causally related (Buddecke and Drzeniek, 1962). The action of polyanions as an ion exchanger to fix extracellular calcium may be pertinent to the importance of calcium in living cells. For example, there is a deficiency of calcium in tumor cells which may affect their adhesion (Carruthers, 1950; Carruthers and Suntzeff, 1944, 1946), arid the removal of calcium from cells results in “surface bubbling” which is similar to what is seen 011 metaphase or exposure of isolated cells to ATP. This is associated with the loss of surface rigidity (Dornfeld and Owczarzak, 1957, 1958; Thomason and Scholfield, 1961; 1,. Weiss et nl., 1966) and bears superficial resemblance to Belkin’s obser-
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vations (Belkin and Hardy, 1961) regarding the effects of -SH inhibitors on cell surface. I n this regard Hultin (1950a,b) fclt that calcium ions enhanced S-S linkagc in sea urchin eggs, and i t is of interest that the inhibition of succinic dehydrogenase appears to be involved in bleb formation (Belkin and Hardy, 1961) which may relate to the loss of -SH groups. Heparin and suramin have also been found to inhibit. dehydrogenases (Bernfeld, 1963; Horn and Bruns, 1959; Wills and Wormall, 1950). Amoeba transformation to flagellate forms is governed by the absence of cations (Willmer, 1961), and the administration of calcium ions to a homogenate of sea urchin eggs caused a transient outburst of oxygen consumption (Hultin, 1950a) as well as an increase in viscosity. Similar effects have been seen in spermatozoa (Tyler, 1953). Calcium ions are necessary for the stability and physiological integrity of the egg cortex. When molluscum eggs are treated with alkali salts, calcium ions are exchanged. When calcium loss is too large, mortality occurs. The lethal action of Cat+ or alkali ions is similar to the "ion spectrum" that results in reversal of charge depending on the concentration of phosphate colloids, but differs from that seen for carboxyl or sulfate colloids (Raven, 1963, 1964). According to Raven, the lethal concentration of alkali ions depends on the affinity of these cations for phosphatides in the cell membrane. Versene separates amphibian gastrulae with the release of ribonucleoprotein with no alteration in cell morphology or viability. This is associated with a clear gel that forms between the cclls a t pH 9.8 but which disappcars in the prcsence of calcium or return to pH of 6.8 (Curtis, 1958). The dissociation of embryos in calciumfree sea watcr (Herbst, 1900, 1904) and the sensitivity of cells to calcium is seen in that amphibian blastocoelic cells, when in a calciumfree solution, show no architectural alteration until calcium induces aggregation (Holtfreter, 1944). Further discussion of the role of calcium ions in morphogenesis has been presented in great detail by DeHaan (1958) and, more recently, Steinberg's data (1962)' have supported the idea that Ca"-binding causes cell aggregation by bridging cells rather than loss of cell surface charges. I n an independent area, hyaline cartilage rich in acid mucopolysaccharides removes calcium ions selectively (Boyd and Neuman, 1951), and Glimcher (1959) suggests that depolymerization increased the calcification of collagen-rich tissues by making free Ca++ ions available for mineralization. Calcium binding is greatest for sulfomucopolysaccharides, and Buddecke and Drzeniek (1962) indicate that this is an ion exchange reaction.
Recently, the rcmoval of calcium ions by chelation resulted in the uncoupling of both nerve (Penn and Lowenstein, 1966) and epithelial membrane junctions (Nakas et al., 1966). Whereas, electrical stimulation of single cells in salivary gland epithelium resulted in changes in neighboring cells ; when calcium ions were removed, cell membrane potentials were isolated from each other. Thus, calcium ions provide an electrical coupling between the reaction surface of cells, and removal of sufficient calcium irreversibly interrupts intercellular communication. The importance of polyanions in inducing or preventing this phenomenon would be of interest. Related may be the observation that the chelator, EDTA, was shown to cause condensation and dchydration of the mitochondrial matrix which is associated with increases in the mitochondria1 concentration of ATP (Lynn et al., 1964) in the absence of Ca++. Protoplasmic gelation was felt by Wilson and Heilbrunn (1957) to be dependent on oxidative processes that would convert -SH groups to -S-S groups. Heilbrunn (1952, 1956a,b) maintained that the vacuolization of intact cells occurs as a result of the release of excessive calcium salts into the cell interior. Aging or iiiaturation of eggs is accompanied by the same phenomenon (Heilbrunn, 1956a,b,c ; Heilbrunn and Wilson, 1955). Like heparin and related polyanions, the chelating agent, EDTA (Borei and Bjorklund, 1953; Nishimura et al., 1955), has antagonistic effects to the presence of calcium ions (Heilbrunn and Daugherty, 1932) on sea urchin eggs, ascites tumor cells, and Amoeba. The viability of sea urchin eggs has been correlated with increased zeta potential, and increased elcctronegativity was associated with increased viability. This was reversed by calcium ions (Dan, 1947a,b). Therefore it is not surprising that the aging of sea urchin eggs and animal cells has been related to tlic coiiccntration of calcium (Schechter, 1937, 1941 ; Heilbrunn, 1956a). Decreased Concentration of calcium to one-half the control increased survival of eggs in sea water, and, similarly, exposure of the cladoceran, Daphnia magna, to heparin increases its survival in spring water a t concentrations of 0.01 to 0.1% (Schechter, 1950) and also increases motility. This might be related to the calciurnbinding capacity of heparin, since chelators of heavy metals prolong the survival of both sea urchin sperm and ova (Tyler, 1953). Rigidity of the cell cortex and the permeability of the cell both depend on the presence of calcium (Yamamoto, 1956). Heilbrunn (1952, 1956a,b,c) has oheerred that when protoplasm changes from a fluid condition to a more or less solid state, tlic primary reaction involved is similar to blood (*lotting which in its early stages requires free calcium. The prp'selicr~of r;ilriiitii ions f ' : i \ m i ~ tlrc rortic:il r~sponscof' eggs, nit11 niito-
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WILLIAM REGELSON
activation of cggs is conditioned by the release of calcium ions (Goldstein, 1953) which may activate proteolytic enzymes (Hultin, 1950b) and ribonuclease (Lindvall and Carsjo, 1955). Cell division and nuclear breakdown in Chaetopterus eggs may be due to calcium ions which probably activate the proteolytic enzymes that dissolve the nuclear membrane (Goldstein, 1953). Under the influence of ATP or cysteine, calcium phosphate is precipitated on the surface of rat and mouse eggs. This is related to the position of the mitotic spindle relative to the cell surface where furrow formation occurs (Dalcq, 1959). I n turn, this relates t o the prior position of metachromatic cytoplasmic granules which possess ATPase activity (Dalcq and Pasteels, 1963). Once more, wc are attempting to equate metachromasia with a morphological role for polyanions. Calcium accumulates to a greater extent in slower-growing than in faster-growing tissues (Lansing et al., 1948). The aging and decrease in viability of eggs is associated with a rise in their calcium content (Tyler, 1953). Versene, other calcium chelators, and a low Ca++environment prolong the motility and fertilizing capacity of sea urchin sperm (Tyler, 1953), Chalkley and Daniel (1934) and Pitts and Mast (1924) found that calcium would permit cell division in Amoeba proteus even under acid conditions and would reverse the inhibitory action of lactic and pyruvic acids and high concentrations of Na and K ions. I n view of the lipolytic-promoting action of heparin and heparinoids, which splits lipid to free fatty acids and glycerides (see Section XXI), the work of Deamer and Cornwell (1965) is of interest in regard to changes in the cell membrane that might be related to polyanion Ca++ interaction. Simple soaps (two fatty acids bound to a single calcium ion) of stearic and palmitic acids can form a copolymeric lattice structure to form rigid nonflowing films between pH 5.5 to 8.5 a t temperatures as high as 90°C. The availability of Cat+ ions and free fatty acid could possibly govern the rigidity of the cell surface or its internal structures. The mitotic apparatus binds calcium strongly (Mazzia, 1957). Similarly, the polyanionic jelly coat substance of unfertilized eggs takes up calcium, as measured by 45Ca uptake as soon as it is immersed in sea water. Calcium is lost from the egg surface with dissolution of the jelly coat on fertilization, and the binding capacity of the jelly coat for Ca++ is such that 8% of the total weight of the jelly coat may be calcium (Rudenberg and Klein, 1953). I n addition, membrane elevation in sea urchin eggs is associated with the release of calcium from the egg cortex accompanied by stiffening of the cortex (Wilson and Heilbrunn, 1952; H e i l b r u n ~and ~ B ~ w 1959) , , C l ~ ~ ~ o ~ t ~ o shoI ~* dn ~: i:lt g crclateil to or in(1rpcndcnt of rndintion lins
I w n s h o ~ ~ton lw iticreastvl i n r~ilriiiin-cleficiciicys t a t c s in both plant and :tniiiial cell s y s t e m (Ptrffenson, 19611 . Iu :mother physiologiral area, diastolic arrest of the frog heart using :I he~):win piq):ir:itioti w:th 1wc~rsc~1 hy calciuin (Kraus et al., 1932; Chaet, 1955; Caprtlro et al., 1958), and Coleman (1958) used diastolic arrest of the frog heart as a method for seeking out synthetic heparinoids in the search for mitotic inhibitors of which polyethylene sulfonate was found to show antitumor activity. This was based on the work of Chaet (1955) who found a dialyzable fraction of heparin which produced diastolic arrest of the frog heart as well as inhibition of Chaetopterus egg development. Further, heparin exerts a permeabilizing effect on the isolated frog heart resulting in rapid loss of calcium (Capraro et al., 1958). Siniilar effects have been seen in calciurn-45 labeled tumor cells (Thomnson and Scholfieltl, 1959 I . Rlore recently, Csaba and Horvath (1963) hare found tliat glucuronic acid can behave similarly, but its action is potentiated by glucosaininc and the presence of sulfate groups. Diastolic arrest results from the negative inotropic affect on the frog heart by heparin which is reversed by calcium ions and protamine (Hegyvary, 1964). Contrary to Csaha and Horvath (1963) , this is partially reversed by adrenaline and serum. Sufficient dosage of heparin inhibited the sensitivity of the isolated heart muscle to Ca++but this sensitivity could be 1-estored with adrenaline, noradrenaline, or caffeine. Hegyvary feels that substances activating phosphorylase enhance the sensitivity of the ventricle to Ca++ and that heparin interferes with calcium transport. The ticinonstration that heparinoids increased the resistance of cats to lethal doses of ouabain (Macht, 1943) may be associated with these observations. Polyphosphates which have in vivo antitumor activity (Regelson e t nl., 1960) can separate plant cells by binding calcium ions (Letham, 1962). The intercellular cement of plants may be a protein gel linked together by two types of metallic ions characterized by cross-linkage through a chelate. In support of this, EDTA also promotes the separation of plant cells (Ginzburg, 1961). Among a series of anionic polysaccharides studied, heparin had more cation bound per clisaccharidc period (Matthcwb, 1964) . Howcvrr, ThomRYOII arid Scholficlcl (19.59, 1961) have not found cvidcncc of heparin extraction of calcium from Ehrlirh ascitcs tumor cells equilibrated with isotopic calcium-45. Desl’itc this, acid mucopolysaccharidc iiicreases precede calcium deposition i n ncphrocalcinosis secondary to vitamin D intoxication in the kidney (Konctzki et al., 1962), and the GI absorption of heparin is a i d d I)y thc simultnnc~ousadministration of calcium-binding subst:tnres such 3.s EDTA, sodiuin citratcb, soaps, :inti phosphates.
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WILLIAM REGELSON
\\%ether the quantity of rxchnngeable calcium a t the cell surface can govern the intracellular penetration of heparin or related polysulfonates has not been determined. The calciurn chelator, EDTA, depressed D N A synthesis of in vitro bone marrow aspirates (Lochte et al., 1960). Treatment of growing onion roots with solutions of EDTA also produce chromosomal abnormalities attributed to alteration in the general metabolism of the cell, possibly via activation of ribonuclease, rather than t o direct breakage of chromosomes through chelation (Kaufman and McDonald, 1956; McDonald and Kaufman, 1958). However, exposure of sea urchin sperm to EDTA results in spreading of DNA fibrils indicating that chromatin linkage is affected by the absence of calcium ions (Solari, 1965). This extension of DNA fibrils could affect the proportion of active euchromatin involved in protein synthesis (Frenster et al., 1963). I n related studies, EDTA causes “surface bubbling” of interphase fibroblasts with retraction of ectoplasmic blebs but with no interference with mitosis (Dornfeld and Owczarzak, 1957, 1958). These authors suggest that morphological changes occurring in prophase resemble EDTA effects and may relate to removal of divalent cations from the cell surface. Bell and Jeon (1963) have been able to induce pseudopod formation in Amoeba with ion exchange resins. Ethylenediaminetetraacetate does not prevent the attachment of T 2 phage to the intact host cells, but does prevent the formation of infective centers by preventing the injection of DNA into the host cell which may be similar to its action in inhibiting contraction of muscle fibers (Watanabe and Sleator, 1957). This may provide a clue as to the antiviral action of polyariions such as hydrolyzed malcic anhydride copolymers, which chelate calcium and other cations (Morawetz e t al., 1954) and possess antiviral (Feltz and Regclson, 1962) and antitumor activity. Although the calcium ion is important for the promotion of phagocytosis, inhibition of phagocytosis by polyglutamic acid was not reversed by calcium (Zwartouw and Smith, 1956). Pyran copolymer and related polycarhoxylic polymers can either block or stimulate phagocytosis depending on the assay (Old, 1959; Muiison et al., 1967). In addition t o effects on calcium ions, heparin has been found to mobilize bound muscle potassium. This bound potassium is mobilized from rat diaphragm when temperature is raised or heparin is added (Hashish, 1958). Furthermore, calcium ions are the chemotropic factors in higher plants that govern the growth and direction of pollen tube formation. In other plants, carboxylic acids act as chemotactic influences governing the direction of motion of the male gamete (Machlis and Rawitscher-
Kunkel, 1963). There is a proteiii gel structure in plant tissue which is cross-linked by metal cations (Ginzberg, 1961). Pretreatment with other than monovalent cations enliariced subsequent plant cell separation in EDTA and several rnultivalent metallic cations sucli as Fe, Cu, Mn, as well as Ca, aided separation. Polycarboxylic polyanions diff’cr in their chelatirig ability in that styrene maleic anhydride copolyniers show increased chelation with increasing charge whereas the reverse is found with vinyl ether copolymers (Morawetz et al., 1954). How this relates to the biological antitumor or antiviral activity of both of these polyaiiions remains to be seen and is under investigation a t this time (Hodnett, 1966). The advantages of isotatic acrylic acid as a Ca++binder over the atatic form have been discussed by Smets (1962), and the use of stereospecific polyanionic copolymers in biology is awaited with interest. X. Adhesion
As discussed previously, a decrease in cell adhesiveness is related to the removal of calcium or magnesium from the medium (Chambers, 1940; Zeidman, 1947). The adhesive role of calcium dates back to the observations of Ringer (1890), Wille (1897), and Herbst (1900) in tadpoles, algae, and sea urchin blast om ere^. Cancer epithelial cells are less adhesive (Coman, 1944, 1946) than normal cells and this has been related to a decrease in Ca++concentration in the tumor cells as compared to normal cell populations (Carruthers, 1950; Carruthers and Suntzeff, 1944, 1946; DeLong e t al., 1950). Isolated cancer cells frequently enter the peripheral circulation from the tumor vascular bed and the problem of metastatic disease is not why it is prevalant, but why so few of the circulating tumor cells find an appropriate environment for subsequent growth. The concept that cell adhesion may be related to malignant growth and the role of calcium in governing this phenomenon has been reviewed by L. Weiss (1960). The hyaline layer substsnce in sea urchin eggs, which make the hlastomeres adhere, consists mainly of polysnccharides which are solubilized when dimlcwt cations arc rernovd from the medium (Vasseur, 1948; Nakano and Ohashi, 1954; Dan, 1960). Similarly, hexametaphosphate is a useful agent in separating plant cells through selected removal of Cat+ (I,ctham, 1962). The siction of lieparinoids in producing viscous gels a t c ~ l sui-faces l (Torti. :m1 Regclson, 1965) may be instrumental to tlie pheiioint~iioii u i c~.ll idhesion. Cell aggregation induced by polyanions stiniulate,s contact inhibition-a phenomenon which is not seen as readily in the case of malignant cells (hbercrombie and Heaysman, 1953, 1954; Abcrcronihie, 1962) . Contact inhibition is asso-
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WILLIAM 11EGELSON
ciated with loss of free cytoplasmic polyribosomes and deprcssion of DNA, RNA, and protein synthesis, and indicates that surface contact between cells can alter the expression of cell function (Levine et al., 1965). This might be pertinent to the observation that acid polysaccharides can disaggregate microsomes (Hoster et al., 1950) and that heparin can displace ribosomal RNA (Anderson and Wilburt, 1950). The earlier observation of Leo Loeb (1922) is of interest in that stereotropic movement of amebocytes is related to their stickiness which leads to aggregation of isolated cells and eventual tissue formation. This earlier work is supported by that of Holtfreter (1947) who has correlated phagocytic activity with cell adhesion. I n further support, Nordling et al. (1965a,b) and Vaheri (1964; Vaheri and Penttinen, 1962a,b) have shown that anionic polymers in low concentration inhibit cell attachment and alter the growth behavior of HeLa cells in tissue culture so that they grow in dense clumps. There is, in contrast to the action of polycations, a relationship between the clumping effect of the polyanions on tissue culture cells and their prevention of attachment of cells to glass. Nordling et al. (1965a,b) found that perum or albumin is necessary for polyanions to exert their inhibition of cellular attachment to glass. This they ascribed to the presence of a cofactor in anti-P-lipoprotein sera; thrombin and hyaluronidaac inhibited this effect. The action of anti-P-lipoprotein was similar to what Saksela (1962) and Saxen and Penttinen (1961) obtained with cells in fresh human serum, whereas 1,. Weiss (1959) and others (Lieberman and Ove, 1957; Fisher et al., 1958) found that both crude serum, a lipid-rich serum fraction, and a G2-rich globulin, increased the adhesiveness of cell surface to glass. Tryptic inhibitors such as fetuin accomplished the same result (Fisher et al., 1958). In contrast to adherence to glass, L. Weiss (1959) found that cells sticking to fibrin or collagen gels did not require serum, but serum was necessary for adherence to calcium alginate, silica, and agar gels. The role of lipoprotein in cell attachment to glass or gels may be of interest in that serum elevations of p-lipoprotein are found with advanced metastatic breast cancer ; levels which are normalized by systemic administration of heparin (Barclay et al., 1955). The presence of heparinlike material in the intercellular cement of vascular cndothclium and the affinity of tlic cndothelial cement lines for exogenous heparin have been suggested IJY the obscrvation of Samuels and Webster (19521 and Ohta P t ul. (1962). The permeability of this vascular intercellular ccnicnt is governed by the level of calcium ion aiid is increased with dccreusing calcium concentration (Zweifach, 1940). This affinity of cement substance for heparin suggests that selec-
tivo I)iii(iiiig of tliis i i i : ~t c ~ i : i l : i t iritc~rc~clliil:~~~ sitw cwiild iilter t l ~ physical character of the c ~ l lsiii-faw and its I)intling properties to neighboring cells and response to calcium concentration. Heparin causes leukocyte agglutination (Wilander, 1938) and, despite similar negative charge, heparin also has fin affinity for the red cell surface and can interfere with polylysine-induced aggregation (Sad e t al., 1962). The importance of the sulfomucopolysacrharide jelly coat and polyanions in the egg and embryo as controlling factors in cell division and morphogenesis has been discussed earlier and has been recently reviewed by Lippinan (1965). The presence or absence of similar material on the surface of the cell and in intercellular cement (Bell, 1960) may be important to the phenomenon of tumor induction, mitosis, or inctastases. The ability of embryonic cells to adhere (“coapt”) to neighboring cells of siniilar origin has becii amply demonstrated (Weiss and Andres, 1952). Normal embryonic cells in a foreign environment die, but this is not as true of tumor cells (Bender et al., 1949), although most disseniinated tumor cells die in proportion to the number freely circulated. The production of colloidal exudates by cells in tissue culture has been shown by Weiss (1945) to govern the orientation arid migration of cells. The production of sulfornuco1)olysaccharides by cells may provide the “control guidance” responsible for morphogenesis or cell localization in normal or malignant states. I n support of this, most recently Gustafson and Wolpert (1963) indicates that tlic anirnalization of sea urchin eggs is associated with increased cellu1:tr colicsion. How this may relatc to the formation of mucopolysaccharitles is not clear as vegetalization of the cmbryo is associated with free sulfate groups. 1,allier (1964) also feels that increased adhesiveness is pertinent to vegetaliz a t‘ion. I n an unrelated area the action of collagen on platelets to induce platelet aggregation (Zucker and Borelli, 1958, 1962) can determine the localization of thrombi in a vascular system. Similarly, the action of anionic polyelectrolytes can cause cells to orient or coapt to a given neighbor provided conditions are appropriate. Thus, the presence of endogenous or exogenous mucopolysaccharides or polyanions could govcrn the direction of cell growth or metastases. XI. Polysaccharides
l‘hc t1ieoretic:il importance of polysaccharides, which may be poly:iiiioiiic in char:icter, as structural components of the cell surface is obvious (Bcll, 1962a,b). Even the single-celled Amoeba possesses metachromatic material a t the surface (Heilbrunn e t al., 1958), and this may be pertinent to locomotion or phagocytosis. In another area, the im-
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WILLIAM REGELSON
pol,t:tllc(: of stllj;lcc l’olys:tct.liiii.itIch ; ~ s roiit i.olling :gents ill (+ation cxchange has alreattly been discussed in relation to the importance of the calcium ion. Nonsulfated, connective tissue mucopolysaccharides may function as cation exchange resins in addition to their structural and water-binding role (Meyer and Rapport, 1951). Of additional importance is the fact that tumor cells may possess surface polysaccharides which may differ from normal. Also, alterations in connective tissue may contribute to changes in stroma which could vary gaseous diffusion and contribute to cell adhesion and metastability. I n the case of hyaluronic acid, the dissociation of carboxyl groups could be supprcsscd hy the more dissociated sulfate groups. The presence of polysulfonates could affcct thc behavior of adjacent carhoxyl groups on the cell surface to govern the exchange of electrolytes and the physical character of the cell surface. The stability constants of acid mucopolysaccharides reveal relationships between ligand structure and calcium-binding capacity. Calcium affinity decreases in the order : heparin-chondroitin sulfate B-chondroitin sulfate A-hyaluronic acid. The high tissue concentrations of chondroitin sulfate and calcium suggest that these polyanions act as ion exchangers to fix extracellular calcium (Buddecke and Drzeniek, 1962). The possible role of acid mucopolysaccharidcs in electrolyte exchange othcr than that involving calcium is seen in the presence of metachromatic material in the salt-secreting glands of turtles, the salt gland of the duck, and the rectal gland of the dogfish (Ellis and Abel, 1964). The acid polysaccharides in the chloride cells of salt water-adapted killifishes (Fundulus heteroclitus) are involved in osmoregulation (Philpott, 1964). Meyer (1947) postulated that the fibroblast first secretes hyaluronic acid, then chondroitin sulfate and a collagen precursor. The mucopolysaccharides form chains with regularly spaced acidic groups which serve as templates on which the globular collagen precursor is denatured. The mucopolysaccharides may act as anionic detergents rolling out the peptide chains along the acidic groups. Hyaluronidase removes hyaluronic acid which blocks the maturation of collagen; thus, enzymes that control the breakdown of particular polysaccharide polyanions can control the physical character of the cell matrix. This is supported by the ohservation of Wood (1960) that low concentrations of heparin and DNA retard connective tissue, fibril formation whereas othcr polyanions could accelerate it, and that of Anderson (1961) in regard to alterations in gastric mucin permeability. Polyanions could also exert their effects by inhibiting hyaluronidase.
CH<)\VTII-RE(;ULAlI~TC ACTII’ITT O F I’OLYAA’IOSS
261
Thc rolc of calcium or cations in goveriiiiig tlie physical character of connective tissue and the intcrccllular matrix may be pertinent to the tumor problem. The subcutaiicous injection of the chelator, EDTA, results in transient increases in urinary excretion of acid mucopolysaccharides independent of local inflammatory response (Smith and Kerby, 19GO), and the structural factors involved in cation binding hy anionic polysaccharides in connective tissue has been analyzed in detail by Matthcws (1964). He found in relation to the anticoagulant effect of connective tissue polysaccharides that, although high binding affinity for Co”+(NH,), was seen, rnoleculitr weight was an independent factor. Increasing the molecular weight contributed to the anticoagulant effect. The latter may be similar to what is involved in the antiviral action of polyanions, since the higher-molccular-weight copolymers are most active (Feltz and Rcgelson, 1962). I n plants, calcium agglutinable, acidic polysaccharides play a role in the changes that occur with germination (Gould et al., 1965). Mucopolysaccharides are also found in the nucleolus (Stich, 1951a,b; King, 1960; Lazzarini and Weismann, 1960), and Dalcq (1959) proposed that a discharge of nuclear vacuoles could contribute to cortical ATPase needed for furrow contraction. A5 discussed earlier, these or related particles referred to as alpha and beta particles are found in invertebrate eggs, stain metachromatically, and contain acid phosphatase (Beams, 1964) and ATPase (Dalcq and Pasteels, 1963), and may be important to the institution of furrow contraction and cell cleavage. Besides containing protein, aster fibers contain neutral and acid polysaccharides (Mom6 and Slautterback, 1950; Stich, 1951a,b; Pollister and Ris, 1947). These acid polysaccharidcs in addition to causing solation of Amebic cytoplasm, disaggregate niicrosomcs (Hoster et nl., 1950) and could effect protein synthesis. Kalckar (1964a,b) hits found that there is a defect in the capacity of malignant cells to convert glucose to galactose (“epimerase choke”). This defect is associated with the relative increased aerobic glycolytic capacity of the tumor with resultant acidic conditions and high levels of reduced pyridine nucleotides which block epimerase activity. Kalckar postulates that the defect results in a “thinning out” of galactosyl compounds on the surface (“ektopolysaccharides”) of the cell. This may alter the inimunogenic specificity of the cells and gives rise to malignant transformation. In support of this idea, but distinct from the immunological place of ektopolysaccharides, is the observation that inactivation of T 2 bacteriophage by antiserum is reduced in the presence of the polyanion, polyglucose sulfate. This effect is reduced by addition of polycations which indicates that there is a reversible electrostatic
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WILLIAM R1X:ELSON
intoraction Ixtwrceii :uitrit)ocIy p o t cins ancl polyclect,rolytes (Rilora, ant1 Young, 1962). Thus, as an addendum to Kalckar’s hypothesis, alteration in the synthesis of polyanions can theoretically affect the cell surface or the surrounding medium not only by altering polysaccharides which provide immune specificity, but by alternatively affecting the balance of polyelectrolytes. Ektopolysaccharides change the surface characteristics of microorganisms. Alteration in these surface polysaccharides may also possess growth-controlling activity through control of enzyme activity, e.g., permeases, or through effects governing the stickiness, rigidity, or porosity of the cell surface. Alternatively, the presence of polysaccharides surrounding cells may alter the availability of oxygen through impeding diffusion or, conversely, through increasing the concentration of carbon dioxide. Using tissue culture systems, McLimans ( 1967) has found that oxygen tension or, conversely, CO, tension, is critical for the survival of normal cells. Decreased oxygen supply selects cells of preferential anaerobic capacity which may result in malignant transformation. Thus, the extracellular mucopolysaccharides could select for cell populations of malignant type. With block to the diffusion of CO?, however, cell division is impaired, which could constitute an important growth-controlling mechanism. Regardless of the theoretical mechanism involved, Kalckar speaks of these postulated altered surface polysaccharides in malignant cells as changes in the “ektobiology” of the cell, and it is appropriate a t this time to consider what we know of the surface of the tumor cell that distinguishes it from normal cells that could relate to the antimitotic and/or antiviral action of polyanions as ektobiological growth controllers. A number of observations have been made in this area. For example, contact inhibition is lessened in tumor cells in tissue culture. This may relate to the absence of the production of extracellular material which may be necessary for cell adhesion. Cell contact results in the depression of DNA, RNA, and protein synthesis and is associated with polyribosome disruption (Levine et al., 1965). Ascites tumor cell aggregation i n viwo induced by intraperitoneal phytohemagglutinin is associated with a decrease in cell volume and marked inhibition of RNA synthesis (Tunis, 1968). The evidence that cliorionic epithelium is vulnerable to fibrinolysin (Thomas et al., 1959) and that there may be a surface barrier around placental cells t o the penetration of antibody, supports Kalckar’s concept of surface alteration for cells particularly adapted for infiltrative growth. Polysaccharides arc in intimate association with polyphenols. The physiological role of polyphenols has been explored by Monn6 (1961) in
cells :md tissue cnvelopes. Changes in the availability of polyphcnols inay govern the susceptibility of tissue to infection or the breakdown of intercellular structure. Polyphenok are potcnt inhibitors of enzymes responsible for connecti\*etissue lweakdown and, bccausc of their tanning effect on proteins, a cuticle develops which can protect cells from the injurious action of outside agents. Vegetable tariniiig agents such as niimoxinc are polysulfatcs, arid similar phy>icocheinical interactions with collagen have been described for sulfoiiated phenols. Even catechol and benzoquinone polymerize to produce polycarboxylic tanning agents which behave as polyanions. These tanning reagents interact with collagen monolayers t o produce coinpaet structures of high surface viscosity (Ellis and Pankhurst, 1952), which may reflect changes in ccll surface or in interccllular cement that could affect membrane permeability and cell granules. Sulfated niucopolysaccharides are found in intestinal mucosa (Kent, 1962) and the polyanion, carrageenin, decreases the permeability of mucin to the penetration of pepsin. Surface alterations can be induced in cells and cellular inucin (Anderson, 1961) by polyanions. Similarly, heparin and other polynnions induce the formation of collagen fibrils when added to collagen solutions a t concentrations as low as 1:80,000 (Morrione, 1952; Gross c t nl., 1952). Differences in the sensitivity of cell lines to polyvalent cobalt based on their production of sulfated mucopolysaccharides have been found by Lazzarini and Weismann (1960). Human aortic intimal cells produced large cytoplasmic metachromatic granules on exposure to as little as 5 pg./ml. of tri- and hexavalent cobalt salts. I n contrast to the malignant HeLa cell line, normal cells did not show prolongation of generation time or inhibition of mitosis. Similar rcsults were obtained for nickel salts. The authors found differences in thc sensitivity of the nucleolus of the normal and malignant cell lines to these polyvalent cations. This indicates t h a t the sensitivity of cell Iines to complex cations depends on intracellular sites containing polyanions. Heparin will transform Pnrnmecizcnt nurelin types 51D and 51E to the stable type 51B (Austin, 19591, and the transformation of sea urchin eggs to the animalized motile form iq governed by the presence of polyanions (Lallier, 1956, 1957n,b,c, 1958, 1959, 1966). The role of polyvalent cations in this rcgard is of intci wt i n that Zn++is a potcnt nnimnlizing agent whicli ciin rci*ttrbe the iwq~t:tlizjogdfect of lithium ions, ant1 nr-ts in additive fashioii with the ~~olvanioiiic dye (Evans Blue). Zinc forms complexes with iiniclazoleb (Gurfl, 1960; Gurd and Goodman, 1952) as docs heparin, which binds with histamine in the mast ccll, and there is evidence supporting a histamine-zinc-heparin complex which represents
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WILLIAM REGELSON
a possible storage form of histamine (Kerp, 1963). As it has been suggested that imidazoles may be growth-promoting factors (Kahlson, 1962), then Lallier’s (1966) observations may provide a clue as to the growth-inhibiting activity of polyanions. It is pertinent that, along with decreases in calcium ion, Carruthers and Suntzeff (1946) found a decrease in the content of zinc in epidermal cancer, and zinc released from the nucleolus of starfish oocytes was found to participate in spindle formation and cell division (Fujii, 1954). L-Tyrosine produces sea urchin vegetalization and potentiates the vegetalizing action of lithium chloride, L-tryptophan, and L-arginine (Fudge, 1959). It would be of interest to see if these amino acids would similarly antagonize the animalizing or antitumor action of polyanions and/or zinc metabolism in this and other biological systems. Exogenous copper and/or ascorbic acid depolymerize the polysaccharides obtained from extracts of aqueous humor (Balazs, 1960) and tumors (Pirie, 1942). These are similar to effects produced by radiation (Balazs, 1960). Copper was bound to the endogenous tumor polysaccharides obtained, and this may be important in view of the decreased levels of copper in tumor (Carruthers and Suntzeff, 1946). Similarly, polyvalent cobalt alters the morphology of aortic intimal cells by combining with polyanions within the cell (Lazzarini and Weismann, 1960). This action of multivalent cations can be compared with similar effects on the protein gel structure of plants in conjunction with chelating agents (Ginzburg, 1961). The importance of polyanions a t the cell surface is possibly reflected in similar alterations which occur in connective tissue. For example, Sokoloff (1963) feels that anionic groups of cartilage, presumably the sulfate and carboxyl groups of the matrix polysaccharides and proteins, govern its physical properties. Reduction in the electrostatic charge of these anionic groups by cations decreases the size of the colloid aggregates and, therefore, permits greater deformation and extrusion of water when cartilage is compressed. Sokoloff feels that chondroitin sulfate binds counterions more like R polyelectrolyte solution than an ion exchange resin. Cartilage can, thus, act as a rapid ultrafilter to exclude selectively long-chain polymers while allowing water and electrolytes to enter the tissue. Similar observations have been made by Balazs and Jacobson (1966) for the interccllular matrix, and it is easy to see how these changes can affect the passage of material between cells which can govern their nutrition. As implied above, the ability of polysaccharides to form films and gels may govern their partitioning of suspended particles or the diffusion of oxygen and CO,. This may be pertinent to the effect of various agents
(;RO~vTI-I-RE(;TI,ATIN(; ACTIVITY O F POLYANIONS
265
on go1 formation among which is acetyltryptophan (Feigen and Trapani, 19.54) which increases the hydration of gels (Ferry, 1948). A polymerization product of t,ryptophan is Iiumic acid which is a con(Itws:ttion pi~orliic~tof tryl~toph:in forniod on esposurc to heparin (Riley and Shepherd, 1961). Humic acid is found in high concentration in epidermal cancer (Fels and Greco, 1961), and this might be related to the polyphenols found in association with acid polysaccharides which have antiviral and antibacterial properties through their antienzyme and/or tanning action on cell surfaces. Polyglutamic acid can increase the viscosity of plasma more than 100% (Blout et al., 1962) with agglutination of red cells (Kenny, 1959). Thus, in considering the effects of polyanions on tumor nutrition, the observation that heparin can induce agglutination of circulating red cells may be of interest. This alteration in the colloidal stability of blood is associated with the increased stickiness of suspended elements with associated intramscular clumping. This was thought by Naidoo and Gilman (1957) to promote phagocytosis; however, polyglutamic acid inhibits 1,olymorplioi~uc.lear ameboid activity and phagocytosis. I n contrast, a copolymer containing glutamic acid and lysine residues in a ratio of 7 :3 increases phagocytic activity. Serum glycoproteins which are altered with tumor growth are protective colloids which enable small amounts of normally insoluble compounds to remain in solution (Anderson, 1965). This could theoretically affect local tumor nutrition. The opposite is seen when heparin and heparinoids alone, or in the presence of bacterial endotoxins, prccipitate fibrinogen with decrease in the colloiditl stability of blood (Godal, 1960; Thomas et al., 1955). Similar effects have been seen for the anionic dye, Congo red, with associated release of scrotonin and the production of shock (Suzuki, 1964). Thus, in similar fashion to bacterial endotoxins, which can induce licparinlike prolongation of clotting time, the antitumor action of heparinoids could relate to circulatory insufficiency in the vascular portion of tlie tumor. Although these effects, which resemhlc 1)actcrial endotosin action, have becn seen clinically on the administration of heparinoids (Regelson and Holland, 1962), it is doubtful that they explain all the antitumor effects seen. However, a consideration of the effects of polyanions on the colloidal stability of a number of systems may be enlightening. XII. Colloidal Effects
Hydrolyzed ethylene maleic anhydride copolymers and related polyacrylic polycarboxylic antitumor and antiviral agents have been found to improve the quality of clay and silty loam soils by breaking them up
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WII,I,IAM REGELSON
illto brrlaller particleh whicll allows for aeration arid water dispersion without, iinduc formation of mud (Hedrick and Mowry, 1953; Quastel, 1952). The ionizable grorlpr on t,hese polyniers provide the hydrophilir portions of t h ( w m:~cron~oIeciiI(,s whirl1 mtlli(>thew water-wliihle. For soil conditioiiing, tllolecular weight must bc ill cxcess of :it least 10,000, and linear polymers are preferred. Used in excess in soils, the reverse effect occurs and soil can become hydrophobic and conglomerated. These changes occurring with rapidity in the circulation could account for changes in the distribution of formed elements in the blood accompanying the administration of certain macromolecules (“colloidoclasmic shock”). Colloidal exudates are responsible for “directed orientation” or adhesion of cells according to P. Weiss (1945). Disturbances in the production or physical structure of these exudates by polyanions could thus alter the growth characteristics of cells. As these exudates may be responsible for cell adhesion (coaption) which may be responsible for cell survival a t a given locus within the animal (Weiss and Andres, 1952), the action of polyanions as colloidal dispersing or aggregating agents could be critical to tumor cell survival and metastasis. On a cellular level, changes in the colloidal character of protoplasm could be utilized to explain Haddow’s paradox of carcinogenic or carcinolytic properties for the same agent, as has been done by Heilbrunn (1956a) and Wilson and Heilbrunn (1957). If polyanions could react with cell surface or cytoplasmic colloids in similar fashion as they do with clays, then similar alterations of the structural character of the cytoplasm or cell membrane could occur which would govern the functional capacity of the cell. This is an elaboration of Bancroft’s older concept (Bancroft and Richter, 1931a,b) which states that successive cell states of (1) excitation, (2) anrsthesia or inhibition, and (3) death are characterized by (1) incipient rcvcrsible agglomcration of protein, ( 2 ) a more advanced state of sgglomcration, and ( 3 ) irreversible coagulation. Crawley (1932) has elaboratcd on this in his interpretation that the cancer process represents an iricrcased peptiaation of cellular proteins. Using this hypothesis, thc nntimitotic action of the polyanion would result in conglomeration of cytoplasmic or surface colloids into hydrophobic bodies which would impede surface or interior exchange. This would be comparable physically to the surface precipitation phenomenon of Heilbrunn, since induced cell injury and/or cell death leads to colloidal changes within the cell that are supcrficially parallel to those observed in soil and, perhaps, related in similar fashion to collagen precipitates induced by polyanions (Morrione, 1952; Gross et al., 1952). In support of this, degraded carrageenin, a sulfated polysaccharide,
hinders the tlift’u~ioiiof pclwin through iriiiciri which serves to protect tlic stomach from ulceration (Anderson, 1961) and precipitates casein p:irticles in cow’s iiiilk (Stoloff, 1959). Also, changes in the character of niucinous material a t the cell surface could protect against enzyme action. Comparable to changes induced in soil by polyanions, Stahman (1955) has found that inhibition of pepsin and trypsin by polyglutamate can be reversed and converted to enhanced enzyme activity by an excess of the inhibitor; similnr findings have been made for polycations. In any colloidal system, particles with opposite charge will tend to flocculate out. Thus, the colloidal stahility of a cell depends on maintenance of liltc charge> for a vast ni:tjority of the particles in the cytoplasm (Anderson, 1956), nnd, as the ~)rcdoniinantcharge within the cell is negative and a typical cell consists of a series of polymerized negative charges (nucleic acids, sulfatcd polysaccharides, and protein with acidic isoelectric points), these can bc influenced by divalent cation and polycations of varying size such as histoncs and polyamines (e.g., spermidine, azgmatine, a r i d other multiply cli~rgedamines) . Anderson feels that solgel transformation in the cytoplasm is secondary to reversible crosslinking of underiatured macromolecules hy charge effects which can be influenced by the introduction of polyanions or a decrease in the quantity of polycations. The interaction between ionizable groups of a polyelectrolyte molecule can also result in contraction of the molecule by twisting of the molecular chain as a result of repulsion between neighboring groups (Lifson, 1958). The importance of the binding of polyanions is seen in the possible role of polyamines, such as agmatine, as promoters of cell division (Anderson, 1956). The polycation, agmatine, causes a reversible condensation of chromatin in living cells (Anderson and Norris, 1960; Whitfield et al., 1962), speeds up cell division in grasshopper neuroblasts (St. Amand e t al., 1960), rabbit epidermal explants (Gelfant, 1963), ascites tumor (Regelson and Hauschka, 1958) , and prevents postirradiation mitotic delay in tissue culture (Whitfield e t nl., 1962) which may be contrary to the action of polyanions. Similar growth-stimulating action has been found for the polyamine, spermine, in mammalian cells (Ham, 1964; Alarcon e t nl., 1961) and bacteria (Hcrbest and Snell, 1949; Mager et al., 1954). The interaction between polysaccharides and other rnacrornolecules is also seen with the changrs in osmotic pressure exerted by mixture of serum albumin with hyaluronic acid (Laurent and Ogston, 1963) . ,41bumin is excluded from the part of the solution occupied by hyaluronic acid wliich c\erts R ~)rofounrlinfluence on the wI2itioiisliips l w t n w n other mnci~omolrciil:~~~ solritcs an11 the solwnt.
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WILLIAM REGELSON
Similarly, neutral dextran solutions can significantly decrease the solubility of serum protein which varies only with the concentration of dextran (Laurent, 1963). Therefore, the concentration and character of the macromolecular sugar moieties a t the cell surface may govern the solubility of protein or other macromolecules that enter into the extracellular space or, a t the surface, where hyaluronic acid or related charged or noncharged polysaccharides are found. This could considerably affect cell nutrition, gaseous diffusion, or the action of surface enzymes, as is seen in the block by mucin to pcptic activity following the atlministration of carrageenin (Andcrson, 1961). Further support for the above is the obscrvation of Moore (1941) that relatively small increases of osmotic pressure prevent blastocoel invagination. Also, rouleaux formation was fclt by Fahraeus (1929) to reflect the action of hydrophilic proteins which desolvate the surface of red cells, raising surface energy and promoting adhesion. In this regard, Chargaff and Ziff (1939) have shown that cephalin forms insoluble emulsions with histone. Tlic strong interaction between the basic and the acidic protcins causes the expulsion of water and the production of an insoluble precipitate. Based on this, a possible role for histone or polycations in cell binding was observed by Schmitt (1941). Histone in similar fashion to polylysinc links rcd cells to form epithelial or morulalike structures. Therefore, displacerncnt of histone by polyanions from within the cell which then react with surfacc phospholipid could change the character of the cell surface and increase cell-to-cell of cell-to-glass or connective tissue binding. As discussed elsewhere, divalent calcium ions have similar effects. XIII. Hydrophilic Gels
Physical alterations of cells occur in the presence of polyanions. Fresh unfixed nuclei when treated with heparin show swelling and the release of DNA (Anderson e t nl., 1960). Nuclei of intact isolated liver eel1 suspensions show swelling on prolonged exposure to heparin. These alterations in the structure of the nucleus with the releaee of DNA have been attributed to displacement of bound nucleohistone from nucleic acid by the negatively charged heparin molecule. Detergents behave in similar fashion (Douncc and Monty, 1955). However, the formation of hydrophilic gels on exposure of isolated nucleated cells to polyanions is not necessarily associated with apparent swelling and disruption of the nucleuh. I n view of enzynic iiilii1)itioii I)y polyanions, n less likely alternative is a block to the tiction of cytop1:ismir or nuclear proteolytic ciizyiiit~sclwril~ctl I I I~ h u ~ ~ r( c Doui~rc.and h l o ~ ~ t y1955; , noiince and
Unama, 1962) which c:tn disrupt gel forn1;iLion or lcatl to loss of normal nuclear structure. The agglutination of nucleated chicken red cells by polyphloroglucinolphosphate was described by Penttinen (1956). More recently, Vaheri (1964) has correlated the antiviral action of a number of synthetic polyanions with their ability to induce clumping of nucleated chicken red cells and to alter the growth characteristics of HeLa cells on glass in the presence of serum (Vaheri, 1964). We have obtained (Tunis and Rcgelson, 1965), on the interaction of isolated washed nucleated cells with polyethylene or polystyrene sulfonate, the formation of gel-like mitcrial which entraps the cells. The interaction is tiiiic and tcmpcraturc dcpendcnt, and as it occurs, polymer is adsorbed to the cells. Unlikc the observations of Vaheri (1964) and Nordling e t al. (1965a,b), protein in the suspending medium interferes with the reaction. The gel has the “slimelike” character t h a t hioscona (1960) has called “extracellular material” (ECM). The gel produced by the polyanions is readily disrupted hy pancreatic DNase (DNase I) in the presence of Mg++ and is similar to the effect of DNase I on E C M produced by exposure of nucleated cells t o trypsin (Steinberg, 1962). Intact nuclcated cells were necessary for gel formation; however, treatment with DNase I readily disrupted the gel, caused morphological changes in the nucleus, and released D N A into the medium. This DNA mntcrial rcmained in the supernatant solution following sedimentation of the cell residues, but, unlike purified D N A digested by DNase I, was completely insoluble in 5% trichloroacetic acid. Exposure of cells to the polyanions results in the leak of ribonucleic acid and protein, with a decrease in Feulgen positivity in the nucleus, and Feulgen-positive material has been seen to leak from the cell surface with contraction of the nucleus. Native heparin could not form gels with isolated cells, and gel formation depended on the high-molecular-weight fractions studied and the electronegativity of the polyanions. This was similar to Vaheri’s (1964) observation regarding the antiviral action of polyanions. The simultaneous presence of nucleated cells and DNase I Mg++ followed by the polyanion resulted in a reduction of cell clumping and the complete inhibition of gel formation, while DNase 11, RNape, Iysozyme, and hyaluronidase were without cffect. Polyanion-induced gels are similar to what has becn seen in E D T A disaggregated amphibian embryos which form clear jelly between the cells a t or above pH 9.8. However, a t lower p H values, the EDTAinduced intercellular jelly in the presence of Ca++is dissolved by trypsin
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WILLIAM REGELSON
:111(1 ]{N:lhC(C:ui.tis, 1958), :111(1 t r y 1 ) h - 01’ i,il)ou~iclcasc-tl.c’:ltctl dih:iggl-eg:itccl ( ~ ( ~f:lil l l ~ to r(ylggi*cg:ttcb.B:ts(vI 011 t l i c + c s ohc~rvatioii~, Curtis felt tllat riboiiucleoprotcin was rcsl)onsiI)le for surface-mediated adhesion. Of possible importance, the gel (ECM) material obtained from calciumfree, saline, disaggregated cclls can induce differentiation of pigmellt cells (Wilde, 1962). The observation of Garber (1962) that ~-glucosaminccan prevent the reaggregation of trypsin neural-retinal cells is of interest. Whether the polyanion-intluced gels have similar character has not 11ecn explored. I n regard to the character of the gel formed by the interaction of polyanions with intact cells, it is difficult to study this independently of nuclear material, as any attempt t o separate the gel from the interiningled and clumped cells results in cell destruction with release of nuclear contents. Polyanions with or without lysozomal action could create stress which produces holes in the surface membrane similar to that produced 1)y antibody or polylysine on the red cell (Katchalsky, 1964). Metachroniatic gels have also been obtained from muscle fibers (Metin) which are precipitated by calcium ions and which are thought to be a part of the actomyosin system (Szent-Gyorgyi and Kaminer, 1963). Whether those gels resemble the homogeneous 7 S soluble proteins isolated from sea urchin eggs which form gels with low concentration of calcium (Kane and Hersh, 1959) or the “plasmosin” of Beazley et al. (1954), the character of which niay be related to the presence of nucleohistonc or related proteins with fibrillar structure similar to myosin, has not been determined (Schmitt, 1941; Mirsky and Pollister, 1943). The fact that gels induced by polyanions require nucleated cells suggests that a similarity to mitotic spindle protein should be looked for if the loss of nuclear DNA is not a factor. T h e correlation of the antiviral action of polyanions with clumping effects on nucleated cells has been made by Vaheri (1964) and is fascinating in view of their interferon-inducing capacity (Regelson, 1967; Merigan and Regelson, 1967). Similar gelling or clumping phenomena may also occur in plants. For example, during the germinative growth of white mustard seetllings, acidic pectic polysaccharides appear which are associated with the ability of cell walls to swell markedly. Some of the acidic polysaccharides formed are readily aggregated in the presence of calcium ions, and cation bridging aftcr germination may result in thc eventual strengthening of the cell wall (Gould e t al., 1965). The gel-likc character of the teleost yolk layer has been thought to be responsible for epiboly governing the growth and development of the
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G R( )R’T I 1 H K(i1111 A T I N (
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I)lastoderrii. Tlic gclh uutlcrlaying cells I i a w bccii sliowii to govcrii their growth and orientation in iiorinal morphogenesis (Holtfreter, 1944; Townes and Holtfreter, 1955), and polyanionic-induced gels could have similar function. The physicochemical charactcristics of gels relates to their ability to iinbibc many times their volume of water without loss of structure. As previously discussed, acetyltryptolhan is a very effective inbibition agent for biological gels, causing an increasc in water-binding capacity and lowering the melting point of gels (Feigen and Trapani, 1954; Fels and Greco, 1961). Humic acid found in tumors is a condensation product of tryptophan aldehyde which is formed on exposure of tryptophan to heparin; thus heparin might act indirectly to affect the physical characteristics of the gel-like matrix that supports cells. Using the precipitation of protein within clear r a t Iiver brei incubated a t 40°C. as the index for sol-gel alteration, Anderson e t al. (1960) found that ATP, heparin, RNA, glucose, high p H , and high NaCl concentration prevented this effect. Based on this, they felt that in vitro “denaturation” within the cell was dcpendent upon the level of polyanions which in tumor activates or inhibits cnzymes which govern first reversible and then irreversible aggregates. In their model system, the maintenance of cellular integrity would depend on the interaction of polyanions vs. the cations of which the latter would work toward denaturation of suspended protein. Support for the above may reside in our recent observation t h a t nucleated cells suspended in polystyrene sulfonate show displacement of histone from the nucleus (Regelson and Roque, 1966). This is seen in cells serially stained with Fast Green safranin (Roque’s stain) which show histone disappearing from the nucleus and appearing within cytoplasmic vacuoles and a t thc cell surface-bridging cells. As indicated by Chargaff and Ziff (1939), this frcc histone can interact with surface phospholipid to bind cells together (Schinitt, 1941). XIV. Surface and Enzyme Activity
The formation of solid enzyme complexes which possess different kinetic qualities from the parent dissociated enzymes has bearing on what may be occurring in cell membranes. Thcsr solid cnzymes fixed to spccifir sitcs 1)y lmidiiig to plyailions might li:ivc bpwific function a t g i t w loctitioiis w t h i i i t Ii(, wII or on tlic. rt.11 ~ i i i f a w .T>isplaccwoit from I)oridiiig \vould i z l t c ~ if‘utictioiinl ~ ciapacity of tlie cc.11 id, pohsihly, produre cell (Ioiitli I)y :wtiiig I)eyontl thc confiiir.s of Iioi*m:tl rcgul:ttion, as has becw proposed for lysozyoinnl ciizyines. Of particular interest was the synthesis of an ethylcnc 1ii:tleic anhydridc copolynicr-trypsin prcpara-
tion. Trypsin was covalently bound to a water-insoluble polyelcctrolyte gel (Levin et al., 1964; Goldstein e t al., 1964). If this were to happen in v i m , following the administration of ainmoniated EMA or vinyl/MA antitumor or MA antiviral copolymers, it may explain their biological activity. For this enzyme complex, the charged carrier protein affects the distribution of charged low-molecular-weight substrates between the gel phase and the external solution. The pH activity profile of this enzyme polyanion complex was displaced 2.5 pH units toward more alkaline pH values a t low ionic strength, whereas a t higher ionic strength, pH optima shifted toward more acid pH values (Goldstein et al., 1964) as compared to the parent enzyme. The authors feel that this results from an effect of the clectrostatic potential of the polyelectrolyte carrier on the local concentration of hydrogen ions and from positively charged substrate inolcculca in the microenvironment provided by the polyanionbound enzyme molecules. L. Weiss (1962, 1963) gives data which suggest that optimal values for enzymes may be different a t the cell surfaces, as compared to an in zritro system dissociated from the intact organism. This relates to postulated differences in p H values that may be found scattered in local regions of high charge density where pH would be lower than in the intermediate spaces on cell surface. L. Weiss (1962) feels that polyanionic cell surface components could hinder the outward diffusion of enzymes. Enzymes may exist as structural components a t the cell surface linked covalently to structural polyanions which exist as part of the cell surface or between the cells. Thus the ethylene maleic anhydridetrypsin complex may provide a model for what is true as relates to carboxylic groups and enzyme complexes a t the cell surface. Changes in the physical characteristics of our postulated cellular solid enzymes would immediately alter enzyme kinctics and affect cell functionality or viability. The work of Katchalski’s group a t RehovotEi (Katchalski, 19621963) and others (Bernfeld and Wan, 1963; Quiocho and Richards, 1964) who are forming solid enzyme complexes is, therefore, of great interest. The effect of polyanions on the enzyme complexes may shed light on the niechanism of action for the biological action of polyanions. The systemic administration of these enzyme complexes and their biological activity have had minimal trial (Sicuteri et al., 1962) and further exploration is cwrnti:tl. The crystal1iz:rtion of tobacco mos:ric virus which was o1)taincd by S. S. Cohen (1942) from solutioii with heparin may be pertinent to the dynamics of solid eilzyme formation. Whetlier polpanions could do the samp to cnzymes a t a given cell site and whether crystalline enzyme
stru(btures \\Tit11 a]terp.tl functional capacity are ~ ~ ~ ~ s :it i i al ~cell l c sllrface, is bpeculatioii, b u t the validity of tlic ideas i h also hiiggehtcd by the resistancc of solid cnzynios to cIohicc:tt 1011 :ind tl(w~tiir:ition i n similar fashion to vii.uscs. Polyethylene sulfonnte can affect chiwicteristic hydrolytic cleavage patterns for peptides and proteins (Kern and Scherhag, 1958). This may be due to the charge density of the polymer which maintains the position of the charged groups in relation to the substrate in the presence of a mineral acid. Polymers react much faster with small molecules than do their lower-molecular-weight monomeric analogs. For example, carboxyls from polycarboxylic acids displace bromine from a-bromoacetamide more quickly than do carboxyls from mono- or dicarboxylic acids (Ladenheim et al., 1959). The effect of one neighboring reactive group on another is influenced by a polymeric configuration, and this action of polymers is similar to factors govcrning enzyme substrate affinity (Morawetz and Westhead, 1955). This observation is supported in Sicgel’s (1963) recent review regarding “The Macromolecular Environment as a Factor in the Control of Chemical Proccsses.” Siege1 presents data regarding a wide variety of proteins, e.g., globulin or albumin, polysaccharides or synthetic polymers on the crihancement of a number of enzymic reactions. This is not restricted to water-dispersible macromolecules; for example, keratin, fibrin, and cellulose all exhibit appreciable activating effects of a peroxidwe-catalase reaction. I n support of this, the colloidal character of the environment can enhance enzyme activity since cytochrome c activity is greatest in phosphate gels (Keilin and Hartree, 1949), and electron transfer is facilitated by the ordered structure of synthetic polyglutamic acid (Kopple e t al., 1962). Intramolecular hydrolysis of some organic compounds is 10‘ and loG timcs as fast as an intermolecular reaction. In regard to factors governing these hydrolysis reactions, the stercochemical structure of polyacids is fundamental, and differences in the internal stiwcturc of copolymers can govern this reactivity. In support of this, equilibrium rncasurements have shown that isotactic polymethacrylic acids bind Ca++ions much more strongly than the atactic form. Therefore, differences in the shape and internal organization of polymers can play an important role in directing the rates of particular reactions (Smets, 1962). The nonspecific replacement of DNA by polyethylene sulfonate which permits the continued amino acid incorporation into isolated nuclei (Allfrey and Milasky, 1959) may be an example of enzyme enhancement by polymeiic surface interactions. This enhancement or inhibition of enzyme reactivity wliicli is governccl hy macromolecules indi-
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WILLIAM REGE1,SON
cates tlic importuncc~of tlic cell m:itris i n bylithetic procesacs. Another example of this may bc bee11 in the ahility of pect,ic acid to mimic 11ormaI plant, poIysncrharicIe surf:zrw in the inductioii of 1'77 ~ l t f r olignill forlllatioli (SipgpI, 1963) . This nncl otIi(8i- (.virhic.r ~ull'ly clclllolbtl'atc tllat specific rc:hctjious c:ill I)c c:ttaIyzctl Ly tllc ininiecliatc 111:~cI~oIl1olccular enviroiirnent prcsentcd by the varied surfaccs within the cell. Polymers of critical size interacting with cell surfaces may provoke lysis of the red cells (Katchalsky, 1964) as described by Charache et aZ. (1962) for silicate polymers both with and without associated rcd cell agglutination. Surface alterations are seen in that synthetic polypeptides, which contain 50% more glutamic acid over lysine residues, increase the rate a t which red cells take up and release oxygen (Blout e t al., 1962). I n addition to effects on enzyme reactions, polyanions can enhance antigenicity if linked to protein or block the action of antibody, depending on its position in relation to the active sites of antibody or antigenic determinants. Polyanions can prevent antibody inactivation of phage (Mora and Young, 1962) or, with lysine (Maurer e t al., 1959; Maurer, 1962), can be made weakly antigenic. Heparin and suramin complex with a variety of proteins (Gorter and Nanninga, 1953), and the complexing of proteins with polyelectrolytes has been regarded as an extension of the use of polyvalent ions for protein precipitation. The stability of the complcxes formed may be related to the flexibility of the polymer chain, permitting a close approach of its ionic charges to the opposite charges of the protein separation (Morawetz arid Hughes, 1952; Whistler and Spencer, 1961). Thc efficiency of protein precipitation can increase with the chain length of the polymer (Polson e t al., 1964). The converse is also truc as concanavalin A has been used to precipitate and isolate polysaccharides (Cifonelli et al., 1956). Hyaluronic acid markedly effects the partition of diffusable niacromolecules between the polysaccharide and buffcr (Laurent and Ogston, 1963). Support of a steric exclusion cffcct is found in a n cspci~inicnt using an uncharged dextran polymer up to a minimum sizc. The larger the protein molecule the less soluble it was in the dextran (Laurent and Ogston, 1963). This may play a role in the ability of heparin and other polysaccharides to precipitate protein and viruses (Cohen, 1942). I n this regard, partition of proteins is readily obtained using a liquid polymer-polymer two-phase system (Albertsson, 1958), a technique which enables one to separate and isolate viruses (Philipson e t al., 1960). This technique has also been usrd to separate whole cells, re11
LWIIS, :tnd inicroso1iics. The separation proccdures depend on the surface properties and size of the particle to be isolated. I n the case of virus isolation, dextran sulfate, niethylcellulosc or polyethylene glycol led to the isolation and purification of T2 phage, adenovirus, influenza, parotitis, Newcastle disease virus and ECHO viruses. B y analogy, this phenomenon may be important for the entry and distribution of viruses and/or protein within the cell or a t the cell surface. Other physical effects of polyanions that are of biological importance might depend on the presence of dipoles in polyanionic macromolecules which could cause directed movements perpendicular to biological memhranes. Katchalsky (1964) has suggested that these dipoles may act as organizing elements which could bring biocolloids together in orderly array. I n support of this, significant electrical potential is developed by the displacement of hyaluronic acid (Christiansen et al., 1961) and piezo- and photoelectric effects are obtained from bonc crystal (Bassett and Becker, 1962); clectric current has been found to dircct the orientation and organization of bone growth (Bassett et al., 1964). B y proper use of high polymer systems, Katchalsky (1964) dcmonstrated that chemical energy can be transformed into mechanical energy. Kuhn (1952) has shown that filaments made of polyacrylic acid and polyvinyl alcohol can produce mechanical energy equal to that of muscle fibers. Contraction occurs on the addition of acid, and relaxation on the addition of alkali. The stretching effect is due to electrostatic repulsion of polycarboxylic charges which are fixcd on the ionized chain of the molecule. This could be pertinent to changes in the physical state of protoplasm, and, if polyanions could adlierr to reactive groups on the cell surface or enter within the cell and these be subjected to mechanical changes, this could result in physical alteration or disruption of the cell or cellular elements. Furtheimore, the extension of polymer groups at a cell surface could impede the entrance of essential nutrients or block viral fixation or release to and fi-om the cell surface. XV. Enzyme Inhibition and Activation
The concept of the mitotic inhibitory role of polyanions was suggested by Heilbrunn and Wilson (1948) to be analogous t o their role in :inticoagulation. However, the antimitotic action of polyanions may be mediated through inhibition or activation of many enzymes independent of those immediately involved in hlootl clotting. Converscly, the biological action of enzyinea roul(1 he exerted I Jtheir ~ rt.Ieaw of liepariii or Iicy:ri*inoitl~(Aloiikhousc, 1955 ; Quick, 1956) . Of 1mrticuI:~ri i i t o t w t i r tlic ~yi~tlicsis of solid enzvnirs, somc of thcin
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WILLIAM REGELSON
attached to synthetic polyanions such as ethylene maleic anhydride copolymers, which possess kinetic properties different from the parent soluble cnzyme (Katchalski, 1962, 1963). I n recent reviews, Engelberg (1963) and Bcrnfeld (1963) have ably discussed the action of polyanions as inhibitors of enzyme action. Bernfeld has classified the macromolecular polyanionic enzyme inhibitors into three main groups: sulfate esters and sulfones, phosphate esters, and polycarboxylatcs. For these compounds, enzyme inhibition has been shown for acid phosphatase, alkaline phosphatase, p-glucuronidase, aamylase, hyaluronidase, lysozyme, RNase, DNase, trypsin, chymotrypsin, pepsin, lipoprotein lipase, fumarase glyceraldehyde phosphate dehydrogenase catalase, elastase, and fibrinolysin. Additional enzymes inhibited by polyanions, but not mentioned in the Engelberg and Bernfeld reviews, are adcnylic deaminase (Diamond, 1955) phosphoprotein phosphatase (Paigen, 1958), lecithinase, arginine esterase (Floch and Groisser, 1962), and deoxyribonuclcotide transferase (Back, 1964). I n addition, alcohol dehydrogcnasc, pyruvate kinase, glutamine dehydrogenase, glutathione reductase, and glucose-6-phosphate dehydrogenase, all NADand NADH-dependent enzymes, are also inhibited (Horn and Bruns, 1959). Vishniac (1950) has found that sodium tripolyphosphate inhibits hexokinase. The anticomplement activity of heparin and related polyanions has been described (Mustaars and Lison, 1948; Storti and Vaccari, 1956; navies, 1963) and may reflect polyanionic inhibition of esterases in this enzyme complex. The action of surface-active agents such as sodium dodecyl sulfate will not be considered in this review, although Bernfeld (1963) feels that they behave similarly to polysulfonates andlor other macromolecular polyanions. Sulfated mucopolysaccharides are secreted in large quantities by gastric mucosa (Schrager, 1963), and antipeptic action is not only a direct anti-enzyme effect but may reflect the action of the polyanion in decreasing the availability of the enzyme by altering the permeability of the mucin through which the enzyme must pass (Anderson, 1961). Thus changes in the availability of enzymes a t cell surfaces could be affected by polyanions. Heparinoids inhibit peptic activity (Nutr. Rev., 1962; Bianchi and Cook, 1964), but, paradoxically, low concentrations of heparin activate and high concentrations inhihit proteases (Ungar and Mist, 1949). Similar fibrinolytic a i d proteolytic activity has hcen srcn for mucopolysaccharidcs (Garber, 1962). Anlolig thc anionic. t1yc.s witli provcn tilnlor iullibitory :kct,ivity,
GROWTH-REGULATING
ACTIVITY O F POLYANIONS
277
surainin has been shown to inhibit a number of enzymes (Town et al., 1950; Wills and Wormall, 1950). Thc behavior of suramin and related polysulfonate dyes resembles heparin or the heparinoids in that fumarase (Quastel, 1931) as well as RNase (Grubhofer, 1955) and hyaluronidase (Caneghem and Spier, 1955; Beiler and Martin, 1958) are inhibited. Suramin inhibits urcase (Town et al., 1950; Wills and Wormall, 1950), hexokinase, carboxylase, succinic dehydrogenase, proteolytic enzymes, choline dehydrogenase (Wills and Wormall, 1950), and trypsin (Beilinsohn, 1929). There are two patterns of enzyme inhibition inherent in the activity of suramin (Wills and Wormall, 1950). One group of enzymes which includes urcase, invertase, peroxidase, and catalase is inhibited on the acid side of their isoelectric points. Wills and Wormall feel that the action of the polyanions is to form a ])ridge between basic groups on the enzyme to block the active center. This activity is entirely related to the size of the polyanion and is extremely sensitive to slight changes in pH. Simihr findings wcre niatlc by Hummel et al. (1958) for inhibition of acid phosphatase hy polyphosphates. The other group of enzymes inhibited by surainin a t neutral pH or alkaline pH includes hexokinase, carboxylase, and succinic and choline dehydrogenase. Morawetz and Westhead (1955) found that the reactivity of p-nitrophenyl ester groups in neutral solution was increased by a factor of a million when the groups were attached to acrylic acid copolymers. They have considered polyelectrolytes with reactive side chains as the models of the enzyme-substrate complex. This model is pertinent where a low degree of enzyme specificity indicates that only a few polar groups of the enzyme, rather than the detailed configuration of its molecular surface, participate in the activities of the substrate. Such a low specificity is seen with many esterases (Hofstee, 1954). In analogous fashion to the observation of Morawetz and Westhead, the acceleration of biological reactions by polyanions could impede normal cellular processes through substrate competition or inhibition, or provide mechanical problems of storage and disposition. The mechanism of action for enzyme-polyanion inhibition is not critical for this review, hut what may be pertinent is the specificity of the inhibition scen. Some polysulfonates are more potent inhibitors of RNase than others (Fellig arid Wiley, 1959). Similar findings have been made for acid phosphatase (Hummel et al., 1958), lysozyme (Mora and Young, 1959), and hyaluronidase (Rcrnfcld e t nl., 1961; Ferno et al., 1953). We littire ~ I i o w n inhihition DNase TI activity hy polyethylene siilfoti:ite :1w1 ctliplc~nctn:lleic niilrp~li~itle copoly~nerswithout inhibition
of DNase I ('runis ant1 Rcgclson, 1965). The relationship between the
acidic character of the polymers arid TlNase I1 inhibition is of interest. On a molar basis, the charge on the molecule apparently enhances the enzyme-inhibiting potential of the polymer. Similar observations wcre made by Back (1964) for RNase and deoxyribonucleotide transferase wherein maximum inhibition for polyethylene sulfonate was found for fractions of mol. wt. above 12,900. The effect of a variety of polyglucose suIfuric ester preparations on the inhibition of lysozyme, hyaluronidase, and RNase were studied by Mora and Young (1959). They found that inhibition was due to electrostatic interaction forces between oppositely charged macromolecules and that inhibition was reversed by polycations stronger than the substrate. Similar findings have been obtained by Vanclendriessche (1956), and, in an excellent review, Person e t al. (1963) discussed the inhibition of cytochrome oxidase by polycations with reversal of inhibition by polyanions. They suggest that electrostatic bonding by polycations vs. polyanions may control oxidative phosphorylation. Papain has been found to release heparin on systemic administration (Monkhouse, 1955; Quick, 1956). The inhibition of enzymes by polyanions suggests that polyanion release by enzymic action could serve as a control mechanism to shut off further enzymic activity. A search for this pattern of enzyme release and inhibition would be of interest. XVI. Respiratory Enzymes
The effect of polyanioris on cell respiration is of interest because of the observations of Roux and Callandre (1950a,b) that ester smide polyphosphates of thiamine substitute for cocarboxylase and that sodium polyphosphates inhibit cocarboxyIase activity. Exposure of red cells to the ester amide thiamine polyphosphate or t o sodium polyphosphate results in a marked decrease in the amount of lactic acid formation on incubation (Roux and Callandre, 19CiOa,b, 1952). This was confirmed in vivo by a decrease in the production of lactic acid and pyruvic acid in the blood of fatigued dogs givcn the thiamine ester amide polyphosphate. In contrast, sodium polyphosphate given similarly, produced lethal convulsions (Roux e t al., 1951). In pursuing these observations, Roux e t al. (1955) found that hexokinase was inhibited and that the character of the polyphosphates governed their action on glycolysis. The diphosphate accelerated glycolysis, whercns the triphosphate inhibited glycolysis. The inhibition of cocarboxylase and hexokinase is sinlilar to what has been seen for the anionic rosaniline derivtitivc IWills n ~ l ( lWorm:lll, 1950), and the :irlthws >glY1rwith ~ ' k 1 ~ 1 r ~ :(~19.50) c ' s hiIggc1.stiou t,h,zt, t?te rncc]rn-
~ ~ R O ~ ~ T I - I - R E ~ ~ UACTIVITY L A T I ~ G O F POLYANIONS
279
nibin of iiiliihitioii of liesoliiiixw iuay be R competition of the polyphobphatc for the I~indiiigof RIq" with ATP. In othrr i ~ ~ r l:rmiuo i, s i i j i a i s w t w most rft'cBc-ti\t s J a1111hyalilronic~aciil, hepiriii, a i i i l choii(lroitiii mlf:tttn I c s h artivc' :IS iiihihtors of :iscites tumor NADH c1i:iplioritscs M hicli, Iliiidiicr (1961) felt, MWC reqoiisible for the cytocidal activity of these compouiids. XVII. Hyaluronidase and Glycosidases
Eridogenous hyaluroiiidase activity in tumors has been associated with tissue necrosis in the study of Balazs and Von Euler (1952). They discuss the importance of the mast cell as a possible factor in the secretion of hyaluronic acid or heparin. The presence of heparin as a hyaluronidase inhibitor has been stressed by Asboe-Hansen (1950) a i d may be of importance to tumor growth in view of the increased presence of these cells around the border of proliferating tumor tissue. Hyaluronidase was shown to affect the spread of tumors in the studies of Hoffman et al. (1931), Gopal-Aycngar and Simpson (19471, and Russo and Terranova (1953). Kamei and Nagoya (1964) maintain that the presence of endogenous hyaluronidase in tumors may play a role in tumor growth and/or invasion. Their contention is supported by the work of Grossfield (1961) and the recent observations of Saldgen (1965). Duran-Reynals and Stewart (1931) found that tumor extracts could enhance the spread of local vaccinia lesions in the rabbit, and later found that local vaccinia infection could enhance and localize the development of methylcholanthrene-induccd skin tumors in mice (DuranReyiials and Duran-Reynals, 1952). Hyaluronidase can account not only for the intracutaneous spreading of colloidal particles but also the spread of vaccinia, fibroma virus, and Virus 111 (Duran-Reynals, 1928; Sprunt et al., 1938; Sprunt, 1950; Pearce and LaSoike, 1954). Shope fibroma virus administered to rabbits simultaneously with hyaluronidase accelerated tumor development, whereas an aiitiliyaluronidase agent decreased or prevented tumor dewlopment through EL local host reaction (Pearce and LaRorte, 1954). Locally, hynluroiiic acid can inhibit granulation reactions (Csermley ant1 Curri, 1958), and in another area hyaluronic acid has bccn found to inhibit influenza and Semiliki Forest virus on in vitro mixing (Warren, 1965). Similar phenomena may be important for virus tumor induction, and, thus, the inhibition of hyaluronidase by polyanions could conceivably play a role in tumor development and growth. However, Pirie (1942) was unable to obtain more virus following tlepolymerization by hyaluronidnse of the viscous hya-
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WILLIAM REGELSON
luronic acid of Rous and Fuginomi tumor. More recently, the administration of a hyaluronidase inhibitor (polyphloretin phosphate) diminished the spread of Rous rat sarcoma to pwitonral lymph nodes (Saldeen, 1963) . However, tiyaluroniditsc did not increitse the perineahility of the peritoneal cavity to Rous sarcoma cells (Saldgen, 1965). The spread of infectious agents can a t times favor the host or the infecting organism (Duran-Reynals, 1942). This has been studied by Sprunt (1950) who found that, whereas hyaluronidase decreased the violence of a local cutaneous staphylococcal infection, i t increased the spread of virus infection. This phenomenon was altered by the hormonal environment which affects the physical characteristics of ground substance. I n addition to polyanions, antihyaluronidase and anticoagulant activity has been shown for phosphorylated hesperidin (Beiler and Martin, 1948; Sheppard e t al., 1954) and related flavones, but these do not possess antitumor activity against the Sarcoma 180 test system (Sheppard, 1956), although flavinoids have direct cytocidal activity. Attempts to correlate antiviral action with inhibition of hyaluronidase and RNase were unsuccessful (Heymann et al., 1958) for polyphenolic polyanions. I n similar fashion, Vaheri (1964) also found that polyanion inhihition of these two enzymes did not coincide with viral inhibition in any meaningful way. The invasiveness of three subcutaneous tumors in contrast to their ascitic intraperitoneal form was found by Carr (1965) to correlate with marked increase in ,8-glucuronidase activity. This enzyme has been shown to be inhibited in vitro by heparin (Bernfeld, 1963). Thus heparin or related inhibitors might exercise a controlling effect on connective tissue breakdown and penetration by tumor. I n addition, the tanning of skin is facilitated by extraction of the ground substance or by prior depolymerization of chondroitin sulfate by testicular hyaluronidssc (Burton and Reed, 1962). Tanning increases the resistance of tissue to bacterial or viral action (MonnB, 1961), and hyaluronidase may have a role in the regulation of this process. Also, as radiation results (Balazs, 1960) in depolymerization of ground substance, the action of radiation on connective tissue andjor malignant growth could represent an acceleration of the tanning process in conjunction with the release of polyphenols. XVIII. Ribonuclease
Heparin (Lorenz et al., 1960) and numerous heparinoids inhibit both the ribonucleases (Fellig and Wiley, 1959; Zollner and Fellig, 1952, 1953; DeLamirande et al., 1954, 1956; Roth, 1953a,b; Vandendriessche,
1956; Houck, 1957; Houck et al., 1957; Huiiimel e t al., 1958; Hcyrnallll et al., 1958; Stock and Dierick, 1959; Mora and Young, 1959; Dickmari, 1955; Littauer and Sela, 1962; Gaerther and Lisiewicz, 1962; Lorcnz at!., 1960; Back, 1964) responsible for depolymerization and for the opening of the ring structure of cyclic nuclcoside phosphates of RNA. Paff e t al. (1952) pobtulated that a possible niechanisiii for the inhibition of mitosis by heparin is due to the inhibition of cellular RNase which was associated with an increasc in intracellular ribonucleic acid. Elevation in RNase and DNase activity has been associated with normal ccll proliferation (Brody and Thorell, 1957; Brody and Balis, 1959), and inhibition of these enzymes may conceivably result in prevention of cell division. However, increascs in these clegradative enzymes are not found in most tumors (Brody aiid Balis, 1958; Brody, 1958), and thus a l t e r n a t i i ~explanations regarding their importance in the inhibition of tumor growth may be necessary. Preincubation of rat liver homogenates with RNase abolishes the capacity of these homogenates to metabolize mevalonic acid. This is reversed by anionic polymers including polyethylene sulfonate, heparin, DNA and even RNA, which can inhibit the RNase enzyme in vitro (Wright e t al., 1960). I n wivo dosage of heparin compatible with survival can change tissue RNasc activity and inhibit serum RNase (Lorenz e t al., 1960). Hepatic alkaline and acid RNase activity was decreased significantly in mice following heparin administration a t anticoagulant dosage, altlioiigh correlation with the anticoagulant effect was not clear (DeLamirande et al., 1956). Ribonuclease produces marked mitotic abnormalities in onion and lily root tips (Kaufmann and DCS,1954). This is of particular interest in view of the possibility that RNA constitutes up to 55% of the isolated mitotic apparatus and may represent a true structural component of the spindle. Brachet (1957) feels RNA is a major component of intercellular matrix in amphibian ectoderm, and Lindberg and Ernster (1954) have suggested that rihonucleic acids may act as a cement substance in mitochondria in view of the degeneration of mitochondria on exposure to RNase (Zollinger, 1950), a finding supported by the observation of Curtis (1958). Reagents that interact with sulfhydryl groups increase the activity of RNase in the liver which, Roth (1953a,b) felt, may be due to inactivation of n liPt,:iiin-cyPtc.inc tissuc inliihitor. If tlw factorh that go\.ern the oi1sc.t of ccll division following fcrtilization of sen urchin eggs bear any relationship to mitosis in general, then RNase and its inactivation 1)y polyanions may l)e of critical importance. Ribonuclease activ:ites proteolytic enzymes ancl IlXnse (T,eh-
et
inail e t ul., 1962) aud c:tn inducc cytolysis ill the oocyte with 1)twipit:ition of the jelly coat substance. Riboriuclcase is present in the developing sea urchin egg (Bernstein, 1949; Lindvall and Carsjo, 1955), and it has hccn found that RNasc remarkably increases the proteolytic activity of ciizymes obtaincd from sea urchin oocytcs. These RNase-activated proteolytic cnzyines :ire activated by the basic protein, clupeinc, and inhibited by heparin, fucus polysaccharitlcs, and the related polyanionic jelly coat substance. Acid phosphatase is found in the spindle and thc metachromatic beta granules which participate in spindle formation. This enzyme has been shown to interact with RNase to accelerate the hydrolysis of RNA (Schmidt et al., 1947), and, thus, the inhibition of acid phosphatase or RNase by polyanions may be pertinent to spindle formation as well as ribosomal integrity. Ribonucleic acid, as is true for heparin and other polyanions, retards the in vitro heat denaturation of liver protein, whereas RNase has the opposite effect (LePage, 1949; Anderson et al., 1960). This may be important to the structural integrity of the cell and changes in the availability of polyanions may affect the functional capacity of the ribosome as heparin can displace ribosomal RNA (Anderson and Wilburt, 1950). Ribonucleic acid uptake by cells has been reported (Schwarz and Rieke, 1961). This has been found to influence embryonic morphogenesis (Ranzi et al., 1963; Niu, 1963; Niu et al., 1962; Hillman and Niu, 1962). Most recently, RNA uptake (Helenine) has been found to induce interferon production (Lampson et al., 1967; Tytel et al., 1967). The uptake of RNA can he enhanced by protamine (Amos and Kearns, 1963) and, theoretically, antagonized by polyanions. Similarly, uptake of DNA with transformation of mammalian cells or interferon production has also been rcported (Szybalska and Szybalski, 1962; Field et al., 1967), and, thus, inhibition of RNase or DNase by polyanions could play a significant rolc in embryogenesis, informational transfer, or host resistance to virus. XIX. Deoxyribonuclease
Deoxyribonuclease activity of cellfrce extracts of Escherichia coli can be increased on exposurc to pancrcatic RNnsc. nnd Lehnxan e t al. (1962) Iiaw (IescriLetl a DNaw with eridoiiucleabe activity in E . coli which is inhibited by KNA. If DNase activity is essential for cell proliferation (Brody, 1958; Brotly and Balk, 1959; Goutier e f nl.. 1960), then inhibition of RN:ise hy polyanions could, as Paff et al. (1952) have postulated, prevent mitosis through the accumulation of
ItNA iii1iiI)itorh of I )N:ihe. IIo~vcvcr,iiihi1)ilion of DNase activity Etas been ohtainc.tl for p01y:iiiioiih iiirlepcmtcnt of cffccts on RNase or increases in RNA (Rotli, 1953a,b; Tunis xncl Regelson, 1963; Heyrnann e t al., 1958), and no correlation was obtaincd by us between the level of DNase I1 nctivity (Regelson e t al., 1960) and the degree of tumor inhibition pi otluced by polyanions. I n the liver and spleen, administration of dextran sulfate or polyethylene aulfonate was associated with an elevation of DNase I1 activity that did not relate to morphological change. I n regard to this observation, it is of interest that heparin results in an increase in hepatic mitosis (Zininierman and Cclazzi, 1961) as well as in a rise in circulating Iyniphocytcs (Cronkite et al., 1962). A growth-stimulating factor has been obtained by Braun (1961) from A variety of bacterial and tiiinial DNA’s degraded by DNase I. This material albo promoted the growth of mammalian cells in tissue culture and, under certain circuinstances, could both promote tumor development or delay the oiisct of spontaneous mammary tumors in C3H mice. Recently (Braun and Fershein, 1967) this has been shown to be due to the action of oligonucleotides. Chevreniont (1961) found t h a t fibroblasts exposed to DNase I1 inhibited DNA synthesis. Bacterial DNase inhibits ascites tumor cell development whcn given iiitraperitoneally (Belyaeva e t al., 1964). More appropriate to tumor growth and induction is the recent observation that DNA transferase obtained from KB cells is inhibited by polyanions (Back, 19641. This enzyme may he important to viral synthesis within the cell. Independent of direct effects, the action of oligodeoxyribonucleotides and synthetic polyiiucleotides in promoting interferon activity, phagocytosis, and immunological reactivity (Ficld e t al., 1967; Braun and Nakano, 1966; Braun and Fershein, 1967; Braun, 1967) may be protected by tlic DNase-inhibiting activity of polyanions. XX. Polyphosphates
Like polysulfonates, polyphosphates inhibit a number of enzyme systems (Ebel, 1959), particularly cocarboxylase (Vishniac, 1950 ; Roux e t al., 1951, 1955; Roux and Callandre, 1950a,b, 1952). Sodium tripolyphosphate inhibits the anaerobic conversion of glucose by intact yeast cells, yeast zymase preparations, or crystalline hexokinase. This inhibjtion can be removed by ATP or Mg”’. In synchronized bacterial systcins the accumulation of polyphosphate is found prior t o onset of logarithmic growtli, and in a wide variety of bacteria, fungi, yeasts, dyes, and protozoans, L‘volutiongranules” are found which stain metachromatically. These are thought to contain polymetaphosphate. The formation
284
\\III,LIAM RECELSON
of this nict:tchromatic iiiatcrial usually occurs during the end of the active growth phase. Polymetaphosphatc has been thought to provide a storage compound for the availability or storage of phosphate or energy in similar fashion to creatine or arginine phosphate, and in Corynbacterium diphtheriae its accumulation correlates with the cycle of cell division. The action of uranium and divalent cation in inhibiting the glucose metabolism of yeast and bacteria is believed to be mediated via complexing with polyphosphates a t the cell surface (Rothstein and Meier, 1951). Polymers of phosphoric acid have detergent activity as they are able to form stable soluble complexes with most cations. Exploration of a possible role for polyphosphates as metal chelators in the treatment of metal poisoning led to a study of their metabolic fate in rabbits. Linear phosphate polymers were hydrolyzed to ortliobiphosphate in rats and rabbits while cyclic polymers were recovered intact in the urine. Pharmacologically, systemic polyphosphates in mammals show similar toxicity to polysulfonates on systemic administration (Ebel, 1959; Gleason et al., 1963), but there systemic degradation (Ebel, 1959) may make them less toxic on prolonged administration. Calcium pyrophosphate is phagocytized by macrophages and can produce lysomal breakdown with subsequent synovial or mesothelial inflammation resembling that seen in gout (“pseudogout”) (Riddle et al., 1966). This has been found to be pertinent to joint destruction and inflammation and similar phagocytic uptake or polyanions and phagosoma1 leakage of enzymes could be responsible for tumor destruction. The action of polyphosphates as inhibitors of respiratory enzymes is discussed in Section XVI. XXI. Lipase and Esterase Activity
Polyanions have both inhibitory and activating effects on lipolytic enzymes (Stahman, 1962; Bernfeld et al., 1963; Cronheim, 1964). In addition, heparin dissociates phosphatides from thromboplastic proteins (Chargaff et al., 1950). Therefore, any study of the antitumor and antiviral action of anionic polyelectrolytes might profitably include an investigation of lipid and phospholipid or lipases, esterases, and the associated problems of lipid metabolism in tumor growth. The role of lipids in maintaining membrane structure has been reviewed by Wolpert (1960), and Bell (1962a,b) has postulated that polysaccharides stabilize the cell membrane. Lipoprotein lipase enzyme inhibition is caused by all those polysaccharide sulfates that contain a t least 0.6 sulfate groups per repeating unit as long as they are free from sulfamino groups. Sulfamino groups
contribute significantly to the ability of polyanions to activate lipoprotein lipase. Low-molecular-weight substances with sulfamino groups fail to activate the enzyme, and N-sulfatyl groups appear to be essential for enzyme activation (Bernfeld, 1963). Inhibition of aliesterase activity has been associated with selective in vitro inhibition of malignant lymphoblasts, germirlating seedlings, tubercle bacilli (Mendel e t al., 1953), and bacteria (Smith e t al., 1949). Esterase activity in cancer patients is depressed, but, following heparin injection, it may be increased substantially beyond that of controls (Skorepa e t al., 1958). In this regard human plasma lipoproteins in women with advanced h i cast cancer differ from normal controls (Barclay et d., 1955; Peterman e t d., 1958; Kaufinann e t al., 1955, 1959). There is less a-lipoprotein in cancer patients with significant elevation of SF 11-20 and 21-100 components, which is altered toward normal in response to castration (Kaufmann e t al., 1955) and heparin administration. The interaction of polyanions with lipoproteins has been described by Gurd (1960). Low density lipoproteins (1.035 and less) are precipitated by anionic polysaccharides. These have incIuded suIfated dextran, amylopectin, dextran, heparin, and polygalacturonate. Factors affecting solubility of polyanion-lipoprotein complexes are the degree of polymerization, the ionic strcngth, and the prescnce of divalent cations. A critical factor appears to be the high inolecular weight of the polyanion as the reaction occurs a t neutral pH valucs where the net negative charge of both the polyanion and the lipoprotein complex is the same. Fatty acids formed following heparin or polyanion activation of lipase action, aside from serving as metabolic substrates, also have an orienting function as monolayer films and, thus, may govern morphogenesis (Rosenberg, 1963). They show narcotic activity (White and Samson, 1956) and also are bacteriocidal (Nieman, 1954), fungicidal (Kenny e t nl., 1944; Kenny, 1959), and hemolytic (Breusch and Bodur, 1950; Greisman, 1955). I n addition, fatty acids also inhibit a number of glyrolytic enzymes in yeast (Samson e t nl., 1955) and anions of the higher fatty acids have R high affinity for lipoproteins as well as serum albumin (Gordon, 1955). ‘In reviewing the action of short-chain fatty acids on yeast metabolism, Samson et 01. (195.5) discusped the possible surfactant action of fatty acicls. IIolhti (1958) obs ctl that olvic arid proniote(1 the cutaneous carcinogenic action of tlimetliylbenza~~thracene in mice. The electroh t i c binding of fatty acids to enzymes or other proteins possibly accounts for their tneclinnisni of action, and Hotcl~kiss (1946) attributed
the disorganization of cell membranes to a binding of fatty acids by components of the cell membrane. I n this regard, butyric acid can induce parthenogenetic cell division in sea urchin eggs (Moore, 1930; Motomura, 1960; Sugiyama, 1953) when followed by hypertonic sea water, and this was associated with induction of ribosomal amino acid incorporation independent of the presence of the nucleus (Denny and Tyler, 1964). Bennett and Connon (1957) found that Ehrlich ascites tumor cells were vulnerable to the lytic action of a variety of fatty acids. Also, fatty acids may give rise to extensive respiratory inhibition in carcinoma cells (Scholefield et al., 1960; Scholefield, 1958; Weinhouse et al., 1953) which could explain some of the action of polyanions on cell surface. Heparin has been shown to inhibit the hemolytic action of lecithinases, and heparin and other sulfated polysaccharidcs can increase resistance t o the lethal effects of Russell’s viper venom (Higginbotham, 1962; Condi et al., 1964) and other snake venoms (Condi et al., 1964) ; phospholipid is an important stabilizer of serum lipid emulsions (Ahrcns and Kunkel, 1949). All of which may be of importance to the integrity of the cell membrane. A structural interrelationship between lipid and connective tissue is found in the observation that the elastase group of enzymes possesses lipase activity with release of polyanions (Saxl, 1957, 1961). Yu and Blumenthal (1958) have shown that an acid polysaccharide attaches itself to elastic tissue and protects it from proteolytic enzymes. I n another area the action of a shark liver lipid fraction which stimulates the RES and increases the resistance of animals to Rous sarcoma and Friend leukemia (Heller, 1965) is of interest in view of the possible effects of polyanions on host resistance. XXII. Clinical Antimitotic Side Effects, a n d Clinical and Experimental Antitumor Activity
I n another review (Regelson, 1968a) we have outlined the clinical side effects of heparin, reflected in antimitotic activity as seen in alopecia, alteration in fingernail growth, or diarrhea, which may represent inhibition of epithelial growth. Similarly, alterations in bone growth by heparin and heparinoids may represent effects of polyanions on a proliferating tissue. Of greater pertinence to our discussion is the antitumor activity of polyanions. This too has been discussed in detail in our previous paper (Regelson, 1968al. This includes anionic dyes as antitumor agents, and the effect of heparin sncl related anticoagulants on metastases. Similarly, wc have discussed t8hc g~owth-inhi\)itingaction of the anionic
inorganic lieterol)olyiiiolyl,dates :IS well as the action of polyphosphates. The antibacterial and antiviral action of polyanions has also been disc u w d hy this author in the same paper. XXIII. Summation
We have outlined the role of polyanions as growth inhibitors and discussed the biological evidence for their role in fertilization, morphogenesis, and control of normal growth. The place of mast cells as sources of the native polyanion, of heparin, and thcir role in malignant transformation and growth deserves a separate review. The evidence has been presented elscwhere for polyanionic antitumor action (Regelson, 1968a), and in this paper we have not discussed their role in controlling metastases nor the place of anionic dyes as antitumor agents, nor the antibacterial and antiviral action of these and related compounds (see Regelson, 1968a). This review has discussed thc action of polyanions as controllers of enzyme action and presented their theoretical relationships to ATP, the cell membrane, surface charge, cell adhesion, and calcium. We have outlined the possible importance of surface polysaccharides and the place of polyanions in colloidal stability and the formation of hydrophilic gels. I n this paper we have not discussed, nutritional, and informational transfer, and mitochondria1 effects nor have we mentioned in detail antihormone action, antiinflammatory, and immunological effects, nor the action of polyanions on biogenic amines (see Regelson, 1968a). The effects of these agents on components of the intercellular matrix, and the dynamics of protoplasmic change have been presented with emphasis as to thc importance of polyanions in the dynamics of cell multiplication in normal and malignant growth. Regardless of the above, the recently rediscovered importance of polynnions in governing both resistance to hacterial and virus infection and thcir enhancing role in immunological respoiisiveness makes ccrtain that further work in this area will lcad to increascd understanding of mechanisnis which will lead to the development of useful clinical agents.
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MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY OF AROMATIC COMPOUNDS. NEW PERSPECTIVES’*’ Joseph C. Arcos and M a r y F. Argus Seamen’s Memorial Research Laboratory, U. S. Public Health Service Hospital, N e w Orleans, and the Department of Medicine (Biochemistry), Tulane University School of Medicine, N e w Orleans, Louisiana
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I. Introduction . . . . . . . . . . . 11. Condensed Polycyclic Aromatic Compounds . . . . . A. Polycyclic Aromatic Hydrocarbons and Hydrogenated Derivatives B. Pyrolytic Formation and Environmental Occurrencc . . . . C. Heterocyclic Compounds . . . . . . . . . D. Metabolism . . . . . . . E. Present Status of the K-Region Hypothesis . . . . . . F. Noncovalent Interactions of Polycyclic Aromatics . . . . G. Evidence for Hydrocarbon Free Radicals . . . . . . 111. Conjugated Arylamines and Compounds Generating Arylamines. Arylhydroxylamines . . . . . . . . A. Arylamines and Arylnitro Compounds . . . . . . . B. Amino Azo Dyes . . . . . . . . . . . . C. Detoxicating Metabolism . . . . . . . . . . D. Activating Metabolism : Present Status of tlie ortho-Hydroxylation Hypothesis; the Carcinogenicity of N-Arylhydroxylamines . E. Free Radicals in Arylaniine Carcinogenesis. Interactions Which Appear to Be Nonwvalent with Prott.ins and Nncleic Acids . . IV. Covalent Binding to Proteins and Nucleic Acids . . . . . A. Polycyclic Hydrocarbons and Tricycloquinazoline . . . . B. 4-Nitroquinoline-N-Oxide . . . . . . . . . . C. Arylamines and Amino Azo Dyes . . . . . . . . Referenws . . . . . . . . . .
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305
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308
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363 363 392 400
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412
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427 433 433 435 436 454
308 320 . 323 .333 . 339 . 344 . 362
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I. Introduction
Since the time of appearance of a previous article on the “Molecular Geometry and Mechanisms of Action of Chemical Carcinogens” (Arcos Reference material available to the authors through October 1967 has been considered in the preparation of this article. For a selective summary of more recent literature up to September, 1968, see Note Added in Proof on p. 448. Investigations in the authors’ Laboratory are supported by the U. S. Public Health Service Research Grants CA-05431 and CA-05793 from the National Cancer Institute, and in part by Grant H-6769 from the National Heart Institute. 305
306
.JOSEPH C. ARCOS AND M A R Y F. ARGUS
and Arcos, 1962) , iniportaiit, dridopniciits h a w twkeii placc iii many study arcas of chemical rarrinogenesis. One of the fields whercl these devc I opnicn t s o s p r i~a I 1y rapid is t hr st ru c t I I r+ac t i v it y ix.1 a t i )n ships arid nietabolisin of conjugatcd aromatic coinpouiids: polycyclic hydrocarbons and heteroaromatics, aromatic amines, and azo compounds. Moreover, study of the biological mechanisms of action of these carcinogens has allowed an everdeepening insight into the nature of their interaction with proteins and nucleic acids, and the structure of their protein- and nucleic acid-bound forms. It may not be undue optimism to say that we are a t present passing the threshold beyond which the effect of carcinogens on cell morphology, gene expression, and templates of macromolecular synthesis may be described in precise molecular terms. The survey of Buu-Hoi (1964) on polycyclic hydrocarbons and related heterocycles brought to a conclusion the first chapter in the search for the mechanism of action of these agents. Subsequent work by Nagata et al. (e.g., 1966a,b) and by E. C. Miller and Miller (1967), Boyland and Sims (1967), and Sims (1967b) appears to give definitive indication that these compounds are active as such without previous metabolic activation. Moreover, the elegant flow dichroism studies of the Japanese investigators resolved an important controversy on the nature of the in vitro interaction of polycyclic carcinogens with DNA. On the other hand, for the carcinogenic activity of the aromatic amines related to 2-naphthylamine, 4-nitroquinoline-N-oxide, 2-aminofluorene, 4-aminobiphenyl, the amino azo dyes, and 4-aminostilbene, the requirement of metabolic N-hydroxylation provides the likely unifying theme (E. C. Miller and Miller, 1966; J. A. Miller and Miller, 1966). Yet, the N-hydroxy derivatives appear not to be the ultimate active metabolites, since various 0-esters of these are more carcinogenic than the free hydroxy compounds themselves and also more reactive to bind to proteins and nucleic acids (Lotlikar et al., 1967a; Poirier et al., 1967). However, as is often the fate of successful hypotheses, the complete generality of the requirement of N-hydroxylation for amines is challenged by such findings as ( a ) the N-hydroxylation of 2-naphthylamine by the bladder mucosa (Uehleke, 1966b, 1967) in which organ this amine is inactive by pellet implantation (e.g., Bonser et al., 1956b), ( b ) the questionable carcinogenicity of subcutaneously administered 2-naphthylhydroxylamine in newborn mice (Roe et al., 1963; Walters et al., 1967) which are notoriously sensitive to carcinogenic stimuli, (c) the potent and ubiquitous carcinogenicity of 3-methyl-2-naphthylamine toward local and distant tissue targets (Shenoy et al., 1964; J. H. Weisburger et al., 1967a), and ( d ) the in vitro N-hydroxylation of 2-aminofluorene by liver microsomes of the guinea pig (Kiese e t al., 1966) which species is refractory to w
m
b
MOLECULAR GEOMETRY A N D CARCINOGENIC ACTIVITY
307
carcinogcnesis by this aniinc. Moreover, Kiese and Wiedemann ( 1966) and von Jagow et al. (1966) showed t h a t guinea pigs injected intraperitoneally or intravenously with 2-amino- or 2-acetylaminofluorene excrete the N-hydroxy metabolite in the urine. An increasing body of evidence indicates t h a t a variety of carcinogens bind covalently t o both proteins and nucleic acids, and the results of two reports suggest an approximate parallelism between carcinogenic activity and binding in the target tissue to proteins (Abell and Heidelberger, 1962) and to D N A (Brookes and Lawley, 1964b). The question whether binding t o one or the other type of macromolecular components is of critical importance is probably a moot one and may be resolved in terms of a cybernetic hypothesis of carcinogenesis (in Arcos, 1961; Arcos et al., 1961; Arcos and Arcos, 1962), as it becomes increasingly evident that carcinogens represent a class of nonspecific cell poisons with affinity to ii wide spectrum of cell components. A corollary of this hypothesis is that, in addition to covalent binding, noncovalent interactions of various types must play major roles in the interactions of carcinogens with cell coniponcnts (cf. Arcos and Arcos, 1956; J. A. Miller and Miller, 1963) which support pathways of metabolic control and information transfer. Following the demonstration by Argus et al. (1 961 ; see also Argus et al., 1966a,b; Bemis et al., 1966) of the effects of carcinogens on the conformation of model proteins, different authors described noncovalent interactions of carcinogens with nucleic acids and proteins (e.g., Kaye, 1962; Boyland and Green, 1963; Troll et al., 1963a; Watters and Cantero, 1967). Just as the 1962 review (Arcos and Arcos, 1962), the present article does not claim to be a detailed presentation of the literature. What it does attempt is t o give a “bird’s eye view”-from the time of closing the references for the 1962 article to the p r e s e n t o f the carcinogenic activity of aromatic compounds and their interactions with cell components, in relation to their molecular geometry. B y ‘(aromatic carcinogens” are meant here those compounds in which the aromatic skeleton is believed to play an exclusive or major role in the tissue interaction leading to carcinogenesis. A4ccordingly,compounds such as nitrogen mustards, lactones, epoxides, and nitroso compounds, bearing aromatic substituents, are not classified as “aromatic carcinogens,” since in these the aromatic moiety merely modulates the reactivity of the critical functional groups and confers to the molecule some degree of tissue target specificity. The need and, indeed, urgency for the periodical scanning of the literature continues to increase not only because of the unprecedented mushrooming of the discovery of new cilrcirrogens, but a h lmause of the consider;il)lo tlccycning : t i i d refinemciit, oi tliffercnt f:twts of tlw
308
JOSEPH C. ARCOS AND MARY F. A R G U S
mechanisms of action. The difficulty arising from the coriiplexity and vast volume of the literature has been compounded by the regrettable interruption of publication of the famous compilations by Hartwell (1941, 1951) and Shubik and Hartwell (1957), and of Ca,rcinogenesis Abstracts which began in 1963 (1963-1965). However, an equally important positive event is the appearance of an enlarged English edition of Clar’s (1964) unique treatise on the chemistry and physical chemistry of polycyclic hydrocarbons with a chapter on carcinogenesis by Schoental. Also, the concepts and techniques of testing chemical agents for carcinogenic activity have been exhaustively reviewed and discussed by J. H. Weisburger and Weisburger (1967) and by Arcos et al. (1968). II. Condensed Polycyclic Aromatic Compounds
A. POLYCYCLIC AROMATICHYDROCARBONS AND HYDROGENATED DERIVATIVES 1. Structure-Activity Relationships
Notwithstanding that the structure-activity relationships of the hydrocarbons have attracted interest for well over a quarter of a century, a number of reports appeared on this subject during recent years. Lacassagne et al. (1962) reported that the 1,2-benzanthracenes, monomethyl-substituted in the angular ring are noncarcinogenic or have trace activity, a t most. This agrees with Dunning and Curtis’ (1960) confirmation of the finding of the early workers (see Shubik and Hartwell, 1957). The inactivating effect of methyl substituents in the angular ring of simple l,2-benzanthracene derivatives has already been known. Recently, Lacassagne and his co-workers have found that this rule holds also in more complex molecules, the steranthrenes, closely related to l,2-benzanthracene. Whereas F-norsteranthrene (I) is highly carcinogenic (Lacassagne
(1)
(11)
e t a,?., 1963cl), its 8-niethyl derivative (11) is an inactive compound (Lacassagne et al., 1966). It is of greater significance, however, that in their 1962 study, Lacassagne et al. confirmed the iippreciable carcinogenicity of 3- and 4-11~etl1yl-l,2-benza11tl~~accnc. Sincc thew positions
MOLECULAR GEOMETRY A N 0 CARClNOGENIC ACTIVITY
309
occur in the meso-phenanthrenic region, this finding has an important bearing on the K-region hypothesis (see also Section 11,E). Heidelberger e t al. (1962) have demonstrated that contrary to earlier data (see Hartwell, 1951) 9,10-dimethyl-1,2,5,6-dibenzanthracene is a strong carcinogen. Thus, tlie general rule that dimethyl substitution of 1,2-benzanthracene-type n~olecules in the 9,lO-positions enhances carcinogenicity seems to hold without exception. Another study of the carcinogenicity of 1,2-benzanthracene and 3,4-benzophenanthrene derivatives is due to Stevenson and von Haam (1965). Unfortunately, the latent periods were not given in their report and the scaling of potency can be qualified a t most as semiquantitative. This report indicates, nevertheless, that the 4'-fluoro derivative of 1,2-bcnzanthracene is a mediumpotent carcinogen to the uterine cervix of the mouse. It has been known for some years that the parent compound, 1,2-benzanthracene, considered by the early workers to bc noncarcinogenic or only weakly active, may initiate skin tumorigenesis or induce sarcomas, hemangioendotheliomas, and occasional hepatomas in special experimental conditions. Recently, Klein (1963) described the production of a high incidence of pulmonary adenomas and hepatomas in BGAF,/J hybrid mice if treatment with the agent was instituted during infancy. Considerable effort has been devoted by Buu-Hoi, Lacassagne, and their associates to the synthesis and testing of fluoranthene hydrocarbons and of large-size hypercondensed benzenoid hydrocarbons composed of a t least six rings. The existence of potent carcinogens in the fluoranthene series was first noted by Wynder (1959) and Wynder and Hoffmann (1959). The more recent results of Lacassagne e t al. (1963a) confirm and extend these observations (Table I). While it is truc that these compounds are nonalternant hydrocarbons and do not possess true K-regions, the compounds which possess an angular distribution of the rings similar to that of the phenes are more active than those which do not, as illustrated below: n ,-----
lball epithelioma index: 59
--
%all epithelioma index: 15
T ~ I U3,4Y , m(1 l O , l l - l e i i z o f l u o i ~ ~ ~ ~(~I V t l ~and ~ i ~V) e :we tlie most active of the series, tlie potency of the former approacliing that of 3,4-benzopyrene. The weak r:ii*cinogenirity of the 11,I 2-isoiner (VI) is abolished wlmi thc 11,12-beiizo ring is methyl substitutcd (VII) or hydrogenated
310
JOSEPH C. ARCOS AND MARY F. ARGUS
TABLE I
Carcinogenic Activities of Fluoranthene Hydrocarbons" Avg. latent period (days)
Tumor incidence
16 14
d ? (20 ?
130 133 120
66 83 95
V
(20 ?
160
95
VI
16 6 14 ? (20 ?
203 210 330
50 45 50
Compoundb
IV
No. and sex of mice used
8 \
(8)
mall index
50
62 79)C
25 21 15)'
311
M O L E C r L A R GEOMETRY AND CARCINOGENIC ACTIVITY
TABLE I (contirzrwd)
Compoundb
No. and sex of mice used
Avg. latent period (days)
Tumor incidence
(W)
ball index
0
0
VIII
M
X
d ?
0
0
14
0
0
16
d
0
0
0
0
d
0
0
14 ?
0
0
15
14
15
312
JOSEPH C. ARCOS AND MARY F. ARGUS
(14 1 4 $?
XII&
265
71
145
7
27 5)
a Except w h e r e otherwise indicated, the data a r e compiled from Lacassagne e l a/. (l863a) and Buu-Floi (1964). The Iball indexes a r e based on the s a r c o m a incidences obtained in XVII nc/Z s t r a i n mice of both s e x e s (Paris Radium Institute).
The compounds listed a r e : fluoranthene 011); 3,4-benzofluoranthene (IV); 10.111lI12-benzofluoranthene (VI); l'-methyl-ll,12-benzofluoranbenzofluoranthene 0 ; thene (VII): 11,12-tetramethylenefluoranthene (VIII); 3,4,11,12-dibenzofluoranthene (IX); 3,4,11,12-bistetraniethylcncfluorant!iene (X): 2,13-benzofluoranthene (XI); 2,3phenylenepyrene (XII), Data summarized from the r e s u l t s of Wynder (l859) and Wyndcr and Iloffmam (1958) obtained using skin painting on random-bred female Swiss mice ; the Ihall
indexes w e r e based
00
the epitheliomn
+ papilloma
incidence.
" Papillomas only.
eSimilar activity level has been observed by Hoffmnnn and Wynder (1966).
(VIII). No comparable information is available on the effect of substitution or hydrogenation of the benzo ring in the 3,4- or l0,ll-positions. Although both 3,4- and 11,12-benzofluoranthene are carcinogenic to a greater or lesser degree, 3,4,11,12-dibenzofluoranthene(IX) and its octahydrogenated analog (X) are totally inactive. That annelation of an additional benzene ring to the highly active (IV) yields the moderately active 2,3-phenylenepyrene (XII) suggests that the inactivity of (1x1 may be related to a molecular size too large in this series. The weak activity of 2,3-phenylenepyrene, established by the Paris group via subcutaneous route, was confirmed by Hoffmann and Wynder (1966) in epithelial testing.
IWOLECIILAR CEOMETRY .\NU C A R C I N O ( X N 1 C ACTIVITY
313
Thc itlea of Scliniidt (1957) tliat thc G- and 12-pobitions in aiithtiiithrene arc coiiiparable to meso-anthraccnic positions such as in 1,2-
e
10
Anthanthrene
1,2-Benzanthracene
benzanthracene has been substantiated. I n fact, the inactive anthanthrene acquires weak carcinogenic activity by one methyl substitution in position 6; activity is further enhanced by n second meso-methylation leading to G,12-dimethylantlianthrene (Lacassagne et al., 1958). As in the 1,2-beneanthracene series, a ,meso-methyl group may be replaced by a formyl group with no loss of carcinogenicity (Lacassagne et al., 1961b). Of great theoretical interest are the recent investigations with largesize polycyclic hydrocarbons. Five dibenzopyrene isomers are possible (Table 11).Of these, 3,4,8,9-dibenzopyrene ( X I I I ) and 3,4,9,10-dibenzoTABLE I1 The Isomeric Dibenzopyrenes
pyrcwe (SIV) have lwen known to Iw highly potent in intlucing subcutaiieous s:wconi:is. L:ic:~ss:~giieet al. ( 19631)) have now sliowii that
1,2,3,4- and 1,2,4,5-(libcnzopyr~iie(XV and XVI) are also potent sarcomatogens, the former bcing less active than the latter. On the other
314
JOSEPH C. ARCOS AND MARY F. ARUIJS
hand, 1,2,6,7-dibenzopyrene (XVII), the only compound having no meso-phenanthrenic region, is inactive (Buu-Hoi, 1964). I n skin painting experiments by Hoffmann and Wynder (1966), the four carcinogenic dibenzopyrenes proved generally to be less active than 3,4-benzopyrene1 both as complete carcinogens and as tumor initiators (with croton oil as promoter). l12,6,7-Dibenzopyrene is inactive also in epithelial application. Naphtho[2,3: l12]pyrene, which may be regarded as an intermediate structure between 1,2,6,7-dibenzopyrene (XVII) and 1,2,3,4dibenzonaphthacene [ (XXII) ; see below] , is noncarcinogenic. It has been known for some time that substitution in the mesoanthracenic positions (especially by methyl groups) considerably increases carcinogenicity in the I,%-benzanthracene series. I n the dibenzopyrene series, however, substitutions in rquivalent positions produce an opposite effect. It has been previously reported that substitution of 3,4,8,9dibenzopyrene (XIII) in position 5 (or 10) and substitution of 3,4,9,10dibenzopyrene (XIV) in position 5 (or 8) by a methyl or a formyl group bring about a decrease of carcinogenic activity. Disubstitution in 5,lO and 5,8, respectively, by two methyls or by a methyl and a formyl group causes total loss of activity (Lacassagne et al., 195710, 1958, 1961b). As in the anthanthrene series, a methyl and a formyl group are about equivalent in terms of their effect on the activity of the parent hydrocarbon. The 5,lO- (or 53-) positions in the two dibenzopyrenes are equivalent to meso-anthracenic positions. Thus, 3,4,8,9-dibenzopyrene may be hydrogenated accordingly :
/ Lacassagne e t al. (1964b) extended the validity of this finding to 1,2,3,4dibenzopyrene (XV) in which substitution by a methyl or especially by a formyl group in the meso-equivalent 5-position causes decrease of activity. In the same study, methyl substituents were also found to cause the decrease of activity of ll!2,4,5-dibcnzopyrcnc when introtluctd in a latrral ring r e g , tlic 1,2-t)enz ring in (XVI) 1. Pyranthrene (XVIIT), 1,2,4,5,8,9-ti~ibt~i~zo~)yr~nr ( X I X ), and its 2’methyl dwivative ( S X ) are t,lw largest-size hytlrocai~l~ons tested for carcinogenic activity, so far (Lacnssagne et nl., 1961a, 1964b). These are highly insoluble compounds, and a large proportion of the subcutaneously
(xvnr)
(XM)R = H (XX)R = CH,
injected material rcmains unresorbed in the test animals even after several hundred (lays. The only inactive coinpound is pyranthrene. The tribeiizopyreiies ( X I N ) and (XX) are weak to moderately potent carciiiogciis toward tlie subcutaneous tissue of XVIIiic/Z strain mice of both sexes routinely u b e d for subcutaneous testing of polycyclic compounds in the Paris Radiuni Institute. Obviously, however, although hydrocarbons of even larger size synthesized and tested later may prove to possess trace carcinogenic activity, a molecular size limit must exist beyond which carcinogenicity totally ceases, as was attested years ago by the total inactivity of subcutaneously implanted graphite (Lacassagne e t al., 1957a). Of the above three large-size hydrocarbons only pyranthrene proved to be inactive, which is, surprisingly, also the only compound having two typical nzeso-plienniithrenic regions. Other large-size hydrocarbons display carcinogenic activity, howerer, dcspite tlie absence of meso-phenanthreriic region (s) (Buu-Hoi, 1964). One of these is 5-methyl-1,2,3,4dibcnzanthracene ( X X I ) . The uiisubstituted lxtrent hydrocarbon has repeatedly been found inactive by previous workers (see Hartwell, 1951; more recently, Heidelberger et al., 1962) or a t most possessing trace activity (see Hartwell, 1951). The structural rationale of the carcinogenicity of (XXI) is unknown. However, since nicthyl substitution in position 5 abolisheb the structural symmetry of the parent compound, this instance recalls the suggcstion of Lettr6 ( 1944) that carcinogenic activity is much more frequent in structures which bhow geometric asymmetry. I n addition to ( X X I ) , two lai,ge-size carcinogenic hydrocarbons (XXII
316
JOSEPH C. ARCOS AND M A R Y F. ARGUS
Incumbrance area
i'
FIG.1. The critical molecular size-range of condensed polycyclic aromatic hydrocarbons for carcinogenic activity. When available, the average of the sarcoma and epithelioma (fpapilloma) Iball indexes were used. I n a number of cases, because of the unavailability of data, the indexes were based on epithelial or subcutaneous tumor incidence only. For the purpose of comparison all hydrocarbons were regarded as being coplanar. Key to numbers (compound) and type of tissue tested: 2 , Naphthalene (skin only) ; 2, anthracenc (skin and subcutanrous) ; 3, acrnaphthenc (skin only) ; 4 , fluorene (skin and subcutaneous) ; 5, phenanthrene (skin and subcutaneous) ; 6, pyrene (skin and subcutanrous) ; 7 , naphthacene (skin only) ; 8, fluoranthene (skin and subcutaneous) ; 9, 2,13-benzofluoranthene (subcutaneous only) ; 10, perylene (skin only) ; 11, 1,Z-benzofluorene (skin and subcutaneous) ; 12, 3,4-benzofluorene (skin only) ; I S , chrysene (subcutaneous only) ; 14, 3,4-benzophenanthrene (skin and subcutaneous) ; 16, 1,2-benzanthracene (skin and subcutaneous) ; 16, 5-methyl-1,2-benzanthracene (skin and subcutaneous) ; 17, 8-methyl-1,Z-
MOLECULAR GEOMETHI’ AND CAHCINOGENIC ACTIVITY
317
and XSIII) Iiiivc been tlescrilecl rccently. Tlicsc compounds, which may be regarded n s highcr benzologs of 1,2,3,4-dihenzarit~iracene,are not s:trcomatogenic in situ, but have protluccd ovarian tumors and leukemia ill mice (Buu-Iloi, 1964). Because of the carcinogenicity found with large-size hypercondensed polyeyclic hydrocarbons in recent years, thc correlation between molecular size and carcinogcnic activity shown in the previous review (Arcos and Arcos, 1962) had to be updated. Figure 1 indicates that the optimum size of 2 1 2 0 A’, first concluded by Arcos and Arcos (1955) appears to be a solidly established molecular parameter. However, the size a t which Immtnthracene (skin and subcutaneous) ; 18, pentacene (skin only) ; I S , triplienylenc (skin only) ; 20, 1,2-benzopyrene (skin only) ; 21, anthanthrene (skin and subcutaneous) ; 22, 3,4-bcnzopyrene (skin and subcutaneous) ; 23, 10,l I-benzofluoranthene (skin only) ; 24, 1,2,3,4-tetramethylphenanthrene(skin only) ; 25, 9-methyl-l,2-benzsnthraccne (skin and subcutaneous) ; 2G, 4’-methyl-l,2-bcnzantliracene (skin and subcut:meous) ; f l , l’-methyl-l,2-bcnzanthra.cene (skin only) ; 28, piccne (skin only) ; 29, 11,12-benzofluoranthene (skin and subcutaneous) ; 30, 3-methyl-l,2-benzantl1racene (skin and subcutaneous) ; 31, 7-methyl-l,2-benzanthracene (skin and subcutaneous) ; 32, l0-mcthyl-1,2-bcnzanthracene (skin and subcutaneous) ; 33, 4’-mrthyl-3,4-benzopyrene (subcutancous only) ; 34, l‘-methyl-11,12-benzofluoranthenc (subcutaneous only) ; 35, 1,2,7,8-dibenzanthracene (skin only) ; 36, 2‘-niethyl-l,2-benzanthracene (skin and subcutaneous) ; 37, 1,2,5,&dibenzophenanthrene (skin and subcutaneous) ; SS, 5-.methyl-3,4-benzopyrene (subcutaneous only) ; 39, 9,10-dimethyl1,a-benzanthracene (skin and subcutaneous) ; 40, 1,2,5,6dibenzanthracene (skin and subcutaneous) ; 41, 3’-methyl-1,2-benzanthracene (skin and subcutaneous) ; 42, 6methyl-l,2-benzanthracene (skin and subcutancous) ; 43, 3,4-benzofluoranthene (skin and subcutaneous) ; 44, 1,2,3,4-dibenzopyrcne (subcutaneous only) ; 45, coronene (subcutaneous only) ; 46, 1,2,3,4-dibenzophcnanthrene(skin and subcutaneous) ; 47, 3(or 4)-methyl-1,12-bmzopeg.lme (subcutaneous only) ; 4S, 6-methylanthanthrene (subcutaneous only) ; 49, 6,12-dimethylanthanthrene (subcutaneous only) ; 50, 4methyl-1,2-benzanthraccnc (skin and subcutaneous) ; 51, 2,3-phenylenepyrene (subcutaneous only) ; 62, 9-methyl-3,4-benzopyrene (subcutaneous only) ; 53, 20-methylcholanthrene (skin and subcutaneous) ; 54, 1,2,6,7-dibenzopyrene (subcutaneous only) ; 66, 3,4,8,9-dibenzopyrenc (skin and subcutaneous) ; 56, naphtho12’,3‘:3,41pyrene (subcutaneous only) ; 57, 3,4,9,10-dihcnzopyrene (subcutaneous only) ; 68, 5methyl-3,4,9,10-dibenzopyrrne(skin and subcutaneous) ; 59, 5,&dimethyl-3,4,9,10dibenzopyrene (skin and subcutaneous) ; GO, 1,2,4,5-dibenzopyrene (subcutaneous only) ; 61, 1,2,3,4-dibenzanthracene (skin and subcutaneous) ; 62, 5-methyl-3,4,8,9dibcnzopyrene (subcutaneous only) ; 63, 5,10-dimethyl-3,4,8,9-dibenzopyrcne(subcwtaneous only) ; 64, 9,10-dimcthy1-1,2,5,6-dibenzanthracene(skin and subcutancous) ; 65, 9,1O-dimethyl-1,2,7,8-dihenzanthracene (skin and subcutaneous) ; 66, 2’,l’-anthra1,2-anthrarrne (skin only) ; 67, 3,4,11,12-dibenzofluoranthenc(subcutaneous only) ; GS, 15,16-~~rii~odt~hydroc~liolsn t,llrcnc (subcutanrous only) ; 69, phenanthro [2’,3’:3,41 pyrene (subcutaneous only) ; 70, 4,5-benzo-10,11[1’,2’-naplitholchrysene(skin only) ; Y l , 1,2,4.5,8,9-tiil~e11zo]iyrrne (subcutaneous only) ; 7 9 , pyranthrenr (subcutaneous ( ~ i i l , c . r t t i t i i c t i i i s oilly) ; ?4. gr:~i~hile only) ; 78, 2’-1~1ctliyl-1,2,4,5,8,’3-1i~il~cr1zc1~~~~r~~1io (s:ul)cii tanroris impl:int,)
318
JOSEPH C. ARCOS A N D MARY Y. ARGUS
carcinogenicity vanishes is notably more flexible than it was thought earlier. This suggests that for very large-size polycyclic carcinogens, such as the tribenzopyrenes (XIX) and (XX), and for the carcinogenic tricycloquinazolines (Section II,C,2) interaction with critical cellular receptor site(s) may involve only part of the polycyclic molecule. There is, indeed, good evidence for the noncovalent interaction of DNA with large polycyclics by intercalation, despite the fact that the space between the purine-pyrimidine base-pairs can accommodate only part of the aromatic molecule (Section II,F,3). An interesting study on the effect of hydrogenation upon the carcinogenic activity of 1,2,5,6-dibenzanthracene to the skin of mice was carried out by Lijinsky et al. (1965) ; Table I11 summarizes the comTABLE I11 Hydrogenated Derivatives of lJ2,5,6-Dibeneanthracene
MOLECULAR GEOMETRY AND CARCINOCiEKIC ACTIVITY
319
pouncls tested i n this study. Their rebults roncur w i t 1 1 tlrc findings ohtaincd on hydrogenated derivatives of the 3,4,8,9- a n d 3,4,9,10-dihenzopyrencs ( w e Hartwc~ll,19.51; T,ac*:issagnt.e t nl., 1960h; rcivicn.cd by Arcos and Arcos, 1962) : c:ircinogciiic-ity is largely niaintainccl as long as molecular planarity and the presence of resonant diphenylnaphthalene segments are preserved. Lijinsky e t al. (1965) have shown that 3,4dihydro-l,2,5,6-dibenzanthracene (XXIV) (also known as 5,6-dihydrodibenz [ a,h] anthracene) and 1’,2’,3’,4’,7,8-hexahydro-1,2,5,6-dibenzanthracene (XXVI) (also known as 1,2,3,4,12,13-hexahydrodibenz [ a,h] anthracene) are comparable, moderately active carcinogens. The hydrogenated derivative (XXIV) has resonance pathways identical to 2-phenylphenanthrene (XXV), and compound (XXVI) to that of 2-phenyl-5,6dimethylnaphthalene (XXVII) . Neither (XXV) nor (XXVII) has been tested biologically; thus the role of the intercyclic -CH, - CH,- groups in carcinogenic activity cannot be assessed. Further degradation of the resonance pathways abolishes carcinogenic activity; thus, 3,4,7,8,-tetrahydro-1,2,5,6-dibenzanthracene(XXVIII) (also known as 5,6,12,13-tetrahydrodibenz [a,h] anthracene) is inactive toward the skin but produced a large increase in the spontaneous lung adenomas in the test animals. This compound (XXVIII) corresponds to a -CH, - CH,- bridged p-terphenyl. No biological activity was observed with compounds (XXIX) , ( X X X ) , and (XXXI) in which the remnant of the aromatic structure corresponds to an acene. 2. T h e Question of Tissue Target Specificity The findings reported in a number of investigations tend to weaken the still strongly held notion of tissue target specificity of polycyclic hydrocarbons. The mass of data published, especially in the early era, created the general impression that their carcinogenic action was overwhelmingly topical. Later work showed, however, that these agents can produce tumors in a variety of distant tissue targets, depending on the route of administration, species, strain, and age. The production of lung tumors by hydrocarbons (c.g., Andervont and Shimkin, 1940-41 ) has been known for some time. Hepatomas have been induced by oral administration of hydrocarbons under special conditions of strain (White and Eschenbrenner, 1945), age (Klein, 1959, 1963), and diet (HochLigeti, 1954). Also, the production of tumors in the alimentary tract (e.g., Lorenz and Stewart, 1948) and more specifically in the forestomach (Saxen et al., 1950) has been reported following the feeding of carcinogenic hydrocarbons. In addition to the topical carcinogenic effect, 100% incidence of leukemia has been obtained in mice by skin painting with 20-methylcholanthrene (Morton and Mider, 1938) and 9,lO-dimethyl-
320
J O S E P H C. ARCOS A N D M A R Y F. ARGUS
1,2-benzanthraceiie (Huggins and Sugiyama, 1966; I’ietra et ul., 1961). A variety of strains of mice respond wit,li the development of a high incidence of lyinpliomas to the adininist,rat,ion of the same polycyrlic 1iyclroc~:~rl~on (Piclttx c t ( I / . , 1961 ; Iic~llyam1 O’~;:LIX, 1961). An cxteiisive literature has devclolm~on the protliidioii of In:inniiary tulnors in young female rats by oral administration of 20-methylcholanthrcne arid 9,lOdimethyl-l,2-benzanthracene (e.g., Huggins et al., 1959a,b) ; the incidence of this tumor localization is reduced in older animals. Also, the production of skin tumors by oral administration of 20-methylcholanthrene to very young rats (Gruenstein et al., 1966) and the production of subcutaneous sarcomas in young (Gruenstein e t al., 1967) and adult rats (Hoch-Ligeti e t al., 1968) has been reported.
FORMATION AND ENVIRONMENTAL OCCURRENCE B. PYROLYTIC There is a vast body of evidence showing that carcinogenic polycyclic aromatic hydrocarbons are formed in the pyrogenation of almost any kind of organic material (e.g., Kennaway, 1930; Kuratsune, 1956; Kuratsune and Hueper, 1960; Lijinsky and Shubik, 1964). High temperatures produce the cracking of carbon compounds and repolymerization of the resulting molecular fragments and also bring about the loss of hydrogen, leading to the aroniatixation of the polycyclic carbon skeletons formed. Extensive studies on the Conditions governing the formation of aromatic hydrocarbons a t high temperatures have been carried out by Badger and his co-workers. They found that 3,4-benzopyrene, 3,4- and 10,11benzofluoranthene, and a number of other polycyclic hydrocarbons are formed in the pyrolysis of simple hydrocarbons such as acetylene, butadiene, ethylbenzene, styrene, vinylcyclohexene, n-butylbenzene, l-phenylbutadiene, and Tetralin a t 700°C. (Badger et al., 1960; reviewed by Badger, 1962). The C,-C, structure, i.e., n-butylbenzene, gave the highest yield of 3,4-benzopyrene (Badger, 1962; Badger et al., 19641)). These studies carried out with 14C-hydrocarbons led Badger and co-workers to propose the reaction sequences shown in Fig. 2 to account for the formation of 3,4-henzopyrene and 10,ll -benzofluorantliene. A t the temperature of pyrolysis (about 700”C.), aromatic ring systems cxhibit the greatest stability of the structurnl typcs prescnt. This stability leads t o the gradual accuniulation of condensed ring structures showing successively greater degrees of condensation as the duration of pyrolysis progresses (cf. Sack, 1951; Hadzi, 1953). Under these conditions, acetylene and butadiene undergo chain lengthening leading first to vinylcyclohexene and then to Tetralin. Following Badger’s scheme, the saturated C-C bonds in Tetralin readily undergo cleavage leading to a radical structure which is in equilibrium with uncleaved Tetralin. A molecule of Tetralin
cti
-.
FIG.2. Hypothetic mechanism of the formation of 3,4-beneopyrene and 10,llbenzofluoranthene from l o w ~ r hydrocarbons during pyrolysis. (Contlensed from Badger, 1962; Badger et al., 1960, 1964a.)
and an n-butylbenzene radical joins to w-tetrahydronaphthyl-n-butylbenzene, which then yields 3,4-bcnzopyrene and 10,ll-benzofluoranthene following ring closure by dehydrogenation (Badger et al., 1964a). That n-butylbenzene used as the starting material gives the highest yield of these hydrocarbons may well have its explanation in the advanced position of the C,-C, fragments in the reaction sequence. Resynthesis via o-tetrahydronaphthyl-n-butylbenzeneis, however, not the only mechanism for the formation of 3,4-benzopyrene (Badger et al., 1960). Dimerization of two vinylcyclohexene molecules to dodecahydropyrene also occurs, followed by addition of a further butadiene unit and aromatization of the molecule. 3,4-Benzopyrene is probably formed by both nicchanisms. Using n-butylbenzene, Badger et al. (1964b) also studied the temperature dependence of the formation of polycyclic hydrocarbons. It appears that for most hydrocarbons, in particular for the potent carcinogens, 3,4-benzopyrene, 3,4- and 10,ll-benzofluoranthene, there is an optimum temperature around 700°C. beyond which the yield rapidly decreases (Fig. 3). Taking up the lead of earlier investigations of the 3,4-benzopyrene content of rural soils (Kern, 1947) and of deep marine sediments (Meinschein, 1959), Blumer (1961) analyzed the solvent-extractable hydrorat*hon content of New Englttnd soil snmplcs in rural areas where the I)oshtl)ility of ititlustrial cotitnininatioti :ippears to bc excludrd (TL~I)Ic IV). IiIutiit~t*’shtiitlies suggest that 3,4-bcnzopyrene and itb iiinctive isomer, I ,2-lxtizopyrctic, arc comnioii and fairly abundant constituents of soils. In addition to the benzopyrenes, the following hydrocarbons were represented in United Statcs soil samples: anthracene, phenanthrene, 1,2-
322
JOSEPH C. ARCOS AND MARY F. ARGUS
.031
J,4-bemoftuoronthem
Temperature of Pyrolysis
FIQ.3. Yield of 3,4-benzopyrene and 3,4-benzofluoranthene formed in the pyrolysis of n-butylbenaene, as a function of the temperature. (Condensed from Badger, 1962; Badger et nl., 196413.)
benzanthracene, pyrene, chrysene, fluoranthene, perylene, triphenylene, benzofluorene, coronene, anthanthrene, and 1,12-benzoperylene. Polycyclic hydrocarbons have been detected in various unprocessed cereal grains (Grimmer and Hildebrandt, 1965). The leading contaminant is, surprisingly, phenanthrene, of which as much as 10 mg. is contained per 100 kg. of certain samples of grain. The 3,4-benzopyrene content is considerably less (0.02-0.41 mg./100 kg.). The variety of othcr polycyclic THECONTENT
OF
TABLE IV 3,4-BENZOPYRENE I N
Type of soil and origin
SOME
RURALSOILSO Conc. (pg./kg.) ~~~
Oak forest, West Falmouth Cape Cod, Mass. Pine forest, West Falmouth Cape Cod, Mass. Mixed forest, West Falmouth Cape Cod, MasH. Mixed forest, eastern Connecticut Garden soil, West Falmouth Cape Cod, Mass. Plowed field, eastern Connectirut a
From Blumer (1961).
40 40 1300 240 90 900
~
323
hfOLECUL.4R GEOME'I'RY AND CARCINOGENIC ACTIVITY
1iytlrouarl)oiis ctettdetl is about thv w i i e as fouiltl i i i b o i l sstinples hy Blunier. Blumer (1961) hypothesized that the occurrence of the high hydrocarbon concentrations in rural soils distant from major highways and industrial sites cannot be ascribed to fallout froni polluted air but is indigenous to thc .oil. The hydrocarbons may be formed in the soil by the same low-tenil)(,raturepi'ocesses which bring about the transformation of plant and animal organic matter into peat and lignite. Hydrocarbons may also be the products of niicroorganisms present in the soil. Indirect evidence for these views was provided by studies (reviewed by Blumer, 1965) on the structure of organic pigments responsible for the intense color of certain fossil materials. Typical fossil pigments are the fringelites (in the Jurassic crinoid, Apowinus sp.) , the simplest of which is fringelite H. All fringelites arc hyclroxy dcrivatives of 7neso-naplitliodiantlirone. O/H.., ,-€i\ 0
0
@ \
/
\
' 1 , I
\ ' \
\
//
/
v
\/
@& \
' ,
\
/
/
/
o,H.,.o...H ,,o Fringelite H
Bisanthene
1,14-Benzobisanthene
The parent hydrocarbon nLeso-naphthodiantlireiie (also called bisanthene) and its benzolog, 1,14-benzobis~tnthene,were also detected in the fossil material. The format,ion of other hydrocarbon types as a result of different routes of geocliemical synthesis in other geochemical deposits is, thus, a definite possibility. The iinportant implication of the presence of polycyclic aromatics in soils and fossils is that man has probably been in contact with carcinogenic hydrocarbons not only during the recent industrial period, but during his entire history (Blumer, 1961).
C. HETEROCYCLIC COMPOUNDS The synthesis and testing of heteroarornatic polycyclic carcinogens was carried out in recent years almost exclusively by Buu-Hoi, Lacassagne, and their associates in France, and Baldwin, Vipond, and their group in England. Because every polycyclic hydrocarbon is the structural analog of a large number of heteroaromatic isomers, it is likely t h a t a great variety of carcinogenic compounds will be found among the hetero-
324
JOSEPH C. ARCOS A N D MARY F. ARGUS
aromatic coiiipounds. For c ' x R I ~ ~ ~considcriiig ~P, only the 1:itcrnl beiizo ring in 3,4-benzopyrene, four nza :in:ilogs are possiblc. Aza substitution in 3,4-benzofluoranthene can yield twelve isosters. Aza polycyclics appear more likely t o be endowed with carcinogenic activity if they are isosteric with hydrocarbons of high potency. 1. Coinpounds with One Nitrogen Heteroatom
The finding t h a t the presence of a nnphthacene grouping in a hydrocarbon molecule is unfavorable for a high level of carcinogenic activity (Lacassagne et al., 1960a) is also valid in the acridine series. Thus, benzo-
/
/
/
R
(XXXVII)
(xxxV111)
[c]-phenaleno-[1,9:i,j]acridine(XXXII) (R = H) and benzo-[a]-phenaleno-[1,9:i,j]acridine (XXXIII) (R = H) are only very weakly sarcoinatogenic in mice (Buu-Hoi e t al., 1965). However, they also produced leukemia in the tumor-bearing animals, not usually observed in the strain used. Methyl substitution in para with respect t o the heteroatom (R =
CH, in X X X I l n i i t l X S X I I I ) does not, britig :ibout c1i:inge in activity which is notably different from the findings with the simple benzacridines. The low carcinogenicity of (XXXII) and (XXXIII) may not be ascribed to an increase of molecular size beyond a high activity range since a number of highly active compounds, which have as large or larger iiiolecular sizes, have been found in the benzo- and naphthopyridocarbazole series. Apart from the benzacridines (reviewed by Lacassagne et al., 1956) , few other aza analogs of the alternant polycyclic hydrocarbons have been tested. Lacassagne et al. (1964a) found that l’-aza-3,4-benzopyrene (also called pyrido [2’,3’: 3,4]pyrene) (XXXIV) possesses appreciable sarcomatogenic activity (Iball index 14) toward the subcutaneous tissue of XVIInc/Z mice. I n the early experiments of Badger e t al. (1940) and Shear and Leiter (1941-1942), this compound was inactive toward the skin and subcutaneous tissue of mice. Lacassagne et al. (1964a) also reported that pyrido [ 3’,2’: 5,6] chrysene (also called pyrido [ 3’,2’: 1,2] chrysene) (XAXXVI,has a potency about equal to that of the isosteric However, removal of the parhydrocarbon 1,2,3,4-dibenzophenantl~rene. ticular benzo ring in (XXXV) corresponding to the 1,a-benzo ring in the hydrocarbon analog brings about total loss of activity, since l’-aza-3,4benzophenanthrene (also known as naphtho [ 1,2-f] quinoline) (XXXVI) is an inactive compound. A typical example of the rule that nlternant aza polycyclics are more likely to be carcinogenic if they are analogs of highly active hydrocarbons has been found among the aza benzofluoranthenes. Pyrido [ 3’,2’: 3,4] fluoranthene (XXXVII), an analog of the potent 3,4-benzofluoranthene (IV) , is highly sarcomatogenic, whereas pyrido [ 3’,2’: 11,121benzofluoranthene (XXXVIII) which is isosteric with the moderately carcinogenic 11,12benzofluorantliene (VI) is an inactive compound (Lacassagne et al., 1964a). Partial hydrogenation in the benzacridine scries appears to affect carcinogenic activity to a much greater degree than among the hydrocarbons. The sarcornatogenic activity of both 10-methyl-1,2,5,6-dibenzacridineand of lO-methyl-l,2,7,8-dibenzacridineis almost totally lost by replacing one of the aromatic benz rings with a hydrogenated ring: 1’,2’,3’,4’-tetrahydro-10-metliyl- 1,2,5,6-dibenzacridine and 1’,2’,3’,4’-tetrahydro-lO-methyl1,2,7,8-dibenzacridirie are almost totally inactive. Hydrogenation in these lateral rings brings about only a small loss of coplanarity which, thus, cannot account for the considerable loss of biological activity. Hydrogenation also brings about, however, change in the resonance characteristics in the polycyclic frame which should consequently be compared to the respective trimethylbenzacridines. The former hydrogenated com-
326
JOSEPH C. ARCOR AND MART F. ARGUS
poiintl is, in fact, isostcric with 1,2,1O-ti~i~nctlryl-~,6-t~e1iz~~~r~~liiic, whicli is iiiactivc (1,acassagnc et nl., 1956). The latter tetrahydro compound is analogous to 1,2,1O-trimethyl-7,8-benzacridine which does not appear t o have been tested ; however, none of the isomeric trimethyl-7,s-benaacridines tested are active (Lacassagne et al., 1956). 2, Conzpounrls with Sevet-a1 Nitrogen Heteroatoins
Extensive work has been carried out in the last 8 years on two groups of heterocyclic carcinogens containing two and four nitrogen atoms, respectively : the benzo- and naphthopyridocarbazoles and the tricycloquinazoline derivatives. It was already evident from the study of the benzacridines that in some instances, the presence of the heteroatom raises the carcinogenic activity of the heterocyclic compound above t h a t of the hydrocarbon analog. Since the presence of nitrogen brings about a decrease of electron charge a t the K-region, the heteroatom itself-by means of its electron doublet-also appears to participate in the interaction with cellular receptor sites. I n compounds with two or more nitrogens all these heteroatoms may partake in the cellular interactions. Polycyclic compounds bearing several nitrogen atoms should be carcinogenic if the position and distance with respect to one another of the nitrogen atoms is maintained. These expectations were generally borne out from the study of structure-activity relationships of naphtho- and benzopyridocarbazoles and tricycloquinazolines. a. Benxo- and Naphthopyridocarbaxoles. Table V compares the structures and activities of the benzo- and naplithopyridocarbaaoles and of thc benzonaphtho-/3-carbolines to those of thc isosteric dibenzocarbazoles. Some of the benaopyridocarbazoles are much inore potent than the structurally analogous dibenzocarbaaoles (Lacassagne e t al., 1961d, 1963c; Buu-Hoi, 1964). Thus, 5,6-benzopyrido [2!,3’: 1,2]carbazole (XL) and 5,6-benzopyrido [ 3’,2’: 1,2]carbazole (XLI) are considerably more active than the isosteric 1,2,5,6-dibenzocarbazole( X X X I X ) for inducing sarcomas by subcutaneous injection. Compounds (XL) and (XLI) also elicit epitheliomas of the forestomach in mice following administration by stomach tube. Ability to induce sarcomas remains, however, a t the low level of the parent diberizocarbazole (XXXIX) if the second nitrogen atom is introduced into the 5,6-benzo ring as in ( X L I I ) . I n both (XLI) and ( X L I I ) , thc noncarhazole nitrogen atom may conjugate with the intercyclic biphenyl bond and is extraneous to the biphenyl moiety. T h e already low activity of (XLII) vanishes by increasing the molecular size as in (XLIII) or (XLIV). Interestingly, activity is totally lost if the noncarbazole nitrogen is-even though in a position of conjugation with the intercyclic bond-within the biphenyl moiety as in compound
MOLECULAR GEOMETRY AND CARCIKUGEKIC ACTIYITI'
327
(XLVI) . Activity is not regained I,y annclation of additional benzene rings (XLVII and XLVIII). Considcrable potentiation of sarcomatogenic activity was also found with the analog (LII) of 1,2,7,8-dibenzocarbazole& I ) . Compound (LII) appears to be, Iiowcver, cxtreincly sensitive to any motlificatioii of thc molecular size since methyl substitution a h in (1,111) or annelation of TABLE V Comparative Activities of Dibenzocarbazoles, Benzo- and Naphthopyridocarbazoles, and Benzonaphthocarbolines a
B
B
T
n
dl0; ? I (XLII)
do; 00
do; 90
(XLIV)
(XLIII)
n
NP n
/ / -
do; 90 (XLVI)
do;
00
(XLVIII)
$0;
90
(XLVII)
25; 73"
d34; 924
(XLIX)
( L)
328
JOSEPH C. ARCOS A N D MARY F. ARGUS TABLE V (continued)
do; 00
trace e
(LIII)
(LO
so; 00
do; 00
60; ?O
(LIV)
(LV)
(LVI)
@Except when otherwise indicated the data were compiled from the revim of Buu-Hoi (l964) and the reports of Lacassagne eral. (l961d,1963c). The nomenclature of compounds is a8 follows: 1.2,5,6dibenzocarbazole(XXXM); 5,6-benzopyrido-[2',3' :I ,2]-carbazole (XL): 5.6-benzopyrido-13',2':1,2]carbazole ELI); l,Z-benzopyrido-[Z',3': 5.61-carbazole (XLII); naphtho-(2',1': 1,2]-pyrido-12",3": 5,6]carbazole (XLIII); naphtho-[l',Z': 1,2]-pyrido-(2",3": 5,6]-carbazoIe (XLIV); naphtho-l1',2':1.21-pyrido[3",2": 5,6]-carbazole (XLV): 1,2,6,7-dibenzo-p-carboline (XLVI) (also known as 3.4,7,P-dibenzo-pcarboline): 3,4-henzonaphtho-11',2': 7,8]-P-carboline (XLVII) (also known as l12-benzonaphtho[2'.1': 6.7]-P-carboline); 3.4-benzonaphtho-[Zf,1': 7 , PI-0-carboline(XLVIII) (also known as 1 ,Z-benzonaphtho-[l',2':6.7]-P-carboline);3,4,5,6-dibenzocarbazole (XLM);5,6-benzopyrido-[3',2':3.41 -carhazole (L); l12,7,8-dibenzocarbazole(LI); 7,8-benzopyrid0-12',3': 1,2]-carbazole(LII); 6'-methyl-7,Pbenzopyrido-(2'.3':1,2]-carbazole (LIII); naphth0-[2',1': I ,2]-pyrido-f3",2": 7,131-carbazole (LIV): naphtho-Ill,2': 1,2]-pyrido-[3" ,2": 7,8]-carbazole (LV); naphtho-(it,2': 1,2]-pyrido-(2" .3": 7 , 81-carbazole (LVI). Except when otherwise indicated the compounds were tested subcutaneously In XVlI nc/Z strain mice of both sexes in the Paris Radium Institute,and the arablc numbers represent the sarcoma indexes (Iball). Index calculated from the data of Badger ef a/. 0942). C Also
elicit epitheliomas of the forestomach.
dlndex calculated from the data of Kirby (1948). This compound possesses borderline carcinogenicity toward the subcutaneous tissue (Boyland and Brues, 1937).
an additional benzene ring as in (LIV) or (LV) brings about total loss of activity. In contrast to some pyrido analogs of 1,2,5,6-and 1,2,7,8dibenzocarbazole discussed above, the potency of 3,4,5,6-dibenxocarbazole (XLIX) is much less altered by introduction of a second nitrogen atom as may be seen from the testing of the sole isoster (L) studied to date. There is, however, modification of distant target specificity since (L), unlike (XLIX), is not carcinogenic toward the liver. b. Tricycloqui7inzoZines. The biological study of tricycloquinaxoline derivatives initiated by Baldwin, Partridge, arid their co-workers (Baldwin e t al., 1958, 1959) is a fascinating and highly promising lead toward the elucidatioii of the mechanism of interaction of polycyclic carcinogens
hlOLECI;LAR GEOMETRY AND CARCINOGESIC .iCTlVITY
329
with tissucl constituents. Tricycloquinazoline [ (LVII) in Table V I j , synthesized first, by Cooper and Partridge (1954), is a highly stahle compoiintl. It inay be siihliined aroriiid 500"-600"C. without decomposition and is highly rcsist;int to oxidation. Tricycloquinazoline inay produce epitheliomas or subcutaneous sarcomas (Baldwin et al., 1959) depending on whether testing is carried out by skin painting (in mice) or by subcutaneous injection (in rats). Added interest to the carcinogenicity of tricycloquinazoline is provided by the realization by Baldwin and his associates that this compound could be foriiied by the combustion of various plant materials (Baldwin et al., 1959). I n fact, they have shown that tricycloquinazoline is readily produced in simple pyrolytic reactions from a variety of derivatives of anthranilic acid, including methyl anthranilate (Baldwin et al., 1958, 1965a) which has a wide distribution in plants (Klein, 1932). This ease of formation may be compounded by the fact that, owing to its exceptional chemical stability and its subliinatioli temperature of 500'-600°C., tricycloquinazoline could escape in substantial amounts during the combustion of plants. Tricycloquinazoline (LVII) is a trigonally symmetrical molecule having one nitrogen atom a t the geometric center and, equidistantly from the central atom, three lateral nitrogen atoms positioned as the vertices of an equilateral triangle. Baldwin and his associates gave a beautiful demonstration (Baldwin and Partridge, 1964; Baldwin e t al., 1962c, 1963a,b, 1964a, 1965a) that ( a ) the presence of all three lateral nitrogen atoms is a n essential requirement for carcinogenicity and ( b ) a t least one of them may be replaced by an oxygen atom without notable loss of activity. Table VI lists a number of the compounds which were tested for obtaining this inforination. Compounds (LXI) and ( L X I I ) represent the replaceincnt of the nitrogen atoms with =CH- groups in the 5 , and 5 and 15 positions, respectively. These compounds are virtually inactive : (LXI) gave a 10% tunior incidence, and ( I X I I ) 3% a s compared to a tumor incidence of 75% for (LVII) when tested for the same period of time. Conipounds ( L X I I I ) , and (LXV) through ( L S V I I I ), which contain :in unsymmetrically distributed or only part of the typical nitrogen tetrad, are slightly active or inactive. Also inactive is the "open" ring isosteric tris-o-xylylamine (LX) having none of the lateral nitrogens. On the other hand, as long as the three equilaterally positioned nitrogens are maintained, carcinogenic activity remains virtually unchanged despite the elimination of the central N-atom, as was indicated by the high activity of (LVIII). Furthermore, one of the three lateral nitrogen atoms may be replaced by an oxygen without loss of activity since compound (LTX) is about as active as the parent compound (T,VII). This
JOSEPH C. ARCOS AND MARY F. ARGUS
330
TABLE VI Structural Analogs and Molecular Fragments Tested to Delineate the Structural Features Critical for Carcinogenicity in Tricycloquinazoline
n
N’
N
4
di / \
I
\
/
\
(LX)
\
O \ N
’
(LXIII)
(LXVIII)
(LXVII) ~~
‘The nomenclature of the compounds is according to the IUPAC system: tricycloquinazoline (LVII)(or 5,10,16,15b-tetraza-tribenzo[a.e,f1 phenalene): Q,5,10,15-tetraza-trihenzo~~,e,f] phenalene (LVIII): 5om-10.15-diaza-tribenzo[ u ,e.f ]phenalene (LIX);tris-o-xylylamine (W; 10,15,15b-triaza-tribenzo [u,e,i]phenalene (LXI):10.15b-diaza-tribenzo [a,e,f1 phenalene (LXII): iso-tricycloquinazoline(LXIII) (or 9,10,15,15b-tetraza-tribenzo [a,e , j ] phenalene); benzo [a]chromeno-I4,3,2-d,el-3,B-phenanthroline (LXIV): benzo+]-4,5-phenylene-3,6-phenanthroline (LXV)(or 9,14-diaza-dIbenzo[ b ,el fluoranthene); 9,10,13,13b-tetraza-dibenzo[o.j] phenalene (LXVI); 5 , €u,l2-triaza-T-hydroxybenz [a] anthracene (LXVII); benzo{o] -3,B-phenanthroline(LXVIII).
MOLECLJLAH GEOMETHY A N D CARCINOGENIC ACTIVI'I'Y
33 1
indicates, then, that tlic requirement is riot for nitrogen atoms per se, but rather for the presence of three hydrogen bonding centers a t these sites. These findings led t o the seemingly obvious conclusions that the three lateral heteroatoms are probably required for hydrogen bonding \N:.
//
.
. H-
and
\: O : . . . H/
to cellular receptor site(s) and t h a t the central N-atom in (LVII) is unimportant for carcinogenic activity (Baldwin and Partridge, 1964 ; Baldwin et al., 1964a). Howevcr, the very rcccnt surprihing finding by Partridge and Vipond (1966) that 5,10,15-triaza-benzo [ a ] naplitli- [ I ,2,3:d,e J anthracene (LXIX) is inactive under identical conditions of testing, prompts a re-
(LXW
exmiination of tlic latter p a r t of this coiiclubioii. As its structural formula shows, uninterrupted conjugation is lost in ( L X I X ) . The tetrahedral orbital configuration of the central carbon atom resulting from this loss increases tlie tliickncss of the inolecule and, thus, tlie distance through which hydrogen bonding iiitcractions with thc lateral hetcroatonis iiiay I)c estahlished. But the inactivity of ( L X I X ) also suggests that in (1,VIII) and ( L I X ) , the role of tlie sp2 hybridieecl central carbon atom may be inore than stereocheiiiical perrnissivcness aiid that tlic fractional reactivity (frre v:tlcnce index) of this atom is essential for interaction by secondary valeiice forces and carcinogenicity. Beyond the dibtribution and symmetry of the hydrogen-bonding Iicteroatoin tetrad, molecular sizc and shape have a strong cffrct on tlic activity of this family of carcinogens. Tricycloquinazoline (LVII) appears to represent an optimum of bize and/or shape, sirice none of tlie btructurill analogs and compounds corresponding t o one or another segment of tlie parciit inolecule liavc show11 higher carcinogenicity than (LVII) itself. That tricycloyuiiinzoliii~represents an optiniuin of molecular geonietry is alho Imrne out from studies O I I t h r carciiiogriiicity of riiig-hLibstitLitt,i~ tlcri\.:itiw> (Table YII) . C'aixinogcnicity i> lo5t outright by iuetliyl s u l ~
332
JOSEPH C. ARCOS AND MARY F. ARGUS
TABLE V I I CAR(:INOGENICITY OF TRICYCLOQUINAZOLINE DERIVATIVES TOWARD THE SKIN OF MICE"
+
Papilloma epithelioma incidence Compound
( %)
Tricycloquinasoline (TCQ) 1-Methyl-TCQ 2-Methyl-TCQ 3-Methyl-TCQ 4-Methyl-TCQ 3,8-Dimethyl-TCQ 3,8,13-Trimethyl-TCQ 2-Methoxy-TCQ 3-Methoxy-TCQ 3-Ethyl-TCQ 3-tert-Butyl-TCQ 2-Fluoro-TCQ 3-Fluoro-TCQ 4-Fluoro-TCQ 3,8-Difluoro-TCQ 2-Chloro-TCQ 2-Bromo-TCQ 3-Bromo-TCQ 2,3-Benzo-TCQ 3,CBenzo-TCQ
81 55 9 59 44 9 17 12 12 55 54 79 76 31 40 58 74 68 2 0
Epithelioma Mean latent Iball index incidence period (epitheliomas (%) (days) only) 75 45 0 48 44 0 9 6 0 24 20 23 65 11 16 53 68 0 0
186 3 16 46 1 296 282 244 277 32 1 464c 53w 544 454c 375 3 10 55P 360" 33OC 274 487" 425c
Compiled from Baldwin et al. (1962a,c, 1963b, 1964a, 1965a,b). The 2-hydroxy derivative is also inactive (cited in Baldwin et al., 1962a, 196513). Total length of experiment (days). Estimated transitory values based on available data: totul tumor incidence and/or totul duration of experiment.
stitution in the 2-position, but the isomeric 1-, 3-, arid 4-methyl derivatives are still appreciably active. The 3-position is surprisingly insensitive to the nature or size of the substituent; appreciable ability to induce malignant epitheliomas is preserved in 3-methyl-, 3-fluoro-, 3-bromo-, 3-ethyl-, and even 3-tert-butyltricycloquinazoline.Annelation of another benzene ring in the 2,3- or 3,4-positions brings about total loss of activity. All these observations suggested to Baldwin and his associates that stereocheniical factors play a preponderant if not exclusive role and that the critical interaction of tricycloquinazoline in the cell requires a highly specific orientation or fit of the molecular frame with respect to or on the receptor site(s) (Baldwin and Partridge, 1964; Baldwin e t al., 1965a). They thought to derive support for this view from the fact that substitution in the. 2-pnsition by n fluoriiic atom, wliich is only slightly larger than
hydrogen (atomic radius 1.35 A, compared to 1.1 A for hydrogen), lowers the epithelioma incidence to less than one-third. Thus, it was held that steric orientation during cellular interaction is critically controlled by the 2- and equivalent positions. However, an important piece of evidence furnished recently by Partridge and Vipond (1966) showed that deactivation by the 2-substituents in the hitherto tested derivatives should be ascribed to electronic rather than to steric effects. Thus, it appears t h a t deactivation is due to the +M effect of the substituent. This follows from the observation that 2-trifluoromethyltricycloquinazoline gives a tumor incidence of 60%. Methyl groups are well known to promote mesomeric shift by hypereonjugation, whereas a trifluoromethyl group gives a n overall -1 effect owing to the electronegativity of the fluorine atoms. With 2-substituents which have an overall -1 effect, activity is retained, whereas a substituent having a +I or +M effect abolishes activity. This also provides a rationale for the substantial activity of 2-bromotricycloquinazoline over that of 2-fluorotricycloquinazoline (Table V I I j which cannot be explained stereochemically.
D. METABOLISM 1. Carcinogenic Activity and Rate of Elimination from the Tissues
The relationship between the rate of elimination from the target tissue and carcinogenic potency has continued to attract interest. A study (Domsky et al., 1963) of the rate of disappearance contributed in drawing attention to the high susceptibility of newborn mice t o carcinogenic chemicals (Kelly and O’Gara, 1961 ; Pietra et al., 1961; Roe et al., 1961), in particular to 9,10-dimcthyl-1,2-benzanthracene, which has been found to induce a high incidence of lymphomas and other tumors in the newborn a t dosages t h a t induce only few such tumors in the adult. I n Fig. 4 are shown the results of Domsky et al. (1963), indicating t h a t the free 9,10-dimethyl-l,2-benzanthracene,solvent-extractable from the homogenized whole carcass, decreases much faster in the adult than in newborn mice. Thus, the greater susceptibility of the newborn to tumor induction is due t o the longer persistence of the compound in the newborn than in adult mice. This is consistent with the earlier finding of Cramer et al. ( 1960a) that young aiiirrials mctabolize hydrocarbons less actively than adults. Correlation between slow rate of elimination from the tissues and a high degree of carcinogenic potency was also the conclusion of Unseren and Fieser (1962) who found t h a t most, if not all the potent 3,4,9,10dibenzopyrene remains unmetabolized in mice a t the site of subcutaneous injection, No aromatic metabolites were detected either in the feces or
334
.JOSEPH C. ARCOS AND M A R Y F. ARGUS
2
4
6
8 10 Days
12
14
FIG.4 . Rate of disappearance of 9,10-dimethyl-1,2-benzanthracene (DMBA) from adult ( X ) anti newborn ( A ) whole Swiss mice. The logarithm of the percentage of injccted hydrocarbon rerovered from thc carcass is ploi t ~ dagainst thc time aftcr injrction. (From Domsky et al., 1963.)
the emerging tumors despite the fact that the hydrocarbon may be readily oxidized chcmically to 5,g-quinone and 5,8-diacetoxy derivatives. This substantiates the earlier findings of Lacassagnc e t al. (e.g., 1961a) on the long persistence of this and other large hydrocarbons in the tissues. I n contrast, Goodall e t nl. (1963) observcd no correlation betwcen tissue localization and rarcinogcnic potency in fcrriale Spraguc-Dawlcy rats when 14C-labeled 20-methylcholanthrcne, which is known to producc mammary tumors in females of this strain (e.g., Huggins e t al., 1959a), was administered orally ; neither labeled carcinogen nor a labcletl metabolite was localized in the mammary tissue. The relation between rate of elimination and the resistance of different tissues to topical carcinogenic action was investigated by Pozdnyakov (1963a,b). This author found that using 9,10-dimethyl-1,2-benzanthracene by direct intramuscular route in roosters there is a higher tumor incidence in the thoracic muscle from which resorption is slow than in the femoral muscle from which resorption is much faster. It seems, therefore, that the higher mctaholic activity of the latter muscle contributes to increased resistance to local carcinogenic effect. The rate of excretion of tricycloquinazolinc arid its persistence in thc target tissue has been briefly investigated by Baldwin e t al. (196313, 1964a). Intravenously injected l-lC-tricyclocluinazoline concentrates very rapidly in the abdominal tract and carcass ; following intraperitoneal administration, over 50% of the radioactivity is excreted in 48 hours through the feces. Twenty-four hours following skin painting, as much
hlOLECIlL.4R (;EOhIETRT AND CARCINO(;ENIC’ ACTIVITY
335
:is 80y0 of thc radioactivity is rccoverable froiii the skin. However, this low rate of ahsorption hliould not ncccssarily be regarded as a limiting factor in carcinogenic activity. One should recall in this regard, the ~ ~ r s u lof t s Bock and Bui*nliaiii (1961) who found no relation hetween skin pcwc’tration ui~lci. stnndnrrl cxpcriincntal conditions and carcinogenic activity in ti ect of twclvc polycyclic hydrocarbons. 2. A‘onbo wit1 M e tci bol ites u . Metabolism of 1,2-Benzantlzracene uiid I t s Relation to Bond Order. The early finding that polycyclic hydrocarbons containing a 1,2-benzantliracene nucleus undergo metabolic hydroxylation predominantly in the 4’-position suggested that the 3’,4’-bond represents a molecular “region” of metabolic perhydroxylatiori p a r excellence; hence the term M region designating this bond (Pullman and Pullman, 1955a). However, in recent years, Boyland and his associates (e.g., Boyland and Sims, 1964; reviewed by Boyland, 1964a,b) carried out exhaustive investigations on the nietabolisni of 1,2-benzantliracene and plienanthrene, and showed t h a t metabolic reactions involve other bonds of tlic iiiolccule as well. What is highly significant is that tlie relative ninouiits of total inetaholites involving thc different bonds vary with tlic chemical reactivity of the latter. A measure of this reactivity is the bond order which, in turn, is an expression of electron presence a t an aromatic double bond (Table VIII) . These TABLE VIII Bond Orders and Relative Amounts of Metabolites of 1,2-Benzanthracene Produced by Rodentsa 2’
10
5
4
Bond order
Relative amount of metabolites
6,7
0.593
-
2‘,3‘
0.628
-
1‘,2’
0.695
-I-
3’,4‘
0.700
++
7,8
0.731
+
Bond
5,6
0.132
3,4
0.783 a From Boyland (1964a).
tit
i
+++ t
336
JOSEPH C. ARCOS AND MARY F. AROUb
and similar data for phenanthrene seem to concur with the notion t h a t a specific molecular region of metabolic perhydroxylation may not be a tenable one. Despite the fact that all metabolites of polycyclic hydrocarbons have proved so far to be less active than the parent compounds or inactive, Boyland (1950, 1964a) hypothesized that epoxides might be proximate carcinogens of the hydrocarbons. Epoxidation may take placc preferentially a t the K-region which has a high bond order. I n an attempt to substantiate this hypothesis, E. C. Miller and Miller (1967) found t h a t 3,4epoxy-3,4-dihydro-10-methy1-1,2-benzanthracene1 the K-region epoxide of the potent carcinogenic hydrocarbon, lO-rnethyl-1,2-benzanthracene,has only low carcinogenic activity when tested subcutaneously in the rat or on thc skin of the mouse. Boyland and Sims (1967), testing subcutaneously in C57 mice, confirmed t h a t the cpoxide is significantly less active than the parent hydrocarbon. I n the same test system, the K-region epoxides of other hydrocarbons, chrysene, 1,2-benzanthracene1 1,2,5,6dibenzanthraceiie (Boyland and Sims, 1967), and of 20-methylcholanthrene (Sims, 1967b), were consistently less active than the parent hydrocarbons. E. C. Miller and Miller (1967) found the K-region epoxide of l12-benzanthracene t o be devoid of tumor-initiating activity in surface application. The K-region dihydrogenated derivative of 20-methylcholanthrene was also inactive (Sims, 1967b). b. Metabolism of J,.&Renzopyrene. With the exception of thc socalled F, metabolite, the identity of the 3,4-benzopyrene biliary metabolites remains unresolved. Falk et al. (1962) reported the isolation of a total of twenty-seven biliary metabolites in the rat. These have been tentatively identified as being various sulfo and glucuroconjugates of the following hydroxy derivatives of 3,4-benzopyrene: 5-, 8-, 5,8-, 5,lO-, 8,9-dihydroxy-8,9-dihydro-, 6,7-dihydroxy-6,7-dihydro-,and 1,5-dihydroxy-l15-dihydro-. Awaiting detailed confirmation of this very complete work, it should bc already noted t h a t Pihar and SpAleny (1956a,b) isolated 8-hydroxy-, 10-hydroxy-, and a small quantity of 5-hydroxy-3,4benzopyrene from the feces of rats rcceiving 3,4-benzopyrene and that rat liver homogenates were found to metabolize the hydrocarbon to the 8- and 10-hydroxy and 5,8-diliydroxy derivatives (Conney et al., 1957). Nevertheless, a t present there is far from general agreement concerning the identity of Weigert and Mottram’s XI, X,, and F, biliary metabolites (reviewed by Boyland and Wcigert, 1947) a s illustrated in Table IX. Moreover, Sinis (1967s) working with liver hornogcnatcs clainrctl that F, corresponds to 8-hytfroxyl)enzo[a ] pyrenc (also known as 3’-hytlroxy-3,4benzopyrene) wliich is actually a n artifact resulting from the deconiposition of 7,8-dihydro-7,8-diliydroxybenzo [ a ]pyrene present probably in its
IDEXTITY O F
Metabolite
BILl.4RY B P X
AND
TABLE IX BPF ~IETABOLITES O F 3,PBENZOPYRENE
Weigert and Mottram (1943, 1946)
Bereiddurn and Schoerital (1946, 1955)
IN
IVTRAVENOUS MICE FOLLOWING
Harper (1958, 1959b)
.4DMINISTR4TlOrrl
Falk ef al. (1962)
8,9-Dihydroxy-8,9-dihydro- 10-Hydrnxy-3,4-benzopyrene, 3,4-benzopyrene, conconjugated with unknown jugated a t the 8-hydroxyl group with unknown group
Sulfoconjugates of l-hydroxy- and 8-hydroxy3,4-benzopyrene
S-Hydroxy-3,4-benzopyrene, conjugated mit,h an unknown group
8,9-Dihydmxy-S,S-dihydro- X-Hydroxy-3,4-beiizopyrene, 3,4-benzopyrene, both hydroxyls conjugated with unknown groups
conjugated with unknown group
(;lucuronides of l-hydroxyand 8-hydroxy-3,4benzopyrene
8-Hydrosy-3,4-t,eiixopyreiie glucositlnronic acid
8-Hydroxy-3,Pbenzopyrene. conjugated with unknown group
10-Hgdroxy-3 4-benzopyrene
1-Hydroxy-3,Pbenzopyrene
Sulfoconjugates of 8-hydroxyand 5,8-dihydroxy-3,4benzopyrene
8-Hydroxy-3,Pbenzopyreiie
8-Hydroxy-3,Pbenzopyrene
8-Hydroxy-3,4-benzopyrene
8-Hydroxy-3,4-benzopyrene
338
JOSEPH C. AHCOS AN0 MARY 17. ARGUS
sulfo-conjuga ted form. Sims found no c~itleiiccfor the presence of 3,4benzopyrenc metabolites hydrosylated in the K-region. Ingenious cxpcriinents by Kotin et n l . ( 1962) gave furthcr support to tlic lwlivf that tlit. 1iydroctirl)ons t h c ~ n w l ~ratlicr ~ ~ h than sonic inetabolitcs are the cruci:il carcinogeiiic. stimuli. In thcse experiments, where mice received simultaneously subcutaneous injections of 3,4-benzopyrene and the hepatotoxic solvent, carbon tetrachloride, a strong enhancement of the tumorigenic response was brought about by impairment of thc detoxicating ability of the liver. Since the XI, X,, and F, metabolites are noncarcinogenic and F, is a wcak carcinogen compared t o the parent compound, the enhancement can be reasonably ascribed to slower detoxication of the hydrocarbon by the carbon tctrachloride-injured liver and, hence, longer persistence a t the injection site. These and subsequent studies with 3 , 4 - b e n ~ o p y r e n e - ~ ~have, C in fact, shown a considerable slowdown of the elimination of total radioactivity through the bile in the carbon tetrachloride-injure(1 animals (Falk, 1963). However, not only is the rate of elimination drastically altered in these animals, but also the hepatic damage results in qualitative and quantitative alterations of the metabolic profile; in particular, there is blockagc of conjugation of the dihydrodiol alcoholic hydroxyl groups. These effects are not uniquc to carbon tetrachloridc, and identical or similar alterations are produced by other hepatotoxic agents such as tannic acid, thioacetamide, and bromobenzene. c. Side-Chain Oxidation of Methyl-Substituted Hydrocarbons. Up to 1959, all the identified nontissue-bound metabolites of polycyclic hydrocarbons were exclusively various typcs of aromatic arid hydroaromatic ring-hydroxy compounds in conjugated or unconjugated form. Testing of the metabolites showcd invariably that they are less activc than the parent compounds or inactive. Identical conclusions have now been reached by Siins (196713) who tested various ring-hydroxy and keto derivatives of 20-mcthylcholanthrene. Harper (1959a) was the first to describe a metabolite corresponding to the oxidation of a substituent carbon chain. H e found t h a t in micc, 20-methylcholanthrenc is metabolized, in addition t o phenolic derivatives, to an acidic product which he tentativcly identified as cholanthrene-20carboxylic acid 011 thc strength of spectral and chemical evidence. The typical biliary metabolite of 9,10-dimethyl-l,2-benzanthracene is the 4'-hydroxy derivative (reviewed by Boyland and Weigert, 1947). However, rat liver honiogenates oxidize the side chains and convert this hydrocarbon into a mixture of 9-hydroxymethyl-10-methyl and 10-hydroxymethyl-9-methyl derivatives. The 4'-, 3-, 9-, and IO-methyl-1,2henzanthrarcncs arc also converted into the corresponding hydroxyniethyl
MOLECULAR GEORIETRY AND CARCIKOGESIC ACTIVlTY
339
derivatives. To a minor extent, ring hydroxylation occurs with all tliese Iiydrocarbons to yield phenols and diliydrodihydroxy compounds. As with 3,4-benzopyrene, no hydroxylation (Boyland et al., 1964c, 1965) was detected a t the K-region. Interestingly, using liomogcnates from animals pretreated with 20-niethylcholanthrene or phenobarbital, both of which induce microsomal enzyme synthesis, the yield of ring hydroxylated products was increased a t the expense of the hydroxymethyl derivatives (Sims, 1966). Boyland and Sims (1967), testing by subcutaneous route in C57 black mice, found that hot11 9-hydroxymethyl-10-methyl- and 10liydroxymethyl-9-methyl-1,2-l~enzanthracene are much less carcinogenic than the parent 9,10-dimethyl compound. Thus, these metabolites may not be regarded as proximate carcinogcns. d . Metabolisnx of Tricycloquinazoltne. Studied exclusively by Baldwin and his colleagues (Baldwin et al., 1963b, 1964a), biliary metabolism yields the 1- and 3-hytlroxy derivatives; the 2- and 4-hydroxy derivatives are definitely absent. On the other hand, in witro metabolism with r a t or iiiousc liver homogcnates appears to yield the 3-hydroxy derivative only. No acid-labilc dihydrodiol-typc compounds were detected. A fraction, h i t apparently not all, of the two hydroxy metabolites formed arc conjugated with glucuroiiic acid ; no bulfoconjugatcs were detected. In interesting contrast to biliary metabolism, metabolism in the mouse skin (which is priniarily the target tissue) hydroxylates tricycloquinazoline in all four positions. However, the significance of these metabolites is questionable as they account for not more than 2% of the tricycloquinazoline-14C applied to the tissue. About 5% of the compound is transformed into nontricycloquinazoline-like but unidentified metabolites. At least 90% of the tricycloquinazoline remains unchanged 6 hours after skin painting.
E.
P R E S E N T %FATI’S OF’ THE
K-REGIONHYPOTHESIS
The multitude and variety of carcinogens discovered in recent years brought about an apparent decrease of interest in the K-region hypothesis. A strong defense of the orthodox point of view, against competing physicochemical liypotlieses, has been voiced by A. Pullman ( 1964). Howevcr, L: possil)ly inore fruitful, eclectic attempt of generalization has b c v n made (in lhiidel and Daudcl, 1966). An early detected inconsistency of the L ‘ e l e ~ t r ~ ntheory i ~ ’ ’ was t h a t sullstitution of 1,2-benzanthraccne in position 10 by either electron-C-N, attracting or clectroii-rlonating subxtituents (e.g., -0CH -G‘HO, or, 011 the other l i : m l , -CH, or -CLH5) inv:triably converted the parent liytlroc~url)oii,v-liicli h l i o ~ r doiily 1)ortlerlinc cai~inogenicity tow:ircl the siil)cutuncour tishue of stock niicc1, into a inow 1)otent agcnt $,
340
JOSEPH C. ARCOS AND MARY F. ARGUS
(e.g., reviewed by Badger, 1954). Yet, in the framework of the theory, electron-attracting substituents should rather decrease carcinogenicity since they withdraw the electron charge from and, hence, decrease the reactivity of the K-region. Rcccnt work shows better accordance with the theory in the 7,8-benxacridine series. Buu-Hoi et al. (1966) reported that substitution of the highly active IO-methyl-7,8-benzacridine with a trifluoromethyl group (which has a powerful -1 effect) in position 2 (LXX) brings about total loss of activity. Substitution of 7,8-benzacridine with a carboxamide or cyano group in position 10 leads to the
CHs
CH,
inactive
weakly active
(LXXIV)
(LXXV)
highly active
(=I)
highly active
weakly active
(LXXVII)
(IZXVIII)
slightly active or inactive compounds (LXXI) and (LXXII). On the other hand, substitution of the inactive 3-methyl-7,8-benzacridine with a formyl group in position 10 yields the highly active 3-methyl-10formyl-7,8-benzacridine (LXXIII) . Since the isosteric 3,10-dimethyl-7,8benzarridinc is alw ii potent c'wrciriogen (Ltwissagne ct ( i l . , 1956) , the
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
341
carcinogenicity of ( L X X I I I ) is in accordance with the observation in the anthanthrene and dibenzopyrcne scries (Section II,A,l) t h a t the effects of a methyl and a formyl group are cquivalcnt. Using methyl-l,2-benzaiithracenes fluoro-substituted a t the K-region, other investigations attempted to providc more specific evidence that covalent interaction via the K-region is a requirement for carcinogenicity. The central idea of these studies was derived iE. C. Miller and Miller, 1960) from the earlier findings that a number of fluoro-substituted derivatives of 4-diniethylaniinoazohenzene a i d 2-acctylarninofluorene are highly carcinogenic. This was interpreted to mean (e.g., ,J. A. Miller et nl., 1953) that the ring position(s) substituted by fluorine is not involved in the process of carcinogenesis but rather, because of the strength of the C-F bond, they are protected from metabolic inactivation. Conversely, loss or decrease of carcinogenicity owing to fluoro substitution was taken as a n indication t h a t the respective positions required for the cellular interactions leading to tumorigencsis are blocked by the fluoro substituent (s). Guided by this concept, E. C. itfiller and Miller (1960), J. A. Miller and Miller (1963), and Bergmann e t al. (1963) tested several methylbenzanthraceiies and niethylbcnzacridines, fluoro-substituted in the K-region, in order to gain insight into the metabolic importance of the 3and 4-positions for tumorigenesis. The results obtained with fluoro derivatives of the highly active lO-methyl-l,2-benzanthracene would seem to indicate that a free 3-position is critical for tumor induction, whereas a free 4-position is not. Both compounds, 3-fluoro-l0-methyl-l,2-benzanthracene (LXXIV) (E. C. Miller and Miller, 1960) and 3-fluoro-9,lOdiniethyl-1,2-benzanthracene (LXXV) (Bergmann e t al., 1963) are inactive or very slightly active, whereas 4-fluoro-l0-methyl-l,2-benzanthracene (LXXVII) is about as carcinogenic as the nonfluorinated parent hydrocarbon. However, no clear-cut conclusion can be made in view of the unexpected activity of two 3-fluoro-substituted polycyclics : 3-fluoro9-methyl-l,2-benzanthracene (LXXVI) is clearly a highly potent carcinogen (Bergmann et al., 1963), and 6-fluoro-2,10-dimethyl-7,8-benzacridine (also called 3-fluoro-7,l O-diniethyl-l,2-benzacridine) (LXXVIII) has well detectable although low carcinogenic activity (Bergmann et al., 1963). Although i t is true that considerably more detail is available in thc full reports of E. C. Miller and Miller (1960) and J. A. Miller and Miller (1963) than in the note of Bergmann, Blum, and Haddow (1963), there is no reason to disregard the important results in the latter. Further complexity is introduced into this picture by the fact t h a t 3,9-dimethyl1,2-benzaiitliraceiie (a stcrir analog of I X X V I ) is an inactive compound (HartwcJll,1951, 1). 151).
342
JOSEPH C. AHCOS AND MARY F. ARGUS
The totality of these data suggest, then, t h a t the inactivity or activity of these methylbenzanthraceiies is not related to the effectiveness of the fluorine atom to block covalent bond formation a t the K-region, but t h a t the activity level depends on the relative position of the hyperconjugating methyl group(s) and the fluoro substituent; the latter may have a net -1 or fM effect depending on its position on the resonant frame. These, in turn, indicate that activity or inactivity depends on the overall x-cloud distribution in the polycyclic frame, modified and shaped by the resultant electronic effect of the substituents. T h a t covalent binding a t the K-region is not involved in the activity or inactivity of these compounds is also borne out from the carcinogenic potency of 4,9- and 4,10-dimethyl-l,2-benzanthracene (Hartwell, 1951, p. 151), since in these compounds the supposedly critical 3-position, although not substituted, is sterically hindered by the neighboring methyl group. Another argument which cautions against a simple interpretation of the results with the fluorinated hydrocarbons is t h a t the strength of the C-F bond is not invariable, but is likely t o be influenced by the electron distribution of the whole moleculc. The metabolic removal of fluorine from p-fluoroaniline has been shown many years ago by Hughes and Saunders (1954). Very recently, Westrop and Topham (1966a,b) reported the enzymatic defluorination of 4’-fluoroaiiiinoazobenzenes. Unfortunately, investigations are lacking on the metabolic removal of fluorine suhstituen ts from polycyclic hydrocarbons. Chalvet and Mason (1961) predicted t h a t in lO-methyl-1,2-benzanthracene, fluorine substitution in 1’,2’,3’,4,6,8, or 9 will lend to carcinogenic coinpounds, whereas in 3 and possibly in 5 or 8 to inactive substances. The studies of the Millers did, however, support the idea that blocking the sites of metabolic inactivation by fluorosubstitution potentiates carcinogenicity. This has been illustrated with 4’-fluoro-l,2-benzanthracene which has considerable carcinogenicity toward the subcutaneous tissue of the rat ( J . A. Miller and Miller, 1963). Here, the substituent occupies the typical position of metabolic ring hydroxylation. The high carcinogenicity of 4’-fluoro-1,2-benzanthracene stands in striking contrast to the virtual inactivity of 4’-methyl-1,2-benzanthracene (Dunning and Curtis, 1960 ; Imassagne e t al., 1962). Studies of the metabolic fate of methyl substituents in the beiiz ring of 1,2-beiizaiithr~cene,as a possible clue for the inactivity of these derivatives, are needed. One of the instances often cited to point out the paramount importance of the K-region is the carcinogenicity of 1,2,5,6- and 1,2,7,8-dibenzanthracene, each having two K-regions, versus the inactivity of 1,2,3,4tlil~crizniitlirncc~ie which lias no K-rcgioii (ci.g., Pullman nnd Pullni:in,
1955:r,l);Hci~lell~erger et nl., 1962).Tile high level of binding of the latter to skin proteins was ascribed to the presence of a reactive L-region ( Heitlelhcrger, 1959 ; Heidclherger and Rloldenhauer, 1956). Considerable doulh h:is I)cc~n tlirown on t h t ~vahlity of t l i i h rc~isoningnow by the important finding of Uuu-Hoi (1964)that it suffices to introduce a methyl group in position 5 to evoke carcinogenicity in 1,2,3,4-dibenzanthracene [see ( X X I ) in Section II,A,l]. Evidence for tlie importance of the clelocalization of the x-electrons in polycyclic carcinogens has been provided hy Buu-Hoi et al. (1963). These workers obscrved tlie total absence of carcinogenic activity in a series of coiijugatcd aromatic polyacctylenes despite the pronounced number of x-electrons in these compounds. The great unsaturation of the conjugated polyacetylenic chain, that is, tlie much greater localization of the x-electrons than in the condensed aromatics, and the long, linear molecular shape are the likely c a u m of inactivity. The much greater localization of the x-electrons is shown by the fact t h a t none of the polyacetyleiies can act as an electron-donor to form colored r-complexes, a behavior typical of the condensed aromatic polycyclics. Thus, it would appear that neither extreme localization of the x-electrons (as in an ethylene or acetylene bond) nor great clelocalization is favorable for carcinogenic activity (cf. Badger, 1954). Evidence which has been accumulating in the last 12 years (see also Arcos and Arcos, 1962) points inescapably to the fact t h a t interaction through a ,neso-plienanthreiiic region is not the exclusive way by which polycyclic carcinogens initiate neoplastic changes. I n many highly active polycyclics, the K-region (9) may bc important zone (s) of interaction. I n other aromatic polyriuclear compounds, different reactive zones produce cellular alterations leading to the same biological result. Buu-Hoi (1950) already envisioned that van der Waals forces may play an important role in kcy cellular interactions. Arcos and Arcos (1956) proposed that covaleiit bond formation, hydrogen arid chargc-transfer bonding, dipole interactions, resoii:ince, arid dispersion forces act siinultancously and that the critical interaction ( s ) between the carcinogenic molecule and the cellular receptor sitc(s) is duc to tlie totality of these forces. A chemical agent is a “potelit” carcinogen when the cliffcrent optimal conditions prevail siinultancously a t the b i t r of action. The ahsence of one of the factors responsible for the intermolecular forces, for example, lack of a K-region, may limit carcinogenirity but not necessarily eliniinate activity altogether, since various types of stable molecular coinplexes can he inaintained hy secondary forces alone. Similar views ivew adopted recently 1)y .J. A. RIiller and Rfiller (1963).
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JOSEPH C. ARCOS A N D M A R Y F. ARGUS
From the mass of sotnetimes contradictory experimental evidence and maze of interpretations, there emerges a newer, more simple picture of the carcinogenic hydrocarbons so aptly described by Buu-Hoi ( 1964) : The conjugated frame offers certain sites and areas of high r-electron densities, hence a greater covalent and noncovalent reactivity, through which the interaction with cell components is facilitated; in many instances, there is a meso-phenanthrenic K-zone, whose involvement in the metabolic degradation of the carcinogen has been experimentally established but whose existence is essential neither for protein-binding nor for carcinogenicity. Where meso-anthracenic L-zones are present (case of naphthacene hydrocarbons), their reactivity does not necessarily preclude carcinogenicity. Replacement of =CH- groups by tervalent nitrogen heteroatoms may have a positive or a negative effect on the carcinogenicity, depending on the number and the position of the nitrogen atoms and on the nature of the molecule ; when several nitrogen heteroato.ms are present, the prime importance of their position in relation to one another suggests that they act as centers for binding with cell components, in place of, or in conjunction with, other zones of biochemical interaction. Where there are substituents, these may be either electron-donating or electron-accepting groups (except acid functions), and their contribution to the carcinogenicity may be positive or negative, depending on the type of molecule. In the case of alkyl substituents, lengthening of the chain has an adverse effect on activity, owing to increase in the encumbranw area of the carcinogen, the degree of loss of activity depending on the site of substitution. The introduction of substituents with acid hydroxyl groups (carboxyl, sulfonic acid, and phenolic functions) invariably results in a sharp deerease or total loss of carcinogenicity-an effect which must be due to a departure from the “normal” molecular orientation of the carcinogen within cellular lipid structures, produced by the strong hydrophilic radical. These, then, are the basic physico-chemical characteristics which are to be borne in mind when formulating or assessing general theories on the mode of interaction of polycyclic aromatic hydrocarbons and their heterocyclic analogs with cell components. The electronic theory of carcinogens which has proved of such great value in the past, as a guide in the search for nctive compounds through the .maze of organic chemistry, can probably continue to play that role if through adequate refinements and/or modifications it, can integrate the new experimenhl data.
F. NONCOVALENT INTERACTIONS OF POLYCYCLIC AROMATICS 1. Solubilization by Surfactants and Proteins Demisch and Wright (1963) investigated the nature of solubilization of polycyclic hydrocarbons by deoxycholate in a study of the partition coefficients of twenty-eight polynuclear aromatic hydrocarbons between aqueous monoethanolammonium deoxycholate and hexane. Their data are
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
345
consistent with the much earlier results of Fieser and Newrnan (1935) who observed that some hydrocarbons, but not others, yield well-defined crystalline addition compounds with deoxycholic acid. The complexes conform to a coordination principle inasmuch as they contain two, three, or four molecules of deoxycholic acid per molecule of hydrocarbon. Solubilization and complexing depend on a favorable molecular shape rather than molecular size proper. It is likely t h a t these complexes represent inclusion compounds since deoxycholic acid even alone tends to form helical aggregates in solution (McCrea and Angerer, 1960). Relevant to the interaction of polycyclic aromatics with cellular lipid constituents is the report of Snart (1967) who studied the surface behavior of mixed monolayers composed of a carcinogenic or noncarcinogenic hydrocarbon and cholesterol or lecithin. This study extends the early investigations of Clowes, Davis, and Krahl (Clowes e t al., 1939; Davis e t al., 1940). Hydrophobic bonding is probably the sole interaction between polycyclic hydrocarbons and proteins and lipoproteins in body fluids during transport. This is suggested by the study of Sahyun (1966a,b) on the solubilization of aromatic hydrocarbons and other nonionic aromatic compounds to bovine serum albumin. Following Sahyun’s solubility model, when a planar nonionic aromatic compound is dissolved in water alone, both surfaces are exposed to the solvent. On the other hand, when the compound is bound in a plane-parallel fashion to planar surfaces of the protein, only one surface is exposed. Hence, the energy of the hydrogen-bonded water cage in contact with the aromatic compound is twice as great in the former case as in the latter. Since the solubilities give a measure of the energy of thc dissolved state, Sahyun could confirm this model by determining the solubilities in the presence and absence of bovine serum albumin. Related to the problem of transport is the brief study of Anghileri (1967a,b) on the effect of different hydrocarbons on the binding of tritiated 3,4-benzopyrene to plasma proteins. 2. Solubilization by Purines
Probably more closely related to the critical cellular interaction of polycyclic hydrocarbons is their noncovalent combination with and solubilization by purines. Boyland and Green (1962a) have extended the investigations initiated by Weil-Malherbe (1946a,b) and Booth and Boyland (1953). The solvent power of purines shows a i l approximate parallelism with the number of N-inetliyl groups. The solvent effect is shared by the ~iucleosideu,adenosine arid guanosine. The l o w r solvent power of these, relative to the component purines, is likely to be a reflection of the fact t h a t the binding ability of the nucleosides is due t o
346
JOSEPH C. ARCOS AND MARY F. ARGUS
the purine moiety only. The pyrimidines, tliyniidine, vytitline, and uracil, have very little solvent powcr compared to the purines (Table X ) . Hydrogen bonding appears unimportant in the solubilization phenomenon since urea and lithium nitrate, both rcgarded as strong hydrogen bond breakers, h a v ~no cffect on the soluhilization of 3,4-hcnzopyrenc by caffeine (Boyland and Green, 1962a). Purines arc also cffirieiit quenchcrs of the typical fluorescence of polycyclic 1iydroc:trbons (Weil-Malherbe, 1946b; Boyland and Green, 1962a). Structural changes in purines which bring about loss of solvent power also bring about loss of quenching effect. The efficiency of quenching incrcascs linearly with purine concentration, but does not increase with the temperature, indicating t h a t collisional deactivation is not involved. The quenching action is not due to solubilization per se since deoxycliolate ion, a powerful solubilizer by hydrophobic bonding, is not a fluorescence quenchcr. Two hypotheses have becn proposed to account for the nature of the TABLE X RELATIVE SOLVENT POWER OF PURINES AND PYRIM IDINES TOWARD 3,4-BENZOPYRENE" Compound Caffeine
Caff eine-Ng-methiodide Caffeine-Ng-methochloride R-€Iydroxy-9-methyl-8,9-dihydrocaffeine 3,7-Dimetliyl-4,5-dihydroxy-2,6,8-trioxypurine 1,3,7-Trimet hyl-4,5-dimethoxy-2,6,8-trioxypurine Tetramethyluric acid 6-Dimet~hyl'aminopuriiie G'uanine Guanosine Adenine Adenosine Hypoxari thine Thymidine Orotic acid Cytidine Uracil Tryptophan
Molecular ratio
2 ,430 24,'LO0 16,050 87 ,400 00
m
468 9,620 18,500 23,130 43,600 50 ,400 92 ,000 110,000 209,000 272,000 1 ,255,000 ;W,4OO
Solvent power ( %)
100 10 0 15 1 2 8 0 0 520 25 2
13 1 10 5 56 48 26 2.2 12 09 0 2 8 0
u Compiled from Weil-Malherbe (1946s)and Boyland arid Green (19CLtt). The niolecular ratio represents the number of moles of purine required to dissolve one mole of hydrocarbon. The solvent power is calculated relative to that of caffeine. In order to obtain romparal)le values for the solvent power, the molecular ratios froin Boyland a ~ d Oreeii's paper were adjusted t hrorigh it coiiversioii factor obtained 1)y dividing the catfeirie molecular ratios from the two reports.
Pu1lni:tn :in(I PuIIiii:in (19.58, 1960) were tlir first to 1)i*opow,on t h v hihis of calculations of the relative electron-donor :tnd acceptor :t Mities of purines and pyrimidines, that charge transfer is involved. In charge transfer, polycyclic hydrocarbons usually play tlie role of electron-donor. However, I,ovelock e t nl. (1962) conclucled from an estimation of the electron affinities that aromatic polycyclics can also act as clcctroii-acceptorh. From the X-ray crystallographic data of DcSantis e t al. (1961) and Leela and Mason (1957), and the investigations of Ts’o and his co-workers (Ts’o e t nZ., 1963; Akinrimisi and Tb’o, 1964) on tlie absor1)tion and fluorescence spectra, it appears that the purine-1iydrocarl)on complexes consid of columnlike aggregates in which purine and hytlrocarbon molecules are alternately stacked. This is consistent with the geometry of aromatic charge-transfer complexes. Furthermore, any structural modification of the purine partner which impairs the planarity of the molceulc and, thus, brings about a decrease of plane-parallel adlineation to the hydrocarbon, produces decrease or loss of solubilizing power. This is exemplified by purine derivatives in which the tervalcnt nitrogen in position 9 is transformed into a quaternary ammonium salt (caffeine nicthiotlidc or methochloride) or by purines in which the double bond bctuwn the 8,9- and/or 4,5positions is lost (Table S ) ;probably for the same reason, the highly carcinogeiiic but nonplanar 9,10-dimethyl-1,2-benzanthracene is only slightly solubilized by aqueous caffeine. On the other hand, Boyland and Green (1962a) pointed out that the purine-hydrocarbon complexes do not show a new almorption band in the visible, which is so characteristic of the charge-transfer complexes of hyilrocarbons with chloranil, trinitrobenzene, iotlinc, etc. The only spectral change observed is a hathochromic shift in the ultraviolet absorption spectrum of the hydrocarbon. Hence, they suggested that solubilization depends on polarization forces or van der W:tals interactions. However, it should be noted that the same structural modifications which bring about decrease of charge transfer also bring about decrease in electrostatic attraction forces which are highly sensitive to tlie increase of intermolecular distance. Furthermore, increasing the molecular size and introducing coplanar mrthyl substituents, which generally enhance electron-donor ability, also increase tlie polarizability. Support for Boylitnd’b thesis was providcd by Pullman et al. (1965) who calculated the bonding energies by dipole-induced dipole attraction and by London dispersion force between 3,4-benzopyrene and various purines and pyrimidines. They found a fair parallelism between tlie order of London force bonding energies and purine solvent power. It is important to note, however, t h a t neither the correlation between London ~~iii~i~ic-liydi~oc.;ll.l,oll inter:ictions.
348
JOSEI’II
C. .211(’OS AND M A R Y
18.
ARGTJS
force and solvent powcr, nor the correlittion between the solvent power and the energy of the highest-filled or lowest-empty molecular orbital (Pullman and Pullman, 1958, 1960) is satisfactory enough to account alone for the total intermolecular attraction. It is more likely that these forces are only components of thc overall binding force which is, in addition, also influenced by the actual geometric orientation of the partners and the physicochemical variables (dielectric constant, ionic strength, viscosity, etc.) of the medium. It is known that the stability of charge-transfer complexing is, in many instances, reinforced by simultaneously operating dipole attraction and London forces (“polarization bonding”). It is perhaps significant in this regard that in the calculations of Pullman et al. (1965) the correlation is improved if the sum of the two bonding energies is considered.
3. Interaction with Nucleic Acids Boyland and his associates resumed their investigations on the solubilization of polycyclic hydrocarbons by DNA (Boyland and Green, 1962b). Solubilization of pyrene and 3,4-benzopyrene by DNA, just as solubilization by purines, is accompanied by bathochromic shifts of the spectra and by fluorescence quenching, and the changes are larger than are observed with the single purines, caffeine, and tetramethyluric acid. Destruction of the double-helix structure of D N A by heat denaturation brings about great loss of the solvent power. Similarly, in the presence of ethylene glycol or formamide, which also abolish the double-helix structure, DNA does not induce the bathochromic spectral shift. Ribonucleic acid, which unlike DNA has a low helix content, has also very little solubilizing power. These results were confirmed by Liquori et al. (1962) with the exception, however, t h a t in their experiments, solutions of denatured DNA had a greater solvent power than solutions of native DNA. T h a t noncovalent binding does take place between the polycyclic hydrocarbons and nucleic acid was also confirmed using a different technique. Robert (1963), taking up the earlier investigations of Brigando (1956), demonstrated interaction between 20-methylcholanthrene and DNA in monomolecular film. Solubilization by DNA can be accounted for by two binding mcchanisms: (a) “internal” binding, i.e., insertion between base-pairs; (6) “external” binding to DNA perpendicular t o the planes of the purine bases and interaction with them a t points not involved in the maintenance of the double helix (Fig. 5 ) . Boyland and Green (196213) showed on a molecular model of DNA that planar polycyclic hydrocarbon molecules, such as 3,4-benzopyrene and 1,2,5,6-dibenzanthracene,can be accommodated between the base-pairs by slight untwisting, “straightening” of
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
349
the sugar-phosphate backbone and intcrcalation of the hydrocarbon molecule into the spacc thus arisen. This stcric accommodation would then he stabilized by polarization bonding betwccn the hydrocarbons arid the purines and by a hydrophobic effect involving the entire doublehelix strand. The ability of hydrocarbons to complex with DNA is dependent on the pH (Ball et al., 1965) and is sensitive to the presence of inorganic ions and small polar molecules. This is consistent with the polyelectrolyte nature of DNA and the intercalation model of the complexing. The
I I
I
A
I
I
B
Fro. 5. Schematic representation of possible spatial orientations of polynuclear aromatic molecules in complexing with DNA. I n ( A ) is seen slight untwisting of the helical backbone and intcrcalation of thc aromatic compound into the spacc aristw, parallel to thc basc-pairs; in (B) is shown external binding to DNA without disturbance of the double helix, the planes of the aromatic molecules lying more or less parallel to the helix axis.
solubilization experiments of Boyland and Green were usually carried out in 0.001 M NaCl to protect DNA against spontaneous denaturation. Increasing the salt concentration reduces the solubility of the hydrocarbons in DNA solutions. Since the solubility of hydrocarbons in water is not affected by such concentrations of salt, an explanation based on simple solubility effect appears to be excluded (Boyland and Green, 196213). Sodium ions, as well as K+, Ca++,Mg++,aliphatic amines, urea, and dimethyl sulfoxide all lower the solubility of hydrocarbons in DNA solutions and can also, upon addition, release the bound hydrocarbon from the soluble complex (Boyland e t nl., 1964~1).The ion effect may be interpreted in terms of the intercalation hypothesis. Increasetl ion conccntr;ition t c ~ n ( l xto clccrc:~sciq)uIsion 1)c~twcwithe pliosp11:ttc~groups in
350
,JOSEPH C. ARCOS AND MARY 1“. ARGUS
the sugar-phosphate backbone and would also increase hydrophobic bonding between the bases by rendering all nonpolar substances less soluble. The overall effect amounts to an increase of the stability of the helix (Boyland and Green, 1962b; Boyland e t al., 1964d). The solubility and spectral studies of Kodama e t al. (1966) indicate, indeed, t h a t the ions bring about a more compact packing of the bases resulting probably in a shortening of the pitch of the helix. Hence, the macromolecule becomes less accessible to intercalation. Boyland and Green (1964a) presented evidence that the ion effect accounts for the conflicting observation of Liquori e t al. (1962) on the greater solubility of 3,4-benzopyrene in denatured rather than nativc DNA. Other evidence for the combination of polycyclic hydrocarbons and D N A was provided by interesting results on the in vitro effect of hydrocarbons on the T, of DNA. Boyland and Green found t h a t anthracene, pyrene, 3,4-benzopyrene, 1,2-benzanthracene, and 9,10-dimethyl-1,2-benzanthraccne appreciably increase the T , of calf thymus D N A (Boyland and Grcen, 1963; reviewed by Boyland, 1964a). Thc increase of the T , could reflect contribution to polarization bonding or, more likely, enhancement of hydrophobic bonding. The latter effect may consist in shielding sites of the D N A molecule-at which fluctuations in the rigid hydrogen-bonded structure occur most readily-against the surrounding water structure. These regions are thereby stabilized causing a n increase of the temperature necessary for “unzipping” the double helix (Boyland and Green, 1 9 6 2 ~ ) . However, Daniieriberg and Sonncnbichler (1965) were unable to bring about a significant increase of the T , of D N A with the above and other polycyclic hydrocarbons, and also with aromatic amines. The very drastic methods used 1)y these authors (consecutive chloroform and cyclohexane extraction) to remove the “unbound” hydrocarbon are likely to be responsible for the negative rcsults. This is actually indicated by their own tabulated values of the spectrophotoinetrically determined levels of compounds in the solution following extraction ; the nonpolar hydrocarbons are extensively removed while the more polar 3-aminophennnthrene (the only aromatic amine studied in this regard) remains in solution a t levels 50-100-fold higher. Finally, from the DNA-proflavinc and DNA-acridine orange complexes, extraction removes even less of the non-DNA component; this is consistent with the highly polar character of these dyes. With these solutions, in which a high level of non-DNA component remained, an appreciable increase of the T, was observed. It is of interest, in view of the possible gene mediation of thalidomide embryopathy, t h a t N-phthalylglutarylimide produced an increasc of thc T,,, comparnhlc t o the above ncridine Iiiutagens.
MOLECI'1,AR
tiEOMETRY AND CARCINOCIESIC ACTIVITY
351
I n Boyland and Green's ( 1962c, 1963) experiments, botli the carcinogenic and the noncnrciiiogrnic hydrocartions gave the same effect in ? ) { f r ophenomenon IJe involved raising t,hr T,. Howeiw, should this in the niwhanisni of hiological :ivtioii, qii:intit:atir.cI diffcwiicoh inay hc of i of the T , was 10°C. critical importance. I n fact, tlic i i i : ~ x i i ~ i u i iiiicrc:w with 9,10-dimethyl-l,2-benzanthracene (one of the most rapidly acting skin carcinogens known), but thc iiicrease was only 6°C. with the less active 3,4-benzopyrene. Also, whereas this latter increase of 6°C. was brought about with as little as 0.3 phf 3,4-l)enzopyrene, when using the inactive pyrene 1.4 piM was needed to bring about the same increase of the T,,&. These quantitative differences bring to iiiiiid and may be related to the investigations of Nagata and his co-workers on the quantzty of charge transfer-as distinct from the stabilization energy due to complex formation (i.e., thc charge-transfcr force) --betuwn a hydrocarbon carcinogen and purine-pyrimidine base-pairs. Nagata e t al. (1963b) found that although there is only a small difference in the stabilization energy calculated for pyrelie and for the potent carcinogen, 3,4,9,10-dibenzopyrene, the quantity of charge transfer is far larger for the carcinogen than for the inactive pyrene. It is interesting, moreover, that with either hydrocarbon, both the quantity of charge transfer and the stabilization energy is greater with the guanine-cytosine than with the adeninethymine base-pair. Results consistent with the latter finding were obtained when these two charge-transfer parameters were calculated for the heterocyclic carcinogens, tricycloquinazoline (LVII) and its oxygencontaining structural analog ( L I X ) (Nagata e t al.,196613). Since aniong all the constituents of the nucleic acids, guanine is the best electron donor, this base has probably a dominant role in charge-transfer complexing with nucleic acids. Experimental cvidencc supporting this is found in the preponderant solvent power of guanine aniong the bases in nuclcic acids (Table X ) . These impressive inrestigations on the bolubilization of polycyclic aromatics by DNA came under strong attack by Giovanella et al. (1964). I n a refutation of the work of the 13oylancl aiitl Liquori groups, Giovanella et nl. reported that when a O.OSC/, solution of DNA is ground with 3,4benzopyrene or 1,2,5,6-dibeiizantlirace1ie, the suspension of the hydrocarbon (nonsedimentable a t low speed) can be completely sedimcnted under their experimental conditions by high-speed centrifugation, or filtered off by uniform 0.45-p pore-size Millipore filters. They concluded that the hydrocarbons are not truly solubilized by D N A hut form finedisperse aqueous suspensions stabilized by the macromolecule, and they reported t h a t this stabilization effect can actually be duplicated with
352
JOSEPH C . ARCOS A N D MARY F. ARGUS
the m e of soap (approx. O . o l y 0 ) instead of DNA. In tlie light of their findings, Giovanella et al. related the lowering of solubility in or release of hydrocarbons froni DNA solutioiis by ion6 and p01m molecules to thc wcll-knowii fact th:it d t s prccipit:itr colloids. O i i the other hand, the concentration of 3,4-benzopyrenc in saturatc>tlcaffeine solutions could not be diminished by high-speed centrifugation for a prolonged period of time; thus, they regarded these as true solutions. I n an examination of the criticism of Giovanella et al. (1964), Boyland and Green (1964b) presented evidence that accounts for the conflicting observations. Pyrenc and 3,4-benzopyrene are, indeed, removed from aqueous caffeine or aqueous DNA solutions if the high-speed centrifugation is carried out in plastic tubes (polypropylene, polypropyleneethylene copolymer, cellulosc acetate). No loss of hydrocarbons occurs, however, upon centrifugation in quartz or glass tubes. Boyland and Green also confirmed the spectrophotonietrically demonstrable loss of hydrocarbons when filtering the solutions (in aqueous caffeine or DNA) through a pad of several Millipore filters. There is consequently no difference in the actual results obtained, but rather in their interpretation. Boyland and his associates attributed the losses of hydrocarbons to adsorption by the filter material or to the formation of solid solution with the polymer material of the centrifuge tubes. I n retrospect, the controversy is even more surprising in view of evidence from the earlier work of Steele and Szent-Gyorgyi (1957) that 3,4-benzopyrene complexes with and is solubilized by caffeine as well as DNA. These authors noted in accordance with the observations of Brock et al. (1938) and Weil-Malherbe (1946b) that the hydrocarbon has a yellow fluorescence either in solid form or in fine-disperse colloidal solution. For example, by adding a drop of acetone solution of 3,4-benzopyrene to water, the resulting colloidal solution emits a greenish-yellow fluorescence under ultraviolet light. Howevcr, the color of fluorescence turns dramatically to blue if the solution is shaken with solid caffeine or with DNA, indicating quite evidently that, in the latter case, a-electron interaction with the nucleic acid took place. 3,4-Benzopyrene is also fairly potent in quenching the phosphorescence emission of frozen aqueous solution of DNA from which i t can reasonably be interpreted that the collective energy conduction through the bases is cut by interposition (i.e., intercalation) of a “dud,” the hydrocarbon molecule (Steele and Szent-Gyorgyi, 1957). The interaction of polycyclic hydrocarbons with DNA brings to mind the well-investigated interaction of nucleic acids and polynucleotides with various planar acridine dyes, acriflavine, proflavine, and acridine orange (e.g., Heilweil and Van Winkle, 1955; Bradley and Felsenfeld, 1959; Steiner and Beers, 1959; Lerman, 1964). The complex of DNA with proflavine was studied by Luzzati et al. (1961)
ILIOLECULAR (XOMETBY A N D CARCINOGENIC ACTIVITY
353
by X-ray crystallography aiid found to have a planar, “sandwichlike,” inclusion compound structure in which proflavine molecules are intercalated between the base-pairs of DNA. The recent work of Ball et al. (1965) provided further support for the solubilization of hydrocarbons by DNA. Heating the DNA-3,4bcnzopyrene complex over a temperature rnnge, which produces the separation of control DNA into single polynucleotide strands, brings about a loss of absorption at 395 nip, the wavelength of iiiaximuni absorption of the complex. The dissociation of the complex into its component parts (over this temperature range) is also indicated by the increase of relative viscosity. Essentially identical conclusions were reached subsequently by Isenberg et al. (1967) from studies of the complexing of hydrocarbons with native and denatured DNA, RNA, aiid single-stranded poly A. Particularly interesting is their finding that the degree of complexing appears to depend on the molecular dimensions of the hydrocarbon. Definitive confirmation of Boyland’s interpretation was provided by the elegant experiments of Nagata et 01. (1966a,b). The Japancse workers applied the method of flow dichroism to dctcrmine the spatial orientation of the polycyclic molecules with respect to the lengthwise axis of DNA. The basis of this important but little-known technique, as applied by Nagata and his co-workers, is as follows. In planar aromatic molecules, such as 3,4-benzopyrene1 polarized light can induce electronic transitions only if the plane of polarization (i.c., the electric vector) is parallel to the molccular plane; in this case light energy is absorbed. No electronic transition and, hence, no light absorption occurs when the plane of polarization is perpendicular to the molecular plane (Fig. 6 ) . Figure 7 gives a schematic representation of the flow dichroism spectrophotometer arI
poloriled light plane of polorimtion
y
0 ABSORPTION
polarimllan
FIG.6. .1 representation of electronic: tr:uiiitions induced by polarized light in 3,4-benzopyrene.
354
JOSEPH C. AIlCOS A N D MARY F. ARGUS L iahl source
mi
mofo mulliplier
FIG.7. Schematic representation of the apparatus of Wada and Kozawa (1964) for the measurement of flow dichroism. C1 and C, are coaxial cylinders made of optical glass. The clearance between them is 0.5 mm. and the total light path in the solution is 1 mm. The inner cylinder is rotated a t or above 1000 rpm. P is the calcite polarieer which can be positioned so that the plane of polarization is either parallel to or perpendicular with the flow line.
rangement of Wada and Kozawa (1964) used by the Nagata group in their studies, and Fig. 8 illustrates the principle of determination of differential dichroism. Owing to the flow of the solution containing the DNA-hydrocarbon complex, in the space between the stationary optical glass cylinder C, and the rotating glass cylinder C,, the double-helix strands of the complex are aligned parallel to the flow line. Therefore, the position of the molecular planes of the bound hydrocarbon molecules, with respect to the plane of polarization, will be different depending on whether they are intercalatcd between the bases (Fig. 8A) or adsorbed to the surface of the double helix (Fig. 8B). Hence, the difference of the light absorption values in the two positions of the polarizer will directly indicate the geonietry of the complex. The differential dichroism, AE, is obtained by subtracting the molar extinction coefficient toward the light polarized perpendicular to the flow line, e,, from the niolar extinction coefficient toward the light polarized parallel to the flow line, ell, that is Ae =
€11
- e,
Since € 1 1 and el are both positive, Ae can be either positive or negative depending on whether € 1 1 or el is greater. If the niolecular planes of the chromophores are oriented perpendicular to the flow line, then e, > t l I and Ae is negative, whereas Ae is positive if the niolecular orientation i s parallel to the [!ow line Iwxaiisc thcii € 1 1 > el. Thus, froin the sign of A€, the orientation of the plaiir of electronic transitions (i.c., the spatial orientation of the nioleculcs) can be tleterinined (k’ig. 8). Nagata d 01. (lSCiGa,l)) showed that Ae is negative for phenanthrene, pyrene, 3,4-benzopyreiie, tric’yclocluiiiazolirle (LYII), ant1 the tricycloquinazoline analog (LIX), whereas A€ has a positive sign for 20-methyl-
cliolantlirene, ( ~ o I ' o ~ I ~iiaplithacwc, wc~, niill pciit:tcc~no. Thus, tlic formcr compounds are oricnted 1)arallcl to tlic p1anc.s of the bases so that interaction hy intercalation is unequivocally established. T h e latter compounds, on the other hand, are oriented perpendicular to the planes of the bases :tnd bound to the external surface of the DNA strands. The cliffcreiitial dichroibtn spectra arc excniplified and compared to the respective absoiptiou spectra in Fig. 9. Distinct dichroism was not observed for 9,10-diniethyl-1,2-benzanthracenc and 1,2,5,6-dibenzanthracene so that their mode of orientation could not be dcteriiiincd. However, the changes in soluhility in the presence of various concentrations of NaCl are similar to the oiics ohserved with thobe hydrocarbons that complex by intercalation. I n addition, the AE spectra provide unainbiguous evidence for the noncovalent interaction with DNA and for the true solubilization of polycyclic aromatics. I n fact, the bathochromic shift produced by thc complexing iii the hydrocarbon absorption spectrum (Boyland and Green, 1962b; 1,iCjuori et al., 1962) also occurs in the dichroism spectrum, and the peaks of the two types of spectra coincide quite accurately (e.g., Fig. 9). Nonspecific soaplike adsorption to D N A molecules or colloid stabilization by DNA, as suggestcd by Giovanclla et al. (1964), can certainly be excluded bccaube 110 tlichroism can occur in such cases (cf. Nagata e t al., 1966a). From the parallel orientation of the hydrocarbons to the bases, as observccl for 3,4-benzopyrene, overlapping of the r-orbitals is a virtual certainty so that charge transfer and polarization bonding are almost to be expectcd. The phenomena of hathochromic shift and fluorescence quenching are entirely consistent with this view. For intercalation into DNA (also to some extent for adlineation to the external surface of the doublc helix) as well as for complexing with purine-pyrimidine base-pairs, conditions of steric fit and geometric similarity must he met. The requirement of geometric similarity between a hydrocarbon moleculc and a single purine or pyrimidine, or a haw-pair, is implicit in the calculations of charge-transfer parameters, since a-type overlap of atomic orbitals is assumed (Nagata e t al., 196311, 1966a,b). I n order to fit into the narrow but flexible and stretchable space between the base-pairs, the moleculc must have within limits an adequate shape. Haddow (1957) has poiiitcd out that scveral carcinogcnic hydrocarbons are of a sizc and shapc >imilar to purine-pyrimidinc pairs; for cxainple, the potent carcinogen, 3,4,8,9-dibcnzopyrei~~~, occupies an area similar to that of an adenine-thyminc pair (Fig. 10A). I n some instaiices, intercalation into and interaction with DNA docs not appear to involve the entire polycyclic moleculr. This is the case with tricycloquinazolinc-a coinpound complexing by intercalation-which is similar to the g u a n i n e
w cn Q,
A. Hydrocarbon intercalated parallel to the bases. therefore A € IS negative >
EL El,
I I
clearance 015 mm.
polorizer
-
Monochromator electric vectors
DN4 dwble helix ___)
flow line
r Photomultiplier
I
plane of polorization
polorizer
Monochrornotor
-@
+El=
osciIli+ory plane porollel to flow line
-
flow line
done of phorizotion
Photomultiplier
0
B Hydrocarbon bound externally and oriented perpendicular to the bases.
< E,I
therefore Ac is positive
I Photomultiplier
,~-./’
hydrocobon h n d to surface of DNA
flow line
’
cross s?ction of M A hbte helix
plane of polarization
I ~
-
Photomultiplier
Monochromotor
oscil latory plane parallel to flow Ime
flow line
plane of polarization
FIG.5. Principle of the determination of differential dichroism of polycyclic hydrocarbons complexed with DNA. In (A) is shown why the negative sign of h e indicates intercalation parallel to the bases; in (B) the basis of a positive value, indicative of external coniplexing parallel to the lengthwise axis of DNA, is depicted.
358
JOSEPH C. ARCOS AND MARY F. ARGUS
370 380 390 400 m p UI
a
380 390 400 410 420rnp
-0.1-
FIG.9. Comparison of absorption spectra to flow dichroism spectra for 3,4benzopyrene and 20-methylcholanthrene complcxcd with DNA. (A) The negative sign of Ae for 3,4-benzopyrene indicates that there is intercalation between the base pairs; (B) 20-methylcholanthrene has a A€ spectrum in the positive region indicating that the hydrocarbon is bound parallel to the lengthwise axis of the double helix. (From Nagata et al., 1966a.) Dotted curves, absorption spectra; solid curves, Aclc spectra.
cytosine pair (Fig. 10B), as pointed out by Baldwin e t al. (1963a), Of special interest in this regard is the recent finding of Isenberg e t al. (1967) mentioned above that molecular size is an important parameter determining the extent of solubilization by DNA. The concatenation of
3,4,8,9-Dibenzopyrene
Tricycloquinazoline
A
B
FIG.10. Geometric similarity of polynuclear aromatic carcinogens with purinepyrimidine base-pairs in DNA. (A) 3,4,8,%Dibenzopyrene and adenine-thymine. (After Haddow, as modified by Boyland, 1964a.) (B) Tricycloquinazoline and cytosine-guanine.
these findings and concepts now provides a beginning for understanding, a t the molecular level, the requirement of an optimum molecular size range for carcinogenic activity. 4. Significance of Hydrocarbon-DNA Interaction for Mutagenesis
and Carcinogenesis: Some Conceptual Advances Watson and Crick (1953) have postulated years ago that spontaneous mutations may occur as the result of amine -+ imine-type tautomeric shift in one of the purine or pyrimidine bases of DNA and that this results in the miscoupling of bases. Subsequently, Lawley and Brookes (1962; reyiewed by Brookes and Lawley, 1964a) and Nagata et al. (1963a) suggested that the ionization of guanine due to alkylation of the base may also lead to anomalous pairing. This would explain the well-known mutagenic effect of alkylating agents. A somewhat different molecular mechanism should account for the mutagenicity of planar aromatic molecules that complex with DNA by intercalation. Brenner et al. (1961) put forth the idea that the mutagenic activity of aminoacridines-which were shown to intercalate between the bases of DNA-may be due to the increase of the distance between certain neighboring base-pairs and, thus, to the creation of a gap in the orderly periodicity of DNA structure. This could conceivably lead to the deletion of a base from or to the inclusion of a randomly selected additional base into the replica DNA, in the region of the gap, during cell division. Hence, the replica would become a mutant DNA species. Intercalation of hydrocarbons and other polycyclic carcinogens could, similarly, bring about alteration of the base sequence (Boyland and Greeii, 1962b). It is likely, moreovcr, that the polarization bonding between the alternately stacked hydrocarbon molecules and bases promotes plane-parallel molecular adlineation and, hence, keto + enol-type, lactam-lactim tautomerism in the latter (Arcos and Arcos, 1962). The observation that there is an approximate parallelism between the mutagenicity and basicity of the aminoacridines (Orgel and Brenner, 1961) and between their mutagenicity and ability to form charge-transfer complexes (Brenner et nl., 1961 ; Pullman, 1962), suggests that this mechanism may be a participant in the niutagenicity, in addition to the purely steric iriterfcrcnce suggeated by Brenncr et nl. Credence to the idea of lactam + 1:ictiin rc:i1.1.nrlgc.lncltit is lent l y thc rarly experiments of Henclricks ( 1941) who hliowcd th:tt (Iiiriiig t l w :icl~otptionof gunnitw by tlie 1nriit~ll:ir t t i o l t ~ u l ~&vc, ~r iiiontiiioi~illoititt~,t l i c ~ w ih :L shift of the tautoirirr eyuililxiuin toward the more plmar enolic f o i ~ i iwhich provides greater contact for molecular adlinentioil. For example, following the Watson-Crick model, guanine and thymine niust be in the keto form for
360
JOSEPH C. A R C 0 6 AND MARY F. ARGUS
providing the required sitcs for hydrogcn bonding. Thus, displacement of the tautomer equilibrium would bring about a change in the hydrogenbonded partners. Hence, guanine (enol) would couple with thymine (instead of cytosine), and thymine (enol) would couple with guanine (instead of adenine) during the formation of the first replica DNA strand (Fig. 11). At the second replication (occurring in the absence of the carcinogen) the bases in the first replica single strand, thymine and guanine, would couple with adenine and cytosine, respectively. Therefore, a t the site of intercalation, the bases in the parent single strand, guanine and thymine, are now permanently replaced by adenine and cytosine, respectively, so that the DNA alteration has become self -perpetuating. Thymine
Hd
Guanine (enol)
\
Thymine (enol)
HO
Guanine
I
\
FIG.11. Miscoupling of the cnol forms of guanine and thymine.
Calculations by the Pullmans (B. Pullman and Pullman, 1962; B. Pullman, 1964) of the resonance energies of the lactam and lactim tautomeric forms provide other support for the participation of this phenomenon in the mechanism of mutations. I n fact, the lactam + lactim rearrangement is paralleled by an increase of resonance energy, and this gain represents the “driving force” toward the increase of the proportion of the lactim form. On the other hand, in the amine + imine-type tautomerism proposed by Watson and Crick (1953), the shift is accompanied by n clccrcnhe of thc rcmianw rnergy ; thcrcforc, tlic snialler this rlectwsc, the grcittcr will IN, tllc proportioil of the iiiiiiie form (Table X I ) . The prediction that cytosine would h~ the greatest tendency to exist in a tautorneric forin (lactiin or imino) beems to be substantiattxl by iiuclear magnetic resonance studies (Kokko et al., 1961 ; Gatlin and Davis, 1962).
361
MOLECIJ1,AR GEOMETRY ZND C' \RC'TKOC:ESIC ACTIVITY
ItESONAXCE
Gurtiiiiie
Cytosine Iiracil
Thymine C; mini rie Adenine
Cytosiiie
TABLE XI ENERGIES OF THE TAUTOhIERIC F O R M S ~
1,acI an1 3 84 2 28 I %2 2 05 Aniine 3 x4 3 89 2 28
irn 4.16 2.69 2.14 2.27 Imine
1,itc.l
3.68 :; . A2
2.15
0.32 0.41 0.22 0.22 -0. 16 -0.27 -0.13
l h m B. Pullniaii and Pullman (196'2). = Variation of resonance energies arcompitnyiiig the transformation from the ~ I a l ~tloe the less stable form. 'I
* AH
Karreman (1962) calculated that charge-transfer coinplexing of 4-nitroquinoline-N-oxide with adenine actually promotes the amine + imine tautomerism by increasing the reboiiance energy of the iiiiino form above that of the aniine. Evidence for the cornplexing of 4-nitroq~inolinc-~Voxide with DNA by intercalation was provided by the flow dicliroisrn studies of Nagata et al. (1966a). Of cowbe, such ti specific mechanistic picture based on amine + imiiie or lactam -+ luctim tautomerism can not directly explain alterations in base-pairing brought about by externally complexed planar aromatics. It should be borne in mind, however, t h a t except for hydrophobic bonding, all types of noncovalent interactions can bring about a redistribution of 7-clectrous. Hence, given an optiinum geometry and charge distribution, :in externally bound polycyclic aromntic molecule may weakeii hydrogen bonding and the stability of the helix a t certain loci. The prohleni of the causal relationship betwcen mutagenicity and cnrcinogenicity retilains unresolved since tlie time of the rcvirws by Boy1:uid (1964) :intl Burclctte (1955). Altliougli :Lgreat nuniher of rcports :~1)poiiredin recent yc:irs on the niutagcwicity of various nit1 osiiiiiinc,~ (rcvicwetl by hlagcc an({ Bariies, 1967) nu(l other :ilkylating cnrcinogenh (e.g., Frccse, 1963; A1ex:indcr :in([ Glnngcs, 1965), the present reviewers feel that these do not constitute unambiguous support for the causal rclationdiip between the two phenomena. In fact, these agents are chemically highly reactive molecular species and, thus, their mutagenicity is likely to be due to randoin alkylations (cf. Trams e t al., 1961). Therefore, their mutngcnicity rnny not be construed as evidence for the relatedness of the two p1ienonien:i. 4 t any rate, the niutagrnirity of these ciircinogens of
362
JOSEPI-I C. ARCOS A N D MARS F. ARGUS
high chemical reactivity certainly does iiot seem to be relevant to the rarcinogenicity of thc rather unreactive polyryclic aromatics, which gave ambiguous and often irreprociucihle rewlts as mutagens in a nuniher of cxperimcnts (rrvicwc(1 hy Boyl;iii(l, 1954 ; B u ~ ~ l r t t 1955). c, TIE spccultltions of Clayson (1962) throw frcbli liglit on this question a l i c l possibly reconcile the opposing views: If cancer is thc result of a mutation at a specific locus or loci in the genetic system, the mere demonstration of the mutagenicity or otherwise of a chemical is of little relevance to thc induction of cancer. I t is necessary to show that carcinogens induce mutations a t the correct loci. That is to say, tissue, species and strain specificity should be partially explicable in terms of the ease of induction of the required mutations.
G. EVIDENCE FOR HYDROCARBON FREERADICALS Szent-GyGrgyi e t al. (1960) claimed that a correlation exists between the carcinogenic activity of a series of polycyclic hydrocarbons, aromatic amines and azo compounds, and the ability of these to form chargetransfer complexes with iodine. They have measured the magnitude of the electron spin resonance (ESR) signals of these complexes and concluded that carcinogenic compounds give strong signals whereas noncarcinogens give wcak or no signals. I n an extension of this study, Damerau and Lassmann (1963) examined the iodine complexes of a greater variety of azo compounds by ESR spectrometry and could not distinguish between carcinogens and noncarcinogens. Similarly, Jones e t al. (1966) used this technique to study the cornplexing between purine and pyrimidine bases, on one hand, and carcinogenic hydrocarbons and aromatic amines, on the other, and failed to detect any ESR signal. However, Wilk et al. (1966) concluded from a new dimerization and tetramerization reaction of polynuclear hydrocarbons that the reaction must pass through an intermediate radical cation species. Nagata et al. (1966d) used ESR spectrometry in a study of the interaction of polycyclic hydrocarbons with animal tissues. They observed a strong signal-indicating the presence of free radicals-in rat liver homogenates following in vitro treatment with 3,4-benzopyrene1 but only very weak signals in control tissue samples and in tissue samples treated with the noncarcinogen, pyrene (Fig. 12). On the other hand, no differencc in ESR signals was detectable in rat skin tissue 2 days following the final painting of a 5-day treatment period with the same two hydrocarbons. It is, therefore, possible that the radicals formed during the in vitro treatment of liver homogenates, and detectable immediately after treatment, are short-lived and are destroyed by metabolism, as seems to be indicated by the results of the in vivo experiments. Thus, there is now a beginning
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
363
5 0 gauss H
C
FIQ.12. Free radicals detccted by ESR spectrometry in rat liver homogcnates. (A) control; (B) following treatment with pyrene; (C) following treatment with 3,4-benzopyrene. The arrow points in the direction of increase of the magnetic field strength H. (From Nagata et al., 1966d.)
of evidence that carcinogenic hydrocarbons may give rise to reactive radical species in the tissues. Ill. Conjugated Arylamines and Compounds Generating Arylamines. Arylhydroxylamines
A. ARYLAMINESAND ARYLNITROCOMPOUNDS 1. Monocyclic Compounds
It is increasingly accepted that aniline itself is not carcinogenic, a t least in man (Scott, 1962). However, the question of the activity or inactivity of its ring-methylated derivatives is not yet definitely settled. There is some suggestive evidence that o-toluidine might be carcinogenic in man (Vigliani and Barsotti, 1962). Deichmann (1967), however, was unable to induce tumors in dogs by administering the three toluidines for 6 years. Carcinogenicity, although still of very low order, is more readily detectable in the ring di- and trimethylanilines. In addition to 3,4-dimethylacetanilide which produces mammary tumors in a scattering of animals (E. C. Miller et al., 1956), 2,4-dimethylacetanilide (Klein and Weisburger, 1966) anti 2,4,6-t,rimethylacetarlilide (Morris and Wagner, 1964; Klein :tnd Weisl)iii*gcr, 1966) were shown to induce malignant or near malignant 1iep:ttic lesions in occnsioiinl nnimals (rats or mice). Consistent
364
JOSEPH C. AHCOR AND MARY
11’.
AllGVS
with thc carcinogenic activity is tlic observcd hepatotoxic effect whcn large amounts of 2,4-xylidine were fed to rats (Lindstrom et al., 1963). Nonetheless, because of the a t most borderline tumorigenic activity of all these monocyclic compounds, it may not be excluded that activity is due to trace amounts of potent impurities. I n view of the industrial importance of the ring methylanilines, further assaying with highly purified compounds, in large test groups and for longer periods of time is urgently needed. It may be of interest to remember, accepting conditionally these amines as truly carcinogenic, that the methyl substitutions raise the conjugating power of the ring so that the aromatic moieties represent intermediates betwecn an unsubstitutcd phenyl and a naphthyl group. Searle (1966b) has shown that another group of monocyclic compounds which may be transformed metabolically to amines and hydroxylamines are active both as tumor initiators and as complete carcinogens. Treatment of the mouse skin with pentachloronitrobenzene or with any of the thrce isomeric tetrachloronitrobenzcnes produces multiple papillomas during subsequent promotion with croton oil. On epithelial application, these compounds did not induce tumors without promotion. However, subsequent work (Searle, 1966a) showed that they act as complete carcinogens when assayed in mice by subcutaneous route. Metabolism of these compounds, with the exception of the 2,3,4,5-tetrachloro compound, results in the formation of mercapturic acids by replacement of the -NOz group to the extent of 36 to 37%. The 2,3,4,5-tetrachloro compound was the most effective, both as a tumor initiator and as a coinplete carcinogen. Furthermore, N-ethylmaleimide, a sulfhydryl reactor par excellence, was found inactive as initiator in these tests. For these reasons, Searle (1966b) discounted the irnportancc of SH- reactivity in the biological activity of the compounds. A nonbenxenoid group of monocyclic aromatic compounds which manifests carcinogcnic activity is the 5-nitrofurans. A variety of 2-sub-
(LXXJX)
stituted 5-nitrofurans (LXXIX) have been known for soinc tirric to he effective antibacterial agents (Eaton Laboratories, 1958) and have found widcsprcd c1inir:il al)plications for trcating infcctioris of the urinary tract and gynecological bacterial disorders. It is assumed that metabolism transforms these nitrofurans to amines and hydroxylamines. Price et al. (1966) and Stein e t 02. (1966) found that several 5-nitrofuran derivntivec arc potent carcinogens having an ubiquitous tissuc spectrum upon atl-
MOLECTTLAR GEOMETRY A K D CARCINOGENIC ACTIVITY
365
iiiiiiist,r;ition to Slmiguc-D:iivley i x t h :it the lcvels of 0.1 to 0.3% i i i Llic tliet. Tumors of the following Iiistologir:~l typcs and tissue localizations were found: maminary :idcnornas arid ;i~Ie~ioc~rci~ioiias, multiplc papillomas, squainous cell carcinomas and adenocarcinomas of the forestomach, adenocarcinomas of the small and large intestine, renal adenomas, and adenocarcinomas. Moreover, a high incidence of fibroaderioinns and occasional ear duct tumors were observed. 2. Diphenyl- and Triphen ylinethane Derivatives
There is a scarcity of systematic structure-activity studies on amines of the diplienylmethane and triphenylmethane series. Case and Pearson (1954) concluded from statistical data that bladder cancer is an orcupatiorial hazard among workers employcd in the manufarture of auraminr
(LXXXI) R = H (LXXXII) R = CH,
(LXXX). Subsequent testing in rats showed that this dye is a fairly potent carcinogen producing exclusively hepatomas when administered in the diet (Williams and Bonser, 1962; Walpole, 1963), and liver and intestinal tumors and local sarcomas when administered by subcutaneous
366
JOSEPH C. ARCOS AND MARY F. ARGUS
injection (Walpolc, 1963). N o tumors were lwoduccd in Walpole’s experiments by repeated injection of dimethylaniline, the starting material in the manufacture of auramine, or of the intermediate tetramethyldiaminodiphenylmethane (LXXXII) . Dogs appear to tolerate high dietary levels of auramine in chronic experiments without adverse effects. However, recent results of Munn (1967) suggest that the complete absence of carcinogenicity of (LXXXII) should be taken with reservation. I n fact, 4,4’-diaminodiphenylmethane (LXXXI) was slightly carcinogenic in Munn’s experiments. With the latter compound, when a total dose of 600 mg./100 gm. body weight was administered by gastric intubation to 24 male Wistar rats in a n 18-month period, and the animals then kept in observation, liver tumors were found in 2 rats. Two subcutaneous fibromas, one pituitary tumor, and one tumor of the small intestine were detected in other animals. Also, Michler’s ketone (tetraniethyl-4,4’diaminobenzophenone) which is thc last step in a synthesis of auramine produces papillomas of the stomach and neoplastic changes in the liver (in Hueper and Conway, 1964). Carcinogenicity of (LXXXI) is preserved or even somewhat augmented by methyl substitution of the central carbon atom. Deichmann (1967) fed 2,2-bis (4-aminopheny1)propane (LXXXIII) a t a total dose level of 178 gni. to 3 female beagle dogs for 6 years and found, upon autopsy, multiple bladder tumors in one dog. Considerably greater potentiation of carcinogenic activity is brought about if methyl substitution is in the two rings instead of the central carbon atom. Munn (1967) administered 3,3’-dimethyl-4,4’-diaminodiphenylmethane (LXXXIV) by gastric intubation, a t a dose level of 1020 mg./100 gm. body weight over a period of 10 months to 24 malc Wistar rats. I n a total observational period of 487 days, among 23 survivors, 20 rats had liver tumors (18 malignant and 2 benign) ; moreover, subcutaneous fibromas were found in 11 animals. Munn suggests a possible connection between the potent carcinogenicity of this compound and the epidemiological finding of Case and Pearson (1954) of cancers that have occurred in workmen employed in the manufacture of Magenta dye. Compound (LXXXIV) is, in fact, the first intermediate, known as the “ditolyl base,” of Magenta manufacture. The closely related 3,3’-dichloro4,4’-diaminodiphenylmethane, increasingly used in industry must also be regarded with suspicion. This compound has not yet been tested for carcinogenic activity. However, Mastromattco (1965) reported hematuria in 2 workmen who had acute exposure to this substance. The simple aminotritane, 4-dimethylaminotriphenylmethane(LXXXV) was tested by Druckrey and Schmiihl (1955). No tumors were obtained by administering a total dose of 7.3 gm. (LXXXV) orally during an
MULECU1,AR GEOMETRY AND CARCINOGENIC; ACTIVITY
367
observational period of 800 days. However, by subcutaneous injection of a total of 360 mg. of the compound, local sarcomas were produced in 5 of 9 surviving rats in 28 months. Comparison of the test data obtained in various conditions suggests that carcinogenic activity probably increases with the number of amino groups on the tritanc nucleus. Druckrey et al. (1956) obtained a 7/12 sarcoma incidence in 11 months following subcutaneous injection to rats of a total of 650 mg. Parafuchsinc (LXXXVI) hydrochloride. Kaump et al. ( 1965) fed (LXXSVI) as the panioatc [ 4,4’-iiietliyleiiebis (3-hydroxy2-naphthoic acid) ] salt to Sprague-Dawley rats and obtained in females, but not in males, a marked incrc:isc in the incidence (and tlecrease of the induction times) of tumors of the skin and of the sebaceous and mammary glands; tumors were also induced in the small intestine, subcutaneous tissue, and auditory canal gland. With the hexamethyl derivative (LXXXVII) , Kinosita (1940) induced, by oral administration, gastric papillomas and slight adcnoinatous proliferation of the liver in the same species. Some investigations have been carried out in the past on the carcinogenicity of complex substituted aminotriphenylmethane derivatives used as biologic stains and industrial dyes. A review of these studies is given in the “Discussion” of the report by Kaump e t al. (1965).
3. Derivatives of Naphthalene and Anthracene Since 1961, evidence continues to accumulate that nonmetabolized 2-naphthylamine is inactive or is at most a very weak carcinogen, and must undergo metabolic conversion t o the proximate carcinogen (s) prior to tumor induction. Intraperitoneal injection of 50 mg./kg. of 2-naphthylamine twice weekly for 3 months to random-bred rats produced abdominal sarcomas in only 2 out of 14 rats surviving for more than 600 days (Boyland et al., 1963a). Nor could the carcinogenicity of the arninc be shown by taking advantage of the special sensitivity of newborn animals to carcinogenic stimuli (Section II,D,l). Roe et al. (1963) and Walters et al. (1967) found no significant increase, relative to the control, in the incidence of lung and other tumors in newborn BALB/c mice injected subcutaneously with 50 or 100 pg. of 2-naphthylamine. These results stand, however, in some contradiction with previous results of Bonser et al. (1956a; rf. Bonser and Ckiyson, 1961) who observed the emergence of n high incidence of hcpntoinas in mice injected sutxutaneously with freshly prep;Lred solutioiis of thv :iniinc. Tested by t l i t i bladdcr iniplaiitatioii tc~cliiiiquc.,it was ~ l i o w ni)reviously that 2-naphthylaniirre is inactive or a t most marginally active (Bonser et ul., 1956b), and, riiore recently, also I-risphtliylamiiie ( u s hydrochloritle) was found inactive by the same route (Bonser ct al., 1963). Tlic
368
JOSEPH C. A R W S AND MART F. ARGUS
inactivity of Z - n ~ ~ ~ h t h y l a n i (as i r ~ etlic acctamide) in testing by bladder implantation in mice was confirrncd by Bryan et al. (1964~).Nonetheless, the inactivity of 2-naphthylamine toward the bladder remains an intriguing problem because ( a ) the bladder mucosa of various species appears to possess a high level of N-hydroxylating activity (e.g., Uehleke, 1966b, 1967), and ( b ) 2-naplithylhydroxylamine appears to be a proximate carcinogen on the grounds that, a t least by intraperitoneal route in rats, the N-hydroxy compound is much more active than the parent amine (Boyland et al., 1963a). However, the superior carcinogenic activity of the N-hydroxy compound in rats could not be unequivocally confirmed by testing via subcutaneous injection in newborn mice. I n the experiments of Roe et al. (1963), a t 50-pg. dose level, both 2-naphthylamine and 2-naphthylhydroxylamine were inactive or a t most marginally active. I n subsequent experiments (Walters et al., 1967), using doses of 100 pg., 2-naphthylhydroxylamine but not the parent amine increased the incidence and multiplicity of lung tumors above those of the control. However, the significance of these results remains open to question since neither local sarcomas (compare to Boyland et al., 1963a) nor hepatomas (compare to Bonser et al., 1956a) were obtained. Brill and Radomski (1965b, 1967) and Belman et al. (1967) investigated the finding of Bonser et al. (1956a) that aged solutions of 2-naphthylamine in oil produce a high incidence of local sarcomas upon subcutaneous injection to mice. Brill and Radomski found that the development of the red coloration of these solutions can be prevented by storing the solutions in the dark, or in vacuo, or under nitrogen atmosphere. This indicates that a photochemical oxidation takes place. Three major products of this oxidation have been identified as 2-amino-l,4-naphtho-
(LXXXVIII)
(LxxxrX)
~uiiione-2-naphtliyliiiiiiie(IXXXVIII), its hydrolysis product, 2-amino1,4-naph thoqui~ionc,and 1,2,5,6-dil~~~1izophcnal,inc (LXXXIX). l-Aniii1o-1,4-1ia~~l1lhoyuinone was fourid noiicarcinogeiiic fur rats (Belman et al., 1966). However, testing by early workers indicated that the closely related compounds, benzoquinoi~e and 1,4-naplithoquinone, are fairly potent carcinogens toward the skin of mice (in Hartwell, 1951).
In view of the mtrtivity of quinoneiiiiinc~toward proteins (Irving a d Gutniaiin, 1959; Belman and Troll, 1962), Brill and Radomski (1967) :ttkrit>iitcrl tho iiirwawl local rarc+iogenirity of the aged soliltions to thp ( ~ ~ i i i i ~ ~ i i ( ~ i (i 1 i i,iS i iS~S V I I I J . Howo~~c.i*, dt~hl)it(~ : i n o:trly rq)ort, ( i n H:tttwell, 1951) 011 tlic in:trtii~ityof I ,2,5,li-clil~cnz01~~1~1i~~zi11~ (LSXXIX) as a carcinogen in cpitlielial application, it should be recalled here that, according to a more recent report, testing by bladder irnplantation into rats (Rudali e t al., 1955) indicates this eonipound to be quite a potent carcinogen. The testing now in progress of the carcinogenicity of the quinoneimine (LXXXVIII) and of 1,2,5,6-dibenxophenazine, in oral application in beaglc dogs, by Brill and Radomski (1967) has not yielded tumors up to 14 to 16 months. Up to recently, the dog was the only experimental species in which malignant bladder tumors were known to arise by oral administration of 2-naphthylamine (Hueper e t al., 1938; Bonser, 1943). Saffiotti e t al. (1967) have found now that 2-naplithylamine is highly potent to induce bladder tumors iii hamsters when fed a t the dietary level of 1%; the induction time was 45-49 weeks. Already a t 0.1% dietary level, proliferative changes in the bladder epithelium can be seen after about 100 days, but no cancers develop. Also, Coiizelrnari e t al. (1967) reported the induction of carcinomas in the urinary bladder of rhesus monkeys by daily oral administration of 200 mg./kg. of 2-naphthylamine. It was already known that in rats, and in rabbits even up to 5 years, administration of the amine produces only papillomatous changes in the bladder (Bonser e t al., 1952). The problematic status of tlic requirement of N-hydroxylation for thc carcinogenicity of 2-naphthylaniine was brought to focus by the finding of Shenoy e t al. (1964) that 3-niethyl-2-naphthylamine (XC) is a potent
mNHa /
/
CH,
(
Oc1
p
Cl
(XC)
(XCI)
(XCII)
carcinogcii wliicli induccs upoii subcutancow iiijcction to mice, suhcutaneous sarcomas with a high incidence and short latent period; no tumors were obtained in these experiments in the control mice injected with 2-naphthylamine. The carcinogenicity of the methyl derivative was confirmed by J. H. Weisburger et al. (1967a) who administered it by stomach tube to male and female rats. Tested by this route, the coinpound produced skin and ear duct tumors, and a high incidence of tumors of the gttstrointestinal tract in males ; rnnmmary gland tumors
370
JOSEPH C. ARCOS AND MARY F. ARGUS
arose in almost all females. In another experiment (Griswold et (~1.)1966) in which this compound (in a single dose) was tested by the same route and found to be carcinogmic toward t0w mammary gland, R kidney carcinoma was found in one rat. Thus, 3-mcthyl-2-naphthylarnine, proposed originally as a substitute for the parent amine, is a. potent and ubiquitously acting carcinogen. The related 3-nitro-2-naphthylamine (XCI) was less active toward the mammary gland in the experiments of J. H. Weisburger et al. (1967a) but somewhat more active to induce tumors of the gastrointestinal tract. No tumors of the skin or ear duct were observed. 1,2-Dichloro-3-nitronaphthalene (XCII) , which expectedly undergoes reduction to the corresponding amine in vivo, produced only mammary tumors with a notably lower incidence. 2-Nitronaphthalene, the nonchlorinated compound corresponding to (XCII), is inactive just as 2-naphtliylamine, when tested by bladder implantation (Bryan et al., 1 9 6 4 ~ ) . Whatever the final outcome of the investigations on 2-naphthylaminej available evidence seems t o suggest that its higher benzolog, 2-anthramine, does not require metabolic activation for carcinogenicity. It has been known for some time that 2-anthramine is carcinogenic when tested in bladder implantation (Bonser et al., 1958). 2-Anthramine is an unusual aromatic amine carcinogen. It is highly active by epithelial application toward the skin of rats, but not of mice. An interesting feature of 2-anthramine carcinogenesis is that croton oil has a retarding effect on the emergence of skin tumors in the rat (reviewed by Arcos and Arcos, 1962). I n several respects, however, the biological action of 2-anthramine resembles that of typical carcinogenic hydrocarbons. Administered orally to female rats as a single dose, i t produces malignant mammary tumors in 6 months in a sizable proportion of the animals (Griswold et al., 1966). Dobson and Griffin (1962) and Dobson (1963a) studied the histogenesis of tumors and the alterations of the pilosebaceous apparatus (Dobson, 1963b) during anthramine-induced skin tuinorigenesis. Zackheim et al. (1959) and Zackheim (1964) made comparative histopathological investigations of the effects of 2-anthramine, 20-methylcholanthrene, and 9,10-dimethyl-1,2-benzanthraceneon the skin of mice and rats of various strains. The conclusions of Dobson and Zackheim agree that mainly basal cell epitheliomas are produced by anthramine and methylcholanthrene, while with dimethylbenzanthracene, squamous cell carcinomas are the predominant type of tumor. Inspite of the commercial availability of 2-anthramine, the biochemical changes and alterations of electronmicroscopic morphology brought about by this interesting carcinogen appear to be totally unexplored.
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
371
In the reviewers’ opinion, this compound which structurally is a typical aromatic amine, yet gives biological responses resembling those of polycyclic hydrocarbons, could possibly hold interesting clues for the mechanism of carcinogenesis. I n the experiments of Griswold et al. (1966), 2-chryseneamine was found to be noncarcinogenic when administered as a single dose (at the maximum tolerable level) by gastric intubation to Sprague-Dawley rats which were observed for 6 months. 4. Derivatives of Biphenyl and Fluorene I n the last 6 years, investigations on the structure-activity relationships of 4-aminobiphenyl and benzidine have been carried out mainly by E. C. Miller et al. (1962) and by Pliss (1963, 1964). The totality of the data available now (also see review by Arcos and Arcos, 1962) gives a fairly complete picture of the structural requirements for carcinogenicity (Table XII) and of the effect of ring substituents in influencing tissue target specificity in the rat (Table XIII). It must be recognized t h a t t i e s p i t e the extensiveness of the accumulated data-no consistent picture can be drawn about the role of the ring substituents, except for those in the 4- and 4’-positions. Although tumor distribution in the target tissues varies, carcinogenicity is maintained if the functional group in 4-aminobiphenyl is replaced by amine- or hydroxylamine-generating groups, such as acetylamino, dimethylamino, nitro, or acethydroxamic acid. 4-Fluorobiphenyl is a noncarcinogenic compound. Surprisingly the ability to produce tumors of the small intestine is not lost by replacing the amine by a methoxy group (Arcos and Simon, 1962). The effect of 4’ substituents on the carcinogenicity of 4-aminobiphenyl brings to mind the analogous situation with 2-acetylaminofluorene and 4-dimethylaminoazobenzene. I n 4’-fluoro-4-aminobiphenyl, carcinogenicity toward the mammary tissue and acoustic sebaceous gland is maintained, but in addition, this compound also produces a high yield of tumors in the liver and the kidney. However, activity is absent if the 4’-substituent is a chlorine or bromine atom, and 4’-methyl-4-aminohiphenyl possebses probably only borderlinc carcinogenic activity (Table X I I , footnote a ) . In this respect also, the pattern resembles that seen with 4-dimethylaminoazobenzene. The 4’-bromo derivative of the azo dye is inactive, and the 4‘-chloro and 4’-methyl derivatives are w r y weak carcinogens. However, similarity apparently ends here since, whereas both benzidine and certain of its derivatives and itlho 4,.l’-diiiitrobi~~t1~~11yl ( T A n l i : i i n et ul., 1964) ure ciircinogenic, thc 4’-nitro, 4’-acety1:iiiiino, :tiit1 4’-tlimetliyla1uiiio deriixtives of 4-tlinictliyl-
372
JOSEPH C. ARCOS AND MARY B’.
ARGUS
TABLE XI1 Synoptic Tabulation of the Structural Requirements for Carcinogenicity of 4-Aminobiphenyl and Benzidine Derivatives
t F o r 4-aminobiphenyl Very active if Active if
Very active if 3-methyl 3,2‘-dimethyI 3,3’-dimethyl 3 -fluoro 3’-fluoro
Very active if ,OC .CH, -N ‘OH Active if -NH * OC * CH, --N(CH,), -NOz -OCHs
Active if Inactive ifasb
-B r
Active if
-S-
Inactive if -NH-
3- chloro 3 - methoxy 3, 2’, 5‘-trimethyl 3, 2’, 4’, 6’-tetramethyl
Inactive if -F
Weakly active if 3-hydroxy Inactive if 2- methyl Z’-methyl 2’ -fluoro 3-amino
R I -CHTransition to diphenylmethane and triphenylmethane amines
For benzidine Very active if 2-methyl 3,3’-dihydroxy 3,3’-dichloro Weakly active if 3,3‘-dimethyl 3,3’-dimethoxy Inactive if 2,2‘-dimethyl 3,3’-bis-oxyacetic acid
a
-CH=CHTransition to aminostilbenes -N=N-
t -
Transition to amino azo dyes
‘E. C. Miller ef a/. (1962)found the4‘-methylderivative inactivewhen fedto rats a t t h e level of 1.62 mmoles for 8 months. However, Walpole and Williams (l958)found this compound hepatocarcinogenic in the s a m e species when it was administered by subcutaneous injection and given at a comparatively very high total dose of 10.1 gm/kg body weight. bWhile 4’-hydroxy-4-aminobiphenyl does not appear to have been tested, it is generally assumed to be an inactive compound.
‘E. C. Miller et a/. (1956)found the2-methylderivative inactive by dietary administration. However, according to Walpole and Williams (1958) this compound is a hepatic carcinogen when tested by subcutaneous route.
aiiiiiio:izobenzenc arc inactivc by oral routc (.J. A. Miller et al., 1957; Arcoh and Simon, 1962). Data are lacking on the testing of the 4’-amino derivatives of 4-dimethylaminonzobe~izene by other routes, in other species, and a t higher dose levels. The rationale appears to be less clear concerning the influence of other ring substituents on carcinogenic activity. In 1952, it was proposed on the grounds of correlations between ultraviolet spectral shifts and carcinogenic activities that 2- (or 6-) or 2’- (or 6’)-methyl groups depress the carcinogenic activity of 4-aminobiphenyl by distorting the coplanarity of (and, thus, lowering the resonance between) the two benzenic nuclei (J. A. Miller et al., 1952; Sandin et al., 1952) ; the generally greater biological activity of 2-aminofluorene derivatives has been attributed to the -CH2bridge which helps maintaining a coplanar arrangement (E. C. Miller et al., 1949). In 1962, this explanation appeared still acceptable, and the high potcncy of 3,2’-dimethyl-4-aminobiphenyl and 2-methylbenzidine versus tlic inactivity of 2-methyl-4-aminobiphenyl could be explained by the contribution of the hyperconjugating 3-methyl group (in 3,Y-dimethyl-4-aminobiphenyl) and of the second amino group (in 2-methylbenzidine) to the 4-4’ resonance which counteracts the crowding effect of the 2-methyl substituent (Arcos and Arcos, 1962). Support for this view was actually provided by the finding of Walpole and Williams (1958) that 3-methyl-4-aminobipheny1, tested by subcutaneous route in rats, is a more potent carcinogen on the basis of the total dose used than either the parent compound or the 3,P-dimethyl derivative. Subsequently, E. C. Miller et al. (1962) tested 3-methyl-4-aminobiphenyl (as the N acetyl) by oral administration and concluded that it is only moderately active. However, analysis of their results clearly indicates that, although this compound has a somewhat different target tissue spectrum (Table X I I I ) than 4-acetylaminobiphenyl (tested simultaneously a t the same dose level), in the target tissues actually affected the 3-methyl derivative produced a much higher incidence of tumors in a notably shorter period of time. Thus, on the strength of the results, obtained either by parenteral or I)y oral routc, 3-nicthyl-4-nminobiphcnyl should be regarded as inarkcdly morc potent than tlic parent compound. However, different consitlcrntions introduce added complexity in this attractive interpretation of the sul)+tituent effects, as mediated through influence on intcrnuclcar conjugation. In fact, in the tcsting of Walpole :LII(I 1Villi:iiiis (1958) ( ~ I T I Itlir Iiiglrly rrowtlr(l 3,~,4’,C,’-tct,i.:in~rthyl-4: I I I I ~ I ~ ~ ~ ~ ~ J\\’ah ~ I I i~~ui~krclly ~ ~ I I ~ I arti\rr. Chi llle other hand, ill tlir oral testing experiment hy E. C AIiller rt ul. (1956) , 2’-fluor0-4-aminobiphenyl, which is only sliglitly iiiore cro\vtlctl than the pnreiit amine (compnre the vitn der Wnals r:itliuh of 1 . 1 A of Irydrogcn to tlic radius of 1.35 A of
TABLE XI11 EFFECTOF RING SUBSTITUENTS ON THE TISSUE TARGET SPECIFICITY OF ~AMINOBIPHENYL AND BENZIDINE DERIVATIVES IN THE RAT’ Substituent (s) PAminobiphenyl* None None 2-Methyl 2-Methyl 3-Methyl 3-Methyl 3-Fluoro 3-ChlOrO 3-Hydroxy 3-Methoxy 3-Amino 2’-Methyl 2’-Fluoro 3’-Fluoro 3,2‘-Dimethyl 3,3’-Dimethyl 3,2’,5’-Trimethyl 3,2’,4‘6’-Tetramethyl
Routec
s. c. Oral
s. c. Oral s. c. Oral Oral
s. c.
Intestine
Liver
Bladder
Mammary Ear duct
Salivary gland
Other sites
Uterus; kidney(?) Thymus & lung(?) -
-
Oral
s. c. Oral Oral Oral Oral
s. c. s. c. s. c. s. c.
-
Uterus(?) -
Bemidine* None None 2-Methyl 2,2’-Dimethyl 3,3’-Dimethyl 3,3’-Dimethyl 3,3’-Dihydroxy 3,3’-Dihydroxy 3,3’-Dimethoxy 3,3’-Dichloro 3,3’-bis-Oxyacetic acid 3,3’-Disulfonic acid
s. c. Oral Oral Oral
s. c. Oral
s. c. Oral
s. c. s. c. s. c. s. c.
+ + + + + + + +
Skin & S. C. sarr. Skin & lung(?) -
-
Uterus(?j Stomach
-
-
Skin Stomach Ovary Skin 8: S. C. sarc. -
-
-
-
a Compiled from Spitz et al. (1950);Walpole and Williams (19%);Xalpole et al. (1952, 1955); Baker (19.53);E. C. Miller 1962); Pliss (1959, 1963, 1964) ; Laham et al. (1964) ; Bremner and Tange (1966). * Nitro, acetylamino, and dimethylamino groups have been regarded as equivalent to an amino group. S. C. = subcutaneous.
f f (I(.
(1356,
376
,JOSEPH C. ARCOS AND MARY F. ARCXTS
fluorine) , was devoid of activity. Contrastiiig with tlic above fin4tig of Walpole and Willianis, the similarly crowded 2,2’-dirnethylhenzidine (as N,N’-cliacet,yl) is inactive hy oral route (E. C. hIiller ct a!., 1956). It, alioiiltl I w n o t c ~ It h t while iii Iho Iwiizifliiicsrriva tlic 3,3’-dihytlrosy derivativc is one of tlic most wtive compounds, in the 4-aininobiphenyl series, hydroxy substitutioii in the 3-position brings about considerable loss of activity. Now, it is well known that among the aromatic carcinogens, in general, ring hydroxylation causes a partial or total loss of activity, and that “shielding” of the free hydroxyl group by methylation brings about a considerable regain of activity, sometimes beyond the activity level of the parent compound itself. Allowance made to the evident possibility that differences in experimental conditions may completely obscure the relative order of activities, the data available suggest that 3,3‘-dimethoxy benzidine is much less potent than the 3,3”-dihydroxy derivative; there is, moreover, a change in target specificity when passing from the dihydroxy to the dimethoxy compound. Change in target specificity is also seen between 3-hydroxy- and 3-methoxy-4-aminobiphenyl, although, here again, the difference in the routes of administration could well be responsible (Table XIII) . The overall picture becomes even less consistent if the structural requirements for carcinogenicity in the xenylamine and benzidine series are related to the structural features of the two compounds tested recently by Munn (1967) (see Section III,A,2). These compounds, 4,4’-diaminodiphenylmethane (LXXXI) and 3,3’-dimethyI-4,4’-diaminodiphenylmethane (LXXXIV) are analogous t o benzidine and 3,3‘-dimethylbenzidine, respectively, with the essential difference, however, that in the diphenylmethane derivatives, conjugation between the two benzenic nuclei is blocked by the sp9 hybridized -CH2group, Moreover, in these diphenylmethane derivatives, the two benzenic nuclei are not colinear as in the biphenyl derivatives, but their lengthwise axes form an angle approxicarbon. Yet, despite the commating the valence angle of the -CH2partmentation of conjugation into two quasi-independent halves and the great difference in molecular shape, methyl substitution in orfho to the amino groups-just as in the 4-aminobiphenyl series, but not in the henzidine series-has a powerful potentiating effect on carcinogenic activity (cf. Walpole e t al., 1952). The totality of evidence summarized above illustrates the scarcity of consistent correlations between carcinogenic activity and ring substitutions in the xenylamine and benzidine series. For a more comprehensive view of the rapidly evolving panorama of the structure-activity relationships, activating metabolism, leading to the formation of N-arylhydroxylamines (Section II1,D), must be considered.
MOLECDLAR GEOMETRY A N D CAI{CINO(:I~:NIC A C ' I ' I V I l Y
377
In an invebtigation of the dosing schedules for bladder tumor induction, Deichmanri (1967) found 4-aminobiphenyl t o be considerably more carcinogenic to the dog than 2-naphthylarnine. Bladder tumors were produced with the former agent in female beagles after a total oral dose of 5.4 to 7.3 gni. per dog over a pcriod of 31 to 37 months; similar doses of 2-naphthylamine failed to induce tumors over thc same period of time. Benzidine, administered to hamsters a t 0.1% dietary level for the entire life-span, induces a high incidence of livcr tumors; no bladder tumors were found. 3,3'-Dimethyl- and 3,3'-dichlorobenzidine are inactive under identical conditions (Saffiotti et ai., 1967). Histopathological studies on aromatic amine-induced tumorigenesis have been carried out with benzidine by Pliss (1964) and with N,N'diacetylbenzidine by Bremner and Tange (1966). Laham et aZ. (1964) reported on the tumor distribution in 4,4J-dinitrobiphenyl-inducedcarcinogenesis. Their preliminary results suggest that 2,2'-dinitrobiphenyl is a weak carcinogen on the basis of the average cumulative intake recluired to produce a significant percentage of malignant tumors above the control level. Spjut and Spratt (1965) and Cleveland et al. (1967) investigated the genesis of intestinal neoplasia by 3,2'-dimethyl-4-aminobiphenyl in defunctionalized intestinal tracts (in cecal pouch and colon segment, respectively) and concluded that direct surface contact of the compound or its metabolite through the fecal stream with the intestinal mucosa is necessary for tumor induction a t intestinal sites. Spjut and Spratt (1965) and Navarette-Rejna and Spjut (1966) injected the amine directly into the isolated intestinal segment and concluded that the amine was active as such since no ncoplastic lesions were observed a t distant sites. It is not impossible, however, that, just as the bladder mucosa (Uehleke, 1966b, 1967), the intestinal iniicosa may possess N-hydroxylating activity. Progress has been made in the study of the structure-activity relationships of derivatives of 2-aminofluorene (XCIII) and related compounds. These are discussed in the following paragraphs.
(XCIII)
n. Amino Subatituents unil Amiire-Generutiny Groups. It appears now to be definitely establislicd that for high level of carcinogenic activity, there must be a t least one aniine or amine-generating group in para
378
JOSEPH C. ARCOS AND MARY F. ARGUS
position with respect to the biphenyl linkage. Replacement of the 2-amino group by CH3*S- brings about total loss of activity; substituting by CH, CO S-, however, the resulting acetylthiofluorene retains trace activity toward the small intestine of Sprague-Dawley rats (E. C. Miller et al., 1962). Also, acetylthiofluorene is inactive in mice by oral administration (Argus and Ray, 1956) ; in Wistar male rats, however, by the same route, this compound was found to induce syuarnous papillomas of the stomach in 9 out of 29 animals (Argus and Ray, unpublished). It was already established that, just as with 4-aminobiphcnyl, the 2-amino group of 2-aminofluorene may be replaced by a nitro group with only partial loss of activity. E. C. Miller et al. (1962) have now shown that also 2,7-dinitrofluorenc is a carcinogen toward the mammary tissue of the rat, having a potency cqual to that of 2-acetylaminofluorene; liowever, this dinitro compound is inactive toward the liver and the ear duct gland and has only trace activity toward the intestinal tract. Activity toward the mammary tissue is reduced to a very low level but not totally abolished by positioning one of the nitro groups ortho to the biphenyl linkage, as in 2,5-dinitrofluorene. The marginal activity of the 2,5-dinitro derivative is the compounded result of ( 1 ) the unfavorable position of the second amine-generating group and ( 2 ) the fact that the nitro groups must be reduced in the animal’s body. This is borne out by the finding (E. C. Miller et al., 1962) that the corresponding 2,5-fluorenylenebisacetamide is a notably active but still a less potent carcinogen than 2-acetylaminofluorene. The noncarcinogenicity of 2-dimethylamino-3-nitrofluorene exactly parallels the situation in the 4-aminobiphenyl series, since both 3-amino-4-acetylamino- and 3-amino-4-dimethylaminobiphenylare virtually inactive (E. C. Miller et al., 1956, 1962). The prediction of Argus and R a y (1959) that the carcinogenic activity of various N-acyl-2-aminofluorenes parallels the ease of hydrolysis of the N-substituent [which was partially verified by Morris and his associates (reviewed by E. K. Weisburger and Weisburger, 1958)j is borne out by the moderate carcinogenicity of 2-formylaminofluorene (E. C. Miller e t al., 1962). b. Ring Substituents. Until 1962, 7-fluoro-2-acetylaminofluorene was the only fluoro-substituted derivative tested. It was found to be much more active in the rat for the induction of liver tumors, but with an activity comparable to the parent amide toward other target tissues. E. C. Millcr et nl. (1962) have ussaycd all the rcniaining isomcrir :iromatic ring-monofluoro tlerivativcs, that is the 1-, 3-, 4-, 5-, 6-, and 8-inonofluoro-2-acetylamiiiofluorenes. With the cxception of the 4-fluoro derivative, whicli is inactive towaid the liver, cnch compound is activc to a level comparable to the parent amide in the four typical target tissues:
ALOLECULAR GEOMFTKY A N D CAKCXNOGENIC A C T I V I T Y
379
liver, inaniniary gland, ear duct, and small intestine. The fact that all aromatic ring-fluoro derivatives of 2-acetylaminofluorene have high levels of carcinogenicities has been construed to mean that covalent bond formation between cell components and the substituted positions is not a prerequisite for carcinogenesis by this amide. The potency of the ring-fluoro derivatives stands in striking contrast with the virtual inactivity of the ring-hydroxylated compounds. It may be recalled that of these, the 5- and 7-hydroxy derivatives are major and the 1- and 8-hydroxy derivatives are minor metabolites. It has been known for sonic time that 1-, 3-, and 7-hydroxy-2-acetylaminofluorene are inactive or a t most slightly active in the rat by oral or intraperitoneal administration, and Boiiser (cited in Clayson, 1962) found l-hydroxy-2miinofluorene inactive also by bladder implantation in iiiice (using crushed paraffin as vehicle). Very recently, Gutinann et al. (1967) found the 1- and 3-hydroxy derivatives inactivc also in the N-benzoylatcd form, administered intraperitoneally. In an attempt to test the validity of the ortho-hydroxylation hypothesis, Irving et al. (1963) have assayed in mice the hydrochlorides of the 1-, 3-, 5 - , and 7-hydroxy derivatives of 2-aniinofluorene by bladder iniplantation, using paraffin as vehicle. None of these compounds proved to be carcinogenic and the only tissue changes noted were squamous metaplasia and epithelial liyperplasia. A possible objection which may be raised against the very careful experimental work of Irving et al. is the unquestioning acceptance of paraffin as a vehicle in an effort to reproduce the exact experimental conditions of Boiiser et nl. (1956b). Since the detection of carcinogenic activity by the bladder implantation technique is highly dependent on the rate of elution from the pellet (in Arcos et al., 1968), the total absence of carcinogenic activity with all the aminofluorenols may have been due to the too slow rate of elution (compare also Bryan et al., 1964a,b). This is, in fact, suggested by the elution experiments with 2-amino1-fluorenol-”C. It is stressed t h a t this should not be construed as a defense of the ortho-hydroxylation hypothesis on the part of the reviewers. The inactivity of the aminofluorenols is due to the hydrophily of the phenolic hydrosyl groups. “Shielding” of these groups by methyl substitution brings about partial or total regain of activity. 7-Methoxy-2-acetyl:miinofluorenc is a highly carcinogenic compound (Morris et al., 1960), whereas l-nietlioxy-2-acctylaminofluorene is moderately active, and 3methoxy-2-acetylaminofluorene inactive (Gutinann et al., 1968). c. Other Aspects. Hackmarin (1956) was the first to show that 2amino-3-niethoxydiphenylene oxide is highly specific toward the urinary bladder of Wistar rats without the requirement of indologenic substances
380
JOSEPH C. ARCOS A N D MARY F. A R G U S
in thr diet. Tliat s1)ccificity tow:ircI Llic I ) l a t l t l ( ~ r is t l u c to the -01)rirlgc is further supportrd now hy thc fintliiig C1i:it tlio p r r c n t ) coIlil)ouli(l, 2aininodipliciiylciic oxide, is :i bladder cart*iiiogcii I)y oral at1iiiiiiistr:itioii to C57 x IF mice (Bonser et al., 1965). In addition to the individuals bearing bladder tumors, all mice developed malignant hepatomas. It was known for a number of years that 2-aminofluorene produces only rarely bladder tumors in rats maintained on a normal high pioteiii diet, and the addition to the diet of indole, indole acetic acid, or tryptophan is necessary to direct target specificity toward the bladder. Recent reports on this subject are due to McDoniild et al. (1962) arid Dunning (1967). In the rabbit, on the other hand, it had already been known that 2-acetylaminofluorene produces tumors of the urinary tract (Bonser and Green, 1950). Their findings have now been confirmed by Irving et al. (196713). Tumors of the liver and mammary gland have not been observed, which stands in marked contrast to the systemic carcinogenicity of the amide in susceptible rodents. Instances of production of tumors in the skin by dietary administration of carcinogens are rare. How and Snell (1967) have now shown that a high incidence of tumors of sebaceous gland origin and a number of epidermal tumors develop in ACI/N female rats following the feeding of 2,7-fluorenylenebisacetamide. Second to the well-studied instance of the guinea pig, macaque monkeys are now found to be completely refractory to the carcinogenic action of 2-acetylaminofluorene and 2,7-fluorenylenebisacetamide tested by oral administration (for periods varying from 10 to 43 months) and by repeated subcutaneous injections (as the cupric chelate of 2-fluorenylhydroxylamine). With a few exceptions, the animals were newborn when the treatment was initiated (Dyer et al., 1966). The failure of these two fluoreneamides to show any carcinogenic effect in monkeys is possibly related to their metabolic behavior. Thus, 98% of the 2,7-fluorenylenebisacetamide given orally to rhesus monkeys is recoverable unmetabolized from the feces within 1 week. Orally or intraperitoneally administered 2-acetylaminofluorene is excreted within 72 hours (97% in the urine and 3% in the feces); 87% of these metabolites consist of the 7-hydroxy derivative (partially conjugated), the rest is a mixture of unchanged amide, free amine, and a small amount of the N-hydroxy metabolite (Dyer et al., 1966).
5. Tryptophan Metabolites Because of the increasingly recognized causal relationship of tryptophan metabolites to spontaneous bladder cancer in humans, there is continued interest in the testing of these metabolites for carcinogenic
activity, i n the seiircli for new nictabolites, and in asccrtaining the increased excrction of carcinogenic tryptophan metabolites in patients with bladder tumors. A careful study of the coiiditions of testing for carcinogenic activity by the bladder implantation technique has been carried out by Bryan et al. (1964b). These workers studied the rate of rlution of tryptophan metabolites and other aromatic nitrogen-containing compounds from cholestcrol pellets implanted into mouse bladders, in order to determine thc probable extent ant1 duration of exposure of the bladder mucosa to potential carcinogcns. The importance and necessity of such elution studies, previous to actual tcsting, have been first pointed out by Allen et al. (1957) and recently reemphasized by Arcos et al. (1968). Price arid his co-workers liavc reported a comparative testing study using paraffin (Bryan et ul., 19644a) and cholesterol (Bryan et nl., 1964c) as “inert” pellet material. They concluded that cholesterol (compressed into pellets in a pellet preqs), but not paritffin, is a satisfactory vehicle for testing urinary tryptophan ~nctabolitcsfor carcinogenic activity. Using cholesterol, five metabolites were found active to various degrees: xanthurenic acid and its 8-methyl ether, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and 8-hytlroxyquinaldic acid. The 8-methyl ether of xanthurenic acid was the most active of all the compounds tested in these experiments and confirms the carcinogenicity of this compound to the bladder first noted hy Allen et al. (1957). Xanthurenic acid was found earlier to he inactive (cited in Boyland, 1958). 8-Hydroxyquinaldic acid had not been previously tested. Using cholestero1 as vehicle, these tryptophan metabolites were as active as the reputedly potent carcinogens, 2-naphthylhydroxylamine and N-acetyl-2-fluorenylhydroxylamine. Three out of the five active tryptophan metabolites are quinoline derivatives and are closely related to 8-hydroxyquinoline which has repeatedly been found (Allen et ul., 1957; Hueper, 1965) to possess a t least weak carcinogenicity. Thus, Bryan et al. (1964~)were led to explore the possible activity of other dihydroxyquinoline derivatives. The 2,8-, 4,8-, and 2,6-quinolinediols are inactive by the same statistical standards. A synoptic tabulation of the structural requirements for carcinogenicity of quinoline compounds is given in Table XIV. The work of Bryan e t al. contributes a needed confirmation of thc carcinogenicity of 3-hydroxyanthranilic acid to the mouse bladder. I n fact, the carcinogenic activity of 3-hydroxyanthranilic acid toward this tissue (tested in clwlrsterol) , w1)urtetl origiiially by Allen et ul. (1957), could not be confirmed b y Clayson et nl. (1958). Inasmuch as doubts hare heen expressed (e.g., ,J. .4. IIiller et nl., 1961; Irving r t al., 1963) ahoiit thc re1i:tI)ility :mti/or validity of tlic hlnd~lcrimplantation tech-
w
00
t3
TABLE XIV Synoptic Tabulation of the Structural Requirements f o r Carcinogenicity of Quinoline Compounds in Rats and Micen
4
1
2
Positions of substituents 3 4 5 6
0
?n
7
8
-OH
-0 -0
-OH
Activity
+ -
-OH
-OH -OH
-OH
-
-COOH
-OH
-OH
+ +?
-COOH
-OH
-OCH,
r '
-OH
-0
Bladder implantationb Bladder implantationb B1adder implant at ionb Bladder implantationb
Urinary bladder
Bladder implantationb
Urinary bladder"
Bladder implantationb
Urinary bladder
Skin, S. C.b Skinb
-NO, -CH3
-0
-c1
-0
-Br -NO,
-
Skinb
+ ?
s. C.b s. c . b s. C . b
z d P
E
8
Skin, S. C.b
-NO,
-0
Bladder implantationb; intrarectal o r intraUrinary bladder? UterusC vaginal instillationC
Bladder implantationb
-OH
-COOH
-0
Target tissue
s. CP
-OH
. -OH
Route
Skin & subcutaneous t i s s u e e
-0
+
-NO,
Skin, S. C b , c ; oral
Skin, s u b c u t a n e o u s tissue
(& all local sites), lung & uterus.6.c M a m m a r y & lymphoid t i s s u e -0
NO,
-0
- NO,
-0
-NO,
-0
-NO,
-0
-NO,
-0
-NO,
-0
--NHZ
-0
NH.OH
-0
-NH.OH
T h e 2-, 5 - , 6 - , 7 - , and 8methyl, 2-ethyl, 5,6-, and 7 - c h l o r o , - 6 , 7 - d i c h l o r o & 6-carboxy der i v a t i v e s are a c t i v e to various d e g r e e s ~
NO,
-NO,
Skin, S. C . b
-
s. c.6 s. c.6 s. C.b
-
-NO2 -NO,
-t
-
-NO2
-
-NO,
-
.4ctive sites
at local & d i s t a n t
0
m 0
S. C.*
s. c.6 s. c.6 s.C.b*C
Papillomas. s a r c o m a s L leukemia.* S a r c o m a s C
Skin
“Compiled from Allen 1 1
In rats. “Royland and his co-workers (see Boyland, 1958) foundxanthurenic acid to be inactive. However, Bryan einl. (1964~) reported that this compound is moderately carcinogenic toward the bladder when implanted using cholesterol as vehicle. “ Nakahara and h i s associates (cited in Nakahara, 1964) found both 4-chIoroq~inoline-.~~-oxide and 4-bromoquinoline-A’-oxide inactive under their conditions. However, Searlp (1965,1966a) retested the 4-chloro compound i n higher concentrations for complete carcinogenicity on skin and in subcutaneous tissue, and for tumor-initiating activitv on the s k i n . I r i s preliminary data indicate that 4-chloroquinoline-N-oxide is a t least weakly carcinogenic. Possibly also 4-bromoquinoline-A’-oxide is active to s o m e degree.
384
JOSEPH C. AItC!OS AND MARY F. ARGUS
nique, studies on the production of solid tumors by tryptophan metabolites through other routes of administration and in other species are iic~rled.It, shoiild he noted in this connection that Ehrhart e t nl. (1959) cl:~iniccItli:it 3-liy1Irosyaiitlir:~iiili~~ :wid is leukeinogciiic i i i niiccb. A l t h o ~ g l i confirmutory expcrimcnts linve not been carried out, it iii:ry I)c of interest in this connection that Rauschenbach et ul. (1963) obtained a low incidence of leukemias by the injection of the following compounds related to tryptophan: indole, 3-indoleacetic acid, 3-indolyllactic acid, 5-hydroxyindole-3-acetic acid, and 5-methyltryptophan; antliranilic acid was inactive. Because of the low incidence and the lack of clear-cut information on the long-term spontaneous leukemia incidence of the strains used, these results should be accepted with reservation. Since the initial report of Price, Brown, and their co-workers (Brown et al., 1955), quite a considerable amount of literature has developed which shows a definite correlation between bladder cancer and abnormally high levels of carcinogenic and noncarcinogenic tryptophan metabolites in humans and different animal species. A recent confirmation was reported by Alifano e t al. (1964). The field has been exhaustively reviewed by Price (1965) and by Price et al. (1965). Although there is some suggestive indication (cited in Kerr e t al., 1964) that the urinary tract itself is possibly a significant source of kynurenine, a precursor of the carcinogenic aminophenols, by far the greatest weight of evidence indicates that the high levels of tryptophan metabolites are due to systemic (probably hepatic) metabolic disorder (s) . In fact, in most cases, removal of spontaneous bladder tumor(s) from patients has no effect on the abnormal tryptophan metabolism (Price et al., 1956; Quagliariello et al., 1961). It is also true, however, that in a few instances, excision of the tumor brought about a return toward the normal of the abnormal tryptophan metabolic pattern (Price et al., 1955, 1956). McDonald et al. (1962), who studied the effect of simultaneous administration of indole and 2-acetylaminofluorene on the production of bladder tumors in rats, noted rz correlation between the rate of tlevelopmcnt of bladder tumors and tlic loss of hepatic function. It lias 1)cen known for soinc time thttt :~dministrationof high levels of tryptophan can incrcasc hepatic tryptopli:cn metabolism as much as tenfold (Knox and Mehler, 1951). Yet, despite the relative frequency of occurrence of spontaneous bladder tumors in dogs (Bloom, 1954; Cotchin, 1956) and the elevated levels of urinary tryptophan metabolites normally present in this species (Brown and Price, 1956), there is preliminary evidence that prolonged loading with high levcls of tryptophan does not induce bladder tumors in dogs (Deichmann, 1967). Deichmann found that feeding dl-tryptophan to beagles in doses of approximately 4 gm. pcr dog,
RIOLECI'LAR (:F:UI\lETHY AND C'ARCIXOGENIC ACTIVITY
385
5 times a wcck tlicl riot intlucc hcinaturia during :L pcriotl of 34 months, and the animals were in apparent good health. The feeding of tryptophan to these dogs is bcing continucd, and the total dose had reached 3.7 kg. of tryptophan per dog a t thc time of the symposium report. The possible implication of this hody of evidence appears to be t h a t the requircment for tuniorigcnesis is not simply an overall increase of tryptophan metabolism, but rather an abnormal increase in the relative amounts of certain metabolites which may he compounded by synergistic or cocarcinogenic effects between them. Of course, the possibility of more potent, still undetected metabolites should be borne in mind. The finding of McDonald et al. (1962) of bladder tumorigenesis concomitant with loss of hepatic function suggests that, nevertheless, the combined effect of tryptophan loading and differcnt types of hepatic injuries is an approach to the induction of bladder tumors worthy of exploration.
6. 4 - N i t r o q z ~ i n o l i n e - N - ~ . and ~ ~ ~ eDerivatives Investigations in a number of laboratories have given considerable extension to the initial discovery of Nakahara et al. (1957) on the carcinogenicity of 4-nitroquinoline-N-oxide (XCV), proposed originally as a fungicide. Table XIV summarizes the known relationships of structure and carcinogenic activity. The simultaneous presence of the coordinatively linked oxygen atom and the nitro group are required for carcinogenic activity. I n fact, both cluinolinc-n'-oxitle and 4-nitroquinoline arc inactive. Furthermore, the nitro group must he specifically in position 4, since 3-nitroquinoline-N-o>ride is inacti1.c ; 3-metliylquinoline-N-oxide and 6-nitroquinoline are also inactive. Just how narrowly are the structural rcqiiircniciits for carcinogenicity delineated is best il1ustr:tted hy the inactivity of 4-nitropyridinc-N-oxidc
6 i
i
0
0
& +
0
inactive
highly active
inactive
(XCIV)
(XCV)
(XCVI)
(SCIV) :ml of 9 - n i t i ~ o ~ ~ c r i d i n c - . ~(XCVI) - ( ~ ~ i ~ l (N:ikali:~ra, (~ 1961, 1964). XI\' sliows tliat alkyl a i d chloro sulJstitutioii of 4-iiitroquinoline-.V-oxide i n various positions does not aholish carcinogenic activity (Nnkali:ir:L, 1961, 1964; Kawazoe et al., 1966). However, as far as it can be ascertained from the rlata reported, these derivatives are ncver more ' l ' a t ) l t b
386
JOSEPH C. ARCOS AND MARY F. ARGUS
active than the parent compound itself. Introduction of a carboxyl group in the 6-position in (XCV) does not abolish carcinogenic activity, but definitely lowers the potency (Kawachi e t al., 1965). This impairment of activity by the -COOH group could be due to the increase of the solubility, but may also be related to the strong -1 effect of the group “syphoning” away n-electrons from the N-oxide-bearing ring. The latter alternative is actually supported by the fact that introduction of a second nitro substituent (which has a stronger net -1 effect than the carboxyl group, and may also redistribute x-electrons by -M effect) in any position in the 4-nitroquinoline-N-oxide molecule abolishes activity ; also thc 4,6,8-trinitro derivative is inactive (Nakahara, 1964; Kawazoc e t al., 1966). There is evidence for both covalent and noncovalent interaction of 4-nitroquinoline-N-oxide with tissue constituents, and these will be reviewed in Sections II1,E and IV,B. The covalent interaction in which 4-nitroquinoline-N-oxide is very reactive is the substitution of the nitro group by nucleophilic physiological constituents, cysteine, glutathione, and protein -SH groups. The reaction:
& -
+ HNO,
+HS-R
4
0
+
0
proceeds nonenzyinatically and a t physiological pH range. The unreacted -SH may be measured by amperometric titration with AgNO1, and the liberated HNO, spectrophotometrically following color development with a diazo reagent (Endo, 1958). The reaction is specific to -SH groups; no reaction takes place between 4-nitroquinoline-N-oxide and -NH, or -COOH groups (Okabayashi, 1953). Carcinogenic activity may be dependent in some way on this arylating reaction of the compound. It is in accordance with the probable biological importance of the arylation reaction that 4-hydroxyquinoline-N-oxide, in which the reactive displaceable -NO, is lacking, is not a carcinogen (Nakahara, 1964). [The inactivity of 8-hydroxyquiaoline-N-oxide toward the mouse bladder (Bryan et al., 1964c) suggests that the mechanisms of action of the carcinogens, 8-hydroxyquinoline and 4-nitroquinoliiie-N-oxidc, must be different. The chelation properties of 8-hydroxyquinoline are greatly modified in the N-oxide derivative.] Surprisingly, the Nakahara group also found 4-chloroquinoline-N-oxide and 4-bromoquinoline-N-oxide to be noncarcinogenic. Yet, these compounds are also arylating agents and,
in fact, give tlic b;iiiie products on rcactioii wit11 --St1 coiiipouiids as tlic 4-nitro compound (Ochiai, 1953). Searlc has retested the two halogen derivatives, and his preliminary data indicate that the 4-chloro compound is a t least weakly carcinogenic (Searle, 1965, 1966a). If such arylation reaction represents the critical cellular interaction responsible for the carcinogenic activity, the relative activities of the different derivativea should depend oil the rcaction rate. The studies of Endo (1958)-although they could be carried out a t that time with only a liniited number of con~l)oundP--provide some support for the involvement of arylation in 4-nitroquinoline-n’-oxide carcinogenesis. Hayashi (1959) has shown t h a t 4-nitroquiiioline-hT-oxide reacts, in fact, with -SH groups in the skin in vivo. Following single application to the skin, thcre is a nisrked decrease of epithelial -SH content ; no such decreasc occurs following app1ic:ition of noncarcinogenic derivatives. Tlic importance of the arylitting activity of 4-nitroquinoline-N-oxide was d s o illustrated in anothcr way by Searle and Woodhouse (1963, 1964). Since the time of the classical cxperiments of Crabtree (reviewed 1947) it was known t h a t a variety of sulfhydryl reagents and mercapturate-forming compounds inhibit 1,2,5,6-dibenzanthracene- or 3,4benzopyrene-induced tuniorigenesis in the mouw skin. All these inhibitors also cause a marked decrease in the skin -SH level, and, therefore, inhibition was explained in terms of reaction between the inhibitors and tissue -SH groups. Searle and Wooclhouse found now that also 4-nitroquinoline-N-oside delays the induction of tumors by these hydrocarbon,c. However, other workers, using minimal amounts of 4-nitroquinolineN-oxide in combination with 20-niethylcholanthrene (Nakahara and Fukuoka, 1960) and 4-dimethylaniinoazobenzcne (Takayama, 1961), observed summation of carcinogenic effects toward the mouse skin and rat liver, respectively. Thc significnnce of these results is not clear. I n addition t o or instead of the arylation reaction, reductive metabolic transformation of the nitro group leading to an N-hydroxy derivative is considered to be of importance in the mechanism of carcinogenesis by 4-nitroquinoline-N-oxide. Reduction of the nitro group to amino and to hydroxylamino groups by microorganisms and by the mammalian liver has been reported. These metabolic routes will be reviewed in some detail in Section III,D,2. Of the two reduction products, 4-aminoquinolineN-oxide is inactive in mice by subcutaneous route (Nakahara, 1964). 4-Hydroxylaminoquiiioline-N-oxide is, on the other hand, a potent carcinogen to induce sarcomas and epithelial tumors in mice (Shirasu, 1963; Shirasu mid Ohta, 1963) and in rats (Shirasu, 1963; Endo and Kume, 1965). A comparative testing (via subcutaneous route and using small doscs of the carcinogens) of 4-nitro- and 4-hydroxylaminoquinoline-
TABLE XV LOCALIZATION OF TUMORS INDUCED B Y ~-NITROQUINOLINE-A;-OXIDE IN DIFFERENTSPECIESA N D STRAINS Species and strain
ROUteO
“dd Strain” mice
Skin
“dd Strain” mice C57Bl/Z mice Swiss mice (females)
s. c.
Random-bred albino mice Swiss mice ICR, C3H/He, and C3H/Fe mice A mice (newborn) Swiss mice (newborn) CF1 mice (males)
Skin
Skin
s. c. s. c. I. P.
s. c. s. c.
Stomach tube or forced drinking (EtOH sol.) Random-bred albino, A, C57B1, Skin C57B1 X IF Fl hybrid, and NZY mice Ratls Skin Buffalo rats (females) s. c.
Golden hamsters
Skin
Guinea pigs
Skin
a
S. C. = subcutaneous; I. P.
=
intraperitoneal.
Tumor site and type
Reference
Epitheliomas; sarcomas (cell type uncertain) Fibrosarcomas, rhabdomyosarcomas Papillomas and epitheliomas Pulmonary adenomas and adenocarcinomas; uterine carcinomas; leukemia Papillomas (only 21 weeks of treatment) Epitheliomas; sarcomas Generalized lymphomas; mammary carcinomas Pulmonary adenomas(?) Pulmonary adenocarcinomas Tongue; pharynx; esophagus; forestomach; skin Papillomas and epitheliomas; sarcomas
Xakahara et al. (1957); Takayama (1960) Nakahara and Fukuoka (1959) Lacassagne et al. (1961~) Mori (196213, 1965)
Fibrosarcomas Local sarcomas; pulmonary adenomas and adenocarcinomas; uterine leiomyosarcomas and adenocarcinomas Squamous cell carcinomas; keratoacanthomas ; melanomas hlelanomas; trichoepitheliomas; Iteratoacanthomas
Takayama (1961) Mori (1962a: 1964a)
Searle and Woodhouse (1963) Shirasu (1963) Tanaka et al. (1963); Kinosita et al. (1964) Kimura and Senra (1964) Hisamatsu et al. (1965) Horie et al. (1965) Searle and Spencer (1966)
Searle and Woodhouse (1962, 1963) Parish and Searle (1964, 1966)
N-oxitlc w s c:iri.iccl out 1)y Shiixsu ( 1965) i i i tliitc btraiiis of niicc. i:~~, and Icukcmia were Dcperitling on tltr straiit, j ~ ~ ~ i l l o n s:iiwinas, observed in different proportions. The turnor yields indicated that tlic 4-hydrosylamino derivative is not:ibly 1noi-e active than the parent 4-11it roqu i no 1inc-S-oxide . ConsitIer:tble effort has beeii devoted to tltc study of thc 1oc:ilizwtio~i of tumors induced by 4-nitroquinoline-~Y-oside in diffei~cntspecies and strains and by different routes. A partial list of these tumor localiz il t'10t1s is giwn in Table S V . There is some evidence that thc relative yields of cpithelial and connective tissue tumors in mice depends on the dose applied to the skin (Seurle and Spcncer, 1966). Mori and Hirafuku ( 1964), and Mori (196414 studied the histogciiesis of pulmonary tumors induced in iriice hy 4-nitroc~uinolinc-N-oxitlc.T h e over:tll potency of tlic compound approsches that of the most potent polycyclic hydrocarhons. Evcn the skin of tlic guinea pig quite readily responds to the carcinogenic action of 4-nitroquinoline-N-oxide (Parish and Searlc, 1964, 1966) ; liowever, Shirasu (1962) was able to induce tumors in only l out of a group of 9 animals of this species by subcutaneous injection. I n addition to its systemic tissue targets (lung, uterus, lymphoid tissue), 4-nitroquinoline-N-oxide is a strong topical carcinogen. Administered to mice by stomach tube or by forced drinking (as an ethanol solution), 4-nitroquinoline-N-oxide produces tumors along the surfaces of tissue contact: tongue, pharynx, esophagus, foi-estoinach (Horic ct nl., 1965). Also, bladder tumors have been inducc(l in rats with this ciircinogcn by direct tissue contact by injecting its suspension int,o tho s u b mucos:il connective tissue of the bladder (Okn,iirna, 1964). 7. Purine-AT-oxides These compounds are considered here because of the presence in the molecule of a n N-oxide grouping as in the quinoline-N-oxide derivatives just, discussed. Allen e t 01. (1957) noted first that xanthine is weakly carcinogenic by bladder imp1:tntation in mice. Subsequently, Haddow (1959) stated t h a t xanthinc produces sarcomata in rats. No attempts were made, however, to either confirm or follow up thcw preliminary findings. Brown e t al. (1965) and Sugiura and Brown (1967) reccwt ly reported the carcinogenicity of certain purine-N-oxides in Wistar rats by subcutaneous injection. Xanthine-7-N-oxide arid guanine-7-Ar-oxide (as carboxymethylcellulose-stslbilized suspensions in saline) induce suhcutaneous tumors of various histological types following repeated weekly administration for 6 months. One hundred percent tumor incidence was obtained
390
JOSEPH C . ARCOS A K D M A R Y F. ARGUS
with an intli~ct~ion pcriod of 182 to 210 (lays (for ol)taiuing the Iirst tumor) with the lowest dosc used in these cxperiments (3 mg. per week). T h e study of the spectra and pK indicates that a t the physiological pH, these compounds exist primarily as the 7-hydroxyxanthine and 7-hydroxyguanine tautomers. On the other hand, the isomeric xanthine3-N-oxide, which undergoes a tautorneric shift to 3-hydroxyxanthine1 is not carcinogenic. Adenine-1-N-oxide was also found inactive under these experimental conditions. These findings indicate specific structural requirements for carcinogenicity in this series. The existence of the purine N-oxides in the tautomeric N-hydroxy form is not a sufficient condition for carcinogenicity since xanthine-3-N-oxide, which does undergo the tautomeric shift, is inactive. Furthermore, carcinogenicity, per se, is not associated with the N-oxide group’s being in the 7-position. I n fact, another compound, the 3-N-oxide of 6-niercaptopurincl did produce a few tumors a t a very high dose level (50 mg. per week). Tumor induction was not attempted with adenine-1-N-oxide a t this high dose. All of the carcinogenic purine N-oxides and also the inactive adenine-1-N-oxide are inhibitory to various rat and mouse tumors (Brown e t al., 1958; Sugiura and Brown, 1967; Frederiksen and Rasmussen, 1967). Under their experimental conditions, Sugiura and Brown (1967) retested adenine, guanine, and xanthine for carcinogenic activity. Although no tumors appeared a t the sites of injection in the guanine- and xanthinetreated rats, some tumors did appear a t distant sites, and the combined incidence was distinctly higher than the spontaneous tumor incidence of the Wistar strain used. At any rate, while xanthine and guanine appear to be slightly tumorigenic, they are much less SO than their 7-N-oxide derivatives. 8. Aminoacriclines The preliminary data on the hepatocarcinogenicity of Acridine Orange (Munn, 1967) represents probably only the first instance of carcinogenicity in the arninoacridine series. It is, indeed, surprising that, despite the recognized mutagenicity of amino derivatives of acridine and the widespread application of acridine dyes as biological stains, aminoacridines have not been adcquately testcd for carcinogenicity up to now.
(XCVII)
(XCVIII)
M u n n (1967) administered Acridine Orangc (XCVIII) as the purified basc to 24 niale Wistar rats for 16 months, a t the levcl of 0.1% in the diet. All of the 14 rats surviving to the end of the experiment dcvcloped mdignant liver tumors. The nonmethylated base, proflavine (as hemisulfate) (XCVII) has been noted by Salaniaii and Glcndenning (1957) to be a weak promoting agent via intradermal injection. The discovery of potent oncogenic properties of certain purine Noxides and the carcinogenicity detected among aminoacridine compounds will probably give powerful new impetus to the study of the relationship between mutagenicity and carcinogenicity. The reason for this belief is illustrated with the following brief survey. Purines and pyrimidines themselves are known to interact with DNA, although apparently not by intercalation between the bases (Ts'o et al., 1962). Nevertheless, Kihlman (1966) showed with twenty-four purine derivatives that there is a parallelism between radiomimetic effects in plant tissues and solubilizing power toward polycyclic hydrocarbons. Various purines and tryptophan have also been shown to produce niutations in bacteria (Demerec e t al., 1951 ; Novick and Szilard, 1951 ; Green et al., 1955) and in Drosophila melanogaster (Plaine and Glass, 1955). Carcinogenicity (and likely also the mutagenicity) of purines is considerably enhanced by a coordinatively bonded oxygen atom with powerful hydrogen-bonding capability. Moreover, although the ability per se to exist in the tautomeric hydroxy form is apparently not required for carcinogenicity of the purine N-oxides, the two potent compounds are present a t physiological pH in the N-hydroxy form. Using information obtained from the study of N-hydroxy azo dyes and aromatic amines (e.g., Poirier et al., 1967; Lotlikar et al., 1967a; Scribner, 1967), one may hypothesize t h a t the tautomers may undergo esterification in order to become the ultimate carcinogenic metabolites. Metabolic N-liytlroxylation is now well established (Section II1,D) , and also metabolic oxidation to nontautomeric N-oxide has been described (Culvenor, 1953 ; Chaykin and Block, 1959; Ochiai, 1967). This led Brown et al. (1965) to suggest that purine N-oxides could be endogenous oncogenic agents. For the aminoacridines, Orgel and Brenner (1961) and Brenner e t al. (1961) have noted a correlation betwcen basicity and mutagenicity, and proposed that the latter property is due to the intercahtion of these planar :irorn:itics lwtwccn the kxises in DNA. The intcrnctioii of aminon 1)y >~vcrtiI n.or1wi.h (cb.g.,T,uaznti :icricliiies with 1)N.i Iias l ~ h iii\xL-tig:itc.tl t l ( ~ l . ,19G1 ; ldeiiii:tiiJ1964) , :iiuI tlic c * u i i w i h i i h of evi(leiict. i. i i i favor of Scctiuii 1 I,E',4). E'ollowiiig tlic discovery 11i e i 1i te r c x 1:t tio I i I i 1 wt Iit+ is ( by hlunn (1967) of the carcinogenicity of Acricline Oixnge, thc :tbove findings and concepts lead directly to the idea that the testing for car%
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JOSEPH C. ARCOS AND MARY F. ARGUS
cinogenic activity of a series of aminoacridiiies of graded mutagenic potencies might prove fruitful. B. AMINOAzo DYES Investigations in recent years on the effect of ring substituents on the carcinogenic activity of alkylamino azo dyes have been carried out mainly by Brown and his co-workers (Brown et al., 1961 ; E. V. Brown, 1963; Napier, 1964; Brown and Hamdan, 1966) and by Arcos and Simon (1962). An excellent review on the methods of study and problems of amino azo dye carcinogenesis has been given by Terayama (1967). 1. Auxocarcinogenic Effect of 4 -Substituents
Poteiitiation of the carcinogenic activity of 4-dimethylaniinoazobenzene (C) by certain substituents in the 3’- (or equivalent) and 4’-positions has been known for some time. In particular, 4’-ethyl substitution brings about a reinarkable enhancement of the hepatocarcinogenic activity of 4-monomethylaminoazobenzene (MAB) and 4-dimethylaminoazobenzene (DAB) in the rat (Sugiura et al., 1954; J. A. Miller et al., 1957) . The 4’-isopropyl-, 4’-n-propyl-, 4’-n-butyl-, and 4’-te&butylDAB are all active in this decreasing order (Brown and Hamdan, 1961). On the other hand, 4’-methyl-DAB is a very weak hepatic carcinogen (J. A. Miller and Miller, 1953), and this could be due to oxidation to the inactive 4’-carboxy derivative or to replacement by a hydroxyl group. Besides an ethyl (or higher alkyl) group in the 4’-position, other groups can enhance the activity of the parent dye. Thus, 4’-fluoro substitution brings about a doubling (.J. A . Miller and Miller, 1953; J. A. Miller et al., 1953), and replacement of the “prime” ring by a 4-pyridylN-oxide brings ahout a three- to fivefold increase (Brown et al., 1954a; Brown and Hamdan, 1966) of activity relative to the parent compound (C). The coordinatively bonded oxygen atom in the N-oxide analogs studied by Brown and his associatcs corresponds sterically to a substituent in the respective positions.
(XCIX)R = H (C) R = CH, (CI)R = C,H,
True potentiatioii of activity, a t least with the alkyl and N-oxide groups, sippears to be specific to thc 4’-position since both P’-ethyl-MAB (Sugiura et al., 1954) and pyridine-1-oxide-2-azo-p-dimethylaniline
MOT,ICCTJLAR GEOMETRY AND C A R C I S O C E N I C ACTIVITY
393
(PO2) (Brown c t a l , 1954a) are inactive, No potentiation is tee11 either i n tlie correspondiiig 2’-fliioro-ei~hstitiited coinpound, since 2’-fliioro-DAB Irws a potency e q i i ~ lto or nt niost slightly Irigher than tlic paicwt dye (,J. A. Miller et al., 1953). The 3’-position is much less sensitive to the nature of the substituents than the 4’-position. I n particular, while the 4’-methyl, 4’-methoxy, 4’cliloro, and 4’-nitro derivatives of DAB are inactive or a t most weakly active, the same substituents in the 3’-position givc rise to medium active or potent hepatic carcinogens. There is an inverse situation with the pyridine-N-oxide analogs: pyridine-1-oxide-3-azo-p-dimethylaniline( P 0 3 ) is only about :is active as DAB, while pyridine-1-oxide-4-azo-p-dimethylaniline ( P 0 4 ) is 3-5 times as potent (Table XVI). On tlie other hand, fluoro substitution, irrespective of whether in the 3’- or 4’-position, brings about doubling of the activity of the parent dye (J. A. Millcr et al., 1953). Similarly, Burkhard et al. (1962) reported that 3’- and 4’-methylthioDAB are about equally active hepatic carcinogens in the rat. Unfortunately, a DAB control group was not included in the testing, so t h a t relative potencies cannot be calculated. However, judging from the tumor incidences (16/19 in 16 weeks for the 3’-isomer and 13/16 in 20 weeks for the 4’-isomer) , their activity levels are betwecn those of the parent compound and the 3’-methyl derivative. The 2’-methylthio derivative mts found inactive when fed to 21 rats for 23 weeks under identical conditions. I n an attempt to gain insight into tlie mechanism of potentiation by 4’- and 3’-substituents, Arcos and Simon (1962) carried out a comparative study of the effect of 3’-methyl, 4’-ethyl, and 4’-fluoro substitution on hepatocarcinogenic activity in Sprague-Dawley rats. The inactive azo compounds, 2-niethyl-DL4B, 4-diethylaminoazobenzene (CI), 4-aminoazobenzene (XCIX) , and 4-hydroxyazobenzene, were substituted with these groups in the respective positions. All derivatives of 4-aminoazobenzene and 4-hydroxyazobenzene were inactive. However, 4’-ethyl substitution was found to confer appreciable cnrcinogcnic activity to tlic inwtive 2-iiiethyl-DAB and 4-dietliylnniiiioazo~eiizeiic (CI) . 4’-Ethyl2-metliyl-DAB and 4’-ethyl-4-diethylaniino:~zohenzene have relative activities of 12 and 5, respectively. 4’-Fhoro substitution is much less effective in bringing about carcinogenic activity, as 4‘-fluoro-2-methylDAB is only weakly active (relative activity 1-2) and 4’-fluoro-4diethylaminoazobenzene is inactive. A methyl group in the 3’-position is the least effective of all three substituents in bringing about carcinogenic activity, as both 2,3’-dimethyl-DAB and 3’-methyl-4-diethylaminoazohenzene are not carcinogenic. Recently, Brown and HRmdan (1966) have provided the iiitcresting
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JOSEPH C. ARCOS AND MARY F. ARGUS
TABLE XVI Carcinogenicity Toward the Rat Liver of "Prime" Ring Analogs of 4-DimethylaminoazobenzeneTested by O r a l Administrationa
Active compoundsC PO 3 P4 2'-Me-P4 PO4 2'- Me-PO 4 3'-Me-P04 2, P-diMe-PO4 3,2'-diMe-P04 a', 6'-diMe-PO4 N, N-Methylethyl-PO q d N,N-Diethyl-P04d 1- Naphthyl Z-NWhthyl 44 QO 4 Q5 QO 5 66 W 6 4 - Isoquinolyl 5- Isoquinolyl 5- Isoquinolyl-N- oxide 5- Isoquinolyl-N- oxide 7-Isoquinolyl
Lowest dietary level
0.06 0.06 0. 06 0.03 0.01 0.06 0.03 0. 03 0.01 0.03 0.03 0.075 0.075 0.03 0.03 0.01 0.01 0.01 0.01 0.03 0.03 0. 03 0. 01 0.03.
(56)
Activityb -6 <6 -6 29 > 50 15 -13 > 18 > 50 > 18 12 Moderate Potent 15 22 150 200 200 100 15 40 > 50
-
zoo -6
Inactive compoundsC P2 PO 2 P3 4' - Me-P 2 4'-Me-P02 6'-Me-P 2 6'-Me-P02 N,N-Dipropyl-PO 4 2-Thiazolyl 4-Xenyl QO 2 Q3 QO 3 Q7 go7 Q8 QO 8 2- Anthryl 1- Anthraquinonyl 2- Anthr aquinonyl 6-Quinoxalinyl 2-Dibenzofuryl 3- Dibenzothienyl 2- Benzothiazolvl
a Compiled from Brown el a/. (1954a,b.1961); E. V. Brown (1963); Brown and Hamdan (1966); Lacassagne e! a/. (1952); Mulay and Firminger (1952); Mulay and Congdon (1953); Napier (1964).
b The values for the quinoline and isoquinoline dyes were taken from the reports of Brown
er a/. (1961) and E.V. Brown (1963). Other values were approximated from other published data of Brown and co-worker8 where 4-dimethylaminoazobenzene controls w e r e available. C The xenyl, quinoline, isoquinoline, quinoxaline, dibenzofuran, dibenzothiophen, and benzothiazol dyes were fed for 6 months. All other compounds were fed for 10 to 12 months or for the time necessary to reach 100% tumor incidence,
These compounds correspond to 4-methylethylaminophenyl-, 4-diethylaminophenyl-, and 4-dipropylaminophenyl-azo-4-pyridine-N-oxide,respectively.
information that also an N-oxide group in 4' confers carcinogenicity upon the inactive 4-diethylaminoazobenzene (CI) . Pyridine-l-oxide-4-azo-pdiethylaniline (N,N-diethyl-P04) is a hepatic carcinogen in the rat with potency roughly comparable to that of 3'-methyl-DAB (relative activity 12). Thus, the totality of the data availahle shows that the relative effec-
R1OLEC;ULAR C;EOMWIIY AND CAIK‘INOGENIC ACTIVITY
395
tivcncss of tlic four substitucirts to confer activity upon iiiactivc carcinophile structurcs is
4 ’ e N - 0 > 4’-CzHS > 4’-F > 3’-CHy The relative ineffectiveness of a 3’-methyl group is also indicated by the fact t h a t such substitution of the parent compound (C) raises activity only twofold. In Table XVI, the N-oxides PO4 and 2’-Me-P04 (the steric analog of 3’-methyl-DAB) show roughly the same ratio of activities. The potentiating effect of a 4’-ethyl group seems to be related t o the conditions t h a t ( a ) it is in thc 4’-position and ( b ) it is linked dircctly to the “prime” ring. In fact, whereas 4’-methoxy-DAB is a weak t o moderately active carciiiogcn, the 4’-cthoxy derivative is inactive (Arcos and Simon, 1962). Thc graded nuxocarcinogenic effect of tlic four substituents strongly suggests that also noncovalent interactions arc involved, in a nonspecific fashion, in binding the dye molccule to critical ccllular site (s) . Speculatively, EN + 0, -C,H,, and -F groups can interact by means of coordination bonding, 1iydi.ophobic bonding, and hydrogen bonding, respectively. The bond cnergy of these interactions (consider the short length of the cthyl group) decreases in this order and parallels the observed carcinogenicities. T h a t a 3’-methyl group is the least active as a n auxocarcinogen may be due to the possibility that, here, potcntiation results froin the positive inductive effect of the group, and this effect increases the electron charge a t tlic 4’-carbon atom. This, then, strengthens the electrostatic interaction bctween the 4’-position and tlic cellular site(s) ( M . Arcos and Arcos, 1958). Neverthcless, such an electrostatic or fractional valence interaction is of lower energy than the above considered bonding types. Hence, a 3’-CH, group is the ]cast effective auxocarcinogen. The inactivity of 4”ethoxy-DAB has been interpreted (Arcos and Arcos, 1962) as due to the loss of hydrophobic bonding ability because of the hydropliilic character of the oxygen atom. I n addition to neutralizing hydrophobic bonding, the electroriegative oxygen atom may act as an “elcctron sink” reducing electrostatic interactions with the 4’-carhon atom. Support for the latter vicw is provided by thc notably higher potency of 4’-rnethylthio-DAB (Burkliard e t ul., 1962), which contains thc less clectronegative sulfur atom, than of 4’-methoxyDAB (*J. A. Miller e t al., 1957; Arcos and Simon, 1962), which contains the more clectroncgative oxygen atom. I n the light of their results, Arcos and Simon (1962) have questioned the current iiiterprctation of the phenomenon t h a t fluoro substitution in (wtaiir Imsitionh increases the activity uf vnrioub carcinogtmic cum1 ) o i i n i l h . AccotS(1iiig to this i~itcrpi~ct:~tion ( c , . ~ . , J . A. Rliller ef u l . , 1953;
396
JOSEPH C. A R C 0 6 AND MARY F. ARGUS
E. C. Miller et nl., 1962; E. C. Miller and Miller, 1960; J. A. Miller and Miller, 1963), carcinogenicity is increased because fluoro substitution diminishes the extent of metabolic ring hydroxylation by virtue of the strength of the C-F bond. While this working hypothesis has led to a number of valuable data, i t appears to be difficult to reconcile with the inactivity of 4‘-fluoro-4-diethylaminoazobe~1zene and the weak activity of 4’-fluoro-2-methyl-DAB. We have scen above t h a t substitution by 4’-ethyl or 4’-N-oxide groupings, instead of 4’-flUorO, leads to potent compounds. Yet, according t o Westrop and Topham (196Ga), a 4’-ethyl substituent protects DAB against 4’-hydroxylation lcss than a 4’-methyl substituent, although thc 4’-cthyl-substituted compound is considerably more activc than the 4’-methyl-substituted compound. Clearly, then, the problcin of potentiation of carcinogenicity is a phenomenon more complex than merely protection of ring positions against metabolic hydroxylation. The work of Westrop and Topham is discussed in more detail in Section III,C,3. At any rate, whatever the ultimate significancc of Westrop and Topham’s work is, their results do not appc:ir to support a correlation between carcinogenic activity and protective cffect of 4’-suhstitucnts (including fluoro) against 4’-hydroxylation.
2. The Requirement of N-Alkyl Groups The reports of Arcos and Simon (1962) and Brown and I-Iamdan (1966) prompt a reformulation of the conclusion of the Millers that a t least one N-methyl group is required for hepatocarcinogenic activity of amino azo dyes (reviewed by J. A. Miller and Miller, 1953; see also Kinosita, 1937). This requirement must now be broadened to include N-alkyl groups other than methyl, in view of the carcinogenic activity of 4‘-ethyl-4-diethylaminoazobenzene and N,N-diethyl-P04. Although i t is true t h a t these compounds are somewhat less active than the corresponding N-methyl dycs, the differences of activities arc insignificant in view of the considerable widening in recent ycars of the activity spcctrum of hepatocarcinogenic azo dyes (Table XVI). Regarding the possible importance of a two-step enzymatic activation mechanism (N-hydroxylation and N-hydroxy esterification) for hepatocarcinogenicity of amino azo dycs, Poirier et nl. (1967) assume t h a t an N-alkyl group may be necessary for fitting the dye on the cnzymc(s) involved. This suggestion appears to be in contradiction, however, with the results of the carcinogenicity tests of N-hydroxy dyes. I n fact, while N-hydroxy-2-ttcctylaii~iiiofluo1~e1ic is a potcnt agent to inducc sarcomas in rats by subcutaneous injection, 4-hydroxylaminoazobenzene, its N acetyl derivative, and the O-acc>tyl ester of the latter are inactive when n i ~ lin long-tcrni fcc~ling(Sato tested in tlic s:me spccies pni~ciitc~r:illy
hIOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
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et al., 1966; Poirier et al., 1967). Yet, N-acetoxy-4-aminoazobenzene, a t least, represents an esterified N-hydroxy compound similar to the end product (s) of the postulated two-step in vivo activation, and, therefore, an alkyl group should logically not be required for activity. At any rate, tliis picture is further complicated by the recent important discovery that amino azo dyes are potent carcinogens also toward extrahepatic tissues of tlie rat (e.g., Fare, 1966). In particular, 4-;21iiinO-, 4-nionomethyl:mino-, and 4-diniethylaminoazobenzene and their 3-niethoxy derivatives are potent carcinogens to the skin of the rat by topical application. Any “all-or-none” requirement for an N-alkyl group is absent for activity toward this target tissue, since all six dyes give 100% tumor incidence; Iiowcvcr, it remains that tlie N-methyl dyes have shorter induction times than the corresponding free amines (Fare, 1966). 3. Heterocgrlic and Lnrye-Sizc Priine Ring Aiinlogs of ~ - D i n z e t h y Z a ~ ~ i i n o a x ~ b e n(zD e ~AiB e)
Tlie extremely high lcvels of carcinogenic activity encountered in certain nieriibers of this class (Table S V I ) represent a n important developnient. Tlie suggestion of J. H. Il’eisburger and Weisburger (1963) should he reiterated here, that, because of the considerable levels of activities, “application of these compounds to studies on the possible existence of a threbhold in cheinical carcinogenesis might prove worthwhile.” Moreover, the investigations of Brow11 and his associates may stimulate new studics on the structure-activity relationships of tlie amino azo dyes and ttic inechaiiim of auxocarcinogen effect. T h e carcinogenicity of the 1- and 2-naphthyl and 4-xenyl analogs has h e m rcvicwed previously (Arcos and Arcos, 1962). Although the 2-anthryl :tnalog has been fed to Sprague-Dawley rats for 1 year a t 0.06% level in :Lcontrolled riboflavin semisyntlietic diet (Napier, 1964), the length of time may have been insufficient and the strain of test animals may have h e n rcnistant t o this agent. In this regard, a similar instance should be recalled in which J. A. Miller and Baumann (1945) found the l-naphthyl analog inactive in Holtzmnnn (Sprague-Dawley) rats before its carcinogenicity in Oshorne-Mendel rats was shown by Mulay and Firminger (1952). It is also possible t h a t differcnt routes of administration may have to be used for tcsting. Nevcrthelcss, conditionally accepting Napier’s testing as :itlcquatc, it is not surpribing that other tricyclic LLprinie’lring analogs, 1- ~ t n d2-:inthraqui1ioiiyl, dibenzofuryl, and dibenzothienyl, are inactive (E. V. Brown, 1963; Napier, 1964). Molecular size may not be, however, a limiting factor hcrc since Dittmar (1942) found 4-nitrobenzeneazo-3,4-benzopyrene quite potent to induce heinangiosarcomas in mice by subcutaneous injection.
398
.IOSEI’H C. ARCOS A K U RIAltT
17.
ARGUS
,Judging from thc f:wt t1i:it N:Lpicr t1escril)ctl no mammary tumors wit11 the 2-tnthryl aii:iIog, it, should IIC :issunid thitt the compound does not undergo significant metabolism involving reductive clcavage of the azo linkage. I n fact, one moiety of such cleavage would be 2-anthramine which has been shown by Griswold e t al. (1966) to yield an 8/18 incidence of malignant mammary tumors in rats, in as little as 6 months, following an oral administratioii of the maximum-tolerated single dose. Another possibility is that the insolubility of this high-molecular-weight dye may have limited its adsorption from the intcstinal tract. Testing thc 2-anthryl analog by subcut,aneous route might prove worthwhile since Mulay and O’Gara (1957) found thc 1-naphthyl analog also active by this route. The pyridine-N-oxide analogs have been discussed in some detail in relation to tlie auxocarcinogenic effect of 4’ substituents. Table XVI shows that the potent carcinogenicity of PO4 is further enhanced by a 2‘-methyl group. (Note that, because of the numbering of the pyridine ring system, 2’-Me-P04 is actually the structural analog of 3”methylDAB.) On the other hand, introduction of the methyl group into the 3’-position (3’-Me-P04) reduces the activity of P04. Here, again, the pattern follows that of the parent compound series, since 3’-Me-P04 is the analog of a’-methyl-DAB, which has a relative activity of only 2 to 3. Potentiation by tlie N-oxide grouping in the 4‘-position (‘overrides” the deactivating effect of unfavorable methyl substitutions ; thus, 2,2’- and 2’,6’-diMe-P04 are potent carcinogcns. Except for the presence of the 4’-N-oxide grouping, these compounds are identical to the inactive dyes, 2,3’- and 3’,5’-dimcthyl-DAB (Arcos and Sinion, 1962; J. A. Miller and Miller, 1953). The inactivity of both N,iV-dipropyl-P04 (Table XVI) and of N,N-dipropyl-DAB (J. A. Miller and Miller, 1953) gives a definite indication that the stereochemical limitations compatible with carcinogenicity are much more stringent for the amino substituent than for a substituent occupying thc 4’-position. The most potent hepatic carcinogcns known to date can be found among the quinoline and isoquinoline analogs and their respective N oxides. It should be noted in passing that Cosgrove e t al. (1965) reported the carcinogenicity of the styryl analog of Q4, 4- (p-dimethylaminostyryl) quinoline, which produces a high incidence of hepatomas in mice following single intravenous injection. Whilc the rationale for the high activity of the azo derivatives remains to be cluciclated, it is clcar that, in the quinoline series, linking of the 4-dimethylaminophcnylazo group to the 2-, 3-, 7-, or 8-position does not give rise to carcinogenic compounds. Speculatively, this is possibly due to ( a ) tlie rcquircment of a certain distance between the dimethylamino group and the region of highest electron density in the heterocyclic nucleus and ( b ) the fact that because of the electronegativity of the nitrogen or N-oxide grouping,
MOLECULAR GEOMETRY A N D CARCINOGENIC ACTIVITY
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conjugation tlirough the heterocyclic nucleus is oriented in a specific fashion. Morcover, obscurc steric eflects arc probably additional determinants of carcinogenic activity. To the reviewers, it seems that investigations with these most unusual compounds have, in truth, barely begun. Correlated biochemical and theoretical studies with them could open new levels of understanding of the relationship between the electronic propcrties and carcinogenic activity of aromatic compounds. 4. Activity of Azo D y e s to ExtTahepatic Tissues For about three decades, the notion pervaded the literature that the amino azo dyes related to 4-dimethylaminoazobenzene (DAB) and oaiiiinoazotolucne have an exclusive spccificity toward the liver. J. A. Miller and hhller (1961) reported that ingestion by the rat of 3-methoxy-4aminoazobenzenc or its N-monomethyl and N,N-dimethyl derivatives a t the level of 2.67 mmoles/kg. diet iiiduccs high incidences of tumors in extrahepatic tissues, in particular, squamous cell carcinomas of the ear duct. However, these dyes manifested only trace activity toward the liver. 3-Methoxy-4-aminoazobcnzene also iiiduccd low incidences of tumors in the small intestine and the mammary gland. Moreover, the dyes induced skin tumors in occasional animals. No tumors were noted in rats which received the corresponding 2-nicthoxy or 2-hydroxy dyes in the diet. Essential features of the Millers’ results were confirmed by Fare and Howell (1964) ; however, these authors found a notably higher incidence of skin tumors with 3-mctlioxy-4-aminoazobenzene. Simultaneous administration of cupric oxyacetatc protected against liver tumor induction by the methoxy dyes, just as it was noted earlier in DAB-induced hepatic tumorigenesis (Howell, 1958) ; the copper salt did not affect, however, the incidence of ear duct and skin tumors. Subsequently, Fare and Orr (1965) showed that 3-methoxy-DAB is a potent agent to induce tumors on the skin of rats. Painting twice wcekly with 1 ml. of a 0.2% solution of the dye in acetone on the shaved intcrscapular region yielded 100% tumor incidence with an average induction time of 46 weeks for the appearance of the first tumor. All rats developed multiple skin tumors, and a number of them also developed ear duct tumors. The sampling of skin tumors was identified mainly as squanious carcinomas, keratoacanthomas, basal cell carcinomas, and trichoepithelionias. Just as in the 1964 report, feeding of cupric osyacctatc to the rats did not protect against skin tumorigenesis, ~ l i i c l110 col~pc’i’;ic.cuinul:ttion was noted in the skin. No bound or free dye mas detected ill thi. li\.cbi*b of thv rats. Previous to this study, only two (unswces>ful) attenipts to iiicluce skin tumors in rats by epithelial a1)pIie:itioii of tliffcrctit :izo compounds appear to have beeii rccordcd in the literature (Ray et nl., 1952; Mulay and Congdon, 1953). Prompted I Jliis ~ results, Fare (1966) undertook a systematic investi-
400
JOSEPH C. AHCOS A N D MARY F. A R G I J S
gation of the carcinogenicity of specially purified amino azo dyes toward the rat skin. As we have briefly mentioned in the discussion of the requirement for N-alkyl groups, the methoxy grouping in the 3-position is not necessary for carcinogenic activity since 4-aminoazobenzene, 4-monomethylarninoazobenzene (MAB) , and DAB are all active and produce multiplo skin tumors of histologic types observed with other skin carcinogens. With or without the 3-methoxy substitucnt, the N-monomethyl dyes proved consistently to bc the most potent. Since all compounds eventually produced tumors in 100% of the animals, the relative activities of the dyes toward the rat skin are defined on the basis of the average induction times and have the following order: 3-methoxy-MAB > MAB > 3-methoxy-DAB > DAB > 3-methoxy4-aminoazobenzene > 4-aminoazobenzene Tumors of thc ear duct arose only with the methoxy dyes, and whereas the 3-methoxy group is not necessary for epithelial activity as i t was believed in early studies, its introduction into the molecule does increase the potency. I n contradistinction to the high susceptibility of the r a t skin to the epithelial carcinogenic action of azo dyes, mice were found to be totally resistant to skin tumorigenesis by 3-methoxy-DAB. Another newly found, extrahepatic tissue target of amino azo dyes is the bladder. Saffiotti et al. (1967) reported that feeding of o-aminoazotoluene to hamsters a t 0.1% dietary level for 49 weeks produces a high incidence of liver tumors as well as a high incidence of bladder tumors. Trypan Blue has been known to produce histiocytic tumors in the liver and other reticuloendothelial organs in rats by prolonged weekly subcutaneous administration. Driessens et al. (1962) and D. V. Brown (1963) described the induction by this dye of subcutaneous tumors a t the injection sites. The dye was injected every 2 weeks (1 ml. of a 1% solution) until subcutaneous tumor formation began; the tumor incidence was 50-60% with a latent period of 7 t o 18 months. Histologically, the tumors were described as being probably histiocytosarcomas.
C. DETOXICATING METABOLISM Except for ortho hydroxylation, which possibly plays some role in the carcinogenic activity of 2-naphthylamine, the metabolic ring hydroxylation of aromatic amines-just as the ring hydroxylation of polycyclic 1iydrocai.bons-invnria~)ly brings about decrease or loss of carcinogenicity. The clcawge of tlic YZO double bond and the removal of N-alkyl groups from amino azo dyes also cause loss of activity. Such metabolic routes of aromatic amines and azo dyes, since they a11 bring about decrease of carcinogenicity, represent pathways of true detoxication. On
MOLECI’LAR (;EOl.II.:TRT AKD C A R C I S O G E S I C ACTIVITY
401
the other hand, -hytlroxylatiou anti sulwqiient v4cmficwtion of the N-hy droxy metaholi t es I’PP I I It 111 111 c rt’n *(+ of c :i r r i 11 ogciiir a c t i vi t y . The latter pathways ni:ty I)c dehigii:itcJ(l:t* rcc.//zw/rrr!/ / u c / a I d r s m . Unfortunately, itii uneyuivocnl clenionstr:itioii that a irietabolite is a proximate carciiiogeii, i.e., that the metabolic path leading to it is a truly essential step in the carcinogenic effect (s) of the parent compound, presents some inherent difficulties. Several metabolites along the pathway (s) originating a t the pareiit compound may be carcinogenic. However, the higher potency of :t metabolite, and even its local carcinogenicity versus the local inactivity of the parent conipouiid-although it furnishes strong circumstantial evidence that the particular metabolite is a proximate carcinogen-does not logically rule out that the parent compound, prior to metabolism, is also carcinogenic. Thc circumstance that a parent compound is active toward certain target tissue (s) when systemically administered, but inactive locally, can certainly mean that the target tissue (s) represent site (s) of metabolic activation. However, activity in specific target tissues, following systemic administration, can also be the result of the particular physicochemical properties of the parent coinpound together with the permeability aiid circulatory characteristics of the tissues affected. That both niechanisms may be responsible for determining the target specificity of carcinogens cannot be excluded a t the present time. In the following, some salient results of investigations on detoxicating metabolism are summarily reviewed. Because the role of ortho hydroxylation for the carcinogciiicity of 2-naphthylamine is still unresolved, this topic is discussed in the section (II1,D) 011 “Activating Metabolism.” It should be recalled in passing, that the carcinogenic benzidine derivative, 3,3’-dihydroxybenzidiiie, is not a riictabolite of benzitline. A\
1. %Acetylanainofluorene, 4-Acetylnnzinobipheny1, and 4-Acetylaininostilbene
A considcrablc amouiit of work has lwei1 carried out in the last 10 years 011 the metabolism of 2-acetylarninofluorene in a variety of species, including man, arid these investigations arc summarized in Table XVII. The 7-position is a major site of rnctabolic ring hydroxylation in all species studied. In the dog, monkey, and iiiaii, the 7-hydroxy metabolite is actually the only urinary ring hydroxy compound detected to date. There appears to be no evidence for hydroxylation in the 9-position in any species. The hydroxy metabolites arc excreted both free aiid conjugated with glucuronic and/or sulfuric acid. Hydroxylation of 2-acetylaminofluorerie in vitro by hepatic microsomes from normal and 20-incthylcholanthrcne-treated mice, rats, ham-
TABLE XVII METABOLIC HYDROXYLATION OF 2-.h2ETYLAMINOFLUORENE IN DIFFERENT SPECIES Species
Rat
Mouse
Hamster Guinea pig Steppe lemming Rabbit Dog Cat Monkey Man Rainbow trout
Positions of ring hydroxylation"
.Y-Hydroxylation
Reference
G, S
E. K. Weisburger and Weisburger (1958); J. H. Weisburger et al. (1958, 1959); J. A. Miller t t nl.
+ +
7, 5, 3, 1 (trace) 7, 5, 3, 1 7, 5, 8, 3 (trace)
G,S Gi S
G, S
+(low)
+ + + +Oow) +
7 7, 5 7 7 7, 5
G G S, G G 0, S G
~
(1960) J. A. Miller et al. (1960) J. H. Weisburger et al. (1964a) J. H. Weisburger et al. (1958); J. A. Miller et al. (1960); Kiese and Wiedemann (1966); \*on Jagow et al. (1966) J. H. Weisburger et al. (1965) Irving (1962, 1963) Poirier et al. (1963); Dyer et al. (1965) J. H. Weisburger et al. (196413) Enomoto et al. (1962); Dyer et al. (1966) J. H. Weisburger et al. (1964~) Lotlikar et al. (1967b)
?
?
7, 5 7, 3, 5
~~
Conjugat.ion6
+
5, 7, 1, 3, 6, 8
~~
(URINARY METABOLITES)
~
~
Bold-faced numbers represent positions of hydroxylation in major ring-hydroxy metabolites. b Glucuro and sulfo conjugation are represented by G and S, respectively. Bold-faced letter indicates the major conjugatiou. a
P
>
8 >
3
sters, mbbits, aiitl guinw pigs has been stutlicd by Lotlikar et al. (19674. There were notable differences in these enzyme activities in normal animals of the species studied. The lowest level of in vitro 7-hydroxylation was found in the rat, the most susceptible among these species, and the highest level in the guinea pig, which is refractory t o the carcinogenic action of the amide. Only rat and mouse liver microsomes showed large increases of ring-hydroxylation activity following trcatment with 20methylcholant,hrene. It is consistent with the preference to the 7-position as a hydroxylation site in 2-acetylaminofluorcne that fluoro substitution in this position notably iitcreases carcinogcnic activity. However, a s alrcady noted in Sections II,E and III,B,l, fluoro substitution does not give complete protection against hytlroxylation. WwtroI) and Topliam (1965) reported preliminary evidence that 7-fluoro-2-acetylaminofluorene undergoes defluorohydroxylation in v i m . Urine from rats and guinea pigs, to which this compound was administcrcd, contained the glucuronide of 7-hydroxy2-acctylaniinofluorcne, although in a much smaller amount than t h a t which was prorluccd from the unsubstitutecl amidc. T h a t similar rationale may account for the potentiation of carcinogenic activity of 4-acetylaminobiphenyl by 4'-fluoro substitution is indicated by the finding of Booth and B o y h i d (1964) that rabbit liver microsomcs ring-hydroxylate this amide in the 3- and 4'-positions besides metabolic conversion to the N-hydroxy form. Similarly, Baldwin et ul. ( 1 9 6 3 ~ )found 4'-hydroxy-4-acetylaminostilbene inactive by oral administration t o rats under conditions in which t,he nonhydrosylated amide is highly carcinogenic. Metabolism studies by Ancicrsen et a l . (1964), Baldwin and Smith (1965), and Baldwin and Romerii (1965) have shown bubsequently t h a t the 4'-hydroxy and (probably to a lesser cxtent) 3-hydroxy derivatives, free and conjugated, are actual urinary metabolites of 4-aminostilbenc (as N-acetyl or N,Ndimethyl), in the rat. Surpi-isingly, in tumor inhibitory studies (which property roughly parallels carcinogenic activity in this series), 4'-fhoro4-acetylaminostilbene had little activity a t the dose used, but the N-hydroxy derivativc-following expectations-was much more active than the parent arnidc (Anderscri et al., 1964).
2. l-Pherz ylnzo-2-naphthol Exhaustive studies 011 thc mc~tnhoIismof l-plic1iylazo-2-n:iphthol have kwen c:ii.rietl out by Daniel (1956, 1962) :i11(1 by Chilcla aiitl Clayson (1966). The IJI'CWntly knowit 1)atliways of this c o ~ i ~ p o uarc ~ ~ dsumm:rriactl ill Tal~lcM ' I I I . I t ~ l ~ o u llw d recalled that 1-~~lic~iyl:~zo-2-~i:~plitho1 is prob:il)ly it wrak c:ircinogcn, but W:LS usc(l c:irlicr for coloring
404
JOSEPH C. ARCOS AND MARY F. ARGUS
TABLE XVIII The Known Pathways of Metabolism of l-Phenylazo-2-Naplithola
0-Gluc
Q I
NHCOCIL,
t pH2
\
SL ,hate of p-am iophenc
/
Probably sulfoconjugate of 4', 6'-dihydroxy-1phenylazo- 2-naphthol
Modified, after Child8 and Clayson (l9fX)
margarine. Certain derivatives are still in use as food colors. Kirby and Peacock (1949) induced hepatomas with this compound by injection into random-bred mice. However, Clayson et al. (1965) found it inactive by oral administration t o :uiolhcr litlo of riuidoin-l~iwl niicc ant1 to CBA
MOLECULAR GEOMETRY AND CARCINOGENIC ACTIVITY
405
mice. Bonser e t al. (1956b) and Clayson and Bonser (1965) found it active by bladder implantation.
3. Ring Hydroxylation of 4-Dimethylanzinoazobenzene Derivatives The mechanism by which amino azo dyes are diverted toward noncarcinogenic pathways has been investigated from different approaches. The work of Westrop and Topham on the removal of 4’-substituents from 4-dimethylaminoazobenzene (DAB) derivatives has been succinctly mentioned in earlier sections, with particular reference to the fluoro substituent. Aromatic ring-linked fluorine atoms have generally been regarded as metabolically inert despite the carlicr work of Hughes and Saunclers (1954), and Kaufman (1961) 011 the removnl of the fluorine atom from p-fluoroaniline and 4-fluorophenylalanine, respectively. I n a first study, Westrop and Tophain (1966b) identified the nonbound mctabolitcs ether-extractable from the livers of rats, intragastrically dosed with 4’-fluoro-nA~. The compounds separatctl hy thinlayer chroinatography w ( w tlie 4’-fluoro and 4’-hydroxy derivativcs of 4-aminoazobciizcuc.ie~ and of ~ ~ 7 - ~ ~ i o ~ i o r nanrl c t l ~N,N-diinethyl-4-aniinoylazobenzcne; 4’-fluoro4-aii1i1ioazot~~1izciic was also present, N-acctylatcd. I n their second study (using tlie same cxpcrimental procedure) , Westrop and Topham (19GGn) mwbiired the extent of 4’-hydroxylation of DAB and various ring-methyl and 4’-substitutcd derivatives. Taking thr 4’-substituted dyes as a separate group, they have rioted in a total of eight compounds that the amount of 4’-hydroxylated metabolites increases in the same order as the carcinogenic activities. No such correlation was observed with dyes having the 4’-position free. On the grounds of the data obtained with the 4’-suhstitutccl dyes, n’estrop and Topham concluded that the 4’-hydroxylation is thc result of the p-p intramolecular rearrangement of metabolically formed, respcctivc N-hydroxy compounds. T h at is, bince S-hydrosy tlcrivativcs (a5 tlic 0-esters) arc considered as likcly candidates to bc the active fornib of amino azo dyes (E. C. Miller and Miller, 196G), the greater amounts of N-hydroxy derivatives formed from the more carciriogenic tlyes bring about, in turn, highcr lcvcls of the 4’-hydroxy metabolites. Although, in any event, the claimcd corrcltition does not support tlie idea t h a t ring suhstitucnts (fluorine or other) incrcasc carcinogcnicity by blocking hyclroxylation, s:oniu aspects of Westrop and Topham’s hypothesis may be qucstioncd. First, the N-hgtlroxy O-(Jsters, assurncd ultimate carcinogc~nic metabolites of nmino nzo dyes, show high reactivity toward proteins and nuclcic acidh ( Poirier e t nl., 1967). Second, Funakoshi a n r l Ter:iy:tnia ( 1965) conclutlerl, 011 tlic 1-m.is of q ~ c t r n evitlencc, l tli:tt 110 plrenolic liytlixxyl gioul) i b present i n tlic (lye moiety of tlic
406
JOSEPH C. ARCOK AND MARY F. ARGUS
“polar dye” extracted from liver tissue. These two findings appear to lessen the significance of the metabolic 4‘-hydroxylation for the mechanism of carcinogenesis and lead t o two alternatives: ( a ) that carcinogenesis and 4’-hydroxylation follow divergent pathways, both originating at and requiring the prior formation of an N-hydroxy metabolite and/or its 0-ester ; ( b ) that the observed partial correlation between carcinogenic activity and amount of 4’-hydroxy metabolites may be entirely fortuitous. That the latter alternative may be valid is strongly suggested TABLE XIX 4’-HYUROXYLATION AND HEPATOCARCINOCiENlClTY I N THE R.ATOF AMINO Azo DYEP
Compoundh 4‘-Nitro-DAB 4‘-Trifluoromethyl-DAB 4-Ethov-DAB 2-Methyl-D AB 4’-Me t hyl-D AB 3-Methyl-MAB 4’-Chloro-DAB a’-Methyl-DAB 4’-Methoxy-DAB DAB 4’-Ethyl-DAB 4’-Fluoro-DAB 3‘-Meth yl-DAB
Relative activity 0 0 0 0 (trace) 1 1 1-2 2-3 3 6 10 10-12 10-12
4’-Hydroxylated metabolites (mpmoleslgm. liver) 0 0 6.6 24.3 6.6 18.7 5.0 20.4 10.0 47.7 21.6 16.2 18.4
a From Westrop and Topham (1966a). The compounds listed in Tables 2 and 3 of these authors have been combined and arranged in order of increasing carcinogenic activities * DAB designates 4-dimethylamiiioarobenzene; MAB designates 4-monomethylaminoazobenzene. 4’-Ethoxy-DAB is an inact,ive compound (Arcos and Simon, 1962).
by the finding that the relationship holds only for 4’-substituted compounds of which only a small number were studied. Actually, if all of thc dyes in Westrop and Topham’s report are arranged in order of increasing carcinogenic activities (Table XIX) , the correlation vanishes (correlation coeff. = 0.44). Biliary metabolites of DAB, retaining the cliromophore group, were examined by Ishidate e t al. (1963) in the rat. 4’-Hydroxy derivatives of DAB, of 4-nionoiriethyla1iiiiioazobeiiaene, and of 4-an1ii1onzobeiizenc were detectetl I)oth as glucuro t i t i d :is sulfo coiijugutes.
MOIXXXJLAR GEOMETRY AND CARC~IKOGENIC ACTIVITY
407
4. Reductive Clenvnge of the A z o Double Bond Rlecke and Schmiihl (19.57) were thr first, to iiivestigatp the possible existence of it rorrelatioii in azo dyrs hetwcw cwcinogrnic activity and the ability to unclcrgo recluctivc, c1e:ivagcI. A similar study was carried out recently by Matsunioto arid Terayama (1965a). These authors measured the rates of azo double-bond reduction using rat liver homogenates fortified with a NADPH,-generating system, in nitrogen atmosphere. As with the yeast suspensions in Mecke and Schmahl’s experiments, no correlation was found between azo reduction rate and carcinogenic activity with liver homogenates.
5 . Methylation and Demethylation of Amino Azo Dyes The problem of the interconversion of primary, secondary, and tertiary amino azo dyes requires further study. In fact, although Matsumoto and Terayama (1965b) appeared to have confirmed the earlier view (J. A. Miller and Miller, 1953) of the rapid metabolic interconvertibility of 4dimethylaminoazobenzene and 4-monomethylaminoazobenzene, the Miller’s group concluded in the same year that-contrary to their previous hypothesis-the metabolic product of 4-monomethylarninoazobenzene, hitherto regarded as the N,N-dimethyl dye, is actually the 3-methylmercapto derivative (Scribner et al., 1965; E. C. Miller and Miller, 1966). Primary amino azo dyes, such as o-arninoazotolucne and the 4’-fluoro and 3’-methyl derivatives of 4-aminonzobenzene, are not methylatecl (Matsumot0 and Terayama, 1965b). Although unlikely to be of any significance for the mechanism of carcinogenic action, it is of interest to note that l’C-labeled N-methyl carbon of 4-dimethylaminoazobenzene is incorporated into purines in nucleic acids (Berenbom, 1962; Terayama and Yang, 1964). This is probably a normal metabolic route for the one carhon fragment liberated by oxidative N-demethylation. 6. Some Special Metabolic Pathways of o-Aminoaxotoluene
In their above-mentioned study on the interconvertibility of azo dyes having tertiary, secondary, and primary amino groups, Matsumoto and Terayama (1965b) noted that primary amino azo dyes, such as o-aminoazotoluene, 4-aniinoazobenzene1 and the 3’-methyl and 4‘-fluoro derivatives of the latter, gave several unknown metabolic products which seemed to be of a complex nature. Subsequent work (Matsunioto and Terayama, 1 9 6 5 ~ )allowed the identification of a high molecular weight metabolite of o-aminoazotoluene (CII) which corresponds to a product of oxidative dimerization of the dye. The tentative pathways leading
408
TABLE XX Tentative Pathways of Metabolic Reductive Dimerization of o-Aminoazotoluenea
I
JOSEPH C. ARCOS A N D MARY F. ARGUS
I
a From Matsumoto and Terayama (1965~).
from o-:iiriirioazotolueii~ to 4,4’-bis (0-tolylazo) -2,2’-dimethylazobenzene ( C I I I ) are given in Table XX. The identity of the metabolically formed ( C I I I ) was ascertained by comparing it to a synthetic sample obtained by oxidative dinierization of the parent dye ( C I I ) with M n 0 2 . There is no experiment:il evidence as yet for the formation of the postulated Nhyclroxy and nitroso interrnediatcs ; however, inference from results with other dyes strongly supports this possibility. The metabolic fate of the ring methyl groups of o-aminoazotolucnc was investigated by S:iniejima et nl. (19437). Interestingly, the 2J-niethyl group alone undergoes oxidation, first to hydroxymethyl and, subsequclntly, to cnrl)oxyl. Tlierr is no evitlencc for the oxidation of the 3-methyl group. 4’-Hydi~oxylation is ;ilsu a major metabolic route for o-amino:tzotolucne. Thebe inetabolitcs appcar in the bile as N-glucuroi d e s and 4’-O-~ulfo conjugates. N-Glucuronidation appears to occur predominantly prior to oxidation of the 2’-hydroxymethyl group. This was inferred from the fact that rats, admiiiistercd the 2’-hydroxymethyl dye, excrete it, largely unaltered :IS the N-glucuronide, and only a notably smaller amount of 2’-carbosy-N-glucuronide is formed. 7. Possible Significance of Amino A m Dye Amine Oxides
The possibility that the amine-oxide form of 4-diinethylaminoazobenzene may be mi intermediate in the oxidative demethylation of the dye has been prol’osed by Terayama. Using, a t first, the amine oxide of the azoxy form of the dye, Terayama and Hanaki (1959) observed a great loss of carcinogenicity and tissue binding. On the other hand, in subsequent work, the ainine oxide of the azo form proved to be significantly more potent than the parent amine dyc in inducing hepatic tumors by oral administration (Terayama antl Orii, 1963) as well as in binding to tissue constituents (Terayama, 1963a,h; Terayama and Orii, 1963). 4-Dimethylamino:izot,enzene-N-oxide (D.4B-N-oxide) is notahly reactivc and decomposes rapidly in the presence of ii,oii-porphyrin coiiipountls to yield mainly DAB antl 4-iiionomethyla1iiiii~~:tzob~rizeiie (MAB) and 3OH-DAB, and in lesser amounts, 3-OH-R9AB, 4-aminoazobenzene, antl its 3-hydroxy derivative (Terayama, 1963n,b). Tcrayaiiia i1967) proposed st scheme (Table X X I ) to account for the products of decomposition. There is evidence for the metaholic formation of niiiine oxides (Baker :mI Chaykin, 1960, 1962; Zieglcr and Pettit, 1964; Tielileke and Stahn, 1966) , and the cnt:tljtic~( iiuu-KAl)PH,-i tquiring) cleiiirthylation of dimethylaniline-h--oxitle hy liver inicrosonics has been shon-n (Pettit and i ~ Zieglei., 1963). The iiiiiine ositle of DAB ib clealkylate(l ( i l l tlic ~ i I ) w uf NADPH?) by isolated rat liver rnicrosomcs niorc rapidly t1i:in tlinictliyl-
~
e
TABLE XXI
0
Hypothetical Sequence for the Decomposition of DAB-mine-N-oxide by Iron Porphyrin Compounds'*b Q/ - \ N = N o f / ;
DAB-N-oxide
\ 0
- .....Fe-Porphyrin
"activation"
I
deoxygenation
? ,OH
a activated oxygen
From Terayama (1967) DAB = 4-dimethylaminoazobenzene; MAB = 4-monomethylaminoazobenzene; AB = 4-aminoazobenzene.
OH
RIOLECULAH (IEOMETRY ANI) CAHCINO(iEN1C ACTIVITY
41 1
: ~ i i i l i i i ~ - ~ - o ~:Lid i i l ( ,t,licw , i h rii1)id I)iiiding to protc4ns rliiring (lw~lkyl:ttion (Uehleko id Slalin, 1966). ,4 comprrlic~nsivetreatisc 011 thc clicmistry, physical chemistry, and biological properties of aromatic arnine N-oxides has been written by Ochiai (1967). The N-oxide of 4-dimethylaminoazobenzene could also represent the intermediate of a pathway leading to an unusual type of active metabolite. Furst (1963) hypothesized that an arigular arrangement of DAB is necessary to fit and complex with hnsc-pairs in DNA. Actually, the possibility of such angular molecular arrangenicnt of the dye-corresponding ( c ) cinnoline (C1V)-has been aftcr ring closure to 2-di1iietliyl:i1~iiiiol~e1izo
(CIV)
considered earlier by the Millers and their associates. In view of testing the cyclized compound for carcinogenic activity, DAB was treated in an AlC1,-NaCI-KCl-NaF eutectic using a procedure from an I. G. Farbenindustrie patent for the synthesis of benzo (c) cinnoline and its amino derivatives (Arcos et al., 1956). The resulting compound-what was then believed to be 2-dimethylaminobenzo (c) cinnoline on the strength of its elementary analysis, the preparatory procedure, and certain physicochemical considerations-was tested by J. A. Miller e t al. (1957) for carcinogenic activity in rats and found to be inactive. However, subsequent work by M. Arcos (1958) on the spectra of benzo(c) cinnoline derivatives led to a questioning of the structure of the assumed cyclized derivative. She has demonstrated that the AlCl, dehydrogenation procedure yields benzo (c) cinnoline derivatives only with RZO compounds having no electron-donor group in para to the azo linkage. If such an electron-donor group is present, as in DAB, the reaction takes a different orientation to yield biphenyl derivatives. Thus, the presumed cyclized derivative of the dye was shown to be actually 4,4'-bis (dimethylaminophenyl) -bisazobiphenyl (CV) . (CHAN-@=N-N=
N-@(CH), (CV)
Recently, Lewis and Reiss (1967) synthesized 2-dimethylaminobenzo(c) cillnoline (CIV) by photochemical cyclization of DAB-N-oxide; the structure of the compound was ascertained by its identity with the con-
412
.JOSEPH C. 4RCOS AND XIART F. ARGTJS
(Lensation product of 2-clilorol~enzo( c )cinnoline and dimethylamine. These authors have also confirmcd the identity of 4,4’-bis (dimethylaminophenyl) -bisazobiphenyl (CV) , This, then, reopens the problem of the carcinogenic activity of 2-dimethylaminobenzo (c) cinnoline and suggests the interesting possibility that an in vivo-formed, azo dye N-oxide could undergo metabolic cyclization to a heterocyclic aromatic amine.
D. ACTIVATING METABOLISM : PRESENT STATUSO F THE Ortho-HYDROXYLATION HYPOTHESIS ; THE CARCINOGENICITY OF N-ARYLHYDROXYLAMINES 1. Metabolites of W-Naphthylamine and Their Carcinogenic Activity
A considersblc amount of ingenious work has bcen carried out to unravel the fascinating jigsaw puzzle which is the carcinogenically significant pathways of naphthylamine metabolism. The validity of the ortho-hydroxylation hypothesis, i.e., that aromatic amines are carcinogenic by virtue of their metabolic oxidation to o-aminophenols (Clayson, 1953), appears now to be limited to 2-naphthylamine. The demonstration since 1953 that synthetic ortho-hydroxylation of 4-aminobiphenyl, benzidine (to 3-monohydroxy), 2-aminofluorene1 and 4-dimethylaminoazobenzene brings about a total or almost total loss of activity, rules out that this metabolic route is a participant in their mechanism of carcinogenic action. Even regarding 2-naphthylamine-especially since the discovery of the metabolic N-hydroxylation of aromatic amines and subsequent studies along those lines-the role of ortho-hydroxylation in carcinogencsis has bcen seriously questioned. Certain aspccts of this problem have already becn touched upon in Section 1111A,3.Metabolic pathways leading to probable proximate carcinogens of 2-naphthylamine are given in Table XXII. Despite the uncertain results of carcinogenicity testing with 2-naphthylhydroxylamine (CVI) in newborn mice by subcutaneous administration (Roe et al., 1963; Walters et al., 1967), repeated intraperitoneal injection into random-bred rats produced a much higher abdominal sarcoma incidence than the parent amine (9/15 versus 2/14) (Boyland et al., 1963a). I n bladder implantation, both 2-amino-l-naphthol and 2-naphthylhydroxylamine showed a highly significant tumor incidence relative to the controls, the latter compound giving a somewhat higher incidence (Bonser et al., 1963). The high level of carcinogenic activity of 2-naphthylhydroxylamine toward the mouse bladder epithelium was confirmed by Bryan et al. ( 1 9 6 4 ~ )Regarding . the above report of Bonser et al., it must be pointed out that 2-naphthylhydroxylamine certainly did not induce “a higher incidence of bladder tumours than any other compound tested” as stated by Boyland et al. (1963a) about that investiga-
41 3
I\IOLECUL.ZR GEOMETRY A N D CARCINOCXKIC A C T I V I T Y TABLE XXII Metabolic Pathways Leading to Probable Proximate Carcinogens of 2-Naphthylamine
_
_
_
_
'
~
Compounds in parentheses represent hypothetical metabolites analogous to those found with other aromatic amines and azo dyes. Solid lines represent established routes of metabolism, and the broken lines are hypothetical pathwaye.
tion. This is a n important point to strehs, since in that study of Uonser et aZ., by far thc highcst tumor incidence among 2-naphthylaminc mctabolites was actually observed with bis(2-amino-l-naphthy1)sodium phosphate, which compound has probably a special significance for 2-naphthylamiiie carcinogenesis. The urinary presence of 2-naphthylhydroxylami~~e has been actually clctected in the dog (Boyland et al., 1960, 1964a; Troll and Nelson, 1961) and in man and the rabbit (Troll et al., 1965; Troll and Belni:tii, 1967). However, 2-n:iphthylamine is also N-hydroxylated in the cat in vivn aiitl mny he detected 111 the 1)lood (Uchlckc, 1963) ; the definite absence of the AT-hyclrosy t1eriv:itivc i l l t h c ui*inc of t,liis species clocs not appcitr to havc heen reported. X mctaholic. pntliwny csi>ts whicli osiclizes 2-1iaplithylli~d~oxyl~mine to 2-nit1~0~011:1~~Iitl1:~le1~~ :111(1, c.onwrwIy, nnother whicli i*educcs the hy-
414
JOSEPH C. AHCOS AND MARY F. ARGUS
clroxylamine to thc parent amine. Boyland c t al. (1964a) detected the preseiicc of 2-nitrosonaphthalene (CVII) in the urine of dogs dosed with 2-naphthylamine ; 2-nitrosonaphthalene does not appear to have been tested for carcinogenicity. Lotlikar et al. (1965) reported that total rat liver homogenates (from weanlings) reduce N-hydroxy-2-acetylaminonaphthalene to the respective acetamide. Recent results of Uehleke (1966a,b, 1967) indicate that N-hydroxylation is not unique to liver tissue but is actively carried out by the bladder mucosa of various animal species. I n view of this observation it would seem surprising that 2-naphthylamine is a t most slightly active in bladder implantation, although the enzymatic modality is present to convert it in situ to 2-naphthylhydroxylamine. Other aspects of the relationship between N-liydroxylation and carcinogenicity of the two naphthylamines complicate this picture further. As late as 1963 it was still assumed that 1-naphthylamine-which has, in comparison with the 2-isomer, very low activity by oral administration in dogs-is not metabolized to l-naphthylhydroxylamine (Clayson and Ashton, 1963). However, the following year l-naphthylhydroxylamine was detected as a metabolite in occasional animals among dogs dosed with l-naphthylamine (Boyland et al., 196411). l-Naphthylhydroxylamine is almost as active in bladder implantation in the mouse as 2-naphthylhydroxylamine (Boyland e t al., 1962a). Surprisingly, by intraperitoneal administration to rats, the former compound appears to be considerably more potent than the latter, to produce abdominal fibrosarcomas (Belman e t al., 1966; Troll and Belman, 1967) ; in fact, although these authors used exactly the same dosing schedule and route of administration as Boyland et al. (1963a), they found after 10 months a tumor incidence of only 1/15 with 2-naphthylhydroxylamine against 11/14 with l-naphthylhydroxylamine. The considerable discrepancy in the findings of the Boyland and Belman-Troll teams on the activity level of 2-naphthylhydroxylamine may be due to the difference in strains (random-bred albino by the former and Wistar by the latter) or the nature of the oil vehicles used. Nevertheless, this discrepancy could raise some doubts about the significance of topical carcinogenicity results as a basis for considering a metabolite a true proximate carcinogen. Paralleling the p a r e n t e d testing results of Belman e t al., Perez and Radomski (1965) found 1naphthylhydroxylamine to be more mutagenic than the 2-isomer. I n connection with the doubtful carcinogenicity of intraperitoneally administered 2-naphthylhydroxylamine, i t should also be noted that Boyland et al. (1964b) failed t o obtain tumors in guinea pigs which received 24 closes (20 nig./kg. body weight) of 2-naphthylhydrosyl~ininc ant1 survived for as long as 26 riiontlis ; howcvcr, c11:uiges descril)cd as
MOLECI’1,AR GEOMETRY A N D CAHCINO(>ENIC ACTlVlTY
41 5
“pnthologir:tI” WCI‘C ol)scrved i n the 1ii.cl.s a n t 1 lii(1iicys. The inactivity of thih compound w:i\ ii\criI)cd to its lwing r:zpicIIy rrducerl to 2-iiaphthylaiiiiiio 111 this +1)0rit’s. (hi(, rhoiilrl a1.o i ~ c a l lI i ( w that the N-hydroxy dcrivtitive of 2 - : ~ ~ c ~ t v l : ~ 1 ~ i i n r ~ fi,l 1raiwiiogtwtti ~ 1 ~ t ~ 1 i ~ ~ i n t l i i h s])ccics (which is refractory to tlic 1):irc‘iit :miinc~) citlrcr by oral or intrapcritoneal administration (E. C. hliller et al., 1964b), and this is regarded as an evidence supporting the view t h a t the N-hydroxy derivative is a proximate carcinogen of the pnrent amine. Regarding 2-amino-l-naphthol (CVIII)-the long assumetl proximate carcinogen of 2-naplitliylaii~ine-its prescncc in the urine of different species, susceptible and nonsusccptiblc, lias been known for some time. The claim t h a t the susceptibility of different species to bladder tumor induction by 2-naphthylamine depends on the proportion of the dose metabolized to 2-amino-l-naphthol slid excreted in the urine (Bonser et al., 1951) has been criticizcd by Arcos and Arcos (1962). However, Conzelman et al. (1963) have shown subsequently that, maintained on the same dosing schedule, dogs are murh inore susceptible to bladder carcinogenesis than monkeys; dogs excrete about 70% of the ingested amine as ortho-hydroxy derivative, whereas monkeys excrete only about 19% in t h a t form. Although earlier authors found 2-amino-l-naphthol active toward the mouse bladder, in recent testing experiments its carcinogenicity could not be consistently demonstrated (Bonser et nl., 1963; coinpare Bryan et al., 1 9 6 4 ~ ) .Moreover, in subcutaneous administration the activity of 2amino-l-naphthol is definitely low (Bonser et a l , 1952) so as to be hardly compatible with the expected activity level of a proximate carcinogen. In line with the demise of this compound from its status of proximate carcinogen, Dewhurst ( 1963) found that young rodents, which arc notoriously more suscepti1)le to various carcinogenic stimuli than adults, convert a smaller perccntagc of a dose of 2-naphthylainine to 2-amino-l-naphthol conjugates th:m do adults. Troll et 01. (1959, 196311) isolated from urine of both dog and man bis(2-amino-l-naphthyl) phosphate (CIX) and a second unidentified phosphate ester as nietaholites of 2-1ia~~htliylai~iinc. Their findings were confirmed fly Boyland et al. (1961) working with dogs only, and thc latter authors also provided a definitive proof of structure for the mctabolite (CIX) by comparing it with n synthetic sample. In R recent investigation (R:idomski et al., 1967) the presence of a siniilar diester could not be detected in the urine of (logs dosed with l-naphthylamine. Bis (2-amino-l-iiaphtliyl) phosphate has the distinction in having been regarded, up to recently, as the ultimnte carcinogenic metabolite of 2-naphthylamine (c.g., Clayson, 1964). There is justification for this
416
JOSEPH C . ARCOS AND MARY F. ARGUS
view (cf. Ratloiiibki ct nl., 1967) since, a t least in bladder implantation, metabolite (CIX) is btrongly carcinogenic, and actually appreciably more bo than 2-naplitliyIIiydroxyl~miii~ ( E3onsc.r ct nl., 1963) . Recently, l'roll nnd Iklinn~i( 1 (367) took u p the bO-ucturc dclrtniination of the :Ibovc-mciitioiictl unitlentifietl pliohpliate ester metabolite. This metabolite has been shown to be bis (2-hydroxylamino-1-naphthyl) phosphate (CX). The two N-hydroxy hydrogen atoms are probably hydrogen bonded to a phosphate oxygen; this explains the ether-solubility of the metabolite, which property may be essential for cell penetration. This compound, if its structure is confirmed in other laboratories and its carcinogenicity demonstrated by different methods of testing, may well prove to be the true ultimate carcinogenic metabolite of 2-naphthylamine. It combines, interestingly, ortho- and N-hydroxylation in one and the same structure, both features regarded a t diffcrent times a s essential for the carcinogenic activity of the parent aminc. 2. N-Hydroxylation of 4-Nitroquinoline-N-Oxide Investigations showing the carcinogenic activity of 4-hydroxylaminoquinoline-N-oxide, the tissue targets thereof, and the inactivity of 4aminoquinoline-N-oxide have been reviewed in Section III,A,6. Both microorganisms and mammalian tissues possess pathways t o reduce the parent 4-nitroquinoline-N-oxide to the hydroxylamino and amino derivatives. 4-Nitroquinoline-AT-oxide is rcduced by microorganisms in the following manner (Oliabayashi, 1962; Okabayashi and Yoshimoto, 1962) :
+
b
0
J
d-d
NH*OH
4
0
The mutagenicity of 4-nitroquinoline-hT-oxide appears t o depend on the relative rates of these pathways. For example, the compound is not mutagenic in Escherichin coli in which the entire process progresses
ra1)idly to 4-an1inoc~uinoline. On the other h m d , tlie conipoun~l is mutagenic toward Aspergillus nigw in which reduction from -NH . O H to NH, is slow; this results in an :mumulation of the former which is the mutagenic form. An interesting survey of the possible role of -SH groups which react with 4-nitroyuinoline-N-oxide in biological systems in relation to mutagenesis and carcinogeiiesis is given in the L ‘ D i s c ~ s ~ i o of n’’ the paper by Hutner et al. (1967). Invcstigations on mammalian tissue enzymes catalyzing thc convcrsion of 4-nitroquinoline-AT-oxideto the hydroxylaniino and amino f o r m have been initiated independently by two Japanese teams (Hashimoto et al., 1964; Sugimura e t al., 1965). Considerable extension t o these initial studies was given by the Sugimura group (Hoshino et al., 1966; Sugimura et al., 1966a,b). The reduction of 4-nitroquinoline-N-oxide t o 4-hydroxylaminoquinoline-N-oxide and t h e reduction of the latter to 4-aminoquinoline-N-oxide are catalyzed by two different enzymes. The enzyme reducing -NO, t o --NH.OH has been identified as the DT diaphorase in the r a t liver supernatant (Sugimura et al., 1965, 1966a,b). Typically, reduction requires NADH, or NADPH,, and it is inhibited by dicoumarol and stimulated by albumin and Tween 80. Normal liver and slowly growing hepatomas contain relatively large amounts of this enzyme, while its level is close to nil in fast growing hepatomas. The enzyme that produces 4-aminoqui~ioline-N-oxideis almost equally distributed in the mitochondria1 and microsonial fractions; NADH,- or NADPH,-generating system can serve as hydrogen donor. Although p chloromercuribenzoate inhibits tlie overall reaction, reduction does not seem to depend on the functioning of the main electron transport chain since 2,2’-bipyridyl, amytal, antimycin A, and cyanide do not inhibit thc reduction (Sugimura et al., 1965). The in vivo conversion in rats of subcutaneously injected 4-nitroquinoline-N-oxide to the --NH.OH and -NH, forms and t o 4-hydroxyquinoline-N-oxide has been shown by Hoshino et al. (1966) and Sugimura c f al. (1966b).
3. N-Hydroay Derivatives of 2-Acetylaminofiuorene, 4-Acetylaminobiphenyl, and 2-Acetylaminophenanthrene Investigations on arylhydroxylamincs aiitl their carcinogenicity began with the important diycovcry of N-ltgclt,osyl:itiori, n new nictnl)olic reus substrate action o l ) ~ . r v c t al t firht i n llir rat witlt 2-~irctylnniiiiofluoi,~,ii~ (Cr;imer e t al., 1960h). -1conqweliensiw r e ~ ~ i rof w tlie hiologjcal oxidation ant1 iecluction of nromatic amiiio and nitro derivatives was contributed by Uehleke (1965) . Varioub natural iV-hydroxy derivative+-
418
JOSEPH C. ARCOS A N D MARY F. ARGUS
hydroxamic acids-play a rolc in iron metabolism and possibly in othcr metabolic processes in microorganisms (revicwed by Neilands, 1967) ; on the grounds of the structural similarity of some of thcse compounds with purine N-oxides, quinoline-type carcinogens, and N-hydroxy urethan, they probably represent a new field of investigation for unsuspected carcinogens which may be present in the normal environment. I n the first extensive report of their finding, J. A. Miller et al. (1960) showed that the product of N-hydroxylation of 2-acetylaminofluorenc, N - (2-fluorenyl) acetohydroxamic acid, is a major metabolite of the amide in the rat. It is excreted in the urine as a conjugate in amounts which increase considerably with the time of administration. This was interpreted by them as probably being related to the progressive liver damage caused by this carcinogen. Since that time, a variety of susceptible species was found to excrete the N-hydroxy metabolite, free and conjugated, following administration of 2-acetylaminofluorene (Table XVII, Section III,C,l). The N-hydroxy metabolite is absent or a t a low level in the urine of species which are resistant or refractory. Thus, in their initial study, J. A. Miller et al. (1960) found no N-hydroxy metabolite (following 2-acetylaminofluorene administration) in the urine of guinea pigs, a species which is notoriously refractory to the carcinogenic action of this amide and to arylamine-induced cancer, in general. Similarly, in the steppe lemming (cited in J. H. Weisburger et al., 1964c), monkey, and rainbow trout, in which 2-acetylaminofluorene is inactive or weakly active, the N-hydroxy metabolite (free or conjugated) is absent or low in the urine. Also, man metabolizes, in uiuo, the amide to the N-hydroxy form. There is no record of human malignancy due to accidental exposure to the amide. The amide is also N-hydroxylated in vitro by isolated human liver microsomes (Enomoto and Sato, 1967). In view of their finding that microsomes from human liver with jaundice or with fatty changes concomitant with acromegaly do not N-hydroxylate, the increase of N-hydroxy metabolite excretion in rats may have been due t o adaptive enzyme synthesis rather than liver damage as interpreted earlier by J. A. Miller e t al. (1960). Contrary to the Millers' findings, Kiese and his co-workers reported the excretion of the iV-hydroxy metabolite in the urine of guinea pigs dosed with 2-acctylnminofluo1,cne (Kicsc nnd Wicdcinann, 1966 ; Kiese f t nl., 1966; voii Jagow f t ul., 1966). Coiisistcwt with their in vi7)o fincling, Kiese e l a l . (1966) idso olxwvecl i/t 7 1 i t ~ oi\'-liy~lrorryIation of tllc. frcc aniiiie by guinea pig liver microso~ncs.Yet, 1,otlikar et (11. (1967~)coulcl find no N-hydroxylation with liver iiiicrosoiiies from intact guinea pigs previously treated with 20-methylcholanthrene; in the rat, hamster,
moube, and rwhhit , gix~ntincwltseb i n A‘-hytlroxylalioii were not~tvlfollowing treatinelit with the hydrocarbon. Hence, further investigations will be necessary to clarify this question. N-Hydroxylation considerably enhances the carcinogenic potency of and the variety of tissue targets affected by 2-acetylaminofluorene. N(2-Fluorenyl) acetohydroxamic acid is more active than the parent arnide in producing tumors of the liver, ear duct, and small intestine by ingestion or by multiple intraperitoneal injection into adult rats of both sexes. When administered by intraperitoneal injection, the hydroxarnic acid also produced a variety of sarcomas in the peritoneal cavity. Administered orally, about 60% of the animals also developed benign tumors, and another 30% of them developed malignant tumors of the forestomach. By injection into weaiiling female rats, N- (2-fluorenyl) acetohydroxarnic acid was much more :ictive than 2-acetylaminofluorene in inducing mamniary tumors (E. C. Illiller et al., 1961). I n parallel experiments, the parent arnide was inactive toward the forestoninch and the connective tissue a t the sites of injection. The hydroxamic acid is also a uhiquitous carcinogen in other species. E. C. Miller et a l . (1964b) have studied the comparative carcinogenicity of the N-hydroxy metabolite in mice, hamsters, and guinea pigs by oral and pareiiteral aclministration. Just as in rats, the compound produces tumors a t sites of tissue contact; on oral administration, it induces tumors of the forestomach in mice and hamsters, and tumors of the small intestine in guinea pigs; by injection, the metabolite induces abdominal sarcomas in all three species. The parent nmide is inactive toward these tissues in mice and hamsters and toward all tissue targets in the guinea pig. On the othcr hand, toward the liver, mammary gland, and urinary bladder in the mouse and toward the liver in the hamster, the hydroxamic acid and the parent amitle have about equal carcinogenic activities. The generally higher activity of the metabolite toward local and systemic tissue targets is, however, surprisingly contrasted by the higher tumor-initiatory activity toward the mouse skin of orally administered 2-acetylaminofluorene upon croton oil promotion. Nevertheless, these findings provide strong evidence (with the reservations pointed out above in connection with the work of Kiese and his associates) that N-(2fluorenyl) acetoliytlroxamic :icid is a major proximate carcinogenic metabolite of 2-acetylaminofluorenc, since “the apparent inability of the guinea pig to N-hydroxylate 2-acetylaminofluorene parallels the failure of 2-acetylaminofluor~~ne to produce tumors in this species.” I n the rabbit, however, N-hydroxy-2-acetylamiiiofluorene is not more active than the parent nmide upon oral administration, and both induce tumors in the urinary tract only (Irving et al., 1967b). On the other hand,
420
JOSEPH C. ARCOS AND M A R Y F. ARGUS
the liyciroxamic wid is inuch more carcinogenic in this species thau the parent alnide when injected intraperitoncally (or intramuscularly in the form of its cupric chelate), and a high incidence of peritoneal sarcomas results, confirming its topical carcinogenicity observed in other species by the Millers' group. Peritoneal sarcomas cannot be produced by intraperitoneal injection of the amide. The potent local carcinogenic action of N- (2-fluorenyl) acetohydroxamic acid was also demonstrated in other ways. Goodall and Gasteyer (1966) obtained a 100% incidence of benign and malignant skin tumors in the rat following skin painting with this agent (as a 2% acetone solution) for 37 weeks; the first skin tumor arose a t 21 weeks. Several rats also developed distant primary tumors arising in the ear duct, mammary gland, ant1 lungs. These authors also found that a single subcutaneous
FIG.13. A possible modality of binding of N-hyclroxy-2-acetylaniinofluorrn~~ iricttil chclates to proteins and nucleic acids. (Froin Poiricr e t id., 1965.)
injection of 5 mg. of the N-hydroxy compound sufficed to induce subcutaneous sarcomas in 7 out of 9 rats in 44 weeks. I n the tumor-induction studies reviewed above, multiple injections were given. Toward the bladder epithelium in mice, however, N-hydroxy-2-fluorenylacetamide is somewhat less carcinogenic than free or AT-acetylated 2-naphthylhydroxylamine (Bryan et al., 1 9 6 4 ~ ) . Paralleling the findings with other types of locally acting carcinogens, the topical carcinogenic action of N- (2-fluorenyl) acetohydroxamic acid is roughly proportional to the length of retention a t the site. This was the conclusion of Poirier e t al. (1965) who studied the carcinogenic activities of various metal cliclates of the N-hydroxy compound in relation to their carcinogenic activities. The greater carcinogenic activities of these chelates a t the subcutaneous injection site are generally associated with a longer persistence, so that the increase of carcinogenicity due to chelation with the heavy metals appears a t first sight, solely as a matter of solubility decrease. However, Poirier e t al. also considered (cf. Furst,
MOLICCIJLAR GEORIETRY AND CARCISOGENIC ACTIVITY
421
1963) tliat tht inctal iiiay :wt a h ~1 coorc1in:iting atoni to facilitatc h i i i c l ing to proteins a i d nucleic acids and, thus, interfere with normal cell metabolism (Fig. 13). Hence, the carcinogenic activity is enhanced. Taking advantage of the prolonged retention time of N - (2-fluorenyl) acetohydroxamic acid in the chelated form, Stanton ( 1967) iiiducrtl primary bone and lung tumors in rats by local depositioii of the cupric chelate. The alterations of electron-microscopic ultrastructure of the rat liver following N-hydroxy-2-acetylarninofluorene administration have been studied by Hartniann (1965). His findings are in general agreement with reports on the effects of other carcinogens on hepatic ultrastructure. However, the disorganization of the parallel arrays of the rough enrloplnsrnic reticulum begins somewhat earlier tliaii with tlic other carciriogens so f:ir studied electroiirriicroscopically, and this ih consistent with tlic generttlly high level of carcinogenicity of this agent. N-Hydroxy-7-f~uoro-2-acetylaminofluorer~eis a urinary mctabolitc of 7-fluoro-2-acetylaminofluoi ene in the rat. The N-hydroxy derivative is considerably more active than the parent 7-fluorinated amide. It is probably the most active of all fluorene carcinogens tested to date. Administered a t the 0.01% dietary level for 10 to 15 weeks, i t produces high incidences of malignant tumors of the forestomach, small intestine, liver, and of the mammary gInn(1 ( i n females). It is also notably active toward the ear duct and urinary bladder (E. C. Miller et nl., 19662~). Thc cause of inactivity of 7-hyrlroxy-2-acetylaniinofluorene appears to be that this compouncl docs not uiiclergo inctabolic &\\’-hydroxyld t’ 1011. This niay be infcrred from thc recent finclings of Gutiiiann et al. (1967) that this compound may be converted by synthetic AT-hydroxylation t o the highly carcinogenic N - (7-hydroxy-2-fluorenyl) acetohydroxamic acid. Since 7-fluoro substitution decreabes hydvoxylation in this position (Westrop and Tophnni, 1965), one reason for the high carcinogenicity of 7-fluoro-2-acctylaminofluorene should be that a greater proportion of the total dose is N-hydroxylated than in the case of 2-acetylaminofluorenc. Howevcr, that an additional factor is involved here is readily cliscerned since synthetically obtained iV- (7-fluoro-2-fluorenyl) acetohydroxamic acid is more carcinogenic than either N - (Pfluorenyl) acetohydroxamic acid or iY-(7-hydroxy-2-fluorcnyl) acetoliydi.oxamic acid. Possible reasons for this will be discussed in the following scction in connection with Scrihner’s theoretical investigations. Again, inferring from the investigations of Gutmann et ul. (1967), 2-aminofluorenes, iV-suhstitutcd with bulky groups, have low activity or are inactive because the substituents sterically hinder N-hydroxylation. Under conditions in which 2-benzoylaminofluorene is a very weak car-
422
.JOSEPH C. ARCOS A N D M A R Y F. ARGUS
cinogen, Gutmauu ct ( I I . fouird the synthetically i\7-lylroxylalecl derivative, N - (2-fluorcnyl) benzohydroxamic acid, to be a highly potent agent; the tumor incidences were 8 and loo%, respectively. This is probably the true rationale for the observation that the ease of hydrolysis of various N-acyl-2-aniinofluorenes roughly parallels carcinogenic activity (Section III,A,4), since the rate of hydrolysis also depends on steric factors. In the same investigation, Gutmann e t al. made some highly interesting observations on metabolic peculiarities of the benzohydroxamic acid which contribute to its unexpectedly high carcinogenicity. Thus, whereas TABLE XXIII Metabolism of 2-Hydroxylaminofluorene in the Rat a
I
I
isomerization
dehydroxylation
t
t
1
\OH
acetylation
A
&&c*'"
h
HO&{o*c%
ydroxylation
H
OH "From J. H.Weisburger eta!, (l966a)
2-fluorenylbenzamicle is comparatively resistnnt to metabolic debenzoylation, the benzoliydroxamic acid is rapidly debcnzoylated to yield 2-hydroxy lamiriofluoreiic. Furthcrniorc, unlike N- (2-fluorenyl) acetohydroxainic acid (Lotlikar et nl., 1965), N - (2-fluorcnyl) bcnzohydroxamic mid is not reduced to thc coi,respontling ncylarylamine. Several reports appcarcd on the metabolic fate (Table XXIII) of N - (2-fluorenyl) acetoliydroxitmic acid in the rat (e.g., E. K. Weisburger et al., 1964; Grantham et al., 1965; Lotlikar et al., 1965; .J. H. Weisburger et al., 1966s). Moreover, Irving (1964) p.csented evidence that
2-iiitrosofluoreiie is formed from N-liydroxy-2-:icetyIxiiiiiioflnore1ic by rabbit liver microsoiiies. 2-Nitrosofluorene is also a highly potcnt, locitlly acting carcinogcn and likely a proximatc carcinogenic metabolite of 2acetylaminofluorene (E. C. Aliller e t al., 1964a). The most receiit investigations from the Millers' laboratory suggest that 2-acetylaminofluorene may undergo a sccond metabolic activation step following N-hydroxylation. Work was begun already in 1964 (E. C. Miller et al., 1964a) in a mmli for further proximate carcinogens. This culminated in the finding that the acetyl (CXI), pliosphatc ( C X I I ) , and
sulfate esters of A'- (2-fluorcnyl)acetohydroxamic acid are all more reactive than the nonesterified N-hydroxy compound toward amino acids and iiucleosicles in v i t r o (Lotlikar e t nl., 1966, 1967a; Kriek e t al., 1967; DeBaun e t al., 1967). The phosphate arid sulfate esters are more reactive than the acetoxy derivative, and they may represent the actual in wivo ester form. The in vivo reactive metabolites may also include carboxylic acid esters and 0-glucuronides. The one 0-rstci. tested for carcinogenic activity so far, N-acetoxy-2-acetyl:~minofluor~~1ie ((2x1) , is a stronger subcut:incous carcinogen than thc nonesterified A'-liytlroxy compound in :iccoid:tnce with the higher chemical reactivity in the in vitm systems (Lotlikar et nl., 1967a). i\'-Iiytlroxy nietaholiteo of otlicr corijugatctl arylaniincs have bcert dcrnoiist ra tecl to he proximate carcinogeiis. Thc respective N-hytfroxy metabolites are present in substantial amounts, mostly as glucuronides, in the urine of rats and dogs fed 4-acetylxminobiphenyl (,J. A. Miller e t al., (E. C. 1961 ') :ml in the urine of rats fed 2-~crtyl:~mi1iopheii~iiitlirr1ie Millcr et nl.. 1966a). Fefcr et al. (1967) have detectrtl the presence of 4-nitrosobiphenyl in the ivine of dogs rlohed with 4-ainiiiobiphenyl. The metabolites, A T - (4-xenyl) acetohydroxamic acid and N - (2-phenanthryl) :icctoliydi~oxnmicacid arc iiiorc potent carcinogcns t h i n the parent amitlcs. I3;v httbeiit:Ltio(>il* or ititi.:il)et*itotic:~ltxoiltr, tllcy pro(lrtc~loc:il si1rcoIii:lP : i t i t 1 :i Iiigli i i i ( * i i l t ~ i i c tof ~ iii:itiiiii:iry tuii~or..:1.1 .I. llillct. P / ( i l . , 19til ; 14:. C. 3Iillt.r tit ( { I . , I !%(j:t) . ?'lit. coiivc.i.tit)ility of iV-l~yd~~uxy-2-at~ctyla~~~i~to~,lit~n;~ritlii~eiie t o tlir 1):iwnt :iiriicle by r a t liver lioiiiogenatcs has Iwen s1ion.n (1,otlikar et nl., 1965).
424
JOSEPH C. ARCOS AND MARY B. ARGUS
4. N-Hydroxy Derivatives of Amino Azo Dycs and 4-Acetylaminostilbene Amino azo dyes and aminostilbenes follow the general pattern of other arylamines in that they are N-hydroxylated in mammalian organisms. Already in 1964, preliminary results were available illustrating that derivatives of 4-aminoazobenzene are N-hydroxylated in the r a t in vivo (J. A. Miller et u,L,1964; for the full report of this investigation, see Sato et al., 1966). Rats which parenterally received 4-aminoazobenzene or its N-acetyl, N-methyl, or N,N-dimethyl derivatives excrete in tlic urine appreciable quantities of N-hydroxy-N-acetyl-4-aminoazobenzenc, mainly as glucuronide, besides 4?- and 3-hydroxy derivatives of 4-acetylaminoazobenzene (also in conjugated form). Extension of these experiments to mice and hamsters indicates that these species follow the same metabolic pattern. Injection of 3’-methyl-4-monomethylaminoazobenzene into rats resulted in the excretion of two metabolites which were tentatively identified a s N-hydroxy- and 3-hydroxy-3’-methyl-4-acetylaminoazobenzene. Surprisingly, unlike the N-hydroxy and N-acetoxy derivatives of 2-acetylaminofluorene, N-hydroxy- and N-acetoxy-4-acetylaminoazobenzene (CXIII) and 4-hydroxylaminoazobenzene are inactive in rats either in long-term feeding, by repeated intraperitoneal injections, or by repeated subcutaneous injections as the cupric chelate of the acetohydroxamic acid form (Sato et al., 1966).
Q-N=N+”s
\
0-C-C,H,
II 0
highly active (CXIV)
The unexpectc(1 inactivity of these derivatives was the first indication that N-hydrouylation and even conversion to an N-acyloxy ester may be a necessary, but not sufficient condition for carcinogenicity and that the structure of and conjugation in thc aromatic moiety largely determines
MOLECIJ1,AR GEOMETRY AND CAIK!Ih’OCENIC ACTIVITY
425
carcinogenic acti\.ity. A’-Hydroxylation is not restricted to carcinogenic aromatic amiites (Uchleke, 1965), and, therefore, i t cannot be regarded as a metabolic stel) leacling inc\it:ihly t o carcinogenic metabolites. For cxaniplr, plirnylliycl~o~yI:i~~iinc~, l)li(~iiyl(~f Iiylliycli,osyl:iiiiitie ( 14. C. 1LIillcr et al., 1966a), aiicl N-plietiylbenzohytlroxwtiiio :kcid (Gutiiiann e t d., 1967) were totally inactive under conditions in wlticli N-hydroxy-2-acetylaminofluorene was highly carcinogenic. Yamamoto et al. (1967) tested the simplest N-hydroxyamine, hydroxylamine and, also, hydroxyurea and methoxyamine in long-term oral administration. None of these compounds appeared to have any carcinogenic effect in C3H/HeN strain mice, and rather had a lowering effect on the spontaneous tumor incidence of the strain. Hydroxylamine is a mutagen which is considered to be highly specific in acting on cytosine (Kihlman, 1966) ; methoxyamine (O-methylhydroxylamine) is reputed to be a more potent mutagen than hydroxylamine (Turbin et al., 1964). A subsequent study by Poirier et al. (1967) showed that the inactivity of the above N-hydroxy and N-acyloxy derivatives of 4-aminoazobenzene must be ascribed to the lack of an N-alkyl group. As attempts to synthesize N-hydroxy-N-niethyl-4-aminoazobenzene were unsuccessful, the 0benzoyl derivative, N-benzoyloxy-N-methyl-4-aminoazobenzene (CXIV) , was prepared. Paralleling the findings with N-acetoxy-2-acetylaminofluorene, the benzoyl ester of the N-hydroxy dye (CXIV) was sarcomatogenic locally in rats. One hundred percent tumor incidence was obtained in 9 t o 12 months by intramuscular injection of twenty-four 3.9-mg. doses. Parallel control experiments with 4-monomethylamino- and 4-dimethylaminoazobenzene, 4-benzoyl-4-monomethylaminoazobenzene,N-hydroxy-4-aniinonzobenzene, 4-dimethylaminoazobenzene-N-oxide,and benzoylperoxide yielded no tumors, while positive control groups injected with N-hydroxy2-acetylaminofluorene reached 50-6570 tumor incidence in 12 months. The carcinogenic activity of N-hydroxy-4-acetylaminostilbene in the rat has been reported by Smith and Baldwin (1962) and Baldwin et al. 41963c,d), and coiifirmctl by Aiitlerseii et aL. (1963, 1964). The only organ in which tumors were observed (following oral administration) by Bnldwin and his associates, is the ear duct gland. In these experiments, the activity of the N-hydroxy derivative appeared not greater than that of 4-acetylaminostilbene, but higher than the activity of 4-diniethylaminobtilbene. On the other hand, in the Millers’ group, Andersen et aL. found the N-hydroxy derivative to be a definitively stronger carcinogen than either 4-amino- or 4-acetylaminostilbene toward the mammary gland, forestomach, subcutaneous tissue, and small intestine in the rat. In agreement with Baldwin’s findings, the stilbene derivatives were about rqually carcinogeiiic toward the etkr tliict glands. ni iickwy et al. (1955)
426
JOSEPH C. ARCOS AND MART F. ARGUS
previously reported the carcinogenicity of 4-nitroetilhene toward the forest,omach of khe rat, and Andersen et al. (1964) suggested in this connection that forcstomnrh tissue may h a w the capacity to rcrliic~the nitro to L: liytlroxyluniiiio grou]). Andersen et al. (1964) liave tilao syntliesized the N-acetoxy and iV-acetoxy-7-fluoro derivatives of 4-acetylaminostilbene.The former but not the latter compound was somewhat more active than the N-hydroxy in inhibiting the growth of the Walker 256 tumor, which may be regarded as an indication of the relative carcinogenicities of these compounds. Smith and Baldwin (1962) were the first to report the detection of N-hydroxy-4-acetylaminostilbene in the urine of rats fed 4-acetylaminoor 4-dimethylaminostillene. The metabolic results (for detailed accounts, see Andersen et ul., 1964; Baldwin and Smith, 1965) show that 4-aminostilbene (free or N-acetylated) follows the general metabolic pattern of fully conjugated arylamines. ortho-Ilydroxylation, N-acetylation, and N-hydroxylation, as well as reduction of the N-hydroxy group occur. Following parenteral administration of N-hydroxy-4-acetylaminostilbene, Andersen et 01. detected an increase in the excretion of 3-hydroxy4-acetylaminostilbene, which they regarded as supporting the thesis that the N-hydroxy compound is an in vivo precursor of the ortho-hydroxy metabolite. On the other hand, Baldwin and Smith observed that the 4'-hydroxy derivative is the only major ring-hydroxy metabolite following oral administration of N-hydroxy-4-acetylaminostilbene. The latter finding appears to lend circumstantial support to the hypothesis of Westrop and Topham (1966a) that the 4'-hydroxy metabolites result from a rearrangement of the N-hydroxy forms. The data available a t present on carcinogenic N-hydroxy arylamines indicate that for conferring carcinogenicity upon a hydroxylamine or a hydroxamic acid by attachment of an aryl moiety alone, this moiety 1967). must have a t least a certain minimum size (cf. Gutmann et d., Hydroxylamine, phenylhydroxylamine, and the N-benzoyl derivative of the latter are not carcinogenic, although the mutagenicity of hydroxylaminc is well known. Carcinogenicity arises with an N-linked 1- or 2nxplithyl moiety, and activity is maintained and even augmented by replacing the naphthyl by a 4-xenyl, 2-fluorenyl, 2-phenanthryl, or 4-stilbenyl group. These are conjugated systems, and, therefore, the resonance in the aromatic skeleton must strongly influence the bond strength of the 0-ester linkage. Hence, the reactivity of the hydroxamic acid group (and the carcinogenic activity of these compounds) depends on the force of conjugation. The more the electrons are withdrawn toward the aromatic skeleton, the greater is the reactivity of the hydroxamic acid ester grouping toward nucleophilic reagents. Evidently, electron withdrawal, i.e., bond activation, is lower with a plienyl than with the higher
:cry1 groups. This is tlic basis for thc inactivity of ~ ~ l i c n y l l i y ~ ~ ~ o x y l : ~ i i i i i ~ ~ and N-phenylbenzohydroxaniic acid. A similar inbtance has been obser~etl with respect to the effect of the aryl moiety on the rate of hydrolybis of N-aryl iiitrogen mustards (reviewed by ROSS,1953). This was excellently demonstrated by Scribner (1967; also Lotlikar e t al., 1967a) who calculated tlic rcsonance activation energicb for certain aryl moieties, labilizing the ester bond. I n parallel with these thcoretic:tl studies, the corresponding AT-arylacetohydroxamic ebters were tested in the Millers' laboratory for sarcoinatogcnic activity in the rat undcr standardized conditions. A good correlation was found hetween tlic calculated aryl resonance activation energies and the sarcoma incidences of the acetoxy esters, which ranged in the following order: 2-fluorenyl > 4-xenyl > 4-stilbenyl > 2-phenanthryl. In this theoretical framework, the very high carcinogenic activity of N - (7-fluoro-2-fluorenyl) acetohydroxamic acid (E. C. Miller et ul., 1966a) is duo to the electronegativity of the fluorine atom, increasing tlirrcby clectron withdrawal toward tlic aromatic nucleus. The samc molccu1:ir mechanism may account for potentiation of carcinogenic activity, ill general, by fluoro substitution a t various points on an amine-linked aromatic skeleton. The unexpected finding that with an azobeneene grouping, the presence of an N-alkyl group is required for carcinogenicity of the hydroxamic acid form should now be considered. It may be speculatively advanced that because the resonance activation provided by azobeneene is too low, a n N-alkyl group is necessary to increase conjugation by the hyperconjugation increment beyond a certain thrcshold value. The influence of steric factors, which may limit the reactions involved in the metabolic activation steps, may account for the absence of carcinogenicity with N-alkyl groups longer than ethyl. The N-substituent and the azobenzene frame both may contribute in linking the molecule during the activation process t o enzyme site(s) (cf. Burkhard et al., 1962). This may account for the fact that, for an increase of chain length as little as passing t o N-ethyl groups (which are sterically less favorable than N-methyl groups), an auxocarcinogenic suhstitucnt is required in the 4'-position for reinforcing noncovalent interactions with the activatioii site (s) , if carcinogenic activity is to be maintained (Section III,B,l) .
E. FREERADICALS IN ARYLAMINE CARCINOGENESIS. INTERACTIONS WHICH APPEARTO B R NONCOVALENT WI'I'TI PROTEINS AND NIlcLIClC ACIDS I , Evidence for and the Possible Role of Soine Arylumine Free Radicals
The ESR spectronietric study of Damerau and Lassninnn (1963) on iodine coriiplexcs of boiiic aniiiio :mo dyes : ~ n dnonJ)asic, largcr molecular
428
JOSISPH C. AllCOS AND MARY F. A R G U S
sizc azo compounds lias becn bricfly mentioned in Scction II,G. Althougll the number of compounds cxamiiied was small and, perhaps, not fortunately selected for such a study, it clearly appears that there is no correlation between carcinogenicity and either spin concentration or bandwidth in their system. Nagata et al. (1966c,e) more recently observed the formation of large amounts of free radicals in solutions of 4-dimethylaminoazobenzene and of 1-amino-2-naphthol and 2-amino-lnaphthol. They proposed that these radical forms may play a role in the interaction of these compounds with DNA. Perhaps the most interesting findings by this approach have been obtained with 4-hydroxylaminoquinoline-N-oxide. At first Nagata e t al. (1966~)showed that 4-hydroxylaminoquinoline-N-oxide gives rise to a large amount of free radicals in the solid state, or in solution in water or certain organic solvents. The ESR signals are strong a t p H 11 to 12, decrease to about a third of that intensity a t p H 7 t o 8, and are absent a t pH 3. The frce radicals are produced by an oxidative process. This was convincingly demonstrated by the facts that ( a ) the ESR signal was absent when 4-hydroxylaminoquinoline-N-oxidewas dissolved in dioxane and rigorously degassed, but as soon as the solution was exposed to air, it appeared instantly, and ( b ) the signal of the free radical was quenched immediately by addition of the reducing agents, bensaldehyde and ascorbic acid, or of catalase. The temperature dependence of radical TABLE XXIV Participation of 4- Hydroxylaminoquinoline-N-Oxide Free Radical in the Oxidation-Reduction Process of 4-Nitroquinoline-N-Oxide Derivatives= N=O
I
0
0
1
0
.N/OH I
b a From Nagata et d.( 1 9 6 6 ~ ) .
1 0
MO1,ECIJLAR GEOMETRY A N D CARCINOGENIC ACTIVI'L'Y
429
foriuation hliows, iirterehtingly, :t +Ii:irl) r i s ~to :i ni:txi~iiuni :It, 30"(:. followed by it grar111:tl ciec.rcase. Howcvcr, tlic aniount of radicals present is still close to thc maxinium up to about 40°C. This free radical formed by oxidation and destroyed by reducing agents is a participant in the oxido-reduction processes of 4-nitro- and 4-hydroxylaminoquinoline-N-oxide, for which Nagata et al. proposed the scheme shown in Table XXIV. Actually, the unpaired electron is delocalized and distributed throughout the whole molecule. That this radical form may play an unusual role in the carcinogenic activity of 4-hydroxylaminoquinoline-N-oxideis suggested by an interesting report by Hozumi e t al. (1967). These authors have shown that glutathione and cysteine are oxidized in the presence of air by 4-hy(lroxylaminoquinoline-N-oxide in vitro a t pH 7 and 37°C. The pH clependence of the oxidation of glutathione by this compound parallel:, the intensity of ESR signals in the experimcnt of Nagata and his associates. There is no detectable chemical combination between the yuinoline compound and the sulfhydryl agents or any chemical alteration of the former. The role of 4-hydroxylaniinoqi~inoline-N-oxideappears to be purely catalytic. The overall reaction is dcacribeci by Hozunii et al. as
H*O
wherc RSH, RSSR, HAQO, and HAQO. are the reduced and oxidized forms of glutathione (or cysteine) and 4-hydroxylaminoquinoline-A'oxide, respectively. It is an interesting possibility that the greater carcinogenic potency of this compound in comparison with the 4-nitroquinoline-N-oxide may be related to the difference in their modes of reaction with tissue sulfhydryls, since the former, unlike the latter carcinogen, is not deactivated by chemical combination with sulfhydryl agents. An investigation with two arylamine carcinogens-which in some respects parallels that of Nagata et al. (1966d) on the presence of free radicals in benzopyrene-treated liver homogenates-was reported by Vithayathil et al. (1965). These workers observed the general appearance of a special type of ESR signal (g = 2.035 signal) in the livers (slices) of rats fed 2-acetylaminofluorene, 4-dimetliylaminoazobenzene, or thioacetamide. The plot of the intensity of this signal (relative to the normal g = 2.005 signal in the same liver samples) against the time of carcinogen administration is shown in Fig. 14. It is of interest to observe that the times of occurrence of the maxima follow the same order as the carcino-
430
JOSEPH C. ARCOS A N D MARY F. ARGUS
20
40 Days on dlel
60
80
FIG.14. Amplitude ratio of g=2.035 over g=2.005 ESR signals in rat liver slices as a funvtion of the time of administration (at 0.06% dietary level) of 2-acetyl4-dimethylaminoazobenzene (O), and thioacetamide ( V 1. aminofluorene (01, (From Vithayathil et al., 1965.)
genicities of these agents toward the liver. Administration of various drugs with no known carcinogenic activity does not bring about the appearance of the g = 2.035 signal. Both the g = 2.005 “normal)’ signal and the g = 2.035 “prccunccr” signal arc absent i n tissue from tumors induced by the azo dye. The generation of the free radicals detccted, suggested to Vithayathil et al. that the primary effect of thcse agents may be on cellular electron transport. In view of the very stnall tissue samples needed, an exploration of the possible diagnostic value of this system was planned. 2. Apparently Noncovalent Interactions of A9,ornatic Amines with D N A
The demonstration by Belman and Troll (1967) that 2-naphthylhydroxylamine brings about a lowering of the T,,, of DNA only a t pH 5, but not a t pH 7, suggests that the N-hydroxy compound reacts via the liydrogen-ion-catalyzed mcchanism described by Heller et nl. (1951), and which is considered to account for the covaleiit interactions of arylIiydroxyla~nines,in general. On the other hand, tlie T,,-lowering reaction of l-amino-2-naphthol (Troll et al., 19G3a) and of 2-amino-l-naphthol (Troll et al., 1963a; Beltnan and Troll, 1967) with DNA (salmon sperm, calf thymus, or Escherichiu coli) does not require specific p H conditions. Testing the T,-lowering effect of aminonaphthol on synthetic polynucleotides suggests that guanine is the base that reacts in DNA. Reducing agents, such as hydrosulfite, inhibit the T,-lowering effect, and this
effect is paralleled hy a strong binding of the amitlonaphthol to DNA. Aniinonaphthol in the presence of hydrosulfite neither binds to nor affects the T , of DNA. Although it is highly probable from the foregoing evidence that 2-amino-l-naphthol is oxidized to ortho-quinoneimine before undergoing interaction with DNA, the evidence advanced by Belman and Troll-that insep:irability during CsCl density gradient centrifugation demonstrates the covalent nature of binding-is not entirely convincing. I n view of the findings of Nagata et nl. (1966c,e), another possibility, which is equally consistent with inhibition by liydrosulfite is t h a t interaction with DNA involves the formation of aminonaphthol free radicals. It seems, howcver, that further work will be necessary on the entire problem of the aminonaphthol-DNA interaction as a consequence of the report of King and Kriek (1965) of their inability to observe the reduction of the T , under the conditions used by Troll e t al. and Belman and Troll. Although the exact nature of the miinonaphthol-DNA interaction remains t o be elucidated, the breakage of some hydrogen bonds in the native helical structure becomes manifest not only in the lowering of the T, (if confirmable), but also in the greater reactivity of the DNA amino groups toward formaldehyde. However, a much more sensitive system for detecting structural changes in D N A is the change in RNApriming ability (Belman et al., 1964). Interaction of 2-amino-l-naphthol with calf thymus D N A resulted in total loss of RNA-priming ability, whereas there is only about 50% loss following reaction with N-hydroxy2-acetylaminofluorene. Structural changes induced in D N A by aminonaphthol are also shown by the decrease of the rate of its hydrolysis by niicrococcal nuclease. The kinetic data indic:ite that aminonaphtholtreated D N A is more tightly bound to nucleabc (Belman and Troll, 1967).
3. I n Vitro Coinplcxing of ~-Nitroqi~ir~olinc-h -Ozide with 1)NA The quantum nicchanical calculations of Karreman (1962) on the alterations of the charge distribution of adenine by 4-nitroquinolineN-oxide have already been discussed in relation to hydrocarbon-DNA interaction, mutagenesis, and carcinogenesis (Section II,F,4) . Nagata et 01. (1966a) were the first to show that, in vitro, 4-nitroquinoline-Noside complexes with DWA by intercalation, and concluded in agreement with Karrenian’s calculations that either adenine or guanine is the principal paitnor of intcmctiotr witliii~tlie Iielis. C:iffeincb coinpetitively inhibits the intertictioil betweell 4-nitroyuiuoliiie-~~~-oxide a i d DNA. A distinct parallel was found i n their aystcm between the carcinogenicity and extent of complcxlng to DNA with $everal 4-1iitroc~uinolin~-~-oxide derivative?. These experimental findings are in good agreement with the earlier theoretical prediction of Nagata e t al. (1963b) that in the 4-nitro-
432
JOSEPH C. ARCOS AND MARY F. ARGUS
quinoline-N-oxidc-DNA complex, the quantity of charge transfer rather than the strength of the charge-transfer bonding is the determinant factor. According to Paul et al. (1967), guanine is the interacting partner in the in vitro interaction of 4-nitroquinoline-N-oxide with DNA. I n their studies, the complex proved to be quite stable, withstanding extensive dialysis without loss of complexed 4-nitroquinoline-N-oxide. There is, however, no evidence so far that covalent binding is involved. This is noteworthy regarding the previously discussed view of Belman and Troll on the nature of binding between 2-amino-1-naphthol and DNA. Both Nagata et al. (1966a) and Paul et al. (1967) have noted the similarity between the 4-nitroquinoline-N-oxide-DNA and actinomycin D-DNA complexes. The thin-layer chromatographic study of this interaction by Malkin and Zahalsky (1966) supports the intercalation mechanism, since neither heat-denatured DNA nor soluble RNA complex. There is slight interaction with synthetic, low-molecular-weight polynucleotides and histone. The great stability of the 4-nitroquinoline-N-oxide-DNA complex (as compared to the weak hydrocarbon-DNA complexes, Section II,F,3) is also indicated in Malkin and Zahalsky’s experiments by the resistance to ionic strength increase and by the lack of competitive replacement of the nitroquinoline compound by proflavine. 4. Noncovalent Interactions of Azo Dyes with Nucleic Acids and Proteins There is some indication for weak noncovalent interactions between 4-dimethylaminoazobenzene (DAB) and RNA (Marmasse, 1964) or DNA (Nagata et al., 1966e) in in vitro systems. Evidence is much more solid, however, for similar interactions between carcinogenic amino azo dyes and proteins. Szafarz and Galy-Fajou (1966) carried out a spectral study of DAB complexed in vitro with various proteins. Complexing with histones resulted in the greatest changes in the 410/452 mp absorbancy ratio in the limited series of proteins examined. Watters and Canter0 (1967) reported a careful and interesting study, using optical rotation and viscometry, on the interaction of bovine serum albumin and eighteen amino azo dyes of graded carcinogenic activities. There is a reasonably good parallelism between the structural features required for carcinogenicity nnd those for inrrease of optical rotation. Thus, optical rotation is little nffectcd by 4-nmino:~xobenzen~and its 3’-methyl Iioniolog, is nollal)ly more so by tlie N-iiiethyl aiitl N,ATdiinethyl derivatives, arid by far the highest optical rotation increase is produced hy 3’-fluoro-DAR. The results tend to suggest that such secondary valence forces (independently from covalent binding) may play a role in azo dye carcinogenesis by changing the helix content and structural rigidity of cellular proteins, thereby influencing their functional
MOLECTJLAR GEOMETRY AND CARCINOGENIC ACTIVITY
433
contributions to cell metabolism. The finding of Whitcutt e t al. (1960) that in the soluble proteins of liver from rats, which received a single oral dose of 3’-methyl-DAR1 there i s a11 immediate qualitative change of ekctrophoretir behavior in a gruul) of m l i i l ~ l vprotcin5 which do not covalently bind the dye, tends to suppor1 the view that structural clia~rgesin proteins may be brought about by direct noncovaleiit interactions. Although :izo dye interaction did not significantly affect the reduced viscosity of ovalbumin in Watters and Cantero’s system (suggesting that there was little or no change in tertiary structure), it is not known whether the changes in protein helicity are accompanied by sulfhydryl4isulfide changes such as observed by Argus e t al. (196610) with ovalbumin in the presence of water-soluble carcinogens. IV. Covalent Binding to Proteins and Nucleic Acids
A. POLYCYCLIC HYDROCARBONS AND TRICYCLOQUINAZOLINE Abell and Heidelberger (1962) reported that protein-bound hydrocarbons in mouse skin are predominantly bound t o a slightly basic fraction of soluble proteins, electrophoretically similar to the h, proteins of rat liver supernatant. I n a series of twelve hydrocarbons of graded carcinogenic activities, a good quantitative correlation was found t o exist between the extent of binding and carcinogenic activity. I n hydrocarbon-induced epithelial carcinomas and subcutaneous sarcomas, a considerable reduction of this h-like protein fraction was observed in analogy with the reduction of h-protein level in liver tumors induced by amino azo dyes and 2-acetylaminofluorene. Daudel et al. (1962) carried out a low-temperature fluorescence spectroscopic study of tissue-bound metabolites of 3,4-benzopyrene following application to mouse skin. The bound metabolites were liberated by a modification of the hydrazinolynis technique used earlier for obtaining bound metabolites of 1,2,5,6-dibenzzanthracene.Comparison of the fluorescence spectra of the metabolites with those of chrysene and 1,2benzanthracene appears to indicate that hincling OCCUI*S through both K-regions:
434
JOSEPI3 C. ARCOS A N D MARY F. ARGUS
Anthanthrene is a typical exception to the K-region hypothesis of hydrocarbon-induced carcinogenesis in that this compound, while it possesses an electronically favorable K-region and no L-region, is not carcinogenic. Daudel et al. (1960) have shown t h a t j u s t as the noncarcinogenic 1,2,3,4-dibenzanthracene-anthanthrene is bound to mouse skin proteins. Actually, the amount of tissue-bound anthanthrene was found by them to be greater than the amounts of bound 9,10-dimethyl-1,2,5,6dibenzanthracene or l0-methyl-7,8-benzacridine, both of which are potent carcinogens. Howell (1958) has shown that administration of cupric oxyacetate gives a good degree of protection against 4-dimethylaminoazobenzeneinduced hepatic tumorigenesis in the rat. Subsequently, Fare (19644 was successful in demonstrating that this protection against tumorigenesis parallels the considerable delaying by the cupric oxyacetate t o attain the maximum amount of bound dye in the liver. A similar study carried out and cupric oxyby Fare (196413) with 9,10-dimethyl-l,2-benzanthracene acetate could not demonstrate an analogous situation in mouse skin tumorigenesis. On the contrary, this fluorimetric study shows that, whereas the addition of 0.15% cupric oxyacetate to the acetone solution of the hydrocarbon does lower the binding to skin proteins, i t also accelerates the rate of appearance of the tumors. Despite the considerable body of evidence that polycyclic hydrocarbons interact with DNA as such and do not require metabolic activation for carcinogenicity, Brookes and Lawley (3964b) reported a remarkable correlation between covalent binding to DNA and the activity of six polycyclic aromatic hydrocarbons (Fig. 15). Covalent binding is not specific t o DNA, and fixation to RNA has also been observed. The DNA-bound hydrocarbon persists for a longer time than the proteinhound hydrocarbon. Goshman and Heidelberger (1966) confirmed these results and provided important additional evidence that the nature of the DNA hydrocarbon combination is, in fact, covalent binding. Among others, Goshman and Heidelberger have ascertained that the binding is not affected by treating the mice and isolating the DNA in the dark. This is a significant point toward determining the metabolic origin of this binding, in view of the observation of Ts’o and Lu (1964) that irradiation of noncovalent DNA-3,4-benzopyrene complex a t the absorption band of the hydrocarbon (above 340 mp) yields a covalently linked photoproduct. Unlike with the hydrocarbons, there is no firm evidence that covalent binding to cell constituents occurs with tricycloquinazoline. An extensive study by Baldwin et nl. (1962b) failed to detect any firm binding to nucleic acid or protein fractions in the mouse skin painted with tricyclo-
MOLECULAR GEORCETRY A N 0 C.AHCINOGICNIC AC'I'IVI'I'Y
435
quinazoline. Suhoequent work has revealed some protein-bouiicl tricycloquinazoline (Baldwin e t d.,1964b) which is, apparently, covalently hound (Baldwin e t aZ., 1965b) ; however, because of the extremely low lcvel of bound material (1 molecule of tricycloquinazoline per 4.3 X lo4 molecules of soluble protein of mol. wt. lo"), the significance of this binding for carcinogenesis is questionable. Attempts to demonstrate in vivo binding to skin nucleic acids have so far given negative results.
Iball's index
FIG. 15. Number of micromoles hydrocarbon bound per mole DNA phosphorus, divided by the dose of hydrocarbon given (in micromoles), a t the maximum cvtrnt of binding. The symbols represent the following hydrocarbons : A naphthalcnr ; A 1,2,3,4-dibenzanthracene ; 0 1,2,5,6-dibenzanthracene; @ 3,4-benzopyrene ; 20-methylcholanthrenc ; 0 Q,lO-dimethyl-1,2-benzanthracene. (From Brookes and Lawley, 196413.)
B. 4-NITROQUINOLINE-N-OXIDE A demonstration that this compound actually becomes covalently bound to cell components in its many tissue targets appears to be lacking despite much in vitro evidence of its high chemical reactivity in both the -NO, and -NH.OH forms (Sections III,A,6 and III,E,l) and the observation by Hayashi (1959) that there is a decrease of the intraepithelial -SH content following a single application of 4-nitroquinolineN-oxide. There is also qualitative in vitro evidence that, in addition to simple sulfhydryl compounds, 4-nitroquinoline-N-oxide interacts with the -SH groups of proteins (Searle and Woodhoube, 1963). Nevertheless, this interaction may well represent a detoxication mechanism rather than a facet of its carcinogenic action, since the rat liver, which is not a tissue target of 4-nitroyuinoline-N-oxide (Table XV) , contains an enzyme that catalyzes its conjugation with sulfhydryl compounds such a~ glutathione (Al-Kassab ct (11.. 1963).
436
JOSEI'II C. AI3COS A N D MARY I?, ARGUS
Just as arylhydroxylamines, in general, condense with sulfhydryl compounds in vitro to give S-aminoaryl derivatives (Boyland et aZ., 1962b, 1963b), 4-hydroxylaminoquinoline-N-oxidecould react with tissue sulfhydryls, and this may well prove to be involved in the mechanism of action. Parallel studies of the sulfhydryl levels in target tissues using the -NOz and the --NH.OH forms would be of importance. It may not be excluded, however, that the catalytic effect of 4-hydroxylaminoquinolineN-oxide free radicals in oxidizing -SH groups (Hozumi et al., 1967) plays a key role, in which case a correlation may not exist between tissue binding and carcinogenic activity of various ring-substituted derivatives.
C. ARYLAMINES AND AMINOAzo DYES 1. 2-Naphthylamine Although the early experiments of Henson et al. (1954) indicated that the bladder epithelium and the red blood cells are the only tissues capable of retaining 2-naphthylamine-14C in the rat and rabbit after iqtraperitoneal injection, Roberts and Warwick (1966b) found that tritiated 2-naphthylamine binds t o liver, kidney, and spleen of the rat. The extent of binding to different cell components in all three tissues ranked in the following order: cytoplasmic proteins > nuclear proteins >> ribosomal RNA. No binding to DNA is indicated by their results. On the other hand, in the urinary bladder of the dog, which is a typical tissue target of 2-naphthylamine, no bound metabolite was detected following feeding this agent (Brill and Radomski, 1965a). Although it may not be excluded that the fluorescence method employed in the latter study is not sensitive enough to detect low levels of metabolites or that metabolites may lose their fluorescence by tissue binding, in appearance this observation agrees with the earlier conclusion of Scott and Boyd (1953) that the carcinogenic action of 2-naphthylamine is related to prolonged physical contact rather than tissue retention. 2. dcetylaminofluorene I n arialogy with their earlier observations on amino azo dyes, Sorof et al. (1960, 1965) found that 2-acetylaminofluorene is localized in the fast h, proteins separated by electrophoresis. The results of Barry and Gutmann (1966) essentially confirm the finding of Sorof et al. despite differences in experimeiital coiiditions. Protein-bound derivatives are not detectable in hepatic tumors induced by this carcinogen, following administration of 2-acetylaminofl~orene-'~C(Sorof et al., 1965), and this is in ugreemeiit with their early finding that in these tumors there is a large decrease of the level of h, protein (Sorof et al., 1958). Although the
MULIK!ULBR GEOMETRY AND CAHCINOGENIC ACTIVITY
43 7
level of total h proteins, as a class, is considerably higher in the “minimal deviation” hepatomas than in 2-acetylaminofluorcne-induced liver tumors, tlie former hepatomas contain little or no h,-fluorenyl proteins following administration of 2-acetylaminofluorerie or its N-hydroxy derivative (Sorof et al., 1966). Following intravenous injection, 2-acetylaminofluorene becomes rapidly bound in an unextractable form to red blood cells (J. H. Weisburger et al., 1966b), but it is tightly as well as loosely bound to plasma proteins and this may represent the modality of its circulatory transport (Bahl and Gutmann, 1964; J. H. Weisburger et al., 1966b). The protein binding to liver proteins of the inactive metabolite, l-hydro~y-2-acetylaminofluorene-~~C, has been shown to occur in vitro (Nagasawa and Osteraas, 1964) and in vivo (Irving and Williard, 1964). Whereas in vitro much more bound radioactivity was observed in this study with the metabolite than with 2-a~etylaminofluorene-’~C,in the in vivo study in most tissues, radioactivity was much higher following administration of the parent amide than following administration of equivalent doses of the metabolite. Even the hydrocarbon corresponding to the aryl moiety of the amide, fluorene, binds to Iiver proteins to a notable extent when administered to rats a t high doses (Grantham, 1963), but with low doses the extent of binding is less than with the amide or the N-hydroxy derivative (Marroquin and Farber, 1965). The essentiality of protein binding for carcinogenesis is illustrated by the observations that chloramphenicol which inhibits liver carcinogenesis by 2-acetylaminofluorene (Puron and Firminger, 1965; Oster and Firminger, 1966), also inhibits the binding of the carcinogen to liver proteins (J. H. Weisburger et al., 1967b). I n view of the often-considered possibility that the antibiotic exerts its effect through the ribosomes, which arc attached to the endoplasmic reticulum membrane, it is of interest that 2-acetylaminofluorene-binding proteins are present in the microsomes (Kitagawa et al., 1966) and the level of these proteins is considerably decreased in the amide-induced hepatoma (Tanigaki et al., 1967). Binding of 2-acetylaminofluorene to RNA was reported by Marroquin and Farber (1962, 1965) and confirmed by Williard and Irving (1964), E. C. Miller et al. (1964a), and Irving et al. (1967a). E. C. Miller et al. (1964a) have a150 shown that the level of binding to RNA of 2-nitrosofluorene, 2-fluorenylhydroxylamine, and N - (2-fluorenyl) acetohydroxamic acid is 2-4 times greater than tlie kvel of binding of tlic parent aiuicle. Binding of 2-acetylaminofl~oreiie-’~Cto rat liver RNA is wveral times higher than to liver RNA of guinea pig$, hamsters, atlid cotton rats (Marroquin and E’iirl~er, 1965) . Nuclcur riboson1:~1RNA and cytoplasmic (soluble) RNA are labeled to :m equal extent and their specific activities
438
JOSEPH C. ARCOS AND MANY I?. ARGUS
are about 3 times higher than that of ribosomal RNA; the pattern of labeling suggests binding to pre-formed RNA rather than incorporation during synthesis (Henshaw and Hiatt, 1963). Investigations by Irving et al. (1967a) have shown that unlike the above carcinogenic fluorene compounds, injection of the noncarcinogenic metabolite l-hydroxy-2-acetylaminofluorene-14C does not result in any binding of radioactivity to rat liver RNA. In a single experiment, 2acetylaminofl~orene-'~Cwas found to bind to liver RNA of the rabbit, and in repeated experiments N - (2-fluorenyl) acetohydroxamic acid-14C was noted to bind to liver RNA of the but not 2-acetylaininofl~oreiie-~~C guinea pig. Neither 2-acetylaminofluorene nor its N-hydroxy derivative are hepatocarcinogenic to these species; however, in all cases the extent of binding was notably low, only about 30% of that found in the rat liver. Experiments with the acetohydroxamic acid labeled with 14Cin positions 9 or 1' indicate that no deacetylation occurs prior to binding to RNA. Although Henshaw and Hiatt (1963) could not find clearly demonstrable labeling of rat liver DNA following intraperitoneal injection of radioactive 2-acetylaminofluorene, significant specific radioactivity in this DNA could be demonstrated by Williard and Irving (1964) following administration of 14C-labeled 2-acetylaminofluorene or its N-hydroxy derivative. Binding of 2-a~etylaminofluorene-~~C to rat liver DNA was fully confirmed by Sporn and Dingman (1966) ; there is no binding of 14C-20-methylcholanthrene to DNA of this organ which is generally not a target of the carcinogenic action of the hydrocarbon. This stands in interesting contrast with the investigations of Brookes and Lawley (196413) (see Section IV,A) showing that 20-methyl~holanthrene-~~C binds appreciably to DNA of the mouse skin which is a highly sensitive tissue target for the hydrocarbon. Binding to RNA and DNA may not be the exlusive mechanism by which 2-acetylaminofluorene metabolites alter cellular information transfer in the target tissues. This is suggested by the finding of Barry e t al. (1967) that after a single intraperitoneal injection of 2-acetylaminofluorene-14C to rats the carcinogen becomes extensively bound to histones in the liver.
3. Amino Azo Dyes Following the well-known classic demonstration in 1947 by the Millers of the binding of amino azo dyes to liver proteins (e.g., reviewed by E. C. Miller and Miller, 1952), the cytoplasmic h-protein components of this combination have been extensively investigated by Sorof and his associates (e.g., Sorof e t al., 1963). Freed and Sorof (1966) have provided evidence that the h, proteins function in normal cells as met,abolic regulators.
MOLECULAR GEOMETRY A N D C A R C I S O G E S I C ACTIVITY
439
Isolated h, protein fraction drongly inhibited the growth of L-strain mouse fibroblasts in suspension tissue culture, and the inhibition of cell multiplication is reversed by rcnioval of the h, proteins. The inhibitory fraction centered a t the slow h, proteins has been recently identified as arginase (Sorof e t al., 1967). Protein-bound dye has also been found in the livers of rats which received the N-oxide of 4-tli1nethylaminoazobellaene (DAB) orally (Terayama and Orii, 1963). Protein-bound amino azo dyes are present in both cell targets in rat liver tissue-the parcnchynial and the bile duct cells (DeLamirande, 1964). The amiiio acid composition of the peptide segment to which the dye is bound was studied following 3’-methyLDAB adininistration and alkaline hydrolysis of the total liver homogenate by Prodi (1963). A simi1:irly oriented and very careful investigation has been carried out by Ketterer et 01. (1967) (following DAB administration) on electrophoretically separated, dye-binding protein preparations submitted to Pronase digestion. Bakay and Sorof (1964) have investigated a small dye-bound, salinephosphate extrartable, soluble, nuclear protein fraction and found that it exhibits electrophoretic properties similar to the cytoplasmic h proteins ; also these nuclear proteins are markedly reduced in dye-induced liver tumors. Dijkstra and Griggs (1967) studied the binding in the rat liver of 3’-methyl- and 2-methyl-DAB to the acid-insoluble nuclear proteins of the chromatin and extrachromatin fractions. The amount of bound 3’-methyl-DAB was significantly higher than the amount of bound inactive 2-methyl isomer, and this differential binding was specific to the chromatin fraction. That dye binding to nuclear proteins may be related to alteration of repression and derepression of gene function is more specifically suggested by the finding of Rees and Varcoe (1967) that, in vivo, histones in the rat liver bind administered, tritiated DAB. The distribution of protein-bound dye in subcellular fractions of thr rat liver, following oral administration of 3’-methyI-DAB, was studied ill inore recent year$ by Y:imnda e t al. (1963). They have reported that a consistently high concentration of protein-bound dye is present in the ribosome fraction (separated by deoxycholate treatment) throughout the whole period of dye administration. On the other hand, J. C. Arcos and Arcos (1958) found previously that practically all the bound form of this dye localized in the deoxycliolate-soluble membrane fraction of the microsomes, and the amount of membrane-bound dye had a sharp maximum a t 2 weeks. This is the same pattern as was noted for whole homogenates (reviewed by E. C. Miller and Miller, 1952). T h a t dye binding in the microsomes is preferentially to the lipoprotein membrane fraction is consistent with the mnrkcd tlcpression of activities of meml,rane-localized,
440
JOSEPH C. ARCOR AND MARY F. ARGUS
microsoinal drug-iiirt:tt~olizirlg riizymcs hy varioiis inicro~oiiic-l,iiitljng amino azo dyes, irrespectii-e of thcir carciuogenic activities (Baldwin and Barker, 1965). Data OII t l i t b inc,ol.l”)i’:itioii iiilo RNA of 1 -co:tr.l)oii f r a y i i i w t h , origitiatiiig from :~niiiio: ~ Z O dyc N-iiietliyl groiil)s, arc 1)id);ibly iiot grmume to the meclianisni of carcinogenic action. Roberts and Warwick (196613) used DAB tritiatcd in the “prime” ring to study the time course of binding to protein, RNA, and D N A in different tissues of rats and guinea pigs. A high level of radioactivity was noted in the livcr RNA from albino rats; the label was also detectablc in guinca pig liver RNA although the level a t the maximum time of binding was a t most one-sixth of t h a t in rat liver RNA. Binding to DNA was comparatively very low in these experiments. Actinoniycin D, which depresses the incorporation of orotic acid into ribosomal RNA does not inhibit the binding of tritiated as with 2-acetylDAB t o RNA (Roberts and Warwick, 1 9 6 6 ~ )Hence, . aniinofluorene-14C (Henshaw and Hiatt, 1963), binding of DAB is to pre-formed RNA. Persistent binding of amino azo dycs to r a t liver DNA was reported simultaneously by Warwick and Roherts (1967) using DAB (tritiated in the “prime” ring) and 1)y Dingmsn and Sporn (1967) using DAB (I4C-labeled in the ‘Lprime”ring or tritiatcd in the amine ring) and the 2- and 3’-methyl derivatives (ring tritiated) . Dingnian and Sporn reported that binding of thc highly active 3’-methyl derivative is 6 times greater than binding with the comparatively inactive 2-methyl isomer, and 9 times greater than binding with the noncarcinogenic, radioactive, 3’-trifluoromethyl derivative. High dietary riboflavin protects against binding. Thus, the bound metabolitc probably possesses a n intact azo linkage. The pattern of DNA labeling with the different radioactive dyes suggests that covalent binding of D N A to both rings may occur. Burkhard et al. (1962) reached an analogous tentative conclusion regarding the binding of DAB derivatives to liver protcins.
4. Oxidation of o-Anainophenols to o-Quinoneimines As a Possible Activation for Binding. Mechanisms of ( ovalent Binding of hT-llyd?.ol?/ Arylamines and Their Esters to Cellular iYucleophiles Nagasawa and Gutmaiin (1959) and Nagasawa et al. (1959) reported that o-aminophenol, 2-amino-1 -fluorenol, and 3-hydroxy-4-aminobiphenyl are oxidized by cytochronie c and cytochrome oxidase to indophenols and isophenoxazones. These oxidative dimerizations pass through highly reactive intermediates, thc corresponding o-quinoneimines (Gutmann and Nagasawa, 1960). Addition of bovine serum albumin and 2-amino-1 -fluoreno1 to the cytochrome c plus cytochrome oxidase system leads to cxtensive protein
hlOLECULAR GEOMETRY A N D CAHCINOCENlC ACTIVITY
44 1
tinding of the transitory oxidation intcrmecliatc, 2-i1iiino-l,2-fluorenoquinone (Nagasawa and Gutmann, 1959). Synthetic 2-iniino-l ,a-fluorenoquinone readily combines with serum albumin nonenzymatically in an in vitro system (Gutmann and Nagasawa, 1960). These findings appeared to provide experimental support for the view (Gutmann e t al., 1956) that quinoneimides and -imines derived from the phenolic intermediates of %aminofluorenc, in particular l-hydroxy-2-aminofluorene, may play a role in the protciii 1)incling and carcinogenicity of 2-acctylaniinofluorenc. 1-Hydroxy-2-acctylaminofluorene appears to be gencrated metabolically from 2-acetylaminofluorene via N - (2-fluorenyl) acctohydroxamic acid (.J. A. Miller et al., 1960), and in in vitro experiments, 1-hydroxy-2-acetylaminofluorene binds to liver proteins much more extensively than 2acetylaminofluorene (Nagasawa and Osteraas, 1964). However, an in vivo study (Irving and Williard, 1964), which is more germane to the actual process of carcinogenesis, indicated more protein binding in most tissues following intraperitoneal administration of 2-a~etylaminofluorene-'~C than following injection of equimolar amounts of the labeled 1-hydroxy metabolite. Furthermore, both o-hydroxy metabolites of 2-acetylaminofluorene are virtually inactive as carcinogcns (Section 111,A,4). For thcse reasons it appears unlikely that o-c~uinoneimincsplay a role in the carcinogenicity of 2-acetylaminofluorene. It is possible, however, that o-quinont~imincintermediates do play a role in carcinogenesis by other arylaminrs. Thc interactions of these interrnediatcs, which are assumed to result in covalent binding to cellular nucleophiles a t it position metn to the amino group (J. A. Miller e t al., 1960), may hc exemplified as follows:
B
@ \
/
S-R
& IOP
R-S:H
\
/
0
I t is still a reasonable assumption a t prescnt that 2-ai~iiiio-l-1i:~plltlio] and/or its bis-phosphate ester is one of the proximate carcinogens of
442
JOSEPH C!. ARCOS A N D M A R Y F. ARGUS
.
2-naphthylamine (Section III,D,l) Morcover, there is evidence that tryptophan metabolites bearing an o-hydroxy group are causative agents in spontaneous bladder cancer (Section III,A,5). The interaction of these with their target tissues may involve the above mechanism following metabolic activation by oxidation to the respective o-quinoneimines. Another reaction in which proximate carcinogens behave as arylating agents is the condensation of arylhydroxylamines with sulfhydryl compounds to give aminoaryl mercapturic acids (Boyland et aZ., 1962b), as exemplified with 2-naphthylhydroxylamine and N-acetylcysteine: OH
NH
a
S--C,H,O,N I
I
HS--C,H&”,
NH
c
Similarly, reaction of phenylhydroxylamine with N-acetylcysteine gives the corresponding o- and p-aminophenyl mercapturic acids. The same mercapturic acids have been detected in the urine of animals treated with the parent amines (Boyland et al., 1963b), and it is likely that these mercapturic acids arise in vivo subsequent to the formation of N-hydroxy metabolites. Boyland et al. (196313) have proposed that N-linkage of the sulfhydryl compound is a possible intermediate in mercapturic acid formation. I n fact, they found that 2-naphthylhydroxylamine, and arylhydroxylamines, in general, react readily with sulfhydryl compounds in neutral solution a t room temperature (cited in Boyland, 1963). On the other hand, the direct formation of a ring-linked nucleophile would require catalysis by hydroge? ions in order to generate, by a Bamberger-type rearrangement, the clectrophilic o-quinolimide ion (Heller et al., 1951). The rearrangement from N-linked to ring-linked acetylcystcine appears to parallel a tnetabolic pattern in which N-hydroxyarylamines are the metabolic precursors of the o-hydroxyarylamines (J. A. Miller et al., 1960). In view of the discovery by the Millers and their associates that the N-acyloxy derivatives of arylamines are more carcinogenic and also more reactive than the N-hydroxy derivatives toward proteins and nucleic acids, it is not clear whether activation of the latter by in vivo esterification is a mandatory step preceding binding to cell structures and carcinogenesis. In fact, arylatioii of cell components by N-hydroxyarylamines could occur in vivo, without previous esterification, in lowpH-gradient regions following the h3’drogen-ion-catalyze~ Bambergertype rearrangement of :irylhydroxylamines described by Heller e t nl. (1951). This is exemplified in Table XXV. The importance of low pH
MOLECULAR G E O M E l R Y AXD CARCIXOGEK I C AC'I'LVI'TY
443
TABLE X X V Hydrogen-Ion-Catalyzed Reaction of 2-Fluorenylhydroxylaine with a Cellular Nucleophile
t
\*
,S'R
for arylation is dramatically illustrated by esl)erinients 011 the in vitro interaction of arylhydroxylamines with DNA. Kriek ( 1965) reported that 2-fluorenylhydroxylamine interacts with DNA and alters its spectrum a t pH 5, but not a t p H 7. Similarly, Belnian and Troll (1967) found that 2- and especially 1-naphthylhydroxylaniine lower the T, and modify the spectrum of DNA following interaction a t pH 5, but not a t neutrality. Considerable effort has been devoted recently to the study of the structure of protein- and nucleic acid-bound forms of acyloxy arylamines. Not only are these esters more carcinogenic and chemically more reactive than the parent N-hydroxy compounds, but what is of particular importance is that, unlike the latter, they rcact with cell components a t neutrality. Lotlikar et ul. (1966, 1967:i), E. C. Miller e t nl. (1966b), and Poirier e t ul. (1967) have shown tli:\t N-acetoxy-2-acc~tyl:i1;iinofluorenc and N-benzoyloxy-4-monomethyl:~1tii11o:tzo~~e1iz~1ic react readily in vitro a t pH 7 with proteins, RNA, and DNA to form niaci~omolecular bound dye. Under similar experimental conditions five nuclcophilic components of proteins and nucleic acids (tyrosine, tryptophan, cysteine, methionine, and guanosine) react with the above two N-acyloxy compounds to form polar, bound carcinogens. No reaction occurs a t pH 7 with sixteen other
444
JOSEPH C. ARCOS AND MARY 11’. ARG‘CTS
ainino acids, nor with thyinicline, cyticlitie, ancl uricliiic. With N-:hcctoay2-acetylaminofluorene (Lotlikar et al., 1967a), but not with N-benzoyloxy-4-monomethylaininoazobenzetie (Poirier e t al., 1967), adenosine gave about 4% as much reaction as guanosine. In accordance with the lower Carcinogenic potency of N-acetoxy derivatives of 4-acetylaminobiphenyl, 4-acetylaminostilbene, and 2-acetylaminophenanthrene, these derivatives are less reactive than the more carcinogenic N-acetoxy-2acetylaminofluorene toward methionine and guanosine in vitro (Lotlikar e t al., 1967a). The reaction of N-acetoxy-2-acetylaminofluorene with guanine in DNA and RNA in vitro a t pH 7 causes a marked increase in absorption from 280 to 320 mp (E. C. Miller e t al., 1966b). These authors confirmed the observation of Kriek (1965) that N-hydroxy-2-acetyla1ninofluorene does not react a t neutrality. Unlike the carcinogenic alkylating agents, which are known to alkylate the 7-nitrogen atom of guanine in nucleic acids in vivo or in vitro (e.g., reviewed by E. C. Miller and Miller, 1966; and by J. A. Miller and Miller, 1966), N-acetoxy-2-acetylaminofl~1orene arylates guanine (as guanosine) in vitro in the 8-position. The site of arylation in guanine (in RNA and DNA), in vitro and in vivo, is invariably the 8-position (De Baun e t al., 1967; Troll and Rinde, 1967). Kriek (1965) assumed that, in the reaction of 2-fluorenylhydroxylamine with guanine derivatives a t pH 5, substitution occurred a t the same position. Expectedly, reaction with N-acetoxy-2-acetylaminofluorene brings about drastic alteration in the T,,, and the functionality of DNA. Troll and Rinde (1967) found that DNA treated for as little as 1 minute loses as much as 50% of its RNA polymerase priming activity, and there is complete loss after 1 hour, as well as lowering of the T,. In elegant experiments, Lotlikar e t al. (1966) and Poirier et al. (1967) in the Millers’ group demonstrated that both N-acetoxy-2-acetylaminofluorene and N-benzoyloxy-4-monomethylaminoazobenzenereact with methionine in an essentially identical way. The detailed mechanism proposed by Poirier et al. (1967) to account for the reaction of the azo dye with methionine is given in Table XXVI. The reaction products of both carcinogens with methionine have been characterized as the 3-methylmercapto derivatives. These same derivatives have also been detected as alkaline degradation products of liver proteins from rats which were fed 2-acetylaminofluorene or 4-monomethylaminoazobenzene (MAB) (Scribner et al., 1965; De Baun et al., 1967). 3-Methylmercapto-MAB has also the distinction of being the artifactual constituent isolated by the alkaline digestion procedure from the liver of rats fed MAB; this mercapto derivative was incorrectly assumed earlier to be 4-dimethyl-
MOLE,ClJL.\R (;EOMETHY A N D CAltCINOGENIC ACTIVITY
TABLE XXVI Possible Mechanisms for the Reaction of N-Benzoyloxy-N- methyl-4-aminoazobenzene (N-Benzoyloxy-MAB) with Methionine a
3-Methylmercapto-MAB' a From Poirier el al. (1967).
b MAR
7
4-monomethylaminoazobenzene .
Homoserine lactone
445
446
JOSEI’H C. A R C 0 8 AND M A N Y 14’. ARGUS
aminoazobenzerie (DAB) and, thus, led to the belief that MAB unclergoes niethylation to DAB in vivo (Section 111,C15). There is significant evidence, a t least for the amino azo dyes derived from DAB that much of the dye is attached to the protein by means of a methionine moiety. This is consistent with the following observations: ( a ) the facile release of the dye from the protein upon treatment with alkali a t room temperature, but not by ethanol, lauryl sulfate, phenol, 01‘ hot trichloroacetic acid; ( b ) treatment of the liver homogenate in 957% ethanol a t 60°C. greatly diminishes the amount of 3-methylmercaptoMAB that can be obtained by subsequent alkaline digestion from the livers of rats admjnistered DAB or MAB; this is presumably due to demethylation of the sulfonium derivative hy the hot ethanol trcatment to an alkali-stable thioether (Scribner et al., 1965) ; (c) the livers of rats administered MAB together with m e t h i ~ n i n e - ~but ~ s , not with cystine-”S, yielded 35S-labeled 3-methylmercapto-MAB (Scribner e t al., unpublished, cited in E. C. Miller and Miller, 1966). Funakoslii and Terayama (1965) investigated thc reaction between 3-hydroxy-4-amino~tzobenzcne or 3-hydroxy-MAB and amino acids or ulkylamines, on the grounds that the o-quinoneimine metabolically generated via the o-hydroxy derivative might be responsible for protein binding. I n the same report, a spectral study of the natural polar dye suggests that the dye moiety does not possess a phenolic hydroxyl group, a t least in the free state. The difficulties encountered in investigating the presence of a phenolic group are discussed by Terayama (1967). In the in vitro reaction of the above two azo dyes with lysinc, histidine, and tyrosine, the a-amino and carboxyl groups of the amino acids do not seen1 to be involved, and the 3-hydroxy group in the dye moiety is preserved (Funakoshi and Terayama, 1965). Thus, in view of the observation with thc natural polar dye, such reactions are unlikely to occur in vivo. In the experiments of Terayama and his associates with the natural polar dye, enzymatic and alkaline hydrolysis yielded four fractions of polar dyes (Terayama and Takeuchi, 1962; Higashinakagawa et al., 1966). Subsequently, Higashinakagawa et al. (1966) and Terayama (1967) detectcd 10 conthe presence of sulfur in their polar dye fractions. Their results alL firm the conclusions of Scribner et al. (cited in E. C. Miller and h/lillcr, 1966) in that methionine-’W, but not cystinc-’Y3, is overwhelmingly incorporated into the polar dye. However, they assigned a different position of binding of the methionyl group to thc dye. I n fact, they found that the polar dye in three out of the four fractions contains a aecondary amino group since it could be methylated by dimethylsulfatc. Furtherinow, comparison of the spertm of the polar dye and of 3 - l n r t h y l - D ~ R
suggested the a l w n c e of a substituent in the 3-position in the polar dye. The latter point ~ ' R Sthen more firmly ascertained by oxidative degradation (liy I,'.roxytrifliioroaretic acid) and siilisequent, reduction (by SnCl,) of llic polnr (lye, which yiclcled p-pheiiylc~ncdiamiiie.Other experiments with MAB and DAB bearing 14C-methyl groups showed t h a t the N methyl carbon of the former dye is completely retained in the polar dye, whereas half of tlie N-iiicthyl carbon of the latter dye is lost during protein binding. Hence, since the polar dye has a secondary amino group in the 4-position and the amine ring is unsubstituted in the 2-, 3-, 5 - , and 6-positions, tlie following formula was assigned to thc polar dye:
Other possible structures for the polar dye were proposed by Terayama, Matsumoto, and Higashinakagawa (cited in Terayama, 1967). The problem of the conflicting assignments of the position of substitution in the polar dye remains unresolved, a t present. However, Lin et al. (1967) concluded in an ingenious study that. the polar dye contains the methyl group of the administered MAB in an intact form. These investigators have prepared labeled MAB using a mixture of CH,I-3H and CH,I-14C. They found that the dyc administcred to the rats and the polar dye isolated from the liver had very similar ?H/I4C ratios, indicating that no hydrogen in the methyl group was replaced by a substituent group. I n tlie discussion of their data, Lin e t nl. implied that the p-phenyleriediamirie obtained by Higashinakagawa e t nl. may have resulted from the removal of the methionine side chain by the reducing agent. The reactivity of the N-hydroxymethyl drrivativcs formed a s probable intcrrnediates in the oxidative tlemethy1:ition of tlie dyes continued to attract interest. It 1ias been known for many years t h a t hydroxymethyl groups combine with rcactive C H and N H groups in proteins by a Mannich base type of binding (E. C. Miller and Miller, 1952). Roberts and Warwick (1963) sliowcd more recently that 4-aminoazobenzene in the presence of formaldehyde binds covalently also to RNA and D N A in vitro. With cytosine derivatives, 4-aminoazobenzene (with two molecules of formaldehyde) undergoes Mannich-type condensation a t two points (with the pyrimidine amino group and the ring N, atom) so as to form a triazine ring structure, stable in a wide pH range around neutrality (Robcrts and TV:irwirk, 1966a).
448
JOSEPH C. ARCOS AND MARY F. ARGUS
N O T E A D D E D I N PROOF: ( 1 ) 1’0 Section II,AJ. The full report on the carcinogenic activity of 5-methyl-l,2,3,4-dibenzanthracene (XXI), also known as 10-methyldibenz[a,c]anthracene has appeared [A. Lacassagne, N . P. Buu-Hoi, and F. Zajdela., Eirrop. ,I. Cancer 4, 123-127 (1968)l. Irpon suhrutaneous injection into tiiicr, this cornpoitiid proditc:cs local sarwnii\s w i t h a m t w i I:il,c$nt pmiocl of 250 days; the noiiniuthylatcd parenl compound is itmtivc i i n tlor i(Ii~iit,ii*:ilc*oiitliiions. A. Lacassagne, F. Zajdela, N. P. Buu-Hoi, 0. Chalvct, and G. €1. Daub [Intern. J. Cancer 3, 238-243 (1968)l have investigated the carcinogenicity of fourteen mono-, di-, and trimethylated 3,4-benzopyrenes. Many of these homologs are distinctly more active than the nonsubstituted parent compound. There is an approximate correlation between carcinogenic activity and the calculated electronic charge of the respective K-regions. I t is predicted that introduction of more than three methyl groups will lead to loss of activity. D. Lavit-Lamy and N. P. Buu-Hoi [Bull. SOC. Chim. France, pp. 2613-2619 (19S6)l have shown that the compound believed to be 1,2,3,4-dibenzopyrene (XV) is, in fact, 3,4,6,7-dibenzofluoranthene,also known as dibenso [a,elfluoranthr~nc.The true 1,2,3,4-dibenzopyrene, also known as dibenso[a,llpyrene, which was synthesized by an unequivocal route, has now been shown to be a potent sarcomatogcnic agent (Iball sarcoma index: 82) by subcutaneous injection in XVII nc/ZE mice [A. Lacassagne, N. P. Buu-Hoi, F. Zajdela, and F. A. Vingiello, Naturwksew9mjten 55, 43 (1968)l. That the problem of the endogenous transformation of naturally occurring steroids to carcinogens cannot yet be ruled out is suggested by the discovery of the potent carcinogenicity of ll,l2-dimethylcyclopentano[alplienanthrene and the 11,12methoxy compound [cited in F. Homburger, Science 161, 190 (1968)l. T. Arata, S. Tanaka, and C. M. Southam [J. Natl. Cancer Znst. 40, 623-627 (1968)1 have shown that the halogenated nucleosides, iododeoxyuridine and fluorodeoxyuridine, and also cliloramphenicol produce a statistically significant doubling of 20-methylcholanthrene-induced skin papilloma incidence in mice. This contrasts with an earlier study [H. V. Gelboin and M. Klein, Science 14& 1321-1322 (1964)l showing that another agent interfering with DNA, artinomycin D, inhibits skin tumorigenesis by 9,10-dimethyl-1,2-benzanthracene. (8) To Sections II,A,2 and II,B. An exhaustive review on chemical carcinogenesis by hydrocarbons and other agents using newborn mice and rats has been given by B. Toth [Cancer Res. 28, 727-738 (1968)l; F. J. C. Roe, R. L. Carter and W. H. Percival [Brit. J. Cancer 21, 815-820 (1967)l reported on carcinogenesis in newborn rabbits induced by 9,10-dimethyl-1,2-benzanthracene.A good survey on “Chemical and Environmental Carcinogcnesis in Man” has been made by D. B. Clayson [Europ. J. Cancer 3, 405-415 (1967)l. (3) To Section ZZ,DJ. The absorption, distribution and cxcretion of 20-methyladminischolantlirene, Q,lO-dirnrthyl-1,2-bcnsunthraccnc and 1,2,5,6-dibcnzantItr~~cne tered by stomach tube t,o rats has been studied by P. M. Daniel, 0. E. Pratt, and M. M. L. Prichard [Nulure 215, 1142-1146 (1967)l. Much of the absorbed carcinogen is taken up and retained for a long period of time by the body fat, but there is only little carcinogen in the brain in which tissue thc lipids are mainly polar. The extensive storage of carcinogen in fat adjacent to the mnmmary gland could explain the systemic specificity of these agents toward this organ. (4) To Section II,D$. P. Sims [Bfochem. J. 105, 591-598 (1967)l and P. Sims and P. L. Grover [Nature 216, 77-78 (1967)l continued to investigate the metabolism of 9,10-dimethyl-1,2-benzanthraccne by rat liver homogenates and the conditions of
MOLECI'L.iR
(iEOMETRY A N D CARCINOGENIC ACTIVlTY
449
animal age and dict influencing this metabolism. D. N. Wheatley and M. S. Inglis [Brit.J . Cancer 82, 122-127 (1908)l found that, in contrast t o the potent mammary t,umor inducing properties of 9,10-dimethyl-1,2-benzanthracene,the 9- or 10-hydroxymethyl derivatives induced tumors in only an occasional animal, and the 9,lOhishydroxymethyl derivative was inactive when given by stomach tube t o SpragueDawlcy rats. This concurs with the previous results of E. Boyland and P. Sims [Intern. J . Cancer 2, 500-504 (1967)l working with C57 black mice and subcutaneous administration, on the relative inactivity of these hydroxy metabolites. W. Levin and A. H. Conney [Cancer Res. 27, 1931-1938 (196711, P. H. Jellinck and B. Goudy [Biochem. Phnimacol. 16, 131-141 (196711 and P. Sims and P. L. Grover [Brit. Empire Cancer Campaign 45, 18-19 (196711 investigated the effect of pretreatment of the animals with polycyclic hydrocarbons on the subsequent It appears that although pretreatmetabolism of 9,10-dimethyl-1,2-benzanthracene. ment, in general, increases metabolism, it alten the pattern of metabolism from sidecalrain to ring hydroxylation. Since the hydroxymethyl metabolites are active in inducing adrenal necrosis, it is likely that this shifting of metabolic pattern is responsible for the protective effect of hydrocarbons and other compounds against adrenal necrosis by 9,lO-diinethyl-l,2-benzanthracene. A study of the structure-activity relationships of flavones, flavonones and chalcones t o induce increased 3,4-benzopyrene-hydroxylaseactivity in the liver and lung of the rat has been carried out by L. W. Wattenberg, M. A. Page, and J . 1,. Leong [Cancer Res. Za, 934-937 (196S)I. One of the very rare instances in which polpcyclic hydrocarbon administration brings about inhibition rather than induction of a microsomal mixed function oxidase is the basis of the finding of C. HochLigeti, M. F. Argus, and J. C. Arcas [ J . Null. Cancer Inst. 40, 535-549 (1968)l that simultanrous administration of 2O-methylcho1,znthrcne inhibits hepatic tumorigenrsis by dimethylnitrosamine. Consistcnt, with this is thr sithsrqurnt ohsrrvation by N. Venkatesan, J. C. Arcos and M. F. Argus [Life Sciences 7, 1111-1119 (1968)l that 20-methylcholanthrene is an inhibitor of the dfmethylation and incrrases the LD,, of dimethylnitrosamine. Also other aromatic polycyclics bring ahout this inhibition which appears to depend on the molecular size of the hydro(-arbon. The inhibition is possibly gene-mediated since 20-methylcholanthrene in vilro, unchanged or after metabolism, does not inhibit the demethylating activity of microsomrs. Study of the biliary metabolism of intraperitoneally injected tricycloquinazolinc by R. W. Baldwin, J. A. Nilrolic, H. C. Palmrr, and M. W. Partridge [Bioehem. Pharmacol. 17, 1349-1363 (1068)I showed the prrsence of the 1- and 3-hydrosy metabolites in the urine and feces; the major metabolites, however, were unidentified polar compounds. Mt~tabolim of Y"tricgc1oquinazolinc in the mouse skin yields all four monohgdrosy dcrivativcs tR. W. Baldwin, M. Moore, J. A . Nikolic, and M. W. Partridge, Biochpna. P h a i m a c d . 17, 1365-1375 (1968)l. A small amount of radioactivity was prescnt in the skin as strongly bound conjugates to protein. This radioactivity was liberatrd only by drastic hydrolytic conditions. (6) To Section II,P. R. Franke and M. Biichncr [A& Biol. Med. German. 19, 1047-1051 (1967)I csrrird out an invrstip:ition on t.he soluhilimtion of pgrcnc and 3.4-I~i~iizopvrenr1 y 1lliin:ui srruni :illwinin, niitl c : i n i t ~ to c.onvliisions rswnti:~llg itlrntic.:il l o tliose of Fal~giin ( r i ~ v i i ~ \ v v t li n TI.F,1) regarding tllc i l ~ ~ ~ t ~ l l a nofi s i ~ ~ s o l i ~ ~ d i z a t i oI i I~ proteins. ~ D. I<. 1,asltoivski [Cancer Res. 27, W3-911 (1967)1 rc~porlc~dtl1:it carcinogrnic electron-donors of widely differing structures form charge-tr;insfer cwiiiplexes with quinones thnt arc simplc hoinologs of a-tocophrrol rluinonc. and with rrlatrd toco-
450
JOSEPH C. ARCOS AND M A R Y P'. ARGUS
pherol quinonrs. This brings t o mind the csrlier finding of S. Liao and H. G. Williams-Ashman [Riochem. Pharmacol. 6, 53-54 (1961)1 on the strong inhibition by certain polycyclic hydrocarbons of a flavoprotein enzyme in the kidney and seminal vesicle of the rat which catalyzes the oxidation of reduced ribosyl nicotinamide by the quinone vitamin K8. B. Green [J. Mol. Biol. 29, 447-456 (1967)l continued his investigations on the noncovalent binding of polycyclic hydrocarbons to DNA. He reported a characteristic change in the intensity of polarized fluorescence when aqueous DNA solutions, in which various polycyclic aromatic hydrocarbons wcre solubilized, were subjected to flow-orientation. These studies confirm and extend the observations of Nagata and his coworkers (reviewed in II,F.3). C. B. Huggins, J. Pataki, and R. G. Harvey [Proc. Natl. Acad. Sci. U.S. 58, 22532260 (1967)l carried out an investigation on the effect of size and coplanarity of polycyclic aromatic hydrocarbons on their carcinogenic activity and ability to induce the synthesis of menadione reductase in the rat liver. This report confirms and extends the earlier findings of J. C. Arcos and M. Arcos [e.g., Bull. Soc. Chim. Belges 65, 5-16 (1956)l and J. C. Arcos, A. H. Conney, and N. P. Buu-Hoi [J. Biol. Chem. '236, 1291-1296 (1961)l. (6 ) To Section III,A,I. The carcinogenic activity of new derivatives of 5-nitrofuran are coming to evidencc. E. M. Erturk, J. M. Price, J. E. Morris, S. Cohen, R. S. Leith, A. M. vonEsch, and A. J. Crovetti [Cancer Res. 27, 1998-2002 (196711 reported the potent carcinogenic activity of N-[4-(5-nitro-2-furyl)-2-thiazolyll formamide. Twenty-nine out of 33 female Spraguc-Dawley rats (which lived 34 weeks or more) developed bladder carcinomas. Fifteen animals also developed mammary tumors. In a standardized study of miscellaneous aromatic and heterocyclic nitro and amino compounds D. P. Griswold, A. E. Cascy, E. K. Wrisburger, and J. H. Weisburger [Cancer Res. 28, 924-933 (1968)1 found 4,4'-thiodianiline, a structurally new type of agent, to possess weak carcinogenicity toward the mammary gland of SpragueDawley rats. (7) 7'0 Section IZZ,A,4. R. Oyasu, H . A . Battifora, R. Eisenstein, J. H. McDonald, and F. M. Hass [J. Natl. Cancer Inst. 40, 377-388 (1968)l have reported the considerable enhancement of tumorigeneris in the urinary bladder of rats by Z-acetylaminofluorene, if administration was begun a t the first day of life. Despite the presence of indolo in the dirt, bladder tumor incidence decrcascs with the age at which the administration of thr carcinogen was initiated. D. B. Clayson, T. A. Lawson, and J. A. S. Pringle [Brit. J . Cnncer 21, 755.762 (1967)l reported the induction of hepatomas in 100% of C57 X IF mice following oral administration of 2-aminodiphcnylene oxide. J. L. Radomski, E. Brill, and E. M. Glass [ J . Natl. Cancer Inst. 39, 1069-1080 (1967)l confirmed that 2-amino-3methoxydiphenylene oxide, also known as 2-methoxy-3-aminodibenzofuran, is highly specific for inducing carcinomas in the bladder of rats. ( 8 ) To Section IZIJ,5. G. T. Bryan [Cancer Res. ,%, 183-185 (1968)l found a significant increase in the incidence of lymphatic tumors in female mice following repeated subcutaneous administration of xanthurenic acid &methyl ether. A study of the tissue distribution of this carcinogenic tryptophan metabolitc was carried out by G. T. Bryan and C. R . Morris [Cnnwer Res. 28, 186-191 (1968)l. ( 9 ) To Sections III,A,B uud III,D,J. The view that derivatives of 4-nitroquinolineN-oxide (XCV) are uevcr more carciiiogenic than t,he parent compound itself must he abandoned in light of the report of A. Jmnsstignc:, N. P. Buu-Hoi, F. Zajtirla, J. P. Hoeffinger, and P. Jacquignon [Life Sciences 5, 1945-1947 (1966)l. These
t v51c.II .I-iii t roqiii nci~iiic.-,~-c~sitl~~ :in( I iI s 6-nirt Ii\.I,6,7-11iiiic~t Iiy 1 : I I I I I 6-flitor0 tlcrivat,ivcls. ‘Thv 6,7-~liinrthylt l i ~ r i v a t i v oprfJVIY1t.o I)(, a very f:ist, :ic.t.ing sarronialogenic agctit, surliiliring in :ic,tivity tliv I~artxitconil)ouncl. The 6-mcthyl and 6-fluoro derivatives were less tictivc, than 4-nitrot~uiiioline-Ar-osidc. The full report of thc preliminary comniunication of 1’.Kawazw, M. Tachibana, I<. Aoki, and W.Nnlcahara a t the Kinth Intrrnational Cancrr Congrclss (Tokyo) has np1w:ircel IBiochem. I hccrttzacol. 16, 631-63G (1967) I . They found that wherras the 4,6- and 4,7-dinitroc~uinoline-N-oxides are not carcinogcnic, the corresponding imino-6 (or 7)-nitro drrivatives are as strongly carcinogenic as other 4-hydrosylaniino drrivativcs tested by tliern. Thcy attributed the noncnrcinogenicity of the two clinitroclriinolinc:-N-oxidcsto their high reactivity toward cellular nucleophilcs such as YH-compounds, so that the &nitro group is removed bcforc met:ibolism to the carcinogenic hydroxylaniino derivatives. I n fact, the rate of reaction oT 4,6-dinitrocluinoline-N-osidewitli tliioglycolic acid is 50 times fastrr than that of 4-11itror~uinoline-N-oxi~~e. On the 1)asis of thcir testing of thirteen 4-hydroxyl:t~iiii~oquinolinc-iV-o~ides preparcd by reduction of the corresponding nitro comI)onnds, Kawazoe et nl. concluded that the 4-hydrosylamino group, unlike the 4-nit10 group, confrrs carcinogenicity to a wide range of structural types of substitutcd quinoline-N-oxide skeletons. Thus, thc inactivity of certain substituted 4-nitroquinoline-N-oxides is probably gcnerally due to thrir inability t o undergo in vivo reduction to the proximate carcinogenic N-hydroxylaniino derivatives. Concluding his earlier experiments C. E. Scarle [Brit. Empire Cancer Campaign 45, 271 (1967) I reported that 4-chloroquinoline-N-oxide and also 4-nitropyridine-N-oxide (XCIV) possess tumor initiating activity on the niouse skin (promoted with croton oil). Moreover, liver tumors and lymphosarcomas have arisen with both compounds. That 4-nitropytidine-N-oxide is a c*omplctc carcinogen for distant sites contrasts the finding of the Nakahara group who rcportcd that this compound is inactive. (10) To Section III,B. G. P. Warwiclc [Brit. Empire Cancer Campaign 45, 24 (1967) 1 reported that partial hepatcctomy is a powerful potentiator of the borderline carcinogenic activity of 2-mothy1-4-dimr thyla~iiinoazol~r~izenc in rats. This dye produced an 8/10 tumor incidence in rats fed at 0.06% level in a 10% protein diet for 12 months and maintained for an additional 2 months on a dye-free diet, when partial hepatectorny was (wried out 1 month after the brginning of dye feeding. No tumors were produced in rats frd thc dye without partial hepatectomy; neither were tumors produccd in rats treated with CCI, as an alternative t o partial hepatectomy. This stands in interesting contrast with the finding (H. H . Gosch, J. C. Arcos, and M. F. Argus, unpublished) that 3’-methyl-4-dimethylnminoazobenzene, adniinisteretl a t 0.12% level for a total of 8 months, is inactivr as a carcinogen in guinea pigs partially hcpatectomized 1 month after the beginning of dye feeding. E. V. Brown and W. H. Kipp (personal communication) determined thc basicity of the amino nitrogen and tlie p-azo nitrogen in a series of 4’-alkylated derivativcs of 4-dinif~tliylan~inoazo~~en~ene. Thrir data support the hypotlic~sis of G. Cilento [Cancer Mes. 20, 12CL124 (1960) 1 that Iiei,ntocarcinogt.nic activity disappears when electron tlcnsity a t the amino nitrogcn is abovf? :L certain value. This finding appears to conform to Scribncr’s t~liroreticnlfranirwork on tlie labilization of the hydroxamic acid ester bond by elrctron withdrawal in the postulated ultimatr carcinogenic metabolitrs of amino BZO dyes (discussed in Section III,D,4). E. 1‘. Brown and J. J. Duffy [ J . Na11. Cancer Znst. 40, 891-893 (1968)l prepared and tcJstrd for mrcinogenic activity in rats seven monomethyl derivatives of the previously tmted 5-~p-diiiiethylaminophenylazo)quinoline ( Q 5 ) ; see in Table XVI. :I i i t Iiors
JOSEPH C. ARCOS AND MARY F. A R G T X
452
Tlie order o f c.:iri~inogc,nic,it,ic.s, rr1:itiw to 4-d~iiir~li~l:rm~Ilon&ol,rnxenr (1l:lB) were: Q5, 2’-methyl-Q5, 7’-methyl-Q5, 8’-niethyl-Q5 >>> 2-met,liyl-Q5, 3-methyl-Q5 >> 3’DAB 6’-mcthyl-Q5. methyLQ5 (11) T o Section III,D,l. E. Brill and J. Radomski [Life Sciences 6, 2293-2297 (1967)1 confirmed that 1-naphthylhydroxylamine is a urinary metabolite of l-naphthylamine in the dog. H. Uehleke, F. Geipert, F. Schnitger, E. Brill, J. L. Radomski, end W. B. Deichmann [Abstract, Naunyn Schmiedebergs Arch. Pharmak. E x p . Puthol. 260, 213 (1968) 1 have shown that in the same species the N-hydroxylation of 2-naphthylamine is enhanced by phenobarbital pretreatment. The full report on the comparative carcinogenicities and mutagenicities of 1- and 2-naphthylhydroxylamine has appeared [S. Belrnan, W. Troll, G. Teebor, and F. Mukai, Carlcer Res. 28, 535-542 (1968)I. (12) To Section III,D,3. New results strengthen the conclusion of H. R. Gutmann, 8. B. Galitski, and W. A. Foley [Cancer Res. 27, 1443-1455 (196711 that synthrtic. N-hydroxylation transforms inactive or weakly active N-substituted derivatives of 2-acetylaminofluorene to highly active compounds. For cxamlilr!, syntlirtic N-llytlroxylation of the inactive 2-benzenesulfonamidofluorene transforms it int,o 11 highly active agent producing 100% mammary tumor incidence in frmalc rats; similarly, N-hydroxylation transforms the inactive 3-acetylaminofluorrnc to the highly active 3-acetohydroxamic acid. The general validity of this principle is also shown by the considerable enhancement of activity by N-hydroxylation of 4-benzenesulfonamidobiphenyl (H. R. Gutmann, personal communication). Linking of an amino, hydroxylamino, or acetohydroxamic acid group to a sufficiently large hydrocarbon-type grouping is, however, not per se a sufficient condition for carcinogenic activity. This is exemplified by the finding of H. Dannenberg, I. Bachmann, and C. Thomas [Z. Krebsforsch. 71, 74-80 (l96S)l and E. Hecker, M. Traut, and M. Hopp [Z. Krebsforsch. 71, 81-88 (1968)l that the introduction of a 3-amino, 3-acetylamino, 2-acetylamino, or 3-acetohydroxamic acid grouping onto the steroid skeletons, A’,*~G(’a)-oe~tratriene and A‘~s~G‘‘0’-oestratrienol-(17~), dors not evoke carcinogenic activity. Thus, in addition to a sufficiently large size (cf. Gutmann, Galitski, and Foley, Zoc. cit.), the hydrocarbon moiety must be planar and aromatic in accordance with Scribner’s theoretical framework (discussed in Srction III,D,4). E. Hecker, M. Traut, and M. Hopp [Z. Krebsforsch. 71, 81-88 (1968)l confirmed the pot.ent carcinogenic act,ivity of 2-nitrosofluorene. Fed by stomach tube to rats for 42 weeks it yielded, a t an activity level equal to that of 2-acetylaminofluorene, ear duct tumors in all animals and liver tumors in males. The nitroso compound also produced squamous epithelial carcinomas of the forestomsch. The results of C. C. Irving, R. Wiseman, and J. T. Hill [Cancer Res. 27, 23092317 (196711 suggest that it is the formation of 0-glucuronide which is the major metabolic reaction of 2-fluorenylacetohydroxamic acid in the rat liver in vivo, rather than reduction or deacetylation. Subsequently, E. C. Miller, P. D. Lotlikar, J. A. Miller, and B. W. Butler [Mol. Pharmacol. 4, 147-154 (196813 demonstrated that the 0-glucuronide is, in fact, reactive at neutral p H in vitro toward methionine, tryptophan, and guanosine. However, the reactions are considerably slower than those with esters of N-hydroxy-2-acetylaminofluorene, such as the N-acrtoxy derivative. I n accordancc with the lesser reactivity of the 0-glucuronide, the conjugate was found to be a notably weaker carcinogrn than the unconjugated arctohydroxamic acid (see “Note added in proof” in the report of E. C. Miller et ul., Zoc. cit.). Thus, whethcr metabolically formed 0-glucuronide is involved in the carcinogenic activity of 2-acetylaminofluorene and its N-hydroxy derivative still remains questionable.
>
>
MOI.ECUI,:\R
GEOMETRY A N D CARCINOGENIC ACTIVITY
453
'l'Ii~* frill rcqwrt, froin Ualtlwin's group 011 coniparat,ivc carcinogenicity in thc rut of 4-acet,amidost,iIhene and its N-hydrosy derivative has appeared [Brit. 3. 1 1 i t b
Cancer 22, 133-144 (196S)l. The N-hydroxy derivative is highly active by three routes of administration and produces invariably ear duct tumors in the majority of animals; local tumors were seldom seen in the subcutaneously injected animals. The parent amide anti the N-hydroxy derivative were equally active orally ; howcvw, by subcrrtnnrous and intrnperitoncxl injection t,he N-hydroxy derivative was more potmt. (13) To Sectwn 111,E,1. M. Hozumi [Bwchem. Phurmacol. 17, 769-777 (196S)I showed that 4-hydroxylaminoquinoline-N-oxideis a powerful inhibitor of sulfhydrylrequiring enzymes, such as catalase, alcohol dehydrogenase, and urease, by virtue of its catalytic property to oxidize -SH groups. Significantly, the inhibition is rwersed by the addition of glutathione. ( 1 4 ) To Section IV,A. In the full report [R. W. Baldwin, M . Moore, and M. W. Partridge, Intern. J . Cancer 3, 244-253 (1968)l of the rccent work of Baldwin's group on the interaction of "C-tricycloquinazoline with mouse skin proteins, i t wu shown that; this cbarcinogcm bocomes covalently bound to soluble and particwlnt c skin proteins, although the binding levels are lower than those obscrved with carcinogenic and noncarcinogenic hydrocarbons. Furthermore, the level of binding reaches a saturation level at the applied carcinogen dose of 0.03 fiM. Thc bound radioactivity can only be liberated by drastic hydrolytic treatment and it is not identihble as unchanged tricycloquinaeoline. The soluble proteins with which tricycloquinazoline is associated in the skin have the electrophoretic mobility of albumin and axe not comparable to h-like proteins. (16) To Section IV,B. Ma. Tada, Mi. Tada, and T. Takahashi [Biochem. Biophys. Res. Commwn. 29, 46-77 (1967)l investigated the complex formation in vivo between 4-hydroxylaminoquinoline-N-oxide,on one hand, and DNA and RNA, on the other hand, during exposure of ascitcs tumor cells to the action of thc carcinogen. The isolated DNA complexes show strongly decreased RNA-priming ability, and there is s reciprocal relationship between the amount of cornplexcd carcinogen and RNA-priming ability. Surprisingly, heating to 100°C brings about a release of the carcinogen in a probably metabolized form and the template activity of the DNA increases to that of heat-denatured control DNA; .this would suggest that the binding to the bases is not of covalent nature. However, when the DNAcarcinogen complex was submitted to enzymatic degradation and the hydrolysatilc: cliromatographed, about one-half of the carcinogen was eluted together with thc nucleotides; these results would indicate that covalent binding between the carcinogen and the nucleotides has taken place. (10) T o Section IV,C. M. Sluyser [Biochirn. Biophys. Acta 154, 606609 (19f38)J studicd the in vitro interaction of 4-dimethylaminoazobeneene and 3,4-benzopyrene with various histones. The association a.ppears to be rather nonspecific and is duc! to noncovalent bonding to large hydrophobic rcgions of the histones. Administration of 2-acetylaminofluorene to rats, however, results in covalent binding of the carcinogen to lysine-rich and arginine-rich histones [E. J. Barry, C . A. Ovechka, and H. R. Gutmann, J . Biol. Chem. 243, 51-60 (1968)l. B. Bakay [Biochem. Phurmucol. 17, 689-698 (1968)l produced rcsults indicating that in the liver nuclei of rats f d 3'-methyl-4-dimethylaminoazobenzene some of the protein-dye conjugates are aseociated with, or are an integral part of, nuclear ribosomes, Regarding the role of the liver h proteins a9 growth regulators, M. F. Argus, J. A. Walder, J. A. Fabian, and J. C. Amos [Brit. J . Cancer 22,330-341 (1968)l have shown that during liver regeneration following partial hepatectomy there is an
454
JOSEPH C. ARCOS A N D MARY F. ARGUS
iiivcrst, rt~l:ttiondii~ibetween total h protvin levcl on one Iland, and mitotic intlcs ant1 solrhlc cytolilasmic sidfhydryl level, on the ot lirr. ITonPvc~r,in 20-metliylcholanthrene-induced liver growth the total h protein lrvel remains uuchanged despite the considerable increase in the mitotic index and the sulfhydryl level. There is now substantial evidence indicating that o-quinoneimines are not instrumental in the mechanism of carcinogenesis by 2-acetylaminofluorene [reviewed in C. M. King and B. Phillips, Sczence 59, 1351-1353 (1968)l. These investigators have shown that a protein fraction of the 105,000 X g supernatant of rat liver catalyzes the reaction of 2-fluorenylaeetohydroxan~ic acid wit11 sRNA, and also with DNA and protein. The cofactor requirements and labeling studies suggest that sulfate and phosphate esters of the acetohydroxamic acid may be the reactive ultimate metabolites. J. K. Lin, J. A. Miller, and E. C. Miller [Biochemislry 7, 188s1895 (196811 cstablished that the major polar dye derived from the liver proteins of rats fed 4-monomethylaminoazobenzene is 3-(lioinocystein-S-yl)-N-mrtliyl-4-aminoazobenzcne. The protein bound sulfonium dye (see Table XSVI), 3-(methion-S-yl)-Nmethyl-4-sminoazobcnzcne appears to be the lilrcly precursor of bolli this polar dye and 3-methylmerc:tpto-4-monomethylaminoazobenzene.
REFERENCES Abell, C. W., and Heidelberger, C. (1962). Cancer Res. 22, 931-946. Akinrimisi, E. O., and Ts’o, P. 0. P. (1964). Biochemislry 3, 619-626. Alexander, M. L., and Glanges, E. (1965). Proc. Natl. Acad. Sci. U . S. 53, 282-288. Alifano, A., Papa, S., Tancredi, F., Elicio, M. A., and Quagliariello, E. (1964). Brit. J . Cancer 18, 386-389. Al-Kassab, S., Boyland, E., and Williams, K. (1963). Biocliem. J. 87, 4-9. Allen, M. J., Boyland, E., Dukes, C. E., Homing, E. S., and Watson, J. G. (1957). Brit. J . Cancer 11, 212-228. Andersen, R. A., Enomoto, M., Miller, J. A., and Miller, E. C. (1963). Proc. Am. Assoc. Cancer Res. 4, 2. Andersen, R. A., Enomoto, M., Miller, E. C., and Miller, J. A. (1964). Cancer Res. 24, 128-143. Andervont, H. B., and Shimkin, M. B. (1940-41). J. Null. Cancer Znst. 1, 225239. Anghileri, L. J. (1967a). Naturwissenschaften 54, 249-250. Anghileri, L. J. (1967b). Ezperientiu 23, 661-662. Arcos, J. C. (1961). Bull. Tulane Univ. Med. Fac. 20, 133-150. Arcos, J. C., and Arcos, M. (1955). Naturwissenschajten 42, 608. Arcos, J. C., and Arcos, M. (1956). Bull. SOC.Chim. Belges. 65, 5-16. Arcos, J. C., and Arcos, M. (1958). Bwchim. Biophys. Acta 28, 9-20. Arcos, J. C., and Arcos, M. (1962). Progr. Drug Res. 4, 407-B1.*
* The
following errors in this review should be noted: p. 419, first line: ref. (749) should be deleted and placed on p. 425, end of 2nd paragraph. p. 427, Table 6: references 56, 58, 59, 60 should read 84, 83, 270, 821, respectively. p. 439, second line: ref. (838) should read (383). p. 462, 29th line: “resting” should read “testing” p. 404, 30th line : triphenyl-stilbene should read triphenylethylene ; 1,1,3-tris-(4methoxyphenyl)-3-chl0r0-stilbene should read 1,1,Z-tris(4-methoxyphenyl)-2chloroethylene. p. 510, 5th line: sentence should read “Selye recorded the induction with croton
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1).
p. p. p. p.
oil, :inotlier agent known origin:illy as tumor promotor, of sarcomas in grannIoiii:Ltoiis ~ O I I ( * ~ I I i~~Hr i - : i t c din 1 I I V rat (748) ,” 510, 3rd paragrapli : 2nd sentriirt~should rc(m1 “Plienol WMS Tuuii(l 1 0 intiiicil ptqJilloirias 111 suriacci application on the I I I O I I S I ~skin (713, 717) ,” 510, line 40: after “to!vald thc 1110usz;(~ skill’’ insrrt “and Y ~ I k J ~ ’ 1 l t i l l l C lissuc.” ~ll~ 511, Section 2.223: second ref. should reaa! (796). 514, 4th line: a t end of first sentence insert ref. (557). 560, 13th line: “hydrolytic” should read “metabolic.”
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AUTHOR INDEX Numbers in italic refer to the pages on which the complete references are listed
A Aaronson, S., 417, 468 Abel, J. E., Jr., 260,291 Abel, P., 197, 219 Abell, C. W., 307, 433, 454 Abercrornbie, M., 257, 287 Acs, T., 233, 234, 235, 236, 237, 239, 240, 290, 291
Adamik, E. R., 240, 296 Adamik, G., 283, 290 Adams, E., 292 Adams, J. M., 5, 69 Adams, P., 2, 66 Adarnson, R. H., 28,35,68 Adenis, L., 37, 400, 460 Adrian, R. W., 18, 19, S9 Afanaseva, T. P., 164, 816 Afzelius, B. A., 226, 287 Agrawal, H. O., 81, 83, 86, 807 Ague, S. L., 30, 66 Ahrens, E. H., 286, 287 Aisenberg, A. C., 47, 58, 70 Aketa, K., 226, 229, 887 Akinrimisi, E. O., 347, 464 Alarn, A., 307, 433, 456 Alarcon, R. A., 267, 288 Albertsson, P. A., 274, 288, 299 Alexander, H. E., 22, @ , Alexander, M. L., 361, 464 Alifano, A., 384, 464 Al-Kassab, S., 435, 464 Allard, C., 280, 891 Allen, A,, SO3 Allen, J. G., 241, 288 Allen, M. J., 381, 383, 389, 464 Alleri, R. A,, 3, 30 bllfre),, V. C., 256, 273, 28s. %k? Allman, V., 62, 68 Almquist, P. O., 234, 288 Alonuo, D., 240, 888
Altmon, K. L., 241, 298 Arnbaye, R. Y., 306, 369, 408 Ambellan, E., 248, 288 Ambrose, E. J., 246, 888, 299 Ambrus, J. L., 74, 807 Amiel, J. L., 241, 89Y Amos, H., 282, 288 Anderer, F. A., 85, 207 Andersen, R. A., 403, 425, 426, 454 Anderson, A. J., 265, 288 Anderson, D. O., 277, 281, 894 Anderson, N. G., 245, 248, 258, 267, 269, 271, 282, 288, 600 Anderson, T. F., 87, 221 Anderson, W., 260, 263, 267, 268, 276, 888 Andervont, H. B., 319, 464 Andres, G., 259, 266, 603 Andrewes, C. H., 74, my, 216 Angerer, S., 345, 464 Angermann, J., 176, 177, 178, 916 Anghileri, L. J., 345, 464 Anken, M., 100, 105, 202, 210, 214 Aoki, K., 383, 385, 386, 461, 468 Aono, H., 105, 816 Appleyard, G., 87, 807 Aposhian, H. V., 122, 129, 166, 167, 807, $20
Arata, T., 448 Arber, W., 140, 143, 144, 145, 146, 207, 210 Archer, H. E., 22, 66 Archer, 0. K., 51, 65, YO Arcos, J. C., 305, 307, 308, 315, 317, 320, 343, 359, 370, 371, 373, 379, 392, 393, 395, 396, 397, 398, 406, 415, 433, 439, 449, 450, $51, 453, $c;S Arvos, M , 306, 307, 317, 319, 343, 370, 371, 373, 379, 395, 397, 411, 439, 460, 464, 466 473
147,
319, 381, 411, 461,
359, 415,
474
AUTHOR INDEX
Argus, M. I-,, 307, 308, 320, 378, 379, 381, 433, 440, 461, 463, 464, 466, 466, 401 Armstrong, J. A,, 74, 216 Arnason, B. G., 59, 6s Arthur, C., 29, 38 Asano, K., 103, 163,216 Asano, M., 398, 459 Asboc-Hnnsrn, G., 238, 279, 288, 292 Ascoli, F., 251, 288, 348, 350, 355, 464 Asello, L., 285, 296 Ashkenazi, A., 80, 198, 207, 213 Ashton, F . T., 22, 37, 228, 238, 259, 294 Ashton, M. J., 414, 469 Astier-Manifacier, S., 152, 207 Astrachan, L., 163, 220 Atanasiu, P., 84, 216 Atchison, R. W., 80, 207 Au, M . H., 254, 296 Auclair, W., 230, 298 Auerbach, R., 5,9,38,58, 65 August, J. T., 152, 156, 158, 164, ZW,216, 21s
Aurelian, L., 95, 96, 97, 207, 218 Austin, C. R., 229, 288 Austin, M. L., 232, 263, 288 Axelrod, D., 176, 177, 212
B Bachelard, H. S., 247, SOf Bachmann, I., 462 Bablanian, R., 167, 168, 169, 207 Back, M. K., 276, 278, 281, 283, 288 Bacq, Z. M., 241, 288 Bader, J. P., 152, 221 Badger, G. M., 320, 321, 322, 325, 328, 340, 343, 456 Bahl, 0 . P., 437, 466 Bailey, H. S., 24, 30 Baird, 9.L., 353, 358, 362, 462, 469 Bakay, B., 439, 463, 466 Baker, J. R., 409, 466 Baker, R. I<., 375, 466 Balandin. I. G., 168, 207 Balazs, E. .t.,233, 235, 242, 243. 264, 279, 280. .?SS Baldini, E., 3/)2 Baldwin, K. W,,328, 329, 331, 332, 334, 339, 358, 403, 425, 426, 434, 435, 440, 449, 453, 466, 466, 469 Balis, M. E., 281, 282, 290
Rail, J. K., 349, 353, 466 Balls, M., 14, 36 Balner, H., 52, 65 Baltimore, D., 152, 153, 155, 156, 167, 168, 207, 210 Aanrroft, W. D., 266, 288 Barclay, M., 258, 28.5, 288, 896, 299 Barrlay, R. I<.,82, 209 Barkrr, C. R., 440, 466 Barkin, M., 384, 4G2 Barner, H. D., 90, 91, 92, 93, 207, 209 Barnes, J . E., 112,220 Barnes, J. M., 361, 464 Barnes, R. D. S., 45, 66 Barnett, L., 359, 391, 468 Barnhart, M. I., 284, 300 Baron, F. A., 2, 35 Barry, E. J., 436, 438, 458, 466 Barsotti, M., 363, 470 Basilco, C., 84, 197, 205, 216 Bassenge, F., 45, 55, 66 Baasett, A. L., 275, 288 Batchelor, J. R., 45, 51, 56, 59, 60, 66, 67 Bates, M. E., 75, 212 Bates, R. R., 21, 38 Battifora, H. A., 460 Bauer, R. D., 393, 395, 427, 440, 468 Baum, J. H., 235, 298 Baum, S. G., 198, 199, 217, 218 Bnumann, C. A., 397, 466 Baumann, M. E., 309, 315, 343, 461 Beelmear, M., 50, 56, 70 Beak, T. F., 80, 217 Beams, H . W., 261, 288 Bearcroft, W. C. C., 74, 208 Beard, D., 81, 82, 208 Beard, J. W., 81, 82, 208 Beaudreau, G., 160, 219 Beazlcy, H. L., 270, 288 Becker, A., 149, 208 Bcckcr, It. O., 275, 288 Beclrcr, R. S., 347, 464 B r c b r , Y., 103, 152, 155, 169, 171, 207, 208, 217, 258, 262, 296 Rrrrs, It. F., 352, j69 Beickert, A,, 22, 23, 24, :
AUTHOR INDEX
Belkin, M., 233, 239, 244, 245, 251, 252, 289, 296
Bell, E., 259, 2X.9 Bell, J. R., 7, 40 Bcll, I,. G. E., 250, 256, 259, 284, 289 Bell, T. M., 63, 65 Bello, L. J., 91, 122, 208 Belman, C., 415, 470 Bclman, S.,307, 368, 369, 413, 414, 416, 430, 443, 462, 466, 470 Bolyaeva, M. I., 283, 289 Bemis, J. A., 307, 433, 456, 45G Brmder, D. H., 259, B 9 Bendich, A., 23, 27, 28, 36, 86, 209 Ben-Ishai, D., 4, 16, 17, 18, 19, 26, 29, SG Bendix, It. M., 236, 289 Benjamin, T. L., 83, 176, 201, 208 Bennett, L. L., 23, 24, 32, 41 Bennett, L. R., 286,289 Ben-Porat, T., 82, 95, 97, 99, 100, 134, 170, 208, 209, 213 Brnsley, R . R., 289 Bmzer, S., 111, 163, 208 Bereczky, E., 176, 177, 178, 216 Berenblum, I., 3, 4, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 22, 23, 24, 25, 26, 29, 30, 31, 3’6, 37, 39, 40, 337, 466 Berenbom, M., 407,456 Berezesky, I. K., 198, 216, 217 Berg, R., 80, 88, 209 Berger, A., 296 Bergrnann, E. D., 341, 466 Berkelcy, W., 75, 210 Borman, L. D., 89, 208 Bernard, C., 81, 211 Berneis, K., 27, 36 Bernfeld, P., 238, 252, 272, 276, 277, 280, 284, 285, 289, 294 Bemo, M., 241, 297 Bernstein, D. S., 238, 2S9, 893 Bernstein, H. M., 282, 289 Bersohn, M., 362,469 Bersohn, R., 353, 358, 462 Bertani, G., 391, 469 Benvald, Y., 23, 36 Brssman, M. J., 91, 93, 105, 115, 122, 123, 208, 210, 214, 216 Bettex-Gallard, M., 249, g89 Bcz, W., 362, 471 Bhide, S. V., 14, 22, 36, 38
47.5
13i:tiit,lii, l'., 178, 216 Rianchi, H . G . , 276, 289 Siitncliini, P., 236, 289 Bichowski-Slommitzki, L., 296 Bieber, S.,20, SS Biclschowsky, F., 45, 46, 56, 62, 66 Bielschowsky, M., 45, 46, 56, 62, 6G Bierbauer, J., 233, 237, 291 Biesele, J. J., 390, 468 Billeter, M. A,, 154, 155, 156, 157, 159, 208, 214, 216, 220
Billingham, R. E., 43, 44, 45, 46, 47, 48, 53, 57, 61, 65, G6 Bing, It. J., 45, 55, 68 Bingley, M., 250, 289 Bishop, M. W.H., 229, 2SS Bittner, J. J., 4, SG Bjorklund, U., 253, 289 Black, P. H., 80, 82, 84, 88, 192, 195, 196, 199, 201, 202, 208, 209, 212 Blair, P. B., 81, 210 Blickenstaff, D. E., 21, 38 Block, K., 391, 468 Bloom, F., 384, 456 Bloom, J. L., 4, 36, 38 Rloiit, E. R., 251, 265, 274, 289, 257 Bluhm, G. B., 284, 300 Blum, B., 3, 36 Blurn, J., 341, 456 Blurnenthal, J., 286, 303 Blurnrr, M., 321, 322, 323, 466 Bo&, J. I,., 44, 50, 67 Bocher, C. A,, 227, 281, 282, 289, 299 Bock, F. G., 335, 456 Bodmer, A., 2,4O Bodur, H. Z., 285, 289 Boeyk, A., 85, 199, 208 Bogdanova, S. L., 164,213 Boiato, L., 10, 30,31, 36 Boiato-Chen, L., 5, 10, 36, 41 Boiron, M., 80, 81, 84, 208, 221, 216 Bollag, W., 27, 36 Bolle, A., 111, 210, 218 Bolognrsi, D. P., 168, N 8 Bonar, R. A., 81, 82, 208 Bond, H. E., 245, 248, 269, 271, 282, 288 Bonner, J., 247, 250, 602 Bonser, C. M., 306, 365, 367, 368, 369, 370, 379, 380, 381, 404, 405, 412, 415, 416, 467, 469, 471
476
ATTTHOR INDEX
Booiw, C. W., 258, 262, %DG Booth, J., 345, 403, 467 Borei, H. G., 253, 289 Borenfreund, E., 23, 27, 28, 3G Borgesp, N. G., 75, 219 Borman, G. S., 80, 171, 210, 218 Born, G. V. R., 289 Boros, T., 57, GO Borrelli, J., 249, 259, 304 Borst, P., 152, 154, 155, 156, 157, 158, 159, 214, 216, 220 Botkin, C.C., 28, $8 BotrB, C., 251, B8, 348, 350, 355, 464 Boughton, B., 53, G6,70 Bourgain, R., 280, 301 Boutwell, R. K., 21, $0 Boyce, R. P., 148, 149, 208, 219 Boyd, E. S., 252, 289 Boyd, H. L., 436, 468 Boyde La Tour, E., 111,210 Boyer, G., 57, 66 Boyland, E., 2, 20, 22, 23, 24, 26, 27, 28, 31, 32, 33, 36, 37, 306, 307, 328, 335, 336, 338, 345, 346, 347, 348, 349, 350, 351, 352, 355, 358, 359, 361, 362, 367, 368, 381, 383, 389, 403, 412, 413, 414, 415, 435, 436, 442, 449, 454, 457, 46s Boyse, E. A., 57, 59, GO, 69 Brachet, J., 281, 2SD Brachetto-Brian, D., 5, 37 Bradley, D. F., 352, 468 Bradley, S. G., 57, 69 Bradshaw, L., 306, 367, 379, 405, 467 Brandon, F. B., 75, 208 Branigan, W. J., 80, 217 Braun, W., 225, 283, 289 Breese, S. S., Jr., 80, 208 Bremner, D. A., 375, 377, 458 Brenner, S., 111, 142, 218, 359, 391, 468, 466
Brent, L., 43, 44, 53, 57, 61, 66 Bresnirk, E., 22, 37 Breusch, T. I,., 285, 289 Brigando, J., 348, 468 Brill, E., 368, 369, 415, 416, 423, 436, 460, 462, 458, 460, 467 Brillantes, F. P., 320, 4G2 Briaiarelli, G., 320, 334, 462 Brock, N., 352, 468
Brody, S.,281, 282, 289, 290 Brooke, M. S., 45, 52, 56, 59, GG Brookes, P., 307, 359, 434, 435, 438, 468, 464 Brown, D. H., 234, 292 Brown, D. V., 400, 465 Brown, E. V., 392, 393, 394, 396, 397, 467, &8 Brown, F., 119, 216 Brown, G. B., 389, 390, 391, 468, 469 Brown, J. B., 44, 47, 66, 236, 299 Brown, R. A., 22, 37 Brown, R. H., 253, 297 Brown, R. R., 368, 370, 379, 381, 383, 384, 386, 412, 415, 420, 468, 4G7 Bruening, G., 81, 83, 86, 207 Brucs, A. M., 328, 457 Brune, H., 14, 41 Brims, F. H., 252, 276, 294 Brunson, J. G., 265, 302 Bryan, C. E., 2, 19, 20, 22, 23, 24, 32, 37, 40, 41
Bryan, E., 75, 221 Bryan, G. T., 368, 370, 379, 381, 383, 386, 412, 415, 420, 450, 458 Bryson, V., 2, 37 Buchanan, J. M., 103, 105, 107, 112, 113, 120, 163, 166, 2V9, 214, 219, 220 Buchta, E., 308,4G4 Buddecke, E., 251, 252, 260, 2*90 Budovskii, E. I., 425, 470 Buchner, M., 449 Burchenal, J. H., 28, 41 Burdette, W. J., 361, 362, 458 Burdon, R. H., 154, 155, 156, 157, 159, 208, 214, ZlG, 220 Burger, D., 62, 66, G7, 68 Burkhard, R. K., 393, 395, 427, 440, 468 Burkitt, D., 63, GG Burnet, F. M., 45, 46, 56, 57, 62, GG, 74, 210 Burnham, M., 335, 466 Burtle, J. G., 441, 4s1 Burton, D., 280, 290 Burton, K., 127, 129, 139, 163, 165, 816, 219
Busby, E. R., 367, 412, 414, 415, 416, 467 Buss, J. M., 303 Butel, J. S., 88, 176, 177, 178, 193, 198, 199, 201, 209, 210, g17
h t l i c l i e r , H. It., Jr., 236, 699 Butler, B. W., 452 Butler, K., 328, 329, 455 Buu-Hoi, N. P., 306, 308, 309, 312, 313, 314, 315, 317, 319, 324, 325, 326, 328, 334, 340, 343, 344, 388, 394, 4/58, 450, 458, 463, 464 Byers, T. F., 254, 293
C Cahill, G. F., Jr., 238, 289 Caillet, J., 348, /tG? Cairns, J., 96, 209 Callantlrr, A., 278, 283, 300 Callender, J., 153, 167, 168, 207 Cambel, P., 399, 467 Cammarata, R. J., 63, 66 Cancghein, P. N., 277, 290 Cantrro, A,, 230, 280, 281, 291, 299, 307, 432, 470 Capitano, J., 8, 11, 12, 13, 37 Cappuccino, J. G., 28, 41 Capraro, V., 255, 290, 297 Carasi, E. A., 85, 209 Caren, L. D., 57, 66 Carnes, W. H., 8, 37 Carney, P. G., 198, 615 Carp, R. I., 89, 173, 174, 175, 176, 177, 178, 187, 192, 209, 211 Carr, A. J., 234, 280, 290 Carr, M. C., 262, 302 Carrier, W. I,., 148, 149, 618 Carruthers, C., 251, 257, 264, 290 Cnrsjo, A., 254, 282, 296 Cartrr, R. L., 448 Casazza, A. M., 5, 37 Case, R. A. M., 365, 366, 458 Casey, A. E., 370, 371, 398, 450, 461 Casrp, M. J., 75, 89, 199, 201, 212 Casida, J. E., 19, 37 Caso, I,. V., 61, 66 Cassingrna, R., 202, 220 Castegnaro, E., 228, 291 Castermans, A,, 47, 66 Casto, B. C., 80, 207 Castnr, C . W., 234, 290 Catchpole, H. R., 231, 238, 292 Catton, A,. 241, 297 Crfis, F., 369, 377, 400, 46’8 Celazsi, G., 235, 283, 303
Crrccedo, L. It,, 392, 393, 394, 468 Chart, A. B., 227, 255, 6-90,294 Chagoya, V., 99, 100,211 Chalkley, H. W., 250, 254, 290 Chalvet, H., 314, 369, 463, 468 Chalvct, O., 308, 309, 312, 324, 325, 342, 448, 458, 463, 464 Chambers, R., 257,290 Champe, S. P., 111, 208 Changeux, J. P., 200, 216 Chanock, R. M., 75, 89, 199, 201, 212 Chany, C., 80, 208 Chapman, B. A., 45, 51, 60, 65 Chapman, D. W., 270, 288 Chapman, L., 22, 36 Charache, P., 274, 290 Chargaff, R., 268, 271, 284, 290 Cliarihr, S., 20, 28, 37 Cliaykiii, S., 391, 409, 455, 458 Chccver, F. S., 75, 219 Clienaille, P., 80, 81, 84, 208, 211 Chcng, P. Y., 83,209 Cheong, L., 82, 209 Chevalley, R., 111, 210 Chevremont, M., 283, 29.90 Cliiba, C., 45, 55, 66 Chieco-Bianchi, L., 7, 8, 9, 10, 11, 14, 25, 37,38 Chihara, G., 428, 431, 466 Child, F. M., 251, 290 Childs, J. J., 403, 404, 469 Chiriboga, J., 241, 290 Christian, J. E., 24, 36 Christiansen, J. A., 275, 290 Christie, G. H., 44, 49, 58, 60, 67 Ciccarone, P., 282, 993 Cifonelli, J. A,, 274, 290 Cilento, G., 451 Cittrrie, P., 282, 699 Cividalli, G., 22, 24, 25, 26, 37, 40 Civltirrt, I. I<., 285, 29'7 Clar, E., 308, 459 Clarke, D. A,, 390, 458 Clarke, J. K. R., 75, 89, 217 Clans, P. E., 249, 293 Claverie, P., 348, 46'7 Clay, A., 37, 400, 460 C h w o n , D. R , 306, 362, 367, 368, 369. 370, 379, 380, 381, 403, 404, 405, 412, 414, 415, 416, 448, 450, 467, 459
478
AUTHOR lNDEX
Cleveland, J. C., 377, 469 Cline, J. C., 225, 296 Clowes, G. H. A., 345, 469 Coffey, C. B., 436, 437,469 Cogin, G. E., 258, 285, 188 Cohen, E. P., 54,66 Cohen, S., 460 Cohen, S. S., 90, 91, 92, 93, 103, 104, 105, 107, 111, 119, 121, 137, 163, 207, 209, 210, 216, 218, 221, 225, 272, 274, 284, 290 Cole, J. W., 377, 469 Cole, L. J., 5, 6, 87, 38, 62, 70 Coleman, L. L., 255, 290 Collins, D. N., 55, 63, 67 Colnaghi, M. I., 8, 12, 97 Colson, C., 145, 146, 209 Coman, D. R., 257,290,291 Commoner, B., 429, 430, 470 Condi, R. M., 286, 290 Condington, J. B., 235, 900 Congdon, C. C., 394, 399, 466 Conney, A. H., 336, 449, 460, 469 Connon, F. E., 286, 289 Converse, J. M., 54, 68 Conway, W. D., 366,462 Conzelman, G. M., 369, 415, 469 Cook, D. L., 276, 289 Cook, J. W., 325, 328, 466 Cooke, C. W., 423, 437, 466 Cooper, F. C., 328, 329, 466, 469 Cooper, G. N., 45,50,56,66 Cooper, S., 154, 161, 209, 216 Coppelson, L. W., 47, 66 Coppey, J., 202, 220 Cordova, C. C., 282, 299s Cornman, I., 1, 19, 23, 87 Cornuet, P., 152, 207 Cornwell, D. G., 254, 291 Corre, L., 394, 463 Cosgove, G. E., 71, 398, 469 Cotchin, E., 384, 469 Coto, c., 100, 209 Cotterie, R., 241, 303 Cowdry, E. V., 249,290 Cowen, P. N., 3. 4, 14, 21, 37 Crabtree, H. G., 387, 469 Cramer, J. W., 17, 26, 30, 40, 333, 402, 417, 418, 441, 442, 459, 465 Crawford, E. M., 81, 82, 209
Crawford, L. V., 81, 82, 83, 84, 95, 97, 100, 129, 132, 209, 218 Crawley, B., 266, 290 Cremer, N. E., 55,62,66 Cren, J., 22,37 Crespi, H. L., 245, 299 Cresswell, R. M., 389, 391, 468 Crevat, A., 278, 283, 900 Crick, F. H. C., 359, 360, 391, 468, 470 Crocker, T. T., 5, 6,38 Cronheim, G. E., 284, $90 Cronin, A. P., 27, 28, 29, 34, 40 Cronkite, E. P., 240, 283, 290, 296 Crosby, D. L., 54, 70 Crosby, 1,. K., 54, 66 Cross, M. J., 289 Crossley, M. L., 392, 469 Crout, D. W., 369, 415, 469 Crovetti, A. J., 460 Csaba, G., 228, 233, 234, 235, 236, 237, 239, 240, 255, 290, 291 Csermley, E., 235, 279, 291 Cubiles, R., 282, SO1 Culbertson, C. G., 75, 219 Culvenor, C. C. J., 391, 469 Cunningham, G. J., 328, 329, 331, 332, 334, 339, 358, 435, 466 Curreri, A. R., 384, 4b7 Curri, S. B., 235, 279, 291 Curtis, A. S. G., 244, 248, 252, 270, 281, 291 Curtis, M. R., 308, 342, 460
D Daescli, G. E., 96, 171, 2lf Dahlgard, M., 379, 466 Dalcq, A. M., 226, 230, 247, 249, 254, 261, 291 Dales, S., 81, 95, 104, 209, 211 Dalgarno, L., 152, 153, 155, 156, 212, 219 Dalmasso, A. P., 51, 62, 66 Dalton, A. J., 80, 209 Damcrau, W., 362, 427, 460 Damesliek, W., 45, 46, 47, 60, 61, 62, 63, 66, 68, 69 Dan, J. I,., 230, 241, 251, 257, 2991 Dan, K., 231, 253, 291, 299 Danen, D., 259, 901 Daniel, G. E., 250, 254, 290 Daniel, J. W., 403, 650
AUTHOR INDEX
Daniel, P. M., 44s Dnnnenherg, H., 350. .;52. 469 Dann-Markeri. A.. 85. 209 1)nrncll. .J. E., 88, 152, 153, 155, 167, 160, 207, 208, 216, 217, 210, 281 Das, N. I<., 281, 296 Daub, G. H., 448 Daudel, P., 339, 433, 434, 469 Daudcl, R., 325, 326, 339, 340, 469, 463 Daudy, A. H., 241, 296 Daugherty, K., 253, 293 Davey, A. T., 329, 358,458 Davidson, D., 291 Davidson, E., 228, 294 Davidson, J. D., 30,35 Davies, A. J. S., 44, 45, 50, 51, 57, 60, 66, 68
Davies, G. E., 276, 291 Davis, J. C., 360, 460 Davis, M. L., 398, 469 Davis, W. W., 345,469 Davison, C. L., 149, 216 Davisson, E. O., 245, 298 Deamer, D. W., 254, 291 Dean, H. G., 329, 332, 435, 456 DeBaun, J. R., 423, 444, 469 DeBenedictis, G., 7, 8, 9, 10, 11, 14, 25, 37,38 Debruyn, .'%1 M., 285, 297 de Burgh, P. M., 51, 60, 62, 68 de Costa, H. C., 280, 291 de Estable, R. F., 75, 209 Defendi, V., 44, 47, 58, G6, 69, 89, 177, 192, 211 DeHaan, R. I,., Jr., 252, 291 Deichmann, W. R., 363, 366, 377, 384, 415, 416, 462, 469, 4W Deinhaldt, G., 111, 210 DeLamirandr, G., 280, 281, 291, 439, 469 DeLerma, B., 348, 350, 355, 4G4 Della Porta, G., 8, 11, 12, 12, 37, 41 deLong, R. P., 257, 201 Demaille, A., 37 Demerec, M., 391, 459 Demisch, R. R., 344, 469 Denans, M., 248,278,283, SO0 Denny, P. S., 286, 291 Deringer, M. I<., 4, 7, 11, 12, 13, 37, 39 Dersjanl., H., 52, 06
479
DeSantis, F., 347, 459 De Sirnone, 1'. 178, 216 d P Sousa. c. P , 28. n? t l r SOIICR, M . A . H., 16, 56, 62, G6 de Tlii., G., 82, N S , 203 de Torres, R. A., 99, 100, 105, 176, 182, 183, 185, 186, 194, 195, 196, 202, 210, 2'14 Dettlaff, T. A., 228, 291 Deuchar, E. M., 248, 291 Deutsch, H . F., 85, 209 Devaux, G., 22, 3Y de Waard, A,, 93, 114, 140, 200 Dewhurst, F., 415, 469 de Wolf, S., 121, 211 Deysson, G., 228, 291 Diamond, E. G., 249, 276, 291 Dickman, S. R., 281, 291 Diderholm, H., 80, 84, 88, 209 Dierick, W., 281, 301 Dijkstra, J., 439, 469 DiMarco, A,, 228, 291 Di Mayorca, G. A., 84, 208, 209 Dingman, C. W., 438, 440, 460, 469 DiPaolo, J. A., 5, 6, 20, 37, 235, 253, 2'98 Dirksen, M. I,., 105, 107, 112, 120, 166, 209, 220 Dittmar, C., 397, 460 Dixon, F. J., 62, 69 Djerassi, I., 240, 241, 303 Dmochowski, L., 46, 62, 63, 66, 82, 176, 177, 178, 209, 216 Doak, S. M. A,, 44, 57, 60, 66 Dobrincr, K., 375, 469 Dobson, R. I,., 370, 4GO Dodd, M. C., 391, 46f Dorll, R. G., 8, 37 DoljanAi, Id., 12, 37 Domsky, I. I., 25, 37, 333, 334, &GO Donlan, C. P., 242, 299 Dorman, D. C., 54, 70 Dornfeld, E. J., 251, 256, 291 Dorfman, A,, 238, 301 Douglas, G. W., 262, 302 Dounce, A. L., 268, 201 Dourmashkin, R. R., 57, 66 Driessens, J., 37, 400, 460 Druckrey, H., 19, 37, 352, 366, 367, 425, 465, 460 Drzrnicyk, R., 251, 252, 260, 2.00
480
AUTIIOH INDEX
Dubbs, D. It., 95, 96, 117, 99, 100, 102, 104, 105, 116, 118, 119, 123, 124, 125, 170, 171, 172, 173, 174, 176, 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 191, 194, 195, 196, 202, 210, 211, 213, 21 4 Dubin, D. T., 236, 291 Duesberg, P. H., 81, 85, 210, 818 Duffy, J. J., 461 Duhig, J. T., 45, 52, 56, 66 Dukes, C. E., 335, 367, 368, 381, 383, 389, 412, 414, 464, 467 Dukor, P., 62, 68 Dulbecco, R., 75, 80, 82, 84, 174, 175, 178, 180, 181, 183, 184, 185, 203, 209, 210, 212, 220
Dultsin, M. S., 63, 66 Dunn, A., 227, 294 Dunning, W. F., 308, 342, 380, 460 Duplan, J. F., 6, 37 Dupont, A., 37 Duran-Reynals, F., 279, 291 Duran-Reynals, M. L., 279, 291 Duran-Reynals, R., 279, 280, 291 Dussoix, D., 143, 144, 145, 207, 210 Dustin, A. P., 2, 20, 22, 37 Dyer, H. M., 380, 402, 460 Daiewiatkowski, D. D., 238, 891
E Eagle, H., 258, 262, 296 East, J., 46, 56, 62, 66 Easty, D. M., 246, B S Ebel, J. P., 283, 284, 291 Ebisuzaki, K., 91, 105, 111, 210, 819 Eckert, E., 51, 68 Eddy, B. E., 45, 54, 70, 75, 80, 84, 209, 210, 219 Edgar, R. S., 111,210 Eggers, H. J., 152, 153, 167, 168, 169, 207, 210
Eggman, L., 247, 250, SO2 Ehrhart, H., 384, 460 Eichwald, E. J., 48, 66 Eidinoff, J. L., 204, 211 Eigner, J., 115, 218 Eisenstein, R., 255, 296, 460 Ekstedt, R. D., 45, 52, 50, 66, '71 Ekwall, P., 319, 468 Elicio, M. A., 384, 464
Elion, G . B., 20, 38 Elis, J., 20, 37 Elkins, W. L., 44, 49, 58, 60, 66 Ellis, D. R.,154, 2ZG Ellis, 11. A., 3 s Ellis, It. A., 260, 291 Ellis, S. C., 263, 291 Emmerson, P. T., 149, 210 Enders, J. F., 80, 218 Endo, H., 386, 387, 417, 460, 469 Eng, J., 203, 210 Engelberg, H., 241, 276, 291 Engelbreth-Holm, J., 238, 292 Engelhorn, R., 16, 17, 38 Enger, M. D., 85, 210 Enomoto, M., 402, 403, 415, 418, 419, 425, 426, 464, 460, 464, 466 Eoyang, I,., 152, 156, 158, 207 Epstein, M. A,, 62, 70 Epstein, R . H., 111, 112, 166, 210, 220 Epstein, S. I., 245, 248,292 Erichsen, S., 203, 210 Erikson, It. L., 82, 142, 156, 157, 210, 219 Ernster, L., 281, 296 Eron, L. J., 132, 210 Ertiirk, E. M., 460 Eschenbrenner, A. B., 319, 471 Escher, G. C., 258, 285, 288, 296, 299 Esping, U., 226, 292 Evans-Anform, E., 44, 50, 67
F Fabian, J. A., 463 Fabrizio, D. P. A., 80, 220 Faessinger, R., 392, 393, 394, 468 Fahraeus, R., 268, 292 Falconer, D. S., 4, 36,38 Falk, H. L., 336, 337, 338, 460, 463 Farber, E., 437, 464 Farber, S., 251, 265, 274, 289 Farc, G., 397, 399, 434, 460 Farquharson, M. E., 436, 461 Fasman, G. D., 251, 265, 274, 289 Faulkner, P., 81, 210 Fedele, L., 384, 467 Fedorko, M. E., 240, 245, 292 Fefer, E., 423, 460 Feigen, G. A., 265, 271, 292 Feldherr, C., 228, 238, 259, 294 Frldman, I,. A., 177, 193, 198, 210, 217
I+lis, G., 161, 220 Fellig, J., 277, 280, 292, 304 Fels, I. G., 265, 271, 292 Felsrnfeld, G., 352, 468 Fcltz, E. T., 74, 207, 256, 261, 292 Fenner, F., 74, 210 Fenton, H., 236, 292 Fenwick, M. L., 156, 157, 167, 210 Ferber, K., 368, 456 Fcrno, O., 277, 292 Ferreber, J. W., 256, 257 Ferry, J. O., 265, 292 Fershein, W., 225, 283, 289 Fetterman, P. L., 436, 438, 469 Fex, H., 277,292 Firld, A. K., 224, 225, 282, 283, 292, 296, 302 Fieser, L. F., 333, 345, 460, 470 Filipponc, D. R., 55, 69 Filippovich, A. S., 425, 470 Finch, J. T., 75, 214 Fine, A. S., 278, 299 Finger, G. C., 341, 373, 392, 393, 395, 411, 466 Fink, I<., 21, 38 Fink, R. M., 21, 38 Fiorc-Donati, L., 7, 8, 9, 10, 11, 14, 25, 36, 57, 38 Firminger, H. I., 394, 397, 437, 466, 466, 467 Fischer, A., 26, 38 Fiscus, W. G., 45, 49, 66 Fishhein, B., 368, 414, 4.56 Fisher, A., 224, 228, 250, 292 Fisher, B., 292 Fisher, E. R., 292 Fisher, H. W., 258, 992 Fisher, W. D., 245, 248, 269, 271, 282, 288 Fiskrm, R.. A,, 367, 462 Fitzhugh, 0. G., 364. 464 Flaks, J. G., 90, 93, 105, 210, 229, 293 Flanagan, J. F., 172, 210 Flandcrs, I,. E., 360, 415, 469 Fleiahrr, M. J.. 226, ?97 Flriniiig, W. II., 115. 210 l'lrtc*her, T. I,., 371, 372. 273, 378. 3!G, 421, 423, 425, 427, jG:i Flint, J., 391, 461) Floch, M. II., 276. 292 Fogli, J., 82, 209
I~olvy,G. I<., 267, 2SS Folcy, W. A,, 5, 6, 37, 38, 379, 421, 425, 426, 452, 461 Foltyn, O., 224, 500 Fortncx, S., 253, 297 Foster, W. J., 270, 288 Fox, M., 44, 49, 58, 60, 6G, 67 Fraenkel-Conrat, H., 83, 210 Francillon, M., 248, 278, 283, 300 Franke, R., 440 Franklin, R. M., 81, 152, 153, 156, 157, 167, 168, 207, 210, 211 Fraser, D., 91, 105, 142, 213, 216 Fraser, K. B., 197, 211 Frearson, P. M., 100, 173, 174, 178, 179, 180, 182, 183, 184, 185, 186, 187, 189, 191, 194, 211, 214 Frederiksen, S., 390, 460 Frederickson, T., 306, 369, 370, 471 Freed, J. J., 438, 439, 460, 469 Freese, E., 27, 38, 361, 460 French, G., 272, 301 Frenltel, E. P., 29, 38 Frenster, J. H., 256, 292 Frick, G., 274, 299 Fried, M., 84, 209 Friedgoed, C. E., 259, 259 Friedman, H., 54, 67 Friedman, R. M., 155, 219 Friend, C., 84, 209 Frisch-Niggemeyer, W., 83, 211 Fruliarn, G . I,., 59, 67 Frichs, H. J.. 255, 296 Fridge. M., 264, 292 Fridgc.-Mastnangele, M., 231, 300 Fujii, T., 264, 292 Fujimoto, J. M., 21, 38 Fujinnga. K., 201, 211 I~ukasawa,T., 142, 143, 211, 21% Fulcrii. K., 19, 38, 351, 355, 359, 431, 466 Frlkrlolin, F.. 385, 387, 388. 417, 462, 466, .$fi!l
l~iikiislii,T,, 231.
Fiiiiakoslii,
?!In
r<., -105, 4-16, 460
Fiiiioii, I<. B., 48, GG Furst,, -I 411, ., 420, 4GO Furth, J., 51, 63, 67
482
AUTHOR INDEX
G Gabuniia, N. A., 239, g92 Gaerther, H., 281, 298 Gaetani, M., 5, 37 Gafford, L. G., 82, 217, 819 Gagnon, A., 226, 292 Galibcrt, F., 81, 211 Galitski, S. B., 379, 421, 425, 426, 452, 461 Gallagher, R. E., 47, 58, 67 Galy-Fajou, M., 432, 469 Garber, B., 239, 270, 276, 292 Garcia, H., 7, 18, 38, 318, 319, 464 Gasteyer, S., 420, 460 Gatlin, L., 360, 460 Gaulden, M. E., 267, 300 Gavordosi, G., 282, 299 Gefter, M., 137 149, ,908, 211 Geipert, F., 462 Gelbard, A. S., 204, 811 Gelboin, H. V., 21, 38, &8 Gelfant, S., 292 Gentry, G. A., 82, 219 Georgii, A., 384, 460 Gerber, G., 241, 892 Gerber, G. B., 241, d92 Gerber, P., 80,84, 201, 202, 211, 213, 292 Gersh, I., 231, 238, 292 Gerschon, D., 176, 178, 196, 211 Ghadially, F. N., 238, 292 Ghione, M., 5, 37 Ghobar, A,, 309, 315, 343, 461 Giao, N. P., 340, 468 Giewking, R., 5, 39 Giglio, E., 347, 469 Gilberti, A., 236, 289 Gilden, R. V., 89, 173, 174, 175, 176, 177, 178, 187, 192, 209, 211 Gilead, Z., 89, 211 Gill, 5. J., 245, 302 Gilman, T., 241, 292 Gillman, T., 265, 2998 Gilmore, C. E., 64, 68 Ginsberg, H. S., 75, 87, 89, 96, 98, 172, 210, 211, 221
Ginsburg, B. Z.,255, 257, 264, 298 Giovanella, B. C., 351, 352, 355, 460 Girardi, A . ,J., 75, 80, 211, 214 Giri, C . P., 22, 38 Githens, S., III., 242, 296 Glanges, E., 361, 454
G I w c ~ , I d . , 234, 292 Glass, R., 391, 467 Glass, E. M., 140 Gleason, M. N., 284, $98 Glendenning, 0. M., 391, 468 Glick, J. L., 242, 898 Glimcher, M. J., 247, 252, 296, 29G Globerson, A., 5, 9, 58, 58, 65 Glovcr, S. W., 141, 145, 146, 200, 211, 219 Gochenour, A. M., 75, 219 Godal, H. C., 265, 892 Godman, G., 232, 293 Gold, E., 95, 97, 100, 132, 211, 213, 21s Gold, M., 135, 136, 137, 143, 211, 212 Goldacre, R. J., 228, 892 Goldblatt, M. W., 436, 461 Goldenberg, H., 46, 63, 69 Goldhaber, G., 10, 41 Goldstein, J. H., 360, 463 Goldstein, L., 254, 272, 292, 296 Gomatos, P. J., 81, 169, 170, ,911 Good, R. A., 45, 47, 49, 51, 57, 59, 62, 66, 66, 67, 68, 69,70, 286,290 Goodall, A. L., 334, 460 Goodall, C. M., 420, 460 Goodman, D. S., 263, 293 Gopal-Ayengar, A. R., 279, 298 Gordon, J. A., 22, 39 Gordon, R. S., 285, 893 Gorer, P. A., 57, 59, 67 Gorham, J. R., 62, 66,67,68 Gorlcnko, Z. M., 164, 213 Gorrod, J. W., 335, 336, 350, 358, 413, 414, 467 Gorter, E., 248, 274, 293 Gosch, H. H., 307, 461, 466 Goshman, L. E., 434, 460 Gosselin, R. E., 284, 298 Gaudy, B., 449 Gould, S. E. B., 234, 261, 270, $93 Goutier, R., 282, 293 Goutier-Pirotte, M., 282, 893 Gowans, J. L., 44, 57, 61, 67 Gowland, G., 67 Chibar, P., 46, 67 ( i m w , J . T., 74, 75, 907, 2?1 OrafIi, A , . 2, 6, 38 Graham, A . F., 167, 170, 212, 214 Graham, S., 38 Gmmmer, M. G., 21, @
AUTHOR INDEX
c;r:anil, 1,. C., 320. .$6? (;rant, ( i . A,, 51, 60, 62, H S
483
,126, 436, 437. 438. 4.10, ,141, 462, $37, .j?X,4,5/;, 461, 462, 466 Grantham, P. H., 402, 418, 432, 437, 460, Guyrr, M. I(’.,249, 293 461,471 H Greco, J., 265, 271, 2% Grrcn, B., 307, 335, 336, 345, 346, 347, Hahcl, K., 89, 176, 177, 198, 201, 212, 215 348, 349, 350, 351, 352, 355, 358, 359, Hnhcr, I<., 149, 216 413, 414, 436, 441. 442, 450, 467, 458 Hackmann, C., 379, 461 Green, G. E., 391, 461 Haddow, A., 2, 3, 10, 20, 38, 59, 67, 341, Green, H. N., 204, 21;?, 238, 292, 380, 457 355, 389, 466, 461 Grecn, M., 82, 96, 98, 99, 100, 171, 201, Hadidian, Z., 306, 369, 370, 471 21 1 , 217, 293 Hadai, D., 320, 461 Grrenbrrg. G . R., 91, 115, 121, 221, 218, Haendigcs, V. A., 75, 221 219 Hagens, S. J., 55, 62, 66 (:rrrnr, E. I,., 80, 511, 220 Haginara, S., 140, 146, 220 Crc.grisova, V., 204, 217 Hagstroin, B. E., 226, 227, 231, 251, ?!!1;3, Grcisnian, S. E., 285, 39.3 300 Grette, K., 249, 993 Hahn, M. A., 28, 58 Grey, C. E., 82, 200 Hall, B. D., 103, 216 Grice, H. C., 371, 375, 377, 464 Hall, C., 169, 317 Griesbach, I,., 309, 315, 343, 461 Hall, D. H., 115, 212 Griffin, E., 63, 65 Halver, J. E., 402, 464 Griffin, M., 370, .$60 Ham, R. G., 267, 203 Griggs, H. M., 439, 459 Hamada, C., 88, 99, 100, 125, 212 Grimmer, G., 322, 461 Hamdan, A. A., 392, 393, 394, 396, 46s Griswold, D. P., 370, 371, 398, 450, 4Gl Hamerman, D., 204, 212 Gritsiute, L. A,, 6, 3s Hamilton, I,. H., 240, 290 Hamilton, T. R., 237, 300 Groisser, V., 276, 292 Hanimon, W. McD., 80,207 Gross, J. A,, Jr., 242, 263, 266, 293, ?!I0 Hamper], H., 352, 468 Gross, I,., 46, 59, 63, 67, 75, 82, 211 Hanafusa, H., 206, 212 Grossfrld, H., 203, 215, 232, 203 Hanafusn, T., 95, 98, 132, 170, 206, 212 Grossfield, H., 239, 279, 293 Hanaki, A., 409, 469 Grosaman, N. S., 238, 294 Handschumacher, R. E., 3,38 Grosso, E., 178, 216 Hansen, W. H., 364, 464 Grossowicz, M., 267, 207 Hanson, R., 281, 303 Grover, P. L., 335, 367, 368, 412, 414, 415, Haran, N., 6, 12, 36, 39 416, 448, 449, 457 Haran-Ghera, N., 3, 4, 6, 7, 9, 10, 12, 13, Grubhofer, N., 277, 205 16, 17, 18, 19, 22, 23, 26, 29, 36, 39 Harding, C. V., 226, 227, 228, 293 Grubbs, G. E., 75, 80, 610, 619 Harding, D., 226, 227, 293, 294 Grubin, A. E., 242, 299 Hardwood, T. R., 235, 298 Gruenstrin, M., 320, 461 Hardy, W. G., 244, 245, 251, 252, 289 Gubarrff, N., 3, 4f Harm, W., 148, 212 GuCrin, M., 13, 40 Harpel, P., 238, 294 Gulick, Z. R., 280, 281, 283, 294 Harper, K. H., 337, 338, 461 Gurd, F. R. N., 263, 285, 293 Harpur, R. P., 239, 293 Guri, C. D., 293 Harris, D. L., 285, 300 Gustafson, T., 249, 259, 203, 300 Harris, R. J. C., 51, 63, 67, 239, 298 Gutmann, H. R., 369, 379, 381, 421, 425, Harrison, G. A., 44, 58, 60, 69
484
AUTHOR INDEX
Hartman, M. M., 293 Hartmann, H. A., 381, 419, 421, 423, 461, 466 Hartmann, J. F., 230, 247, 193 Hartree, E., 273, 296 Hartwell, J. L., 308, 309, 315, 319, 341, 342, 368, 369, QGl, 4G8 Hartwcll, L. H., 174, 175, 178, 180, 183, 184, 185, 210, 212 Haruna, I., 152, 157, 159, 160, 112, 119 Harvey, R. G., 460 Haselkorn, R., 155, 156, 219 Hashimoto, Y., 417, 4 G l HaSek, M., 45, 48, 67 Hashish, S. E. E., 256, 203 Hass, F. M., 460 H a s , G. M., 380, 384, 385, 404 Hatanka, M., 181, 183, 212 Hathaway, R. R., 229, 293 Hattman, S., 115, 142, 143, 145, 212, 217 Hauschka, T., 267, 300 Hausen, P., 155, 156, 167, 168, 196, ell, 216, 220 Hausmann, R., 135, 136, 137, 143, 111, 216
Hay, A. S., 373, 468 Hay, J., 123, 150, 171, 212, 213, 219 Hayashi, Y., 387, 435, 461 HRY~S L., I,., 71 Haymann, H., 280, 281, 283, 294 Heaysman, J. E. M., 257, 26'7 Hecker, E., 462 Hedrick, R. M., 266, 293 Heeter, M., 167, 221 Hegyvary, C., 255, 293 Heidelberger, C., 307, 309, 315, 343, 351, 352, 355, 434, 4G4, 4GO, 4 G l Heilbrunn, L. V., 224, 227, 228, 238, 243, 253, 254, 259, 266,275,203,294,303 Heilweil, A. G., 352, 461 Heine, V., 81, 82, 208 Heller, H. E., 442, 4 G l Heller, J. H., 286, 294 Helger, B. J., 45, 56, G7 Helmboldt, G. F., 62, 67 Helmkamp, G. K., 391, 470 Hempelmann, L. H., 241, 292 Hendricks, A. G., 230, 238, 296 Hendricks, J. B., 359, / G I Hendry, J. A., 20, 40
l i c d i a w , E. C., 438, 140, d/;l Henshaw, P. S., 1, 3, 39, 40 Henson, A. F., 436, 461 Henson, J. B., 62, 67, G8 Heppleston, A. G., 38 Herbest, E. J., 267, 294 Herbst, C., 252, 257, 294 Herde, P., 132, 216 Herriott, S. T., 93, 123, 216 Hersh, R . T., 270, 296 Hertlein, W., 241, 303 Hery, H., 6, 37 Heslop, B. F., 44, 46, 47, 53, 57, 59, 60, GS Hcss, S. M., 30, 35 Heston, W. E., 11, 12, 39 Hcwett, C. L., 325, 328, 456 Heyde, M., 48, G9 Heztrin, S., 237, 501 Hiatt, H. H., 438, 440, 461 Higashi, T., 26, 40 Higashinakagawa, T., 446, 4 G l Higashino, S., 253, 298 Higginbotham, R. D., 286, 894 Highberger, J. H., 263, 266, 293 Hildebrandt, A., 322, 4 G l Hildemann, W. H., 44, 46, 47, 58, 60, 61, 67, 70 Hilgard, H. R., 45, 49, 59, 6'7 Hill, J. T., 462 Hill, W. T., 235, 253, 295 Hillcman, M. R., 75, 80, 211, 214, 210, 224, 225, 282, 283 892, 296, 302 Hillmnn, N. W., 282, 284 Hinuma, Y., 88, 220 Hirafuku, I., 389, 466 Hiramoto, H., 230, 294 Hirata, Y., 383, 386, 462 Hisamatsu, T., 388, 4 G l Hitchings, G. H., 20,57 Ho, P. P. K., 152, 154,212 Hoch-Ligeti, C., 319, 320, 449, 461 Hodas, S., 29, 42 Hodes, M. E., 244, 246, 294, 299 Hodge, H. C., 284, 292 Hodnett, E. M., 257, 894 Hoffert, W. R., 75, 212 Hoffman, D. C., 279, 294 Hoffman-Berling, H., 249, 250, 251, 204 Hoffmann, D., 309, 312, 314, 402, 471 Hoffmann, F., 2, 6, 38
485
AUTHOR ISDEX
Horffiiigcr, J. I'., 460 Hofstw, B. H. J., 277, 294 Hogberg, B., 235, 277, ZSS, 292 Hoggnn, M. D., 80, 83, 88, 192, 199, 212, ,018
Holland, I. B., 160, 219 Holland, J. F., 265, 300 Holland, J. J., 167, 168, 212 Hollowray, B. W., 140, 146, 212 Holm, C., 54, 67 Holmes, M. C., 45, 46, 56, 62, 66, 67 Holmgren, H., 235, 243, 288 Holsti, P., 285, 294 Holtfreter, J., 231, 244, 252, 258, 271, 294, 502
Holtser, H., 232, 244, 248, 294 Homburgcr, F., 238, 294, 448 IIoninia, M., 167, 170, 212 Hooprr, J. L., 153, 216 Hopp, M., 462 Hordr, S., 226, 298 Horie, A,, 388, 389, 462 Home, It. W., 82, 21G, 221 Homing, E. S., 381, 383, 389, 464 Horvath, C., 233, 234, 235, 236, 237, 239, 240, 255, 290, 291 Horvath, J., 233,237,291 Horn, H. D., 252, 276, 294 IIorstanius, S.,231, 300 Norton, E., 152, 153, 156, 212 Hoshino, H., 417, 462, 469 Hosliino, T., 8, 9, 39 Hoster, H. A., 258, 261, 294 Hoster, M. S., 258, 261, 294 Hotchin, J., 45, 54, 55, 63, 67 Hotchkiss, R. D., 285, 294 Houck, J. C., 281, 294 House, W., 62, 68 How, S. W., 380, 4G2 Howard, J. G., 44, 45, 48, 49, 50, 56, 58, 60, G5, 66, 67, 68 Howard-Flanders, P , 148, 149, 208, 210 Howatson, A . F., 80, 208 Howe, A. F., 242, 288 Howr, C. D., 58, 60, 69 Howell, J. S., 399, 433, 4GO, 462 Howic, J. B., 45, 56, G7 Hozunii, M., 429, 436, 463, 462 Hraba. T.. 45, 48. 67 Hsu, T. C., 95, &, 170, 177, $13, 817
IIuang, A. S., 168, 212, 220 Huaiig, T., 431. 456 Hubhartl, I<. W., 277, 280 Huebnrr, R. J., 75, 88, 89, 192, 199, 201, 208, 212, 21s Hueper, W. C., 17, 19, 39, 320, 366, 369, 381, 383, 4G2, 463 Huggins, C. B., 320, 334, 460, 462 Hughes, E. D., 442, 4 G l Hughes, G. M. K., 342, 405, 4G2 Hughes, R., 176, 177, 178, 216 Hughes, W. L., Jr., 274, 298 Hull, R. N., 75, 212, 213 Hultin, E , 297 Hulton, H. O., 248, 294 Hultin, T., 229, 252, 294 Human, M. L., 140, 215 Hume, D. M., 55, 69 Hummel, J. P., 247, 277, 281, 294, 301 Hurrphrey, J. H., 57, 66 Humphries, A. A., Jr., 232, 296 Hunter, H. S., 81,217 Hunter, W. S., 84, 209 Hurwitz, J., 135, 137, 149, 164, 208, 211, 916 Husemann, E., 241, 303 Hutchison, 0. S., 22, 24, 40 Hutncr, S. H., 417, 462 Hutson, J. C., 120, 209 Hytlr, J., 82, 217 HyI:ind, R., 255, ,9996
I Ibanes, M. L., 9, 12, 89 Ida, N., 8, 39 Imagawa, D. T. S., 89 Imamnra, A . , 19, 38, 306, 350, 351, 353, 354, 355, 358, 359, 361, 362, 363, 428, 429, 431, 432, 4G3, 466 Immers, J., 224, 226, 227, 228, 229, 231, 296, 300, 302 Indcrbitzin, T., 245, 296 Inglis, M. S., 449 Ingold, C. K., 442, 461 Ingram, B. J., 5, 6, 88 Inuzuka, 429, 436, 462 Irving, C., 306, 369, 379, 380, 381, 391, 402, 419, 422, 423, 425, 437, 438,~.441, 443, 444, 462, 462, 464, 471 Iscnberg, I., 353, 358, 362, 462, 469
s., c.
486
AUTHOR 1NI)IEX
Tslii(lti((>, M., 406, 400, 462, d(;S IsliiL:r\vn, M., 226, 2!Lj Ishinioto, N., 204, 213 Isidor, P., 236, 2.96 Ito, T., 8, 9, 39 Ito, Y.,84, 200, 215, 218
J Jarobsen, 1,. O., 241,288 Jacobson, B., 264, 283 Jacquiqnon, P., 319, 326, 328, 460, 463, 464 Jaffc, W. G., 13, 59 Jahn, U., 18, 19,39 James, A. M., 246, 288 Janieson, M. F., 74, 20s Jamison, R . M., 80, 81, 82, 173, 213, 215, 216 Janoff, A., 240, 296 Janscn, C. R., 240, 283, 290, 296 Jaqurt-Francillon, M. L., 278, 283, 300 Jarova, E. I., 384, 4GY Jellinck, P. H., 449 Jencks, W. P., 22, 39 Jenscn, C. E., 238, 275, 290, 296 Jenscn, E., 57, 70 Jensen, F. C., 80, 202, 211, 214 Jcnsen, W. N., 63, 66 Jeon, K. W., 250, 256, 289 Johnson, I. S., 75, 213 Joklik, W. K., 82, 99, 100, 102, 103, 104, 105, 106, 110, 123, 132, 133, 171, 808, 213, 215 Jolad, S. D., 321, 455 Jones, J. B., 362, 462 Jones, P. C . T., 246, 288 Jones, R. N., 373, 468 Jones, V. E., 61, 68 Jordan, L. E., 80, 81, 173, 215, 216 ,Joske, R. A,, 44, 45, 46, 54, 60, 61, 63, 64, 07, YO Jossr, J., 91, 92, 93, 138, 139, 166, 213, $14 Joubert, F. J., 274, 299 J d i l , U., 28, 40, 423, 443, 444, 463, 465 Jull, J. W., 306, 367, 368, 369, 370, 379, 381, 405, 412, 415, 416, 457, 459 Jung, M. L., 399, 467
LOO, 102, 103, 105, 106, 110, 12.7, 132, 133, 2 f q l Jiirkicwim, M. J., 236, 299 Jutila, J. W., 45, 52, 56, 69, 71 J ~ i i \ ~ w i i l lC., i,
K ICacsberg, P., 85, 210 Kahan, F. M., 94, 166,215 Kahlson, G., 264, 295 Kahn, M. F., 63, GY Kaiser, A., 27, 3G Kajima, M., 46, 63, G9 Kakcfudn, T., 383, 388, 463, 4G9 Kalcknr, H. M., 142, 219, 242, 261, 296 Kalla, 1%.L., 233, 239, 296 Kallen, R. G., 93, 213 Kamahora, J., 75, 95, 97, 613, 216, 210 Kamci, H., 239, 279, 295 Kameyamn, S., 75, 97, 216, 219 Kaminc,r, B., 270, 302 Kamiya, T., 99, 100, 125, 134, 212, 213 Kane, J. F., 307, 454 Kanr, R. E., 270, 295 &no-Sneoka, T., 150, 219 Kapa, E., 228, 233, 234, 239, 890 Kaplan, A. S., 82, 88, 95, 97, 99, 100, 125, 134, 170, 208, 209, 212, 213, 216 Kaplan, D., 241, 297 Kaplnn, H. S.,8, 9, 10, 12, 39, 45, 46, 49, 63, G7 Kaplsn, L., 390, 458 Karasaki, S., 80, 211 Karrcman, G., 361, 431, 462 Kass, S. J., 82, 214 ICataoka, N., 428, 431, 466 Katchalsky, A,, 259, 270, 274, 275, 296, 501
Katchalski, E., 272, 274, 276, 292, 296, 296, 2 9 ~ Kates, J. R., 133, 134, 216 Kato, S., 74, 75, 97, 170, 213, 216, 219 Katz, A. M., 285, 300 Katzberg, A. A., 230, 238, 295 Kaufman, R. J., 258, 285, 288, 299 Kaufman, S., 405, 462 ICaufmann, B. P., 256, 281, 2.95, 297 Kaufmann, R . J., 285, 296 Kaump, D. H., 367, 462 Kawachi, T., 383, 386, 462 Kawamoto, S., 8,9, 12, 39
I
Kirchskin, R . I,., 75, 80, 201, 209, 211,
Kaye, A. M., 3, 9, 14, 21, 22, 23, 24, 26, 30, 31, 36, 38, 30, 40, 164, 178, 180, 216, 221, 307, 462 Iiearns, K. E., 282, 288 Krast, D., 44, 45, 46, 47, 53, 54, 61, 62, 63, 67, 68, 69, 70, 71 I
Kirschbaum, A,, 4, 8, 9, 12, 39, 41 Kish, V. M., 439, 469 Kit, S., 82, 95, 96, 97, 99, 100, 102, 103, 104, 105, 106, 108, 109, 116, 117, 118, 119, 123, 124, 125, 170, 171, 172, 173, 174, 176, 177, 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 191, 194, 195, 196, 202, 209, 210, 211, 213, 214, 216 Kitagawa, M., 437, 463, 469 Kitahara, 88, 177, 193, 199, 217 Kiviniemi, K., 239, 302 Kizer, D. E., 234, 296 Kjellen, L., 96, 98, 214 Klaasrn, D. H., 393, 395, 427, 440, 458 Kyarner, P., 5, 39 Klein, A., 148, 214 Klein, E., 251, 265, 274, 289 Klein, G., 246, 254, 209, 300, 389, 463 Klein, M., 4, 8, 11, 13, 21, 25, 38, 39, 309, 319, 363, 448, 463 Kleinschniidt, A. K., 82, 214 Klcinschmidt, W. J., 224, 225, 295, 2 9 Kline, I., 233, 239, 296 Klingenhcrg, H. G., 236, 295 ~ i ~ A., ~ 75, g ,214 Knight, C. A,, 82, 85, 155, 156, 208, 214, 220 I h o x , W. E., 384, 463 Koch, M. A , , 85, 88, 89, 193, 202, 207,
213, 217
218
Kodama, M., 306, 350, 351, 353, 354, 355, 358, 361, 362, 363, 428, 429, 431, 432, 463, 466 Koerner, J. F., 105, 107, 126, 127, 128, 165, 166, 209, 214, 216, 219 Kofler, M., 27, 36 Kohclii, S., 388, 389, 462 Icojinia, h l . K., 230, 243, 295, 296 Kokko, J. P., 360, 463 Kollcr. 1'. C., 2, 20. 23, 36 T G ~ I I : ~ ~ , w,(3s lioiiininos. %. I),, 4;. 63, c;!l T i o t i t l o , h l . J . , 245, 248, ?!I/; Iioiit.teki, W., 255, :?!h7 Kopplc, K. D., 273, 2llG Koprowslti, H., 80, 202, 211, 214
iw.,
485
A U'I'HOR 1 N D16X
Korn, D., 126, 128, 130, 131, 214, 217, 220 Kornberg, A., 91, 92, 93, 122, 138, 139, 166, 207, 213, $14, 221 Kornberg, S. R., 91, 92, 93, 138, 139, 166, 214, z2t Korngold, G. C., 27, 36 Korosi, J., 233, 235, 236, 240, 291 Korzis, J., 425, 471 Kotchekov, N. K., 425, 470 Koteles, G. J., 150, 171, 818 Kotin, P., 336, 337, 338, 460, 463 Kotliar, A. M., 256, 257, 298 Kozawa, S., 354, 470 Kozinski, A. W., 105,8l4 Kozloff, L. M., 127, $14 Krahl, M. E., 345, 469 Krane, S. M., 247, 296 Kraus, F., 255, 296 Kreis, W., 28, 41 Kriek, E., 423, 431, 443, 444, 463 Krim, M., 23, 27, 28, 36 Kriszat, G., 224, 227, 249, 250, g36, SO0 Kudo, H., 170, 214 Kuhar, S., 255, 283, 300 Kuhn, W., 275, 896 Kume, F., 387, 460 Kunii, A., 51, 63, 67 Kunkee, R. E., 127, 163, 214,816 Kunkel, H. G., 63, GS, 286, 2S7 Kuno, S., 137, 214 Kuratsune, M., 320, 388, 389, 46'2, 463 Kyune, M. F., 283, 2S9
1 Lachman, A. B., 244, 289 Lacawagne, A., 308, 309, 312, 313, 315, 319, 324, 325, 326, 328, 334, 342, 388, 394, 448, 460, 463, 464 Lacroix, G., 433, 469 Ladenheim, H., 273, 296' Lahsm, S., 371, 375, 377, 464 Laipis, P., 82, 220 Lalich, J. J., 364, 467 Ldlier, R., 224, 226, 227, 232, 242, 248, 259, 263, 264, 296' I,anipson, G. P., 224, 225, 282, 283, 996, 302 Landau, J. V., 249, 250, 296, 304 Lane, M., 8, 11, 14, 39
314, 340,
243, 292,
Lane, W. T., 75, 89,201, 2f2,218 Langemann, A., 27, 36 Langridge, R., 155, 814 Langs, L., 370, 471 Lankford, E., 285,296 Lansing, A. I., 254, 296 Lapidot, A., 4, 16, 17, 18, 19, 26, 29, 36 Largier, J. F., 274, 299 Larsen, A. E., 62, 69 Lanen, C. D., 3, 5, 14, 16, 17, 18, 19, 25, 39, 40 Larsen, V. M., 75,214 Larson, D. M., 379, 381, 46'2 Lamson, L. G., 302 Laakina, A. V., 239,296 Laskowski, D. E., a 9 La Sorte, A. F., 279, 299 Lasamann, G., 362, 427, 469 Lastargues, E. Y., 232, 236, 899 Laurent, T. C., 235, 242, 267, 268, 274, 288, 296 Lausing, E., 234, 288 Laver, W. G., 86, 216 Lavit-Lamy, D., 309, 312, 313, 314, 325, 334, 448, 463, 46'4 Law, L. W., 4, 10, 11, 12, 14, 39, 41, 51, 66
Lawley, P. D., 307, 359, 434, 435, 438, 468, 464 Lawrence, H. S., 54, 68 Lawrence, J. S., 241, 296 Lawson, T. A., 380, 404, 460, 467, 4660 Tazaro, E. J., 281,294 Lazzarini, A. A., 245, 261, 263, 264, 296 Lazzarini-Robertson, B., Jr., 245, 896 Lea, A. J., 39 Leader, R. W., 62, 66,67, 68 Leak, P. J., 44, 45, 46, 54, 55, 60, 61, 63, 64, 67, 69, 70 LeBoeuf, B., 238, 289 Lebowitz, J., 82, 220 Lederberg, S., 143, 144, 146, 216 Lec, A. J., 82, 209 T m , €1, F., 259, 283 T m , I<. Y., 21, 22, 39 r.rlc,, s. s.,336, 337, 460 r,pcia, M., 347, 464 Lehman, I. R., 93, 114, 126, 128, 137, 209, 214, 916, 282, 896 Leidy, G., 22, 4
489
ATJTIIOR INDEX
Lcighton, J., 233, 230, 296 Leilniisis, A., 111, 210 Lriter. J., 468 I , c ~ i t I i , K . S., 460 I t i w , S.,7, 38 J,(:ngc:rov:i, A,, 45, 48, 6" TJc:nnettc, E. H., 89, 218 Leong, J. I,., 449 LePage, G. A., 248, 282, 296 Lerman, L. S., 352, 391, 464 Leskowitz, S.,61, 68 Letham, D. S., 255, 257, 296 LettrC, H., 247, 249, 296, 315, 464 Leutze, C. J., 307,464 Lcvene, L., 22,S9 Leveque, T. E., 235, 300 Levin, A. P., 163, 216 Levin, W., 449 Levin, Y., 272, 292, 296 Levine, E., 307, 430, 431, 466, 470 Levine, E. M., 258, 262, 296 Levinthal, J. M., 74, 216 Levintow, L., 153,215 Levvy, G. A., 234, 295 Lewis, F. B., 63, 68 Lcwis, G. E., 411, 464 Lewis, N., 112, 113, 121, 220 Lewis, R. M., 64, 68 Liao, S., 460 Lichtenstein, J., 90, 92, 93, 105, 137, 209, 210, ,215 Liebclt, A. G., 8, 11, 14, 39 Liebclt, R. A , , 8, 11, 14, 39 Licbcrman, I., 246, 258, 296 Lieberman, M., 9, 10, 39 Lifson, S., 267, 296 Light, R. A., 236, 297 Iijinsky, W., 25, 37, 318, 319, 320, 330, 334, 460, 404 Tin, J.-K., 447, 464, 4G4 rindberg, O., 281, 296 Linderot, T., 277, 292 Iindner, J., 239, 240, 279, 296 Lindstrom, H. V., 364, 464 Lindvall, S., 254, 282, 296 Linker, A,, 232, 293 Linker-Israeli, M., 5, 4 l Lippman, M., 228, 244, 259, 296'
Liqiinri, A. M., 347, 348, 350, 355, 469, 4G.S
1,isimvivz. J.. 281. 292 Tlis~n.I,,, 276, 996 I,it,t:tlw, 11. %., 281, Z!//j Jilt,li,livld,J. W., 82, 197, Jla', 220 I,itv:Lk, S. F., 377, 469 Liu, S. T,., 152, 153, 156, 212, 349, 458 Lo, H. W., 367, 460 Lobel, E. M., 273, 296 Lochte, H. L., Jr., 256, 297 Lodish, H. F., 154, 155, 157, 161,616 Loeb, L., 226, 258, 297 Loed, M., 281, SO3 Loge, J. P., 2, 39 Longeviallc, M., 228, 291 Lorch, I. J., 228, 292 Lorenson, M. Y., 193, 216 Lorenz, B., 280, 281, 297 Lorenz, E., 319, 464 Lorcnz, R., 280, 281, 297 Lotlikar, P. D., 28, 40, 306, 391, 402, 414, 418, 421, 422, 423, 425, 427, 443, 444, 462, 46’0, 464, 465 Lotterle, R., 303 Loufit, J. F., 45, 48, 60,68 Love, R. L., 245, 248, 297 Lovelock, J. E., 347, 464 Lowenstein, W. R., 253, 298, 299 Lowick, J. H. B., 246, 288 h i , P., 434, 470 T,ubnrslri, S. W., 81, 216 Lundblad, G., 229, 231, 282, 287 Lunger, P. D., 80, 216 I,unt, M. R., 139, 216 T,riongo, L., 439, 469 I m i a , S. E., 112, 115, 140, 166, 216, 220 Lnscher, E. F., 249, 289 Luscombe, M., 237,29Y Lustgraaf, E. C., 48, 66 Luzzati, V., 352, 391, 464 J,yn, G., 149, 20.8 Lynn, W. S., Jr., 253,297 Lyon, H. N., 226,297 Lyons, M. J., 81, 82, 216
M Mimsnb, H. F., 81, 216 Mabillc, P., 325, 343, 4G8,464
350,
403, 437,
227,
490
AUTHOR INDEX
M(*.iuslnn, l3. R., 99, 100, 102, 105, 108, 110, 124, 125, 132, 133, 134, 210, 215 h l ~ ~ B e€3e , G , 258, 259, 261, 294 Mc*13rlde,H . A , 71 McHntlt-, It. Z , Uti, [(i'j M~CiLll(lll~hs, lC. I,., 237, 297 McCartcr, J. A,, 349, 353, 456 McCarthy, P., 392, 393, 394, 458 McCleary, R. S., 236, 297 McClurg, C. B., 226, 297 McCoy, T. A., 234, 296 McCrea, J. F., 82, 217, 345,464 McCue, M. M., 438, 469 McDearman, S., 279, 301 McDonald, J. H., 380, 384, 385, 450,464 McDonald, M. R., 256, 295, 297 McFarland, V. W., 81, 216 Machlis, L., 256, 297 Macht, D., 234, 255, 297 McIntire, K. R., 50, 56, 68 McIntyre, M. H., 334, 460 McIver, F. A., 384, 467 McIwain, H., 247, 301 MrKinney, G. R., 22, 40 McKinney, L. E., 351, 352, 355, 460 McLean, I. W., Jr., 75, 208 McLear, J. H., 249, 296 Macleod, C. M., 274, 290 McLimans, W., 262, 297 McLoughlin, C. B., 232, 297 Magee, P. N., 19, 41, 361, 464 Magee, W. E., 96, 99, 100, 123, 215, 218 Mager, J., 267, 297 Maggio, R., 229, 231, 297, 298 Maguigan, W. H., 375, 469 Mahler, H. R., 91, 105,213 Maiorano, G., 8, 9, 14, 25, 37, 38 Mnitra, U., 135, 137, 211 Maizrl, J. V., 85, 87, 88, 215, 219 Maley, F., 91, 193, 215 Malcy, G. F., 91, 193, 215 Malkin, M. F., 432, 4Gd Mallett, J. M., 13, 40 Malloy, P. L.,392, 393, 394, 455 Malmgren, R A., 4, 40, 176, 198, 216, 219 Malngren, H., 239, 293 Malpoix, P., 54, 68 Maltonj, C., 4, 8, 12, 13, 14, 40, 41 Mnndel, H. G., 155, 215, 217
Mnnclc-I, M., 1!)8, 217 hl:il1tlrll, l,.. 360, .SS3 ilIansl~r~rg(~r, A . R., ,Jr., 235. 300 Mnnsnn, I).. 335, 336, 350, 358, 413, 414, 415, ,136, 442, 457 N., 306, 369, 370, 471 M:uil,~~l, Miirgrctli, A., 371, 372, 373, 378, 396, 414, 422, 423, 464, 46'5 Marikeusky, Y., 259, SO1 Mark, H., 256, 257, 298 Markus, G., 245, 248, 297 Marmnssc, C., 432, 464 Marinur, J., 93, 94, 213, 219 Marro, F., 255, 290, 297 Marroquin, F., 437, 464 Marsland, D. A., 230, 249, 250, 296, 297, 298, 304
Martin, E. M., 81, 152, 153, 155, 156, 167, 210, 212, 215, 219 Mart,in, G. J., 277, 280, 268 Martinez, C., 45, 47, 49, 51, 59, 62, 6'6, 67, 68
Mason, F., 352, 391, 464 Mason, R., 342, 347, 458, 464 Massie, A., 63, 65 Mast, S. O., 254,299 Mastromatteo, E., 366, 466 Math6, G., 241, 297 Mather, G. C., 240, 283, 290, 296 Mathcws, C. K., 91, 93, 115, 119, 121, 216 Mathison, J. H., 307, 433, 456 Matis, P., 235, 299 Matsubara, F., 258, 299 Matsumoto, M., 407, 408, 439, 446, 461, 4G5, 471 Matsuenwa, T., 46, 63, 69 Matthcws, M. B., 255, 261, 297 Matthcws, It. E. F., 155, 225, 217 Matus, A . I., 155, 215, 217 Maurcr, P. H., 274,297 Mayhew, E., 246, 297 Maylicw, G., 251, 303 Mayor, H. D., 80, 81, 82, 173, 199, 213, 215, 216 Mazsrirelln, I,., 11, 38 Mazzia, D., 250, 254, 297 Mecke, It., 407, 425, 460, 465 Mcdawar, P. B., 44, 65 Medina, D., 80, 2 f 8 Mrhlcr, A. H., 384, 463
Misic,r, It., 284, 9011 Mi~inscIi(~in, W. G., 321, &6 Mekler, 1,. 13., 64, GS Melby, R., 373, 468 Mellow, R. C., 56, 62, 68 Mclnick, J. L., 75, 80, 82, 88, 173, 176, 177, 178, 179, 182, 183, 184, 185, 187, 189, 191, 193, 194, 196, 198, 199, 201, 207, 20S, 209, 213, 214, 216, 216, 217 Melvin, I. S., 347, 470 Menczyk, Z.,384, 4G2 Mendel, B., 285,237 Menrs, G. E. F., 274, 200 Mrranzc, D. R.. 320, 461 Merignn, 'r. C., 224, 225, 226, 241, 270, 297, ms, 300 Mcrlandcr, R., 255, 230 Messrlson, M., 144, 215 Mrssina. I,,, 226, 6.07 M(~sIiarcl,I'., 22, 37 h l r t d f , D., 51, 60, 68 Meta, C. B., 229, 207 Metzger, I<., 114, 221 Meyer, H. L., 1, 3, 39, 40 Meyer, K., 232, 241, 260, 293, 297 Meyer, R. I,., 280, 281, 283, 294 Mirhel, M. R., 178, 181, 2’70 Michelacci, S., 272, 301 Miclric, D., 44, 47, 49, 58, 60, 66, G7 Micklem, H. S., 45, 48, 68 Midrr, G . B., 319, 4G5 Mihailovich, N., 8, 14, 25, 41, 42 Mihiclr, E., 240, 297 Miller, A,, 338, 403 Miller, E. C., 17, 26, 28, 30, 40,306, 307, 333, 336, 341, 363, 371, 372, 373, 375, 376, 378, 391, 396, 397, 402, 403, 405, 407, 414, 415, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 437, 443, 444, 445, 446, 447, 452, 454,469, 4G0, 463, 4G4, 4G.5, 467, 4GY Miller, J. A,, 17, 26, 28, 30, 40, 306, 307, 333, 336. 341, 342, 343, 363, 372, 373. 375, 376, 378, 381, 391, 392, 393, 395, 396, 397, 398, 399, 402. 403, 405, 407, 411, 414, 415, 417, 418, 419. 420, 421, 422, 423, 4 3 , 425, 426. 427, 437, 438, 439, 141, 4-19, 44?,. 444, -145, 446, 347, @", $54, , j Z , ,$C!), j/;o, ,$/id, .j/;,$, ,$/;5, #{W,4ns
M i l h , -1. 11'. A. I)., 45, 50, 51, 56, 60, 62, G3, GS Miller, 0. V., 123, 215 Millrr, It. R., 273, 296 Miller, T. E., 240, 297 Millican, D., 13, 40 Millington, R. H., 303 Mills, D., 160, 219 Minis, C. A., 55, 62, 68 Minagawa, T., 105, 216 Minner, J. R., 75,212 Miiiowada, J., 178, 216 hlirsliy, A. E., 256, 270, 273, CSS, 292, 297 Mirvish, S. S., 22, 24, 25, 26, 27, 30, 31, 32, SG, 37, 40 Mist, S. H., 276, 302 Mitc:hcll, J. H., 22, 23, 24, 37, 40 Mitchell, V., 81, 216 Mitchley, B. C. V., 306, 333, 367, 368, 412, 468, 470 Miyimoto, H., 74, 75, 170, 213 Mixuno, N., 105, 2lG Modest, E. J., 267, 288 Mijller, E., 54, 68 Moller, G., 54, 68 Moesclilin, S., 2, 40 Mohler, W. C., 29, 40 Mold, K., 228, 233, 235, 236, 237, 239, 240, 290, 291 Moldenhauer, M. G., 343, 461 Molholt, B., 142, 216 Moloney, J. B., 46, 63, G.3 Molteni, P., 178, 21G Monkhouse, F. C.,241, 275, 278, 297, 298 MonnC, L., 226, 244, 261, 262, 280, 298 Monnot, I>.,6, 37 Monroy, A , , 224, 227, 228, 229, 231, 297, 295, 300 Montagnicr. I,., 84, 155, 207, 209, 216, 220 Montesano, 12., 369, 377, 400, 468 Montgomt.ry, P. O'B., 432, 466 Montgonirry, R., 274, 290 Montipo, W., 8,11, 12, 13, 37 Monty, K. J., 268, gt91 M o o r i ~ ,-4.It., 286. 2!18 hloorc., L). H., 81, 82, 215 R I i i o i i A . F. D., 268, 2!)S hloow, (;. I<.. 178. hfoorc~.11. F., 81, ?1S
492
AUTHOIl INDEX
Moore, M., 199, 200, 201, U S , 435, 449, 45.3, 456
Moor?, M. A. S., 44, 58, 60, 69 Moorhead, P., 80, 214 Mora, P. T., 81, 216, 262, 274, 277, 278, 898, 299 Morawetz, H., 256, 257, 273, 274, 277, 20G, $98 Morgan, H. R., 203, 210 Morgan, M. A., 440, 441, 466 Mori, K., 383, 388, 389, 461, 465 Mori-Chaves, P., 6, 40 Morrione, T. G., 263, 266, 29s Morris, B. T., Jr., 45, 49, G6 Morris, C. It., 379, 381, 460, 468 Morris, H. P., 363, 379, 402, 437, 460, 466, 4~9,4~1
Morris, J. E., 364, 450, 467 Morris, R. K., 281, 294 Morrison, J. M., 123, d l 3 Morse, S. I., 240, 245, 292 Morton, J. J., 319, 466 Moscona, A., 244, 269, 298 Most, S., 298 Mostofi, F. K., 5,4O Motomura, I., 231, 286, 298 Mottram, J. C., 337, 470 Mowry, D. T., 266, 203 Mudd, S., 234, SO0 Muel, B., 433, 469 Muller, M., 19, 37 Mukai, F., 368, 414, 452, 456 Mulay, A. S., 394, 397, 398, 399, 465, 666 Mulhern, A. I., 240, 297 Muller, T. C., 273, 296 Mulnard, J., 226, 230, 201, 298, 299 Munn, A., 366, 376, 390, 391, 466 Munson, A,, 225, 256, 208 Munyon, W. H., 99, 103, 104, 106, 108, 109, 117, 176, 177, 178, 216 Murphy, E. B., 225, 295 Murphy, M. I,., 20, 28, 37 Murray, J . C . , 45, 48, 69 Murray, M. R., 232, 236, 299 Mustaars, W., 276, 298 Myers, D. K., 285, 8.97 Myers. J., 51, 69
N Xndknrni. M . V., 361, 4CO Nagao, M., 417, 469
Nagasaw:i, 1%.T., 437, 440, 441, 46f, QGG Nagata, C., 19, 38, 306, 350, 351, 353, 354, 355, 358, 359, 361, 362, 363, 428, 429, 431, 432, 463,466 Nagaya, H., 55, 68 Nagington, J., 82, 216 Nagoya, J., 239, 279,295 Naidoo, S. S., 265, 298 Nakahara, W., 383, 385, 386, 387, 388, 451, 462, 466 Naltano, E., 257, 298 Nakano, M., 225, 283, 289 Nakas, M., 253, 298 Nakata, Y., 26, 40 Nanningn, I,., 248, 274, 203 Napicr, D. G., 394, 397, 466 Narrod, M., 251, 265, 274, 289 Nathan, A , , 336, 337, 460 Navarette-Hcgina, A., 377, 466 Naylor, B., 234, 290 Nccco, A., 228, 201 Necheles, H., 236,889 Nectoux, F., 434, 469 Negroni, G., 63, 68 Neilands, J. B., 418, 466 Nelson, A. A., 364, 46'4 Nelson, J. B., 45, 55, 68 Nelson, N., 413, 415, 470 Nemes, M. M., 224, 282, 296 Nery, R., 22, 23, 26, 27, 28, 31, 32, 33, 34, 3G, 3'7, 40, 436, 442, 467 Nespoli, M., 240, 29s Nettlcship, A,, 1, 3, 40 Ncuman, W. F., 252, 289 Newcomh, R. W., 54, 66 Newman, M. S., 345, 460 Newton, M. A,. 23, 24, 32, 41 Niece, G. C., 362, 462 Nielscn, N. D., 240, 283, $90, 295 Nieman, C., 285, 298 Nieper, H. A,, 367, /,GO Nii, S., 95, 97, 216 Nikolic, J. A., 449 Nisbet, N. W., 44, 46, 47, 53, 57, 59, 60,
GS Nishiuiura, E. T., 45, 52, 59, 66, 235, 253, 298
Niu, 1,. C., 282, 2% Niu, M. C., 282, 294, 5.98 Nivcn, J. S. F., 74, 216
493
AUTHOR INDEX
Noliara, H., 99, 216 Nomura, M., 103, 136, 163, 216 Nordling, S., 247, 258, 269, 298 Norris, C. B., 245, 248, 267, 269, 271, 282, 288 Notario, A., 240, 298 Novack, R. M., 392, 394, 45s Novak, S., 285, 301 Novick, A., 391, 46G Novogrodsky, A., 216 Novotny, J., 320, 321, 322, 465 Nowell, P. C., 58, 69 Noyes, W. I?., 75, 216 Numanoi, H., 229, 29s Nunn, J. R., 433, 471 Nuzhina, A . M., 283, 289
0 Ochiai, E., 387, 391, 411, 466 Ochoa, S., 152, 154, 155, 156, 157, 158, 159, 208, 216, 220 O’Connor, T. E., 82, 200 O’Conor, G. T., 80, 198, 216, 217 Odeblad, E., 229, 295 O’Gara, R. W., 320, 333, 380, 398, 407, 460, 40.2, 466 Ogawa, M., 170, 213 O’Gorman, P. O., 57, 59, G7, G0 O’Grady, E. A,, 246, 297 Ogston, A. G., 267, 274, 296 Ohashi, S., 257, 298 Ohlwilcr, D. A., 236, 299 Ohta, A,, 387, 468 Ohta, G., 258, 299 Okabayashi, T., 386, 466 Okabe, K., 417, 46.2, 469 Okagawa, K., 26, 40 Okajima, E., 389, 4G6 Okamoto, K.,103, 163, 21F, 388, 461 Okamoto, T., 105,216 Okano, H., 51, 63, 67 Okazaki, K., 231, 299 Old, I,. J., 57, 50, GG, Gn, 240, 256, 299 Oleson, A. E., 12G, 127, 128, 165, 216 Oliner, H., 47, 60, 69 Olson, A. C., 347, 4YO Omura, H., 95, 97, 100, 132, 218 Orgel, A., 359, 391, 458, 4G6 Orii, H., 409, 469
Orr, C. W. M., 93, 123,216 Ow, H. C., 244, 2S9 Ow, J. W., 399, 4/30 Orth, G., 84, 200, 216 Ortiz, P. J., 164, 216 Ory, A. A., 270, 285 Cster, W. F., 437, 466 Osteiaas, A. J., 437, 441, 4G6 Ott, M. G., 436, 4G9 Otto, H., 3, 40 Ove, P., 246, 258, 296 Ovechka, C. A., 438, 453, 45G O\vcz:trzak, A,, 251, 256, 291 Owen, J. J. T., 44, 58, GO, 6.9 Owens, A. H., Jr., 44, 69 Owens, G., 74, 207 Oyasii, It., 380, 384, 385, 450, 464 ozZc.iio, rJ., 232, 236, 299
P Padgett, F., 82, 209 Padgrtt, C. A,, 62, G7 Paff, G. H., 281, 282, 290 Page, A. R., 240, 2!)7 Page, M. A,, 449 Paigen, K., 209 Paletta, F. X., 249, 290 Palmer, C. G., 246, 299 Palmer, H. C., 434, 449, 45G Paluslta, 1). J., 240, 200 Pankhurat, K. G. A,, 263, 291 Pansc, T. B., 306, 369, 4GS Paoletti, C., 84, 216 Papa, S., 384, 454 Papadimitriou, J. M., 44, 46, 54, 55, 61, 62, 63, 64, GY, 63, 69, 70 Papadopoulos, D., 20, 24, 37 Papermastcr, B. W., 57, 69 Papirmrister, B., 149, 21G Paranchych, W., 154,216 Ptlrdee, A. B., 127, lG3, 214, 216, 249, 299 I’urisli, D. J., 388, 389, 4fiG Parker, F., 279, 2.94 Parmeggiani, A., 14, 40 Parmi, L., 8, 11, 12, 13, 37 Parrott, D. M. V., 45, 46, 50, 56, 62, 66, 69 Partridge, M. W., 328, 329, 331, 332, 333,
494
AUTHOR IKDEX
334, 339, 358, 403, 434, 435, 449, 463, 466, 450, 4SG Partridgc, W. W., 329, 403, 456 Pasmer, C. G., 244, 246, 294 Passen, S., 200, 916 Pasteels, J. J., 230, 247, 254, 261, $91, 299 Pataki, J., 460 Patel, C. P., 277, 281, 294 Paul, A. V., 93, 114, 209 Paul, F. J., 75, 198, 215, 916, 217 Paul, J. S., 432, 466 Pawluk, R. S., 275, 2SS Payne, F. E., 80, 217 Peabody, R. A., 249,296 Peacock, P. R., 404, 463 Pearce, J. M., 279, 299 Pearson, J. T., 365, 366, 466 Pecht, M., 272, 206 Peggie, K. S., 23, 28, 32, 37 Penman, S., 152, 169, 208, 817, 221 Penn, R. D., 253, 299 Pennelli, N., 11, 38 Penttinen, K., 247, 258, 269, 298, 299, 301, 302
Percival, W. H., 448 Pereira, H. G., 74, 75, 87, 89, 816, 217, 220 Pereira, M. S., 75, 89, 217 Pcrez, G., 414, 466 Pbrin, F., 326, 328, 463, 464 Periti, P., 272, 301 Perrault, A., 244, 289 Perrier, M. T., 13, 40 Person, P., 278, 299 Peterman, M. L., 258, 285, 28S, 205, 299 Peters, J. H., 399, 441, 461, 467 Peters, K., 235, 299 Peterson, R. D. A., 51, 6s Petroliunas, F., 364, 469 Petit, L., 324, 468 Pett, D., 132, 216 Pettit, F. H., 409, 466, 471 Pfaff, J. P., Jr., 48, 66 Pfau, C. J., 82, 217 Pfefferkorn, E. R., 81, 217 Philips, F. S., 27, 28, 29, 34, 40 Philipson, L., 274,299 Phillips, B., 454 Philpott, C. W., 260, 999 Pichat, L., 434, 469
Pickctt, G. E., (53, 69 Pictra, G., 8, 13, 14, 40, 48, 320, 333, 466 Pillar, O., 336, 466 Pinckard, R. N., 51,54,69 Pickarski, L. J., 99, 102, 104, 105, 116, 176, 183, 184, 185, 194, 196, ,015, 914 Pifia, M., 82,99, 100,211, 217 Pirie, A,, 242, 264, 279, 299 Pisciotta, A. V., 46, 63, 69 Pitltanen, A,, 81, 218 Pitot, H. C.,421, 423, 425, 427, 466 Pitts, It. F., 254, 299 Plaine, H., 391, 467 Planinsek, J., 437,463 Pliss, G. B., 371, 375, 377, 467 Plots, D., 3, 40 Poirier, L. A., 306, 391, 396, 397, 402, 405, 424, 425, 443, 444, 445, 466,46’7, 468 Poirer, M. M., 420, 467 Polasa, H., 98, 171, 217 Polissar, M. J., 5, 41 Pollack, H., 250 299 Pollak, R. D., 29, 40 Pollard, M., 46, 63, 69 Polli, E., 178, 216 Pollister, A. W., 270, 297 Pollist>er,F. W., 261, 299 Polson, A,, 274, 299 Ponsford, J., 54, 70 Ponten, J. A., 80, 84, 209, 214 Pope, J. H., 88, 201, 217 Porter, D. D., 62, 69' Porter, K. A., 45, 47, 48, G9 Possick, P. A., 245, 248, 202 Potgieter, G. M., 274, 290 Pound, A. W., 7, 40 Posdnyakov, 0. M., 334, 467 Pratt, E. A., 137, 216, 282, 296 Pratt, 0. E., 448 Precerntti, A., 4, 10, 11, 12, 14, 39, 41 Pressman, D., 437, 4G3, 4G9 Preston, R. G., 80, 217 Prcussmann, R., 19, 37 Price, J. M., 364, 368, 370, 379, 381, 383, 384, 386, 412, 415, 420, 460, 468, 467 Pricer, W. E., 93,217 Prichard, M. M. L., 448 Pringle, J. A. S., 380, 460, 467 Probst, G . W., 225, 296 Prodi, G., 14, 40, 433, 434, 439, 469, 467
AUT€fOK INDISX
I'rol~~rss, J . J.. 131, 217
Protnsova, T. G., 384, h/i7 Puck, T. T., 258, %I? Pugh, W. E., 199, 215 Pullman, A,, 335, 339, 342, 347, 348, 360, 361, 467 Pullman, B., 335, 342, 347, 348, 359, 360, 361, 467 Purcell, R . H., 81, 203 Purdom, L., 246, 299 Puron, R., 437, 467 Putong, P. B., 235, 298 Pyrah, L. N., 367, 368, 369, 467
Q Quagliariello, E., 384, 454, 467 Quastel, J. H., 230, 266, 277, 693, 299 Quick, A. J., 275, 278, 299 Quinn, P. A,, 176, 181, 185, 219 Quiocho, F. A,, 272, 299
R Rabson, A. S., 75, 80, 198, 209, 216, 216, 217
Rada, B., 204, 217 Radbill, C. I,., 282, 288 Radding, C. M., 131, 132, ,017 Radike, A. W., 391, /,el Radloff, R., 82, 220 Radomski, J. I,., 368, 369, 414, 415, 416, 423, 436, 450, 452, 45S, 460, 46G, 467 Rafferty, K. A., Jr., 80, 217 Ragan, G., 242, 299 Itai, K., 240, 695 Itall, D. P., 402, 418, 471 Ralph, R. K., 155, 215, 217 Ranadive, K. J., 14, 36 Randall, C. C., 82. 217, 21;) Ranzi, S., 282, S99 ltapaport, F. T., 54, GS Raper, J., 279, ,901 Itnpin, A. M. C . , 142, ? I n Rapp, F., 88, 95, 96, 176, 177, 178, 193. I!W iw. 201, .?os, o m . ?zo, :m.9116, 11; *
m,!/ , ._
l{a1q):lport, [ I . , s, /,u I~aplraport,11.. 330, 333. lial)poit, M. N., 3ti0,2.W
;tic
Iiask-Nic?lson, R., 46, 55, 61, 09 Rasmusscn, A. F., Jr., 203
495
I(asiniissi:n, A . If., 3!)0, 460 Ilauschcnbaclr, M. O., 384, 467 Ravdin, R. G., 80, 214
Raven, C. P., 226, 228, 244, 252, 239 I~awitsclrcr-I(iiLtkc1,E., 256, 257, 297 Ray, B. R., 245, 299 Ray, F. E., 378, 379, 399, 4.55, 465, 467 Rebhun, L. I., 230, 299 Rebiere, J. P., 84, 216 Rechcr, L., 46, 62, 63, 66 Reed, N. D., 45, 52, 56, 69, 71 Reed, R., 280, 290 Rees, D. A., 234, 261, 270, 298 Rees, K. R., 439, 467 Regelson, W., 224, 225, 240, 241, 242, 244, 255, 256, 257, 261, 265, 267, 269, 270, 271, 278, 283, 286, 287, 292, 297, 298, 299, 900, 30z
Reich, P. R., 171, 198, 217, 618 Reimer, C. B., 75,913 Reinhold, R., 368, 414, 456 Reiss, J. A,, 411, 464 Reisaig, M., 97, 208 Renner, G., 306, 307, 402, 418, QGZ, 470 Revel, H. It., 115, 217 Rcwdd, F. E., 10, 36 Reynolds, B. I.., 235, 300 Reynolds, R. C., 432, 466 Rhoden, E., 22, 23, 24, 31, 36, 37 Rich, A , , 169, 217 Rich, M. A., 63, 09 Richards, F. M.. 272, 2". l
Richardson, N . G., 234, 261, 270, 293 Rirhartlson, S. H., 248, 294 ltichter, GI.H., 266, 3% Richter, M., 51, 69 Riddle, J. M., 284, 300 Rieke, W'. O., 51, 69, 282, 801 Riggs, J. L., 89, S l S Riley, J . I?., 265, 500 Rin:il(li, A. M., 231, 29s RiniI(>,E.. ,113, 444. 470 Riiigt>r, S.. 257. YO0 I ~ i ~ ~ ~ m i o.i., n t:M7, i , {5!/ I(i3. 11.. 2ti1, :?!I!) Iiisclr, by, 11.. 2, 19, 20, 41 Iiitclrie, A. C., 7, 40 Rive, D. J., 28, 41
496
AUTIIOR INDEX
RiviAre, M. R., 13, 40 Rivon, R. H., 267, 303 Roanc, P. R., Jr., 97, 170, 218 Robrrt, F., 348, 467 Robcrts, D. C., 375, 376, 470 Roberts, J. J., 436, 440, 447, 467, 470 Robinson, D. W., 237, 300 Robinson, W. S., 81, 220, 228 Ilochlitz, J., 362, 471 Roden, L., 235, 300 Rodman, B. P., 63, 66 Roe, F. J. C., 2, 3, 6, 7, 12, 13, 16, 18, 21, 40, 42, 306, 333, 367, 368, 412, 448, 468, 470 Rogers, S., 2, 5, 14, 21, 23, 24, 25, 40, 199, 200, 201, 218 Roizman, B., 95, 96, 97, 170, 171,207,228 Rolfe, B., 146, 612 Rolfe, U., 85, 218 Rolnick, H. A., 258, 261, 294 Romerii, M. G., 403, 466 Roque, A., 271, 300 Roscoe, D. H., 93, 167,218 Rose, B., 51, 69 Rose, F. L., 20, 28, 40 Rose, J. A., 80, 83, 171, 198, 217, 218 Roscnberg, E., 93, 218 Rosenberg, L. T., 57, 66 Rosenberg, M. D., 285, 300 Rosenbcrg, T., 277, 292 Rosenblatt, M., 45, 55, 66 Rosenkrantz, H. S., 29, 40 Roscnlof, R. C., 63, 69 Rosenthal, M. C., 46, 63, 60 Rosenlhal, T. B., 254, 206 Rosin, A,, 5, 10, 12, 37, 41 Ross, J., 132, 216 Rosa, M. G. R., 63, 66 Ross, W. C. J., 427,468 Ross-Mansell, P., 439, 468 Rosston, B. M., 45, 49, 67 Roth, d . S., 280, 281, 282, 283, 299,300 Rotlistcin, A., 284, 300 Rott, Ii., 81, 218 Rous, P., 14, 25, 4f Ibussrl, O., 324, 465 Roussos, G. G., 282, I'RG Ro~istii,M. K., 171, 216 Roux, H., 248, 278, 283, 300
Rowe, W. P., 75, 80, 84, 88, 89, 192, 195, 196, 198, 199, 201, 208, $12, 217,228 Rowson, K. E. K., 306,367, 368,412,468 Royer, R., 394, 463 Rubin, B. A., 199, 212 Rubin, H., 81, 82, 206, 212, 618, %lI, Ruckewwrt, A,, 63, 67 Rudali, G., 369, 468 Rudenberg, F. H. N., 254,300 Rueckert, R. R., 85, 228 Rundles, R. W., 2,39 Runnstrom, J., 224, 226, 227, 228, 229, 231, 249, 250, 296, 300 Rusch, H. P., 363, 372, 373, 376, 378, 466 Ruschmann, G. I<., 178, 181, 220 Russell, W. C., 82, 95, 97, 100, 132, 218, 221
Russo, G ., 279, 300 Rutman, R. J., 228, 294 Ruys, A. C., 285, 297
S Sabin, A. B., 88, 89, 193, 202,218 Saccone, C., 384, 4Sr Sachs, J. H., 55, 69 Sachs, L.,23, SG, 80, 176, 178, 196, 211, 21 8 Sack, H. A,, 320, 468 Saffiotti, V., 318, 319, 369, 377, 400, 464, 468 Saliyun, M. R. V., 345, 46$ St. Amand, G. A., 267, SO0 Saito, H., 351, 355, 359, 431, 466 Sxito, S., 142, 212 Sakamoto, Y., 26, 40 Saksela, E., 80, 214, 258, 300 Salaman, M. H., 2, 3, 6, 7, 12, 16, 18, 40, 4 1 , 306, 367, 368, 391, 412, 465' Sddeen, T., 279, 280, 300 Sall, T., 234, 300 Salvi, M. L., 195, Sf4 Salzman, N. P., 95,96, 97, 103, 171, 618 Sanicjiinn, I<., 406, 409, 462, 408 Satnprr, I,., 75, ?91 S:tnisoii, 14'. E., Jr., 285, 300, 3OJ SLtmuels. P. B., 258, YO1 Sandel., c’ , 391, 470 Sanders, 17. I<., 155, 216 Sandin, R. B., 363, 372, 373, 375, 376, 378, 465, 408
497
AUTHOR INDEX
Santana, S., 404, 4.5:) Santos, G . W.. 44, (8 Sarabliai. A . S.,111, 218 Sargent,, A. U., 51, G9 S:irma, P. S., 75, 89, %IS, 21,s’ S a d i i , II., 258, 2U! Sato, H., 244, 289 Sato, K., 306, 391, 396, 397, 402, 405, 418, 424, 425, 443, 444, 445, 460, 465, Am, 468 Sato, S., 286, 301 Satoh, P. S., 200, 91s Sauerbier, W., 148, 149, 214, 215 Sauerbuch, F., 48, 69 Saunders, B. C., 342, 405, 462 Saundcrs, E. L., 80,220 Sawauchi, K., 8, 9, 39 Saxen, E. A., 4, 40 Saxen, E., 247, 258, 269, 208, 301, 319, 468 S a d, H., 259, 286, 301 Schabel, F. M., 21,4l Schaffarzick, W. R., 236, 297 Schabel, F. M., 370, 371, 398, 4 G l Schafer, W., 86, 215 Schaffer, F. L., 81, 218 Schardein, J. I+ 367, 462 Schecter, V., 241, 253, SO1 Schell, J., 141, 145, 211, 219 Schell, K., 75, 89, 201, 212 Shemyakin, M. F., 164, 213 Schcrhag, B., 273, 295 Schemer, K., 169, 217 Schiltl, H. O., 53, 70 Schillrr, S., 238, 301 Schlumbergcr, H. D., 85, 207 Schuiahl, D., 14, 19, 37, 4 1 , 366, 407, 425, 460, 4G5 Scliiiiitl, F. A,, 28, 41 Sclimiclt. A. J., 236, 301 Schmidt, G., 282, 301 Schmidt, H., 313, 4GS
Sclimitt, F. O., 263, 266, 268, 270, 271, 293, 301
Schmitz, A., 228, 250, 292 Schneider, M., 241, 297 Schneider, M. C., 156, 220 Sclrnitgert, F., 459 Schoental, R., 19, 28, 41,337, 456 Scholefield, P. G., 286, 301 Sdiolficld, R . , 251, 255, SO2
S~.lrollir;st~l;. C., 81, 21s S~~lrriigrr, .I.. 376, 311 81~1111PIC’r.F. 21, 38 SI:lllllt<’. l i . 343, $65 Scliiiltz, TI. li,, 200, 216 Schulx, I ., 2, 6, 3s Scliwartx, A ., 247, 301 Schwartz, H. S., 27, 29, 34, 40 Schwartz, R. S.,44, 45, 46, 47, 60, 61, 63, 64, 66, 68, GU Schwars, M. R., 282, 301 Schwsrzcnberg, L., 241, 207 Sch\vcrdt, C. E., 81, 218 Scott, T. S., 363,468 Scott, W. W., 436, 4GS Scribner, J. D., 28, 40, 391, 407, 423, 427, 443, 444, 446, 4G4, 468 Searle, C. E., 364, 383, 387, 388, 389, 435, 451, 466, 46s Sebring, E. D., 103, 171,218 Sekiguchi, hl., 103, 104, 105, 107, 111, 121, 218 Se h, M., 281, 296, 2996 Sell, S., 50, 56, GS Sellakumar, A. R., 369, 377, 400, 468 Scnra, Y., 388, 463 Session, J., 45, 49, GG SctB11, K., 319, 468 Seud, D. W., 285, 295 Setlow, R. B., 148, 149, 218 Sexton, W. A,, 2, 20, 38 Shapiro, D. M., 115, 218 Shapiro, J. R., 4, 41 Shapiro, L., 152, 156, 158, 207, 218 Shatkin, A. J., 80, 83, 103, 171, 218 Shear, M. J., 465 Shcdden, W., 123, 215 Shcdlovsky, A., 142, 215 Sheche, P. R., 5 , 20, 37 Sheek, M. R., 96, El8 Shein, H. M., 74, 80, 215, 218 Sheinin, R., 176, 177, 178, 181, 183, 185, 191, 196, 219 Shenoy, K. P., 306, 369, 468 Shephcrd, D. M., 265, 300 Shcppard, E., 280, SO1 Sherwin, B. E., 402, 460 Shile, M., 237, 301 Sliimkin, M. I<., 3, 5 , 13, 14, 41, 319, 320,
w,*
Id;..
454, 61il
498
AIJTIIOR INDEX
Sliipl), W., 155, 156, 213 Shirasu, Y., 383, 387, %S, 38‘3, 437, dGS, 471 S l l O p P , K. k;,, 75, 21!1 Short, E. C., Jr., 126, 128, 166, d f ! ) Shouler, J. A,, 435, 45G Shreffler, D. C., 132, 217 Shubik, P., 8, 12, 13, 14, 21, 25, 37, 39, 40, 41, 308, 320, 333, 334, 460, 464, 466 468 Shuster, R. C., 148, 149, 219 Sicuteri, F., 272, 301 Siebke, J. C., 139, 215 Siegel, M., 273, 274, SO1 Siegler, R., 63, 69 Sieker, H. O., 55, 68 Sigidin, A., 63, 66 Silman, H. I., 295 Silver, S., 141, 219 Silvers, W. K., 44, 47, 66 Silverstone, H., 12, 13, 41 SiminofF, P., 95, 97, 219 Siminovitch, L., 95,209 Simon, E. H., 4, 16, 17, 18, 19, 26, 29, 36, 114, 219 Simon, G., 249, 289 Simon, J., 307, 371, 373, 392, 393, 395, 396, 398, 406, 455 Simon, L., 152, 156, 158, 159, 220 Simon, M., 93, 213 Simonsen, M., 43, 44, 47, 49, 53, 57, 58, 60, 61, 67, G9 Simpson, C. I,., 240, 297 Simpson, D. I. H., 63, 65 Simpson, L., 23, 24, 32, 41 Simpson, W. L., 279, 292, 370, 471 Sims, P., 306, 335, 336, 338, 339, 445, 449, 457, 468, 4G8,469 Sinclair, J. W., 371, 375, 377, 404 Sinclair, W. K., 29, 41 Sinkovics, J. G., 45, 46, 55, 58, 60, 62, 63, 69
Sinohara, H., 238, 301 Sinsheimer, R. L., 85, 155, 156, 209, 213, 218
Sipe, C. R., 240, 283, 290, 295 Siskind, G. W., 43, 57, 59, 69 Sjodin, K., 50, 56, 70 Skipper, H. E., 2, 19, 20, 21, 22, 23, 24, 32, 37, 40, 41
Sktiltl, O., 103, 163. 319 Skowpx, J., 285,301 Hkypwk, 11, H , 238, cIcI1 S I a ~ ~ t t ~ r l - )D. : ~ B., ~ ~ 226, l i , 261, 2%’ SliiJwr, M., 463 Sleator, W., Jr., 266, 308 Slotnick, V. B., 80, dil Slover, G. A., 238, 301 Small, M., 5,41 Smets, G., 245, 257, 273, 301 Smillie, R. M., 417, 469 Smith, A., 197, 811 Smith, B., 181,220 Smith, F., 274, 290 Smith, G. N., 285,301 Smith, H., 256, 304 Smith, J. M., 47, GS Smith, J. W., 75, 212 Smith, K. O., 96, 199,216,219 Smith, M. F., 349,353, 456 Smith, M. S., 166, 214 Smith, P. K., 361, 469 Smith, R. T., 265,302 Smith, W. E., 14, 25, 41 Smith, W. R. D., 329, 332, 403, 425, 426, @5, 469 Smith, W. S., 261,301 Smithers, D. W., 45, 46, 63, 67 Snart, R. S., 345,469 Snell, E., 267, 294 Snell, K. C., 380, 4CS Sodergren, J. E., 27, 29, 34, 40 Soehner, R., 82, 219 Sokoloff, L., 264, 301 Solari, A. J., 256, 301 Somerville, A. It., 436, 4 G l Somciville, It. L., 91, 121, 211, d l 9 Sonnabend, J., 155, 219 Sonnenbichlcr, J., 350, ht59 Sorof, S., 436, 437, 438, 439, 455, 460, 469 Southam, C. M., 448 SpAleny, J., 336, 4GG Spector, W. G., 53,70 Spencer, A. T., 388, 389, 4G8 Spencer, K., 25, 37, 40, 333, 334, 460 Spencer, W. W., 274,303 Spiegelman, S., 103, 152, 157, 159, 160, 212, $16, 210
Spicr, H. W., 277, 290 Spitz, S., 375, 469
499 SI)jiil,,H. J., 377, $/X,$/Cl Sporii, M. B., 438, 440, 4Ci0, d(j9
Spotswood, T. M., 320, 321, 456 Sprakr, J. M., 435, 456 Spratt, J. S., 377, 469 Spriggs, T. 1,. B., 29, 41 Springrr, K., 369, 415, 459 Sprunt, D. H., 279, 280, 301 Stab, E. V., 286, 2.90 Staccy, K. A,, 141, 145, 146, 209, 211, 218 Stahman, M. A . , 267, 284, 293, 301 St,ahn, V., 409, 411, 470 St,anisla\vslti, Ii.. 3S4, 460 Stanlcy, N. F., 44, 45, 46, 47, 53, 54, 55, 60, 61, 62, 63, 64, G7, GS, 60, 70, 71 Stanton, M. F., 45, 54, 70, 421, 469 Staslny, P., 47, 57, 61, 70 Stntr, G., 258, 202 Sl,rc~lc,I. W., 234, 261, 270, 2.95 Stcclr, R. H., 352, 46.9 Stc,ffrnson, D. M.. 255, 501 Strigbigrl, N. H., 402, 418, 471 Striglrdcr, G. Ii., 241, 207 Strin, 11. J., 364, 4/22 Stcint)clrg, C. M., 111, 210 Stcinherg, M. S., 252, 269, 301 Strint.r, K.I?., 352, 469 Steinmullrr, D., 44, 47, G5 Stelzenmdler, A., 2, 19, 20, 41 Stembridgr, V. ,4.,47, 57, 61, 70 Stenkvist, B., 80, 84, 209 Steplrwvski. Z., 202, 214 Strrn, P., 20, 41 Strrnbrrg, S. S.,27. 28, 29, 34, 40 Stevens, M. A,, 390, 45s Strvrnson, J. I,., 309, 469 Strivart, F. W., 279, ?91 Stewart, H. L., 319, 4/74 Stcirnrt, S. IS., 45, 51, i 0 , 75. 84. 209, Pl0, 212 Stetson, G. A,, 57, 70 Sticli, H., 261, 501 Ytincbaugli, S. E., 173, ,015 Stovk, J., 281, 301 Stoc~krit,IC., 57, 59, /7C, li!l S l ~ l i ~ l < l l : l lM l ~.. .I.. 2!),41 S i i ~ i ~ ~ ~ l , i , i l i i \V,$ i * , X I , ?I1 Stolcvr. A l . ( i . I<.. 84, 107, !?//.9, : ? l I , ? I 9 Slolhr, \ . , 178, 180, ,!,?I SlololT, l,.,237, 267, 301
Sloilc-, A , 13., 127, 129, 165, 21.9 Storti. IC., 276, Yllf Straumfjord, J. V., Jr., 247, 501 Strctton, A. 0. W., 111, 21s Stromingcr, J. I d . , 204, 213 Stryzak-Mitchell, D., 13, 41 Stylrs, J. A,, 58 Suarez, H., 202, 220 Snbak-Sharpc, H., 123, 150, 171, 212, 219, 210 Srieolto, S., 150, 210 Sugiinurrt, T., 383, 385, 386, 388, 417, 429, 436, 462, 4GG, 460 Suyino, H., 103, 21G Sugiura, K., 389, 390, 391, 392, 458, dG8 Srigiy:una, M., 286, 501 Siigiyaiiia, T., 320, 4G2 Sugiira, H. T., 281, 282, 980 Srimincw, D. F., 88, 169, 217, 210 Simdrtrarajan, T. A,, 142, 219 Suntzclff, V., 3, 41, 251, 257, 264, 290 Surtecs, S. J., 329, 332, 403, 425, 45G Susmnn, M., 111, 210 Sntherland, D. E. R., 51, 65, 68, 70 Sut,lirrland, K. E., 121, 215 Sul,ton, D. A,, 433, 471 Sutton, H., 320, 334, 462 Siivralimanyan, D., 274, 297 Suzuki, T., 265, 302 Sved, S.,81, 210 Svoboda, D. J., 5, 41 Swanson, A. L., 285, 301 Swerk, W. O., 226, 2.97 Sweet, U. H., 80, 211, 219 Sykcs, J. A,, 46, 62, 63, 66, 82, 209 Sylvcn, B., 239, 241, 993, 302 Synicys, M. O., 59, 70 Symonds, N., 141, 145. 146, 209, 2ff, 218 Sgmons, C., 280, 301 Sznfarz, D., 432, 4G9 Szent-Gyiirgyi, A,, 270, 302, 352, 362, 469 Seilard, I,., 391, 466 Szirniai, E., 238, 302 Szybalska, G. H., 282, 302 Seylxilski. W., 82, 142, 210, 21.9, 282, 302
T T:tcliiban:t, hl., 383, 385, 380, 451, 462
'rLt(iLL, M : ~ . 463 , 'l'aila, Mi., 4 5 3
500
AUTHOR INDEX
Tngashira, Y., 19, 38, 306, 350, 351, 353, 354, 355, 358, 361, 362, 363, 428, 429, 431, 432, 463, 464 Taguchi, F., 89, 177, 192, 211 Takahashi, I., 94,219 Takagi, A., 234, SO0 Takahashi, M., 75, 213, 219 Takahashi, T., 463 Takayama, S., 387, 388, 469 Takemori, N., 89, 218 Takemoto, K. K., 176, 177, 212, 219 Takeuchi, M., 446, 469 Tamm, I., 81, 152, 153, 167, 168, 169, 170, 207, 210, 211, 220 Tamura, Z., 406, 409, 462, 468 Tan, E. M., 63,68 Tanaka, S., 448 Tanaka, T., 46, 62, 63, 66, 383, 388, 463, 469 Tancredi, F., 384, 464, 467 Tange, J. D., 375, 377, 468 Tanigaki, N., 437, 463, 469 Tanishima, K., 258, 299 Tannenbaum, A., 2, 3, 4, 8, 12, 13, 34, 41 Tarnowski, G. S., 28, 41, 45, 55, 68 Tartar, V., 298, SO2 Tatsumi, K., 32, 33, 41 Taylor, A. C., 246, 302 Taylor, D. 0. N., 55, 62, 66 Taylor, G., 8, 9, 12, 39, 75, 220, 221 Taylor, R. B., 51, 60,70 Teebor, G., 368, 414, 462, 466 Teir, H., 239, 302 Temcs, G., 22, 39 Temin, H. M., 82, 203, 204, BlS, 219 Terasawa, M., 417, 461 Terayama, H., 246, 302, 392, 405, 407, 408, 409, 410, 439, 446, 447, 460, 461, 466, 469, 471 Ternberg, J. L., 429, 430, 470 Terracini, B., 318, 319, 464 Terranova, T., 279, 300 Tessler, A., 413, 415, 470 Tessman, E. S., 85,219 Tessman, I., 85, 114, 115, 212, 219 Tevethia, S. S., 198, 219 Thannhauser, S. J., 282, 301 Thind, K. S., 53, 96 Tlmmns, C., 14, 41, 57, 66, 465 Tliornris, I<.I)., Jr., 256, 297
Thomas, L., 43, 57, 59, 69, 262, 265, SO2 Thomas, M., 80, 84, 208 Thomason, D., 251, 255, 302 Thorell, R., 281, 290 Thoren, M. M., 153, 216 Thorn?, H. V., 85, 220 Tillet, W. S., 54, 68 Tischendorf, F., 235, 2.91 Todaro, G. J., 199, 204, E N , El2 Toforovicova, H., 285, 301 Tomatis, L., 9, 13, 14, 41 Toolan, H. W., 80, 220 Topham, J. C., 396, 403, 405, 406, 421, 426, 471
Toriyama, N., 417, 461 Toro, I., 228, 233, 234, 237, 239, 290 Torok, G., 233,237,291 Torok, L. J., 233, 237, 291 Tosteson, T. R., 228, 294 Toth, B., 8, 12, 13, 41 Toth, W. H. B., 448 Tourniw, P., 202, 220 Town, B. W., 277,302 Townes, P. L., 271, SO2 Toyama, S., 140,146,220 Trainin, N., 3, 4, 5, 8, 9, 10, 11, 12, 14, 16, 17, 18, 19, 21, 23, 26, 29, 30, 31, 36, 39, 41 Tralb, A., 267,297 Trams, E. G., 361, 469 Trapani, I. L., 265, 271, 292 Trasciatti, M., 348, 350, 355, 464 Traut, M., 462 Travers, J. J., 392, 393, 394, 468 Tregier, A., 238, 294 Trentin, J. J., 9, 12, 39, 45, 48, 49, 66, 70, 75,220,221 Tridente, G., 7, 9, 10, 11, 14, 25, 37, 38 Trilling, D. M., 129, 220 Troitskii, N. A., 425, 470 Troll, W., 307, 368, 369, 413, 414, 415, 416, 430, 431, 443, 444, 462, 466, 470 Ts’o, P. 0. P., 247, 250, Sod, 347, 391, 434,
464,470 Tsukamoto, H., 32, 33, 41 Tsuru, K., 74, 213 Tucker, R . G., 93, 167, 218 Tuffley, M. A., 45, 59, 65, 0’7 ‘Tiinis, M., 233,251. 457,%3. 369, 378, 283, X/O, 302
50 1
A U T H O R INDEX
Turbin, N. V., 425, 470 Turner, H. C., 88,196, 201, 808, 812 Turolla, E., 5,37 Tuttle, L. P., 277, 289 Tyan, M. L., 62,70 Tyler, A., 252, 253, 254, 286, 291, 302 Tytell, A. A., 224, 225, 282, 283, 293, 296, 303
U Uchida, A,, 105, 21C Uehleke, H., 306, 368, 377, 409, 411, 413, 414, 417, 425, &2, 470 Uetake, H., 140, 146, 230 Ullrey, D., 247, 308 Umama, R., 268, 291 Ungar, G., 276, 30%' Unsercn, E., 333, 470 IJpton, A. C., 71 Usenil, E. A., 283, 290 Ulrick, I<., 251, 303
V Vaccari, F., 276, 301 Vaheri, A., 226, 247, 258, 269, 270, 280, 298, 302 Valentine, R. C., 74, 81, 87, 210, 216, 820 Valentine, W. W., 241, 296 Vallardares, Y., 100, 174, $14 Valselli, G., 255, 290 Van Bibbrr, J. J., 91, 122, 308 Van Bibber, M. J., 91, 122, 205 Vandendrirssche, I,., 278, 280, $08 Van Escli, G. J., 7, 17, 41 Van Gcnderen, H., 7, 17, 41 Vanhorn, E., 402, 418, 471 Vanierenberghe, J., 37 Vanlerenberghr, J., 400, 4GO Van Winkle, Q., 352, 4 G l Varcoe, J. S., 439,467 Varga, L., 242, 288 Vargues, R., 57, 70 Vassalli, P., 249, 259 Vasseur, E., 226, 257, 296, 302 Vaughan, T. M., 309, 315, 343, 461 Veasey, R. A,, 437, 438, 4G8 Velat, C. A,, 379, 4GS Venkatesan, J. C., 4 ( 0 Verrrtt,, M. ,J., $8 F'cr\vocwl, L). IV., 107, 168, JZ.?, ?2fl
Vesselinovitch, S. D., 8, 13, 14, 25, 41, 42 Vidrbaek A,, 45, 63, 70 Vielle, F., 200, 2lG Vigicr, P., 207, 820 Vigliani, E. C., 363, 470 Vilstrup, T . H., 275, 890 Vidal, P. M., 27, 28, 29, 34, 40 Vingicllo, F. A., 448 Vink, H. H., 7, 17, 41 Vinograd, J., 82, 220, 247, 250, 302 Vipond, H . J., 329, 331, 332, 333, 358, 435, 456, 466 Vischer, T. L., 47, 57, 61, 70 Vishniar, W., 248, 278, 283, 302 Vithayathil, A. J., 429, 430, 470 Vlahakis, G., 11, 12, 59 Vlamynck, E., 2, 6, 38 Vogt, M., 2, 42, 80, 82, 174, 175, 178, 180, 181, 183. 184, 185, 2f0, 212, 230 Volcani, B. E., 296 Volkin, E., 163, 220 vonEsch, A. M., 364, 460, 469 Von Euler, J., 279, 888 von Haam, E., 309, 469 von Jagow, R., 307, 402, 418, 470 Von Winkle, Q., 258, 261, 294
W Wada, A,. 354, 470 Wagner, B. P., 363, 379, 466 Wagner, R. R., 168,212, 220 Wakonig-Vaartaja, R., 26, 42 Walburg, H. E., Jr., 7f Walder, J. A,, 463 Walford, R. L., 46, 47, 58, 60, 61, 67, 70 Walker, T. T., 279, 894 Wall, F. T.. 245,302 Wallach, D. H. F., 247, 302 Walpole, A. I,., 20, 40, 365, 366, 372, 373, 375, 376, 470 Walsh, A., 306, 367, 368, 412, 470 Walters, C. P., 152, 154, 212 Walters, M., 306, 333, 367, 368, 412, 468, 470 Walters, M. N.-I., 44, 45, 46, 53, 54, 60, 61, 63, 64, 67, 69, 70,71 Wan, J., 272, 284, 259 Wang, L., 9, 41 1V:trcl. J . , 251, cYfl:2 FF':lrd(*ll, I).. 85, ??/I
502
All'l'lHOR INDEX
Waring, H., 47, 70 Warner, H. It., 112, 113, 121, 220 Warren, A. K., 244, 246, 294, 299 Warren, F. L., 22, 36 Warren, G., 279,302 Warren, L., 229, 293 Warner, R. C., 155,205 Warwick, G. P., 436, 440, 447, 461, 467, 470 Wasastjerna, C., 239, 502 Watanabe, I., 87, 220 Watanabe, K., 88, 220 Watanabc, M., 164,216 Watanabe, S., 258, 2!l9, 502 Watanabc, Y., 88, 220 Watkins, J. F., 203, 220 Watson, D. H., 74, 95, 97, 100, 123, 132, 211, 215, $18, 221 Walson, D. W., 57, 69 Watson, J. D., 82, 220, 359, 360, 470 Watson, J. G., 381, 383, 380, 454 Watson, It., 82, 220 Wattenberg, L. W., 440 Watters, C., 307, 432,470 Wear, J. B., 384,458 Weatherall, J. A. C., 25, 42 Weber, G., 280, 281, 281 Weber, H. H., 249, 250, 302 Webster, D. R., 258, 301 Wechselberger, F. V., 235, 302 Wecker, E., 153,220 Weiderheim, M., 241, 305 Wcigert, F., 336, 337, 338, 467, 470 Weil, R., 82, 84, 178, 181, 620 Weil-Malherbe, H., 345, 346, 352, 470 Weinfield, H., 234, 302 Weinhousp, S., 286, 301, 503 Weir, D. M., 51, 54, 09, 70 Wcisburger, E . K., 3, 28, 41, 42, 306, 308, 363, 369, 370, 371, 378, 397, 398, 402, 418, 422, 425, 437, 460, 461, 4G5, 470,
4Y 1
Wrisburgcr, J. H., 3, 28, 41, 42, 306, 308, 369, 370, 371, 378, 397, 398, 402, 418, 422, 437, 460, 461, 470, 471 Wcisinaiiri, G., 245, 261, 263, 264, 2.90 Wciss, 13., 149, ?20 i v L k , IJ., 47, 58, ? U , 212, 2#i, 2.17, 351, 257, 258, 272, 303 W'cirn, l'., 233, 259, 260, 303
Weissbacli, A., 03, 126, 128, 130, 131, 224, 217, 220 Wcissman, S. M., 171, 108, 217, 218 Weissmann, C., 152, 154, 155, 156, 157, 158, 159, 161, 208, $14,216, 220 Welch, A. D., 3, 38 Welfling, J., 63, f37 Welty, M., 2, 19,20, 41 Wessels, N. K., 232, 303 Wesslen, T., 80, 84, 88, 209 West, G. B., 236, 296, 303 Wrsthead, E. W., Jr., 273, 277, 298 Westrop, J. W., 396, 403, 405, 406, 421, 426, 471 Westwood, J. C. N., 87, 207 Wheatley, D. N., 449 Wheeler, G. P., 21, 42 Wheelock, 13. F., 167, 168, 169, 220 Whistler, R. L.,274, 303 Whitcutt, J. M., 433, 471 White, 17. R., 319, 471 White, I,., 23, 24, 32, 37, 4 1 White, P., 274, 290 White, R. P., 285, 303 Whitehead, J. K., 439, 462 Whitfield, J. F., 267, 303 Wiberg, J. S., 105, 107, 112, 113, 165, 166, 209, 220 Wicker, R., 202, 220 Wicklund, E., 224, 226, 227, 228, 250, 300, 303 Wiedemann, I., 306, 307, 402, 418, 4/22 Wiedershcim, M., 303 Wigsell, H., 57, 70 Wilandcr, O., 240, 259, 303 Wilburt,, K. M., 258, 282, 288 Wikox, W. C., 87, 06, 98, 172, 221 Wilde, C. E., Jr., 232, 244, 270, 303 Wildy, P., 74, 82, 95, 97, 100, 123, 132, 211, 213, 218, 221 Wiley, C. E., 277, 280, 692 Wiley, F. H., 369, 462 Wilk, M., 362, 47i Willr), N., 257, 303 M ' i l l ~ ~ t i i sM., , 169, 2t'f Williams, I., 127, 216 \Yilli:iiiis, li., 2 S , 20, :<2,$7, .$?, 339, 435, 4.54, 45s
503
ATiTfIOR INDE X
M. C., G3. 05, 365, 372, 373, 375. X6, $0, 4 7 t IVi]li:iiiis, H . (It.. 62. 70, 82. 21 j ~ ~ ' ~ I ~ ~ : ~ I I ~ S - A SH. ~ I IG.. ~ I ~W II. wi\li:ir(l, 13. V., ,W, 4hS. 441. 4 2 , 471 Williiii:r, 15. S., 232, 248, 2YJ, :U'l Wilncr, B. I., 74, 221 Willoughby, D. A., 53, 70 Wills, G . D., 252, 277, 278, 302, $03 Wilson, D. B., 60, 70 Wilson, D. E., 168, 208 Wilson, E. J., 277, 301 Wilson, G. J., 243, 303 Wilson, R. E., 50, 54, 56, 70 Wilson, R. G., 152, 221 Wilson, W. L., 224, 227, 228, 238, 253, 254, 259, 266, 275, 204, 303 Winnick, R., 22, 23, 3G Winnick, T., 22, 23, 36 Winocour, E., 178, 180, 196, 211, 221 Wintcrnitz, F., 369, 468 Wiseman, It., 380, 419, 462, 4G2 Wissler, F. C., 245, 248, 297 Withers, H. R., 7, 40 Woislawski, S., 399, 467 Wolf, G., 308, 379, 381, 466 Wolf, P. L., 45, 55, GG Wolfe, 13. D., 369, 462 Wolman, B., 237, 301, 303 Wolman, M., 237, 305 Wolpcrt, I,,, 259, 284, 2.9.3, 303 Wood, G. C., 260, 30s Wood, W. B., 145, 146,221 Woodhousc,, D. L., 387, 388, 435, 4GS Woodruff. M. F. A., 59, 70 w o o s ~ c I<:. ~ , T., 367, 402 Work, T. S., 81, 85, 152, 153, 150, 167, 210, 213, 215, 221 Wormall, A , , 243, 252, 277, 278, SO?, :WJ Worrel, C. S., 285, $01 Wright, (i,IT., 314, 4.59 Wright, II. I?., 75, 213 Wright, I. S., 280, 301 Wright, L. D., 281, YO3 WuH, D. L., 114,221 Wyatt, C. s., 381, 423, 4G5 Wyatt, G. It., 90, 221 Wyndcr, F:. T,., 309, 312, 314, 4G2, ,471 \\'illi:iii~s,
Y Tatit-, Y.,i 5 . "70. 221 Y:&v, RI.. l i , 70 l';igi, Y.,137. $/3, h/;!/ Y ; i i i i : i i I i ~ , 'I,., 231, 246. $08, 439, 471
Y:tniainoLo, I<. S., 28, 42, 226, 253, 303, 425, 471 Yamanaka, J., 45, 55, 66 Yang, H.-Y., 407, 4Gn Yrw, N., 384, 4G7 Tohn, D. S., 75, 221 Yondare, T., 267, 303 Yoshida, T. O., 200, 218 Yoshinioto, A., 463 Yoshimnra, H., 32, 33, 41, 240, 241, 305 Yoshimori, M., 5 , 39 Yost, D., 364, 469 Young, B. G., 262, 274, 277, 278, 298 Young, C. W., 29, 46 Young, E. M., 436, 437, 438, 439, 469 Young, J. M., 380, 419, 462 Young, L., 46, 62, 63, 66 Young, R. A., 80, 210 Yu, C., 286, 303 Yumoto, T., 46, 62, 63, GG
Z Zachariae, F., 238, 296, 303 Zackheim, H. S., 370, 471 Zahalsky, A . C., 417, 432, 462, 464 Zajdela, F., 308, 309, 312, 313, 314, 315, 319, 324, 325, 326, 328, 334, 340, 342, 343, 388, 44S, 460, 468, 463, 464 Zak, 9. J., 61, G 8 Zainenhof, S., 22, 42 Zeigcl, It. F., 80, 209 Zcidman, I., 257, 503 Zritlnian, T., 257, 291 Zickdoose, D.. 307, 455 Zicglrr, TI. M., 409, 46G, 471 %iff, M., 47, 57, 61, 70, 2G8, 271, 284, 200 Zillig, W., 85, 209 Zimmerman, A. M., 230, 235, 236, 240, 250, 283, 296, 304 Zimmerman, E. F., 167, 221 Zinimernian, S. B., 91, 92, 93, 138, 139, 166, 214, 621 Zinder, N. D., 154, 155, 157, 161, 163, 209, 215, 221 Zlstkis, A,, 347, 464
ATJTHOR INDEX
Zollingcr, II. V., 281, $04 Zollner, N., 280, 281, ,997, $04 Zurker, hl. B., 249, 259, 804
Zw:wtouw, H. T., 256, $04 Zmeifach, B. W., 240, 258, 905, 804 Zmickey, R. E., 76, 8’14
SUBJECT INDEX 0-8ininolizotoluenc, N-hydroxy tlerivaA tives, 417-423 4-AcctyluininohipItcny1, N-liyclroxy (I(,metabolic pathways of, 407409 rivatives, 417423 4-Aminobiphenyl, carcinogenic activity, metabolism of, 401403 372-376 2-Acetylaminofluorene, metabolism of, derivatives, 371 401-403 nuclcic acid and protein binding to, 2-Aminofluorene derivatives, carcinogenic activity, 377-380 436-438 o-Aminophenols, oxidation to o-quinone 4-Acc tylaminostilbcne imines, 440447 N-hydroxy derivatives, 417-423 Aminopterin, effects, on viral induction carcinogcnic activity, 424427 of enzymes, 110-111 metabolism of, 401-403 Actinomycin D, viral-induced enzyme Animal viruses DNA-containing, DNA synthesis, 96inhibition by, 100-102 98 Adenomas, lung, see Lung adenomw enzymes induced by, 94-100 Adenosatellite virus, DNA properties, 83 growth curves of, 94-96 Adenosine 5’-triphosphate, polyanions metabolism of infected cells, 170-172 and, 247-251 effects on, cell enzymc activity, 172Adenoviruses 173 DNA of, biosynthesis, 97-98 host-cell nucleic acid and protein properties, 76, 82, 83 synthesis, 162-173 enzymes induced by, 98-100 general properties of, 82-83 ncoant,igens induced by, 89 RNA-containing, 81 pot,entiation of SV 40 virus, 198-199, effects on host biosynthetic proc206 esses, 167-169 propertics of, 82 replication mcclranism, 157 replication cycles of, 96 Anthanthrene, derivatives, as carcinotiimorigenic, 74-75, 78 gens, 313 Adhesion, polyanions’ effect on, 257-259 Anthracene derivatives, carcinogenic acAmber mutants of bacteriophage T,, see tivity, 367-372 Bacteriophage T4 Aminoacridines, carcinogenic activity, 2-Anthramine, carcinogenic activity, 370, 372 390-392 Amino azo dyes, ainine oxides of, 409- Antigen, viral-induced synthesis of, 8489 412 p-D-Arabinofuranosylcytosine, effect on Carcinogenic activity, 392-400 enzyme synthesis, 187-189 in extrahrpatic t,issues, 399-400 ;lrlmviriisrs, RNA-rontnining, 81 hydrosylation mid, 406 Arginase, vird intlnction of, 199-201 N-hydroxy derivirtives, 424--227 tls, acmrthvlntion ; i i i t l tl~.nic.tli~lnt,ion of, Aiwniatit- c o i n ~ ~ o u ~ ~c~ircinogt~iiciiiolrt*iiliir gmmetrp of, tivity anti 407 305471 nuclcic acid and prolein binding of, condensed polycyclic type, 308-363 432-433, 436447 reductive cleavage of, 407 conjugated arylamines, 363-392 505
Ary laniincs
c:arcinogc~riic::iclivity, 363-433 activiling mechanism, 412-427 clct,oxicating incchanisni, 400412 free radicals in, 427-433 covalent binding to proteins arid nucleic acids, 436-447 mechanism, 440447 Arylhydroxylamines, carcinogenic activity, 363-392, 412427 Arylnitro compounds, carcinogenic activity, 363-392 Autoimmunity, runting syndromes and, 43-7 1 Avian leukosis complex viruses, RNA of, 81 tumorigenic, 79, 82
B Bacillus subtilis, viral-enzyme induction in, 129-130 Bacillus subtilis phages, enzymes induced by, 93-94 Bactrrial infcction, runting and, 52 Bacteriophage (s) effects on host-cell nuclria acid and protein synthesis, 162-163 enzyme induction by, 135-137 host-controllrd modification and, 140-143 genes controlling UV sensitivity, 148151 1 -even, cnzynics induced by, 89-92, 94, 137140, 165-166 in E. coli, 127-129 infection of bacteria, effects, 162-163 Bacteriophage f2, mutants, rnzyrne induction studies, 161-162 Bacteriophage A, rnzyrne induction by, 130-132 liost-cont,rollerl modification and, 143144 SI'3, r n z y n i r iiuliicdinn hy, n:t(~rc~riolili:i~!.c~ 1 z - 13') Titlcl.rl.iol,ll:tKc, T.-inf(vslvcl ~ ~ l l "wdy s,
,.
R:wtcriophsge T, dcfccts in cnzynie indirct,ion of, 111115
aIIll)l!l. Inuliulls, 1I I--113 ts inut,ants, 113-114 cnzynic induction by, 134-135 B3ttctcriopliage Ta, cffccts on bact,c:rial metabolism, 162-163 enzyme induction by, 127-12!) Bcnzacridincs, carcinogenic activity, 325326 1,2-Benzanthracenes dimethylated, as carcinogens, 309 metabolism related to bond order, 335 niethylated, as noncarcinogens, 308-309 Bcnzidine derivatives, carcinogenic activity, 371-376 1,14-Brnzobisanthene, in fossils, 323 l0,ll-Benzofluoranthene, pyrolytic formation, 320-321 Benzonaphthocarbolines, carcinogenic activity, 327 3,4-Renzopyrene covalent binding to proteins and nucleic acids, 433-434 flow dichroisin stndics on, 353-354 mrtabolism of, 336-338 pyrolytic formation, 32&322 in soils and marine srdiinents, 321-322 Benzopyrido varbazoles, carcinogrnic activity, 326-328 Biphenyl derivativcs, carcinogcnic activity, 372-380 Bisanthenc, in fossils, 323 Bit,tner moiisr t,umor virus, 79 RNA of, 81
C Calcium, role in growth rogulation of polyanions, 251-257 Capsid proteins, of viruses, 84-87 Carbamatcs, structural formulas of, 15 Carcinogencsis free radicals in, 362-363 hydrocarbon-DNA interaction in, 3593B2 by Ilrrthnn, 1-15 ('nrcinogttns :in)maIi 1. c*oiiiporinds 11s. 305-471 ~'Il\'~r~)lllll('II~:1~ C J ~ ' C ' l l ~ l ' ~ ' ~ l C320-323 f~, K-rvgion Iiylwtlic4s of, 339-314 pyrolylic forimtion, 320-323 in soils and marine sediments, 321-323
StTR.JICC’L’ INDEX
Ckll
i i i o t i i l i r ~ t n i ~Iwl>.anioiis’ , t v l v ill, 244245 ClSI,O viriis, :is l[irrtor-lir~tl[icirlgv i i , i l s , 75 (‘liitnrras (exprrimcntal), ritnting sjmdronir.s and, 45, 48-50 cxpeiimental parabiotic intoxication, 48 parrntal splcc~ncr.11 injection, 4!)-50 r;ttliation chimrrw, 48 Colipliagc Ts, cnzyincs iiitluccd hj-, (3293 Clondt~nsetl polycyc.lir :ironiatir coinpounds carcinogenic activity anti molrcular grometry of, 308-363 lirt,erocyclic compounds, 329-333 Ji-rr.gion Iiypothcsis of, 339-344 of litrgc-size types, 313-318 pyrolytic formation, 320-323 structiirr-act,ivitB relat,ionships. 308319 t,issiie t argtxt spccifici by, 319-320 differrntial dichroisni strttlirs on, 356357 noncovalent intcractions of, 344-362 with nuclcic acids, 348-359 with purines, 345-348 with surfactants and proteins, 344345 mctaholism of, 333-330 pyrolytic format,ion, 320-323
D I h r fibroma virus, as tiimor-producing virus, 78 Dcoxycytidine triphosphatasc, induction by T-even phage, 165-166 Deoxycytidplate deaminasc, proprrtics, 193 rcnrtions ca.talyzcd by, 90 viral indnrtion of, 91, 94 drfccls in, 115 Dcoxvcytirlylatr hydroxymrtIiyI:ise rcwtions catalyzrd by, 00 vim1 induvtion of. 90 distinrt,ivu proprrtic.~,119-120 Dcos~ribonuclt~asrs of E. coli, 126-127
507
iiitliiclinii of I J ~ ~) : i ~ l ~ ~ r i ~ ~ i I 127i:t~c,s, 12!) i n t l ~ i c ~ ~ i cby in
Iiwpcs simlJlcx :mtl poxvirus, 132-134 Ilolymions’ ryffect on, 282-284 D(’osyribonuc:leic acid (DNA) I)nctcxrial, phage-controlled brc.nkdown of, 165 intcraction, with aromatic aininc.s, 430432 \vitli polj.cyclic :troniatics, 348-350 nictliylation. in Iiliagr multi~~lication, 147-118 DSA4-r~ont ainiiig viniscs of animds, 81 groups of, 74 tumor-producing, 7%79 Deoxyribonuclric-acid nictliylasc, viral induction of, 135-137 Deoxyrihonuclcic acid polymc.rasc properties, 192, 103, 196 viral indnction o f , 93, 100 distinctive l)ropcrties, 122-123 vaccinia and hrrpes simplex induction, 123 D(,osyribonuclcoside monophosphate kinase. virally inducrd, distinctive propcrties, 121 Droxyuridylate dcaminase, viral induction of, 94 Dcoxyuridylate kinme, viral intluct.ion of, 94 Dihcnzanthracene derivatives, carcinogtanic activity, 318-319 Dibenzocnrbazoles, carcinogenic activity, 327 Dihrnzopyrene isomers, as carcinogrns, 3 13-3 14 Dihydrofolat,e reductase propertics, 193 rcactions rntnlyzed by, (30 viral induction of, 91, 93 distinct,ivc proprrtics, 121 l-T~imct,hylaminonxol~~~tizcn~ nnnlogs nminc oxidcs of, 400412 carcinogenic activity, 397-398 ring hydroxylation of, 405406 Diphrnylmcthnne drrirntivcs, rarcinogenic activity, 365-367 Dyes, as growth regulators, 224, 243
508
SUR.TECT INDEX
E Egg, jelly coat of, see Jelly coat of cgg Embryogenesis, polyanions’ role in, 226234 EMC virus, ItNA of, 81 Endonucleases, of E . coli, 126-127 Enzyme ( 8 ) inhibition and tictivation of, polyanions in, 275-278 viral-induced, see Vir XI-induced cnzymes Erythroblastosis virus, 79 RNA of, 81 Escherichia coli deoxyribonuclcases of, 126-127 enzyme induction in, 134-135 host-controlled modification and, 140-143 Escherichia coli, B, viral enzyme incltiction in, 135-137 Escherichia coli K12(X), viral enzyme induction in, 130-132 Esterase, polyanions’ effect on, 284-286 Ethyl carbamate, see Urethan Ethylene maleic anhydride, in growth control, 240-241 Exonuclcnscs, of E . coli, 126-127
F Fibroma-myxomn virus, as tumorproducing virus, 75, 78 Flow dichroism apparatus, 354 Fhoranthene hydrocarbons, as carcinogens, 309-312 Fluorene derivatives, carcinogenic nctivity, 372-380 Fluorodeoxyuridinr, effects on viral induction of enzymcs, 110-111 Free radicals, role in carcinogenesis, 362363 of arylaminrs, 427-433 Pringelites, ns fossil pigmrnts, 323
G Gliicosyl transferases, viral induction of, 137-140 Glycosidases, polyanions’ effect on, 279280
Growth control polyanions in, 234-241 ATP and, 247-251 calciiain and, 251-257 radiation effects on, 241-242
H Hemnngiomas, Iibcr, from urcthan, 12 €kparin in fertilization, 226-227 as growth regulator, 224-225, 236-238, 240, 243, 245 Hepatoencephalitis virus, see Reovirus type 3 Hcpatomas, from urethan, 11 Herpes simplex virus enzyme induction by, 123 in mutants, 116-119 infection by, effrct on sRNA, 150-151 replication cycles of, 95-06 Herpes virus(es) DNA of, biosynthesis of, 97 properties of, 76, 83 enzymes induced by, 98-100 DNases, 132-134 properties of, 82 replication cycles of, 95-96 as tumor-producing virus, 78 Hctcrocyclic compounds, cnrcinogenic activity, 323-333 HMC a-glricosyl transferasc-inducing activity, viral induction defwts, 115116 Homologous discase, clinical syndrome of, 46-48 Hpaluronidase, polyanions’ effcct on, 279-280 Hydrophilic gels, polyanions and, 268271 2-Hydroxylaminofluorene, inetabolism of, 422 4-Hydroxylaminoquinoline-N-oxidc, carcinogenic activity, 428-429 N-Hydroxyurethan, 26-31 carcinogenic effects of, 29-30 chemistry of, 27-28 metabolites in blood and tissues, 3031 noncarcinogenic effects of, 28-29 urinary metabolites of, 3 2 3 4
I Influcnza \ inis, c.nzyme induction in, 153-154 Influenza A virus, RNA of, 81 Intcrferon, induction by polyanions, 224225
J Jelly coat of cgg, polyanionic character, 226, 229 role of, 232
K I<-region hypothesis, of carrinogenicity, status, 339-344 Kilham rat virus, DNA propertics of, 83
1 Leukemia(s) viruses and, 63 Leukemia viruses, properties of, 82 Leukopenia, runting and, 55-56 Lipase, polyanions' effect on, 284-286 Luck6 renal carcinoma virus, as tumorproducing virus, 78, 80 Lung adenomas, from urethan, 3-6 Lymphomas, malignant, from urethan, 8-11 Lymphomatosis virus, 79
M Mammary carc.inomas, from urethan, 12 Menstruation, heparin role in, 238 Methyl-substituted hydrocarbons, sidechain oxidation, 338 Minute mousr virus, DNA properties, 83 Mitomycin C, effect on mzyme induction, 186, 187-189 Molluscum contagiosum virus, as tumorproducing virus, 75, 78 hlncopolpsnrcharidrs, in growth control, 238-240, 245 hlurine sarcniua virus, 79 Muljiie leultcniia virus. 79 M i i r i i r ~ iiimor \fii,iisca, RNA-cnnt,nining, 81
hIiilagrnesis, 1iydroc.arhoii-lNA intrraclion in, 359-362 hlj~rloblastosisvirus, 79 RNA of, 81 Myxoviruses, propertics of, 82 RNA-containing, 81, 82
N Naphthalene derivatives, carcinogenic activity, 367-372 Naphthopyridocarbazoles, carcinogenic activity, 326-328 2-Naphthylamine carcinogenic activity, 367-370 metabolites, carcinogenic activity, 412416 protein and nucleic acid binding to, 436 Ncoantigens, by oncogenic viruses, 8889 Neoplasia, runting syndromes and, 43-71 NZB mice, autoimmune reactions in, 45, 56,62 Newcastle disease virus, RNA of, 81, 83 5-Nitrofurans, carcinogenic activity, 364365 $-Nitrocpinoline-N-oxide and derivatives carcinogenic artivity, 385-389, 428 complexing with DNA, 431432 N-hydrosylation of, 416417 protein and nucleic acid binding to, 435436 Nuchic acids covalent binding with, 4-nitroquinoline-N-oxide, 435436 polyryrlic hydrocarbons, 433-435 tric~.cloqriiiiazoline, 433-435 interaction with polycyclic aromatics, 348-359 nith azo dyes, 432-433 phnsphorylation by viral-indurrtl enzvme, 134-135 of viruscs, molecular weights of, 83
P I’apillonia virusc~s, :IS tiimor-protliic.ing viruses, 79, 80 I’apillomas, skin, from urcthan, 6-8 I’apovavirus(es) DNA, genetic information of, 201-206 infectivity of, 84 proprrties of, 77, 82-84 enzyme induction by, 186, 187 grnrs, fnnctbns of, 198-204 -infected cclls, biochrniical changes in, 194197 RNA a n d IJrotein synthesis in, 174175 D N A synthesis, 178-182 metabolic ant,agonists, 177 virus-specific prot,eins, 176-177 viriis-specific RNA, 175-176 proper1irs of, 82 tumorigrnic:, 75, 7!) l-Phcnylazo-2-naphthol, inc~t:tl)olisni of, 403-405 Picodna viruscs, 80 properties of, 82-83 of DNA, 77 Picornavirusrs, D N A properties, 83 enzyme indiicntion in, 153 Poliovirus, -infected cclls “early protein” synthesis in, 88 RNA of, 81 Polyadenylate polynirrase, loss, after phage infrrtion, 164-165 Polyanions :xdhesion and, 257-259 antitumor activity of, 286-287 ATP and, 247-251 re11 mr,mhranc and, 244-245 in rrll rrspiration, 278-270 colloidal effcrts, 265-268 tlrosyribonucleasc and, 282-284 in cmbryogenesis, 226-234 in enzyme inhibition and activation, 275-278 growth-regulating activity of, 223-304 biologiral rvitlrncc for, 226-234 Iiyaliiroiiid:tst~ : i n i t glyrosidasrs nntl, 279-280 hydrnphilir grls f i i i i l - 268-271 in infrrI,ion, 224-225
286 rihonnrlensc: and, 280-282 surfacc charge and, 246-247 in surface and enzymo act,ivily, 271 as virus inhibitors, 225-226 I’olycyclic hydrorarbons, covalent binding to proteins and nuclcic acids, 433-435 Polynucleotide kinase, viral induction of, 134-135 1’0 1yoni:L virus DNA of, 84 -infrctcvl ccslls, DNA synthcsis in, 196197 tlT Itinase tlrfirirncy, 197 viral gcnome continuance in, 201203 ircw:Lnt,ipcns intluccd by, 89 rt3l)lication cyc:lc of, 173-174, 204-205 tumors from, 75, 79, 80 I’olysaccharicl~~s,role in growth c o n h l , 259-265 Poxviruses DNA propcrt,ics, 76, 83 enzymes indricrd by, 98-100 DNmrs, 132-134 properties of, 82 repliration cycles, 95 trimorigenic, 74-75, 78 Protrins, covalent binding with, 4nitroqiiinolinc-N-oxide, 435-436 polycyrlic hydrorarbons, 433-435 tricycloqninazoline, 433435 polycyclic aromatic solubilization by, 344-345 Psrudorahics virus DNA biosynt,hrsis in, 97 rnzymrs intlnrrd by, 99 -infrcted cclls, “early protein” synthrsis in, 88 rrplication of, 95 Pnrinr-N-oxidrs, carcinogcnic activity, 389-390 Piirines, soluhilization of polycyclic aroi n n tics by, 345-348 1’yr:Lii (~o~JoI>wI(T, interferon induction by. 2% I’yr:inIhrrnc, 314 -315
csnrc*inogrnic stiidirs
on,
I'yrnlysis, carciliogtan ftmn:lfion IJ,, 320323
Q Q/3 rcliliwsv, 160--161 Qriinolinc compounds, cxrvinogenic activity, 381-383 o-Qninoncimines, froin o-aininophrnols, 440-447
R It:ilhit, p:~pillonia virus, enzyme induction hy, 109-201 tumors froni, 75 Ratliation, in growth control, 241-212 lt:idi:ttion i-hiiiicra~.rrmting and, 48 Rausrhcr lciikcniia factor virus, RNA of, 81 Rroviriis(es) -infcctcd cells, metabolism in, 168-170 ItNA-cont,aining, 81 Iteovirus Type 3, autoimmunity from 54-55, 59, 65 Itcplicase, see Riboniiclcic arid synthctase Respiratory rnzymrs, polyanion effects on, 278-270 Itibonuclcic acid ( R N A ) doablestranded, from RNA synthetasc, 155-157 sRNA, viivs-infdion effvcts on, 150-151 RXA-containing vir.usf>s. tnmorigcnic, 79, 82 I1NA polymerase, in ec,lls infi'ctrd with DNA viriiscs, 102-104 with T-cvrn phage, 163-164 I?ilmnrirlc:isc, poly:inions' effect on, 280282 l?it)oniii*lvic :ic.itl syntlic~tasr~ 1irn per t irs of , 157-16 1 \.ir:il iniluc~liono f , 151-102 c.linr:ic.lcristii's, 151-154 tloul)l~~-strnnrlc.tlRNA froni, 155-157 virus rcplicntion and, 157 Roiis sai'coiiia virus, 79 nc>onntigcns induced by. 89 RNA of, 51 ltoris sarcoma virus-infrcted tissiirs, 206207 rnnynirs in, 203-204
R i i i i ~ i i i g syndruiiic's,
~ i ~ i ~ o i i i i i i i ~ and, iiiit~~
13-i I
I>:il,teriaI infrctir,n in. 45. 52 52-53 t ~ l ~ t ~ i r ~ intliirlioii i ~ ~ : i l of, 45, 51-52
l ~ : i ( ~ l c ~ r i :i,:ivriiirs il :iiiaI.
Iri~liopcniaand, 45, 55-56 ncoplasia and, 43-71 NZB mice and, 45, 56, 62 splcnomegaly and, 57-58 sti,ril(>vaccines and, 45 siilii*rlliil:u fractions and, 45, 53-54 tliynirctoniy nnd, 45, 50-51, 60, 64-65 viriiscs and, 45, 54-55, 59, 65
S Skigella dysenteiine, phage enzyme induction in, 142-143 Sliope fibroma virus, tumors produced by, 75 Simian adenoviruses, as tumor-producing viruses, 75 Sindbis virus, R N A of, 81, 83 Skin papillomas, from urethan, 6-8 Soils, hydrocarbons in, 321-323 Statolon, 225 Surface chargr, polyanions' effect, on, 246-247 SV 15 virus, rrplicat,ion of, 05, 96 SV 40 virus DN.4 infcclivity of, 84 rnzymr intluction by, 155-186, 187 'l'-nntigrn.q and. 102-103 -infrctcd rrlls, I~iocheinic~alchangcs in, 191-196 lintentiat inn l)y ailcnoviriis, 198-109, 206 viral gc~nomr continn:ini:e in, 201202
:IS
iriiiioi.-lii,o~iii'ingvirus,
79, 80
T T-antigens, 198 by oncopenic viruses, 88-89 prol)rrt,ics of, 193 SV 40-indurcd rnzymcs and, 102-193, 1955196
512
SUBJECT INDEX
1; phagc, we Bartcrio1)hagc Tz Thymedomy, runting syndromes and, 45, 50-51, 60 Tliyniidinr inetalrolihiii, I iral-inducrd enIllPS for, 182-193 Tltyiniilini~Liiiaw
prolic*i11i.5 of, 1%-l!J", 193, 1% viral indiic*lion ol, 99, 123, 132-133, 186-189 drug inhibition of, 101 Thymidylate kinase, propertirs, 193 viral induction of, 99-100 Thymidylatc nucleotidase, induction by phage, 166-167 Thymidylate phosphatasr, viral induction of, 94 Thymidylate synthrtase properties, 193 reactions catalyzed by, 90 viral induction of, 90, 93, 166-167 defects in, 114-115 distinctive properties, 121 Toluidincs, carcinogenic activity, 363 Tribenzopyrcnes, caicinogenic activity, 314-315, 318 Tricycloquinazolincs carcinogenic activity, 328-333 covalent binding to proteins and nucleic acids, 433-435 metabolism of, 339 Triphenylmethane derivatives, carcinogenic activity, 365-366 Tryptophan metabolites, carcinogenic activity, 380-385 Tumor-producing viruscs, 74-82, sep also Viral oncogencsis list of, 78 Tiimora. morpliologicnl altcrntion in, 24224 4
U TJrcthnn rarriiiogenic action of, 1-15 hemangiomas, 12 hepatomas, 11-12 lung adenomas, 3-6, 16 lymphomw, 8-11 mammary carcinomas, 12 16 skin papillomas, a, in nrwborn mire, 14-15, 35
nuclcic acid metal>olisin and, 20-22 (*:itabdizingciizvrne for, 26 cornpounds rrlated to, biological a(#tlvlty. 15-20 l l ~ J l l ~ ~ ~ ~ r l ' l uIS o~f'nl~, t l l ' t ( ' l 1 1 1 1 1 1 : ~ ~ 1 ~ J 1Or, 1 2" N-liytlro\yiirelliaii froni, 26-27 N-substitiit ed, rarcinogenic action, 1617 metabolism of, 22-26 rate of elimination, 24-26 urinary metabolites of, 31-34 UV light, bacteriophage genes controlling sensitivity to, 148-149 UV radiation, effect on viral induction of enzymes, 105-110
V Vaccinia virus DNA of, biosynthesis of, 96-97 enzyme induction by, 123 in mutants, 116-119 replication cycle, 95 UV effects on infectivity, 106 Viral-induccd enzymes distinctive properties of, 119-125 of DNA-containing animal viruscs, 94100 of DNA metabolism, 89-125 host-controlled modification and, 140148 DNA methylation, 147-148 hcat sensitivity, 146-147 of phage A, 143-144 of T-cvm phage, 14CL143 hydrolysis or modification of DNA or RNA by, 126-151 induction system rharartcristics, 100111 induction by UV-irradintcd virus pnrticles, 105-110 inhibitors of, 104-105 mutant virus strains and, 111-119 of thymidine metabolism, 182-193 Irinetics, 182-185 viral onrogenesis and, 73-221 Viral oncogenesis, biochrmical asprcts of, 173-207 genetic factors, 204-207
Virus (cs) animal, see Animal viruscs -induced antigen syntliesis, 84-89 “early protein” synthesis, 87-89 viral-capsid proteins, 8 4 8 7 -infected cells, “early protein” syntliesis in, 87-89 nucleic acids of, iiioleculsr weights of, 83 polyanion inhibition of, 225-226 runting and, 45, 54-55 -transformed cells, continued viral genome in, 201-203
l,ririior-protliiciii~, sco Tunlor-protlucing viruses
W Wasting tliscuse, runting syndroinc and, 50-51
X Xenylaniincs, carcinogenic activity, 376377
Y Yabs monkey poxvirus, as tumor-producing virus, 74, 78
CUMULATIVE INDEX VOLUMES 1-1 1 A Akylating agcnts, cytotoxic, chemistry of, 1, 397 Amino acid transport, in tumor cells, 9, 143 Aminoazo dyes, carcinogenic, 1, 339 Anemia in cancer, 5, 199 Animals, experimental, pulmonary tumors in, 3, 223 Anticancer agents, mechanisms of resistance to, 7, 129 Aromatic compounds, molecular geometry and carcinogenic activity of, 11, 305 Aromatic molecules, electronic structure and carcinogenic activity of, 3, 117 Autoimmunity, 11, 43 Avian virus growths, their etiologic agents, 7, 1
B Benzacridines, angular, and carcinogenic activity, relation between, 4, 315 enzymcs~ in and othrr diseases, 6, 1 Bone marrow, normal and lrirlteinic, hiochemistry of, 9, 303
C
cross resistancc and collateral scnsitivity in, 7, 235 experimental, 2, 425 in man, 4, 1 Cancer production, role of viruses in, 2, 353 Cancer toxin, newer concept of, 5, 157 Carcinogenesis, application of radioisotopes to studies of, 1, 273 aspects of, 3, 171 and tumor pathogenesis, 2, 129 electronic configuration and, 1, 1 epidermal, 1, 57 ethionine, 7, 383 related to contaminated foods, 8, 191 sulfhydryl group and, 10, 247 tobacco, experimental, 8, 249 Carcinogenic activity, and angular benaacridines, 4, 315 of aromatic compounds, 11, 305 of aromatic molecules, 3, 117 Carcinogenic aminoaao dyes, 1, 339 Carcinogenic nitroso compounds, 10, 163 Carcinogcnicity of 2-flnorenaminr and rclntcd compounds, 5, 331 Carcinogens enzyme induction, and genc action, 10, 1 with macromolecules, reactions of, 2, 1 Carcinoma, primary, of the liver, 5, 55 Cell physiology, activity of polyanions in, 11, 223 Chrmical constitution, and carrinogenic activity, 2, 73 Chirkm tumor virnws, and pnssmgrr
Cancer energy and nitrogen mctabolism in, 2, 229 experimental, genetic studies in, 2, 28 1 human, urinary enzymcs in, 9, 1 ionizing radiations and, 2, 177 lipids in, 4, 237 niitrition in relation lo, 1 , 151 viriiws, 7, 51.5 1 ) l r l h i n i t prolc,ins i n , 1, 503 icalatiori of iinniiiiic. r c w t i o i r l o , 9. E 47 Electronic strnctnrc, of niomntic molrCanccr chemothernpy, cules, 3, 117 by perfusion, 6, 111 Energy metabolism in cancer, 2, 220 514
I ~ n z ~ . n iiiiiIu(~t,iun, c gi'iic~:I(,( i o i i , varciiiogl'lls ~ l l i t l , 10, 1 l~~nzymicpat,lrriis I ) ( ncol)laalic t,issuc, 10, 117 Etliioninc carcinogonrsis, 7, 3S3 Etiology of lung cancer, 3, 1 ol nioiiw loukcmia, 6, 291
F 2-l;luoren:iniine, chemistry, carcinogenicity, and nic~t;ibolisrii of, 5, 331 Folic acid, antagonists of, 6, 369 Fowls, clieniically induccd l.uniors of, 5, 179 Fungal iiietat)olit,c~s,foods contaminakd by, :ind carcinogc>nr&, 8, 191
G Gene action, carcinogr~na,enzyme induction and, 10, 1 Growth liroccwcs, 1)rotrin synthcsis referc~nce to, 5, 97 H
Hepatocarcinogoncsis, behavior of livcr enzymes in, 6, 403 Hrpatomns, expc~rimc~ntiil,development of, 9, 227 bioclic.mistry of, 9, 227 biology of, 9, 227 Hormonal aspects, of i~xperimi~nt:il tumorigencsis, 1, 173 Hormonal gcnesis, of mammary cancchr, 4, 371 N-Hydroxyurethan, carcinogenic action, and metaholisni of, 11, 1
I , t i i ki:III i:t cliroiiit,, iisc ol i ~ i y l ~ ~ r iin, i n 4, 73 cliroiiic Iiiycloid, cytogenic studies in,
7, 351 Lilids in cancer, 4, 237 Liver, primary carcinoma of, 5, 55 Liver enzymes, beliavior, in liepatocarcinogcncsis, 6, 403 Lung cancer, etiology of, 3, 1 Lung cancer pathogenvsis, atmos1)heric fuctors in, 7, 475
M P\/lacrornoleculcs, reactions of carcinogens with, 2, 1 Malignant cclls, in U ~ N J studies on protein synthesis by, 10, 83 Mammalian organism, normal and tumor-bcaring, inhibition analysis in, 4, 113 Mainmary cancer, hormonal genesis of, 4, 371 Mamrn:iry tumors in micc, milk agent in, 1, 103 M[yt.abolism, of 2-fluorenamine and relatcd compounds, 5, 331 Motabolites, purine and pyrimidine, antngonists of, 6, 360 Milk agent, in mammary tumors, 1, 103 Molecular geometry, of aromatic compounds, 11, 305 Mouse leukemia, etiology and pathogrnc,sis of, 6, 201 viral etiology of, 6, 140 Myleran, use of, in chronic I(,rikemias, 4, 73
I
N
Immune rcaction, relation of, t,o cancer, 9, 47 Inhibition analysis in normal niainmalinn organism, 4, 113 in tu,mor bearing I 1i:iiii i i i ali:in orgnnism, 4, 113 Ionizing radiations, ant1 cxnccr, 2, 177
Neoplasia, 11, 43 Neoplastic cells, nurlcar protc,ins of, 8, 41 Neol jlastic tissue (s) rnzyinic pattern of, LO, 117 oxidative metabolism of, 3, 269 Nitrogen metabolism in r a n w r , 2, 229 Nitrogen muqtards, cliniral use of, 2, 255 Nitroso compounds, carcinogenic, 10, 163 Nuclear proteins, of neoplastic cells, 8, 41
1 Leucocytes, normal and Icultrmic, biochemistry of, 9, 303
516
CUMULATIVE INDEX VOLUMES
Nuclcolrtr cliromosomcs, structiircs, in1.wa.ctions, and pi’rslwotivcs, 8, 121 Nutrit,ion, relation to wnner, 1, 451
0 Oxidative metabolism, of neoplastic tissues, 3, 269
P Patliogencsis, of nioiise Icukcniin, 6, 291 Perfusion, cmccr chemolhcritpy by, 6, 111 Plant tumor problem, 6, 81 Plasma cell myclorna, treatmcnt of, 10, 311
Plasma proteins in cancer, 1, 503 Polyanions, in intercellular cnvironinmt and cell physiology, 11, 223 Protein synthesis, in vitro studies by malignant cells, 10, 83
1-1 1
T Tlii oiiiboc~i,i*s,iioriiial auil Icukcinic, biuc4ieinistry of, 9, 303 Thyroid gland tumors, development and mctabolism, 3, 51 Tissue, inductive interaction in development, 4, 187 Tobacco carcinogenesis, experimental, 8, 249
Tumor antigens, specific, 5, 291 Tumor cells, amino acid transport in, 9, 143
Tumor-host relations, 5, 1 Tumorigenesis, experimental, hormonal aspects of, 1, 173 Tumor immunity, recent work on, 4, 149
Tumor metabolism, npplirntion of radioisotopes to studics of, 1, 273 Tumor pathogenesis, carrinogenesis and, 2, 129
with reference to growth processes, 5, 97 Pulmonary tumors, in experimental animals, 3, 223 Purine and pyrimidine metabolitcs, antagonists of, 6, 369
Tumor viruses of chickens and mammals, 7, 515 structure of, and relation to general viruses, 8, 1 Tumors, fowl, chemically induced, 5, 179 Tumors, frozen, survival and preservation of, 2, 197
R
U Urethan, carcinogenic action and metaholism of, 11, 1 Urinary rnaymcs, and human cancer, 9,
Radiation chimeras, 6, 181 Radioisotopes, application to cnrcinogenesis and tumor metabolism, 1, 273
Rous no. 1 sarcoma agent, properties of, 1, 233 Runting syndromes, 11, 43
S Sulfhydryl group, and carcinogencsis, 10, 247
1
V Viral-induced cnzymcs, and viral oncogenesis, 11, 73 Viral etiology, of mouse leukemia, 6, 149 Viruses, tumor, of chickens and mammals, 7, 515
