Advances in Cancer Research, Volume 36

ADVANCESINCANCER RESEARCH VOLUME 36

Contributors to This Volume Gianni Bonadonna

Philip D. Lipetz

Clark M. Edson

Janet D. Rowley

Maria E. Ferioli

Armando Santoro

Steinar Funderud

Giuseppe Scalabrino

Alan G. Galsky

Ralph E. Stephens

Kathi Geilinger

Joseph R. Testa

Tore Godal

David A. Thorley-Lawson Robert A. Weinberg

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

SIDNEY WE INHOUSE Fels Research Institute Temple University School of Medicine Philadelphia, Pennsylvania

Volume 36-1982

ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers

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LIBRARY OF CONGRESS CATALOG CARDNUMBER: 52-13360 ISBN 0-12-006636-X PRINTED IN THE UNlTED STATES OF AMERICA 82 83 64 85

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CONTENTS CONTRlBUTORSTOVOLUME36

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ix

Polyamines in Mammalian Tumors: Part II GIUSEPPESCALABRINO AND MARIAE . FERIOLI I . Polyamine Biosynthesis and Concentrations in Different Lines of Cultured Neoplastic Cells . . . . . . . . . . . . . . . . . . . . I1. Polyamines in Human Oncology . . . . . . . . . . . . . . . . . . . . . 111. Diamine Oxidase Activity in Human and in Experimental Neoplasms . . . . . IV . Physiological and Pharmacological Inhibitors of Polyamine Biosynthesis in Neoplastic Tissues or Cells . . . . . . . . . . . . . . . . . V. Concluding Remarks and Speculations . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 20 56

62 86 88

Chromosome Abnormalities in Malignant Hematologic Diseases JANET D . ROWLEYA N D JOSEPH R . TESTA I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . Chronic Myelogenous Leukemia (CML) . . . . . . . . . . . . . Acute Nonlymphocytic Leukemia (ANLL) . . . . . . . . . . . . Acute Lymphocytic Leukemia (ALL) . . . . . . . . . . . . . . Polycythemia Vera . . . . . . . . . . . . . . . . . . . . . . Implications of Nonrandom Changes for Malignant Transformation . References . . . . . . . . . . . . . . . . . . . . . . . . .

I1. I11. IV . V. VI . VII .

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103 105 107 116 126 132 . . . . . 139 . . . . . 143

Oncogenes of Spontaneous and Chemically Induced Tumors ROBERTA . WEINBERG I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . I1. A Model of Cellular Oncogenes . . . . . . . . . . . . . . . . . I11. The Retrovirus-Associated Oncogenes . . . . . . . . . . . . . . IV . Oncogenes Present in Cells Transformed by Chemical Carcinogens .

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. . . 149 . . . 150 . . . . 150 . . . . 153

V . Multiplicity of Transforming Genes in 3-Methylcholanthrene-Transformed Cells 155 VI . Types ofTransformed Cells Yielding Focus-Induced DNA . . . . . . . . . . 156 V

vi

CONTENTS

VII . Multiplicity of Different Human Oncogenes . . . . . . . . . . . . . . . . VIII . Analogies between Virus- and Non-Virus-Induced Cellular Oncogenes . . . . . IX . The Process of Activation of Oncogenes . . . . . . . . . . . . . . . . . . X . The Role of Oncogenes in Carcinogenesis and Maintenance of Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI . The Proteins Encoded by Activated Oncogenes . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

157 158 159 160 161 162

Relationship of DNA Tertiary and Quaternary Structure to Carcinogenic Processes PHILIP

I. I1 . I11. IV .

D . LIPETZ. ALANG . GALSKY. A N D RALPHE . STEPHENS

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer and DNA Superstructure . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . .

165 167 189 202 204 210

Human B-Cell Neoplasms in Relation to Normal B-Cell Differentiation and Maturation Processes TOREGODALA N D STEINAR FUNDERUD I . Introduction . . . . I1 . The B-Cell System . 111. B-Cell Neoplasms . References . . . .

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211 212 234 247

Evolution in the Treatment Strategy of Hodgkin’s Disease GIANNIBONADONNA A N D ARMANDO SANTORO I. I1 . 111. IV . V. VI . VII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Radiation Therapy: From the Kilovoltage to the Megavoltage Era . . Chemotherapy: From Single Agents to Multiple Drug Treatment . . New Treatment Strategies . . . . . . . . . . . . . . . . . . . . Prognostic Factors Influencing Current Strategy . . . . . . . . . Morbidity Influencing Current Strategy . . . . . . . . . . . . . Conclusions: Toward the Total Conquest of Hodgkin’s Disease . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . 257 . . . . . 258 . . . . . 263 . . . . 267 . . . . . 277 . . . . . 283 . . . . . 287 . . . . 290

CONTENTS

vii

Epstein- Barr Virus Antigens-A Challenge to Modern Biochemistry DAVIDA . THORLEY.LAWSON. CLARKM . EDSON.A N D KATHIGEILINGER I . Introduction . . . . . . I1 . Transformation Antigens I11 . Early Antigens . . . . . IV . Late Antigens . . . . . V . Conclusions . . . . . . References . . . . . .

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295 298 309 319 336 342

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF PREVIOUS VOLUMES. . . . . . . . . . . . . . . . . . . . . .

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CONTRIBUTORS TO VOLUME 36 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

GIANNI BONADONNA, Division of Medical Oncology, National Tumor Institute, Milan, Italy (257) CLARK M. EDSON,Sidney Farber Cancer Institute, Boston, Massachusetts 02115 (295) MARIAE . FERIOLI, Institute of General Pathology and C.N.R. Centre

for Research in Cell Pathology, University of Milan, 20133 Milan, Italy (1) STEINAR FUNDERUD, Laboratory for Immunology, Department of Pathology and The Norwegian Cancer Society, Norsk Hydro’s Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway (211) ALANG. GALSKY, Department of Biology, Bradley University, Peoria, Illinois 61625 (165) KATHIGEILINGER,’ Sidney Farber Cancer Institute, Boston, Massachusetts 02115 (295) TOREGODAL,Laboratory for Immunology, Department of Pathology and The Norwegian Cancer Society, Norsk Hydro’s Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway (211) PHILIPD. LIPETZ,Department of Radiology, The Ohio State University, Columbus, Ohio 43210 (165) JANET D . ROWLEY, Department of Medicine and The Franklin McLean Memorial Research Institute, The University of Chicago, Chicago, Illinois 60637 (103) ARMANDO SANTORO, Division of Medical Oncology, National Tumor Institute, Milan, Italy (257) GIUSEPPE SCALABRINO, lnstitute of General Pathology and C.N.R. Centre for Research in Cell Pathology, University of Milan, 20133 Milan, Italy (1) RALPHE . STEPHENS, Department of Radiology, The Ohio State University, Columbus, Ohio 43210 (165) ‘Present address: Department of Pathology, and Department of Medicine, Division of Geographic Medicine, Tufts University Medical School, Boston, Massachusetts 021 11. ix

X

CONTRIBUTORS TO VOLUME 36

JOSEPH R. TESTA,^ Department of Medicine and The Franklin McLean Memorial Research Institute, The University of Chicago, Chicago, Illinois 60637 ( 1 0 3 ) DAVIDA. T H O R L E Y - L A W S O N , ~Sidney Farber Cancer Institute, Boston, Massachusetts 02115 ( 2 9 5 ) ROBERTA. WEINBERG, Massachusetts Institute of Technology, Center for Cancer Research and Department of Biology, Cambridge, Massachusetts 02139 ( 1 4 9 )

'Present address: NCI-Baltimore Cancer Research Program, Baltimore, Maryland 21201. 3Present address: Department of Pathology, and Department of Medicine, Division of Geographic Medicine, Tufts University Medical School, Boston, Massachusetts 02111.

ADVANCES IN CANCER RESEARCH VOLUME 36

POLYAMINES IN MAMMALIAN TUMORS PART Ill Giuseppe Scalabrino and Maria E. Ferioli Institute of General Pathology and C N R Centre tor Research in Cell Pathology, University of Milan. Milan, Italy

Nil minus est hominis occupati quam vivere: nullius rei difficilior scientia est. Professores aliarum artium vulgo multique sunt, quasdam vero ex his pueri admodum ita percepisse visi sunt, u t etiam praecipere possent: vivere tota vita discendum est et, quod magis fortasse miraberis, tota vita discendum est mori. SENECA, “De Brevitate Vitae,” 7,3 L’ignorance qui estoit naturellement en nous, nous I’avons, par longue estude, confirmee e t averee. MONTAIGNE, “Essais,” L. 11, C. 12

I. Polyamine Biosynthesis and Concentrations in Different Lines of ........ Cultured Neoplastic Cells . . . A. Responses to Microenviron perature, PO,) and to the Presence of Different Exogenous Molecules (Amino Acids, Di- and Polyamines, Antipolyamine Antibodies) . . B. I n Relation to the Growth Rate and the Phase of the Cell Cycle . . . . . . . C . Two-way Relationships between Polyamines and Cyclic Nucleotides. Inducibility of the Two Polyamin D. Effects of Infection with Nononcogenic Vir E. Miscellaneous Effects of Polyamines . . . . . . 11. Polyamines in Human Oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ A. Patterns of Polyamines in Human Neoplastic Tissues B. Levels of the Chief Polyamines a Urines of Normal Subjects and of C. Levels of the Chief Polyamines and Their Conjugated Forms in Blood, Plasma, Serum, Formed Blood Elements and Bone Marrow of Normal ................ Subjects and of Cancer Patients D. Levels of the Chief Polyamines ....................... Bloodand Urine . . . . . . . E. Levels of Activity of P Neoplastic Tissues in Relation to the Degree of Malignancy F. Metabolic Conjugation .............. Normals and in Cancer Patients 111. Diamine Oxidase Activity A. In Human Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. In Experimental Tumors ................

2 2 11 12 18 18 20 24

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49 53 55 56 57 60

I Part I of this review (see Volume 35 of this series) covered polyamines and their metabolism in normal tissues and in chemical, physical, and viral carcinogenesis.

1 ADVANCES IN CANCER RESEARCH, VOL. 36

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any fonn reserved. ISBN 0-12-006636-X

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GIUSEPPE SCALABRINO A N D MARrA E. FERIOLI

IV. Physiological and Pharmacological Inhibitors of Polyamine Biosynthesis in Neoplastic Tissues or Cells ............................... A. Physiological Inhibitors and Related Compounds ..................... B. Pharmacological Inhibitors .......................................... V. Concluding Remarks and Speculations ................................... References .............................................................

62 63 68 86

88

I. Polyarnine Biosynthesis and Concentrations in Different Lines of Cultured Neoplastic Cells

A. RESPONSES TO MICROENVIRONMENTAL CONDITIONS (OSMOLARITY, TEMPERATURE,

p o z ) AND TO THE PRESENCE OF

DIFFERENT EXOGENOUS MOLECULES(AMINOACIDS, POLYAMINES,

D I - AND

ANTIPOLYAMINE ANTIBODIES)

The need for expediency in experimental cancer studies has made the cultured neoplastic cell the principal tool for cancer research. There are obvious advantages in working with uniform cell lines that can be prepared as clean cell suspensions. However, the artificial conditions ofin vitro culture tend to change the characteristics of the cells. Several authors have studied different neoplastic cell lines growing in culture in order to delineate the metabolic pathways of the polyamines and to affect growth of these cells by selectively inhibiting polyamine synthesis or sequestering polyamines in order to define their roles (see also Section IV). It is particularly interesting to modify the culture conditions in order to clarify the influence of the environmental milieu on the activities of the polyamine biosynthetic enzymes by enhancing or decreasing the levels of these enzymes to see the role of polyamines in the cell growth process. To this purpose, several studies were carried out with rat hepatoma cells growing in culture. Studies of the effects of growth conditions on ornithine decarboxylase (ODC) activity presented evidence that dilution of high-density hepatoma cell cultures with fresh medium resulted in a very large and transient increase in enzyme activity, reaching a peak about 4 hr after dilution (Hogan, 1971; Hogan and Blackledge, 1972; Hogan e t d.,1973; Hogan and Murden, 1974). This increase was abolished by cycloheximide but not by actinomycin D, suggesting that ODC induction is controlled under these experimental conditions at the posttranscriptional level (Hogan, 1971; Hogan and Blackledge, 1972). The aforementioned increase of ODC activity appeared to be in part concomitant with a decrease in the rate of ODC degradation, i.e., with an increase in the half-life of ODC, hinting at a

POLYAMINES IN MAMMALIAN TUMOHS

3

causal relationship between the two phenomena (Hogan and Blackledge, 1972; Hogan et al., 1973; Hogan and Murden, 1974). The supplementation of high-density hepatoma cell cultures with glutamine or serum or nonessential amino acids, but not with essential amino acids, produced an ODC stimulation of severalfold, at least partially due to an increase of the half-life of ODC (Hogan et al., 1973, 1974; Hogan and Murden, 1974; Fong et ul., 1976). On the contrary, very high concentrations of pyridoxal do not affect the apparent halflife of ODC (Hogan and Murden, 1974). Other investigators followed ODC activity and putrescine levels through two generations of rat hepatoma cells cultured in the presence of serum (McCann et al., 1975). Biphasic ODC induction was noted during the first 24 hr. The intracellular putrescine concentration was found to correlate with rises in ODC activity (McCann et al., 1975). On the contrary, only one broad peak of ODC activity was observed over the same period in diluted hepatoma cell cultures without serum, with no parallel increase in the cellular putrescine content (McCann et al., 1975). Therefore, these authors concluded that only growing and dividing hepatoma cells have biphasic ODC induction that parallels increased putrescine levels, whereas a single peak of ODC stimulation can be achieved in nongrowing cells. Among the factors affecting the growth conditions of the cultured cells, the addition of fresh medium or serum to the culture has been demonstrated to be one of the most important for a variety of other cell lines. Induction of ODC activity, followed by a significant elevation of cellular putrescine concentration, in a rat glioma clone and in a mouse neuroblastoma cell clone when fresh medium was added to confluent cultures was reported (Bachrach, 1976c, 1977, 1980a; Bachrach e t al., 1978). In more detail, ODC activation following the addition of fresh serum was preceded by similar responses in both adenosine 3’ : 5’cyclic monophosphate (cyclic AMP, CAMP)-dependent and CAMPindependent protein kinases of glioma cells (Bachrach et al., 1978). However, in this case no difference in the half-life of ODC before and after the addition of fresh medium was observed (Bachrach, 1 9 7 6 ~ ) . Moreover, the induction of ODC activity appears to be specific for this enzyme, since the activities of other enzymes that decarboxylate other amino acids were not stimulated by the addition of fresh medium (Bachrach, 1 9 7 6 ~ ) This . study also suggests a correlation between growth rate and ODC activity in cultured glioma and neuroblastoma cells, since the enzyme activity was high when the cells were proliferating rapidly (Bachrach, 1 9 7 6 ~ )Interestingly . enough, the addition of serum to culture medium containing mouse neuroblastoma cells or

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GIUSEPPE S C A L A B R I N O A N D MARIA E. FERIOLI

rat glioma cells also greatly increased formation of y-aminobutyric acid (GABA) from putrescine (Kremzner et al., 1975; Sobue and Nakajima, 1977). The S-adenosyl-L-methionine decarboxylase (SAMD) activity was also increased in glioma and neuroblastoma cells shortly after the addition of complete fresh medium (Bachrach, 1977, 1980a). As expected, the enhancements of the activities of the polyamine biosynthetic decarboxylases were found to be paralleled by increases in cellular concentrations of polyamines and of y-aminobutyric acid formed from putrescine (Bachrach, 1980a). In HeLa cells, in response to the addition of serum to quiescent cells not only ODC activity increased but also SAMD activity (Prouty, 197613; Maudsley et al., 1978). Under the same experimental conditions, putrescine and spermidine levels markedly increased as well (Maudsley et al., 1978). When labeled ornithine was added to the cells during the period of the serum stimulation and its uptake was measured, a marked and rapid increase in polyamine levels above that normally observed in resting cells was noted (Maudsley et al., 1978). It appeared that most of the cytosol ornithine was decarboxylated to yield putrescine, which in turn was quickly converted to spermidine (Maudsley et al., 1978). More or less analogous observations were made in KB cells (Pett and Ginsberg, 1968) and in hepatoma cells (Bondy and Canellakis, 1980). In HeLa cells growing in suspension culture, ODC activity was also found to be potently stimulated by the addition of glutamine to the medium; this stimulation was due, at least partly, to a decrease in the rate of decay of the ODC activity (Prouty, 197613). In cultures of L1210 mouse leukemic cells, of hepatoma H35 cells, of neuroblastoma cells (Chen et al., 1976a,b), of virally induced glioma-like hamster brain tumor cells (Hsu et al., 1977), and of Friend erythroleukemia cells (Tsiftsoglou and Kiriakidis, 1979; Gazitt and Friend, 1980), the addition of fresh medium plus serum also resulted in an increase in ODC activity. Additionally, when Friend leukemia cell cultures were stimulated to proliferate by dilution of stationaryphase cultures with fresh medium, both their nucleolar RNA synthesis rates and ODC levels were increased (Dehlinger and Litt, 1978).The addition of putrescine at the time of dilution with fresh medium blocked the increase in ODC levels, but did not prevent the increase in nucleolar RNA synthesis (Dehlinger and Litt, 1978).As observed by Ferioli et al. (1980) in postischemic liver repair, ODC induction in Friend leukemia cells can be dissociated from the stimulation of RNA synthesis.

POLYAMINES IN MAMMALIAN TUMORS

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A proliferative stimulus for the cultured cells, such as the addition of serum, was followed by a dramatic increase in the rate of putrescine transport into both normal human fetal lung fibroblasts and the same cell line transformed by SV40 (Pohjanpelto, 1976). Conversely, the removal of serum resulted in a rapid decrease in the rate of putrescine transport. The magnitude of the increases or of the decreases in the rates of putrescine transport in these two cell lines in response to the addition or the removal of serum were nearly the same (Pohjanpelto, 1976). For studies of the effects of the addition of fresh serum, 12-0tetradecanoylphorbol-13-acetate(TPA), and/or a combination of the two on ODC activity of cultured malignant cells, the reader is referred to Section III,C,2,b, Part I, Vol. 35. Besides the previously mentioned glutamine, another a-amino acid with nonionic polar side chains, asparagine, is a powerful inducer of ODC activity in confluent neuroblastoma cells (Chen and Canellakis, 1977). Among the natural amino acids tested, asparagine led in ability to induce ODC, with L-glutamine second, half as effective as asparagine (Chen and Canellakis, 1977). This ODC induction was neither concomitant with nor followed by an increased rate of incorporation of precursors into DNA, RNA, or proteins (Chen and Canellakis, 1977). What is really astonishing is the finding that asparagine and glutamine play a “permissive” role in ODC induction by N 6 , 0 2 ’ dibutyryl cAMP or by prostaglandin E l (PGEl) plus S-isobutyl1-methylxanthine, since none of these three molecules alone stimulated ODC activity at all in confluent neuroblastoma cells in a medium devoid of asparagine or glutamine (Chen and Canellakis, 1977). The “stabilizing” effect of asparagine on ODC was demonstrated b y the very great lengthening of the enzyme’s half-life when asparagine was in the medium (Chen and Canellakis, 1977). Moreover, in mouse neuroblastoma cells induced to differentiate by any of several stimuli, the regulation of ODC induction by asparagine in undifferentiated and in differentiated cells was studied comparatively (Chen, 1979, 1980). The addition of asparagine to a salts-glucose medium elicited a maximal increase of ODC activity in undifferentiated cells with further addition of fetal calf serum or of N6,02’-dibutyryl cAMP not resulting in any additional increase (Chen, 1979, 1980). In contrast, the addition of asparagine alone caused a small increase in ODC activity in differentiated cells, and this increase was potentiated and reached a maximum after addition of fetal calf serum or of N 6 , 0 2 ’ dibutyryl cAMP (Chen, 1979, 1980).

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GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

In the presence of asparagine, confluent glioma cells exhibited an increase in ODC activity whereas the SAMD activity of the same cells under the same experimental conditions remained at the basal levels (Bachrach, 1980a). That asparagine stabilizes ODC, leading to a lengthening of the enzyme half-life and to an apparent increase in its activity, was confirmed (Bachrach, 1980a). These results are in good agreement with those of Chen and Canellakis (1977). Furthermore, the extents of ODC induction by asparagine were compared in normal and in transformed fibroblasts (Costa, 1979; Costa and Nye, 1979). Enhancement of ODC by this amino acid was much greater in cells transformed either by SV40 or by Ni& than in the counterpart normal cells (Costa, 1979; Costa and Nye, 1979). Despite the numerous investigations, the exact role of asparagine in influencing cellular polyamine metabolism remains to be elucidated, and further studies are needed to achieve a better understanding of the mechanisms by which this amino acid specifically affects ODC activity inside the cells. However, among the various inducers of ODC activity in cultured cells mentioned so far, GABA has been shown to be remarkably more effective in enhancing ODC activity, at least in cultured rat hepatoma cells, than the amino acids asparagine and glutamine (McCann et al., 197913). Like asparagine, GABA seems to have a direct stabilizing effect on ODC with a consequent slowing down of the enzyme’s turnover and a concomitant lengthening of its half-life (McCann et al., 1979b). The cellular putrescine levels after addition of GABA to the culture medium increased in parallel with the increases in ODC activity (McCann et al., 1979b). However, addition of GABA modified neither the cellular spermidine and spermine concentrations nor the SAMD activity of the tumor cells (McCann et al., 197913). Whether or not GABA has a general role in the comprehensive complex regulation of ODC activity in eukaryotic cells or, on the contrary, has only a limited role in particularly specialized cells, such as brain cells, with elevated GABA concentrations, remains a matter for speculation. Another environmental factor influencing basal ODC activity and polyamine contents in cultured cells is the osmolality of the surrounding medium. In HeLa cells the polyamine contents were found to be inversely related to the osmolality of the growth medium (Munro et al., 1975). A sudden increase in NaCl concentration of the medium causes a rapid fall in putrescine and spermidine concentrations. A sudden decrease in NaCl in the medium causes a rapid increase in putrescine (Munro et al., 1975). The levels of ODC activity in relation to external osmolality behaved like the polyamine contents and were, therefore, consistent with the changes in the polyamine levels (Munro

POLYAMINES IN MAMMALIAN TUMOHS

7

et al., 1975). The ODC soon declined when the external NaCl concentration rose and increased when the osmolality decreased. Interestingly enough, these variations in ODC activity were accompanied by similar variations in the half-life of the enzyme, since the half-life decreased when the enzyme activity decreased and increased when the enzyme activity increased (Munro et al., 1975). Nevertheless, suitable concentrations of intracellular cations are also important for regulating ODC activity, at least in L1210 mouse leukemic cells. It has been demonstrated that several ionophore antibiotics (which are compounds produced by microorganisms that specifically increase the permeability of the cell membrane to ions), such as valinomycin, nigericin, and monensin (valinomycin belongs to the group of the neutral ionophore carriers, and nigericin and monensin are carboxylic carriers) have the ability to strongly inhibit ODC activity, with only a slight decrease in protein synthesis (Chen and Kyriakides, 1977). The extracellular cations, in addition to regulating basal intracellular ODC activity, play essential roles in influencing ODC induction. Accordingly, the addition of suitable amounts of MgClz or NaCl or KCI completely or nearly inhibited the rises in ODC activity induced in cultured H35 or neuroblastoma cells by the addition of fresh medium with or without a serum supplement (Chen et al., 1976b). These cations, when present in the induction medium, prevented ODC enhancement (Chen et d.,197613). And, what is of more interest, if the L1210 cells have been grown for several generations in a medium containing a high Mg2+ concentration, the ODC induction produced by diluting these cells during the stationary phase with a medium of high Mg2+concentrations, i.e., with a medium theoretically inhibiting the ODC response, surprisingly reached the same levels as in the controls (Chen et al., 197613). This means that the leukemic cells are able to adapt progressively to new environmental conditions, at least in ODC induction. Furthermore, these cations did not significantly modify macromolecular synthesis when they were present in the medium at the same concentrations at which they prevented ODC induction by serum or fresh medium (Chen et al., 197613). Hitherto we have considered mainly those external factors able to induce ODC activity inside cells in culture. There are other factors that can cause the converse effects. The exposure of cultured hepatoma, W256 carcinosarcoma, leukemic, or neuroblastoma cells to different substances, chiefly to putrescine added to the culture medium, greatly decreased the ODC activity inside the cells (Clark and Fuller, 1976; Fong et al., 1976; Heller et al., 1976a,b, 1977a, 1978;

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GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

McCann et al., 1977a; Heller and Canellakis, 1980). As discussed in detail in Section I,B,l, Part I, Vol. 35, under such experimental conditions putrescine elicits the synthesis of the ODC antizyme. Among the neoplastic cell lines tested, the only negative reports in this respect are those of Clark and Fuller (1976), who did not detect this ODC inhibitor in cultures of polyoma-transformed 3T3 cells after exposure to serum and putrescine, and of Kudlow et al. (1980),who did not note the presence of the soluble inhibitor of ODC activity in cultured cells of a mouse adrenocortical tumor, but without any pretreatment whatever with polyamine. In addition to putrescine, spermidine, spermine, cadaverine, and other unphysiological polyamines can elicit the synthesis of ODC antizyme in a great variety of cultured cell lines, both neoplastic and normal (Heller et al., 1977a, 1978; McCann et al., 1977a). It must be recalled here once again that the concentrations of di- or polyamines in the medium that are required to stimulate the ODC antizyme are several orders of magnitude smaller than the amounts of di- and polyamines present inside cultured cells (Canellakis et al., 1978; Heller et al., 1978). The half-life of the ODC antizyme in various cultured neoplastic cell lines has been shown to vary roughly proportionally with the variations of the half-life of ODC measured under the same experimental conditions (Heller et al., 1976b). Last, it should also be noted that ODC antizyme is normally present in cultured hepatoma cells, not stimulated for synthesis of this ODC inhibitor (Heller et al., 1977b). Under these resting conditions the antizyme exists as an inactive protein bound to subcellular components from which it can be liberated b y treatment with very low concentrations of polyamines, noticeably of putrescine, at concentrations far less than those usually found inside the cells (Heller et al., 197713). The use of heat alone for treatment of cancer patients dates back to the late nineteenth century. However, there has been renewed interest, and considerable emphasis has been placed on using this old treatment for cancer patients, i.e., hyperthermia, either alone or in combination with other types of antineoplastic therapy, usually with radiation (Manning, 1979). It is now widely known that virtually every fundamental phase of cellular biochemistry (respiration, glycolysis, DNA, RNA, and protein synthesis) can be disrupted by sufficient exposure of mammalian cells to hyperthermia, resulting first in a loss of proliferative capacity and ultimately in cell death. In spite of this, the molecular mechanisms by which hyperthermia kills cells or causes prolonged cell cycles are still not fully understood. Nonetheless, some interesting studies have dealt with this topic, elucidating some aspects of the problem. In synchronous Chinese hamster ovary cultures, pro-

POLYAMINES IN MAMMALIAN TUMORS

9

gressing through the cell cycle after exposure to 43°C for 1 hr during either the GI or the S phase, there is a remarkable leakage of polyamines, mainly spermidine and spermine, into the culture medium (Gerner and Russell, 1977; Gerner et al., 1980). Obviously, this has as natural consequence the depletion of intracellular spermidine and spermine (Gerner et al., 1980);this depletion was reversed when the temperature was reset at 37°C (Gerner and Russell, 1977). In contrast, the intracellular putrescine concentration was not affected b y exposure of the cells to heat shock (Gerner and Russell, 1977). It is reasonable to connect the depletions of intracellular spermidine and spermine, most probably due to membrane damage by heat, with the alterations in DNA synthesis observed in the same cell line under the same experimental conditions (Gerner and Russell, 1977). Again, polyamines have the property of potentiating the killing of the cells by heat. In fact, exposure of cultured Chinese hamster cells to hyperthermia plus a polyamine (cadaverine or putrescine or spermidine or spermine) in the growth medium resulted in dramatic, synergistic cell death, regardless of the order of the two treatments (Ben-Hur et al., 1978; Gerner et al., 1980). Spermine was the most effective polyamine for potentiating thermal cell killing, followed by spermidine, cadaverine, and putrescine, in order of effectiveness (Ben-Hur et al., 1978). When there was a long time interval between the two treatments, this synergism disappeared (Ben-Hur et al., 1978). This enhancement of thermal killing by polyamines is dependent on the time of exposure and on the concentration of the exogenous polyamines (Gerner et al., 1980).The minimal polyamine concentrations that enhance the thermal sensitivity of the cells were by far lower than those normally found intracellularly, strongly suggesting a membrane effect (Gerner et al., 1980). Moreover, prolonged hyperthermia caused an increase in the uptake of exogenous polyamines (with the exception of putrescine) added to the growth medium by the same cultured cell line (Ben-Hur and Riklis, 1978). Generally speaking, the polyamines that penetrated into the cells were metabolized into the same products at both the physiological and the high temperature, indicating that the enhancement by polyamines of cellular sensitivity to heat shock is due to these molecules as such, not to their metabolites (Ben-Hur and Riklis, 1978). Another line of evidence that the potentiation of hyperthermia-induced cytotoxicity by polyamines is specific for these polycations is that the effect was not obtained with mono- or divalent inorganic cations, such as KC1, CaCI2, and MgC12 at equimolar concentrations (Gerner et al., 1980). The importance of all the foregoing observations lies in the fact that neoplastic cells usually contain

10

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

larger amounts of polyamines, and this may be one reason for the wellknown higher sensitivity of cancer cells to heat. Hyperthermia also produced in cultured Chinese hamster fibroblasts a drastic decrease in ODC activity, which occurred rapidly and exponentially as a function of continued exposure to heat (Fuller et al., 1977; Ben-Hur and Riklis, 1979a; Gerner et al., 1980).When the temperature reverted to the physiological level, ODC activity recovered and returned to control levels, after overshooting (Ben-Hur and Riklis, 1979a). The activity of SAMD was affected in the same way as that of ODC b y hyperthermia (Fuller et al., 1977). Polyamines have been shown to potentiate the killing effect of heat on mammalian cells, and they can also amplify the synergism between heat and radiation. In fact, spermine enhanced the synergistic interaction between hyperthermia and y-radiation in cultured Chinese hamster fibroblasts (Ben-Hur and Riklis, 1979b).This property of spermine to further strengthen the radiosensitizing effect of heat on the cells resulted in (a) enhanced cell death in the presence of radiation plus heat plus tetraamine, as compared to radiation plus heat; and (b) a drastic inhibition of cell repair of radiation-induced sublethal damage (Ben-Hur and Riklis, 1979b). Unfortunately, the effects of exogenous polyamines on .cultured neoplastic cells exposed to hyperthermia have received little, if any, attention so far, and almost nothing is known about this topic. In addition to hyperthermia, severe hypoxia induces an arrest of the cell cycle, so that it has been, and might presently be, considered a potentially useful tool for cancer therapy. In this regard, there is an interesting report that Chinese hamster cells exposed to severe and prolonged hypoxia and then reoxygenated slowly reenter the cell cycle and progressively increase their protein and DNA syntheses, but, astoundingly, the ODC activity fails to increase when oxygen is supplied (Kehe and Harris, 1978). In this instance, as in hyperthermia, the effect of hypoxia on polyamine biosynthesis and metabolism in cultured neoplastic cells has never been tested. Cellular ODC activity ca'n be greatly inhibited by some mitotic poisons, such as colchicine and vinblastine, that disrupt the cellular microtubule system. Indeed, when these two drugs were added to the medium of cultured L1210 cells, the activation of ODC activity by the dilution of the cells was prevented (Chen et al., 1976a). In contrast, lumicolchicine, an isomer of colchicine without any effect on the microtubular system, did not inhibit the ODC rise under the same experimental conditions (Chen et a1., 1976a). Vinblastine and colchicine also blocked O D C induction in rat glioma cells by dibutyryl CAMP

11

POLYAMINES IN MA.MMALIAN TUMORS

(Gibbs et al., 1979, 1980). In this cell line, too, lumicolchicine had no effect on either basal or stimulated levels of ODC activity (Gibbs et al., 1979, 1980). Furthermore, the integrity of the cytoskeleton seems to b e of great importance for ODC stimulation by added serum, since cytochalasin B inhibited ODC induction in L1210 cells (Chen et al., 1976a; Gibbs et al., 1979, 1980). Last, using an immunological approach, Quash and his collaborators (1971, 1972, 1973, 1978) have demonstrated that antipolyamine antibodies are cytotoxic for baby hamster kidney cells transformed b y the polyoma virus and growing in cell culture, and that complement is a necessary factor for the cytolytic effect, indicating the involvement of the cell membrane in the phenomenon. Moreover, cytolysis was inhibited and the cells recovered if the antipolyamine antibodies were removed or if putrescine, but not spermidine or spermine, was added to the culture medium containing the antiserum. Finally, evidence was provided that cytolysis of BHK-transformed cells is caused by the interaction of antidiamine antibodies with putrescine-containing sites on the cell membrane. This stresses once again the importance of the cell membrane in regulating ODC activity inside the cell.

B.

I N RELATIONTO CELL CYCLE

THE

GROWTHRATE

AND THE

PHASE OF

THE

Cultured cells of experimental neural neoplasias have been widely employed to investigate the connections between polyamine contents, combined or not with enzyme levels, and the growth rate of the cells. The activities of ODC and SAMD in a rat brain tumor cell line reached their maximum levels during the exponential growth phase and decreased as the growth curve reached a plateau (Heby et al., 1975b). The correlation coefficients obtained for the relationship of the enzyme activities to the specific growth rates were highly statistically significant (Hebyet al., 1975b).In studies of the correlations between cellular polyamine levels and the specific growth rate of the tumor cells, putrescine and spermine were not correlated, whereas spermidine and the spermidine : spermine ratio showed a direct positive linear correlation (Heby et al., 1975a,b). Parenthetically, it must be stressed that the rate of cell multiplication was maximal when the spermine content 1975b). Last, the compartmentalization of the was lowest (Heby et d., polyamines between nucleus and cytoplasm in this brain tumor cell line strongly indicates that spermidine and spermine act at the nuclear level, because the concentrations of these two polyamines were much

12

GIUSEPPE SCALARRINO AND MARIA E . FERIOLI

higher in the nucleus than in the cytoplasm (Heby, 1977).There were no significant changes in putrescine levels between the two cellular compartments, although the ODC activity was located mainly in the cytoplasm (Heby, 1977). In mouse neuroblastoma cells and in rat glioma cells, the spermidine : spermine ratio was found to decrease when growth was less rapid, and the putrescine content decreased as the cells entered the stationary phase (Kremzner, 1973; Kremzner et al., 1975; Sobue and Nakajima, 1977). However, the metabolism of the polyamines in these two kinds of neural neoplastic cell lines was found to be different, since in neuroblastoma cells the formation of GABA from putrescine was low during the logarithmic phase of cell growth and increased astoundingly during the stationary phase, whereas in glioma cells this metabolic conversion was always low throughout both phases (Sobue and Nakajima, 1977). There seems to be an inverse correlation between the rate of polyamine biosynthesis and the size of the polyamine pool in HeLa cells. The contents of polyamines were the highest during mitosis and the late GI phase, while at these times polyamine biosynthesis was minimal (Sunkara et al., 1979~). On the other hand, the polyamine contents were the lowest during early the GI and S phases, while the polyamine biosynthesis was maximal (Sunkara et al., 1979~).However, conclusions drawn from studies carried out with synchronized cell populations have to be drawn with caution, since it has been demonstrated in HeLa cells that the synchronization protocols, which yield large numbers of synchronized cells, can deeply affect both the basal cell content of polyamines and the polyamine accumulation during the cell cycle (Goyns, 1980). Polyamine biosyntheses and their levels in normal cultured cells in the different phases of the cell cycle have been reviewed briefly by Pardee et al. (1978)and exhaustively by Heby and Anderson (1980).

c. TWO-WAYRELATIONSHIPSBETWEEN POLYAMINES AND CYCLIC NUCLEOTIDES. INDUCIBILITY OF THE TWO POLYAMINE BIOSYNTHETIC DECARBOXYLASES As mentioned and discussed in Section I,E,l, Part I, Vol. 35, there are accumulated lines of evidence for CAMP as mediator of ODC induction in both in vitro and in vivo systems, with some arguments against it. We will now describe the experiments connected with this aspect carried out in cultured cells plus those experiments emphasizing the reverse aspect of the problem, i.e., the effects of polyamines on

POLYAMINES IN MAMMALIAN TUMORS

13

biosynthesis and metabolism of the cyclic necleotides in some cultured cell lines. ODC activity has been induced by cAMP and by dexamethasone (which is thought to act without implicating cAMP as second messenger) in logarithmically growing hepatoma cells originated from a Morris rat hepatoma and maintained in suspension culture (Canellakis and Theoharides, 1976).The induced enzyme was characterized by its immunoprecipitation and heat-stability patterns and shown to be identical with the enzyme purified from untreated hepatoma cells (Canellakis and Theoharides, 1976). ODC induction by these two drugs is similar in that the rates of ODC synthesis are markedly enhanced in both responses over that in controls (Canellakis and Theoharides, 1976). However, these two types of ODC induction have been shown to differ from each other in several aspects, namely, in their time courses and in their responsiveness to different types of inhibitors, such as actinomycin D and polyamine (Canellakis and Theoharides, 1976). First, the time course of ODC induction after dexamethasone was much slower than that after CAMP, but the enzymic levels were steadily elevated for many more hours. Second, actinomycin D completely inhibited induction by the glucocorticoid but only partially inhibited induction by CAMP.Third, the reverse is true for the effects of spermine and spermidine, since either these two polyamines depressed the ODC levels in the presence of cAMP even below basal control levels, whereas they were without effect on dexamethasone induction (Theoharides and Canellakis, 1975; Canellakis and Theoharides, 1976). Therefore the control of ODC activity in cultured hepatoma cells implies two paths, one CAMP-dependent and one CAMP-independent. All these results are in substantial agreement with those obtained with the same two drugs on another cell line of cultured hepatoma, i.e., the Reuber H35, by Byus et al. (1976) at the same time. ODC activity was induced in the H35 cells not only by dexamethasone and CAMP, but also by a series of 8-substituted cAMP analogs (Byus et al., 1976). Addition of insulin to H35 cultured cells was not followed by any increase in enzyme activity (Byus et al., 1976). Furthermore, the inducibility of ODC activity in H35 hepatoma cells has also been shown to be dependent on the composition of the culture medium (Liu and Chen, 1979). In vitro incubation of slices of rat adrenocortical carcinoma in the presence of cAMP resulted in significant ODC induction (Richman et al., 1973). More recently, a genetic approach has been used to explore whether hormonal activation of ODC activity is mediated by cAMP

14

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

and CAMP-dependent protein kinase (Kudlow et al., 1978, 1980). These authors used a cell line of a mouse adrenocortical tumor and its mutant clones, defective in either the adenylate cyclase response to ACTH or the response of the CAMP-dependent protein kinase to the same hormone. Both ACTH and cAMP induced ODC activity in the intact cell line, whereas only cAMP was able to enhance the enzyme levels in the mutant clones defective in adenylate cyclase (Kudlow et al., 1978, 1980). In the mutant clones defective in CAMP-dependent protein kinase, the magnitude of ODC response to ACTH was greatly reduced, but not totally abolished in some instances (Kudlow et al., 1978,1980). Both cAMP and nerve growth factor (NGF) induced ODC activity in a clonal cell line that originated from a rat adrenal pheochromocytoma, although the two types of induction were shown to be not causally interdependent, since NGF added to the culture medium did not produce any significant increase in cellular cAMP levels, even in the presence of theophylline (Hatanaka et al., 1978).The addition of either insulin or epidermal growth factor (EGF) (which both share some structural analogies with NGF) or of N2,02’-dibutyrylcGMP caused only little or no stimulation of ODC activity in this type of cultured neoplastic cell (Hatanaka et al., 1978). A concurrent report confirmed that ODC activity can be induced in these pheochromocytoma cells b y NGF and that the phenomenon requires new protein synthesis (Greene and McGuire, 1978). Furthermore, it was demonstrated that other noteworthy biological effects of NGF in these responsive cells, i.e., the stimulation of cell survival and of neurite outgrowth, were not impeded by total suppression of the cellular ODC activity by treatment with 1,3-diaminopropane or 5-hexyne1,4-diamine (Greene and McGuire, 1978). In contrast with the earliest report of Hatanaka et al. (1978), it was shown that E G F induced ODC in the rat pheochromocytoma clone PC12 and that preincubation of these cells in the presence of NGF largely prevented the ODC response to addition of E G F (Huff and Guroff, 1979). Both E G F and insulin stimulated putrescine transport into KB cells, but only insulin significantly enhanced the ODC levels of this type of cultured neoplastic cell (Di Pasquale et al., 1978). Cultures of tumors of the central nervous system are a good experimental model for clarifying the links between cyclic nucleotides and polyamine biosynthetic decarboxylases. The ODC activity of a rat glioma clone was quickly stimulated by addition of norepinephrine or isoproterenol or 3-isobutyl-1-methylxanthine (IBMX) or dibutyryl cAMP (Bachrach, 1975). In a mouse neuroblastoma clone, ODC activity was induced by PGE, or adenosine, but preincubation with IBMX

POLYAMINES I N MAMMALIAN TUMORS

15

was absolutely necessary to obtain the stimulatory effect (Bachrach, 1975).Complementary to this observation is another provided by Bachrach and his co-workers (1979), which demonstrates that morphine (an opiate that inhibits adenylate cyclase activity in some neural cell lines) almost completely antagonized the stimulating effects of IBMX with or without PGEl on the ODC activity and of the combination of the two drugs on CAMP levels in neuroblastoma x glioma hybrid cells (Bachrach e t al., 1979). In this case morphine also inhibited the stimulation of the activity of CAMP-dependent protein kinase elicited b y PGEl plus IBMX in the same cell hybrids (Bachrach et al., 1979). The assumption that CAMP is involved in SAMD induction too, was made and verified in the same cultured neoplastic lines (Bachrach, 1977). In fact, the level of this second polyamine biosynthetic decarboxylase was eleveted in glioma cells by the phosphodiesterase inhibitor IBMX or by catecholamines, and in neuroblastoma cells by PGE, and IBMX (Bachrach, 1977). All the foregoing results strongly support the idea that inductions of both the polyamine biosynthetic decarboxylases in cell cultures derived from neoplasms of the central nervous system of the rat or the mouse are mediated by CAMP and probably imply a wide cascade of biochemical events. However, Chen and Canellakis (1977) demonstrated that the stimulation of ODC activity in cultured mouse neuroblastoma cells brought about by the addition of N6,0Z’-dibutyrylcAMP or by PGE, plus IBMX was completely dependent on the presence of optimal concentrations of asparagine in the medium. Even more interesting, these authors also demonstrated that ODC could be induced in this cell line also b y high concentrations of asparagir,,. without any CAMP (Chen and Canellakis, 1977). An attempt to reconcile these seemingly contradictory reports of the true role of cAMP in ODC induction in neoplastic neural cell lines has been made by Gibbs et al. (1979,1980),who showed that there are separate pathways of ODC induction in rat glioma cells, i.e., one involving CAMPmediation and one not, and that these two pathways have as a common biochemical feature an absolute requirement for Ca’+. In fact, both isoproterenol and dibutyryl CAMP induced ODC activity in these neural cells, but both these inductions were completely abolished by the presence in the culture medium of ethylene glycol bis(p-aminoethyl ether)-N,N,N N ’-tetraacetic acid (EGTA), a well-known and powerful Ca2+chelator (Gibbs e t al., 1979, 1980); EGTA alone reduced the basal level of ODC activity (Gibbs et d., 1979, 1980). On the other hand, EGTA was also able to prevent ODC induction in the same cellular line by fetal calf serum, which last had little, if any, effect upon intracellular CAMP content (Gibbs et al., I,

16

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

1979, 1980). The addition of calcium alone without serum did not modify ODC activity to any appreciable extent, and the specifity of Ca2+requirement for ODC induction has been demonstrated by the fact that this ion cannot be substituted for magnesium (Gibbs et al., 1979, 1980). The hypothesis that cAMP mediates the increase in ODC activity has also been tested in mouse S49 lymphoma cells (Insel and Fenno, 1977, 1978), with results opposite to those hitherto described. Incubation of cells of the wild-type of this lymphoma with dibutyryl CAMP, after an initial fleeting increase, profoundly and progressively decreased both ODC and SAMD activities to barely detectable levels (Insel and Fenno, 1977, 1978). Inhibition of ODC activity in S49 lymphoma cells is an early response to other agents that increase intracellular cAMP level, i.e., isoprotereno1, cholera toxin, or PGEl (Honeysett and Insel, 1980). These decreases in cellular ODC and SAMD levels were causally connected with a parallel decrease in the activity of CAMP-dependent protein kinase, since in “kinasenegative” mutant cells, that is, in a cell line totally lacking in CAMPdependent protein kinase activity and therefore in response to dibutyryl CAMP, no decrease in the levels of the two polyamine biosynthetic decarboxylases was observed after addition of this cyclic nucleotide to the culture medium (Insel and Fenno, 1977, 1978). On the contrary, the fall in ODC activity induced in the wild-type S49 cells by cAMP was not accompanied by a progressive decrease in protein synthesis, demonstrating that the two biochemical events are dissociable (Insel and Fenno, 1977, 1978). An analogous split between the decrease in the activities of the polyamine biosynthetic decarboxylases and the cell killing induced by cAMP is also possible, using the “CAMP-deathless” mutants, that is, mutants phenotypically resistant to the cytolysis induced by cAMP (Insel and Fenno, 1977, 1978; Kaiser et al., 1979). Furthermore, treatment with dibutyryl cAMP caused similar decreases in ODC activity in the ‘‘CAMP-deathless” mutants, regardless of the phase of the cell cycle at which the distinct cell populations were examined (Kaiser et al., 1979). Instead, Bachrach (1980b) found that ODC activity was induced by cAMP in cultures of S49 lymphoma cells, but not in a mutant line defective phenotypically in CAMP-dependent protein kinase activity. The aspect, which is converse and complementary to that hitherto analyzed, of the connections between polyamine biosynthesis and cyclic nucleotides, i.e., whether polyamines can modulate the metabolism of the cyclic nucleotides inside the eukaryotic cells, has been scarcely investigated. In spite of this, some evidence is emerging that pol yamines really can regulate the synthesis of the different cyclic

POLYAMINES IN MAMMALIAN TUMORS

17

nucleotides. Indeed, spermine dramatically inhibited the activity of CAMP-dependent protein kinase activity in glioma cells, and the inhibition was shown not to be d u e to an interaction of the tetraamine with the regulatory subunit of the enzyme (Bachrach et al., 1978; Bachrach, 1980b). Addition of any of the three chief polyamines has been shown to cause a decrease in cAMP concentrations in cultured glioma or neuroblastoma or neuroblastoma x glioma hybrid cells, either unstimulated or stimulated with hormones or drugs (such as norepinephrine, isoproterenol, PGEI, adenosine, IBMX), which are well-known agents for inducing cAMP accumulation inside the cells (C16 e t al., 1979). It is of special interest that a decrease occurred when the exogenous polyamine was added even at low concentrations in the range of those found in physiological fluids (Cl6 e t aZ., 1979). Paradoxically, polyamines at higher concentrations caused a slight increase in the intracellular cAMP levels of the cultured neural neoplastic cell lines (C16 e t al., 1979). Last, a report shows that spermidine and spermine and, to a lesser extent, putrescine are effective inhibitors of the activity of specific cCMP phosphodiesterase obtained from leukemic L1210 cells (Bloch and Cheng, 1979). Friend erythroleukemia cells are a relatively pure population of virus-transformed mouse hematopoietic cells. These cells can be induced by a variety of chemical agents with different biological properties to differentiate to orthochromatic or polychromatic normoblasts and are a suitable experimental system for studying the biochemical events involved in cell differentiation. Interestingly enough, ODC activity can be rapidly induced in this cell line by some inducers of differentiation, such as dimethyl sulfoxide (Tsiftsoglou and Kyriakidis, 1979; Gazitt and Friend, 1980). The ODC induction was observed when the cell differentiation process was blocked or when the inducers were added to cell lysates (Tsiftsoglou and Kyriakidis, 1979). However, the cell differentiation process caused by the inducers in the Friend erythroleukemia cells appears to be not at all mandatory or causal for ODC induction, since some potent inducers, like actinomycin D or aminonucleoside of puromycin, do not stimulate ODC (Gazitt and Friend, 1980). Two final conclusions seem to us to be appropriate. First, the induction of O D C activity appears to be a common and easily observable phenomenon in cultured neoplastic cells, in striking contrast with what has been observed in uivo in neoplastic organs and in organs undergoing chemical carcinogenesis (Scalabrino e t al., 1978). Second, the mediation of CAMP claimed to be a general and possibly obligatory step in ODC induction appears, on the basis of the studies carried out with in uitro systems, to be so in some instances, but to be totally

18

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

extraneous in some others, and this is in quite good agreement with what has emerged from in vivo studies of ODC inducibility.

D. EFFECTSO F

INFECTION WITH

NONONCOGENICVIRUSES

Infection of HeLa cells with vaccinia virus brought about both quantitative and qualitative changes in ODC activity inside the cells, as was demonstrated by Hodgson and Williamson (1975). The ODC was induced and rose quickly following viral infection and, what is even more important, the mean K , value for ODC was significantly lower in infected than in uninfected cells (Hodgson and Williamson, 1975). On the contrary, at later postinfection times the biosynthesis of all the polyamines, including cadaverine, was greatly reduced, but not completely inhibited, in HeLa cells infected with vaccinia virus (Lanzer and Holowczak, 1975). Substantially the same thing was observed in KB cells infected with type 5 adenovirus, at long intervals after infection (Pett and Ginsberg, 1975). This time course for polyamine biosynthesis and concentration in neoplastic cells infected with nononcogenic viruses, with first an increase during early infection and then a decrease at late postinfection times, was also seen in Ehrlich ascites tumor cells infected with mengovirus (Egberts et al., 1977). By way of conclusion, let us tentatively compare the polyamine response of neoplastic cells infected by nononcogenic viruses with the responses observed in normal cells undergoing neoplastic transformation by oncogenic viruses. We can state that (a) there is an increase in cell polyamine biosynthesis immediately after the infection in both types of viral cell infection; (b)there is a clear dichotomy in late phases of postinfection time between the two types of viral cell infection, since the cell polyamine biosynthesis remains at high levels in the neoplastic viral transformation process (see Section IV, Part I, Vol. 35) and decreases progressively after nontransfonning infection of neoplastic cells by nononcogenic viruses.

E. MISCELLANEOUS EFFECTSO F POLYAMINES Most reports on the effects of polyamine addition to cultures of some neoplastic cell lines deal with protein synthesis and cell proliferation. Spermine stimulated poly(UG)-dependent phenylalanine incorporation in a subcellular protein-synthesizing preparation obtained from L1210 mouse ascites leukemic cells, and the stimulation was beyond

POLYAMINES IN MAMMALIAN TUhIOHS

19

that achieved with optimal magnesium concentrations, suggesting that spermine may act as more than merely a substitute for magnesium (Ochoa and Weinstein, 1964). All three chief polyamines (with spermine the most effective) had stirnulatory effects on tRNA methylases in extracts of L1210 cells (Hacker, 1973). Exogeneous spermidine and spermine stimulated the incorporation of orotic acid into RNA and considerably decreased the degradation of the newly synthesized RNA in Ehrlich ascites cells (Raina and Janne, 1968; Khawaja and Raina, 1970). The presence of spermine was essential for the translation in a cell-free system derived from wheat germ of tyrosine aminotransferase mRNA from hepatoma cells (Rether et al., 1978). Again, spermine could partially substitute for soluble factors present in dexamethasoneinduced hepatoma tissue culture that stimulate in a homologous cellfree system the translation of mRNA coding for tyrosine aminotransferase (Beck et al., 1978). In Walker 256 carcinosarcoma cells, putrescine and spermidine preserved the ultrastructural morphology of all nuclear structures, including the nucleolus (Busch et al., 1967). In HeLa cells, polyamines were present in abundant quantities in the chromosome cluster region (Goyns, 1979) and have been shown to stimulate the nuclear synthesis of the histone Hl-poly(ADP-ribose) complex (Byrne et al., 1978).The intercellular adhesiveness of HeLa cells harvested from densityinhibited suspension cultures was markedly enhanced by the addition of putrescine to the medium in which the cells were resuspended (Deman and Bruyneel, 1977). In contrast, the diamine did not modify the mutual adhesiveness of cells harvested from fast-growing cultures (Deman and Bruyneel, 1977). Polyamines have been found also to have some inhibitory effects on cultured neoplastic cells. Spermine depressed protein synthesis in Walker 256 carcinosarcoma cells (Goldstein, 1965). This tetraamine is distinctly cytotoxic for different hepatoma cell lines, and the effect was noticeably enhanced by the presence of fetal calf serum in the growth medium (Katsuta et al., 1975). Among the cytotoxic metabolites released from rat ascites hepatoma cells into culture fluid, some closely resembled spermine in chemical nature (Katsuta et al., 1974). Spermidine, putrescine, and cadaverine all inhibited replicative DNA synthesis in mouse ascites sarcoma cells (Seki et al., 1979).The addition of spermidine or spermine to the medium inhibited the growth of cultured human meningioma cells, whereas putrescine had a slight opposite effect (Duffy et al., 1971). Granulocytic chalone, but not the polyamines, inhibited [3H]TdR uptake in rat chloroleukemia cells in short-term cultures (Foa et al., 1979). This result favors the idea that

20

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

this activity of granulocytic chalone does not depend on its possible polyamine content. The reciprocal connections between synthesis of polyamines and that of 5’-methylthioadenosine (MTA) were evidenced in a human leukemic cell line lacking 5‘-methylthioadenosine phosphorylase (Kamatani and Carson, 1980). The addition of spermine or spermidine markedly depressed the synthesis of MTA, whereas the addition of MTA stimulated putrescine synthesis at low concentration or inhibited it at high concentrations (Kamatani and Carson, 1980). Exogenous MTA also depressed the intracellular levels of spermine in leukemic cells but not, very surprisingly, those of spermidine (Kamatani and Carson, 1980), although MTA is a well-known inhibitor of both spermidine and spermine synthetases (see Section I,D, Part I, Vol. 35). Nevertheless, these relationships between polyamine and MTA metabolisms in neoplastic cells await further experimental elucidation. Finally, spermine precipitated a cell-surface protein, fibronectin, from the culture medium into which it had been secreted by a human rhabdomyosarcoma cell line (Vuento et d.,1980). This observation raises the possibility that polyamines have a role in the deposition of fibronectin in vivo. This is of mounting interest, since many types of malignantly transformed cells, unlike the normal adherent cells, generally deposit small amounts of the surface fibronectin in the pericellular matrix, and this scarcity has been causally correlated with some malignant behavioral properties, which have been overcome by the addition of fibronectin to cultures of tumor cells. Whether elevated polyamine production and secretion (when present) and fibronectin scarcity are interconnected with other factors in determining the well-known poor adhesiveness of neoplastic cells, remains hypothetical, speculative but very attractive. II. Polyamines in Human Oncology

Theoretically, the need to diagnose and subsequently to locate a tumor as early as possible has always been considered to be a key goal for antineoplastic therapy. The expectation that this would be possible was kept alive by new findings, in both experimental and clinical oncology, which showed that certain kinds of neoplasias produce unusual metabolites. Many tumor-cell products, collectively called neoplastic markers,” or “tumor-associated markers” or “oncodevelopmental markers,” have been identified in blood, effusions, urine, and cerebrospinal fluid of tumor-bearing patients and in neoplastic tissue extracts. “

POLYAMINES IN MAMMALIAN TUMORS

21

Measurements of these products have been employed extensively in clinical medicine for both initial diagnosis of neoplasia and monitoring tumor recurrence after different types of therapy. In fact, it was hoped that changes in the amounts of these “markers” in one or more of the different physiological fluids of cancer patients or in the neoplastic tissue could be used to reflect changes in the body’s tumor burden, since these products can be either secreted into the surrounding milieu or kept within the neoplastic cells. The most widely employed “markers” for neoplastic growth and tumor dedifferentiation or differentiation are listed in Table I. As new metheds for the measurement of tumor “markers” were introduced and became ever more sensitive, and clinical studies ever larger, the expectation that these “markers” would be of key importance or, at least, very useful for early diagnosis of neoplasia did not fully materialize. In fact, elevated levels of “ markers” were observed in a variety of nonneoplastic diseases, and, conversely, some neoplasms were not accompanied by any known marker.” Some “markers” have even been found in a percentage of normal healthy adults. In addition, some of these “markers” are also found in the earliest stages of human development, and this association has led to the widely accepted practice of referring to most neoplastic “markers” as oncofetal proteins and antigens (Sell, 1980). The divergence between expectation and practical results was explained after extensive studies of the biochemistry of cancer, which have taught as that tumor cells do not synthesize tumor-specific substances, i.e., substances never found in normal cells at any step in their differentiation (Wolf, 1979a). What is characteristic of tumor cells is that they either express certain normal gene information at the wrong time or in the wrong place or in the wrong amount, or completely fail to express some normal genes (Wolf, 1979a,b). Moreover, during recent decades, it has become ever clearer that neoplasia is not one single type of disease, but a group of very many diseases, each utterly different from the others from the clinical and biochemical points of view, with the only common features that they are lethal to the host and have a cell growth type that is invasive and can never b e stopped definitively. Some frequently found discrepancies between the amount of tumor “marker” present and the degree of growth of the neoplasm must be connected with phenotypic expression of these “markers,” which varies from cell to cell within the neoplasm. In fact, quantitative and qualitative variations in the production of “markers” may occur during the natural course of the malignancy. In other words, during the development of a tumor from preneoplasia to early neoplasia to advanced ‘I

MAIN DIFFERENT BIOCHEMICAL AND

TABLE I “MARKERS”OF NEOPLASTIC GROWTH AND TUMOR DEDIFFERENTIATION DIFFERENTIATION USED I N CLINICAL ONCOLOGY

IMMUNOLOGICAL OR

Products Acute-phase reactant proteins (APRPs): a,-Antitrypsin, a,-antichymotrypsin, ceruloplasmin, C-reactive protein, haptoglobins, fibrinogen Chromosomal abnormalities: Ph’, 13 q-, 14 q+ Cyclic nucleotides: CAMP, cGMP, ratio CAMP: cGMP Enzymes or isozymes: Leucine aminopeptidase, y-glutamyl transferase, copper oxidase, creatine kinase BB, histaminase (DAO), muramidase, galactosyl transferase 11, lysozyme, ribonuclease, arylsulfatase A, reverse transcriptase, terminal deoxynucleotidyl transferase (TdT), superoxide dismutase. Glycolytic isozymes: (a) glucose phosphate isomerase, (b) aldolase (shifting vs A form), (c) LDH (shifting in the isoenzyme pattern from LDH-1 to LDH-5 part of the isoenzyme spectrum). Phosphohydrolases: (a) acid phosphatase, (b) alkaline phosphatase, (c) 5’-nucleotidase Hormones, isohormones, fragments or catabolites of hormones: Ectopic production of hormones (paraneoplastic syndromes): ACTH, gonadotropins, ADH, PTH, ILA, TSH, erythropoietin, MSH, HGH, HPL, HCG, PL, PGA, PGE, CT; catechqlamines, metanephrine, vanilly1 mandelic acid; 5-HIAA, 5-HT, 5-HTP, bradykinin.

References Cooper and Stone (1979) Purtilo et al. (1978) Pardee et al. (1978); Pastan et al. (1975) Bodansky (1975); Bollum (1979); Fishman (1974); Fishman and Singer (1975); Goldberg (1979); Kaplan (1972); Oberley and Buettner (1979); Ruddon (1978); Schapira (1973, 1978); Uriel (1975, 1979); Weber (1977);Wolf (1979b); Yam (1974)

Hall (1974); Ode11 and Wolfsen (1975); Rees and Ratcliffe (1974); Ruddon (1978); Seyberth (1978); Sherwood and Could (1979); Wolf (1979b)

Immunoglobulins: Homogeneous (monoclonal) immunoglobulins (M components); Bence Jones proteins ( K or A light chains); abnormal or incomplete heavy chains: (a) y-chain subclasses (yl, yz. y3, y4) of IgG; (b) a-chain subclasses ( a l , a z )of IgA; (c) p-chains Miscellaneous proteins: Fetal hemoglobin, EDC1, milk casein. Placental and pregnancy proteins: (a) SP, pregnancy-associated a,-glycoprotein (aZPAG); (b) SP, pregnancy-specific &glycoprotein; ( c ) PPTPP8 (ubiquitous tissue) proteins; (d) PPs, placental protein five. Plasminogen activators Oncofetal proteins and antigens: a-FP, CEA, FSA, a2H-ferroprotein; pancreatic oncofetal antigen (POA), p-oncofetal antigen (BOFA), OFA, glial fibrillar acid protein (GFAP)

E3

w

Polyamines and their biosynthetic decarboxylases: Pubescine, spermidine, spermine, ornithine decarboxylase, S-adenosyl-Lmethionine decarboxylase Sterols: Desmosterol (cholesta-5,24-dien-3-P-ol, or 24-dehydrocholesterol)

Bodansky (1975); Solomon (1977); Waldenshom (1976)

Bohn (1980); Ruddon (1978); Rudman et al. (1976, 1977)

Fritsche and Mach (1975); Lehman (1979); Loewenstein and Zamcheck (1977); Martinet al. (1976); Ruoslahti and Seppala (1979); Seidenfeld and Marton (1979b); Sell and Becker (1978); Uriel (1975, 1979); Wikstrand and Bigner (1980) Bachrach (1976a); Janne et al. (1978); Milano et al. (1980); Russell (1977); Russell and Durie (1978); Savory and Shipe (1975); Scalabrino et al. (1980); Seidenfeld and Marton (1978, 1979b) Seidenfeld and Marton (197913);Wikstrand and Bigner (1980)

24

GIUSEPPE SCALARRINO AND MARIA E . FERIOLI

metastatic tumor, heterogeneous subvariant cell populations emerge within single clones. Heterogeneity of tumor cell populations may lead not only to the loss of some “marker(s)” but also to the emergence of new ones (Wolfe, 1978; Wolf, 1979a,b). Last, but not least, it is worth mentioning here that some naturally occurring cell labels, such as the glucose-6-phosphate dehydrogenase (G-6-PD) system and the surface-associated immunoglobulins, generally used for identifying different normal cell subpopulations, can also be employed to determine whether a given neoplasm has a single or multiple cell origin, providing an important clue to the initiating event (Fialkow, 1974). The polyamines, although they suffer from the same drawbacks listed above for the other neoplastic “markers,” are widely considered to b e clinically useful “markers” of neoplastic growth and for cancer diagnosis, and particularly for evaluation of the success or failure of an antineoplastic therapy. It is largely accepted to differentiate the different tumor cell “markers” into (a) those produced by dedifferentiation of neoplastic cells (e.g., carcinoembryonic antigen, a-fetoprotein, alkaline phosphatase isozyme) and (b) those produced as a result of overproduction by tumor cells or of the increased tumor cell multiplication (e.g., acid phosphatase, those hormones secreted by specific endocrine-gland neoplasms). Polyamines, for reasons discussed later, have to be included in the second group of neoplastic “markers.” The importance and the clinical significance of polyamines in human oncology have been well reviewed several times by Russell (1973a, 1977), Savory and Shipe (1975), Bachrach (1976b), Cohen (1977), Janne et al. (1978), Russell and Durie (1978), Seidenfeld and Marton (1978, 1979b), Buehler (1980), Durie (1980), and Milano et al. ( 1980). Therefore, we aim here to outline the current “state of the art” about the connections between polyamines and human cancer, together with some recently obtained advances, and to draw particular attention to the use of the levels of activity of the polyamine biosynthetic decarboxylases (PBD) as biochemical indicators of the growth rate, and consequently of the malignancy, of some types of human neoplasias. A. PATTERNS OF POLYAMINES I N HUMANNEOPLASTICTISSUES Hamalainen (1947) made the pioneering observations in this field, systematically screening spermine contents in a great number of organs obtained postmortem from patients who had died of different types of neoplasia. He found an elevated spermine content in the lung

POLYAMINES IN MAMMALIAN TUMORS

25

of a patient who died of lung carcinoma, in the uterus of a patient who died of uterine carcinoma, and in livers, spleens, and bone marrow of two patients who died of leukemia. These observations were subsequently extended to a variety of human malignancies by other authors. Including the more recent reports on the polyamine content of human tumors, it has become ever more evident that there is no general or unique pattern for the polyamine content of human neoplasias. In fact, brain tumor tissues (e.g., neurofibroma, meningioma, glioblastoma, astrocytoma, glioma) have as their particular biochemical feature very high putrescine concentrations in comparison with both gray and white areas of normal human brain (Kremzner, 1970, 1973; Kremzner et al., 1970).On the contrary, the levels of spermidine and spermine in the tumors studied did not greatly differ from levels observed in normal brain, the only exception being high concentrations of spermidine and spermine in astrocytoma and in glioma (Kremzner, 1970, 1973; Kremzner et al., 1970). Additionally, human tumor tissue in vitro and meningioma cells grown in culture actively incorporated [I4C]putrescine into spermidine and spermine, but showed low deaminating activity (Kremzner et al., 1972). The high levels of putrescine in several central nervous system-related tumor tissues have been confirmed (Harik et al., 1978; Harik and Sutton, 1979). Furthermore, it has also been demonstrated that the magnitude of the elevation of putrescine content in the astrocytoma groups is proportional to the degree of malignancy of the tumor as determined by conventional histopathological criteria. A variety of slowly growing and relatively benign intracranial or intraspinal tumors (such as meningioma, cerebellar hemangioblastoma, chordoma, neurofibroma, schwannoma) had low levels of putrescine that in many instances did not exceed the range in samples from normal brain tissue (Harik et al., 1978; Harik and Sutton, 1979). On the other hand, the tissue concentrations of spermidine and spermine varied broadly within normal cerebral cortical samples and within the various tumor types, with no obvious correlation with the degree of malignancy of the tumor (Harik et al., 1978; Harik and Sutton, 1979). Therefore, it can be tentatively concluded that, at least among the astrocytomas, the putrescine level may be a reliable biochemical “marker” not of the tumor per se, but of the degree of malignancy of the tumor. However, amazingly enough, high putrescine levels have been detected in papillary adenocarcinomas of the thyroid, which are the most clinically benign and extremely slow growing of all thyroid malignancies (Matsuzaki et al., 1978). Among the renal cell carcinomas, the concentration of spermidine in

26

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

the poorly differentiated types (grades 3 and 4) was significantly higher than in the well-differentiated types (grades 1 and 2), and in both it was always higher than in normal renal tissue (Matsuda et al., 1978). Thus, in renal carcinomas the concentration of spermidine correlates well with the degree of tumor malignancy ascertained by histopathological known criteria, particularly the nuclear atypia. In the same types of renal tumors, another supposed “marker” for the growth rate of neoplastic cells, i.e., the spermidine : spermine ratio (Russell, 1973b), progressively increases from the normal renal tissue to the poorly differentiated type of renal carcinoma, with the ratio for the well-differentiated type of tumor in the middle (Matsuda et al., 1978). This significant increase in the spermidine : spermine ratio in human renal adenocarcinomas was also confirmed by other authors (Dunzendorfer and Russell, 1978,1979,1980).The concentration of spermidine in neoplastic tissue was significantly higher than in the histologically normal areas of the same kidneys, while the spermine content of the tumor was generally lower than that of normal tissue (Dunzendorfer and Russell, 1978, 1979, 1980). On the contrary, thyroid adenocarcinomas have ratios of spermidine to spermine very close to those found in normal thyroids or those affected by other nonneoplastic diseases (Matsuzaki et al., 1978). The cellular content and secretion of polyamines, in relation to the cell cycle and proliferation kinetics, have been investigated in uitro with cultured cells of Burkitt’s lymphoma (Woo et al., 1979),which is a rapidly growing malignant tumor characterized by a high rate of cell proliferation combined with a high growth fraction. As for the time course of the intracellular contents of polyamines during the growth of Burkitt’s lymphoma cells, large amounts of spermidine and spermine and lower amounts of putrescine were observed during the lag and early exponential growth phases (Woo et al., 1979). This trend was reversed when the cultured cells entered the exponential growth phase and early plateau growth, since spermidine and spermine contents markedly decreased, while the putrescine content tripled (Woo e t al., 1979). The ratio of spermidine to putrescine and that of spermine to putrescine were significantly and positively correlated with both the labeling index and the specific growth rate, whereas there was no significant variation in the spermidine : spermine ratio throughout the growth period (Woo et al., 1979). As for the changes in the cellular polyamine content during the cell cycle, the cell fraction in GI showed a significantly high positive correlation with the intracellular content of putrescine, while negative correlations were calculated for spermidine and spermine (Woo et al., 1979).All these results

POLYAsfINES I N MAMILIALIAN TUMORS

27

seem to suggest that all the chief polyamines actively participate in the process of proliferation of Burkitt’s lymphoma cells. Last, a phospholipid-the so-called malignolipin-containing spermine has been found in some human malignant tumors [e.g., seminoma, gastric cancer, cancer of the colon, uterine cancer, breast cancer (Kosaki et al., 1958)] and in bloods of cancer patients (Kallistratos et al., 1970), but never in normal tissues. This phospholipid contains, in addition to spermine, choline, phosphoric acid, and fatty acids. Although this malignolipin was discovered several years ago, the function and the biological significance of such a compound in tumor development and in tumors of high malignancy remains to be determined and awaits more rigorous demonstration (Bachrach and Ben-Joseph, 1973). B. LEVELSO F THE CHIEF POLYAMINES A N D THEIR CONJUGATED FORMS I N URINES OF NORMAL SUBJECTS A N D OF CANCER PATIENTS Observation of the high polyamine contents in neoplastic tissues at once stimulated looking for increased quantities of these polycationic substances in the extracellular fluids of patients with malignancies. Unlike the neoplastic tissues, the human body fluids generally contain small quantities of polyamines, so that the quantification of these substances in these fluids requires highly sensitive methods. In the last decade noticeable improvements in the assay methods for polyamines have been achieved (Seiler, 1977, 1980). The assay methods most widely used at present for quantitative determinations of polyamines and their derivatives are thin-layer chromatography of dansylated polyamines, automated ion-exchange chromatography, high-pressure liquid chromatography, gas chromatography, and radioimmunological assay. All these methods are sensitive and accurate enough to detect very small amounts of polyamines in both physiological fluids and in biopsy material. Therefore, routine screening for polyamine levels in fluids of human beings with different types of pathologies, whether or not characterized by uncontrolled cell proliferation, is now possible. The data available in the literature on the daily urinary excretion of polyamines and of their conjugated forms by normal subjects are reported in Tables I1 and 111, where the values have been divided into groups according to the units of measurement used by the various authors to express their results. Those data reported by some authors as control levels, but taken from hospitalized patients with nonneoplastic diseases, have deliberately not been reported in Tables I1 and 111,

TABLE I1

NORMAL DAILYCONTENTS Unit mgl24-hr U

rmoll24-hr U

pmollkgl24-hr U

POLYAMINES IN HUMANURINE"

N

Putrescine

Spermidine

Spermine

2 50 50 10 5 12 8 6 42 NR 42 50 56 21 8 20 9

2.5 2.7 2 0.53 2.7 2 0.5 2.5 f 0.6 2.0 1.4 0.94 2.2 3.52 0.2-2.84 0.8-6.2 3.52 4.21 f 0.41 0.89 1.52 0.98 0.49 1.6 2 0.4 1.6 (9) 1.57 2 1.05 21.9 2 7.6 9.8 2.0 0.5 0.38 2 0.017 0.2 0.4 0.38 2 0.17 0.7

2.7 3.1 0.56 3.1 2 0.6 2.4 f 0.4 1.5 1.3 0.86 1.6 2.44 0.36-2.1 0.9-3.9 2.44 1.12 0.11 0.53 0.83 4.57 2 1.02 0.2 2 0.04 0.3 (7) 0.51 0.16 8.4 f 2.1 7.6 2 2.5 0.2 0.1 f 0.003 0.12 0.11 0.11 f 0.04 1.5

2.5 3.4 2 0.67 3.4 2 0.7 0.4 2 0.2 <0.4 0.4

12 12 5 13 28 35

44 pg/ml U

OF THE CHIEF

50 8

*

*

*

*

*

-

0.4 2.59 0.0-0.87 1.0-4.2 2.59 3.4 2 0.67 0.14 0.20 0.31 f 0.09 2.1 2 1.0 2.0 (7) 0.71 f 0.57 2.5 f 1.2 2.5 2 0.8 0.07 0.01 f 0.002 0.02 0.01 0.01 f 0.006 -

Remarks

Reference Russell (1971) Russell et d. (1971a) Schimpff et QZ. (1973) Marton et QZ. (1973a) Marton et ~ l (1973b) . Gehrke et QZ. (1973) Gehrke et ~ l (1973) . Gehrke et QZ. (1974) Kessler et aZ. (1974) Tormey et QZ. (1975) Sanford et QZ. (1975) Lipton et 01. (1975, 1976) Fujita et QZ. (1976) Adler et QZ. (1977) Makita et QL. (1978) Brown et QL. (1979) Abdel-Monem and Ohno (1978) Abdel-Monem et QZ. (1978) Rattenbury et QZ. (1979) Rattenbury et ~ l (1979) . Swendseid et QZ. (1980) Waalkes et QZ. (1975a) Waalkes et QZ. (1975b) Waalkes et QZ. (1975b) Waalkes et aZ. (1975b) Woo et QZ. (1978) Veening et QZ. (1974)

10 12 15 16 NR NR 12 13 10 6 5 10 11 NR 7 61 56 9 10 NR

pmol/mg CR mglg CR nmol/mgCR pmol/g CR pmol/kg/24-hr U mg/24-hr U pmollg CR mglg CR

28 21 10 61

3.58 f 0.99 1.8 2.5 2 0.13 2.1 f 0.62 1.31 1.79 3.0 2 0.84 3.5 2.5 f 0.8 2.73 2 0.59 2.09 2 0.57 2.9 3.4 1.4 1.44 1.68 2 0.62 125.3 f 17.7 13.5f 2.16 0.89 f 0.76 0.45 0.02 f 0.003 0.55 0.93 f 0.50 0.86 f 0.60

1.3 1.7 2 0.10 1.2 f 0.18 0.92 1.02 2.2 f 0.44 2.6 1.1 f 0.5 1.56 2 0.42 1.79 2 0.49 1.4 2.1 0.5 0.81 1.32 f 0.41 20.3 f 2.4 6.3 f 0.35 0.67 f 0.57 0.28

0.5 f 0.24 0.5 f 0.06 0.04 2 0.007 0.27 0.19 0.51 0.0-5.5 0.05 f 0.007 0.06 f 0.01 0.14 f 0.08 8.4 4.5 0.18 0.18 f 0.29 46.4 f 12.5 0.9 f 0.15 -

Cadaverine Cadaverine Cadaverine Cadaverine

-

-

In(PUT U/g CR) = 0.013(age + 3/4) - 0.031 ln(age + 3/4) ln(SPD U/g CR) = - 1.454 - O.O12(age + 3/4) - 0.071 In(age + 3/4) ln(SP U/g CR) = 4.787 - 0.027(age + 3/4) - 0.013 ln(age + 3/4) ~~

~

Rennert et al. (1976a) Russell et al. (1975) Townsend et al. (1976) Russell (1977); Durie et nl. (1977a) Nishioka et al. (197813) Nishioka et al. (1978b) Heby and Andersson (1978a) Osterberg et al. (1978) Russell et al. (1978) Russell et al. (1979) Russell et al. (1979) Tsuji et al. (1975) Tsuji et al. (1975) Slanina et al. (1979) Makita et al. (1978) Fujita et al. (1980) Fujita et al. (1976) Proctor et al. (1979) Berry et al. (1978b) Berry et al. (197813) Waalkes et al. (197%) Adler et al. (1977) Berry et al. (1978b) Fujita et al. (1980) Rudman et al. (1979) Rudman et al. (1979) Rudman et al. (1979) ~~

"The results reported are expressed as means alone, or means 2 SE, or means 2 SD (when specified). N, number of subjects; NR, not reported; FP, free polyamine; TP, total polyamine; M, men; W, women; C, children; U, urine; CR, creatinine; kg, kilogram of body :veight; R, range; PUT, putrescine; SPD, spermidine; SP, spermine.

TABLE 111 NORMALDAILYCONTENTS OF THE CONJUGATED FORMS OF POLYAMINES IN HUMANURINE" Unit

N

Ac-PUT

NI-Ac-SPD

NMcSPD

pmo1/24-hr U

3 3 9 12 5 5 10-11 10 9

2.10 11.7 ? 1.5 14.2 22.0 t 3.9 21.0 t 5.6 N-Ac-S PD N-Ac-SPD Ac-cadaverine

0.382 0.382 2.9 t 0.6 2.9 6.6 ? 2.4 4.7 ? 1.4 5.4 M; 7.1 W 3.48 t 2.62 C; 0.62 1.9 .9

0.306 0.306 2.84 f 0.5 2.8 5.3 ? 1.2 4.1 t 1.3

pmol/mg CR pmollg CR pmo1/24-hr U

*

Remarks

-

SD, M SD, W

Reference Abdel-Monem et al. (1975b) Abdel-Monem and Ohno (1977b) Abdel-Monem and Ohno (1978) Abdel-Monen et al. (1978) Seiler and Knodgen (1979b) Seiler and Knodgen (197913) Tsuji et al. (1975) Berry et al. (197813) Abdel-Monem and Ohno (1978)

"The results reported are expressed as means alone, or means ? SE, or means t SD (when specified). N, number of subjects; M, men; W, women; C , children; U, urine; C R , creatinine; Ac-PUT, acetylputrescine; N1-Ac-SPD, N*-Ac-SPD, N'- and N 8 acetylspermidine; N-Ac-SPD, N-acetylspermidine ; Ac-cadaverine, acetylcadaverine.

POLYAMINES IN MAMMALIAN TUMORS

31

since it has been well demonstrated that urinary levels of one or more polyamines can be abnormal in many nonneoplastic diseases (e.g., cystic fibrosis, infectious diseases, psoriasis, anemias of different etiology, rheumatoid arthritis, systemic lupus erythematosus, polymyositis, cardiovascular diseases, pulmonary tuberculosis, hepatitis, hereditary muscular dystrophies, cystinuria, some inborn errors of metabolism, some inborn defects of renal transport of amino acids) (Kessler et al., 1974; Dreyfuss et al., 1975; Waalkes et al., 1975b; Durie et al., 1977a; Berry et al., 1978b; Janne et al., 1978; Russell and Durie, 1978; Rudman et al., 1980). Even in physiological states, such as normal pregnancy (Russell et al., 1978), or during therapeutic treatments, such as in growth hormone (GH)-deficient children after GH treatment (Rudman et al., 1979), elevations of total polyamines or of at least one or two polyamines have been observed. It appears from Tables I1 and 111 that the values for daily urinary polyamine excretion by healthy humans are quite erratic and sometimes conflicting. It is difficult to compare the normal values reported by the different authors, even within each group of values, for several reasons. First of all, the authors used different units of measurement to calculate and express the daily amounts of urinary excretion of polyamines and of their conjugated forms. Second, another fundamental source of the great variability of the values in Tables I1 and 111, is the large number of different assay methods employed, beginning with the not entirely specific methods of the earliest reports to the most sophisticated and most reliable of the most recent reports. Third, there are also some physiological sources of the variability in the normal data published so far. One of these is the age range of healthy volunteers, since this range is quite wide in most of the reports. However, an influence of age on the amounts of the urinary polyamines excreted per day has been demonstrated and carefully investigated in humans, from 0 to 70 years of age (Rudman et al., 1979).The fact that the determinations of polyamines were usually carried out on the urine of only one day seems to be of minor importance, since normally the excretion of polyamines is quantitatively quite constant, with only small variations from one day to another (Waalkes et al., 197513).Another source of variability is the sex of the control subjects. The majority of the mean values published by the authors were obtained by averaging the levels of polyamines for men and women. However, women have been demonstrated to excrete more putrescine than men, and men have urinary spermine contents greater than those of women (Tsuji et al., 1975; Waalkes et al., 1975b; Nishioka et al., 1978b). However, a report has confirmed this influence of the sex on the daily

32

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

urinary excretion of putrescine, whereas higher values of excretion for spermine were found in woman than in man (Beninati et ul., 1980).No difference between the sexes was found in urinary elimination of spermidine and cadaverine (Tsuji et al., 1975; Waalkes et al., 1975b; Nishioka et al., 1978b; Beninati et al., 1980). As for the conjugated forms of polyamines, it has been shown that men excrete N ' acetylspermidine and N8-acetylspermidine in higher quantities than women (Seiler and Knodgen, 1979), but there is no significant variation between the sexes in daily excretion of acetylputrescine (Seiler and Knodgen, 1979). Interestingly, the urinary excretion of all three chief polyamines has been demonstrated to be enhanced during menstruation, and sometimes it remained increased during the early follicular phase (Osterberg et al., 1978). This increase usually has been connected with endometrial necrosis (Osterberg et d.,1978),since several reports have shown that the extracellular polyamine contents are very often augmented as a consequence of cell death (Heby and Anderson, 1978b; Woo et al., 1979), though there may also be some effects of the concomitant changes in hormonal levels. In 1971, simultaneous studies of Russell (1971), Russell et al. (1971a,b), and Bremer et al. (1971)demonstrated elevated daily excretion of some polyamines by cancer patients or by patients with cystinuria. This immediately emphasized that it is not possible to consider an increase in urinary polyamine excretion to be a biochemical feature characteristic of the neoplastic state. Those reports by Russell and her co-workers, mainly concerning hematologic tumors and some solid tumors, raised the question of whether analysis of polyamines in urines of cancer patients would be useful diagnostic tools for cancer detection and aroused much interest in this field. Since that time, many other authors have carried out urinary polyamine analysis in cancerous subjects and have confirmed that the polyamine content is frequently high. Nevertheless, the hopes stirred up by the initial findings have been partially disappointed, because later findings, particularly in more recent years, have clearly shown that the magnitude of the increases in urinary polyamine levels in cancer patients differ not only in relation to the type of neoplasia but also within the same type of neoplasia. Moreover, the extent of the increases are sometimes no larger than those observed in other nonneoplastic ill subjects, and, what is more, there are even some reports in which no differences between the daily amounts of excreted polyamines in patients with cancer and normal persons were observed (Gehrke et al., 1973, 1974). Last, but not least, it has also been found that the percentage of pa-

POLYAMINES IN MAMMALIAN TUMORS

33

tients with localized malignant tumors who show an elevation of urinary polyamines was not much different from that of patients with benign tumors (Lipton et al., 1976). In the following listing, the different types of human neoplasms are grouped on the basis of the location of the primary tumor. Then, despite the aforementioned drawbacks, for each group of tumors we will cite the papers published so far, showing enhancements of daily urinary excretion of one or more polyamines in some patients with the various tumors. Those papers in which the types of tumors were not clearly specified, have been deliberately omitted from the following list. 1. Neoplasias of the digestive system and associated glands. Tumors of the esophagus (Lipton et al., 1975, 1976; Waalkes et al., 1975b; Fujita et al., 1976), the stomach (Dreyfuss et al., 1975; Tsuji et al., 1975; Waalkes et al., 1975b; Fujita et al., 1976) or the small bowel (Waalkes et al., 1975b), the colon (Dreyfuss et al., 1975; Waalkes et al., 1975b; Fujita et al., 1976; Durie et al., 1977a), the rectum (Russell, 1971; Russell et al., 1971a; Kessler et al., 1974; Dreyfuss et al., 1975; Fujita et al., 1976; Lipton et al., 1976; Nishioka et al., 1978b), the liver (Abdel-Monem et al., 1975b; Waalkes et al., 1975b; Fujita et al., 1976; Abdel-Monem and Ohno, 1977a,b, 1978), the gallbladder (Fujita et al., 1976), the bile duct (Fujita et al., 1976), and the pancreas (Dreyfuss et al., 1975; Waalkes et al., 1975b; Fujitaet al., 1976; Lipton et al., 1976). 2. Neoplasias of the respiratory system. Malignant lung tumors (Marton et al., 1973a; Kessler et al., 1974; Dreyfuss et al., 1975; Lipton et al., 1975, 1976; Waalkes et al., 1975b; Fujita et al., 1976; Heby and Anderson, 1978a; Woo et al., 1980). 3. Neoplasias of the female reproductive system. Cancer of the uterus (Russell et al., 1971a; Fujitaet al., 1976; Lipton et al., 1976), the ovaries (Russell, 1971; Russell et al., 1971a,b; Schimpff et al., 1973), and the vagina (Lipton et al., 1976). 4. Neoplasias of the male reproductive system. Malignant tumors of the prostate (Dreyfuss et al., 1975; Fair et al., 1975; Sanford et al., 1975; Waalkes et al., 1975b; Durie et al., 1977a) and the testicles (Russell et al., 1971a; Marton et al., 1973a,b; Schimpff et al., 1973; Russell and Russell, 1975; Sanford et al., 1975; Durie et al., 1977a). 5. Neoplasias of the urinary system. Renal tumors, such as hypernephroma or carcinoma (Dreyfuss et al., 1975; Sanford et al., 1975; Waalkes et al., 197513; Lipton et al., 1976), and bladder carcinomas (Dreyfuss et al., 1975; Sanford et al., 1975; Lipton et al., 1976; Durie et al., 1977a; Heby and Anderson, 1978a).

34

GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

6. Neoplasias of the hematopoietic system. Generally speaking, the greatest elevation in urinary polyamine levels has been found in hematologic malignancies: leukemias (Russell, 1971; Russell et al., 1971a,b, 1975; Gehrke et al., 1973; Schimpff et al., 1973; Tsuji et al., 1975; Fujita et al., 1976; Heby and Andersson, 1978a), lymphosarcoma (Russell, 1971; Russellet al., 1971a,b; Gehrke et al., 1973; Dreyfuss et al., 1975; Heby and Andersson, 1978a), Hodgkin’s disease (Russell, 1971; Russell et al., 1971a; Denton et al., 1973a; Gehrke et al., 1973; Marton et al., 1973a,b; Heby and Anderson, 1978a), reticulum cell sarcoma (Russell et al., 1971a,b; Gehrke et al., 1973; Tsuji et al., 1975; Fujita et al., 1976; Heby and Anderson, 1978a), multiple myeloma (Gehrke et al., 1973; Dreyfuss et al., 1975; Fleisher and Russell, 1975; Russell and Russell, 1975; Russell et al., 1975; Tsuji et al., 1975; Durie et al., 1977a; Heby and Andersson, 1978a), various forms of nonHodgkin’s lymphomas (including Burkitt’s lymphoma) (Gehrke et al., 1973; Schimpff et al., 1973; Russell et al., 1975; Waalkes et al., 1975a,b). 7. Neoplasias of the integumentary system. Cutaneous malignancies: melanoma (Russell et al., 1971a; Kessler et al., 1974; AbdelMonem et al., 1975b; Fleisher and Russell, 1975; Lipton et al., 1975; Rodermund and Moersler, 1975; Townsend et al., 1976; Abdel-Monem and Ohno, 1977b; Durie et al., 1977a; Gittins and Cooke, 1978; Heby and Andersson, 1978a), basal cell epithelioma (Rodermund and Moersler, 1975), mycosis fungoides (Rodermund and Moersler, 1975), and Sezary syndrome (Rodermund and Moersler, 1975). 8. Neoplasias of the mammary gland. Urinary polyamines are high in only a small percentage of patients with breast cancer (Russell et al., 1971a; Kessler et al., 1974; Fleisher and Russell, 1975; Lipton et al., 1975, 1976; Tormeyet al., 1975,1980; Tsujiet al., 1975; Waalkes et al., 1975b; Durie et al., 1977a; Gehrke et al., 1977; Heby and Anderson, 1978a; Nishiokaet al., 1978b; Wooet al., 1978). This is in keeping with the biological features of these types of neoplasia, which usually have small growth fractions and slow growth rates. 9. Neoplasias of the central nervous system (CNS). There are very few reports on patients with CNS tumors: neuroblastoma (Wall, 1971), glioblastoma (Dreyfuss et al., 1975), and astrocytoma (Waalkes et al., 1975b). 10. Neoplasias of the endocrine system. Thyroid carcinoma, the only one studied (Abdel-Monem et al., 1975b; Abdel-Monem and Ohno, 197713). 11. Neoplasias of bone. Osteogenic sarcomas (Russell, 1971; Russell et al., 1971a; Tsuji et al., 1975; Waalkes et al., 1975b; Heby and Andersson, 1978a).

POLTAMINES I N M A M M A I J A N TUMORS

35

From the survey of the vast literature on this topic, four main conclusions can be drawn.

1. The quantities of polyamines excreted by cancerous patients do not always correlate directly with the growth rate of the tumor. Very frequently, patients with Burkitt's lymphoma (a rapidly proliferating neoplasia with large growth fraction) or with certain other hematologic malignancies excrete very large amounts of polyamines, but a high percentage of patients with carcinoma of the colon (a tumor with a small growth fraction and a slow growth rate) also have increased polyamine excretion. 2. The remarkably high incidence of false-negative values (i.e., the number of patients with advanced malignant disease who have normal levels of urinary polyamines) and the remarkably high incidence of false-positive patients (i.e., the number of patients with diseases other than cancer who have elevated urinary polyamine contents) suggest that the determination of urinary polyamine levels only in cancer patients is of little, or perhaps even no, importance for cancer diagnosis, particularly for early cancer diagnosis. 3. Nevertheless, the satisfactorily high percentage of cancerous patients (with a rather wide variety of types of advanced neoplasias with both scarce and widespread metastases), who have elevated urinary levels of one or two of the chief polyamines causes us to feel that when polyamines are elevated, this can be viewed simply as a general epiphenomenon of neoplastic growth. 4. It is well known that in normal human urines, unlike in the contents of the mammalian cell, free polyamines are present in lesser amounts than conjugated polyamines, which are mostly acetylated derivatives. In turn, among the acetylated forms of polyamine, the acetylspermidines and acetylputrescine are quantitatively predominant over the acetylated forms of spermine and cadaverine. These products have been found in cancer patients too (Denton et al., 1973b; Walle, 1973; Abdel-Monem et al., 1975b, 1978; Tsuji et al., 1975; Abdel-Monem and Ohno, 1977a,b, 1978; Rosenblum, 1980). Furthermore, an interesting series of papers by Abdel-Monem and his co-workers dealing with the acetylated forms of spermidine excreted by normal and cancer patients have demonstrated that the great majority of cancer patients have a higher urinary N'-acetylspermidine : N8-acetylspermidine ratio than normals, and the elevation of the ratio is essentially due to an increase in the amount of N'-acetylspermidine excreted (Abdel-Monem et al., 1975b, 1978; Abdel-Monem and Ohno, 1977b, 1978). Whether the increase of this molecule in urines of cancerous subjects is due to a higher rate of formation or to lesser

36

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

degradation, relative to NB-acetylspermidine, remains to be established. Moreover, the specificity of the increase in that ratio for cancer patients must be confirmed, since there are no analogous clinical studies in patients with diseases other than cancer. The urinary levels of acetylputrescine and acetylcadaverine have also been found to be higher in cancer subjects than in normal ones (Abdel-Monem et al., 1978), although this finding also awaits further confirmation in noncancer patients before it can be claimed as a characteristic of the neoplastic state. All together, these results indicate that the specific abnormality, if any, of the urinary profiles of all the polyamines [including the acetyl derivatives and lY3-diaminopropane (Walle, 1973; Heby and Andersson, 1978a; Slanina et al., 1979)] in cancer patients might consist in a shifting of the quantitative ratios between the various molecules rather than in mere quantitative elevation. Other fundamental problems in clinical oncology are to determine as exactly as possible the stage of the tumor, the prognosis, the response to therapy, and the remission-relapse status. On present evidence, the use of polyamine profiles in urines of cancer patients has given the most promising results in this particular field, especially in short-term evaluation of the efficacy of therapy and in the assessment of the activity status of the neoplasm. In fact, several reports have shown that a successful chemotherapy or immunotherapy or the surgical removal of a tumor very frequently brings about a marked decrease in the urinary polyamine levels of cancer patients in the days and weeks after treatment, whereas the polyamine levels were changed much less or not at all in patients unresponsive to the different types of chemotherapy when the neoplasm recurred or became terminal, or one or more or all of the polyamines rose once again (Russell, 1971; Russell et al., 1971a,b; Denton et al., 1973c; Schimpff et al., 1973; Fleisher et al., 1974; Dreyfuss et al., 1975; Lipton et al., 1975; Sanford et al., 1975; Takeda et al., 1975; Waalkes et al., 1975a,b; Fujita et al., 1976; Townsend et al., 1976; Slanina e t al., 1979; Nissen et al., 1980; Tormey et ul., 1980). However, more detailed and careful studies carried out during or immediately after the beginning of treatment have demonstrated that acute elevations in urinary polyamine levels occur in response to effective chemotherapy, before those levels return to near normality during the remission phase (Denton et al., 1973a,b, 1974; Russell and Russell, 1975; Russell et al., 1975; Waalkes et al., 1975a; Durie et al., 1977a; Russell, 1977; Russell and Durie, 1978).Accordingly, a specific time course for the changes in urinary polyamine values in cancer

POLYAMINES IN MAMMALIAN TUMORS

37

patients responding to effective therapy has been tentatively established (Russell and Russell, 1975; Russell et al., 1975; Durie et al., 1977a; Russell, 1977; Russell and Durie, 1978; Woo et al., 1980).This time course consists, during the first days after the initiation of successful chemotherapy, of a rise of the spermidine levels that is thought to reflect the killing of tumor cells. More in detail, the free spermidine level changed only slightly, while the level of conjugated spermidine was markedly augmented (Rosenblum, 1980). This is generally followed by a return to near normal spermidine values or at least to those existing before therapy. Usually, this decrease in urinary spermidine levels is accompanied by remission of the neoplastic disease. Furthermore, enhancement of urinary putrescine levels in cancer patients after the end of the chemotherapy reflects recruitment of tumor cells into the proliferative compartment, and therefore reflects the tumor load and a recurrence of the neoplastic disease. As for spermine, it was suggested that an abnormal elevation of this tetraamine could reflect cellular aging leading to spontaneous cell loss, i.e., not cell destruction by chemotherapy (Tormey et al., 1980). Thus, in long-term surveillance of cancerous patients, changes in urinary polyamine levels seem very often to correlate with the clinical status of the patient, and monitoring could predict the clinical decline of the patients. However, we still do not know the extent of the contribution of the polyamines produced from tissues, other than the neoplasias that are also affected by cytotoxic agents. Once the urinary spermidine level was held to be a reliable marker of the response to the effective chemotherapy, another clinically useful indicator was introduced for differentiating the patients into nonresponders, partial responders, and complete responders to the different types of chemotherapy. This indicator is the posttreatment :pretreatment spermidine ratio (Durie et al., 1977a; Woo et al., 1978). In nonresponding patients with hematologic or solid malignancies, the mean value of the ratio was found to be around 1.2 and never to exceed 2, whereas the mean values of the ratio for partially or completely responsive patients are between 3 and 4. Nevertheless, in evaluating the real clinical importance of all these urinary parameters connected with polyamines and their conjugated forms in the assessment of the disease status in cancer patients, it must always be borne in mind (a) that marked changes in polyamine excretion may be caused by factors other than anticancer chemotherapy (Denton et al., 1973a; Nishioka et al., 1980);(b)that the clinical malignancy of a tumor is widely thought to be dependent on and connected with not only some biochemical parameters, even though theoretically

38

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

appropriate and suitably evaluating biological aspects of the malignancy of the tumor, but also with other important factors, such as the localization of the tumor, its ability to invade vessels and consequently metastasize, and so forth. Finally, since the urinary polyamine levels, considered alone, are not at all satisfactory markers for neoplastic disease and inadequate for the needs, combinations of urinary polyamine levels plus other relatively tumor-specific molecules present in blood and/or urines (e.g., urinary nucleosides, CEA) might considerably improve diagnostic specificity and the evaluation of tumor changes during treatment (Tormey et al., 1975; Woo et al., 1978; Nishioka et al., 1978a). Combinations of neoplastic “markers” may have remarkable advantages over single “markers,” because different neoplastic “markers” often seem to reflect different biological aspects of the neoplasia.

c. LEVELSOF T H E C H I E F POLYAMINES AND THEIRCONJUGATED FORMSIN BLOOD, PLASMA, SERUM, FORMED BLOODELEMENTS, BONE MARROWOF NORMALSUBJECTS AND OF CANCER PATIENTS

AND

1. Polyamines in Whole Blood and Its Liquid Parts Determinations of polyamine contents in whole blood, in plasma, and in serum from normal and oncopathic subjects have accumulated since early reports of Tokuoka (1950, 1956), who proposed the cupric carbonate test no longer used (Bachrach and Robinson, 1965) for the diagnosis of malignancy, based on the reaction of this substance with the spermine present in the sera of cancerous patients. The data available in the literature for the levels of putrescine, spermidine, spermine, and cadaverine in whole blood, plasma, and sera of normal men and women are reported in Table IV. In this table, we see once again that the values are quite erratic and sometimes even conflicting. Some of the reasons listed earlier to explain in part the great variability of the polyamine levels in normal urines, e.g., the different units of measure used, the different assay methods employed, the physiological source of variability aforementioned, are valid also to account, at least in part, for the great variability in normal polyamine levels in whole blood, plasma, and serum. Therefore, none of these will b e discussed further here. Some general but quite certain conclusions can be drawn about the levels of polyamines in normal human blood and in its liquid parts.

1. Whole blood has a higher polyamine content than plasma or serum because nearly 95% of the polyamines in whole blood are in the

POLYAMINES I N MAMMALIAN TUMORS

39

blood cells (Cohen et al., 1975, 1976; Lundgren e t al., 1975; Cooper et al., 1976, 1978; Rennert and Shukla, 1978; Saeki et al., 1978). 2 . No remarkable differences have been found between polyamine levels in plasma and serum. This means that the clotting process does not subtract polyamines from plasma. 3. Polyamines in normal sera are present in the unconjugated forms, with a minor part conjugated to a peptide carrier (Seale et al., 1979) or to fibronectin (Roch et al., 1980). As for the acetyl-derivative forms of polyamines present in normal sera, N '-acetylspermidine has been identified, but not NX-acetylspermidine (Smith et al., 1978), unlike in urines. Monoacetylcadaverine and monopropionylcadaverine have also been identified in normal blood (Dolezalova et al., 1978). 4. In normal plasma and serum, there is less spermine than putrescine and spermidine, the only exception being reported by Chaisiri et al. (1979). 5. Like urinary polyamine levels, spermidine and spermine concentrations in whole blood fluctuate in women during the menstrual cycle (Lundgren et al., 1976; Rennert et ul., 1976a; Campbell et al., 1977), but not in men observed over the same length oftime (Lundgren et al., 1976), suggesting the existence of a sex-related hormonal influence on blood spermidine and spermine levels. 6. Among the possible different analytical assay methods for polyamines in the blood, the choice and the reliability of a method depends on what blood compartment is to be assayed. For instance, for serum, there are three independent methods, i.e., high pressure cation-exchange chromatography, radioimmunoassay, and gas chromatography-mass spectrometry, that are highly sensitive and allow polyamine analysis of very small amounts of serum (D. Bartos et al., 1975; F. Bartos et al., 1977; Bonnefoy-Roch and Quash, 1978). Despite the initial promise, the same positive and negative comments listed above apropos the real clinical significance and usefulness of urinary polyamine determinations in human oncology have to be repeated and kept in mind when one is evaluating the real importance of polyamine determinations in whole blood, plasma, and serum of cancer patients in relation to cancer diagnosis and treatment. 1. The frequently observed increases in polyamine levels in blood, plasma, or serum from cancerous subjects are not at all specific for this kind of disease, since other analogous elevations have been recorded in some nonneoplastic illnesses, such as cystic fibrosis (Lundgren et al., 1975; Arvanitakis et al., 1976; Rennert et al., 1976a; Berry et al., 1978a), systemic lupus erytheinatosus (Puri et al., 1978), schizophrenia (Pfeiffer et al., 1970; Dolezalova et al., 1978), sickle cell anemia

TABLE IV NORMALLEVELSOF THE CHIEF POLYAMINES AND THEIRCONJUGATED FORMS IN HUMANBLOOD,PLASMA, AND SERUM' Specimen and unit Whole blood pglml pg/ml d m l Lrdml Wml Pdml nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml nmollml nmollml nmol/ml nmollml Plasma nmol/ml nmol/ml nmol/ml d m g CR nmollml n m o1/ m 1 nmol/ml pm ol/l iter nmollliter nmollliter

N

Putrescine

Spermidine

Spermine

Remarks

13 17

1.1 f 0.14 -

1.4 f 0.12 1.25 2 0.06 0.26 1.27 2 0.48 1.48 0.34 1.10 f 0.27 9.97 f 0.87 7.25 0.82 4.28 2 1.4 4.01 f 1.37 2.60 2 1.25 1.93 2 0.33 4.68 7.08 3.92 f 0.36

W M

3 10 19 NR 14 13 NR 4 30 11 2 22 17

0.95 2 0.05 0.97 2 0.04 1.26 0.99 2 0.18 1.0 0.19 1.17 0.57 10.3 2 1.01 8.47 0.56 4.87 -t 0.37 3.87 2 1.29 2.54 5 1.51 3.3 f 0.29 7.07 9.69 6.56 f 0.39 50.12 0.07 0.01 0.08 f 0.02 0.29 2 0.1 0.13 f 0.04

S O . 16 0.08 0.02 0.03 0.01 0.01 0.04 0.015 0.20 2 0.10 0.19 2 0.09 0.06 -

10 20 17 10 37 66 96 3 61 98

-

-

0.21 2 0.02 0.9 0.35 0.9 0.35 0.08 2 0.01 0.23 2 0.1 0.1 2 0.03 -

*

0.13 -

* * *

*

-

0.22 200 f 137.7 201 2 116.8

*

* *

-

W M SD M W SD -

-

-

SD M, SD W, SD

-

M , SD W, SD

Reference

Raina (1962) Raina (1962) Shimizu et al. (1965) Iliev et al. (1968) Iliev et al. (1968) McEvoy and Hartley (1975) Lundgren et al. (1975) Lundgren et al. (1975) Arvanitakis et al. (1976) Chun et al. (1976) Rennert et al. (1976a) Rennert and Shukla (1978) Saeki et al. (1978) Berry et al. (1978a) Cooper et al. (1978) Cooper et al. (1976) Rennert and Shukla (1978) Cooper et al. (1978) Russell et al. (1978) Takami et al. (1979) Chaisiri et al. (1979) Chaisiri et al. (1979) Shipe et al. (1979) Chaisiri et al. (1980) Chaisiri et al. (1980)

*

nmollml nmol/ml Serum nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml

NR 11

0.11 0.03 0.11 2 0.03

0.12 0.03 0.13 2 0.04

NR NR 10 7 7 6 23

0.23 0.09 0.31 0.17

0.32 0.07 0.25 0.33 2 0.09 0.33 0.10 0.39 5 0.06 0.23 t 0.05

pmollml pmollml nmol/ml nmollml nmollml pmol/ml

10 10 2 17 16 23

130 14.7 270 2 23.9

*

-

0.17 t 0.08

*

-

0.51 2 0.06 0.15 0.04 98.9 t 45.2

*

*

*

70 18.4 240 2 30.6 0.78 0.63 2 0.07 0.14 2 0.03 88.5 -+ 47.4

0.03 0.04

* 0.01

" 0.02

SD SD

Takami e t QZ. (1980a) Takami and Nishioka (1980)

-

SD TP FP SD SD

Marton et d. (1973a) Marton e t ~ l (1973b) . Nishioka and Romsdahl (1974) Saniejima et d. (1976) Samejima et d. (1976) F. Bartos et QZ. (1977) Nishioka et al. (1977, 1978a); Nishioka and Romsdahl (1978) Kai e t d. (1979) Kai et QZ. (1979) Hospattankar et aZ. (1980) Baylin e t d. (1980b) Baylin et al. (1980b) Desser et al. (1980)

0.04 0.02 -

*

-

0.12 2 0.008 0.03 t 0.02 20 50

* 9.3

0.33 2 0.08 0 29.1 -+ 26.0

Miscellaneous reportsb Cadaverine In blood 10.9 (24) (pmol/g wet wt.) In serum 0.14 (7) TP; 0.03 (7) F P (nmol/ml) 10 (10) FP; 60 17.33 (10) T P (pmol/ml) N'-Ac-SPD in serum 0.008-0.05 (nmol/ml) FPs in serum 28 2 4.7 (8) (ng/ml) 43.4 2 14.8 (40) (ng/ml) C, SD = 0.206 - 0.008(age + 3/4) - 0.320 ln(age + 3/4) ln(PUT serum) = 0.473 - 0.004(age + 3/4) - 0.142 In(age + 3/4) In(SPD serum) = -1.532 - O.O16(age + 3/4) - 0.296 ln(age + 3/4) ln(SP serum)

*

14.5

FP TP C SD

Dolezalova et ~ l (1978) . Samejima et d. (1976) Kai et d. (1979) Smith et d. (1978) Bartos e t al. (1975) Puri et d. (1978) Rudman et aZ. (1979) Rudman et al. (1979) Rudman et aZ. (1979)

"The results reported are expressed as means alone, or means 2 SE, or means 2 S D (when specified). N, number of subjects; NR, not reported; FP, free polyamine; TP, total polyamine; M, men; W, women; C, children. *N'-Ac-SPD, N'-acetylspermidine; PUT, putrescine; SPD, spermidine; SP, spermine.

42

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

(Chun et al., 1976; Cooper et al., 1978; Rennert and Shukla, 1978), psoriasis (Cooper et al., 1978; Rennert and Shukla, 1978), and hereditary muscular dystrophies (Rudman et al., 1980). 2. In those patients with tumors with small actively growing fractions and slow growth rates, the blood polyamine contents do not always change in parallel with, or reflect the stages of, the development of the disease (Russell, 1977). 3. Plasma or serum polyamine contents, like those in urines, have been shown to decrease when antitumor therapy (either chemical or surgical) is effective, but to remain nearly unchanged with the patients are unresponsive to the chemical treatments. Furthermore, the time course of the changes in plasma or serum polyamine levels following successful therapy appears to b e roughly identical to that of the urinary contents (Marton et al., 1973b; Russell et al., 1973; Russell and Russell, 1975; Nishioka et al., 1976, 1978a; Durie et al., 1977b; Nishioka and Romsdhal, 1977, 1978; Savory et al., 1979; Hospattankar et al., 1980). 4. It must also be stressed that increases of blood polyamine concentrations can result either from an increase in their production rate or a decrease in their removal from the blood, as a consequence of failure of hepatic conjugation andlor of renal clearance. In connection with this, hepatic and renal damage are often observed in patients with advanced metastatic cancer, and serum polyamines have been demonstrated to be high in patients with uremia (Campbell et al., 1978; Swendseid et al., 1980). 5. As has been previously stated about increases in the urinary polyamine concentrations, for each of the different types of human neoplasias enhancement, when present, of polyamine levels in plasma or serum of cancerous patients is not always equally great, and we do not know which increase in polyamines is characteristic of cancer, although putrescine and spermidine are those whose levels have been shown most frequently to be augmented. Therefore, as we did for urinary polyamine levels, we have listed the papers published so far that give polyamine contents (whether increased or not) for whole blood, plasma, and serum in oncopathic humans. a. Whole Blood. Elevations of spermidine and/or spermine have been found in some patients with leukemia (Shimizu et al., 1965), gastric cancer (Saeki et al., 1978), lung cancer, non-Hodgkin’s lymphoma, and chronic lymphocytic leukemia (Cooper et al., 1978). b. Plasma. Infrequently, an elevation of plasma levels of spermidine or spermine or both has been recorded in patients with

POLYAMINES IN MAMMALIAN TULIOHS

43

laryngeal tumors (Savory et al., 1979), breast carcinoma (Chaisiri et al., 1979, 1980; Savory et al., 1979), chronic lymphocytic leukemia (Cooper et al., 1978), polycythemia Vera rubra (Desser et ul., 1975), prostatic cancer (Chaisiri et al., 1979, 1980), teratoma of the testes (Chaisiri et al., 1980), and multiple myeloma (Russell and Russell, 1975). High spermidine concentrations have occasionally been reported also in benign neoplasias of the prostate or breast, although the elevations were of a lesser magnitude than those observed in cancer patients (Chaisiri et al., 1980). In patients with either of these two types of neoplasias there were no significant differences in spermine levels between benign and malignant forms (Chaisiri et al., 1979). No correlation between elevated plasma spermidine or spermine concentrations and the tumor stage or the clinical status of the patient was possible in any kind of neoplasia. c . Serum. Total polyamines were high in some patients with melanoma (D. Bartos et al., 1975), hepatoma (D. Bartos et al., 1975), gastric carcinoma (D. Bartos et al., 1975), Wilms’s tumor (D. Bartos et al., 1975), Hodgkin’s disease (Hospattankar et al., 1980), acute myeloid leukemia (Hospattankar et al., 1980), and non-Hodgkin’s disease (Hospattankar et ul., 1980). Putrescine and spermidine were high in one patient with multiple myeloma (Russell and Russell, 1975). High spermidine was found in some patients with pancreatic carcinoma (Marton et d., 1973a,b), breast cancer (Marton et d.,1973a,b), Hodgkin’s disease (Marton et a1., 1973a,b), acute nonlymphocytic leukemia (Marton et al., 1973a,b), and acute lymphocytic leukemia (Marton et al., 1973a,b). In an interesting series of papers, Nishioka and his co-workers described the polyamine levels in patients with different types of tumors, but particularly in patients with carcinoma of the colon and the rectum. They demonstrated that patients with melanoma, Hodgkin’s disease, or carcinoma of the kidney had higher putrescine concentrations than the normal controls (Nishioka and Romsdahl, 1974; Nishioka et al., 1978a), while the spermidine level in patients with breast carcinoma was also enhanced (Nishioka and Romsdahl, 1974; Nishioka et al., 1978a). In a high percentage of patients with colorectal carcinomas, elevated concentrations of one or more polyamines have been reported (Nishioka and Romsdahl, 1974,1977,1978; Nishioka et al., 1976,1977, 1978a), while other patients with different types of benign bowel diseases had normal polyamine contents (Nishioka et ul., 1977, 1978a). Interestingly enough, careful longitudinal studies of surgical patients with colorectal carcinomas demonstrated that this is one of the few situations in which serum polyamine levels correlate fairly well, despite certain limitations, with the clinical course, especially in long-

44

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

term assessment of the disease’s activity after surgery, with or without adjunctive radiotherapy (Nishioka and Romsdahl, 1977, 1978; Nishioka et al., 1978a). Results obtained by Desser et al. (1980)are in good agreement with those of Nishioka and his co-workers. 2. Polyamine Levels in the Various Types of Formed Cells of the Blood The data available in the literature about the normal concentrations of the chief polyamines in the various types of formed cells in human blood and in human bone marrow are reported in Table V. As shown in this table, the mean concentrations of the polyamines were found to be by far higher in leukocytes than in erythrocytes (Chun et al., 1976; Cohen et al., 1976; Cooper et al., 1976, 1978; Rennert and Shukla, 1978; Saeki et al., 1978). There were no significant differences in spermidine and spermine levels among the various leukocyte types (Cooper et al., 1976, 1978; Rennert and Shukla, 1978), but polymorphonuclear cells contain more putrescine than mononuclear cells (Rennert and Shukla, 1978).Last, platelets have polyamine concentrations roughly equivalent to those of red blood cells (Cooper et al., 1976, 1978; Rennert and Shukla, 1978). This kind of quantitative distribution of polyamines cannot readily be considered to be casual, but is consistent with the fact that leukocytes are nucleated cells, whereas erythrocytes and platelets are not. In addition to quantitative differences between red blood cells and white blood cells, there is another difference in the polyamine profiles of these two cell types, since erythrocytes were found to contain more spermidine than spermine, while the opposite is true for leukocytes (see Table V) (Chun et al., 1976; Cohen et al., 1976; Cooper et al., 1978; Rennert and Shukla, 1978; Saeki et al., 1978).Furthermore, as is well known to happen in many other mammalian tissues and cells, the polyamine content in red blood cells markedly decreases with age of the cells (Cooper et al., 1976; Rennert and Shukla, 1978). When we compare the polyamine contents in the red cells and white cells of the blood not for equal numbers of cells, but in relation to the relative amounts of each of these two cell classes in the blood, since there are about 700 erythrocytes for each leukocyte in circulating blood, the most significant contribution (nearly 80%) is that from the erythrocytes, with only 20% from leukocytes (Cohen et ul., 1975, 1976; Cooper et al., 1978; Rennert and Shukla, 1978; Saeki et al., 1978). In recent years, quantitative assays of polyamines (noticeably spermidine and spermine) in erythrocytes from cancer patients have been

POLYAMINES I N MAMMALIAN TUMORS

45

suggested as a useful tool in clinical oncology. In fact, abnormally high concentrations of these two polyamines have been significantly frequently observed in erythrocytes from patients with different types of neoplasia, such as breast cancer (Savory et al., 1979; Takami et al., l979,1980a,b; Takami and Nishioka, 1980),colorectal tumors (Saeki et al., 1978; Savory et al., 1979; Takami et al., 1979,1980a7b;Takami and Nishioka, 1980; Ueharaet al., 1980a), pulmonary cancer (Cooper e t al., 1978; Savory et al., 1979; Takami et al., 1979, 1980a,b; Takami and Nishioka, 1980; Uehara et al., 1980a), melanoma (Takami et al., 1979, 1980a; Takami and Nishioka, 1980), pancreatic cancer (Saeki et al., 1978; Ueharaet al., 1980a), gastric cancer (Saekiet al., 1978; Ueharaet al., 1980a), duodenal cancer (Saeki et al., 1978), ovarian cancer (Saeki e t al., 1978), myeloblastic or lymphoblastic leukemias (Cooper et al., 1978; Savory et al., 1979), lymphoma or lymphosarcoma (Cooper et al., 1978; Saeki e t al., 1978; Uehara et al., 1980b), tumors of larynx (Savory et al., 1979)or of prostate (Savory et al., 1979), hepatoma (Saeki e t al., 1978; Uehara et al., 1980a), and reticulum cell sarcoma and Hodgkin’s disease (Uehara e t al., 1980b). No correlation was observed between leukocyte counts and increased polyamine levels in cancer patients whose leukocyte counts were within the normal range (Takami et al., 1979, 1980a). In some careful follow-up studies of cancerous patients, the concentrations of spermidine and spermine in red blood cells were markedly reduced after successful chemotherapy or after surgery in a quite satisfactory percentage of patients (Savory et al., 1979; Uehara et al., 1980a,b). Moreover, the polyamine levels in erythrocytes from patients with different types of malignant lymphoma were well correlated with the stage of the disease (Uehara et al., 1980b). However, when we consider the degree of specificity of such a new “marker” (i.e., the polyamine content in erythrocytes of oncopathic humans) for malignancy, we are once again disappointed, since increased contents of at least one or more polyamines have been found in red blood cells from patients with chronic hepatic disease (Savory et al., 1979), elliptocytosis (Cooper et al., 1978), sickle cell anemia (Chun e t al., 1976; Rennert and Shukla, 1978; Natta et al., 1980), sickle-hemoglobin C disease (Natta et al., 1980), and Duchenne muscular dystrophy (Mollica et d., 1980) and from nondialyzed patients with advanced renal failure (Swendseid et al., 1980).Nevertheless, we can answer the question as to whether determination of polyamine concentrations in red blood cells taken from cancer patients is a more sensitive and useful neoplastic “marker” than the same determination in plasma of the same patients affirmatively, since several reports have shown that the

TABLE V NORMALLEVELSOF THE CHIEF POLYAMINES IN HUMAN ERYTHROCYTES, LEUKOCYTES, PLATELETS, AND BONEMARROW Specimen and unit

N -

Putrescine

Erythrocytes

+dml +dml nmol/lOBcells nm0l/l0~cells nm0l/l0~cells nm0l/l0~cells nmol/ml nmol/lO1O cells nm0l/l0~cells nmoVmg protein nmol/rng protein nmol/lO'Ocells nmol/lO" cells nmol/ml

4 7 9 9 17 20 27 37 11 7 7 18 22 6

0.02 0.007 0.07 0.01

*

0.17 0.05 0.05 1.08

*

Spermidine

* * * * * * *

1.6 0.3 1.02 0.08 1.48 1.39 0.46 0.82 0.07 1.06 0.19 14.1 3.1 15.04 3.63 11.76 ? 2.74 0.55 2 0.55 3.21 5 1.82 14.42 3.2 15.58 ? 3.9 24.8 2 6.3

*

Spermine

Remarks

Reference

0.2 0.89 2 0.28 0.61 0.9 0.27 0.48 0.04 0.46 0.09 8.4 2 2.8 8.8 3.12 7.21 2.29 1.13 2 0.54 0.53 2 0.75 8.72 2.7 8.85 2 2.8 12.4 3.4

SD

Shimizu et al. (1965) Cohen et al. (1976) Cooper et al. (1976) Chun et al. (1976) Cooper et al. (1978) Rennert and Shukla (1978) Saeki et al. (1978) Uehara et al. (1980a) Takami and Nishioka (1980) Natta et al. (1980) Natta et al. (1980) Uehara et al. (198Ob) Uehara et al. (198Ob) Swendseid et al. (1980)

* * * * * * *

SD SD SD Stm LY s SD, M SD, W

M, SD

Leukocytes nmol/108 cells nmol/ 10’ cells nrnol/1O9 cells nmolll0’ cells nm01/10~cells nmol/109 cells nmol/io9cells nmoUl0’ cells nmol/lO9 cells nmol/lO’ cells Platelets nmol/lO’ cells nmol/lO’ cells nmolll0’ cells Bone marrow nmol/ml

* 1.4 2.99 * 1.5

3.0 2 0.9 126 t 31 15.3 0.5 253.6 94.9 241.3 155.5 95 26 226 28 266.5 t 28.2 207.3 45.5 207 45

* * * * * *

12.9 3.8 357 t 105 35.95 0.91 490.2 208.7 547.6 183.4 387 5 61 440 61 440.3 2 61.4 493.1 56.3 493 2 56

* * * * * *

L SD, M N SD, PMN SD MN MN PM N PMN

Desser et al. (1975) Cohen et al. (1976) Chun et al. (1976) Cooper et al. (1976) Cooper et al. (1976) Saeki et al. (1978) Cooper et al. (1978) Rennert and Shukla (1978) Rennert and Shukla (1978) Cooper et al. (1978)

0.11 0.05 0.21 0.03 0.11 * 0.05

0.12 5 0.06 0.44 0.05 0.12 * 0.02

*

0.04 2 0.03 <0.043 0.04 0.01

SD

Cooper et al. (1976) Cooper et al. (1978) Rennert and Shukla (1978)

2.04

27.9

31.6

1.8

3 4 9 10 10 7 17 20 20 17

140 60.6 467.5 2 244.3 220 50 140 60.6 467.5 244.3 410 2 80

6 17 20 5

-

* * *

* *

*

*

Miale et al. (1977)

*

“The results reported are expressed as means alone, or means SE, or means SD (when specified). N , number of subjects; L, lymphocytes; MN, mononucleated leukocytes; PMN, polymorphonucleated leukocytes; Lys, lysate; Stm, stroma; M, men; W, women.

48

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

percentage of oncopathic subjects with elevated polyamine levels in their erythrocytes is far higher than that of patients with elevated plasma polyamine levels (Savory et al., 1979; Takami et al., 1979, 1980a,b). White blood cell preparations from patients with chronic myelocytic leukemia contain more SAM than normal peripheral white cells or thoracic duct lymphocytes (Baldessarini and Carbone, 1965).

3. Polyamine Levels in Bone Marrow There have been very few studies concerning this particular aspect, carried out mainly with bone marrow aspirates from children with different forms of leukemia, and a few cases with other malignancies or other hyperproliferative hematologic nonmalignant disorders, such as infectious mononucleosis, sickle cell anemia, and histiocytosis X have been included (Rennert et al., 1976b; Miale et al., 1977). Even fewer are the data available on the polyamine contents in normal bone marrow from control subjects (Miale et al., 1977). What is available is reported in Table V. In various nonneoplastic diseases, very low levels of putrescine, even lower than normal values, have been found (Rennert et al., 1976a; Miale et al., 1977). However, surprisingly enough, very elevated contents of all the polyamines were detected in bone marrow taken from patients with hyperproliferative nonneoplastic hematologic diseases, such as infectious mononucleosis, or with nonhematologic neoplasias, such as retinoblastoma (Rennert et al., 1976b; Miale et al., 1977). Marked elevations of the concentrations of putrescine, spermidine, and spermine in a high percentage of patients either with acute lymphocytic or with acute myelocytic leukemias in relapse were observed (Nishioka et al., 1976; Rennert et al., 1976b; Miale et aZ., 1977). On the contrary, remission in the leukemic patients, regardless of the type of leukemia, was accompanied by an impressive fall in the polyamine concentrations (Nishioka et al., 1976; Rennert et al., 1976b; Miale et al., 1977). Very noteworthy is the observation of Miale et al. (1977) that in some patients there was a much more prominent increase in putrescine level than in spermidine and spermine that preceded by several weeks detection of fulminating systemic relapse of the leukemia. Therefore, in the bone marrow as well, monitoring of the putrescine content is a good biochemical indicator of the clinical status of a leukemia patient (Miale et al., 1977). However, in conclusion, from the few data available in the literature it is apparent that increased polyamine levels in bone marrow cannot be considered to be pathognomonic for neoplastic disorders, whether hematologic or not.

POLYAMINES IN M A M M A L I A N TUMORS

49

The polyamine levels in bone-marrow plasma obtained from patients with different types of leukemias have been measured (Nishioka et al., 1980). Untreated patients with chronic leukemias showed higher polyamine levels than the untreated patients with acute leukemias (Nishioka et al., 1980). Moreover, patients who were in remission showed low polyamine levels, whereas patients who responded to chemotherapy showed high polyamine levels, as a consequence of the release of polyamines from tumor cells killed by chemotherapy (Nishioka et al., 1980). Finally, the topic of polyamines in blood, bone marrow, and other physiological fluids as possible “markers” of malignancy for human leukemias and other hematologic tumors has been reviewed by Desser (1980).

D. LEVELSOF THE CHIEF POLYAMINES I N PHYSIOLOGICAL FLUIDS OTHER THANBLOODAND URINE Polyamines from neoplastic cells could be released into human extracellular fluids other than blood and urines, causing elevated concentrations of these compounds. These other physiological fluids are the cerebrospinal fluid, bile, duodenal fluid, sweat, saliva, and amniotic fluid. The studies available so far on normal polyamine levels in these body fluids are very few, and all the data available on this topic are summarized in Table VI. There have been no studies of changes in polyamine contents in these physiological fluids from patients with tumors of the organ or tissue from which the liquid arises or in which the liquid is retained, or which are immersed in the liquid, with the noticeable exception of cerebrospinal fluid. Polyamine patterns in the cerebrospinal fluid of patients with tumors of the central nervous system (CNS) or tumor not arising from the CNS but involving it with meningeal carcinomatosis have been determined. In patients with meningeal carcinomatosis from breast or lung or colon or bladder cancers, marked elevations of putrescine and spermidine values were observed in comparison with the reference group, which consisted of subjects with non-CNS tumors, but tumors with no evidence of CNS involvement (Yap et al., 1979). Additionally, in children with acute lymphocytic leukemia with or without involvement of the CNS, the free polyamine concentrations of the cerebrospinal fluid were measured (Rennert et al., 1977). Significant increases of spermidine and spermine, but not of putrescine, were found in patients with florid CNS leukemia in relapse, as compared with patients with extramedullar leukemia but without cytologic evidence of CNS in-

NORMAL LEVELS

OF THE

Specimen

Unit

Cerebrospinal fluid Bile Duodenal fluid Sweat Saliva Amniotic fluid

pmoUml Ccdml Ccg/ml Ccg/ml Ccdml pglrng CR

CHIEF POLYAMINES

N 5 2 5 4 4 230

IN

TABLE VI HUMANPHYSIOLOGICAL

FLUIDS

OTHER THANBLOODAND URINE"

Putrescine

Spermidine

Spermine

Remarks

Reference

182 2 79 -

1 2 0 2 34 14.6 1.9 5 0.3 C0.25 <0.05 0.33 2 0.1

ND 16.1 1.27 2 0.6 c0.25 (0.05 0.76 0.2

SD SD -

Marton (1978); Marton et al. (1979) McEvoy and Hartley (1975) McEvoy and Hartley (1975) McEvoy and Hartley (1975) McEvoy and Hartley (1975) Russell et al. (1978)

-

0.96

* 0.2

*

-

-

a The results reported are expressed as means alone, or means 2 SE, or means 2 SD (when specified). ND, not detectable; N, number of subjects; CR, creatinine.

POLYAMIKES IN MAMMALIAN Tl1MORS

51

volvement (Rennert et al., 1977). Strikingly enough, leukemia patients with extramedullary localization, but without cytologic and syniptomatic evidence of CNS involvement, had the highest levels of putrescine of all the groups of patients (Rennert et al., 1977). Certainly, assays for polyamines in the cerebrospinal fluid are of particular clinical importance when they are connected with pathologies of the CNS, whether neoplastic or not. Without any doubt, we owe a debt to Marton and his co-workers, who studied very carefully and thoroughly the derangements of polyamine profiles in the cerebrospinal fluids of patients with neoplastic and nonneoplastic disorders and have tried to establish some connections between these abnormalities and the clinical status of the patient. In a series of patients with different types of CNS tumors, such as glioblastonia, medulloblastoma, astrocytoma, pituitary adenoma, meningioma, ependymoma, and acoustic neuroma, Marton and his co-workers (1974a,b, 1976; Marton, 1977) found elevated putrescine and spennidine concentrations in cerebrospinal fluid of most cases, particularly those with glioblastoma and medulloblastoma, whereas patients with astrocytoma had less consistent increase in these polyamines. But all the patients with any one of these three types of tumor always had polyamine levels higher than those found in either normal controls or the humans with various nonneoplastic disorders of the CNS (namely, infectious diseases, demyelinating diseases, stroke) and used as the reference group (Marton et al., 1974a,b, 1976; Marton, 1977, 1978). Meningiomas are a variable group of tumors, without any characteristic pattern of polyamine elevation in cerebrospinal fluid (Marton et al., 1976; Marton, 1977). Elevation of spermine levels in the spinal fluids of tumor patients was sporadic (Marton et al., 1976; Marton, 1977). Furthermore, patients who underwent successful therapy showed an immediate rise in putrescine concentration, followed later by a marked decrease in polyamine content (Marton et al., 1974a, 1976; Marton, 1977,1978). In the assessment of the disease’s activity in several patients with CNS tumors, who exhibited significant new increases in putrescine concentration, it was observed that recurrence of the neoplastic disease and a decline in the clinical state of the patients followed (Marton et al., 1976; Marton, 1977, 1978). It is of interest that the fluctuations in the concentrations of polyamines in cerebrospinal fluid from patients with CNS tumors do not have any relationship to changes in protein concentration in the same liquid, suggesting that these changes are not merely mirroring some alteration in the bloodbrain barrier (Marton et al., 1976). Polyamine assays of cerebrospinal fluids from CNS cancer patients

52

GIUSEPPE SCALABRINO A N D MARIA E . FERIOLI

have been demonstrated to be helpful in short-term evaluation of the efficacy of a specific course of therapy. They may also, at least in some particular types of CNS neoplasias, be h e l p h l for long-term evaluation of tumor relapse or regression. This is true for medulloblastoma patients, where Marton et d. (1979)demonstrated an absolute correlation between polyamine (particularly putrescine) levels and the clinical status, evaluated b y different radiographic techniques and by cytological criteria. In fact, almost all the patients exhibited appropriate decreases in polyamine values in response to chemotherapy or radiotherapy, and 50% of the same patients showed appropriate increases in polyamine levels several weeks before the recurrence of the disease. This is of great interest, since medulloblastomas, because of their localization, pose particular difficulties for assessment of their activity and progress, with radiographic techniques frequently of little use (Marton et al., 1979). On the contrary, in patients with glioblastoma multiforme or anaplastic astrocytoma, it has been shown that cerebrospinal fluid polyamine level determinations were not as helpful as in patients with medulloblastoma for monitoring tumor progression and for forseeing tumor recurrence (Fulton et aZ., 1980). In fact, the putrescine and spermidine levels in all the patients with glioblastoma multiforme or anaplastic astrocytoma were significantly higher than those of the reference group of patients with nonneoplastic CNS disorders, but there was no difference in polyamine levels between the two groups of tumor patients, in spite of the fact that the degree of malignancy and the fraction of proliferating cells in glioblastoma multiforme are higher than in anaplastic astrocytoma (Fulton et al., 1980). Moreover, no significant relation was found between the enlargmenet of the tumor and the polyamine levels in cerebrospinal fluids of patients with these two kinds of CNS neoplasia (Fulton et d.,1980). The striking discrepancy in the correlation of cerebrospinal fluid polyamine levels with tumor relapse between medulloblastoma and malignant supratentorial gliomas may be connected with the fact that polyamines produced by malignant hemispheric gliomas cannot reach the cerebrospinal fluid or reach it with difficulty (Fulton et al., 1980). In fact, malignant supratentorial gliomas are distant from the cerebrospinal fluid pathways, whereas medulloblastomas are generally located adjacent to the cerebrospinal fluid pathways (Fulton et al., 1980). On the whole, we can conclude that increases in polyamine contents in whatsoever physiological fluid or in more of the physiological fluids of beings ill with cancer (regardless of the type of cancer), when present, cannot be considered to be biochemical “markers” specific for tumor cell proliferation, but rather are merely biochemical “markers”

POLYAMINES IN MAMMALIAN TUMORS

53

of cell growth. In other words, the increases in intra- and extracellular polyamine levels may reflect cell multiplication, whether the type of growth is controlled or uncontrolled.

E. LEVELSO F ACTIVITYOF POLYAMINE BIOSYNTHETIC DECARBOXYLASES I N HUMANNEOPLASTICTISSUES I N RELATIONT O THE DEGREE O F MALIGNANCY Because of all the doubts about the significance of polyamine determinations in biological materials from cancers and because of the futile question as to which polyamine it would be the best to measure in each of the various types of human neoplasia, we decided to measure the levels of polyamine biosynthetic decarboxylase activities in different types of primary human neoplastic tissues. In fact, in experimental oncology research with Morris rat hepatomas with vastly different growth rates (Williams-Ashman et aZ., 1972), and with chemical carcinogenesis in mouse skin (O’Brien, 1976; Boutwell et al., 1979), it has been found that the degree of ODC enhancement can be a useful biochemical indicator of neoplastic growth, with some few exceptions for certain kinds of rat hepatomas (Pariza et aZ., 1976). We chose human tumors that could be easily obtained from the operating room or from circulating blood. Accordingly, we studied cutaneous epitheliomas, brain tumors, and leukemias. In the cutaneous epitheliomas, the assays of the enzymes were carried out only in the neoplastic epidermal layer, after its separation from the dermis (Scalabrino et al., 1980). Metastases of tumors to the brain or to the skin were deliberately excluded from our research, because of their mixtures of normal cells and neoplastic cells, which, coming from the organ site of the primary tumor, have a different histogenetic origin. Furthermore, it is well known that the cell type and cell viability are often different in metastases than in primary tumor cells. Our major finding is the difference in levels of activity of polyamine biosynthetic decarboxylases in various tumor types. The degree of enhancement of ODC activity correlates well with the neoplasm’s growth rate, since among cutaneous epitheliomas it is greater in squamous cell carcinomas than in basal cell epitheliomas (Scalabrino et al., 1980).It is well established, in fact, that basal cell epithelioma is a slow-growing tumor, whereas the squamous cell carcinoma has a faster growth rate and more widespread local invasiveness. Again, the magnitude of the elevation of ODC activity in the CNSrelated tumors was also proportional to the malignancy. In fact, the levels of ODC activity are higher in the group of dedifferentiated

54

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

astrocytomas (i.e., astrocytomas of grades 3 and 4,astroblastoma, and glioblastoma multiforme) than in the group of differentiated astrocytomas (i.e., astrocytomas of grades 1 and 2 ) (Scalabrino et al., 1981).The same is true for the meningiomas, among which the typical forms have lower ODC activity than the atypical forms (Scalabrino et al., 1981).Polar spongioblastoma, which is generally held to be a quite slowly growing intracranial tumor, has the lowest ODC activity among the brain tumors we have tested. Medulloblastomas, highly malignant brain tumors, show the highest ODC activity of all the other tumors of the CNS tested (Scalabrino et al., 1981). Our data are fairly in agreement with the data of Harik and Sutton (1979), who found a higher content of putrescine in CNS tumors in relation with increasing malignancy. However, although the number of patients screened by Harik and Sutton (1979) is undoubtedly larger than ours, medulloblastomas were not tested and no differentiation within the meningiomas was made in that report. We also reported a dramatic fall in ODC activity in leukocytes taken from patients with chronic myeloid leukemia after successful chemotherapy (Scalabrino et al., 1981). When we considered instead the course of the changes in SAMD activity in the same different types of tumors, we saw that the degree of enhancement of this enzymic activity also followed well the degree of malignancy of the tumor. The only exception to this general statement is the surprisingly low activity of the enzyme in the medulloblastomas we have studied. We have no explanation for this finding. Moreover, unlike ODC activity, SAMD activity does not reflect the effectiveness of antineoplastic chemotherapy, since its level in leukemic leukocytes is not changed immediately after treatment (Scalabrino et al., 1981). At present it is quite difficult to interpret this discrepancy in the behaviors of the levels of the two polyamine biosynthetic decarboxylases in response to the same type of therapy. In conclusion, our data, although limited in number for certain types of tumors, prove the clever forecast of Bachrach (1976b), who asserted that “it is not unlikely that the activity of this enzyme (i.e., ODC) in biopsy material may aid the pathologist in the diagnosis of malignancy.” In fact, we have shown that the degree of ODC enhancement correlates well with the degree of malignancy in different types of human tumor, even those with different histogenetic origins. However, we cannot consider the elevation in ODC activity in neoplasias to be specific for the processes of neoplastic transformation and growth, since it has been demonstrated that similar elevations occur with many other processes of nonneoplastic growth, i.e., of controlled growth

PO1,YAMINES IN . M A M M A L I A N TUMORS

55

(Janne et al., 1978; Russell and Durie, 1978). Rather, on the basis of our findings, we consider the degree of elevation in ODC activity observed in human tumors to be clinically useful as an indicator of a neoplasm’s growth rate, which means of the degree of malignancy of the tumor. This correlation also agrees with idea of Helson et al. (1976, 1977), based on their findings in cultured human neuroblastoma cells (1976) and human melanoma tumors grown in Swiss nulnu mice

(1977). In certain experimental tumors, such as Morris hepatomas (Williams-Ashman et al., 1972) and epithelial tumors of mouse skin (O’Brien, 1976; Boutwell et al., 1979),a dichotomy between ODC and SAMD activity has been demonstrated, with SAMD levels increased not at all or only a little. On the contrary, in the human tumors we tested, SAMD activity increases in parallel with, although to a lesser extent than, the increase in ODC activity. However, the idea that SAMD activity levels also mirror neoplastic growth rates must be taken with caution, because of the exception of the medulloblastoma and, theoretically, of other human tumors not yet assayed. Last, when we compare the reliability of determinations of polyamine contents with that of determinations of the levels of the polyamine biosynthetic decarboxylases as diagnostic and chiefly prognostic tools in different areas of clinical oncology, our conclusions are that (a) measurements of polyamine levels in physiological fluids can be useful for short-time evaluation of a specific course of therapy and for detection of remission or relapse of the neoplastic disease, but are of little or no use in evaluating the degree of malignancy of the tumor, even when combined with assays of other tumor “markers” in neoplastic tissue or in physiological fluids from cancer patients; (b) the levels of the polyamine biosynthetic decarboxylases are by far the better indicators of the degree of malignancy of the tumor, but of no use in evaluating the effectiveness of therapy, except for hematologic neoplasias, in which the assays of enzyme levels in tumor cells are obviously repeatable.

F. METABOLIC CONJUGATION O F POLYAhlINES AND ANTIPOLYAMINE ANTIBODIES I N NORMALSA N D I N CANCER PATIENTS The polyamine levels in physiological fluids are clearly sustained by regulatory interplay of several pathways: (a)de nouo synthesis; (b) release from the intracellular compartment; (c) metabolic transformation into conjugated forms; (d) catabolic transformation into degradation products; (e)excretion. Among these pathways, the in uivo metab-

56

GIUSEPPE SCALABRINO A N D MARIA E. FERIOLI

olism of injected labeled putrescine and labeled spermidine have been studied in normal volunteers (Rosenblum et al., 1977, 1978a,b; Rosenblum, 1980) as well as in cancer patients with high endogenous plasma levels of polyamines (Rosenblum et al., 1977, 197813; Rosenblum, 1980). These studies demonstrated that the radiolabeled compounds very rapidly disappear from the plasma of both normal subjects and cancer patients, with mean half-life times that were very short and very similar for the two groups. This was because of the quick transformation of the labeled polyamines into their conjugated forms, which in turn have prolonged half-life times in plasma. Accordingly, most of the injected radioactive polyamines was excreted in the days after injection almost exclusively in conjugated form (Rosenblum et al., 1977,197813; Rosenblum, 1980). All these results suggest that in neoplastic patients, unlike in patients with cystic fibrosis (Rosenblum et al., 1978a; Prussak and Russell, 1980), the metabolic transformation capacity is not impaired, in spite of the fact that extensive production and marked release of polyamines into physiological fluids by neoplastic cells frequently occurs. Therefore, one can conclude that the conjugating pathways for polyamines have high functional capacity and a high saturation level (Rosenblum, 1980). As for natural antibodies to polyamines in sera of normal or cancer patients, recent preliminary results from Roch et al. (1979) suggest that the titers of such antibodies may be quantitatively lower in the sera of tumor patients than in the sera of normal controls. It is desirable to expand this kind of research, to obtain more data, and to clarify the pathophysiological significance of such immunological differences. Ill. Diamine Oxidase Activity in Human and in Experimental Neoplasms

One explanation for the elevated putrescine concentrations observed during neoplastic growth is the increased activity of ODC (see Sections I1 and 111, Part I, Vol. 35). But another enzyme that must also play a role in determining its concentration is diamine oxidase [amine : oxygen oxidoreductase (deaminating) (pyridoxal containing); E C 1.4.3.61 (DAO), which converts putrescine into y-aminobutyraldehyde (see Section I,E, Part I, Vol. 35). Diamine oxidase has been proved to be the same as histaminase, as this enzyme was called several years ago. Because histamine is not the only substrate for this enzyme, which also catalyzes the deamination of putrescine and of several polyamines, including cadaverine, diaminopropane, and a series of aliphatic diamines, the more inclusive term “diamine oxidase” has been proposed. We will use the term “diamine

POLYAMINES IN MAMMALIAN I‘UMORS

57

oxidase” to indicate this enzyme cven when the authors reviewed used the old term “histaminase.” A. IN HUMANTUMORS Increased DAO activity has been shown to b e associated with several types of human cancer. In a series of papers, Baylin and his co-workers (1970, 1972a,b) provided evidence that some patients with medullary carcinoma of the thyroid had abnormally high DAO activity in both the serum and tumor specimens. Although the tissue samples were obtained at autopsy and the controls, instead of being normal healthy subject, had died from other neoplastic diseases (breast carcinoma, osteosarcoma, Ewing’s sarcoma), Baylin and his co-workers should still be considered the pioneer workers in this field. After these first results showing high DAO activity in serum of patients with medullary carcinoma of the thyroid, Baylin and his co-workers and other authors continued the studies of this tumor and compared the usefulness of measurement of DAO activity with that of measurements of calcitonin levels to see whether also the measurement of the activity of the enzyme in serum of patients might be useful for early detection of localized tumor (Baylin et al., 1970) as well as for metastases and residual tumor after surgery (Baylin et al., 1970, 1972a,b; Keiser e t al., 1973). After removal of tumor, the serum DAO activity in patients with localized tumor declined to normal, but it remained high in many patients with metastatic tumor (Keiser et al., 1973). This confirms the previously reported correlation between high serum DAO activity and metastatic disease (Baylin et al., 1972a). However, unlike calcitonin, serum DAO levels are not elevated in all patients with medullary carcinoma of the thyroid, and for this reason measurements of serum-calcitonin levels seem to be a more reliable test for the diagnosis of this neoplasm (Baylin et d., 1972a; Keiser et al., 1973). In a more recent paper, it was confirmed that high plasma DAO levels are not characteristic of patients with occult thyroid tumors or C-cell hyperplasia (Mendelsohn et al., 1978). On the contrary, there is DAO within the tumor cells, and it is almost certainly produced by them (Mendelsohn et al., 1978). In addition, DAO has been shown to be present only in certain cells of medullary carcinoma of the thyroid, but not in normal or hyperplastic C cells (Mendelsohn et al., 1973). Earlier studies also showed that this thyroid tumor contained many times more DAO activity than the normal adjacent thyroid tissue, suggesting that the large amounts of DAO in serum had originated from

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the tumor (Baylin et al., 1970).In fact, the reappearance of DAO activity in serum after the administration of aminoguanidine (a known specific inhibitor of DAO) coincides with new synthesis of the enzyme in the tumor (Baylin et al., 1970, 197213). This has been proved by administration of aminoguanidine to patients with high serum levels of the enzyme: the enzyme disappeared and then reappeared over a period of days at a rate that indicated total turnover of the enzyme in the serum in about 2 days (Baylin et al., 1970). In other words, the tumor continuously produced and released the enzyme into the circulation. This release was not affected by heparin, which does promote the entry of DAO from normal tissues into the circulation (Ettinger et al., 1978). Elevated DAO activity in effusion fluids was reported by Lin et al. (1975), associated with a number of other human cancers, including those of the ovary, breast, stomach, colon, and lung. These authors found an increase in the enzyme activity in ascites fluids from cancer of endometrium, stomach, colon, and in pleural fluids obtained from subjects with cancer of the lung and breast. An elevation of DAO activity was also found to concur with the presence of the Regan isoenzyme of alkaline phosphatase, which is known to be associated with a number of human tumors (Lin et al., 1975). The results of Lin et al. (1975),and other earlier results showing high DAO activity in plasma of patients with endometrial adenocarcinoma, uterine myosarcoma, and granulosa cell carcinoma (Borglin and Willert, 1962), were further evidence that the idea of Baylin et al. (1970, 1972a,b) that increased DAO activity in serum is a specific marker for medullary thyroid carcinoma is not correct. However, Baylin and his co-workers and other authors proposed that measurement of serum DAO activity might be diagnostic for this kind of tumor if combined with simultaneous measurements of serum calcitonin levels (Baylin et d., 1972a; Keiser et d., 1973; Mendelsohn et al., 1978). Lin et al. (1979)extended their studies to evaluation of DAO activity in over 400 malignant effusion fluids (pleural, peritoneal, or pericardial) collected from 162 cancer patients. Elevated DAO activity was found in a larger percentage of the effusion fluids of patients with cancers of the ovary, colon, and stomach (Lin et al., 1979).In patients with cancer of the colon and stomach, the elevation of DAO was found to be correlated with the production of carcinoembryonic antigens in the majority of the cases; whereas among those with cancer of the ovary, the elevation of DAO tended to go along with the production of the &subunit of human chorionic gonadotropin (Lin et al., 1979). Results of Ettinger et a2. (1980) confirmed those of Lin et al. (1979)and

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demonstrated that the ovarian carcinoma cell appears to be the source of the increased DAO activity in the ascitic fluid from some of these patients. Furthermore, in this type of oncopathic subject, DAO activity is consistently greater in ascitic fluid than in plasma (Ettinger et al.,

1980). The results of Kusche et al. (1980) contrast with the results above. They reported that the distribution of DAO activity in the gastrointestinal tract of patients with adenocarcinoma of the large bowel or of the stomach did not indicate that the enzyme was all produced by tumor cells. There was less enzymic activity in the tumor tissue itself than in the adjacent, histologically normal mucosa (Kusche et al., 1980). Nevertheless, the DAO activity of the gastrointestinal mucosa adjacent to tumor was influenced by tumor growth (Kusche et al., 1980). Because of its supposed origin from the neural crest and its embryological relationship to medullary thyroid carcinoma, another tumor whose DAO activity was well studied is small-cell carcinoma of the lung. This tumor was also found to have higher DAO activity than normal lung (Baylin et al., 1975). This activity has been found to be frequently elevated also in plasma of patients with small-cell carcinoma of the lung (Baylin et d.,1975). Further studies confirmed that the increase in plasma DAO activity only partially reflects the increased DAO activity in neoplastic tissue of patients with this kind of tumor, since despite the high DAO activity in the neoplastic tissue, the majority of these oncopathic subjects did not have elevated blood levels of DAO activity (Ettinger et al., 1978). Consequently, the preceding idea that the monitoring of DAO in plasma of patients with small-cell carcinoma of the lung and elevated plasma levels might have prognostic importance, indicating the state of differentiation of the tumor tissue (Baylin et al., 1975), should be discarded. In addition, a specific association of the elevation of DAO activity in the effusion fluids of patients with small-cell carcinoma of the lung was not observed in a more recent study (Lin et nl., 1979). On the other hand, Baylin et al. (1978) reported that the circulating levels of DAO, like the levels of such other markers as L-DOPA decarboxylase and calcitonin, cannot necessarily be expected to mirror tumor growth in patients with small-cell carcinoma of lung. It is possible that the variable biochemical patterns in different tumors from different patients and even in a single neoplasm may reflect heterogeneity of cell population and metabolic activities in the neoplastic tissue, because small-cell carcinoma can originate in more than one clone of cells, each with different patterns of biochemical expression and storage content of markers (Baylin et d., 1978).

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In a study to ascertain whether small-cell carcinoma of lung might have a separate histogenesis from the other major types of human lung tumors, it was shown that the differences in DAO activity in the major forms of human lung cancer are quantitative rather than qualitative (Baylin et al., 1980a). The occurrence of increased DAO activity is roughly the same in the various histopathological types of lung neoplasms in humans (Baylin et al., 1980a). In contrast to L-DOPA decarboxylase activity, which appears to be a valuable marker for differentiating small-cell carcinoma cells from other lung cancer cells in uitro, DAO activity is generally low in all types of lung carcinoma cells in culture (Baylin et al., 1980a). Although the factors responsible for the increased DAO activity in tumors have not been fully delineated, it has been proposed that DAO activity in placental and neoplastic tissues is an expression of a mature genome, but not a unique expression of a fetal genome (Baylin, 1977). Although human placental histaminase is identical with histaminase of the medullary thyroid carcinoma in several biochemical features, the placental enzyme also has similarities with the histaminases of human kidney and of human intestine (Baylin, 1977). This is consistent with earlier suggestions that placental DAO is associated not with trophoblastic tissue, but with maternal decidual elements in the placenta (Lin et al., 1975). The study of Baylin (1977) also emphasizes that high DAO activity in human neoplasms might or might not be considered as an “ectopic protein production.” Last, in addition to those mentioned above, the findings about DAO activity are widely variable for other types of human cancer. In one patient with choriocarcinoma not associated with mole, an initial highly elevated serum level of DAO, comparable to the elevation found in late normal pregnancy when values are at the peak (Torok et al., 1970), was found. One patient with adenocarcinoma of the breast also had enzyme activity similar to that in a normal 8-ll-week gestation (Torok et al., 1970). There was no enzyme activity in three patients with teratocarcinoma of the testes (Torok e t al., 1970). TUMORS B. I N EXPERIMENTAL Studies by Quash and his co-workers demonstrate that there are variations in the activities of DAO and polyamine oxidase (PAO) associated with the growth of the normal and transformed cells. As cells approach confluence, normal cells have about twice the DAO activity of their transformed counterparts. This has been reported for rat kidney cells, both normal and transformed by avian sarcoma virus (Quash

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et al., 1979). Normal and virus-transformed BHK cells grown in cultures have differences in asparagine decarboxylation, which is increased in BHK cells transformed by either hamster sarcoma virus or polyoma virus (Quash et al., 1976). Asparagine not only stimulates DAO activity (Quash et al., 1976), but has been demonstrated to increase the activity of ODC in cultured neuroblastoma cells (Chen and Canellakis, 1977) (see Section 1,A). Asparagine, indeed, may exert two crucial and opposite effects on cellular polyamine metabolism, namely, stimulation of synthesis by activating ODC and enhancement of degradation by activating DAO. Because a decrease of DAO activity similar to that reported for transformed cells has also been observed in rat mammary tumor induced chemically with 9,10-dimethylbenz[a]anthracene, in leukemic myeloblasts and in a human epithelioid carcinoma cell line, the differences in DAO activity do not seem to be linked to viral transformation only, but rather to the neoplastic state (Quash et al., 1979). However, a much larger systematic study with different types of tumor will be necessary before we can conclude that diminished DAO activity is a characteristic of all types of transformed cells. However, an exactly opposite finding has been reported with an astounding elevation of DAO activity in 4-DAB hepatoma and in Yoshida ascites hepatoma cells (Perin et al., 1979). A study b y Bachrach (1980a) demonstrated that the addition of complete serum-containing medium to confluent cultures of glioma cells increased not only the activities of the polyamine biosynthetic decarboxylases, but also that of DAO. This last enzyme increase was maximal at 8 hr, when the ODC activity also reached its peak, and was followed b y the accumulation of y-aminobutyric acid, which was detected in the cells and in the medium (Bachrach, 1980a). Asparagine caused an increase in DAO activity of glioma cells in culture and enhanced the formation of y-aminobutyric acid from putrescine (Bachrach, 1980a). This can be explained by the activation of DAO by 2-oxosuccinamate, which is an intermediate of aspargine decarboxylation (Quash et al., 1976, 1979) (see below). Regarding its intracellular regulation, DAO activity can be affected i n uitro by metabolites of naturally occurring amino acids: 2-0x0succinamate, which is derived from asparagine by transamination, was found to b e an activator; oxaloacetate, which can be formed from aspartate by transamination or from 2-oxosuccinamate by enzymic deamination, was found to be an inhibitor, as is pyruvate, formed by decarboxylation of oxaloacetate (Quash et al., 1976). The activation of DAO b y 2-oxosuccinamate seems to be of limited physio-

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logical significance, because a relative high concentration is needed to produce a comparatively small activation (30-50%) of putrescine oxidation (Quash et al., 1976). Its real physiological role may be as an allosteric effector of the enzyme for substrates other than putrescine (Quashet al., 1976). On the other hand, the DAO inhibition could have physiological significance. In fact, almost total inhibition (94%)occurs with pyruvate (Quash et al., 1976), and it is well known that an elevated pyruvate concentration results from an increased rate of glycolysis, which is exhibited frequently by some types of malignant cells. IV. Physiological and Pharmacological Inhibitors of Polyamine Biosynthesis in Neoplastic Tissues or Cells

In these last years, some physiological and/or pharmacological inhibitors of polyamine biosynthesis have been used to investigate the metabolic consequence of depriving normal or neoplastic cells of polyamines. We believe that this is only one way, and a rather marginal one, to elucidate the role of polyamines in the different types of cellular growth process, both controlled and uncontrolled. Moreover, in our opinion, it would be more useful to attempt to clarify the functions of polyamines using physiological inhibitors rather than to further screening for new molecules able to inhibit polyamine synthesis. As has been reported, the depletion of polyamines in neoplastic cultured cells b y induction of ODC antizyme production provides an alternative to the pharmacological competitive inhibitors for investigating polyamine-dependent metabolic pathways in cells (Branca and Herbst, 1980). Ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase are the most suitable target enzymes for inhibition of polyamine biosynthesis because they are rate-controlling enzymes in the pathway. Substances that can control the enzyme synthesis of ODC and SAMD can be categorized first by whether they occur physiologically. Among the physiological substances, polyamines, especially putrescine, play important roles. On the other hand, most of the pharmacological inhibitors now available and used in attempts to block the accumulation of polyamines in vivo or in vitro are inhibitors of these two decarboxylases. We will consider the physiological inhibitors and the pharmacological ones separately. The reader can obtain more detailed information about the different inhibitors of the polyamine biosynthesis from the excellent reviews provided by Williams-Ashman et al. (1976),Janne e t al. (1978), Mamont et al. (1980), and Stevens and Stevens (1980).

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PHYSIOLOGICAL INHIBITORS AND

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RELATED COMPOUNDS

The naturally occurring polyamines, putrescine, spermidine, and spermine, acting as negative regulators of their biosynthetic decarboxylases, can inhibit cell growth b y themselves or through their oxidized products. We will consider both these aspects, and, because the oldest reports are devoted to the cytotoxic or antiproliferative properties of polyamines, we shall begin by reviewing the inhibitory effects of polyamines on various types of tumors, both in vivo and in vitro. Boyland (1941) found that cadaverine and spermine were effective in inhibiting the growth of grafted sarcomata and spontaneous carcinomata in mice. He demonstrated that these aliphatic bases were the factors responsible for the inhibition of tumor growth by a muscle extract. Subsequently, the effects of biogenic polyamines, including cadaverine, on the growth of cancer cells were examined in vitro in Ehrlich solid tumor and Yoshida sarcoma. None of the amines examined had any effect on the Ehrlich solid tumor, while all the amines were inhibitory for Yoshida sarcoma, spermine having the strongest effect (Miyaki et al., 1960). This inhibitory effect on cell growth is a property of polyamines in the presence of calf serum, in which situation they have a potent cytotoxic effect on tissue culture cells. It must be calf serum, since no such effect is observed with horse or human serum (Alarcon et al., 1961; Alarcon, 1964). The interpretation has been that polyamines are not cytotoxic by themselves, but become so through the action of polyamine oxidase, which is present in calf serum but not in human or horse serum (Hirsch, 1953a,b; Taboret al., 1954). In the absence of amine oxidase, the polyamines did not affect the multiplication of the Ehrlich ascites cells, whereas the toxicity of purified oxidized spermine for cells of this type has been confirmed in an in vitro-in vivo system (Bachrach e t al., 1967). Allen et al. (1979) further confirmed that the inhibition of growth is due to a bovine plasma oxidase that converts the polyamines to the inhibitory factors. They found that spermidine and spermine are potent in vitro inhibitors of proliferation of phytohemagglutinin-stimulated lymphoma cells and human lymphoblastic leukemia cells only in media supplemented with fetal calf serum. In addition, putrescine, which was not an inhibitor in the presence of fetal calf serum, become so in the presence of human pregnancy serum, possibly due to its content of DAO (Allen e t al., 1979). Diamine oxidase degrades its substrates to aldehydes, which in general are cytotoxic, and it is probably in this way that DAO inhibits proliferation of Bri8 human leukemic lymphocytes (Gaugas, 1980). This is supported by the fact that amino-

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guanidine can reverse the DAO-induced inhibition (Gaugas, 1980). However, the products arising from spermine oxidation are not the sole factors responsible for the inhibition of cell growth by spermine, since Higgins et al. (1969) obtained inhibition in KB cells by spermine in the presence of some sera with no spermine oxidase activity. In addition, the cytotoxic effect of polyamines-to be precise, of their oxidatively deaminated products-is not the same for every cell type. It has been proved that oxidized spermidine is cytotoxic in the presence of calf serum only to normal cells, while polyoma virustransformed cells, adenovirus type 1Ztransformed cells, or spontaneously transformed cells are considerably more resistant (Otsuka, 1971). A “spermidine index” for a cell culture, defined as the highest level of spermidine that does not have cytotoxic effects in a standard test system, has been proposed (Otsuka, 1971).This index measures the ability of a cell culture or line to neutralize the cytotoxic effects of spermidine (Otsuka, 1971). The cytotoxic effects of spermidine can be neutralized by the serum’s albumin content, which adsorbs spermidine molecules (Otsuka, 1971). Dioxidized spermine, either in its free cationic form or bound to an unidentified serum component, potently arrested cell proliferation in the GI phase of the cell cycle (Gaugas and Dewey, 1979; Gaugas, 1980). This occurs when polyamines are added to cultures of murine leukemic T lymphocytes, of human leukemic lymphocytes, or of Harding-Passey mouse melanoma and is probably a consequence of the interaction of polyamine oxidase with the exogenous polyamines (Gaugas and Dewey, 1979; Gaugas, 1980). Addition of the diamines putrescine and cadaverine did not produce inhibition (Gaugas and Dewey, 1979). The fact that the cells were arrested in the GI phase suggests that this inhibition was not due to acrolein, which is instantaneously cytotoxic at all phases of the cell cycle (Gaugas and Dewey,

1979).

As has been previously reported (see Section I,E,l, Part I, Vol. 35), the oxidation products of polyamines are aminoaldehydes, which can react with thiol groups. Addition of thiols to culture containing amine oxidase in the medium can protect against polyamine toxicity. This was found in a cell line derived from Harding-Passey mouse melanoma, which was protected against spermidine toxicity by addition of L-cysteine (Dewey and Gaugas, 1980).This protective effect is specific for L-cysteine, because the unnatural isomer D-cysteine enhances the inhibition by spermidine (Dewey and Gaugas, 1980). There is some relationship between bound serum aldehydes and cell growth. In the sera of patients with early malignancies, the amount

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of bound aldehyde that could be released by treating the serum with As203is significantly lower than that in normal sera, but, as metastases occur, it rises above normal values (Quash and Maharaj, 1970). This difference no longer exists when the patients with malignant disease have responded to an antineoplastic treatment (Quash and Maharaj, 1970). Administration of nontoxic doses of putrescine along with the 3,4benzopyrene almost completely prevented tumor development in mice (Kallistratos, 1975a,b; Kallistratos and Fasske, 1976). Although the mechanism of this inhibition of carcinogenesis is still unknown, it is probable that putrescine comes between 3,4-benzopyrene and cell components, hindering binding of the carcinogen (Kallistratos, 197513). Topically applied putrescine inhibits both the induction of epidermal ODC activity and the promotion of mouse skin tumors by TPA (Weekes et al., 1980). This inhibition is not due to some general cytotoxic effect of the diamine, since the application of putrescine did not inhibit the induction of activity of SAMD by TPA (Weekes et al., 1980). Treatment with putrescine before TPA application has little effect (Weekes et al., 1980). It is not clear whether putrescine regulates ODC activity by decreasing its rate of synthesis or by accelerating its rate of degradation. It has been shown that putrescine does not inhibit TPA-induced epidermal ODC activity via production of ODC antizyme (Weekes et al., 1980). This was true also for a clone of rat hepatoma cells in culture (McCann et al., 1979a). Therefore, there might be two separate mechanisms for ODC regulation by putrescine: one through the induction of the inhibitory antizyme that complexes with the enzyme (see Section I,B,l of Part I, Vol. 35 and of Section I,A of this volume) and another without induction of any significant amount of antizyme (McCann et al., 1979a; Weekes et al., 1980).Antizyme induction or noninduction depends on the concentration of putrescine in a rat hepatoma cell line partially resistant to a-methylornithine. Low concentrations of putrescine have an effect on ODC activity similar to that seen of general inhibitor of protein synthesis cycloheximide, i.e., block of new ODC synthesis (McCann et al., 1979a). On the contrary, high concentrations of putrescine elicit the induction of ODC antizyme (McCann et al., 1979a). In spite of the results of McCann, the comparable dose response curves for inhibition of endogenous ODC and for induction of ODC antizyme by diamines and polyamines in HeLa cells suggest that in this culture system there is no need to invoke the existence of separate and distinct mechanisms for regulation of ODC activity (Branca and

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Herbst, 1980).The induction of ODC activity elicited in HeLa cells by arginine was diminished by increasing amounts of canavanine, an arginine analog, in the medium (Prouty, 1976a). Ornithine has an effect analogous to that of putrescine in decreasing tumor incidence. Addition of this amino acid to water and food decreases tumor incidence in mice inoculated with either transplantable breast adenocarcinoma (C3HBA) or MuSV-M virus (Rettura et al., 1978). The mechanism by which ornithine exerts its antitumor action is also not known. It was found that the naturally occurring polyamines potentiate thermal inactivation of mammalian cells (see Section 1,A). The combined effect of heat and polyamines on transplanted B16 melanomas was studied in mice (Hazan, 1980). Synergistic interactions between heat and spermine or cadaverine were demonstrated, but the effects were weak because of the toxicity of the polyamines, which prevent giving high doses (Hazan, 1980).These data agree with those of studies on cultured Chinese hamster cells (Ben-Hur et al., 1978)(see Section I,A), and there is a good correspondence between the in vitro and in vivo results. An interesting effect of spermine was reported by Beck (1977), who found that addition of the tetraamine to cultures of human Wilms’s tumor or rat hepatoma cells reduces the cytotoxic effects of vincristine and vinblastine, which interact with microtubule structures. Vinblastine, vincristine, and colchicine, all of which possess microtubuledisrupting activity, also inhibit ODC induction by TPA in mouse skin (O’Brien et al., 1976). Last, among the different ways by which one or more polyamines might exert their inhibiting effects on cell growth, we must consider the compounds formed by polyamines with other chemical substances. Some of these compounds, the acridines connected by the naturally occurring polyamines to form putrescine diacridine, spermidine diacridine, and spermine diacridine, were studied, and their effects on the growth of HeLa cells and of P388 and L1210 leukemia cells were compared to those of the parent compound 9-aminoacridine (Canellakis and Bellantone, 1976). The diacridines are more effective growth inhibitors than 9-aminoacridine, and the inhibition is not due to the toxicity of oxidized polyamines formed in the medium, since it is seen with leukemic cells P388 and L1210 grown in the presence of horse serum, which does not contain polyamine oxidase (see above). In the case of HeLa cells, which are grown in a medium containing calf serum, the number of cells in the presence of spermine diacridine reaches a plateau and remains at this plateau for many days, while

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exposure of cells to 9-aminoacridine for the same periods of time and under identical conditions results in slower rates of growth but not in a complete arrest of the growth of the cells (Canellakis and Bellantone, 1976). Synthesis of RNA is inhibited immediately, and synthesis of protein and DNA is inhibited later in exposure to both 9-aminoacridine and spermine diacridine (Canellakis and Bellantone, 1976). When P-388 and L1210 cells were exposed to spermine diacridine and then inoculated intraperitoneally (i.p.) into mice, cells that would have been inhibited from subsequent growth in culture can grow when placed in the animal (Canellakis and Bellantone, 1976). In culture, however, spermine or spermidine can reverse the inhibition, probably by the displacement of endogenous bound diacridines (Canellakis and Bellantone, 1976). Another series of compounds, the ferrocenyl polyamines, was screened for antitumor activity against lymphocytic leukemia P388 (Fiorina et al., 1978). These compounds were synthesized with the intent to produce materials that would interact strongly with the tumor surface nucleic acids and, through the hapten portion of the molecule, stimulate antibody formation. The target ferrocenyl polyamines were inactive, but the R-substituted compounds, such as diamide, triamide, and tetraamide, exhibited low, but significant, antitumor activity (Fiorina et al., 1978). It appears that the incorporation of the ferrocene group into an appropriate polyamide carrier might provide an agent with enhanced antitumor activity (Fiorina et al., 1978). Israel et al. (1964) synthesized a series of substances related to spermine and spermidine that showed significant antitumor activity in viuo against four transplantable mouse tuniors-L1210 ascitic lymphatic leukemia, P1534 lymphatic leukemia, C1498 myelogenous leukemia, and DBRB mammary carcinoma. Against human epidermoid carcinoma KB cells in a culture system containing calf serum, the triamines and tetraaniines synthesized demonstrated, in general, the same high degree of inhibitory activity as spermine and sperniidine (Israel et al., 1964). Furthermore, in a systematic examination for growth-inhibitory activity against KB cells, it was found that incorporation of a 2-aminoethyl terminal group in polyamine derivatives is essential for inhibitory activity (Israel and Modest, 1971). Another product, a homolog of spermine, N,N’-bis(3-aminopropyl)nonane-1,9dianiine, when administered in the form of its tetrahydrochloride, inhibits the growth of a variety of leukemias and solid tumors in mice, rats, and hamsters (Israel et ul., 1973). This compound also requires bovine plasma amine oxidase and oxidative deamination, as shown by its not being inhibitory against KB cells in horse serum (Israel et

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al., 1973). It should be noted that the dialdehyde products of this compound inhibited the growth of KB cells in vitro both in the presence and in the absence of bovine plasma amine oxidase and without a need for generation of acrolein (Israel et al., 1973). The effect of some steroidal amines (irehdiamine A, malonetine, 3a-amino-5a-androstane, and 3a-amino-5a-pregnane) on membrane structure and permeability has been investigated in human KB cells (Silver et al., 1970). Some of these amines caused a rapid loss from the cells of more than 95% of accumulated **Mg,but this effect is not a characteristic of tumor cells, since normal mouse fibroblasts are also sensitive to these substances to approximately the same extent (Silver et al., 1970). This membrane effect, although it has been found to be a property of these polyamines not present in mammals, could indicate that spermidine and spermine inhibited growth of KB cells in culture (Israel et al., 1964; Higgins et al., 1969) in the same way.

B. PHARMACOLOGICAL INHIBITORS 1. Znhibitors of ODC Most experimental efforts to deplete cellular polyamines have been focused on the development of inhibitors of the rate-limiting enzyme ODC. Among these are diamines, such as 1,3-propanediamine, 1,5pentanediamine, and 1,6-hexanediamine. These diamines dramatically decreased both tumor growth and ODC activity in neuroblastoma cells, whereas glioma cells continued to grow (Chapman and Glant, 1980). Differences in sensitivity to growth inhibition probably are not due to differences in drug accessibility since the enzyme was inhibited in both cell lines (Chapman and Glant, 1980). The ODC activity in C1300 neuroblastoma was also inhibited significantly in the presence of cytolytic concentrations of bromoacetylcholine and bromoacetate (Chapman et al., 1978), which inhibited cell replication, and RNA and protein synthesis in parallel. Because similar inhibition of neuroblastoma cell replication was obtained with the ODC inhibitor 1,3-diaminopropane, it seems that the mechanism of the potent cytolytic action of bromoacetylcholine and of bromoacetate may be related to inhibition of this enzyme (Chapman e t al., 1978). Among the most potent diamines able to inhibit the formation of putrescine and spermidine, Janne and his co-workers described an analog of putrescine, the l,Sdiaminopropane, and some of its deriva-

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tives, such as the hydroxylated derivative 1,3-diamino-2-propanol, which all inhibit ODC activity. Once again, Ehrlich ascites carcinoma cells, which contain substantial amounts of polyamines and have typical growth-dependent fluctuations in the activities of their biosynthetic decarboxylases (see Section 11, Part I, Vol. 35), have been a suitable model system for studies devoted to the elucidation of the role of polyamines in tumor cells. It was demonstrated that the polyamine patterns in mice with Ehrlich ascites carcinoma could be modified by injection of diaminopropane or cadaverine (Kallio e t al., 1977). Disappearance of ODC activity was accompanied by depletion of cellular putrescine and markedly reduced concentrations of spermidine (Kallio et d., 1977). Repeated injections of diaminopropane virtually abolished any increase in ODC activity in the Ehrlich ascites cells (Alhonen-Hongisto et al., 1979b). In contrast to ODC, there were insignificant changes in the activity of SAMD in response to the diamine injections (Kallio et al., 1977; Alhonen-Hongisto et al., 197913). In a further investigation of the diamine-induced inhibition of ODC, derivatives of 1,3-diaminopropane were tested in cultured Ehrlich ascites cells. 1,3-Diamino-2-propanol appeared to be as potent or even more potent an inhibitor of ODC than the parent compound, whereas 1,2-diaminopropane and 1,2-diamino-2-methylpropanewere less active (Alhonen-Hongisto et al., 1979a). During the growth phase of ascites cells, diaminopropanol permanently abolished increases of ODC after dilution of stationary cell cultures with fresh medium, whereas the activity of SAMD was unaffected by the inhibitor (Alhonen-Hongisto et al., 1979a). Exposure of Ehrlich ascites cells to 1,3-diamino-2-propanol resulted in decreased polyamine accumulation and marked disturbances in cell metabolism and proliferation, as judged by impaired syntheses of DNA and protein and decreases in cell numbers (Alhonen-Hongisto et al., 1979a, 1980a) or cell mass (Alhonen-Hongisto et al., 1979b). Inhibition of DNA and protein synthesis occurred after depletion of spermidine and spermine has been established (Alhonen-Hongisto et al., 1979a). Severe inhibition of polyamine accumulation and of cell proliferation by diaminopropanol was seen in cultures of HeLa cells, as well induced the (Branca and Herbst, 1980). 1,3-Diamino-2-hydroxyropane formation of ODC antizyme, and, like other diamines that cannot serve as precursors of the naturally occurring polyamines, decreased intracellular concentrations of the polyamines, which in turn blocked the proliferation of the HeLa cells. The antiproliferative effect of the ODC

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antizyme inducer does not destroy the viability of the arrested culture, since cell replication occurs after removal of the antizyme inducer from the media (Branca and Herbst, 1980). Although it is strongly suggestive, close parallelism between druginduced growth inhibition and a decrease in polyamine content cannot be taken as definitive proof that inhibition of polyamine synthesis alone is responsible for the antiproliferative effects of the drug. The most straightforward evidence that an inhibitor is specific for the prevention of polyamine synthesis is the reversal of the antiproliferative action of the drug b y exogenous polyamines. Reversal experiments are, however, complicated by the fact that inhibitors of polyamine synthesis may interfere with the cellular uptake of natural polyamines. In fact, tumor cells depleted of putrescine and spermidine take up extracellular polyamines much more effectively than do untreated cells (Alhonen-Hongisto et al., 1980b). In contrast with the data demonstrating a causal relationship between depletion of polyamines and inhibition of DNA synthesis (Alhonen-Hongisto et al., 1979a), it has been found that despite its more pronounced inhibition of polyamine accumulation diaminopropanol has less striking antiproliferative action than the diguanidines (Alhonen-Hongisto et al., 1980a). In addition, the inhibition of DNA and protein synthesis by diaminopropanol was diminished but not abolished by simultaneous addition of putrescine to the culture medium (Alhonen-Hongisto et al., 1979a). The inability to completely reverse the action of diaminopropanol on cell growth with natural polyamines was apparently due to the fact that it is remarkably difficult or even impossible to increase intracellular polyamine concentrations by adding exogenous polyamines in the presence of the inhibitor. Nevertheless, the diaminopropanol-induced arrest of growth is reversible, as judged from the rapid increase in ODC activity followed by restoration of DNA synthesis (Alhonen-Hongisto et al., 1979a). It has been found that 1,3-diaminopropane also induces ODC antizyme in rat hepatoma cells and, like putrescine, can act on ODC by two distinct regulatory mechanisms (McCann et al., 1977a, 1980). Various congeners of ornithine have been found to be potent inhibitors of ODC activity in eukaryotic cells. The first inhibitor of polyamine synthesis tested for elucidation of the metabolic consequences of inhibition of putrescine synthesis, was a-hydrazinoornithine. This drug prevents the increase in intracellular putrescine that occurs when hepatoma cells grown to high density in culture are diluted with fresh medium (Harik et al., 1974a). Doses of the drug that markedly reduce putrescine do not appreciably affect the syn-

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thesis of either RNA or DNA (Harik et al., 1974a). Surprisingly enough, it was also shown that when added to the rat hepatoma cells at the time of dilution with medium, a-hydrazino-ornithine evoked a dose-related increase in ODC activity as much as threefold and prolonged the apparent half-life of the enzyme from 10 min to 28 min (Harik et al., 1974b). The racemic form of a-hydrazino-ornithine, DL-a-hydrazino-6aminovaleric acid (DL-HAVA),appeared to be a potent and fairly specific inhibitor of ODC in transplanted sarcoma 180 in mice (Kato et ul., 1976). This drug greatly suppressed putrescine concentration and its formation from ornithine but did not significantly affect the concentrations of spermidine and spermine (Kato et ul., 1976). On the contrary, DL-HAVA efficiently prevents the accumulation of spermidine and spermine in BKT-1 tumor cells (Raina et al., 1978). A single i.p. injection of DL-HAVA into mice bearing sarcoma 180 also caused strong inhibition of DNA synthesis, which was reversed by administration of putrescine, but not of cadaverine or 1,7-diaminoheptane (Kato et al., 1976). Abdel-Monem and his co-workers (1975a) found that ODC was strongly inhibited in L1210 leukemic cells by a-methylornithine, another competitive inhibitor of the enzyme. Complete disappearance of putrescine and a 50% decrease in spermidine content not accompanied by inhibition of growth were evident in these cells treated with a-methylornithine for two generations (Newton and Abdel-Monem, 1977), whereas in zjiuo administration of the drug to mice with L1210 leukemic cells did not alter the increases in polyamine levels normally observed during tumor growth (Weeks and Abdel-Monem, 1977). There was also no in uiuo effect of the tert-butyl ester of a-methylornithine (Weeks and Abdel-Monem, 1977). In uitro, a-methylornithine did not alter DNA synthesis, indicating that a large portion of the polyamines are not essential for cellular growth in these cells (Newton and Abdel-Monem, 1977). However, a later study of Mamont et al. (1978a) showed that a-methylornithine does inhibit L1210 cell proliferation. a-Methylornithine markedly inhibited the proliferation also of Bri8 human leukemic lymphocytes, not the parent cells present at the onset of incubation but only the daughter cells and their progeny (Gaugas, 1980).The inhibitory effect could be prevented b y adding putrescine or other biogenic polyamines (Gaugas, 1980). DL-a-Methylornithine prevented the biphasic increases in putrescine concentration in cultures of rat hepatoma cells induced to proliferate by dilution with fresh medium (Mamont et al., 1976, 1978c) and blocked the proliferation of rat hepatoma cells and mouse leukemia

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L1210 cells in culture (Mamont et d., 1978a). Increases in cellular spermidine concentration were prevented as well, and inhibition of DNA synthesis and cell division was observed (Mamont et al., 1976). Addition of polyamines results in an immediate resumption of cell proliferation, which is also restored by L-ornithine, presumably due to in situ competitive inhibition of ODC (Mamont et al., 1976, 1 9 7 8 ~ ) . Also in this case, the specificity of the reversing effects of the polyamines was confirmed by the finding that neither cadaverine nor l,Sdiaminopropane, the higher and the lower homologs of putrescine, could overcome the inhibition of cell proliferation by a-methylornithine (Mamont et al., 1976). Another effect of a-methylornithine which was reversed by spermidine is inhibition of cytokinesis with induction of the formation of binucleate HeLa cells (Sunkara et al., 1979a). Treatment of 9L rat brain tumor cells with a-methylornithine resulted in cytostasis when the cells were plated in monolayer cultures at an initial cell density of 5 x 105 per flask but not of 1 x 10' (Seidenfeld and Marton, 1980). This could be explained by the fact that the amount of a-methylornithine entering the cells depends only on its concentration in the medium. As in hepatoma cells and in mouse leukemic cells, the inhibition of 9L cell proliferation by the drug appears to be a specific result of polyamine depletion and can reversed by addition of exogenous putrescine to the culture medium (Seidenfeld and Marton, 1980). a-Methylornithine did not prevent the cells from initiating DNA synthesis. Thus, in these cells too increases in ODC activity and in intracellular polyamine content do not appear to be the signals for initiating DNA synthesis (Seidenfeld and Marton, 1980). Despite the fact that a-methylornithine has been found markedly to inhibit growth of rat hepatoma cells and mouse L1210 cells, it did not affect the growth pattern of Ehrlich ascites tumor cells grown in culture (Oredsson et al., 1980a). A possible explanation is that the latter have putrescine and spermidine contents that are considerably higher than those of the other neoplastic cells. On the other hand, a-methylornithine induced a significant increase in vivo in the cell lethality of Ehrlich ascites tumor cells, suggesting that the drug produces its antiproliferative effect, at least in part, by cytocidal action (Linden et al., 1980). When added to hepatoma cells in culture, a-methylornithine causes an increase in ODC activity, probably stabilizing the enzyme and slowing its degradation (McCann et al., 1977b). This is a common disadvantage of all competitive inhibitors of ODC, which stabilize the

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enzyme in both cultured cells and in uiuo (Harik et al., 1974b), probably because the enzyme bound to the inhibitor is less susceptible to degradation than the free enzyme. This binding to the enzyme and this protection from degradation, resulting in an increase in the half-life of ODC, was also found in neuroblastoma and glioma cells in culture after addition of a-methylornithine (Chapman and Glant, 1980). A clone of rat hepatoma tissue culture cells with a rate of cell proliferation partially resistant to a-methylornithine has been isolated and designated HMO, (Mamont e t al., 1978b). It appears that the reason for the partial resistance of HMOAcells to the ODC inhibitor is their overproduction of putrescine and spermidine (Mamont et al., 1978b), which lends an initial resistance of these cells to the antiproliferative drugs. Significantly, when spermidine depletion has finally occurred, i.e., within 2.5 to 3 generations, cell growth slows (Mamont et al., 1978b). A series of synthetic structural analogs of ornithine were tested as competitive inhibitors of ODC obtained from rat hepatoma cells in culture (Bey et al., 1978). This study clarified the structural features of L-ornithine that are required for binding to mammalian ODC. There is a requirement for an L configuration of the ligand. (+)-aMethylornithine, which was assigned the L configuration on the basis of rotational criteria, was found to be the most effective inhibitor of ODC (Bey et al., 1978). A primary terminal amino group in the ligand appears to be important for enzyme inhibition, and the distance between the two nitrogen atoms of the ornithine analogs is of major importance for inhibition (Bey et al., 1978). Last, hydrophobic interactions with the side chain of the ligand are important for the binding, since hydrophilic functions on the a- and the P-carbon atoms abolish all affinity for the enzyme (Bey et al., 1978). a-Difluoromethylornithine (DFMO), a dihalogenated derivative of a-methylornithine, is one of the most specific inhibitors of ODC because of its mode of action as an enzyme-activated irreversible inactivator (Mamont et al., 1980; Oredsson et al., 1980a). At variance with a-methylornithine, DFMO proved to be an affective inhibitor of growth of Ehrlich ascites tumor cells in culture (Oredsson et al., 1980a) or in uiuo (Alhonen-Hongisto et al., 1979b). However, even though DFMO is an irreversible inhibitor of ODC, there was not total inhibition of the enzyme activity (Oredsson et al., 1980b), and when the drug was added after the initial surge of ODC activity, growth proceeded as in untreated control cultures (Oredsson et al., 1980a). Once the polyamine content has been significantly lowered by the drug, however, the cells grow slowly and synthesize their mac-

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romolecules very poorly, suggesting that at least putrescine and spermidine are essential for maximum rates of cell proliferation (Oredsson et al., 1980b). Another illustration of a greater effectiveness of DFMO than of a-methylornithine was the finding that DFMO, but not a-methylornithine, inhibited the growth of human prostatic adenoma cells (Mamont et al., 1978a). DFMO decreases the concentrations of putrescine and spermidine, but not of spermine, in rat hepatoma cells (Mamont et al., 1978a,c) and in mouse leukemia cells cultured in vitro (Mamont et al., 1978a) and in Ehrlich ascites cells in vivo (Alhonen-Hongisto et al., 1979b). Cell growth inhibition was partially overcome by L-ornithine (Mamont et al., 1978a,c) or prevented b y polyamines, 1,3-diaminopropane, and cadaverine (Alhonen-Hongisto et al., 1979b). It is worth noting, however, that prolonged treatment of hepatoma cells and L1210 cells with these ODC inhibitors did not completely arrest cell growth, suggesting that depletion of putrescine and spermidine does not totally block the cell cycle (Mamont et al., 1978a,c), although in hepatoma cells the most immediate and the predominant consequence of putrescine and spermidine depletion is a decrease in DNA synthesis, with later depression of RNA and protein synthesis (Mamont et ul., 1978c, 1980). Because an inhibition of L1210 cell growth in culture by DFMO has been demonstrated, the effects of the drug were examined in vivo in mice bearing L1210 leukemia (Prakash et al., 1978). Unlike the reversible inhibitor a-methylornithine, DFMO can prolong the survival of mice (Prakash et al., 1978; Seiler et al., 1978). Like a-methylornithine, DFMO also inhibits cell proliferation of 9L rat brain tumor cells at high concentrations but not at low concentrations (Seidenfeld and Marton, 1979a). Both the high and the low concentrations cause equal degrees of depletion of intracellular putrescine and spermidine content, but have no effect on spermine content (Seidenfeld and Marton, 1979a). This is the first demonstration of continued proliferation of a wild-type cell line at the same rate as controls in spite of depletion of more than 95% of its normal complements of two of the three polyamines. In addition, the lack of correlation of the degree of polyamine depletion with the degree of cytostatic action at the concentrations of DFMO tested implies that inhibition of proliferation by this drug is not due to effects on polyamine content of 9L cells, but rather to some non-ODC specific action (Seidenfeld and Marton, 1979a). No effects on DNA synthesis were noticed when DFMO was given in vivo to L1210 leukemic mice (Seiler et al., 1978) or to mice injected with Ehrlich ascites cells (Alhonen-Hongisto et al., 1979b) or

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in vitro in cells infected with human cytomegalovirus (HCMV) (Isom and Pegg, 1979). Isom and Pegg (1979) found that although HCMVinduced stimulation of ODC was prevented by either a-methylornithine or DFMO, the replication of HCMV was not significantly altered for 8 days. In HeLa cells, DFMO caused a rapid inhibition of growth and arrested a majority of the cells in the S phase (Sunkara et al., 1980). In this case too, the growth inhibition was readily reversible b y an exogenous supply of putrescine (Sunkara et aZ., 1980).An analogous inhibitory effect by DFMO was found in Ehrlich ascites tumor cells, which also were arrested in the S-G, phase of the cycle (Heby et al., 1978b). The growth rate of a murine mammary sarcoma EMTG in tissue culture was slowed by DFMO (Prakash et al., 1980).When mice were inoculated with EMTG cells, administration of DFMO beginning 5 days after tumor inoculation resulted in an 80% inhibition of tumor weight (Prakash et al., 1980). Only minimal cytostatic effects of DFMO were observed in neuroblastoma cultures (Chapman, 1980). The effect of continuous oral administration of DFMO on the growth rate of experimental rat hepatoma 5123 was investigated (Kellen et aZ., 1980).The drug caused a significant retardation of growth rate, but did not completely prevent growth (Kellen et ul., 1980). A phenomenon observed during DFMO-induced spermidine deprivation was the enhancement of SAMD activity (Mamont et al., 1978a,c; Seiler et al., 1978).This explains the continuing accumulation of spermine in cells treated with the drug (Mamont e t al., 1978a,c; Seileret al., 1978; Alhonen-Hongisto et u l . , 1979a,b, 1980a).The enhancement of SAMD by inhibitors of ODC could not be a result of direct stabilization b y the drugs, since the drugs are said not to have any affinity to the enzyme. However, DFMO and dianiinopropane appeared to protect SAMD from normal degradation, seen as a marked prolongation of the half-life of the enzyme (Alhonen-Hongisto, 1980; Alhonen-Hongisto et al., 1980a). As revealed in the kinetic data, the mechanism of action of the drugs also involved an enhanced synthesis of SAMD (Alhonen-Hongisto, 1980). In addition to the enhancement of SAMD activity, another mechanism contributing to the compensation for polyamine deprivation has been observed in Ehrlich ascites cells grown in the presence of DFMO. The disappearance of putrescine and spermidine in the cells was accompanied by an appearance of cadaverine, which was rapidly converted to aininopropylcadaverine, an analog of spermidine (Alhonen-Hongisto and Janne, 1980). Supplementation of DFMO-treated cultures with spermidine abolished the enhanced SAMD activity and prevented the formation of

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cadaverine and its aminopropyl derivative (Alhonen-Hongisto and Janne, 1980), suggesting that the two events may be coordinately regulated by polyamines. Under circumstances in which the accumulation of natural polyamines is inhibited, compounds not normally found in cells are synthesized and accumulated to take over the functions of natural polyamines and to compensate for the polyamine deficiency. a-Acetylenic putrescine, another synthetic irreversible inhibitor of ODC, has been found to be more potent than DFMO in rat hepatoma cells in vitro (Mamont et al., 1980). Other analogs of ornithine than a-methyl-substituted ones have also been found by Abdel-Monem and co-workers (1975a). A number of a-alkyl- and benzylornithine analogs were tested as possible inhibitors of ODC in L1210 leukemic cells. The idea was that since the substitution of the a-hydrogen in the ornithine molecule with a methyl group provided a potent competitive inhibitor of ODC, replacement of the a-hydrogen with other alkyl or aralkyl groups might also provide potent inhibitors of the enzyme. However, these new compounds were very poor inhibitors of the enzyme (Abdel-Monem et al., 1975a). It has been found that tumors of neural origin are sensitive to retinoids. Retinol, retinal, and retinoic acid arrested the proliferation of C1300 neuroblastoma cells in culture (Chapman, 1980; Chapman et al., 1980). Glioma cells were less sensitive to all three analogs (Chapman, 1980; Chapman et al., 1980).A correlation was shown between the ability of retinol to inhibit ODC activity and its potency as an inhibitor of cell proliferation. When vitamin A was tested in combination with DFMO, the antiproliferative effects of the two drugs in both neuroblastoma and glioma cells were additive (Chapman, 1980). In two-stage carcinogenesis, TPA induces a very rapid increase in ODC (see Section III,C, Part I, Vol. 35), with the subsequent accumulation of polyamines. It is therefore of great interest that retinoids have been shown to inhibit these effects of TPA. Verma, Boutwell, and their colleagues found that treatment of mouse skin with retinoic acid, prior or soon after application of the tumor promoter, resulted in a much lesser induction of ODC (Verma and Boutwell, 1977, 1980; Verma et al., 1979). Since the retinoid affected neither enzyme activity in cellfree extracts nor the in vivo production of ODC antizyme, it was suggested that the retinoid interferes with enzyme induction (Verma and Boutwell, 1977).Subsequent studies with other retinoids revealed that 13-cis-retinoic acid, 5,6-dihydroretinoic acid, and two cyclopentenyl analogs of retinoic acid were also potent inhibitors of the development of skin papillomas and that this inhibitory property correlates well with their relative potencies in inhibiting ODC induction (Boutwell

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and Verma, 1978; Verma et al., 1979). 5,6-Epoxyretinoic acid, a biologically active metabolite of retinoic acid, is also active in mouse skin, where it inhibits both the induction of ODC activity and skin tumor Retinoic acid inhibits ODC promotion b y TPA (Verma et al., 1980~). activity induced in normal rat kidney cells not only b y TPA but also by epidermal growth factor and sarcoma growth factor (Paranjpe et al., 1980). When skin tumors were induced by DMBA, retinoic acid failed to inhibit either the induction of ODC activity or the tumor formation (Verma et d.,1980b). This indicates that retinoic acid’s protection against skin carcinogenesis is not universal, since it inhibits skin tumor formation by some agents but not by others. In addition, although retinoic acid is not a skin tumor promoter, when given in conjunction with DMBA treatment it even potentiated the formation of skin papillomas by DMBA (Vermaet al., 1980b). Another proof of the nonuniversality of the protective effect of retinoic acid is the fact that retinoic acid only partially inhibited the induction of ODC by germicidal UV light (Lichti et al., 1979). Formation of skin papillomas after DMBA was inhibited by 7,8benzoflavone, which also effectively inhibited the DMBA-induced ODC activity, but not TPA-induced ODC activity (Verma et al., 1980b). The induction of ODC activity in mouse epidermal cells after TPA treatment has been supposed to have the characteristic of a cell surface receptor-mediated process (see Section III,C, Part I, Vol. 35). Local anesthetics can modify a variety of cellular responses mediated by membrane receptors. On these grounds, local anesthetics such as lidocaine, tetracaine, and procaine, which are tertiary amines specifically acting on polyamine biosynthesis, have been used in order to clarify the role of the polyamine pathway in tumor promotion (Yuspa et al., 1980a,b). When added to mouse epidermal cells in culture, the anesthetics inhibited ODC inducation by TPA. In uiuo, lidocaine essentially abolishes ODC induction only when applied shortly after TPA (Yuspa et al., 1980a,b). These results are consistent with local anesthetics inhibiting at the site of interaction of TPA and its putative epidermal receptor. Local anesthetics also inhibit ODC induction b y UV light, which is probably not membrane mediated (Yuspa et al., 1980a,b). In addition, sulfur mustard, a potent inhibitor of two-stage skin tumorigenesis, did not alter TPA-induced ODC activity in mouse epidermis (De Young et al., 1977). The antipsoriasis drug anthralin has been tested for inhibition of TPA-induced ODC activity. Anthralin alone is an inducer of ODC and a moderate tumor promoter. However, when applied 2 hr before TPA,

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anthralin inhibited ODC induction and the TPA-induced epidermal cell proliferation (De Young et al., 1980).In addition, in a tumor promotion experiment with DMBA as initiator, anthralin given before TPA inhibited the number of tumors per animal. This inhibitory response may be explained by synergistic toxicity or by competition for binding sites b y the weaker tumor promoter anthralin with the stronger promoter TPA, or both (De Young et al., 1980). In contrast to their effects in vivo, anthralin and 7,12-dimethylbenz[a]anthracene have little effect on ODC activity in cultures of newborn mouse epidermal cells (Lichti et al., 1978). Not only anthralin, but also ethylphenylpropiolate inhibited TPA-stimulated ODC by 70% or more, whereas other inflammatory agents such as iodoacetic acid and cantharidin were able to shift the peak time of ODC activity after TPA (Di Giovanni and Hoel, 1980). All four compounds effectively inhibit TPA-promotion of DMBA-initiated skin papillomas (Di Giovanni and Hoel, 1980). A nonpharmacological molecule inhibiting TPA-induced ODC activity is interferon (Sreevalsan et al., 1980). This molecule was able to inhibit the increase in ODC activity in 3T3 cells stimulated b y other agents, such as epidermal growth factor, vasopressin, insulin, or fibroblast-derived growth factor, used either singly or in combination (Sreevalsan et al., 1980). An inhibition of TPA-induced stimulation of DNA-synthesis by interferon was also shown (Sreevalsan et al., 1980). The role of prostaglandins in the induction of ODC activity by TPA has been reviewed (Verma and Boutwell, 1980). A number of inhibitors of prostaglandin synthesis, such as indomethacin, naproxen, flufenamic acid, and acetylsalicylic acid, inhibited ODC induction by TPA in mouse skin (Verma et al., 1977, 1980a). The inhibition was overcome by application of prostaglandins concurrently with the TPA, thus indicating that PGE1, PGE2, PGDZ, and PGIz play roles in the induction of enzyme by TPA. Although prostaglandins mediate ODC induction by TPA in mouse epidermis, application of prostaglandins alone did not induce epidermal ODC activity, suggesting that prostaglandins are necessary but not sufficient for ODC induction by TPA (Verma et al., 1980a). In contrast to many data that have indicated that ODC induction is an essential component of the mechanism of skin tumor promotion (O’Brien et al., 1975a,b; O’Brien, 1976; Verma et al., 1977; Verma and Boutwell, 1980), a recent study suggested that polyamines are possible mediators of tumor promotion and therefore the decreases in polyamine levels better reflect the potencies of inhibitors of tumor promotion (Weeks et al.,1979). Changes in ODC activity after coincident treatment with TPA and either a-methylornithine or fluocinolone acetonide (FA), are not representative of the alterations in the in vivo

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polyamine level changes nor related to effectiveness of tumor promotion inhibition in mouse skin (Weeks et al., 1979). In fact, both a-methylornithine and FA provide paradoxical enhancements of the TPA-induced ODC activity. However, a-methylornithine causes the TPA-stimulated increased epidermal putrescine levels to be lower, and FA specifically inhibits the accumulation of spermidine (Weeks et al., 1979). Among the anti-inflammatory steroids, dexamethasone was shown to have little, if any, inhibiting effect in vivo on TPA-induced ODC activity (Vermaet al., 1977), while FA, the most potent steroidal inhibitor of mouse skin tumor promotion, paradoxically increased the TPAenhancement of ODC activity in vivo and in mouse epidermal cells in culture (Lichti et al., 1977a,b; Yuspaet al., 1978).This enhancement of the TPA-induced ODC activity by FA has been confirmed by other authors (Weeks and Slaga, 1979). On the contrary, in the Sencar mouse, a mouse stock selected for increased sensitivity to carcinogenesis, FA inhibited the TPA-induction of ODC activity (Weeks and Slaga, 1979). In numerous systems, inhibition of cell proliferation follows treatment of cells with polyamine antimetabolites, which seems to indicate that natural polyamines play an essential role in cellular proliferation processes (Janne et al., 1978). Nevertheless, the data available on this field are quite controversial. Administration of DAP and DFMO to mice with Ehrlich ascites carcinoma resulted in markedly less accumulation of putrescine and spermidine, associated with a striking depression of DNA synthesis (Alhonen-Hongisto et al., 1979b). DFMO reduced the concentration of spermidine much more than did DAP, but caused only marginal depression of DNA synthesis (AlhonenHongisto et al., 1979b). This could be explained in terms of increased concentrations of spermine compensating for the loss of spermidine in cells treated with DFMO (Mamont et al., 1978a,c; Alhonen-Hongisto et ul., 1979b). Injection of mice bearing Ehrlich ascites carcinoma 3-aminoguanidine) with 1,l’-[ (methylethanediylidene)dinitrilo]bis-( (MBAG) potentiates the effects of DAP and diaminopropanol in depressing incorporation of thymidine into DNA (Alhonen-Hongisto et d.,1979b). The potentiation of the antiproliferative effects of DAP or diaminopropanol by MBAG seemed to be specific for the diamines, since no such synergism was found when DFMO and MBAG were injected together (Alhonen-Hongisto et al., 1979b). Pertinent to this was the finding that MBAG even reversed instead of potentiating the antiproliferative effects of DFMO in mice bearing EMT6 tumor (Prakash et al., 1980). Comparing the inhibition of polyamine synthesis and of cell prolif-

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eration by diaminopropanol or by some diguanidine derivatives in cultured Ehrlich ascites cells, it gives rise to the ideas that the antiproliferative action of the diguanidines is not entirely based on the polyamine-depleting properties of the drugs and that diaminopropanol partly takes over the functions of natural polyamines in ascites cells (Alhonen-Hongisto et al., 1980a). On the other hand, it has been observed that inhibition of DNA and protein synthesis in Ehrlich ascites cells by diaminopropanol did not become evident before severe polyamine depletion had developed (Alhonen-Hongisto et al., 1979a, 1980a). In L1210 cells treated with a-methylornithine for two generations, DNA content was not altered to any appreciable extent (Newton and Abdel-Monem, 1977), suggesting that putrescine and spermidine are not essential for DNA synthesis in these cells. Inhibition of hepatoma cell polyamine accumulation by a-methylornithine affects neither DNA synthesis nor the mitotic activity during the first round of cell division, but once spermidine depletion has been achieved, there is a striking decrease in DNA synthesis and the cell multiplication rate (Mamont et al., 1976). A complete dissociation between the inhibition of DNA synthesis and ODC activity has been found for the antipromoter steroid fluocinolone acetonide (FA) (Lichti et al., 1977a,b; Yuspa et al., 1978). Under conditions in which FA induces a maximum inhibition of TPAstimulated DNA synthesis in mouse epidermal cell culture, it potentiates promoter-stimulated ODC activity, which is also enhanced when FA is present only during and after the TPA treatment, although FA itself does not significantly stimulate ODC activity (Lichti et al., 1977a,b; Yuspa et al., 1978). Similar results were obtained in uiuo. When FA and TPA are applied simultaneously to mouse skin, a treatment that completely inhibits the tumor promotion process, the steroid prevents promoter-stimulated DNA synthesis, but it paradoxically potentiates the induction of ODC activity observed after promoter treatment (Lichti et al., 1977a,b; Yuspa et al., 1978). In vivo also FA itself did not induce ODC activity (Lichti et al., 1977a,b; Yuspa et al., 1978). The antipromoter steroid FA did not act as an inducer, yet it accelerated and enhanced TPA stimulation of ODC activity (Lichti et al., 1977a,b; Yuspa et al., 1978).This demonstrates that a rise in ODC activity is not enough to trigger the DNA synthesis response unalterably and suggests that FA exerts its antipromoter effect distal to ODC activation, assuming that this enzyme is indeed involved in the tumor promotion process (Lichti et al., 1977a,b; Yuspa et al., 1978).The in vitro FA-mediated prevention of stimulated DNA synthesis after TPA exposure can be reversed by the addition of putrescine to the culture

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medium, suggesting that putrescine and perhaps other polyamines are mediators in tumor promoter-stimulated proliferation and that FA exerts its effect by reducing intracellular polyamine levels (Lichti et al., 1977a,b; Yuspa et al., 1978). 2. Inhibitors of SAMD The changes in polyamine pattern following inhibition of SAMD are often quite different from those obtained after inhibition of ODC. While the latter mainly decreases concentrations of putrescine and spermidine, inhibitors of SAMD deplete cells of spermidine and spermine, usually associated with a paradoxical accumulation of putrescine. The best and the oldest known inhibitor of SAMD, i.e., methylglyoxalbis(guany1hydrazone) (MGBG), was first examined because its antiproliferative activities against human acute myelocytic leukemia and leukemia L1210 were reported by Mihich (1963a,b, 1964, 1965, 1975) and were found to be prevented in vivo by simultaneous treatment with spermidine. A similar antagonistic effect of spermidine against MGBG on L1210 cells was also found in vitro (Pathak and Dave, 1977). The growth of lymphocytic leukemia P388, B82T, and B8174, mast cell leukemia P815, reticular cell sarcoma P329, lymphomas 4, P288, P1798, and sarcoma 180 ascites in mice and of Dunning-Schmidt leukemia in rats was also inhibited by MGBG (Mihich, 1975). However, the drug was not active against some solid sarcomas and carcinomas or against mouse lymphocytic leukemia A&, L4946, and L5178YF(Mihich, 1965).Thus, the majority of the sensitive tumors were Ieukemias. Many aspects of MGBG inhibition of animal tissue SAMD have been described in detail b y Corti et al. (1974), Janne et al. (1978), and Gaugas (1980). The reader is also referred for information on MGBG and its biological effects to several excellent reviews (Mihich, 196313, 1965, 1975; Williams-Ashman et al., 1976). Here we will summarize only the specific actions of MGBG on polyamine biosynthesis in mammalian tumors. The inhibition of SAMD activity by MGBG was competitive with respect to adenosylmethionine. The SAMDs of many normal and malignant tissues were inhibited to about the same extent by MGBG; there was no difference in the inhibition of the enzyme purified from a subline of mouse L1210 leukemia whose growth was sensitive to the drug in vivo, as compared with that of the enzyme purified from another subline whose growth was resistant to MGBG (Corti et d . ,1973, 1974). The drug rapidly increases the putrescine content and de-

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creases the net accumulation of spermidine and spermine content in sensitive (but not in resistant) sublines of L1210 leukemia cells in V ~ V O (Mihich et al., 1974). Since MGBG inhibits equally well SAMD activity from sensitive and resistant L1210 cells, the fact that spermidine levels were not reduced in drug-treated resistant cells would seem to be related to a reduced uptake and retention of drug by these cells (Dave and Caballes, 1973). MGBG has been shown to interfere with the transport of spermidine and spermine (Dave and Caballes, 1973; Dave et al., 1974; Mihich et al., 1974; Seppanen et al., 1980a,b) and with biosynthesis of spermidine (Corti et al., 1974). In addition, polyamines in turn inhibit cellular uptake of the drug (Dave and Caballes, 1973; Mihich et al., 1974; Seppanen e t al., 1980a,b,c), which was remarkably effectively concentrated inside some tumor cells (cultured Ehrlich ascites carcinoma cells, and human lymphocytic leukemia cells both cultured and circulating) (Seppanen et al., 1980a,b,c). In human leukemic cells the uptake of MGBG was critically dependent on their growth rate (Seppanen et d., 1980~). The drug MGBG is a rather specific inhibitor of the SAMD step in polyamine biosynthesis, inasmuch as millimolar concentrations of it do not inhibit mammalian ODC or spermidine and spermine synthases (Corti et al., 1974). However, MGBG markedly stimulates ODC activity in spleens of leukemic mice (Heby et al., 1973). Paradoxically, the spleen SAMD activity was also markedly increased in the same animals, when assayed 24 hr after the injection of MGBG (Heby et al., 1973; Heby and Russell, 1974). In mouse leukemia L1210 cells treated in vivo with MGBG, intracellular pools of spermidine and spermine are considerably depleted (Heby and Russell, 1974; Mihich et al., 1974). In accord with this, the activity of SAMD was profoundly inhibited when crude cytosolic fractions from Ehrlich ascites cells were incubated in the presence of MGBG (Alhonen-Hongisto e t al., 1980a). Because of many obviously nonspecific effects of MGBG in whole animals, it has been used more successfully in neoplastic cell cultures. The drug effectively blocked the accumulation of spermidine and spermine but caused an increased accumulation of putrescine in L1210 cells (Heby et al., 1977; Newton and Abdel-Monem, 1977; Dave et al., 1978). In L1210 cells these changes were accompanied by a greater decrease in the DNA content (Newton and Abdel-Monem, 1977). Since a decrease in the spermidine level similar to that caused by MGBG was found after a-methylornithine treatment, and this drug failed to inhibit DNA synthesis (Newton and Abdel-Monem, 1977), it seems that the inhibition of DNA synthesis by MGBG is not a result of a decrease in the cellular level of spermidine.

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In cultured murine leukemic L1210 cells, MGBG produced selective and extensive ultrastructural damage to mitochondria (MiklesRobertson et al., 1977; Dave et al., 1978). Similar damage has been observed in ascites L1210 cells treated i n vivo with MGBG (Porter et al., 1979),and it is reversible after the cells are removed from the drug. In P288 mouse leukemia and in NALM-1 human chronic myelocytic leukemia cells, MGBG also produced ultrastructural damage nearly identical to that seen in L1210 cells (Mikles-Robertson e t al., 1979). In Bri8 human leukemic lymphocytes, MGBG completely arrests cell proliferation, and once again the arrest could be reversed by the addition of exogenous spermine (Gaugas, 1980).The cells inhibited, as after a-methylornithine, were not the Fo generation but their progeny (Gaugas, 1980). In HeLa cell cultures the decrease in spermidine and spennine levels caused by MGBG preceded a drop in incorporation of labeled thymidine, uridine, and leucine into DNA, RNA, and protein (Krokan and Eriksen, 1977). When putrescine, spennidine, sperinine, or cadaverine was added simultaneously with MGBG, the drug had no detectable effect on the synthesis of macromolecules (Krokan and Eriksen, 1977). The inhibited synthesis of DNA was not restored in nuclei isolated from cells treated with MGBG by addition of spermidine or spermine (Krokan and Eriksen, 1977). MGBG has been found to inhibit cytokinesis and to induce the formation of binucleate cells in a variety of mammalian cells in culture, including HeLa cells, transformed CHO and SV3T3 cells (Sunkara et al., 1978a,b, 1979a).The effects were reversed by spermidine (Sunkara et al., 1978a,b, 1979a). Studies with MGBG have shown that the molecule in the extract of Harding-Passey mouse melanoma that inhibits the proliferation of the same cell line grown in culture is spermidine (Dewey, 1978). In addition, MGBG has been found to be a powerful noncompetitive inhibitor of the enzymic oxidation of sperinidine to acrolein in the same line of cultured melanoma (Dewey, 1979). Treatment of neoplastic cell cultures with MGBG results in different distributions of the cells in the phases of the cell cycle, depending on the cell type. Polyamine-depleted rat brain tumor cells accumulate in the GI phase (Heby et al., 1977, 1978a), while Ehrlich ascites tumor cells accumulate in the S and Gz phases (Heby and Anderson, 1976; Anderson and Heby, 1977; Heby et al., 1978a,b). In contrast to normal rat fibroblasts, which are arrested in the GI phase by MGBG, SV40transformed fibroblasts (Rupniak and Paul, 1978, 1980) and transformed CHO cells, HeLa cells, and SV3T3 cells (Sunkaraet al., 197913)

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continue through the cell cycle, although at slightly slower rates, after inhibition of spermidine and spermine synthesis. These data substantiate the proposal of Rupniak and Paul (1978) that normal cell retains a polyamine-sensitive growth “regulation point” or “restriction point” in GI at which the “polyamine status” of the cell is or is not sufficiently favorable to enable it to enter into and complete a new cell cycle. In contrast, this polyamine-dependent regulatory mechanism is largely lost in tumorigenic cells. The compound 1,l’-[(methylethanediy1idene)dinitrilolbis(3-aminoguanidine) (MBAG), closely related to MGBG, retains the irreversible inhibitory activity of the parent compound on SAMD activity in Ehrlich ascites tumor cells (Alhonen-Hongisto et al., 1979b) and in mouse mammary EMTG cells (Prakash et al., 1980). MBAG, like MGBG, is an extremely potent inhibitor of DAO in Ehrlich ascites tumor cells (Alhonen-Hongisto et al., 197913). According to Corti and co-workers (1974), two other derivatives of MGBG, dimethylglyoxalbis(guany1hydrazone)and di-N” ’-methylglyoxalbis(guany1hydrazone)also strongly inhibited SAMD in vitro from L1210 cells. has been found to The adenosine analog 9-P-D-xylofuranosyladenine be a competitive inhibitor of SAM synthesis in L1210 cells (Glazer and Peale, 1979). 3. Combination of lnhibitors of ODC and SAMD Agents that have been shown to be potent inhibitors of ODC or SAMD have been combined to achieve simultaneous cellular deprivation of all three chief polyamines. In addition, some unwanted effects of the single drug can sometimes be avoided when the drugs are administered together. The addition of MBAG and a-DFMO to the drinking water of mice with mammary EMTG tumors resulted in an unexpected restoration of normal putrescine and spermidine concentrations in the tumor cells (Prakash et aZ., 1980). MBAG also antagonizes the effect of the ODC-inhibitor on tumor growth (Prakash et al., 1980).In combination with DAP, or diaminopropanol, but not with DFMO, MBAG exhibited profound inhibition of DNA synthesis in Ehrlich ascites cells (Alhonen-Hongisto et al., 1979b).

4. lnhibitors of Spermidine and Spermine Synthases The synthesis of specific inhibitors of spermidine and spermine synthases is greatly hampered by the fact that the catalytic mecha-

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nisms of these enzymes are not precisely known. However, some progress in this field has been made. In addition to finding that putrescine acts as a natural inhibitor of spermine synthase (Kallio et al., 1977), some unphysiological diamines have been shown to inhibit spermidine and spermine synthases in the presence of their natural substrates. Cadaverine appeared to compete with putrescine in the synthesis of spermidine, while a lesser effect on spermine synthase was observed in Ehrlich ascites cells (Kallio et al., 1977). 5. Miscellaneous Molecules

A minimal cellular level of glutathione has been found to be required for ODC activity and polyamine synthesis. When diazenedicarboxylic acid bis(N,N-dimethylamide), a relatively specific oxidant of CSH, is added to cultures of H35 rat hepatoma cells, it causes a fall in the cellular level of GSH and inhibits the stimulation of ODC activity b y serum (Beck, 1978). Exogenous CSH can reverse the diamide effect (Beck, 1978). The effect of leupeptin, a microbial protease inhibitor, on carcinogenesis was examined during the early stage of tumorigenesis induced by DMBA and croton oil (Coto e t al., 1980). Leupeptin inhibited tumor development and the increase in spennidine content in treated mice, probably because it protects a protein inhibitor of ODC from destruction by protease, which is increased by croton oil. Tumor necrosis factor (TNF), a substance known to have anticancer activity against murine meth A tumors in uiuo and human melanoma in cell culture, has been found to inhibit ODC activity when injected into mice or added to melanoma cells in culture (Helson et al., 1977). Because T N F did not change the ODC activity in the assay when added to the reaction mixture nor affect ODC activity in the spleens of TNF-treated mice (Helson et al., 1977), the measurement of ODC is a mirror of the changes in tumor proliferative activity and of the specificity of an antitumor activity. Tosylphenylalanine chloromethylketone (Tos-PheCHzCl) and tosyllysine chloromethylketone (Tos-LysCH2C1)stimulate in cultured rat hepatoma the loss of ODC that follows inhibition of protein synthesis (McIlhinney and Hogan, 1974). A single dose of cis-dichloro(dipyridine)platinum can inhibit the elevation in ODC activity that occurs during development of the L1210 leukemia in mice (Morns et al., 1976). Finally, there are reports that the concentrations of polyamines and the activities of their biosynthetic decarboxylases in mouse L1210 leu-

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kemia cells (Russell, 1972; Heby and Russell, 1973a,b) and in a methylcholanthrene-induced fibrosarcoma in mice (Sukumar and Nagarajan, 1978) are lowered by administration of antineoplastic drugs such as methotrexate, cytosine arabinoside, and 5-azacytidine. But no evidence was provided by these studies that such cancer chemotherapeutic agents directly inhibited any of the polyamine biosynthetic enzymes in the tumor, and the alterations in polyamine formation evoked by these drugs seem to be indirect. V. Concluding Remarks and Speculations

The reader who hoped to find some total differences between the polyamine metabolisms of normal and neoplastic tissues and thereby some promising perspectives for study of both human and experimental oncology sees that none have been found as yet. In spite of this, some comments can be made at this time. The investigation of the links between polyamines and tumors can be divided historically into two facets; in the first of these, quantitative differences between the polyamine levels of neoplasms and those of normals were looked for, and in the second, which began more recently, differences between neoplasms and normals in polyamine biosynthesis regulation were investigated. This dichotomy is quite recurrent in oncology, representing almost a general trend in this discipline. We think that research into the qualitative differences of several aspects of polyamine biosynthesis regulation is the most promising at present and in the near future, since the principal characteristic of the phenotype of neoplasms is a lack, or at least an alteration, of the regulation of some metabolic activities and pathways, including those for polyamines. Qualitative differents have been demonstrated in etiologically different types of tumors, both human and experimental. In several experimental tumors or cultured neoplastic cells, isozymes of ODC or SAM synthetases have been identified. A lack of ODC antizyme formation, loss of the ODC circadian rhythm, decreased responsiveness of ODC to putrescine inhibition, and an imbalance in ornithine metabolism have also been found. It is quite hard to believe, although it is b y no means theoretically impossible, that all these qualitative derangements may turn out in the near future to be a general rule for all the neoplasms. However, all these results are phenomenological descriptions of some specific neoplastic abnormalities, and we cannot be satisfied until we know their causes. Nonetheless, the picture obtained when we assemble these single results might well be helpful for indicating the causes of these abnor-

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malities. Therefore, we think that all these qualitative differences merit maximal attention from researchers and that they will have great importance in the near future, when other analogous findings have been added to our knowledge of polyamines and tumors. At present, in spite of the decades of research, we are well aware that most of the role of polyamines in eliciting the peculiar neoplastic behavior and in permitting invasive neoplastic growth remains almost totally obscure. It is tempting to speculate that polyamines are one of the tools, but by no means the only one, available to the neoplastic cells for their uncontrolled growth. It is consistent with this view that the increases in the activities of the two polyamine biosynthetic decarboxylases can very frequently, although not always, be correlated with the degree of malignancy of certain experimental or human tumors, but not at all with the neoplastic status per se. Furthermore, another quantitative aspect to be looked into further in human research is the changes in the amounts of polyamine derivatives, and therefore the changes in their ratios, in physiological fluids from oncopathic subjects, as compared to subjects with other diseases. In addition, it is once again quite hard to accept, although it is by no means theoretically impossible, that studies of polyamines alone will be a conclusive turning point in solving the oncological puzzle. In this regard, it seems to us that studies of the connections between polyamines and pericellular fibronectin would be very interesting and stimulating and might possibly explain some features of neoplastic behavior. Greater integration of the different fields of oncological research are not only desirable but mandatory. Finally, we realize that studies of polyamines and tumors have disclosed a number of problems much larger than the number solved. Therefore, if this review becomes obsolete in a short time, it will mean that new studies have appeared and have introduced new trends and new perspectives about the connections between polyamines and neoplasms. We hope that the result of our present toil will at least evoke new ideas on this topic. ACKNOWLEDGMENTS

First, we are very grateful to Professor S. Weinhouse (Philadelphia) for his understanding of our delays. Thereafter w e wish to thank those authors who kindly sent us manuscripts of unpublished, but accepted, papers: Dr. E. S. Canellakis (New Haven), Dr. K. Y. Chen (New Brunswick), Dr. S. S. Cohen (Long Island), Dr. H. Desser (Vienna), Dr. J. M. Gaugas (Northwood), Dr. 0. Heby (Lund), Dr. U. Lichti (Bethesda), Dr. P. S. Mamont (Strasburg), Dr. P. McCann (Cincinnati), Dr. D. Morris (Washington), Dr. K. Nishioka (Hous-

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ton), Dr. G. Quash (Lyons),Dr. A. M. Roch (Lyons), Dr. N. Seiler (Strasburg),and Dr. T. Slotkin (Durham). Although we have continued to reevaluate the topic while writing and have included new papers as they appeared, we know that we have not avoided all errors or lacunae. We apologize to those investigators whose works we have inadvertently not cited. We are deeply indebted to Professor E. Ciaranfi (Milano), who several years ago introduced us to this field and taught us to love polyamines. We thank also Professor A. Bemelli-Zazzera (Milano) for his interest and advice. We gratefully acknowledge the helpful criticism by Professor U. Bachrach (Jerusalem) of our outline for this work. One of us (G. S.) also thanks Professor J. Janne (Helsinki), in whose laboratory he had the opportunity several years ago to deepen his understanding of some modem aspects of polyamine biosynthesis regulation. To our young co-workers, Dr. M. Puerari and Dr. D. Modena, we express our gratitude for their patient help in organizing and revising the manuscript. Last, but not least, to Dr. B. Rubin (Milano) we extend our warmest thanks for her invaluable editorial assistance in revising the English of the manuscript.

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Verma, A. K., Shapas, B. G., Rice, H. M., and Boutwell, R. K. (1979). Cancer Res. 39, 419-425. Verma. A. K., Ashendel, C. L., and Boutwell, R. K. (1980a). Cancer Res. 40,308-315. Verma. A. K., Conrad, E. A., and Boutwell, R. K. (1980b). Carcinogenesis 1, 607-611. Verma, A. K., Slaga, T. J., Wertz, P. W., Mueller, G. C., and Boutwell, R. K. (1980~). Cancer Res. 40,2367-2371. Vuento, M., Vartio, T., Saraste, M., von BonsdorfF, C. H., and Vaheri, A. (1980).Eur. J . Biochem. 105,33-42. Waalkes, T. P., Gehrke, C. W., Bleyer, W. A., Zumwalt, R. W., Olweny, C. L. M., Kuo, K. C., Lakings, D. B., and Jacobs, S. A. (1975a). Cancer Chemother. Rep. 59, 721727. Waalkes, T. P., Gehrke, C. W., Tormey, D. C., Zumwalt, R. W., Hueser, J. N., Kuo, K. C., Lakings, D. B., Ahmann, D. L., and Moertel, C. G. (1975b). Cancer Chemother. Rep. 59,1103-1116. Waldenstrom, J. G. (1976). Eur. J. Cancer 12,413-418. Wall, R. A. (1971). J. Chromatogr. 60, 195-202. Walle, T. (1973).In “Polyamines in Normal and Neoplastic Growth” (D. H. Russell, ed.), pp. 355-365. Raven, New York. Weber, G. (1977). N. Engl. J. Med. 296,486-493, 541-551. Weekes, R. G., Verma, A. K., and Boutwell, R. K. (1980).Cancer Res. 40,4013-4018. Weeks, C. E., and Abdel-Monem, M. M. (1977).J. Pharm. Sci. 66,1586-1589. Weeks, C. E., and Slaga, T. J. (1979). Biochem. Biophys. Res. Commun. 91,1488-1496. Weeks, C. E., Bracken, W. M., and Slaga, T. J. (1979).Proc. Am. Assoc. Cancer Res. 20, 155 (abstr.). Wikstrand, C. J., and Bigner, D. D. (1980).Am. /. Pnthol. 98, 515-567. Williams-Ashman, H. G., Coppoc, G. L., and Weber, G. (1972). Cancer Res. 32, 19241932. Williams-Ashman, H. G., Corti, A., and Tadolini, B. (1976). Ital J. Biochem. 25, 5-32. Wolf, P. L., ed. (1979a). “Tumor Associated Markers.” Masson, New York. Wolf, P. L. (1979b). I n “Tumor Associated Markers” (P. L. Wolf, ed.), pp. 1-19. Masson, New York. Wolfe, H. J. (1978).N. Engl. J. Med. 259, 146-147. Woo, K. B., Waalkes, T. P., Ahmann, D. L., Tormey, D. C., Gehrke, C. W., and Oliverio, V. T. (1978). Cancer 41, 1685-1703. Woo, K. B., Perini, F., Sadow, J., Sullivan, C., and Funkhouser, W. (1979). Cancer Res. 39,2429-2435. Woo, K. B., Waalkes, T. P., Abeloff, M. D., Ettinger, D. S., and Gehrke, C. W. (1980). Proc. Am. Assoc. Cancer Res. 21, 170 (abstr.). Yam, L. T. (1974).Am. J . Med. 56,604-616. Yap, B. S., Yap, H. Y., Nishioka, K., and Bodey, G. P. (1979).Proc. Am. Assoc. Cancer Res. 20, 187 (abstr.). Yuspa, S. H., Lichti, U., Hennings, H., Ben, T., Patterson, E., and Slaga, T. J. (1978).In “Carcinogenesis-Mechanisms of Tumor Promotion and Cocarcinogenesis” (T. J. Slaga, A. Sivak, and R. K. Boutwell, eds.), Vol. 2, pp. 245-255. Raven, New York. Yuspa, S. H., Lichti, U., and Ben, T. (1980a). Proc. Am. Assoc. Cancer Res. 21, 114 (abstr.). Yuspa, S. H., Lichti, U., and Ben, T. (1980b). Proc. Natl. Acad. Sci. U.S.A. 77, 53125316.

CHROMOSOME ABNORMALITIES IN MALIGNANT HEMAT0 LOGIC DISEASES Janet D. Rowley and Joseph R. Testa' Department of Medicine and The Franklin McLean Memorial Research Institute, The University of Chicago. Chicago. Illinois

I. Introduction .................................... 11. Methods . . . . . . . . . .. . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . 111. Chronic Myelogenous Leukemia (CML) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chronic Phase of CML . . . . . . . . . . . . . . . . . . . . B. Acute Phase of CML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............... C. The Ph' Chromosome as a Biological Marker IV. Acute Nonlymphocytic Leukemia (ANLL) A. ANLL d e Novo . . . . . . .. . . . . .. . . . . . . . . . . . . . . . ... . . . .. . . . . . . . . . . . . . . . .

........... V. Acute Lymphocytic Leukemia (A A. The 8;14 Translocation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... B. A 14q+ Chromosome Not Involving No. 8 C. The 4;11 Translocation . . . . . . . . . . . . . . . . . . . . . . . . . . D. Near-Haploid ALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. E. Hyperdiploidy with 50 to 60 Chromosomes F. The Phi Chromosome in ALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Polycythemia Vera .......................... ............

B. Modal N u m b e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Nonrandom Abnormalities ..........................

A. Production of Consistent Translocations .............. B. Function of Nonrandom Changes . . . . . . . . . . . . C. Conclusions ............................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

103 105 107 107 111 114 116 116 125 126 127 129 129 130 130 131 132 132 134 135 138 139 140 141 142 143

I. Introduction

The study of the chromosome pattern in the affected cells of a number of human tumors has been one of the most exciting areas in cancer research over the last 20 years. Major advances in our understanding of the specificity of some of the abnormalities have occurred in the last 10 years with the application of new chromosome banding techniques. I Present address: NCI-Baltimore Street, Baltimore, Maryland 21201.

Cancer Research Program, 655 West Baltimore 103

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These techniques allow the identification of each human chromosome and of parts of chromosomes as well. Thus the hypothesis put forward by Boveri at the turn of the century, namely that an abnormal chromosome pattern was intimately associated with the malignant phenotype of the tumor cell, can now be tested with substantial hope of obtaining a valid answer (Boveri, 1914). The study of the chromosome pattern in human leukemias can be divided into two periods, each one covering 10 years. The first lasted from 1960 to 1970, and the second from 1970 to 1980. During the first period the chromosome abnormalities seen in leukemic cells were identified without banding, and therefore they include the changes in morphology that were detectable in unbanded preparations as well as abnormal modal chromosome numbers. The most significant observation was the identification of the Philadelphia (Ph') chromosome in leukemic cells from patients with chronic myelogenous leukemia. In 1960, when this abnormality was discovered by Nowell and Hungerford, it appeared to represent a deletion of about half of the long arm of one G group chromosome; whether pair No. 21 or No. 22 was affected was not determined. This observation led to a search for similar abnormalities closely associated with other types of malignant hematologic diseases. The results were quite disappointing in that although the abnormalities seemed to be consistent in any particular patient, the patterns varied greatly from one patient to another. Moreover, about half of the patients with acute leukemia of both the myeloid and lymphoid types appeared to have a normal karyotype in their leukemic cells (Rowley, 1980a; Sandberg, 1980a). Thus the accepted notion was that the Ph' was a unique example of a consistent karyotypic abnormality, and the general rule was one of marked variability in karyotype. This, in turn, led most investigators to assume that chromosome changes were a secondary phenomenon not fundamentally involved with the process of malignant transformation. The evidence obtained during the second period showed that these assumptions were probably not correct. With the use of banding techniques, other specific abnormalities were found to be associated with certain leukemias and lymphomas (Rowley, 1980a). Moreover, banding techniques revealed that the gains and losses of chromosomes were distinctly nonrandom. The specific abnormalities that have been identified in human leukemia and in polycythemia Vera during the 1970s are described in detail in this review. The data obtained prior to 1970 have been reviewed in a number of reports, and they will not be described here (Sandberg, 1980a). It should be emphasized that the data presented here have been

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gathered primarily during the period 1974- 1980. The observations and conclusions are based on our increased ability to identify abnormal chromosomes with banding. However, most of the studies during this period used chromosomes that were relatively contracted, and the banding pattern often was fuzzy and poorly defined. For this reason subtle abnormalities, such as a deletion or a duplication of one-third of a chromosome band, involving about 3 x lo6 nucleotide pairs, would be undetectable. We are now embarking on a third period of analysis that will be characterized by substantial improvements in the quality of the chromosome preparations that are available for analysis. These technical improvements have already been used to detect very subtle deletions in cells from some patients with retinoblastoma (Yunis and Ramsey, 1978) and with aniridia-Wilms’ tumor syndrome (Francke et al., 1979). The deletion in the former involves a band in the long arm of No. 13 (13q14), and that in the latter involves a band in the short arm of No. 11(llp13). More recently, these techniques have been adapted for use with bone marrow cells. Yunis et al. (1981)have reported that with the use of elongated (prophase) chromosomes from patients with acute nonlymphocytic leukemia (ANLL) every one of 24 patients had an abnormal karyotype. In six patients, abnormalities were found only with the use of longer chromosomes, and they were not detected with preparations that are used in most laboratories. Thus the future emphasis will be to identify the abnormalities that we have all overlooked in the past. I n this article, however, when the acute leukemias are described, current data will be used that indicate that about 50% appear to have a normal karyotype. Clearly these observations and the correlations that are made with various karyotypic patterns will have to be revised if the observations of Yunis et al. (1981)are confirmed by others. This is a major challenge for the future. II. Methods

An analysis of chromosome patterns, to be relevant to a malignant disease, must be based on a study of the karyotype of the tumor cells themselves. In leukemia, the specimen is usually a bone marrow aspirate processed immediately or cultured for 24-48 hr (Testa and Rowley, 1981). I n patients with a white blood cell count higher than 15,500, with about 10% immature myeloid cells, a sample of peripheral blood can be cultured for 24 or 48 hr without adding phytohemagglutinin (PHA). The karyotype of the dividing cells will be similar to that obtained from the bone marrow.

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When a consistent abnormal karyotype is found in the bone marrow, it is important to analyze cells from normal tissues, such as skin fibroblasts or peripheral blood lymphocytes stimulated to divide with PHA. In most instances, cells from these unaffected tissues will have a normal karyotype. The chromosome abnormalities observed in the malignant cells thus represent somatic mutations in an otherwise normal individual. Chromosomes obtained from bone marrow cells, particularly from patients with leukemia, frequently are very fuzzy, and the bands may be indistinct. These problems can be overcome with new techniques, particularly the use of amethopterin to synchronize dividing cells. These new procedures also provide cells with elongated chromosomes that have a larger number of bands (Yunis et d., 1981).In patients with complex chromosome changes, multiple staining techniques are required for correct identification of the chromosomes involved in the rearrangements and for more accurate definition of the chromosome bands affected by the breaks. In my laboratory, we are able to obtain a precise identification of the chromosome abnormalities in more than 90%of leukemic patients. Unused material can be stored in fixative in the freezer; usable quinacrine-fluorescent bands can be obtained from such material even after 12 years. The observation of at least two “pseudodiploid” or hyperdiploid cells or three hypodiploid cells, each showing the same abnormality, is considered evidence for the presence of an abnormal clone; patients with such clones are classified as abnormal. Patients whose cells show no alterations, or in whom the alterations involve different chromosomes in different cells, are considered to b e normal. Isolated changes may be due to technical artifacts or to random mitotic errors. In the following discussion, the chromosomes are identified according to the Paris Nomenclature (Paris Conference, 1972) and the International System for Human Cytogenetic Nomenclature (1978),and the karyotypes are expressed as recommended under these systems. The total chromosome number is indicated first, followed b y the sex chromosomes, and then b y the gains, losses, or rearrangements of the autosomes. A plus sign (+) or a minus sign (-) before a number indicates a gain or loss, respectively, of a whole chromosome; a plus or a minus after a number indicates a gain or loss of part of a chromosome. The following abbreviations are used: p and q, the short and long arms of the chromosome, respectively; i, isochromosome; r, ring chromosome; mar, marker; del, deletion; ins, insertion; inv, inversion. Translocations are identified b y “t” followed by the chromosomes involved in the first set of brackets; the chromosome bands in which the breaks

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occurred are indicated in the second brackets. Uncertainty about the chromosome or band involved is signified b y a question mark (?).

Ill. Chronic Myelogenous Leukemia (CML)

A. CHRONIC PHASE

OF

CML

Chromosome banding techniques were first used in the cytogenetic study of leukemia for identification of the Ph' chromosome. Caspersson et al. (1970) and O'Riordan et al. (1971) reported independently that the Ph' chromosome was a No. 22s-. Since quinacrine fluorescence revealed that the chromosome present in triplicate in Down's syndrome was No. 21, the abnormalities in Down's syndrome and CML were shown to affect different pairs of chromosomes. The question of the origin of the Ph' (229-) chromosome was answered in 1973, when Rowley (1973a) reported that the Ph' chromosome results from a translocation, rather than a deletion as many investigators had previously assumed. The first report presented data on nine Ph'+ patients, in all of whom there was additional dully fluorescing chromosomal material at the end of the long arm of one No. 9 (9q+). This additional material was approximately equal in length to that missing from the Ph' chromosome, and it had staining characteristics similar to those of the distal portion of the long arm of No. 22. It was proposed, therefore, that the abnormality of CML was an apparently balanced reciprocal translocation (9;22)(q34;qll) (Fig. 1).Subsequent measurements of the DNA content of the affected pairs (9 and 22) have shown that the amount of DNA added to No. 9 is equal to that missing from the Ph' (Mayall et al., 1977); thus, there is no detectable loss of DNA in this chromosome rearrangement. Other studies with fluorescent markers or chromosome polymorphisms have shown that, in a particular patient, the same No. 9 or No. 22 is involved in each cell (Gahrton et al., 1974). These observations confirm earlier work, based on enzyme markers, indicating that CML cells originated from a single cell (Fialkow, 1974). The original report on the translocation, and a number of reports confirming it, noted that the translocation occurred only between No. 9 and No. 22 (Rowley, 1973a; Van Den Berghe, 1973). The karyotypes of 1129 Ph'+ patients with CML have been examined with banding techniques and have been reported by a number of investigators, and the 9;22 translocation has been identified in 1036 (92%) of these patients (for review, see Rowley, 1980b; references not included there

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R G . 1. Karyotype of a metaphase cell from a bone marrow aspirate obtained from an untreated male with chronic myelogenous leukemia. The chromosomes were stained with quinacrine mustard and were photographed with ultraviolet fluorescence. The Ph' (22q-) chromosome is on the right in pair 22; the chromosome 9 on the right (9q+) has an additional pale band that is not present on the normal chromosome 9.

are: Stoll and Oberling, 1979; Bernstein et al., 1980b; Hagemeijer et al., 1980; Olah et al., 1980; Carbonell et al., 1980; Kohno and Sandberg, 1980; Alimena et al., 1981).

1. Variant Ph' Translocations It is now recognized that, in addition to the typical t(9;22), variant translocations may occur. These appear to be of two kinds. One is a simple translocation involving No. 22 and some chromosome other than No. 9, which has been seen in 42 patients. The other is a complex translocation involving three or more different chromosomes; except in two cases, two of the chromosomes involved were No. 9 and No. 22. This type of translocation has been observed in 46 patients. Five patients have been reported who are said not to have had a translocation. The data on these translocations were reviewed by Mitelman and Levan (1978) and by Sandberg (1980b) and will only be summarized here.

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The chromosomes most commonly involved in simple variant translocations are 12p, 17q, and 19q. Forty-six other complex translocations have been reported; as mentioned earlier, these always involve Nos. 9 and 22 except in the patients described by Ishihara et al. (1974) and Geraedts et al. (1977). Many chromosomes are involved in these complex rearrangements; however, l q , 3p, and 49 appear to be involved more often than other chromosomes. At least three of these patients appeared to lack a Ph' chromosome. The patient of Engel et al. (1974) had a complex rearrangement that involved translocation of all of No. 22 to the recipient chromosome, No. 17. In the other two patients (Lawler et al., 1976; Tanzer et al., 1977), a complex translocation of material to the end of the Ph' chromosome occurred. Five Ph'+ patients have been reported whose chromosomes showed no evidence of a translocation (Mitelman, 1974; Sonta et al., 1976; Olah et al., 1980). The experience of Hossfeld and Kohler (1979) is pertinent here, since the initial impression was that they, too, had a Ph'+ patient who lacked a translocation. Examination of the chromosomes of this patient with R-banding revealed a t(X;22) (Hossfeld and Kohler, 1979). The consistency of the break points in chromosomes 9 and 22 is a matter of considerable biological importance. It cannot be established at present because of the relatively contracted chromosomes that are studied in many laboratories. Verma and Dosik (1980) suggested that the break point in No. 22, determined with the R-banding technique, appeared to be variable. It would seem very premature to accept this conclusion without further confirmation. A more precise answer will be obtained from the study of elongated chromosomes and from an analysis of the segregation of genetic loci in hybrid somatic cells obtained from fusion of Ph'+ CML cells. Data from one such study reported by Geurts van Kessel et al. (1981)showed the consistent segregation with the 9q+ chromosome of 3 genetic loci known to be located on No. 22. There was no evidence for a variable break point in the leukemic cells of the six patients whose cells were used in these experiments. It is difficult to ascertain the precise incidence of variant translocations in patients with CML because some reports do not include information about the total number of patients studied; therefore the percentage of variants appears to be higher than it actually may be. Among all of the 1129 Ph'+ patients whose cells have been examined with banding, however, 88 (8%) showed an unusual chromosomal rearrangement, and the translocation may be absent in 5 of them. Chromosome No. 9, with a break in band 934, was involved in 44 of these 93; therefore only 49 (4%) of 1129 Ph'+ patients had a normal

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JANET D . ROWLEY AND JOSEPH R. TESTA

No. 9. The great specificity of the translocation involving Nos. 9 and 22 remains an enigma. An immediate question that arises is whether the presence of a variant translocation has any influence on the clinical course of the disease, According to the summary of Sandberg (1980b), the survival curves for patients with variant translocations appeared to b e the same as those for patients with the standard t(9;22). These studies are based on survival data for unusual translocations covering a 4-year period. These data need continual review so that it can be determined whether this conclusion is valid when more patients have been studied for a longer period of time. A few patients have a mixture of Ph'+ and Ph'- cells when they are first examined. Brandt et al. (1976) reported on one such patient who had only 16% Ph'+ cells when a karyotype was studied 16 years after the initial diagnosis of CML. Sakurai et al. (1976) noted that those of their patients who had some normal cells had a better prognosis than patients who were 100% Ph'+. Attempts have been made to use aggressive therapy with splenectomy and chemotherapy to eradicate the Ph'+ cell line. The results reported by Cunningham et al. (1979) show that 33% (12/37) of patients who were previously 85- 100% Ph' + converted to 0-30% Ph'+ cells after such treatment. Such reduction lasted less than 4 months in 8 patients; 2 patients with slowly progressive disease had a sustained reduction for more than 3 years. To date, however, they have not been able to prevent blast crisis or to prolong the overall survival of the group with this approach. Patients who responded, however, have a longer median survival. 2. Other Chromosome Abnormalities Chromosome abnormalities in addition to the Ph' translocation are found in up to 30% of the patients with CML (Mitelman and Levan, 1978; First International Workshop on Chromosomes in Leukaemia, 1978). In a report of Bernstein et al. (1980b), only 5 of 55 patients in the chronic phase (9%)had other abnormalities. This is similar to the observations of Hagemeijer et al. (1980), who noted abnormalities in 5 of 53 patients in the chronic phase. These include other translocations in addition to the 9;22, and an extra No. 8, i( 17q), a second Ph', and loss of the Y chromosome. Many of these changes are identical to those observed in the acute phase of CML. The occurrence of second translocations adds to the difficulty of distinguishing the typical 9;22 from variant translocations. A second Ph' chromosome results from a duplication of the original 22q- chromosome and is not accompanied by a second translocation. The presence of an additional abnormality de-

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tected at the time of diagnosis of the chronic phase of the disease does not seem to carry a substantially poorer prognosis than the 46, Ph'+ pattern (Whang-Peng et al., 1968). The frequency of -Y males varies considerably. In the series reported by Lawler et al. (1974), 8 of 74 males were lacking a Y chromosome; none of the 55 male patients that I have studied was -Y, and there was only one -Y patient among 107 males in the data reported at the First International Workshop on Chromosomes in Leukaemia (1978); only one - Y patient was noted by Bernstein et al. (1980b)in 34 males, and by Kohno and Sandberg (1980) in 38 male patients. There has been continuing controversy regarding the survival of patients whose Ph'+ cells are -Y. In the series of Sakurai and Sandberg (197613) - Y males had a longer survival, whereas, Lawler et al. (1974) observed no difference. In both series, the majority of Ph'+, - Y patients were younger than the Ph'+, XY group, an observation that led Lawler et al. (1974) to suggest that the lack of the Y chromosome in these patients represents an expression of premature aging of the bone marrow cells. The analogous abnormality in females, namely, loss of one X chromosome, has not been reported. B. ACUTE PHASEOF CML When patients with CML enter the terminal acute phase, about 20% appear to retain the 46, Ph'+ cell line unchanged, whereas other chromosomal abnormalities are superimposed on the Ph'+ cell line in 80% of patients (Rowley, 1980b). Prior to the use of chromosome banding, a change was detected in only about 70% of patients (review in Rowley, 1977a). A change in the karyotype is generally considered to be a grave prognostic sign. The median survival from the time of change until death was found by Whang-Peng et al. (1968)to be 2-5 months; the same group reported similar results in a new series of patients (Canellos et al., 1976). Those patients whose cells had a hypodiploid modal number during the acute phase appeared to respond better to treatment with prednisone and vincristine than patients with other changes (Canellos et d., 1976). As can be seen in Table I, very few patients (18 of 303, or 6%) were found to have a hypodiploid clone when their cells were examined during banding. Ninety-two of the 303 patients (30%)listed in Table I had a change in their karyotype at the time of blast crisis that was not reflected in a change in the modal chromosome number. There is, thus, no substitute for accurate karyotyping, with banding, if one wants to correlate the karyotype with the patient's clinical status. For example, we have

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JANET D. ROWLEY AND JOSEPH R. TESTA

TABLE I MODALCHROMOSOME NUMBERIN 303 PATIENTS IN THE ACUTE PHASE OF CHRONIC MYELOGENOUS LEUKEMIA Modal chromosome number

44

45

46"

47

48

49

50

51

>51

3

15

92

97

41

20

12

9

14

Number ofpatients

" All have new abnormalities in addition to the Philadelphia (Phl) chromosome.

one patient who had a -7, +8 karyotype at the time of blast crisis that would not have been detected without banding (J. D. Rowley, unpublished). Bone marrow chromosomes from 392 patients with Ph' + CML, who were in the acute phase, have been analyzed with banding techniques (for summary, see Rowley, 1980b; Stoll and Oberling, 1979; Bernstein et al., 1980b, Olah et al., 1980; Hagemeijer et al., 1980; Kohno and Sandberg, 1980; Alimena et aZ., 1981). Eighty-nine (22%) showed no change in their karyotype, whereas 303 patients had additional chromosome abnormalities. The gains or structural rearrangements of particular chromosomes observed in 303 patients who had relatively complete analyses are summarized in Table 11. The most common changes frequently occur in combination to produce modal numbers 47 to 50. 1. Common Chromosome Changes Different abnormal chromosomes occur singly or in combinations in a distinctly nonrandom pattern during the acute phase of CML. The significance of these patterns will be understood only when we have TABLE I1 MOST COMMON CHROMOSOME CHANCES1N 303 Phl+ PATIENTSIN ACUTEPHASEOF CHRONIC MYELOGENOUS LEUKEMIA Chromosome Number of patients with

No. 8

No. 17

No. 19

Phl

Gain Rearrangement

119 9

31 89"

45 2

113

i(17q) in 79. All were i(Ph').

ab

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

113

more information about the genes carried on these chromosomes. When patients had only a single new chromosome change, this most commonly involved a second Ph', an i(17q), or a +8, in descending order of frequency. An extra No. 8 and i(17q) occurred together as the only changes in 28 patients. In 9 patients, it was possible to tell which chromosome change occurred first; i(17s) was the initial change in 7 of them. An i(17q) was seen with another C group chromosome only in the presence of No. 8. Two patients have been reported who are mosaic for a +8 line and an i(17q) cell line (Olah et al., 1980; Hagemeijer et al., 1980).A + 8 and + 1 7 were never seen as the only changes. On the other hand, if the patient's cells also had a second Ph', then a +8, i(17q) was seen in 7 cases and a +8, +17, +Phl was seen in 7 cases. Only one patient had a + 8, i( 179) and + 19, and no patient had an i( 17q) with + 19. Two patients had an i( 17q) and a + Ph', and five patients had a + 17 and + Ph'; except for one of the latter, these patients were all included in the series of Stoll and Oberling (1979). As discussed earlier, the frequency of chromosome loss is very low. Of 297 patients, 8 were lacking one No. 7, 7 were lacking one No. 17, and 6 each were lacking one No. 8 or a Y chromosome. Structural rearrangements, other than the Ph' translocation and the i(17q), most often involved No. 1, usually the long arm (12 patients), No. 11 (11 patients), and No. 8 (9 patients). Abnormalities of the sex chromosomes in the acute phase occur relatively rarely; a gain or a loss of the Y chromosome was noted in 5 and 6 patients, respectively. Two patients had lost an X chromosome, whereas a gain was seen in 5 patients, 3 of whom were female. The nonrandom patterns of change in the acute phase have also been discussed by Mitelman and Levan (1978), who noted that 80% of patients show one or more of three specific aberrations: a +8, a +Phl, or an i(17q). This is in good agreement with the data summarized here. Many of the other changes involve structural rearrangements, which frequently are balanced translocations, that occur in association with blast crisis. 2. Clinical Correlations Since most of the patients who are studied in the acute phase of CML have been treated, usually with busulphan, it is impossible to determine whether this therapy affects the pattern of abnormalities described earlier. Alimena et al. (1979) presented evidence that aggressive chemotherapy in the chronic phase may alter the pattern of chromosome abnormalities seen in the acute phase. Of 34 patients with clonal abnormalities in the acute phase, 23 had been treated with

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JANET D . ROWLEY AND JOSEPH R. TESTA

busulphan only, whereas 11 had received combinations of drugs including cytosine arabinoside, thioguanine, and daunorubicin. Both groups of patients had a similar high frequency (85%)of one or more of the common changes such as +8, i(17q), and +Ph'. Additional structural rearrangements were seen in only 3 of the 23 patients treated with busulphan, but were present in 5 of 11 of those treated with intensive chemotherapy. Furthermore, aberrations of chromosome No. 1were seen in all 5 of the latter patients, whereas they were never seen in those treated with busulphan. The type of prior therapy of all patients who are studied in the acute phase should be recorded, so that the observations reported by Alimena et al. (1979) can be extended and confirmed. The data regarding the prognostic significance of particular karyotypic changes in the acute phase are conflicting. Thus Sonta and Sandberg (1978) stated that the survival of patients who developed additional chromosomal abnormalities was similar to that of patients without further changes. This view differs from that of Prigogina et al. (1978), who reported a higher remission rate and a longer survival in patients who remained only Ph'+ compared with those whose cells had additional changes. Alimena e t al. (1981) have reviewed the data from 69 patients in blast crisis; no difference in survival was noted in patients whose karyotype did not change. They did observe that survival was longer in patients with a lymphoid rather than a myeloid blast crisis. AS C. THEPh' CHROMOSOME

A

BIOLOGICALMARKER

1. Ph'-Positive Acute Leukemia Our interpretation of the biological significance of the Philadelphia chromosome has been modified over the course of the last 9 years, as our clinical experience with this marker has widened. Thus, WhangPeng et al. (1970) proposed that cases of acute myeloblastic leukemia (AML) in which the Ph' chromosome was present should be reclassified as cases of chronic myeloid leukemia presenting in blast transformation. This notion was broadened to include the cases that appeared to be acute lymphoblastic leukemia (ALL) at diagnosis and was generally accepted until about 1977. More recently, however, the tendency has been to refer to patients who have no prior history suggestive of CML as having Ph'-positive acute leukemia (reviewed in Rowley, 1980b).It is becoming increasingly evident that the observed interrelations of Ph' + leukemia are complex indeed, and that the dis-

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

115

tinctions between some categories, which are difficult to make, will be determined by the arbitrary judgment of the investigator. Moreover, although the Ph' chromosome is used as the marker that defines these leukemias, the cytogenetic studies on patients have often been woefully inadequate. When one reviews the descriptions of the patients who are first seen with what appears to be acute leukemia and a Ph' chromosome, it is quite apparent that a clear demarcation between morphological types is difficult. Thus, some patients have a high percentage of lymphoblasts, others have a high percentage of myeloblasts, and still others have a mixture of myeloblasts and lymphoblasts. In some instances, cells from the latter patients have been analyzed for cell surface markers. In case 7 of Chessells et al. (1979), for example, 30%of cells, including the smaller blasts, reacted positively with common ALL antiserum; 60%, including larger blasts, did not react with this antiserum, but were positive with an anti-myeloid antiserum. The patient did not respond to vincristine and prednisone, but achieved a remission when daunorubicin, cytosine arabinoside, thioguanine, and L-asparaginase were given in addition. 2. Phl-Positive ALL Evolving to C M L There are 12 patients reported, 2 children and 10 adults, who were first seen with what appeared to be typical ALL that subsequently evolved to CML; these patients have been discussed in an Annotation in The British Journal of Haematology (Catovsky, 1979), and Rowley (1980b). There were 3 female and 9 male patients. The diagnosis of ALL was made on the basis of morphology and cytochemistry, although a few patients had elevated terminal deoxynucleotidyltransferase activity (TdT) or were common-ALL antiserum-positive. When cytogenetic studies in the ALL phase were done, patients had between 50 and 100% Phi-positive cells; where banding was done, the t(9;22) was found. All these patients entered complete remission of ALL and then clearly had CML at intervals ranging from the next clinic visit (1month) to 2 or 3 years (median, 5 months). A cytogenetic study with banding was done on 2 patients during this period (Forman et al., 1977; Gibbs et al., 1977), and bone marrow cells showed only a normal karyotype. When the chronic phase of CML was diagnosed, patients were said to have 75-100% Ph'+ cells; the chronic phase lasted from 3 weeks to 3 or 4 years. Nine patients developed a terminal blast crisis; myeloid morphology or lymphoid morphology was noted with about equal frequency. The evidence obtained from all these studies shows that in some

116

JANET D. ROWLEY AND JOSEPH R. TESTA

patients the Ph' translocation is associated with an alteration in function of the myeloid cells that is expressed as CML. Evidence indicates that some B lymphocytes in one patient were Ph'+ since IgG secreting lymphoblastoid lines were shown to be Ph'+ (Martin et al., 1980). In other patients, the Ph' + leukemic cells appear to have surface markers indistinguishable from those detected on pluripotent stem cells. That is, these cells are positive for TdT, Ia, and the common ALL antigen (Janossy et al., 1977). More recently, cytoplasmic immunoglobulin has been detected in leukemic cells from some patients indicating that the blast cells are pre-B (Vogler et al., 1979). Clinically, these patients either have a lymphoid blast crisis of CML, or have what is currently called Ph'+ ALL. The factors that influence the expression of these various clinical forms, which can sometimes be seen in the same patient at different periods in the disease, are currently unknown.

IV. Acute Nonlymphocytic Leukemia (ANLL)

A. ANLL

DE NOVO

Abnormal karyotypes have been reported in approximately 50% of all patients with ANLL de novo whose bone marrow cells were examined with banding techniques (First International Workshop on Chromosomes in Leukaemia, 1978). However, the incidence of cytogenetic abnormalities will be significantly greater when techniques for obtaining prophase and prometaphase chromosomes are generally applied. The report of Yunis et al. (1981) shows that elongated chromosomes can reveal small deletions and other structural rearrangements that are too subtle to be detected with the procedures used in most laboratories. Generally, in ANLL karyotypic abnormalities are present prior to therapy and disappear when the patient enters remission. The same aberrations reappear in relapse, sometimes showing evidence of additional karyotypic change superimposed on the original abnormal clone (Testa and Rowley, 1980; Sandberg, 1980a). There have now been a considerable number of reports on cytogenetic analyses, with banding techniques, of relatively large series of unselected patients with ANLL as well as of single cases or selected patients. The distribution of modal chromosome numbers in a total of 308 chromosomally abnormal ANLL patients studied with banding is summarized in Table 111. Of the 248 adult patients, 207 (83%)had modal numbers in the diploid range of 45-47. Similarly, 54 of 60 (90%)children aged 15 years or younger had modal numbers of 45-47.

TABLE 111 DISTRIBUTIONOF MODAL CHROMOSOME NUMBERSIN 308 ACUTE NONLYMPHOCYTIC LEUKEMIA PATIENTS WITH ABNORMALKARYOTYPE ESTABLISHED WITH BANDING TECHNIQUES~

AN

Modal chromosome number of the abnormal cells Patients Adults (No.)

42

43

44

45

46'

47

48

49

50

2

6

9

57

85

65

12

4

0

0

13

(34%) 25

16

3

0

1

L

(30%) Children (No.) 0

0

L

(22%)

(41%)

"

51

52

53

55-65

2

2

0

4

0

1

0

(36%) "

1

(37%)

References: Kaneko et d. (1978); Teerenhovi et QZ. (1978);Van Den Berghe et QZ. (1978); Benedict et QZ. (1979); Berger et d. (1979a, 1980b);Hagemeijer et aZ. (1979);Kondo and Sasaki (1979);Morse et QZ. (1979);Padre-Mendoza et oZ. (1979);Petit and Van Den Berghe (1979); Prigogina et QZ. (1979); Abe et QZ. (1980); Bernstein et d. (1980a); Golomb et QZ. (1980); Hossfeld et d. (1980); Hustinx et QZ. (1980);Hsgemeijer et ~ l (1981); . and including 191 abnormal patients reported by various investigators and summarized previously in Testa and Rowley (1980). All patients were pseudodiploid.

118

JANET D . ROWLEY AND JOSEPH R. TESTA

Fifteen other patients, who had what appeared to be ANLL at the time of diagnosis, were subsequently found to have a Ph' chromosome, identified as a 22q- with the use of banding (reviewed in Rowley, 1980b).The Ph' translocation was identified in 13 cases; it involved No. 9 in 9 of these cases. In the remaining 4 patients, the translocation involved Nos. 3, 11, 17, or 19. These 15 patients, reported on between 1975 and 1979, have been excluded from the present summary, since the information provided about some of the cases is too sparse to allow a definitive diagnosis. 1. Banding Patterns in Adults Although the karyotypes of patients with ANLL may be variable, certain nonrandom patterns are evident. Figures 2 and 3 summarize the chromosome changes in 248 adult patients from Table 111, plus changes in one other adult who was not listed in Table I11 because the modal number was not specified in the original report. The nonrandom distribution of chromosome gains and losses (Fig. 2) is particularly evident in the cases with a single abnormality. Thus, a gain of No. 8, the most frequent abnormality seen in ANLL, was found in 61 patients (24.5% of the 249 abnormal cases), in 29 of whom a + 8 was the sole abnormality present. Similarly, loss of No. 7, another frequent numerical change, was observed in 29 patients, 13 of whom showed only this change in the karyotype. In contrast, gains or losses of the other autosomes seldom occurred as the sole abnormality. Thus, these abnormalities were likely to represent secondary events occurring in clonal evolution, rather than primary chromosome changes. Losses of Y, the second most frequent numerical change in this summary, and X often occurred in association with an 8;21 translocation (see below). Of the 8 patients with a loss ofX, 5 had a +(8;21); in each of these there were no other abnormalities. Of the 30 males with a missing Y, 12 had a t(8;21); in 9 of the 12 there were no further changes. In 9 other patients loss of the Y was the sole abnormality; however, the significance of the latter finding is somewhat uncertain, because a missing Y has also been reported in hematologically normal males, particularly in those over the age of 60 (Sandberg and Sakurai, 1973), and Pierre and Hoagland (1972) have suggested that the missing Y may represent a normal aging phenomenon of human bone marrow cells. The median age is 62.5 years in ANLL patients from Table I11 who had loss of the Y but without a t(8;21), whereas the median age is 24 years in patients having both a missing Y and an 8;21 translocation. As with gains and losses, the nonrandom distribution of structural rearrangements is especially apparent in the cases with a single abnormality (Fig. 3). The most frequently rearranged chromosome was

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

119

0 114 Cases with Multiple

Abnormalities

I35 Cases with Single Abnormality or with t(8;21) and lass of X or Y only

a W

m

I 3

z

I

2 3 4 5 6 7 8 9 10 I1 1 2 1 3 14 15 1617 18 1 9 2 0 2 1 2 2 X Y CHROMOSOME IDENTIFICATION

FIG. 2. Histogram of clonal gains and losses ofchromosomes seen in initial cytogenetic samples from 249 adult patients with acute nonlymphocytic leukemia de novo.

No. 17, which was observed in 48 patients, 27 of whom showed only one abnormality. Likewise, the majority of cases with rearrangements of Nos. 8, 15, or 21, the next three most frequently altered chromosomes, involved a single abnormality or a t(8;21) with loss of a sex chromosome only. Thirty-one of the cases with rearrangements of No.

120

JANET D. ROWLEY AND JOSEPH R. TESTA

50

0114 Coses with Multiple Abnormalities

45

135 Coses with Single Abnormolity or with t(8;211 ond loss of X or Y only

40

35 v)

H

a

H

10

5

0

.I

I

1.1.1.1.1.1.1.1.1.1

I .1.1.1.1.1.1

1.1.1

I

cE l

2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16 17 18 1 9 2 0 2 1 2 2 X Y

CHROMOSOME I DENTlFlCATlON

FIG.3. Histogram of clonal structural rearrangements seen in initial cytogenetic samples from 249 adult patients with acute nonlymphocytic leukemia de novo.

15 and No. 17 involved an apparently identical 15;17 translocation; in 22 of these cases the t(15;17) was the sole change present. An 8;21 translocation was observed in 25 patients, in 7 of whom this was the only abnormality; in 14 of the 25 there was a t(8;21) with loss of an X or Y as the only other change. In contrast to Nos. 15, 17, 8, and 21, rearrangements of other chromosomes rarely existed as the sole abnormality in ANLL. For instance, a rearranged No. 5 was seen in 21 patients, 13 of whom had a 5q-; a single abnormality was present in only 2 of these patients, neither of whom had a 5s- anomaly. These data support the contention of Van Den Berghe et al. (1979a) that, whereas a 59- alone may be associated with refractory anemia or preleukemia, a 5q- combined with other abnormalities is almost always associated with overt leukemia. Similarly, a 7q-, another relatively frequent deletion, occurred in 9 adult patients, all of whom had multiple abnormalities. Although earlier studies had indicated that clonal karyotypic evolution seldom occurred in ANLL, we have reported that evolution of the karyotype could be documented in 17 of 60 (28%) ANLL patients for whom serial samples of leukemic cells were obtained for chromosome

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

121

banding analysis (Testa et al., 1979). The pattern of cytogenetic evolution was nonrandom; the most frequent change was a gain of a No. 8, being found in 10 of 17 (59%) patients whose karyotype evolved. The incidence of evolution and the type and frequency of specific evolutionary changes were similar in patients who were initially normal and in those who were initially abnormal. In all but one of the initially abnormal patients, karyotypic evolution involved the original cytogenetically abnormal clone. a. The 8;21 Translocation in Acute Myeloblastic Leukemia ( A M L ) . In 1968 Kamada et al. recognized that a subgroup of ANLL patients may be characterized by an abnormality most likely representing a translocation between a C- and a G-group chromosome. The exact nature of this abnormality was resolved b y Rowley (1973b), who used the Q-banding technique to determine that it is a balanced translocation between chromosomes 8 and 21 [t(8;21)(q22;q22)]. The frequency with which this translocation occurs seems to vary from one laboratory to another, but it amounted to 10% (25/249) of the abnormal cases summarized in Fig. 3. A similar incidence of the t(8;21) was reported in patients reviewed at the First International Workshop on Chromosomes in Leukaemia (1978); 11 of 139 (7.9%) ANLL patients who had abnormal karyotypes had a t(8;21). The abnormality appears to be restricted to patients with a diagnosis of M2 (acute myeloblastic leukemia with maturation) according to the FAB classification (Bennett et al., 1976). At the Second International Workshop on Chromosomes in Leukemia (1980), all 43 cases with a t(8;21) and adequate bone marrow material available for cytological review had a diagnosis of M2. Overall, the percentage of all patients with an M2 marrow who had a t(8;21) was 9.3% (or 17.9% of those with abnormal karyotypes). Patients with a t(8;21) have also been reported to have a low leukocyte alkaline phosphatase level (Kamada et al., 1976) and a high incidence of Auer rod-positive cells (Kamada et al., 1976; Trujillo et al., 1979). These patients also have a relatively long median survival (Kamada et al., 1976; Sakurai and Sandberg, 1976a; Trujillo et al., 1979). Data on survival of 48 patients with an 8;21 translocation were reviewed at the Second International Workshop on Chromosomes in Leukemia (1980); the median survival of the whole group was 11.5 months, which was much longer than for patients with other chromosome abnormalities. The 8;21 translocation is also of interest for two other reasons. First, chromosomes 8 and 21 can participate in three-way rearrangements similar to those involving chromosomes 9 and 22 in CML. Lindgren and Rowley (1977) reported on two patients with three-way translocations in whom the third chromosome was either a No. 11 or a No. 17.

122

JANET D. ROWLEY AND JOSEPH R. TESTA

Second, the t(8;21) is often accompanied by the loss of a sex chromosome; of the cases reviewed at the Second International Workshop on Chromosomes in Leukemia (1980) 32% of the males with the t(8;21) were -Y, and 36% of the females were missing one X. This association is particularly noteworthy because sex chromosome abnormalities are otherwise rarely observed in ANLL. b. The 15;17 Translocation and Acute Promyelocytic Leukemia (APL).A structural rearrangement involving chromosomes 15 and 17 in APL was first recognized by Rowley et al. (1977). The breakpoint in No. 15 appears to be distal to band q24, and in No. 17 it appears to be in q22 [t(15;17)(q25?;q22)] (Testa et al., 1978a; Second International Workshop on Chromosomes in Leukemia, 1980). Of the 80 patients with APL who were reviewed at the Second International Workshop on Chromosomes in Leukemia (1980), 33 (41%) had a t(15;17) alone (23 cases) or with other abnormalities, 7 had other types of chromosome changes, and 40 had a normal karyotype. The rearrangement was not found in patients with any other type of leukemia. Since APL is a relatively rare form of ANLL comprising only 4% of all cases, the incidence of the t(15;17) as summarized in Fig. 3 seems to be an overrepresentation. The high incidence of this abnormality here may be due to preferential reporting of these interesting cases. Two cases with complex translocations involving Nos. 15 and 17 and either No. 2 or No. 3 were reported (Bernstein et al., 1980a). Thus, the same pattern of variation of a specific translocation can involve the t( 15;17) as well as the t(9;22) and the t(8;21). In some cases, the granules typically seen in the leukemic promyelocytes may be too small to be seen by light microscopy, although they are present when the cells are examined ultrastructurally (Testa et al., 1978a). The FAB Co-operative Group recognized that not all APL patients have coarse granules and has thus added a category called the M 3 variant (Bennett et al., 1980). The variant category was identified largely on the basis of the clinical features and the presence of the t( 15;17). There appears to be an unusual geographical distribution of the APL cases with a 15;17 translocation. The abnormality was observed in 7 of 7 patients in Chicago (Golomb et al., 1979, 1980), 11 of 16 patients in Belgium (Van Den Berghe et aZ., 1979b), and 0 of 12 patients in Finland and Sweden (Teerenhovi et al., 1978). The cause of this variation in incidence is presently uncertain, but the particular methodology used may be a factor. Berger et al. (1980a) have provided evidence that APL patients who appear to b e chromosomally normal based on “direct” marrow preparations may actually show the t(15;17) in preparations from cells cultured for 24-48 hr.

123

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

023 Coses with Multiple

37 Cases with Single Abnormolily or with t(8,21) ond loss of X or Y only

Abnormalities

R

A

f

W U

Y

0

a

10

W

:% 3 Z

8 w

I

W

6

0 ZZ

U 4

a U

U Y

4

2 0

I

2 3

4 5 6 7

8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 X

Y

CHROMOSOME IDENTIFICATION F I G . 4. Histogram of clonal karyotypic abnormalities observed in initial samples from 60 children with acute nonlymphocytic leukemia de novo.

2. Banding Patterns in Children Figure 4 summarizes the chromosome changes seen in 60 children with ANLL from Table 111. Although the number of children examined cytogenetically is relatively small, it is possible to make some preliminary comparisons between the patterns seen in adults and children. Whereas specific abnormalities (e.g., t(8;21), t(15;17), + 8 ) are common to each age group, there appear to be differences with regard to the incidence of these changes in the two groups. Thus, a +8 has been reported in only 6 of 60 (10%) children with abnormal karyotypes, and in only 1 of the 6 was this the sole abnormality. In adults 24.5% of the patients with abnormal karyotypes had a +8, in about half of whom this was the only change. In childhood ANLL a gain of No. 19 was a frequent finding, being seen in 6 cases (sole abnormality in 2), whereas in adults a + 19 was rare (3.6%of abnormal cases) and never occurred as the only change. Loss of No. 7 was observed in 4 children; however, 3 of these were reported as selected cases. Three series describing ANLL in children have been published (Benedict et al., 1979; Hagemeijer et al., 1979; Morse et al., 1979). Overall, 30 of 49 (61%)untreated patients were aneuploid. Only one of

124

JANET D. ROWLEY A N D JOSEPH R. TESTA

these 30 aneuploid patients was missing a No. 7. Thus, unlike in adults, a -7 may be a rare occurrence in children. Abnormalities of any kind seldom involve No. 5 in children. Abnormalities of No. 5 were present in only 2 children, but were found in 31/249 (12.4%) adults. It may be of interest that both of the children with an abnormal No. 5 had a 5q- anomaly, and in each case this was the only abnormality present. Thirteen adults had a 5q-, but all of these patients showed multiple abnormalities. Rearrangement of No. 11, like gain of No. 19, appears to have greater significance in children. Thus, 7 children had a rearranged No. 11; in 5 (8.3% of aneuploid cases) this was the sole abnormality. In contrast to this, only 4 (1.6%) of the aneuploid adults had a rearranged No. 11 as the only change. The 8;21 translocation was the most frequent abnormality in children with ANLL, being reported in 10/60 (16.7%) karyotypically abnormal cases. All 5 males with the t(8;21) were missing a Y, whereas 2/5 females with this rearrangement were -X. Five children with APL had a 15;17 translocation.

3. Prognostic Signi6cance of Chromosome Abnormalities in ANLL Sakurai and Sandberg (1973), using conventional staining methods, demonstrated that the karyotypic pattern of the bone marrow cells is correlated with survival in patients with ANLL. Patients with only normal metaphase cells (NN) had a median survival of 11.5 months from the onset of symptoms as compared to 10.3 months for patients with a mixture of normal and abnormal metaphase cells (AN) and only 3.2 months in those with only abnormal metaphase cells (AA). More recent studies, based on series of ANLL patients studied with banding techniques, have shown similar results (Nilsson et al., 1977; Golomb et al., 1978; Hossfeldet al., 1979; Lawleret al., 1980).Ofthe AML ( M 1 and M2 according to the FAB classification) patients reviewed at the First International Workshop on Chromosomes in Leukaemia (1978), a substantially longer median survival (8 months) was found for NN patients as compared to those who were AA (3.5months). Patients who were AN had an intermediate survival (5 months). No such differences were found in patients with acute myelomonocytic or monocytic leukemia (M4 or M5). Benedict et al. (1979) demonstrated that the karyotypic pattern can also correlate with prognosis in childhood ANLL. They reported a median survival of 20.5 months in patients who were NN, whereas those with chromosomal abnormalities had a median survival of only 7.1 months. Three of their patients were AA; none of the three survived longer than 4.5 months from diagnosis. It may be that it is the lack of cytogenetically normal cells, rather

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

125

than the presence of aneuploid cells, that is unfavorable in AA patients. Golomb (1980) has suggested that the cytogenetically normal cells may represent normal stem cells that are required to repopulate the marrow after the leukemic cells have been destroyed by chemotherapy. This may explain why AN, as well as NN, patients tend to have a longer median survival than AA patients.

B. ANLL AS

A

SECOND NEOPLASM

It is now well recognized that ANLL can be a late complication in a number of malignant and nonmalignant diseases treated with radiation and/or chemotherapy, which are mutagenic and potentially carcinogenic agents. Rowley et d. (1981a) reported on the karyotypic patterns of bone marrow cells of 27 ANLL patients who had received prior treatment for a primary cancer (26 cases) or for a renal transplant (1 case). Fifteen of the patients had previously had both radiotherapy and chemotherapy, 8 had only chemotherapy, and 4 had only radiotherapy. The median times from diagnosis of the initial disease to the onset of ANLL for these three treatment groups were 61,59, and 59 months, respectively. Only 1 of the 27 patients had a normal karyotype. Most (19/27)of the patients had a clone with a hypodiploid modal number. One or both of two consistent chromosome abnormalities were found in marrow cells from 23 of the 26 aneuploid cases: loss of No. 7 (18 patients) or part of the long arm of No. 7 (1 patient); and loss of No. S (11 patients) or part of the long arm of No. 5 (3 patients). Although this karyotypic pattern is quite different from that found in lymphomas, it is similar to that found in about 25% of aneuploid patients with ANLL de novo. The data regarding nonrandom changes of chromosome Nos. 5 and 7 in secondary ANLL appear to be especially pertinent to the question of whether or not we can identify patients with ANLL de novo who may have been exposed to environmental mutagens. Although this question cannot be answered as yet, two lines of evidence suggest that the answer may be positive. The first of these comes from a retrospective study of the correlation between karyotype and occupational exposure in ANLL, and the second from studies of childhood ANLL. Mitelman et al. (1978) reported on a retrospective study of 56 patients with ANLL de novo; 23 had a history suggesting occupational exposure to chemical solvents, insecticides, or petroleum products, whereas 33 had no such known exposure. Only 24.2% of the nonexposed group had clonal chromosome abnormalities, as compared to 82.6%in the exposed group. There was a distinctly nonrandom pattern

126

JANET I). ROWLEY AND JOSEPH R. TESTA

of changes in the exposed group, with 84.2% of these cases having at least one of four specific abnormalities: -5 (or Sq-), -7 (or 7q-), +8, or +21. In the nonexposed group, only two patients had any of these changes: one was - 7 and the second was +21. As mentioned earlier, there have been three series describing the karyotype pattern in ANLL de novo in children (Benedict et al., 1979; Hagemeijer et aZ., 1979; Morse et al., 1979). Only one of 30 aneuploid patients from these studies was missing a No. 7, and this was from a child who was also +8. Five patients had partial deletions of 7q, but only two had a deletion at 7q22, which is the abnormality seen in adult ANLL de novo and in secondary ANLL. None of these children was - 5 and none had a 5q-. Furthermore, none of the 30 aneuploid patients had an abnormal clone with a hypodiploid modal number. The consistent finding of a loss of all or part of chromosomes 5 or 7 in patients with secondary ANLL, in conjunction with the frequent occurrence of these same abnormalities in patients with ANLL who may have an occupational exposure to mutagenic agents, and the absence of these aberrations in childhood ANLL all provide support for our proposal that these specific chromosome changes may identify ANLL associated with exposure to mutagens (Rowley et al., 1981a). V. Acute Lymphocytic Leukemia (ALL)

It has been reported that the most useful prognostic factors in childhood ALL are age, WBC count (Miller et al., 1980),and immunologic markers (Chessells et al., 1977). Patients who are between 3 and 7 years old, with a WBC count of less than 10,000/mm3 (Miller e t al., 1980), and whose leukemic cells have non-T, non-B surface markers (Chessells et al., 1977) have the best prognosis. Because of the difficulty of obtaining adequately banded chromosomes, reports describing the banding pattern have been fewer in ALL than in ANLL. Remarkable improvements, however, have been obtained. It is now possible to correlate the karyotype with other recognized prognostic factors and to show that data on chromosome patterns can increase the precision of previously recognized prognostic features. This review includes data on the chromosome patterns of 33 ALL patients studied at the University of Chicago (Cimino et al., 1979; Kaneko and Rowley, 1981), on patients described in recent reports (reviewed in Kaneko and Rowley, 1982), and on 330 patients evaluated at the Third International Workshop on Chromosomes in Leukemia (1981). Based on earlier studies, it appeared that chromosome abnormalities occurred in about one-half of the patients with ALL, and

CHHOhlOSOME ABNORMALITIES IN MAIAGNAKT DISEASES

127

hyperdiploidy was thought to be predominant among aneuploidies (Oshimura et al., 1977b; Whang-Peng et al., 1976). The study of 330 ALL patients (adults, 173; children, 157) at the Third Workshop revealed that 65% of the patients had clonal abnormalities. Of the 213 aneuploid patients, 34.8% had pseudidiploidy; 24.5%, hyperdiploidy; and 6.7%, hypodiploidy. Our study on 33 ALL patients (Kaneko and Rowley, 1981) also showed a high incidence of aneuploidy (70%); pseudodiploidy was predominant (13 of the 23 aneuploid patients) (Table IV). Although the karyotype in many of these patients may be very complex, certain patterns recur. It is now possible to analyze the clinical features of these patients, together with the morphology of the leukemic cells and the results of cell surface marker studies; this provides additional insight into the derivation of these malignant lymphoid cells. A. THE 8;14 TRANSLOCATION A translocation involving the long arms of No. 8 and No. 14 has been detected in a large number of Burkitt tumors of both African and nonAfrican origin, independent of whether they are Epstein-Barr virus (EBV) positive or negative (Zech et al., 1976b; Kaiser-McCaw et al., 1977). An apparently identical translocation has been observed in ALL patients with B-cell markers and in patients with L3-type leukemia cells (Slater et al., 1979; Mitelman et al., 1979; Berger et d., 1979b), indicating that Burkitt lymphoma and most B-cell ALL of the L3 type are probably different manifestations of the same disease. Sixteen patients with this rearrangement were reported at the Third Workshop. There was an excess of males over females, and of adults over children. This group of patients had a high incidence of central nervous system involvement at diagnosis and a poorer prognosis (a median survival of 5 months) than any other group of patients classified according to chromosome patterns. With one exception, all patients in whom the immunologic markers of leukemic cells were identified had B-cell markers, and all but one had L3-type cells. In the exceptional patient, the leukemic cells had a pre-B-cell phenotype and were of the L1 type (Kaneko et al., 1980);the morphology of the leukemic cells, however, changed to L3 type at relapse. Variant translocations have been reported in Burkitt lymphoma; these variants include a t(2;8)(~12-13;q24)(Miyoshict ul., 1979; Van Den Berghe et al., 1979d) and a t(8;22)(q24;qll)(Berger et al., 1 9 7 9 ~ ) . Chromosome No. 8 was always involved in the translocation, with the

C o R R E L A n O N OF

CHROMOSOME SUBGROUP

TABLE IV FAVORABLE PROGNOSTlC FACTORS AND WITH

WITH

WBC

N patients (diploidy) Al patients (pseudodiploidy) A2 patients (hyperdiploidy 47-49) A3 patients (hyperdiploidy 50-59)

SURVIVAL'

WBC

No. of patients

Age 57


<20X

103/jd

103/p1

Non-T,non-B markers

FAB Llb

Survival

Patients alive

11 17 8 9

4 5 2 8

6 5 2 6

8 6 4 8

8V10d 9'114 314 8'18

6 7 5 4

75% at 2545 dayse 674 days' 458daysg All patients alive

9 6 2 9

These data are for combined patients from the University of Chicago (33)and Saitama Cancer Center (17); reported in Kaneko et ~ 1 . (1981). * FAB L1: FAB classification L1 subtype. One patient was tested only for T-cell marker. The denominator indicates the number of patients who were tested for immunologic markers. Actuarial survival. 'Two patients were tested only for T-cell markers. Actuarial median survival. * Three patients were tested only for T-cell markers.

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

129

breakpoint in band 8q23 or 24. This indicates that the consistent chromosome abnormality in Burkitt lymphoma is a rearrangement of 8q rather than that of 14q. The variant translocation t(2;8) was also found in an ALL patient with the L3 type (Rowley et al., 1981b). The occurrence of variant translocations in Burkitt lymphoma may be analogous to that in CML; 92% of CML patients have a t(9;22), and the remaining 8% have variant translocations. Chromosome No. 22, however, is involved in each translocation, with a break in band q l l . NOT INVOLVING No. 8 B. A 14q+ CHROMOSOME A 14q+ chromosome is frequently observed in malignant lymphomas, particularly, although not exclusively, in those of B-cell origin (Rowley and Fukuhara, 1980). Fifteen ALL patients at the Third Workshop were reported to have a 14q+ chromosome that was not involved in a translocation with the terminal segment of chromosome No. 8. The excess of males over females and of adults over children is similar to that in the patients with t(8;14). One-half of the patients, however, had L2-type leukemic cells, and of the other half, one-fourth each had L1- and L3-type cells. The leukemic cells in about one-half of the patients had non-T, non-B immunologic markers, and the other one-half had B-cell markers. The 14q+ chromosome was due to a balanced translocation in 6 patients, in 4 of whom the donor chromosome was identified as No. 11. The t(11;14) is one of the common abnormalities seen in malignant lymphoma, poorly differentiated lymphocyte type (Rowley and Fukuhara, 1980),suggesting that there is a relationship between it and ALL with the 14q+ chromosome. The complete remission rate in the patients with a 14q+ chromosome was 5370, with a median survival of 9 months. C. THE4;11 TRANSLOCATION A translocation involving the long arms of Nos. 4 and 11 was observed in 4 of 52 ALL patients of Van Den Berghe et al. (1979c),4 of 34 patients of Prigogina et al. (1979), and one of 31 patients of Oshimura et al. (1977b),but in none of our 33 ALL patients (Kaneko and Rowley, 1981). According to the report on 18 patients with this change who were evaluated at the Third Workshop, these patients had very high leukocyte counts (median WBC, 183,000/mm3).The leukemic cells were L1 type in 7 patients, L2 type in 7, and L3 type in 1 patient. Of 8 patients in whom immunologic markers were tested, 7 had non-T, non-B ALL, and 1 had T-cell ALL. These patients had a very poor

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JANET D. ROWLEY AND JOSEPH R. TESTA

prognosis; the complete remission rate was 67%, and the median survival was 7 months. One-half of the patients were adults; the other one-half were children most of whom were less than 1 year old. The association of the 4; 11 translocation with neonatal or early-childhood ALL is particularly interesting in view of the low incidence of ALL in this age group.

D. NEAR-HAPLOID ALL The occurrence, in ALL, of leukemic cells with a near-haploid number of chromosomes is rare. Five such cases have been published (Kessous et al., 1975; Oshimura et al., 1977b; Prieto et al., 1978; Shabtai et al., 1979; Kaneko and Sakurai, 1980), and two new cases were presented at the Third Workshop. There were four males and three females; five were children or adolescents, and two were adults. These seven patients had a remarkably consistent chromosome pattern. The chromosome number of the near-haploid clone ranged from 26 to 36 (median, 28). A boy reported b y Prieto et al. (1978) is presumed to have had the karyotype 26,XY,+ 14,+21, although the chromosomes were not banded. I n addition to a haploid set, +21 was seen in all patients, 10 and + 18 in six, +X or + Y in five, + 6 in four, and + 1, + 19, and +22 in three. Patients in four (Kessous e t al., 1975; Oshimura et al., 1977a; Shabtai et al., 1979; Kaneko and Sakurai, 1980)of the five published reports had a variable percentage of cells that contained double the number of chromosomes of the near-haploid line. Of the two patients for whom immunologic markers were tested, both had non-T, non-B ALL (Oshimura et al., 1977a; Kaneko and Sakurai, 1980); one was identified as having common ALL (Prieto et al., 1978). Two patients had no response to chemotherapy (Prieto et al., 1978; Shabtai et al., 1979), whereas three others achieved a complete remission (Kessous et al., 1975; Oshimura et al., 1977a; Kaneko and Sakurai, 1980);however, all three had a relapse after 6-16 months of remission. The median survival was 9 months. Thus, ALL with near-haploidy may be a unique subgroup of ALL, with a prognosis that is poor compared with that for other types of non-T, non-B ALL.

+

E. HYPERDIPLOIDYWITH 50 TO 60 CHROMOSOMES The karyotype of some patients is characterized by many extra chromosomes and few structural abnormalities. Chromosome numbers usually range from 50 to 60, and a few patients may have up to 65 chromosomes. Although identical karyotypes are rarely found, certain

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131

additional chromosomes are commonly seen. Among 30 patients including 22 children and 8 adults evaluated at the Third Workshop, +21 was seen in 22, + 6 in 15, +18 in 14, + 14 in 11, +4 or +10 each in 10 patients. If we compare the additional chromosomes that were common in this group with the additional chromosomes in the 7 patients reviewed in the preceding section who had the haploid complement, the similarities are remarkable, since the most consistent changes in the latter group are + l o , +18, and +21. The median age of the 22 children with this abnormality was 3 years, and that of all 30 patients was 5 years, which was less than that of patients with other abnormalities. The WBC count in patients with hyperdiploidy was low, with a median of 6000/mm3; it was below 10,000/mm3 in more than half of the patients. The L1 and L2 types were seen in about equal numbers, and all patients had non-T, non-B ALL. A good prognosis for this group was reported by Secker Walker et al. (1978) and Kaneko et al. (1981). Hyperdiploidy with 50 to 60 chromosomes was detected in 3 of the 33 ALL patients at the University of Chicago; all continue to be in their first remission (46 days to 1095 days) (Kaneko and Rowley, 1981). The complete remission rate and the median survival of the 30 patients evaluated at the Third Workshop were 86% and 34 months, respectively. Thus, in patients who have hyperdiploidy with more than 49 chromosomes, all of the previously recognized factors, including age between 3 and 7 years, low WBC count, and non-T, non-B markers, are present that indicate a good prognosis. It should be emphasized that the median survival of the hyperdiploid patients, including both children and adults, is longer than that of patients with a normal karyotype.

F. THEPh' CHROMOSOME IN ALL A Ph' chromosome, which is formed by a reciprocal translocation between No. 22 and No. 9 or various other chromosomes, is seen in patients with ALL, as well as in patients with CML. Of the 39 patients evaluated at the Third Workshop, 30 were adults and 9 were children. The incidence of Phl-positive patients with ALL was 5.7% for children and 17.3% for adults; the incidence previously reported was 2.0% for children (Chessells et al., 1979) and 25% for adults (Bloomfield et al., 1978). Thus, the Ph' chromosome is the most frequent rearrangement in adult ALL. Thirty-six patients at the Workshop had the typical t(9q+ ;22q-), and the remaining 3 had variant translocations; the incidence of the variant form was 8%, which is similar to that observed in CML patients. About one-half of the patients showed

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JANET D. ROWLEY A N D JOSEPH R . TESTA

abnormalities in addition to the Ph' chromosome. These additional changes were quite variable, and the usual changes seen in the blast crisis of CML were absent except for +8 in one case. The patients had a high leukocyte count (median WBC, 34,000/mm3),and all had non-T, non-B ALL. The complete remission rate was 55%. The median survival was 9 months, reflecting the poor prognosis for these patients. By identifying this chromosome abnormality, one can detect individuals in the non-T, non-B category who have a poor prognosis. Based on the evidence gathered at the Third Workshop, plus that of individual reports, the cytogenetic study of leukemic cells from patients with ALL clearly can provide important information. At present, these data are clinically useful, providing another independent variable that has prognostic significance. Of more importance, however, is the ability to define subsets of patients with ALL on the basis of certain genetic (chromosome) changes and then to relate these changes to various functional and immunologic studies. In this way, we will gain a much more sophisticated and accurate understanding of the interrelationship of the various subsets of lymphoid cells.

VI. Polycythemia Vera

A. INCIDENCE

OF

ABNORMALKARYOTYPES

There have been two large studies, one by investigators at the Royal Marsden Hospital (Kay et al., 1966; Millard et al., 1968; Lawler et al., 1970) and the other by Visfeldt and associates (Visfeldt, 1971; Visfeldt et al., 1973),of more than 50 polycythemia Vera (PV) patients whose marrow cells were examined cytogenetically with conventional stain. Several series of PV patients have been reported in which at least some of the patients were studied with banding techniques (Shiraishi et al., 1975; Westin et al., 1976; Wurster-Hill et al., 1976; Zech et al., 1976a; Nowell and Finan, 1978; Shabtai et al., 1978; Testa et al., 1981).Of 404 untreated and treated PV patients from the above studies (excluding patients analyzed in the leukemic phase only), 93 (23%) had cytogenetically abnormal clones in their initial analysis. The incidence of chromosomal abnormalities differs between untreated patients (including those who had had phlebotomies only) and those who were treated with cytotoxic agents prior to the first cytogenetic examination (Table V). Patients from the series by Nowell and Finan (1978) are not included in this table because the treatment status (i.e., pre- or posttherapy) at the time of their cytogenetic analysis was not given in

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CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

TABLE V INCIDENCE OF CLONAL KARYOTYPIC ABNORMALITIES IN INITIAL SAMPLES FROM VARIOUS POLYCYTHEMIA VERA (Pv) PATlENT GROUPS“ Untreated PV patients

Treated PV patients

Treated PV patients in a leukemic phase

5(Ub/33

13(1)’(2)d/42

515

2/17 013 7/50 0(2)d/3 14131 114 3/10 413 361264 (14%)

19/50 519 111 -0 45 0/4“ 8(1)’(2)d/18 501129 (39%)

References ~

313 -

~~~

Kay et al. (1966). Millard et al. (1968), Lawler et al. (1970) Visfeldt (1971), Visfeldt et al. (1973) Shiraishi et al. (1975)p,’ Westin et al. (1976)’ Weinfeld et al. (1977)’ Wurster-Hill et al. (1976)’ Zech et al. (1976)’ Shabtai et al. (1978)’ Testa et al. (1981)‘

17/20 (8570)

a Paiients are listed in groups according to the clinical status at the time of their initial cytogenetic analysis. Number in parentheses refers to a chromosomally normal untreated patient whose karyotype became abnormal after treatment began. Number in parentheses refers to a treated patient who was chromosomally normal in the first sample obtained after therapy had begun, but showed an abnormal karyotype in a subsequent sample. Number in parentheses refers to patients who had normal karotypes in initial samples, but showed abnormal karyotypes later, when they developed leukemia. ‘Series in which at least some patients were studied with banding techniques. ’One patient with a normal karyotype is excluded here because the treatment status (i.e., pre- or posttherapy) was unclear. Although treated patients with abnormal karyotypes were observed, the total number of treated patients who were analyzed was not given in the original report. * O n e patient with an abnormal karyotype is excluded here because the time of analysis (i.e., polycythemic or leukemic phase) is unclear.

that report. Only 14%of the untreated patients summarized in Table V had abnormal karyotypes, compared to 39% of the treated patients. The incidence of chromosomally abnormal clones is even higher in treated PV patients whose disease had transformed to a leukemic phase (Table V). An abnormal karyotype was seen in 85% of the patients who were first studied after they had developed leukemia. Many of these leukemic patients had very complex karyotypes. None of the leukemic patients with a normal karyotype was studied with banding techniques.

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In PV, cytogenetic abnormalities do not necessarily predict leukemia. Westin and Weinfeld (1978) reported that none of their patients with initially abnormal karyotypes who were followed for 3-9 years developed acute leukemia. Similarly, we reported that four of our patients who had abnormal clones in the first cytogenetic sample have been followed for 5- 15 years, without leukemic transformation; two were untreated prior to the first analysis (Testa et al., 1981). Nowell and Finan (1978) have proposed that in PV, in contrast to cytopenic diseases, chromosomally abnormal clones are of little predictive value, except that leukemia may develop sooner in those who have karyotypic abnormalities. An evolutionary change in the karyotype during the disease course, however, may be an important prognostic sign. This was illustrated in 4 of our patients ( 3 with a previously normal and 1 with a previously abnormal complement) whose karyotype evolved at the onset of leukemia (2 cases) or when the disease had become more aggressive (Testaet al., 1981). In addition to our 2 patients above who developed leukemia, 4 other PV patients have been reported on who had a normal karyotype in their first sample in the polycythemic phase, either prior to or following therapy, but who subsequently had cytogenetic abnormalities when they developed leukemia (Lawler et al., 1970; Weinfeld et al., 1977) (Table V). All 6 patients had rather complex karyotypes, with at least one marker or bizarre rearrangement in each case, during the leukemia phase. Four other PV patients who were karyotypically abnormal in the polycythemic phase acquired additional cytogenetic changes when they became leukemic (Lawler et al., 1970; Weinfeldet al., 1977; Berger and Bernheim, 1979). The most frequent evolutionary changes seen in these 10 patients were markers (6 patients) and chromosome gains (4 patients).

B. MODAL NUMBER In addition to studies on series of PV patients, there have been a number of reports on chromosomal analyses, with banding techniques, of single cases or selected patients. Table VI summarizes the distribution of modal chromosome numbers in a total of 105 karyotypically abnormal untreated and treated PV patients (excluding analyses done during a leukemic phase), 53 of whom were studied with banding techniques. Of the 105 patients, 87 (83%) had modal numbers of 46-47. Hypodiploidy was rare, accounting for only 10%(10/105) of the abnormal cases. There appears to be a marked difference between untreated and treated PV patients with regard to the distribution of

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C H R O M O S O M E ABNORMALITIES I N MALIGNANT DISEASES

TABLE VI DISTRIBUTIONOF MODALCHROMOSOME NUMBERS I N 105 UNTREATED AND TREATED POLYCYTHEMIA VERA (PV) P A TI EN TS~ .~ Modal chromosome number of abnormal cells

Number of patients Untreated Untreated?" Treated

44

45

46'

47

48

>48

0 1 -1 2

5 1 2 -

6 0

22 4 9 -

6 1 0 -

35

7

0 0 1 1

8

46 52

" References: Reeves et al. (1972); Knight et ul. (1974);Hsu et ul. (1974); Tsuchimoto et al. (1974); Berger (1975); Westin (1976a,b); Hsu et al. (1977); Nowell and Finan (1978); Berger and Bernheim (1979); Van Den Berghe et a / . (1979a); D. Van Dyke

(personal communication); plus references listed in Table V. * Analyses done during a leukemic phase were excluded. 'All patients were pseudodiploid. These patients are from studies in which clinical details were sparse and the time of cytogenetic analysis (i.e., pre- or posttreatment) was not reported.

modal numbers. The abnormal cells from 72% (28/39) of the known untreated patients had hyperdiploid modal numbers, whereas abnormal cells from 78% (46/59) of the known treated patients were pseudodiploid.

C. NONRANDOMABNORMALITIES 1. Polycythemic Phase The pattern of chromosomal changes in PV is nonrandom. Figure 5 summarizes the karyotypic abnormalities seen during the polycythemic phase in 48 patients from Table VI who were studied with banding techniques, as well as changes in two other patients, who are not listed in Table VI because the modal numbers were not included in the original report. Five patients from Table VI who were studied with banding are excluded in Fig. 5: 4 were known to have developed myelofibrosis prior to the initial analysis; the time of analysis was unclear in a fifth patient who developed myelofibrosis. In the polycythemic phase, the nonrandom distribution of chromosomal changes is particularly evident in the gains, which usually involved chromosomes 8 and 9. Eleven patients had a gain of No. 9, and 9 patients had a gain of No. 8. Four of these patients showed gains of

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JANET D. ROWLEY AND JOSEPH R. TESTA

FIG.5. Histogram of clonal karyotypic abnormalities seen in 50 untreated and treated polycythemia Vera patients. Analyses done during a transitional or a leukemic phase are omitted here. Each box represents a clonal abnormality seen in a single patient. “Untreated?’ patients are those for whom the time of cytogenetic examination (either pre- or posttreatment) was not specified by the authors in the original report.

both No. 8 and No. 9 (Westin et al., 1976; Testa et al., 1981).Clones containing both + 8 and + 9 are seldom observed in other hematologic diseases and may be unique to PV (Testa, 1980). Chromosomes No. 20 (17 patients) and No. 1 (10 patients) were the most frequently rearranged chromosomes seen in the polycythemic phase (Fig. 5). All but one of the rearrangements of No. 20 involved a 2Oq-. Thus, a 20q- was present in 32% (16/50)of the aneuploid PV patients summarized here. It had been thought initially that the 20qabnormality might have some diagnostic value in PV (Wurster-Hill et al., 1976); however, whereas a 20q- is a relatively frequent finding in PV, it has now been observed in patients with various other myeloid disorders as well (Testa et al., 1978b). Most (8/10 shown in Fig. 5) of the reported abnormalities of No. 1 in PV have consisted of trisomy of all or part (1q21-qter) of the long arm. Rowley (197%) and Gahrton e t al. (1978) have noted that trisomy of l q , especially of bands 1q25-32 and 1q23-25, can be found frequently in various hematologic diseases.

137

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

aLeukemic phose (I5cosesl Tront~l~onol (I1 cases) Unhealed Observed In polycylhemc phose 0s well

8 6

z

0 a

4

2

2

41 6

10 8 6 4

2 I

2

3 4

5

6

7

8

9 10 II 12 13 14 15 16 17 I8 19 20 21 22 X

Y

CHROMOSOME IDENTI FlCATlON

FIG.6. Histogram of clonal karyotypic abnormalities seen in 26 polycythemia Vera (PV) patients (25 treated) during a transitional or a leukemic phase. Abnormalities that were first observed earlier during the polycythemic phase in 5 patients (of 12 examined during that stage) are indicated by an asterisk (*). Unlike the pattern seen in the polycythemic phase, rearrangement of No. 5 and loss of No. 7 are quite common in the more advanced stages of PV.

Structural rearrangements were seen primarily in treated patients. For example, 14 of 17 patients with a rearranged No. 20 were treated prior to their first cytogenetic analysis. In contrast, most chromosome gains are found in untreated patients.

2. Transitional and Leukemic Phases There have been a number of PV patients with advanced disease who have been studied with banding techniques (Hoppin and Lewis, 1975; Stavem et al., 1975; Nowell et al., 1976; Westin e t al., 1976; Zech et al., 1976a; Weinfeld et al., 1977; Berger and Bernheim, 1979; Van Den Berghe et al., 1979a; Hagemeijer et al., 1981; Testa et al., 1981). Figure 6 summarizes the karyotypic abnormalities observed in 26 patients during the advanced stages of PV. Included here are abnormalities found during the leukemic phase or during a “transitional phase.” As used here, “transitional phase” refers to the development, during disease progression, of myelofibrosis, myelofibrosis with myeloid metaplasia, and/or increased granulocytic immaturity.

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JANET D. ROWLEY AND JOSEPH R. TESTA

Whereas the cytogenetic pattern seen during the advanced disease stages shows some similarities (e.g., +8 and +9) to that found in the polycythemic phase, there are certain striking differences as well. For example, loss of No. 7 was never observed in the polycythemic phase, but was seen in 5 of 26 patients in transitional or leukemic phases. Rearrangements of No. 5 were the most frequent change seen in advanced disease stages, but seldom occurred in the less aggressive polycythemic phase. Rearrangements of No. 20 were less frequent in advanced disease and were found in only I patient who developed overt leukemia. Moreover, structural rearrangements of No. 12, which are rare in the stable phase (Fig. 5), are more frequent in the leukemic phase (Fig. 6). A 5q- may be a specific abnormality frequently associated with the terminal stage of PV (Testa et aZ., 1981). Nine of the 10 structural rearrangements of No. 5 shown in Fig. 6 were 5q- anomalies. In contrast, only one of three rearrangements of No. 5 observed in the polycythemic phase (Fig. 5) was a 5q-, and this was observed in a patient whose chromosomes were examined only very late in the disease course by Testa et al. (1981). A 5q- has been observed in the leukemic phase of five PV patients. Van Den Berghe et aZ. (1979a) reported on three patients with long-standing PV who first showed a 5q- late in the disease course;the abnormality accompanied the transition to myelofibrosis with myeloid metaplasia and the appearance of a preleukemic disorder. However, Testa et al. (1981)observed a 5q- in only two of eight PV patients with documented myelofibrosis. Thus, it appears that the 5q- anomaly may be associated more with disease progression in PV than with the actual development of myelofibrosis. Whereas the 5q- abnormality may be a marker of refractory anemia or, possibly, of preleukemia or of an early nonproliferative leukemia (Van Den Berghe et al., 1976), the presence of a 5q- plus other chromosome changes is nearly always associated with overt leukemia (Van Den Berghe et al., 1979a). In each of the 5q- patients summarized in Fig. 6, multiple chromosomal abnormalities were present. Thus, in PV currently available data indicate that a 5q-, in combination with other karyotypic changes, signals a terminal phase of the disease, which may involve anemia and other progressive changes, or transformation to myelofibrosis or overt leukemia. D. RELATIONSHIP OF TREATMENT TO ABNORMALKARYOTYPES

The reasons for the higher incidence of chromosome abnormalities in previously treated, as compared to untreated, PV patients are unknown. Westin (1976b) suggested that many of the treated patients

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

139

have had the disorder for a longer time than untreated ones prior to cytogenetic study, and data of Testa et al. (1981) support this suggestion. Furthermore, our study also showed that clonal abnormalities were seen primarily in patients who had had prior radiotherapy, and the incidence tended to correspond to the total prior dose of 32P.Similarly, Visfeldt et al. (1973) observed earlier that abnormal karyotypes occurred almost exclusively in their patients who were treated with 32P, and generally only after treatment with more than 2.5 mCi per year over more than 2 years. Follow-up studies done by Westin and Weinfeld (1978) showed that patients with initially normal karyotypes did not develop chromosome changes when they were treated with phlebotomies alone. In contrast, during therapy with 32Por chlorambucil, new abnormalities occurred in some patients whose karyotypes had been initially normal, as well as in others whose karyotypes were initially abnormal. Westin (1976b) noted that uncertainty exists as to whether these evolutionary changes are part of the natural history of the disease, or whether the myelosuppressive therapy induces the changes or accelerates a susceptibility to their spontaneous development. This question may eventually be resolved as a result of investigations such as the already ongoing study of the Polycythemia Vera Study Group (Wurster-Hill et al., 1976), in which cytogenetic analyses are done throughout the disease course on patients randomized for therapy with 32P,drugs, or phlebotomy.

VII. Implications of Nonrandom Changes for Malignant Transformation

The evidence presented demonstrates that nonrandom chromosome changes are closely associated with a variety of human hematologic disorders. Similar associations have been identified in other human The tumors and in animal tumors as well (reviewed in Rowley, 1980~). changes consist of gains or losses of part or all of certain specific chromosomes and of structural abnormalities, most frequently relatively consistent translocations, that are presumed to be reciprocal. The nonrandom translocation that we observe in malignant cells would represent those that provide a particular cell type with a selective advantage vis-a-vis the cells with a normal karyotype. There is very strong evidence that many cancers, CML and Burkitt lymphoma, for example, are of clonal origin. This means that a particular translocation in a single cell gives rise to the tumor or to the leukemia that ultimately overwhelms the host. Other rearrangements may be neutral, and the cells therefore will survive, but will not proliferate differentially; and still others may be lethal and thus would be eliminated.

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JANET D. ROWLEY AND JOSEPH R. TESTA

In such a model, the chromosome change is fundamental to malignant transformation. Two questions are raised by these observations. First, how do such chromosome changes occur; and second, why do they occur? There is very little experimental evidence that is helpful in answering either of these fundamental questions. They clearly provide a focus for future research.

A. PRODUCTION O F CONSISTENT TRANSLOCATIONS

The mechanism for the production of specific, consistent reciprocal translocations is unknown. Chromosome breaks and rearrangements may occur continuously at random and with a low frequency, and only those with a selective advantage will be observed. Alternatively, certain chromosome regions may be especially vulnerable to breaks and therefore to rearrangements. Nonrandom breaks occur in certain human chromosomes exposed to various mutagenic agents. In the rat, Sugiyama (1971) showed that a particular region on No. 2 was broken when bone marrow cells from animals given DMBA were examined. In man, however, trisomy for l q is not necessarily related to fragile sites (Rowley, 1977b).Thus, a comparison of the break points seen in hematologic disorders that involve balanced reciprocal translocations with those leading to trisomy l q revealed a clear difference in preferential break points, depending on whether the rearrangement resulted in a balanced or an unbalanced aberration. Other possible explanations depend on either (a) chromosomal proximity, since translocations may occur more frequently when two chromosomes are close together; or (b) regions of homologous DNA that might pair preferentially and then be involved in rearrangements. Many of the affected human chromosomes, e.g., Nos. 1, 9, 14, 15, 21, and 22, are involved in nucleolar organization that would lead to a close physical association. All partial trisomies that result from a break in the centromere of No. 1involve translocations of l q to the nucleolar organizing region of other chromosomes, specifically Nos. 9, 13, 15, and 22 (Rowley, 1977b). In the mouse, chromosome No. 15 also contains ribosomal cistrons (rRNA) (Henderson et al., 1974). Sugiyama et al. (1978) noted that, in rat neoplasms, translocation trisomies, other markers, and aneuploidy frequently involve Nos. 1, 2, 13, and 19, which are chromosomes with late-replicating DNA, and Nos. 3, 11, and 12, which have rDNA and late-replicating DNA. They have suggested that nucleolus-associated late-replicating DNA rather than rDNA is involved in the origin of nonrandom chromosome abnormalities.

CHROMOSOME ABNORMALITIES IN MALIGNANT DISEASES

14 1

On the other hand, if chromosome proximity or homologous DNA sequences were the mechanism, this should lead to an increased frequency of rearrangements such as the 9;22 or 8;14 or 15;17 translocation, in patients with constitutional abnormalities, but this has not been observed. One of us (J. D. R.) wrote to all investigators who listed patients with these and other consistent translocations seen in leukemia and lymphoma in “The Repository of Chromosomal Variants and Anomalies in Man (Borgaonkar and Lillard, 1980).” Of the 39,971 patients with anomalies listed in this registry only one had one of these consistent translocations as a constitutional abnormality (Ferro and San Romin, 1981).It is possible that either or both of these mechanisms are subject to selection; a translocation might occur because the chromosomes are close together, but only certain specific rearrangements might have a proliferative advantage which results in neoplasia and thus allows them to be detected. One other possible mechanism that should be considered concerns transposable genetic elements that can cause large-scale rearrangements of adjacent DNA sequences. These consist of controlling elements that have been found in maize (McClintock, 1961) and in Drosophila (Green, 1973), and of insertion sequences in bacteria (Nevers and Saedler, 1977). Not only do these elements exert control over adjacent sequences, but the type of control, that is, an increase or a decrease in gene product, is related to their position and orientation in the gene locus. Whereas they can cause nonrandom chromosomal deletions adjacent to themselves, these controlling elements can also move to another chromosomal location, and they may transpose some of the adjacent chromosomal material with them. The evidence for the presence of transposable elements in mammalian cells is tenuous, but a more precisely defined gene map is required for the detection of such nonhomologous recombinations. B. FUNCTION OF NONRANDOM CHANGES Our ignorance of how nonrandom changes occur is matched by our ignorance as to w h y they occur. Two points should be emphasized; one concerns the genetic heterogeneity of the human population, and the second, the variety of cells involved in cancer. There is convincing evidence from animal experiments that the genetic constitution of an inbred strain of rats or mice plays a critical role in the frequency and type of neoplasms that develop. Some of the factors controlling the differential susceptibility of mice to leukemia not only have been identified, but also have been mapped to particular chromosomes, and their behavior as typical Mendelian genes has been demonstrated

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JANET D. ROWLEY AND JOSEPH R. TESTA

(Lilly and Pincus, 1973; Rowe, 1973).These genes have been shown to be viral sequences that are integrated into particular sites on chromosomes; these sites vary for different inbred mouse strains and for different murine leukemia viruses. Thus, the sites in AKR and C3H mice are two different loci on chromosome 7 (Rowe, 1973), and that in Balb/c mice is on chromosome 5 (Rowe and Kozak, 1979; Ihle et al., 1979). Certain genetic traits in man predispose to cancer, especially leukemia and lymphoma, such as Bloom syndrome, Fanconi anemia, and ataxia-telangiectasia (German, 1972). How many gene loci are there in man that, in some way, control resistance or susceptibility to a particular cancer? We have no way of knowing at present. These genes may influence the types of chromosome changes that are present in malignant cells. The second factor affecting the karyotypic pattern relates to the different cells that are at risk of becoming malignant, and the varying states of maturation of these cells. The catalog of the nonrandom changes in various tumors maintained by Mitelman and Levan (1978) provides clear evidence that the same chromosomes, for example, Nos. 1 and 8, may be affected in a variety of tumors. On the other hand, some chromosomes seem to b e involved in neoplasia affecting a particular tissue; the involvement of No. 14 in lymphoid neoplasms and of No. 20 in myeloid-particularly red-cell-abnormalities might be suitable examples. All of the consistent translocations are relatively restricted to a particular cell lineage. Given the great genetic diversity, the number of different cell types that might become malignant, and the variety of carcinogens to which these cells are exposed, it is surprising that nonrandom karyotypic changes can be detected at all.

C. CONCLUSIONS The relatively consistent chromosome changes, especially specific translocations, that are closely associated with particular neoplasms provide convincing evidence for the fundamental role of these changes in the transformation of a normal cell to a malignant cell. In some tumors, these changes are too small to be detected, and the cells appear, with present techniques, to have a normal karyotype. When one considers the number of nonrandom changes that are seen in a cancer such as ANLL, it is clear that not just one gene, but rather a class of genes, is involved. Our knowledge of the human gene map has developed concurrently with our understanding of the consistent chromosome changes in neoplasia (McKusick and Ruddle, 1977). It is now possible to try to correlate the chromosomes that are affected with

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the genes that they carry. Clearly, these efforts are preliminary, since relatively few genes have been mapped, and since some of the chromosomes that are most frequently abnormal have few genetic markers. In such a preliminary attempt, Rowley (1977b) observed that chromosomes carrying genes related to nucleic acid biosynthesis, and also the specific chromosome region associated with these genes, were frequently involved in rearrangements associated with hematologic cancers. In the future, we will be able to determine the break points in translocations very precisely, to measure the function of genes at these break points, and to compare the activity of these genes in cells with translocations with their activity in normal cells. Such information will be the basis for understanding how chromosome changes provide selected cells in certain individuals with a growth advantage that results in malignancy.

ACKNOWLEDGMENTS The results presented in this article were obtained during research supported in part by the Department of Energy, No. DE-AC02-80EV10360, and by grants supported by PHS Grants Nos. CA-16910, CA-19266, CA-23954, and CA-25568 awarded by the National Cancer Institute, DHHS.

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ONCOGENES OF SPONTANEOUS AND CHEMICALLY INDUCED TUMORS Robert A. Weinberg Massachusetts institute of Technology. Center for Cancer Research and Department of Biology, Cambridge. Massachusetts

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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111. The Retrovirus-Associated Oncogenes .................................. IV. Oncogenes Present in Cells Transformed by Chemical Carcinogens V. Multiplicity of Transforming Genes in 3-MethylcholanthreneTransformed Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Types of Transformed Cells Yielding Focus-Induced D N A . . ............. VII. Multiplicity of Different Human Oncogenes VIII. Analogies between Virus- and Non-Virus-Ind IX. The Process of Activation of Oncogenes . . . . X. The Role of Oncogenes in Carcinogenesis and Maintenance of Phenotype. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. The Proteins Encoded by Activated Oncogenes ......................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The molecular basis of nonviral tumorigenesis is poorly understood, in large part because pertinent experimental approaches have been lacking over the past several decades. Indirect experiments have suggested that DNA damage is a central step in transformation, largely because DNA-damaging agents tend to be carcinogens (McCann and Ames, 1976). However, this realization of the importance of DNA damage in no way facilitates an understanding of which DNA sequences must be damaged in order to induce transformation. Moreover, one still is ignorant of the importance of any such sequence changes compared with a number of nongenetic alterations whose role in inducing transformation may greatly overshadow that of the altered DNA. In the present review, we shall discuss how the process of gene transfer, also termed transfection, makes possible the resolution of some of these issues. The introduction of this experimental approach into the investigation of the molecular basis of oncogenic transformation has already yielded important insights into several central issues in this field, 149 ADVANCES IN CANCER RESEARCH. VOL. 36

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II. A Model of Cellular Oncogenes

The present discussion is based on a model that invokes a cellular oncogene as being centrally important in the process of oncogenic transformation (Comings, 1973; Todaro and Huebner, 1972). According to this model, such an oncogene derives from a normally innocuous cellular DNA sequence whose inappropriate activation during oncogenesis confers a novel transforming potency on the altered gene. This paradigm of oncogenesis contains two elements important for the present discussion. First, the oncogenic sequence arises as an alteration of preexisting cellular information. As such, the resulting oncogenic sequence is of endogenous cellular origin and is therefore not a foreign sequence imposed on the cellular genome from outside the cell. Second, the DNA sequences encoding transformation comprise a discrete unit of function and may represent an allelic variant of a normal cellular gene. This in turn implies that the activation of critical cellular genes, not the global activation of large portions of the genome, is important for oncogenic transformation. Ill. The Retrovirus-Associated Oncogenes

A partial vindication of this model has come from tumor virology. Among four classes of oncogenic viruses that have been intensively studied, it would appear that three-adenoviruses, papovaviruses, and herpesviruses-have evolved specific viral genetic sequences that they use to transform virus-infected cells. In contrast, many members of the fourth class, the retroviruses, induce transformation by expropriating cellular genetic sequences whose presence in the retrovirus genomes confers a transforming potential on the viral genome. Having incorporated a cellular gene into the viral genome, the virus liberates the cellular gene from control mechanisms that governed its expression in the normal cell chromosome and places the cellular gene under viral regulation. Moreover, having become allied with the viral genome, the cellular gene acquires the genetic mobility normally associated with infectious virus. This intimate relationship between retrovirus-associated oncogenes and normal cellular genes has been most thoroughly documented in the case of avian (Rous) sarcoma virus (ASV). The ASV viral gene inducing transformation has been termed the src and encodes a protein of 60,000 daltons (Brugge and Erikson, 1977). Mutations in this src gene affect the transforming competence of the ASV genome. It is clear that the DNAs of normal, uninfected cells carry sequences that are

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strongly homologous, if not identical to the viral src gene (Stehelin et al., 1976). Perhaps most relevant is the fact that a homolog of the src protein is present in normal, uninfected cells (Collett et al., 1979; Oppermann et al., 1979; Sefton et al., 1980). The model of retroviruses acting as transducing agents of cellular genes has been extended to as many as 10 other acutely oncogenic retroviruses (Coffin et d.,1981; Klein, 1980), each of whose genomes appears to carry a different cellular sequence. These retrovirus-associated cellular sequences are termed generically onc genes. With most of these viruses, the presence of a normal cellular gene within the viral genome is indicated only by nucleic acid sequence homology studies (Frankel and Fischinger, 1977; Stehelin et al., 1976). However, more direct demonstrations of the functional and structural affinities of the viral oncogenes and their cellular counterparts have been forthcoming. For example, mutants of ASV whose onc genes have been largely lost through genetic deletion can be shown to reacquire both the missing sequences and oncogenic potency by passage through normal, hitherto uninfected cells (Karess et al., 1979).The restoration of function depends on genetic recombination between the deleted ASV genome and a bloc of normal cellular sequences (Karess and Hanafusa, 1981). This shows that the missing src sequences preexisted in the cell prior to infection by the ASV deletion mutant. A totally different experiment using the onc gene of Moloney murine sarcoma virus (MSV) leads to supporting conclusions for this model. Molecular cloning has allowed the isolation of the normal cellular sequences whose homolog is found as the onc sequence of the MSV genome. This cellular sequence would appear to be very similar if not identical to its viral counterpart, yet the cloned DNA of the cellular sequence is biologically inactive when applied to cells in culture using transfection. However, when the cellular gene is juxtaposed to a viral transcriptional promoter, then DNA carrying the fused sequence exhibits strong transforming competence (Blair et al., 1981). This experimental manipulation recapitulates the outlines of a genetic recombination event between viral and cellular sequences which led originally to creation of the hybrid MSV genome. It indicates that at least one cellular onc gene, that related to M-MSV, possesses an intrinsic transforming competence whose expression awaits appropriate activation, in this case, an activation achieved by an alliance with a viral transcriptional regulator. A third example of affiliation of retrovirus and cellular oncogene derives from work on avian leukosis virus (ALV)-induced tumors (Hayward et al., 1981). This virus appears to carry no transforming

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genes in the viral genome, and its ability to induce a variety of tumors, especially leukoses, has therefore been puzzling. Analysis of the DNAs of a series of avian lymphomas has revealed that the ALV genome, or a fragment thereof, is frequently seen to be integrated next to a specific cellular sequence. This cellular sequence had, fortuitously, been studied previously, since it is identical to the sequence expropriated from the cellular genome during the recombination events that led to the formation of avian myelocytomatosis virus. It appears that the ALV genome, by integrating next to this cellular oncogene, is able to activate the gene transcriptionally. In this case, the alliance between retrovirus and cellular oncogene exists only within the chromosomal DNA of a tumor cell, not within a transmissible genome of an infectious virus particle. It remains to be proved that this ALV-induced gene-oncogene activation is the sole cause of transformation of the avian lymphocyte precursors. Others, using the gene transfer procedure, have detected a transforming gene in these tumors that is unlinked to the ALV genome and would appear to represent a second, independently acting, transforming sequence (Cooper and Neiman,

1980). In principle, activation of cellular oncogenes b y retroviruses might be achieved by two different strategies. The cellular oncogene could suffer alterations in structural sequences encoding a transforming protein, which might in turn confer novel properties on the normal cellular protein. Alternatively, the new gene may differ from the old in its degree of expression. Thus, the novel oncogene may depend on enhanced dosage of a normal cellular protein to elicit a phenotype which low level expression could not achieve. The retrovirus models examined to date favor this latter alternative of increased expression of a structurally normal sequence. For example, cells transformed by ASV or Harvey sarcoma virus (Coffin et al., 1981; Hughes et al., 1979; Oppermann et al., 1979; Sefton et al., 1980) exhibit 50- to 100fold higher levels of the respective onc proteins than one found in normal, uninfected cells. It would appear that the virtually identical, cellular onc proteins, present in low level in normal cells, have no oncogenic effects on cellular phenotype. It is important to note that the several cellular onc genes that have been characterized share properties with many other types of cellular genes studied over the past several years. These onc genes are present in low or single copy number in the genome and have no apparent affiliation with any viral sequence in the normal, uninfected cell (Hughes et ul., 1979). They are conserved evolutionarily like cellular genetic sequences, and they would appear to be as central to normal

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cellular function as would any other gene encoding required cellular enzymic functions. IV. Oncogenes Present in Cells Transformed by Chemical Carcinogens

By providing proof of the existence of cellular onc genes, the retrovirus model has given great impetus to the search for transforming genes present in cells transformed by nonviral agents. Rather than invoking exogenous activation of cellular oncogenes via viral regulation, one may presume that an equivalent activation can be achieved by rearrangement of blocs of normal cellular sequences after carcinogen-induced DNA damage. This as yet unproved model is embedded in much of the discussion that follows. Such a model need not imply that precisely the same genes that are expropriated and activated by retroviruses will also be activated after nonviral insults to cellular DNA. Thus, nucleic acid sequence onc probes, developed from chimeric retrovirus genomes, may not be useful reagents to identify chemically activated cellular oncogenes. Rather, an alternative experimental approach is required to illuminate these genes. Gene transfer represents a direct strategy to detect transforming genes in these transformed cells, and the use of the procedure in no way depends upon reagents developed from retrovirus genomes. This same gene transfer procedure was used with great benefit in resolving a variety of problems surrounding the transforming genes of tumor viruses (Graham, 1977). The gene transfer or “transfection” procedure depends technically on the coprecipitation of DNA with calcium phosphate crystals. These crystals settle onto monolayers of cultured cells and are taken up by the cells via a poorly understood mechanism. A small but significant proportion of the applied DNA succeeds in entering a cell intact and in being expressed stably in a recipient cell and its descendants (Graham and van der Eb, 1973). One exploitation of this transfection procedure has occurred in the area of tumor virology. The DNAs of a variety of tumor viruses are able to induce foci of transformed cells on fibroblast monolayers (Andersson, 1980;Graham, 1977;Graham and van der Eb, 1973).These monolayers usually display these transformed foci several weeks after exposure to the donor DNA. These foci arise as a direct consequence of the uptake and fixation of the transforming genes carried by the transfected viral genomes. Introduction of DNAs of 3-methylcholanthrene-transformedmouse fibroblasts into untransformed, recipient fibroblasts also results in

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transformation of the recipient cells (Shih et al., 1979). Use of DNA from untransformed donor cells does not induce foci of transformation in a recipient cell monolayer. This result provides direct proof that the DNA of a 3-methylcholanthrene-transformed cell is structurally altered with respect to the DNA of an untransformed control cell. This alteration relates directly to an important phenotype, since it is likely that the oncogenic information passed by DNA transfer is partly responsible for the transformation phenotype in the donor cell from which DNA has been prepared. The results of this experiment provide direct proof that DNA alteration occurs in chemically transformed cells in regions of the cellular genome that can elicit a transformation phenotype. As described below, many analogous experiments have been performed, using different types of tumor cells, that further support this conclusion. However, all such experiments give no insight into the mechanisms whereby a mutagen, interacting with cellular DNA, is able to induce activation of cellular oncogenic sequences. The biologically active sequences in the transforming DNAs behave very much like discrete segments of nucleotides. These segments may eventually be defined as genes by conventional genetic criteria. These transforming sequences are transferable from cell to cell by serial passage of DNA. Such serial passaging from donor to recipient uses DNA of the transformed recipient cell as donor DNA in a subsequent cycle of transfection. These repeated manipulations d o not result in the diminution of the transforming activity of the DNAs. Therefore, the transforming sequences do not behave like a group of unlinked, cooperating genes whose association is readily disrupted and diluted b y gene transfer (Shih et al., 1979, 1981). Rather, a transforming sequence behaves like a discrete, compact bloc of information, and is thus reminiscent of a gene. Moreover, the biological activity of a given DNA preparation may be destroyed by certain sequence-specific endonucleases (restriction enzymes) and may be left unaffected by other endonucleases (Krontiris and Cooper, 1981; Shilo and Weinberg, 1981). This means that within a given chemically transformed cell, a discrete, definable segment of DNA carries the transforming potential that is observed upon cell-to-cell transfer of DNA. One can rule out an activation of large numbers of scattered gene blocs whose concerted actions are required for specifying the transformation trait. The transforming activities of these DNAs are not associated with readily detectable retrovirus genomes. Repeated attempts at demonstrating transmissible type C retroviruses in association with the donor cells and derived recipients have yielded no trace of titrable virus

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(Krontiris and Cooper, 1981; Shih et al., 1979, 1981).This has led to the tentative conclusion that the observed transforming genes represent activated versions of normal cellular sequences and are not associated with retrovirus genomes. Such a conclusion will be totally credible only when transforming sequences have been isolated and analyzed in structural detail. V. Multiplicity of Transforming Genes in 3-MethylcholanthreneTransformed Cells

These experimental manipulations allow one to provide preliminary answers concerning the multiplicity of transforming genes that are targets of activation upon treatment with 3-methylcholanthrene. Since the mammalian genome may carry several tens of thousands of genes, it becomes possible that any one of several hundred of these genes will serve as a suitable precursor of the activated transforming genes seen in the 3-methylcholanthrene-transformed cells. Experiments have been performed with the DNAs of four independently transformed mouse fibroblast lines, each of whose transformation derived from exposure to 3-methylcholanthrene. These experiments were designed to measure whether the same gene, or four different transforming genes, was activated in the four transformed lines (Shilo and Weinberg, 1981). As probes for the structures of the different genes, the transforming DNAs were treated with site-specific endonucleases (restriction enzymes), which recognize specific hexanucleotide sequences at the site of cleavage of DNA. Each of the DNAs was treated with one of a series of restriction enzymes and then tested by transfection for retention or loss of biological activity. For example, a transforming gene carried entirely within an EcoRI endonuclease-generated DNA fragment should have its activity unaffected by treatment with this enzyme. Conversely, a transforming gene whose sequence contains the hexanucleotide recognition cleavage site of this enzyme will be split and inactivated after EcoRI endonuclease treatment. Transforming genes associated with different nucleotide sequences should exhibit differing patterns of resistance and inactivation to restriction enzymes, depending upon the nucleotide sequences carried by the gene. This type of experiment yields a specific signature of each gene tested, since the pattern of restriction enzyme sites within a gene is a reflection of the structure of that gene alone and is not shared by other genes of the chromosomal DNA. Because the presence of such a cleavage site depends solely on statistical happenstance, each gene will have a different array of restriction enzyme sites in its DNA.

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The results indicate that all four 3-methylcholanthrene-induced transforming sequences behave identically to one another by these tests. Thus, all four genes are resistant to BamHI endonuclease cleavage, but are inactivated by EcoRI endonuclease. Use of three other endonucleases extends this pattern of identical behavior among the four DNAs. In the case of these four independently transformed mouse fibroblast lines, one concludes that the same cellular sequence was repeatedly activated to yield a transforming gene (Shilo and Weinberg, 1981). The result is perhaps unexpected when considering the multitude of cellular genes, the activation of any one of which might lead to creation of a potent oncogene. This result may pertain only to the small number of 3-methylcholanthrene-transformed mouse fibroblasts tested in these experiments. The same target cells, transformed by other oncogenic agents, might well carry an alternative set of transforming sequences. Perhaps more importantly, nonfibroblastic transformants, such as carcinoma cells, may carry totally unrelated active oncogenes in their DNA (see below). VI. Types of Transformed Cells Yielding Focus-Induced DNA

The utility of these types of experiments is not limited to the DNAs of 3-methylcholanthrene-transformed mouse cells. Rather, a variety of different types of transformed cells yields DNAs that are active when introduced into mouse fibroblasts. This group includes cell lines of rat neuroblastoinas and gliomas (Shih et al., 1981), human neuroblastomas (A. Cassill and R. A. Weinberg, unpublished observations), mouse, rabbit, and human bladder carcinomas (Krontiris and Cooper, 1981; Shih et al., 1981), mouse and human lung carcinomas (C. Shih and R. A. Weinberg, unpublished observations; Shih e t al., 1981), human leukemia (Murray et al., 1981), and colon carcinoma (Murray et al., 1981). It is apparent that the NIH3T3 mouse fibroblasts used as recipients in these gene transfers serve as sensitive indicators of transforming genes from a variety of sources. Since human carcinoma and leukemia DNAs are active in these transfection-focus assays, it is clear that these genes are able to act across tissue and species barriers. Also, it seems that tumors originating via a number of carcinogenic stimuli, including “spontaneous” tumors of unknown etiology, carry transforming genes detectable in these assays. Of more than passing interest is the fact that such experiments can be performed to study transforming genes present in a variety of frequently occurring human neoplasms.

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The successful transfer of these various tumor-transforming genes will shortly make possible the isolation and detailed characterization of these genes. These advances should not obscure the fact, however, that the DNAs of most types of human and nonhuman tumors are inactive upon transfection (Krontiris and Cooper, 1981; Shih et al., 1981). It is possible that these nontransferable genes represent a distinct class of sequences whose nature will differ markedly from those discussed here. Alternatively, their unsuccessful transfer may only reflect a technical shortcoming in the transfection-focus assay system.

VII. Multiplicity of Different Human Oncogenes

The experiments on 3-methylcholanthrene-transformedmouse cells suggested that the same oncogene was activated during four different fibroblast transforming events. The possibility remained that other, as yet unstudied, fibroblasts would carry other unrelated, active oncogenes. Additionally, transformation of different tissue types might also depend on different distinct oncogenes being activated. In fact, work on transforming genes of a human colon carcinoma, a bladder carcinoma, and a myelogenous leukemia cell line indicates three different structures for these three genes (Murray et al., 1981). These analyses depend upon transferring the respective human genes through two cycles of transfection in mouse cells, until the transforming gene represents virtually the only human DNA in the final transformed recipient. The structural outline of each gene could then be analyzed, since all human genes are embedded in complex arrays of highly repeated DNA sequences (Houck et al., 1979). Since the structure of these repeat sequences is species-specific, these sequences can be readily detected and analyzed in mouse cells using nucleic acid sequence probes specific for highly repeated human DNA. This type of analysis, which can be readily extended to other human transforming genes, indicates that each of the human genes present in the mouse cells is embedded in a distinct matrix of repeated sequence blocs in the human genome. This in turn provides strong support for the conclusion that one is studying three unique, different genes. Such work establishes the principle that a number of different human oncogenes exist. Following the result with the 3-methylcholanthrene-induced fibroblast lines, one might speculate that each type of tumor carries a characteristic tissue-specific oncogene, and that all tumors of a given type bear the same activated

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oncogene. This hypothesis remains to be vindicated by further work analyzing the DNAs of an extensive series of different types of tumors. Of related interest is the behavior of mouse fibroblasts that have acquired these various transforming genes. The appearance of these transfected cells in culture is quite similar and is independent of the gene that they have acquired. Also, these transfected cells all grow into fibrosarcomas when seeded into young mice. For example, the transforming gene of a carcinoma converts an untransformed fibroblast into the precursor of a fibrosarcoma. Thus, the transfer of these genes results only in the passage of a transformation phenotype, not in the additional cotransfer of other, tissue-specific characteristics. A series of these genes from different types of tumors would appear to act very similarly. Although some of these oncogenes are associated with different sequences, and likely encode different gene products, the consequences of the presence of these active genes are similar if not identical. Such a pattern of a common, convergent phenotype induced by many genetically distinct oncogenes has been observed when studying the onc sequences of retroviruses (Klein, 1980). VIII. Analogies between Virus- and Non-Virus-Induced Cellular Oncogenes

The various data on nonvirally activated oncogenes suggest a series of analogies or parallel properties between the two groups of transforming sequences. The first, and most salient, feature concerns the structural features of the sequences of both groups. In both cases, the oncogenic sequences behave as though they were discrete blocs of sequences and they segregate upon gene transfer as though they were definable elements that could be termed genes. The virus- and nonvirus-induced oncogenes each constitute groups of genes. The size of each of these groups is not yet discernible. With the virus-associated oncogenes, the gene group contains at least eight members (Coffin et al., 1981). A further, and as yet speculative, analogy stems from the behavior of these two groups of genes. Both groups of oncogenes would seem to derive from the normally innocuous cellular sequences with no viral affiities, which have become subverted by virus or mutagenmediated genetic rearrangements. This conclusion is increasingly well substantiated for the retrovirus onc genes and still rests on only indirect data related to the nonvirally activated genes. Finally, one can mention a striking parallel between ALV-induced chicken leukosis and 3-methylcholanthrene-inducedmouse fibroblast transformation. In a series of avian leukosis tumors, the virus is seen

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repeatedly to be integrated next to the same cellular oncogene, even though the ALV genome is capable of integrating at a virtually infinitely large number of sites in the cellular genome (Hayward et al., 1981). In the case of the 3-methylcholanthrene-transformedfibroblasts, analysis of four different transformants appears to reveal repeated activation of the same cellular gene, even though the responsible carcinogen is likely capable of affecting millions of different sites on the cell genome (Shilo and Weinberg, 1981). In both cases, one suggests that the oncogenic pathway within a given tissue or cell type repeatedly leads to the activation of a specific gene. The parallel properties of the virus-associated and non-virusassociated oncogenes may indicate that the two groups overlap or are congruent with one another. Thus, each retrovirus-associated onc gene may eventually be shown to have a counterpart in a certain type of spontaneous tumor that carries the same activated sequence. Alternatively, the two sets of oncogenes may represent mutually exclusive, nonoverlapping groups. In any case, it is already apparent that the virus-associated onc genes represent an extremely useful experimental model for the as yet unexplored genes of non-virus-induced tumors. IX. The Process of Activation of Oncogenes

The repeated activation of the same mouse fibroblast cellular oncogene after 3-methylcholanthrene carcinogenesis raises an important question. It is clear that the carcinogen can interact with a multitude of sites on the cellular genome. The work on the human tumor genes indicates that several, and likely many, latent cellular oncogenes exist in the genome awaiting appropriate activation. Why then does a nontargeted agent (a carcinogen), when interacting with a multitarget genome, repeatedly activate the same transforming sequences? It is apparent that the process of oncogene activation cannot follow classic models of mutagenesis. Rather, tissue- or cell-specific factors must predispose certain genes to be activated and protect other genes from activation. One such predisposing factor might be the state of expression of a gene in the normal cell prior to the carcinogenic insult. Other paradoxes have been reported that also make it unlikely that classic models of mutagenesis will explain the activation of these genes (Kennedy et d., 1980; Reznikoff et al., 1973).The most recent of these paradoxes derives from in vitro transformation of C3H 1OT1/2 fibroblasts by X-rays. These mouse fibroblasts are of the same cell line that yielded the 3-methylcholanthrene-transformedlines discussed above. The work on X-ray carcinogenesis makes it clear that there is no appar-

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ent proportionality between the number of C3H10T1/2 cells exposed initially to the carcinogen and the number of foci of transformants that appear in descendant cultures. This and earlier work by others (Armitage and Doll, 1957; Nordling, 1953;Whitternore, 1978)leave one with unorthodox hypotheses on the mechanisms of carcinogenesis, all of which are incompatible with a simple target theory of gene activation. One such hypothesis would argue that it is unnecessary for the carcinogen directly to alter the target oncogene in a treated cell in order for that oncogene to become activated in a descendant of that cell. Rather, the carcinogen may induce a metabolic state in the cell, whose presence is required for a second event-the subsequent activation of an oncogene such as those described here (Kennedy et al., 1980). The isolation and detailed nucleotide sequence of an activated oncogene will be forthcoming over the next several years. The sequence information may shed no light on the mechanism by which any novel sequence arrangements of the oncogene were achieved. It is quite possible that the novel nucleotide sequence arises many weeks after the mutagenic activities of a carcinogen. In such cases, the mechanisms that intervene between the initial carcinogenic stimulus and the final creation of novel sequences may not be illuminated by studies on isolated activated oncogenes. X. The Role of Oncogenes in Carcinogenesis and Maintenance of Phenotype

A problem raised by the experiments described here concerns the multiplicity of alterations that are responsible for the achievement of a transformed phenotype. It would be simplistic and likely incorrect to assume that the activation of a cellular oncogene is the only necessary alteration occurring during the conversion of a normal cell into a tumor cell. Rather, a variety of other genetic and epigenetic controls may also need to be perturbed in order to fully realize the oncogenic phenotype. These other controls, whose nature is obscure, will likely be seen to cooperate with the oncogene in eliciting the tumor phenotype. Transformation would appear to be a multistep process (Armitage and Doll, 1957; Nordling, 1953; Whitternore, 1978), and the activation of the oncogene likely represents only one of several necessary alterations that make possible the final transformed phenotype. Such a postulated multistep process would seem to be inconsistent with the results of the gene transfer experiments described here. In these experiments, it appears that a single genetic determinant is able to effect the conversion of the recipient cell from a normal, untrans-

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formed fibroblast into a highly tumorigenic fibrosarcoma cell. These existing experiments would suggest that this single genetic factor has allowed the recipient cell to achieve the total conversion from normalcy to malignancy. The resolution of this dilemma may be provided by detailed study of the characteristics of the recipient cells, which in this case are an established line of mouse fibroblasts that have been immortalized for tissue culture. It is possible that these recipients have already undergone many of the necessary predisposing alterations that normally occur during carcinogenesis in vivo. These cells may therefore be poised for transformation and highly responsive to the introduced active oncogene. These predisposing alterations may therefore eliminate the need for all but the final alteration, which in this case is provided by the transfected oncogene. XI. The Proteins Encoded by Activated Oncogenes

The use of molecular cloning techniques will make possible the isolation of a series of different activated oncogenes. However, structural analysis of the various molecular clones will provide few insights into the mechanisms used by these genes to convert a normal cell into a tumor cell. Analogy with tumor viruses would suggest that these oncogenes specify transformation proteins whose continuous presence is required to maintain the oncogenic phenotype. Such transforming proteins play a central regulatory role in the oncogenic phenotype, and therefore contrast with a myriad of other proteins whose levels are altered only as secondary consequences of oncogenic conversion. These other, secondarily regulated proteins have been extensively catalogued over the past decades. Study of the complex catalog of “transformation specific” proteins of tumor cells has provided relatively few insights into the central mechanism of oncogenic conversion. The exploitation of gene transfer should make possible the detection of a variety of centrally acting transforming proteins. This detection will come via two types of experiments. Some of these oncogenes will be isolated as molecular clones, whose detailed structural features will be resolved by nucleotide sequence analysis. Using techniques pioneered by Walter et al. (1980),the proteins encoded by these genes may then be isolated in a relatively straightforward fashion (Lerner et al., 1981; Walter et al., 1980). An alternative path for the isolation of transforming proteins is less dependent on sophisticated molecular technology and is already being

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followed. This approach depends upon the fact that the transfection procedure allows one to create a mouse fibroblast line that contains the transforming gene as the only foreign tumor gene in an otherwise totally murine genetic background. Such a cell line can be expanded into a mass culture and used to seed a tumor in a young mouse. The serum of a tumored animal may then be analyzed for reactivity to any xenogeneic protein encoded by the introduced transforming gene. The immunogenicity of such a protein is not predictable. One is aided, however, by the fact that the foreign protein may be of foreign species origin and may contain antigenic determinants to which the immune system of the host mouse is not tolerant. This strategy has already been attempted for the detection of any protein encoded by the transforming gene present in the DNA of ethylnitrosurea-induced rat neuroblastomas and glioblastomas. This DNA readily induces transformation of mouse fibroblasts (Shih et al., 1981), and these fibroblasts all contain a phosphoprotein of 185,000 daltons mass. The protein is precipitated specifically by the immune sera of animals carrying neuroblastoma DNA-induced tumors (Padhy et aZ., 1982). Mouse fibroblasts transformed by a variety of other tumor DNAs do not contain the protein, nor do a variety of types of tumor cell lines, with the exception of rat neuroblastomas and glioblastomas (Padhy et aZ., 1982). The behavior of the 185,000 dalton phosphoprotein satisfies many criteria of a protein whose structure is directly encoded by the neuroblastoma-glioma transforming gene. A conservative interpretation, which is justified by currently available data, is that the protein is induced specifically by this transforming gene and by no other, transferable, transforming gene. A rigorous proof of the genetic origins of this protein depends upon detailed structural analysis of the protein, which is not yet at hand. This example is cited to illustrate an experimental strategy that will make possible the detection of a variety of tumor-specific transforming proteins over the next several years. These proteins will be of central importance in understanding the intracellular metabolic alterations that initiate and maintain the oncogenic phenotype. Antisera to these proteins should as well serve as useful reagents in the detection and diagnosis of a variety of specific tumors.

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Brugge, J. S., and Erikson, R. L. (1977).Nature (London) 269,346-348. Coffin, J. M., Varmus, H. E., Bishop, J. M., Essex, M., Hardy, W. D. Jr., Martin, G. S., Rosenberg, N. E., Scolnick, E. M., Weinberg, R. A., and Vogt, P. K. (1981).J . Virol. 40,953-957. Collett, M. S., Erikson, E., Purchio, A. F., Brugge, J. S., and Erikson, R. L. (1979).Proc. Natl. Acad. Sci. U.S.A. 76, 3159-3163. Comings, D. E . (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 3324-3328. Cooper, G., and Neiman, P. E. (1980).Nature (London) 287,656-659. Frankel, A. E., and Fischinger, P. J. (1977).J.Virol. 21, 153-160. Graham, F. L. (1977).A d o . Cancer Res. 25, 1-46. Graham, F. L., and van der Eb, A. J. (1973).Virology 52, 456-467. Hayward, W. S., Neel, B. G., and Asbin, S. M. (1981). Nature (London) 290,475480. Houck, C. M., Rinehart, F. P., and Schmid, C. W. (1979).J.Mol. Biol. 132, 289-306. Hughes, S. H., Payvar, F., Spector, D., Schimke, R. T., Robinson, H. L., Payne, G. S., Bishop, J. M., and Varmus, H. E. (1979).Cell 18,347-359. Karess, R. E., and Hanafusa, H. (1981). Cell 24, 155-164. Karess, R. E., Hayward, W. S., and Hanafusa, H. (1979).Proc. Natl. Acad. Sci. U.S.A. 76, 3 154- 3158. Kennedy, A. R., Fox, M., Murphy, G., and Little, J. B. (1980). Proc. Natl. Acad. Sci. U.S.A. 77,7262-7266. Klein, G., ed. (1980). “Viral Oncology.” Raven, New York. Krontiris, T., and Cooper, G. M. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 1181-1184. Lemer, R. A,, Green, N., Alexander, H., Liu, F. T., Sutcliffe, J. G., and Shinnick, T. M. (1981). Proc. Natl. Acad. Sci. U S A . 78,3403-3407. McCann, J., and Ames, B. M. (1976). Proc. Natl. Acad. Sci. U.S.A. 73,950-954. Murray, M., Shilo, B., Shih, C., Cowing, D., Hsu, H. W., and Weinberg, R. A. (1981).Cell 25,355361. Nordling, C. 0 . (1953). Br. J . Cancer 7, 68-72. Oppermann, H., Levinson, A., Varmus, H., Levintow, L., and Bishop, J. M. (1979).Proc. Natl. Acad. Sci. U.S.A. 76, 1804-1808. Padhy, L. C., Shih, C., and Weinberg, R. A. (1982). Cell (in press). Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. (1973).Cancer Res. 33,32313238. Sefton, B. M., Hunter, T., and Beemon, K. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 2059-2063. Shih, C., Shilo, B., Goldfarb, M. P., Dannenberg, A., and Weinberg, R. A. (1979).Proc. Natl. Acad. Sci. U.S.A. 76, 5714-5718. Shih, C., Padhy, L. C., Murray, M., and Weinberg, R. A. (1981).Nature (London) 290, 261-264. Shilo, B., and Weinberg, R. A. (1981). Nature (London) 289, 607-609. Stehelin, D., Varmus, H. E., Bishop, J. M., and Vogt, P. K. (1976).Nature (London) 260, 170-173. Todaro, G . J., and Huebner, R. J. (1972). Proc. Natl. Acad. Sci. U.S.A. 69, 1009-1015. Walter, G., Scheidtmann, K. H., Carbone, A., Laudano, A. P., and Doolittle, R. F. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 5197-5200. Whittemore, A. S. (1978).Ado. Cancer Res. 2 7 , 5 5 4 8 .

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RELATIONSHIP OF DNA TERTIARY AND QUATERNARY STRUCTURE TO CARCINOGENIC PROCESSES Philip D. Lipetz, Alan G. Galsky,' and Ralph E. Stephens Department of Radiology. The Ohio State University. Columbus, Ohio

I. Introduction 11. Background

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A. The DNA Superstructure B. Nucleoid Measurement of DNA Strand Breaks ....................... C. Possible Chromatin Origins of Nucleoid DNA Supercoiling . . . . . . . . . . . D. Physiological Relevance of Decreased Nucleoid DNA Supercoiling . E. Novobiocin and Nalidixic Acid as Probes of DNA Supercoiling . . . . . ......... F. Significance of Prokaryotic DNA Supercoiling G. Significance of Eukaryotic DNA S 111. Cancer and DNA Superstructure . . . . ......................... A. Crown Gall Tumorigenesis .................... B. Chronic Lymphocytic Leukemia ..................................... C. TPA Alterations of DNA Superstructure in Normal Human Cells .......

167 169 173 175 178 185 187 189 189 193 197 . . . . . 199 IV. Conclusion . . . . . 202 References ............................ . . . . . . . . . . 204 Note Added in Proof ................................................... 210

I. Introduction

Eukaryotic DNA possesses at least four subchromosomal levels of organization. Cancer studies have focused upon the primary (nucleotide sequence) and secondary (double-stranded helix) levels of DNA organization. This chapter reviews (a) DNA superstructure as studied in nucleoids (permeabilized and protein-depleted nuclei); and (b)possible correlations between modification of the tertiary (DNA supercoiling) and quarternary (supercoiled domains) levels of DNA organization and carcinogenic processes. When the eukaryotic genome is isolated as a nucleoid, the DNA is supercoiled (DNA negative superhelicity) (Cook and Brazell, 1975; Benjayati and Worcel, 1976; Lipetz, 1981). Most eukaryotic DNA supercoiling results from the wrapping of DNA around histone core Department of Biology, Bradley University, Peoria, Illinois 61625.

165 ADVANCES IN CANCER RESEARCH, VOL. 36

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12006636-X

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particles to form nucleosomes (McGhee and Felsenfeld, 1980). DNA supercoiling may also represent conformational stress induced by the condensation of nucleosomes into 25-30 nm chromatin fibers (Worcel et al., 1981).Since there is no compelling evidence to necessitate the existence of a eukaryotic topoisomerase that induces generalized DNA supercoiling in a manner analogous to the prokaryotic gyrase (Denhardt, 1979; Champoux, 1978), physiologically relevant alterations of DNA supercoiling may represent a probe of chromatin structure. A causal relationship between alterations of DNA superstructure and carcinogenesis has not been demonstrated, although the following suggestive correlations have been noted.

1. Agrobacterium tumefaciens-induced tumorigenesis may be modulated by DNA supercoiling (Lipetz et al., 1981a). 2. Lymphocytes isolated from chronic lymphocytic leukemia (CLL) patients have abnormally high DNA supercoiling (Yew and Johnson, 1979a; Lipetz et al., 1981b), and differentiation of such cells (Totterman et al., 1980) is accompanied by renormalized DNA supercoiling (Lipetz et al., 1981b). 3. Treatment with the carcinogenic promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alters normal and CLL human lymphocyte and human fibroblast DNA supercoiling and the size of normal human lymphocyte DNA domains (Lipetz et al., 1981b). 4. Many carcinogens alter DNA supercoiling (Drinkwater et al., 1978; Lipetz, 1981; Lipetz et al., 1982).

The purpose of this review is to stimulate research; therefore, we will report these intriguing correlations before causality is fully established. Interpretation of nucleoid results has been traditionally retarded by two uncertainties: (a) the origin of nucleoid DNA supercoiling; and (b) the relevance of nucleoid DNA supercoiling, since DNA strand breaks result in decreases in nucleoid DNA supercoiling without corresponding decreases in in vivo DNA supercoiling. Herein w e resolve the latter problem and discuss proposed solutions to the former problem. We also review evidence that the nucleoid technique can be used to quantitate extremely low levels of DNA strand breaks (less than one per 4.4x 10" daltons of DNA). This article is not intended to be an exhaustive review of DNA superstructure and topoisomerase enzymology, but rather to impart enough information so that the reader can form an appreciation of the possible significance of DNA superstructure to the cancer problem.

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It. Background

A. THE DNA SUPERSTRUCTURE O F NUCLEOIDS The DNA superstructure is conveniently studied in permeabilized and protein-depleted nuclei called nucleoids (Cook and Brazell, 1975). Gentle lysis of cells in the presence of nonionic detergents and high salt results in nucleoids that contain nuclear DNA and small quantities of bound protein and RNA (Cook and Brazell, 1976b, 1978). Nucleoids are very similar to nuclear matrix structures consisting of membrane and attached DNA (Nelkin et al., 1980). Nucleoids possess DNA supercoiling (DNA negative superhelicity) (Benyajati and Worcel, 1976; Cook and Brazell, 1975, 197613) and are partitioned into multiple supercoiled DNA domains (Benyajati and Worcel, 1976; Cook and Brazell, 1975, 1976b, 1978). Nucleoid DNA superstructure is indirectly demonstrated by techniques originally developed by Vinograd to analyze supercoiled, closed circular, double-stranded viral DNAs (for a complete review, see Bauer and Vinograd, 1974). Eukaryotic DNA supercoiling can be quantitated by sedimentation of nucleoids in ethidium bromide (EB) containing neutral sucrose gradients (Benyajati and Worcel, 1976; Cook and Brazell, 1975; Lipetz et al., 1982). The intercalation of EB into DNA tends to introduce DNA-positive superhelicity into a closed circular molecule whose DNA twist is held constant by high ionic concentration. As the EB concentration increases, these tendencies counter the preexisting negative superhelicity and force nucleoid DNA into a more extended conformation resulting in a decreased sedimentation rate. When the average preexisting negative superhelicity is countered b y equal but opposing forces of EB intercalation, then nucleoid DNA is maximally extended and the sedimentation rate of the nucleoids is minimal. Higher concentrations of EB induce positive superhelicity, and nucleoid DNA again assumes a compact conformation with a corresponding increase in the rate of sedimentation. The EB concentration required to induce minimal sedimentation is proportional to the preexisting average DNA negative superhelicity (DNA supercoiling). This quantitation of DNA supercoiling reflects the conformation of DNA from which most constraints imposed by chromosomal proteins have been removed by high salt. High salt also increases DNA supercoiling, and so the observed DNA supercoiling and any alterations due to strand breaks in DNA supercoiling will be magnified. When the NaCl concentration is lowered from the generally used concentration

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of 2 M to a more physiologically relevant concentration of 0.2 M, nucleoid DNA supercoiling is halved (Cook and Brazell, 1978). Preliminary reports indicated that DNA supercoiling was similar to HeLa, chicken, insect, and amphibian nucleoids (Cook and Brazell, 1976a). Ethidium bromide titrations of HeLa nucleoids indicate that there is one supercoil every 90-180 base pairs (bp) (Cook and Brazell, 1977). Electron micrographs of HeLa nucleoids indicate the presence of a supercoil every 200-750 b p (Mullinger and Johnson, 1979); however, this latter methodology is subject to artifacts of mounting. At the time these demonstrations were performed the eukaryotic nucleoid technique was in the early stages of development, and therefore small variations would have been beyond the range of experimental error. As increasing DNA strand breaks are introduced, the rate of nucleoid sedimentation in neutral sucrose gradients decreases (Cook and Brazell, 1975). One DNA strand break completely relaxes the supercoiling of covalently closed circular DNA (cccDNA). One DNA strand break however, does not completely relax the supercoiling of the entire eukaryotic nucleoid genome. Therefore, there must be limits that prevent a DNA strand break from acting as a relaxation swivel for the entire genome. Cook and Brazell (1976b) proposed that eukaryotic nucleoid DNA is organized into multiple domains and that each DNA domain is constrained by nonhistonal protein and RNA in such a manner that the DNA supercoiling of only one domain can be relaxed by a single DNA strand break. (The terms “DNA loops” and “chromosome folds” are used by some authors in preference to the term “DNA domains .”) Cook (1974) originally hypothesized that a DNA domain corresponded to a replicon or chromomere unit. Vogelstein et al. (1980) have noted that DNA replication appears to start at the point where a DNA loop is attached to the nuclear matrix and then continues throughout the entire DNA domain. However, early evidence suggests that nucleoid DNA domains are too large to be single replicon units. DNA domain size appears to be approximately lo9 daltons of DNA. Cook and Brazell (1975) utilized the induction of DNA strand breaks by ionizing radiation, the assumption of single-hit kinetics, and nucleoid sedimentation in neutral sucrose gradients to estimate HeLa DNA domain size to be approximately lo9 daltons. Previously published sedimentation patterns of nucleoids exposed to ionizing radiation can be similarly analyzed to obtain DNA domain sizes of 1.8 x lo9 daltons DNA for human dermal fibroblasts (Lipetz et al., 1982) and 3.8 X log daltons DNA for rat spleen cells (lymphocytes) (Egg e t al.,

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1977). We have also used this methodology to calculate a DNA domain size of approximately 8.4 x los daltons for normal human lymphocytes. Other estimates of nucleoid DNA domain size have resulted in much lower values. Cook and Brazell (1978) more recently used isolated HeLa nucleoids to obtain an estimate of 1.8 x 10' daltons. Mullinger and Johnson (1979) used electron microscopy of isolated nucleoids to calculate that a supercoiled domain of HeLa is 0.4 to 3 X lo* daltons. Benyajati and Worcel (1976) utilized alkaline sucrose sedimentation of DNase I-generated DNA fragments to estimate a Drosophila DNA domain size of 5.7 x lo7 daltons. Pinon and Salt (1977) used a similar technique to estimate a DNA domain size of 1.5 x 10' daltons in yeast. One possible resolution of these differences in DNA domain size involves the extreme sensitivity to mechanical disruption of nucleoid DNA (Lipetz et al., 1982). We have noted that handling (or isolating) nucleoids results in sufficient DNA strand breaks to account for the differences between results obtained with previously isolated nucleoids, and those results obtained with nucleoids created by lysis on top of the analytical gradient (as in the preceding paragraph). Further experimental variation could have been introduced either by alkaline sucrose gradients cleaving DNA at alkaline-labile sites (Brash and Hart, 1978) or by nonrandom access to nucleoid DNA by DNase I under the conditions utilized. Thus, we conclude that an average DNA domain size is approximately los daltons of DNA in length.

B. NUCLEOIDMEASUREMENTOF DNA STRAND BREAKS The nucleoid technique can be used qualitatively (Cook and Brazell, 1976c; Weniger, 1979) and quantitatively (Lipetz et al., 1982) to measure the relative increase in DNA strand breaks when control and treated samples are compared. Single-hit kinetics are used to calculate the number of evenly distributed DNA strand breaks that must be introduced to relax the DNA supercoiling of all domains and thereby results in minimal nucleoid sedimentation. The difference between the number of strand breaks required to completely relax a treated sample and the number required to relax a control is the number of preexisting DNA strand breaks in the treated sample. Thus, the nucleoid technique quantitates the relative increase in DNA strand breaks between two samples; it does not quantitate the absolute number of DNA strand breaks.

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The sensitivity of the nucleoid technique as a measurement of DNA strand breaks can be determined by the minimum number of DNA strand breaks that induce a reproducibly detectable decrease in nucleoid sedimentation. Increments as low as that induced by 30 rad X ray [one DNA strand break per 2.2 x 10" daltons of DNA (Brash, 1979; Dean et al., 1969)] in normal human dermal fibroblasts (Fig. 1A) (Lipetz et al., 1982) and less than 17 rad X ray in normal human lymphocytes (greater than one DNA strand break per 4.4 X 10'" dal-

"0°

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FIG. 1. Nucleoid sensitivity to X-ray induction of DNA strand breaks. The minimum number of DNA strand breaks that can be resolved by the nucleoid technique was determined by inducing DNA strand breaks with X-irradiation. FS1, normal human dermal fibroblasts (foreskin), PDLp(A) and normal human mixed lymphocytes (B) were One DNA exposed to X ray and examined as previously reported (Lipetz et al., 1981~). strand break per 2.2 x 10"' daltons DNA (30rad X ray) is resolvable in FS1 cells, and less than one DNA strand break per 4.4 x 10'O daltons DNA (less than 17 rad X ray) is resolvable with mixed normal human dermal fibroblasts. Error bars indicate standard error of the mean. Our nucleoid sedimentation technique has been described elsewhere (Lipetz et al., 1982). It is essentially the method of Cook and Brazell (1976a) with several important variations: (a) the presence of DNA in individual gradient fractions is detected by postsedimentation labeling of DNA with the fluorescent dye 4',6-diamidino-Bphenylindole (DAPI); (b) gradients are fractionated with a Buchler miniscus following fractionator, thus avoiding the artifacts first described by Weniger (1979) that are created when gradients are fractionated from the bottom of the tube; (c) the lysing solution is prepared fresh daily because old lysing solution results in erratic sedimentation behavior; (d) gradients containing freshly lysed nucleoids are handled extremely gently because the method is sensitive to the extremely low numbers of DNA strand breaks induced by mechanical disruption. The migration of human dermal fibroblast DNA was examined by ultraviolet techniques as detailed in Cook and Brazell (1975).

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B

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tons) (Fig. 1B) can be detected. The greater sensitivity of lymphocyte nucleoids to DNA strand breaks may be due to their unusually large DNA domain size acting to amplify the effect of a single DNA strand break by resulting in the relaxation of the DNA supercoiling of a greater length of DNA. In our system, the nucleoid technique approaches minimal nucleoid sedimentation at an X-ray dose (500 rad) that is at the lower limit of accurate detection by alkaline sucrose gradient techniques; indicating that the nucleoid technique should be considered only when low levels of DNA strand breaks are anticipated. The nucleoid technique is perhaps the most sensitive of the four commonly used methods of quantitating DNA strand breakage; the other three are alkaline sucrose gradients, alkaline elution, and Sarma gradients (Brash and Hart, 1978).Yew and Johnson (1979b) utilized a variant of the nucleoid technique to measure strand breaks induced by the endonuclease recognition step of DNA excision repair and demon-

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strated that successful detection utilizing alkaline sucrose gradients required at least one order of magnitude more UV irradiation to stimulate measurable DNA repair than did the nucleoid technique. Alkaline elution techniques apparently rely upon a strand break acting as a focus from which the DNA molecule can unwind, denature, and assume a conformation capable of passing through a membrane filter (Kohn et al., 1977). More recent calculations indicate that alkaline elution techniques may not be quite as sensitive as originally thought and may be capable of resolving only approximately the number of DNA strand breaks induced by 300 rad X ray (Brash and Hart, 1978). Sarma-type gradients are alkaline sucrose gradients in which incomplete lysis of the nuclei occurs; thus, these partially lysed nuclei retain many of the features of DNA superstructure that are ordinarily removed in alkaline sucrose gradients (Zubroff and Sarma, 1976; Cox et al., 1973). We suggest that the latter two techniques rely on hydrodynamic and biophysical principles similar to the nucleoid technique but, unlike the nucleoid technique, do not fully exploit the basis of this similarity, namely, DNA tertiary and quaternary structure. Another advantage of the nucleoid technique over the three other techniques is that it utilizes neutral sucrose gradients. Therefore, what is measured is more likely to have been a DNA strand break in vivo than are DNA strand breaks measured under alkaline conditions [such alkaline conditions may have induced the breaking of alkali labile bonds, such as apurinic sites and phosphotriesters, during lysis and sedimentation (Brash and Hart, 1978)l. Examination of DNA from tissue samples or mitotically inactive cells requires techniques that do not rely on the use of radiolabel. Cook and Brazell (1975, 197613) used ultraviolet (UV) absorption to detect the location of nucleoid DNA in gradients; however, the Triton X-100 detergent used to lyse nucleoids also absorbs UV. Therefore, to avoid masking information contained in the detergent-containing upper portion of the gradient, it was proposed that nucleoids be lysed and isolated separately from the analytical procedure (Cook and Brazell, 1977), a procedure likely to introduce DNA strand breaks and reduce the sensitivity of the method (Lipetz et al., 1982). A wide variety of fluorescent indicators of DNA were examined by Lipetz e t al. (1982) as possible alternatives to UV detection of nucleoid DNA in nonradiolabeled cells. Ethidium bromide was rejected as a fluorescent agent due to formation of fluorescent complexes with non-DNA components and a lower DNA sensitivity. 4’,6-Diamidino-2phenylindole 2 HC1 (DAPI) was found to be well suited to this application since it does not significantly interact with Triton X-100 or non-DNA cellular components (Brunk et al., 1979; Kapuscinski and

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Skoczylas, 1977) and is a sensitive indicator of double-stranded DNA (Lipetz et d., 1982). The sensitivity of DAPI is such that only 15,000 human dermal nucleoids (100 ng of DNA) are required to produce a peak signal of 2.5 times background fluctuation (Lipetz et al., 1982). The nucleoid technique combined with DAPI fluorescent labeling of centrifuged nucleoid DNA has been used as a technique to examine the in vivo response of rat liver cells to carcinogen exposure (Lipetz et al., 1982). The advantages of a technique that can quantitate extremely low levels of DNA damage as well as DNA supercoiling from whole animal tissue samples is obvious. Low levels of non-strandbreak forms of DNA damage can be detected by treating isolated nuclei with repair endonucleases that induce a DNA strand break at the DNA damage site (Brash, 1979) and then lysing the nuclei on top of the analytical nucleoid gradient. It is hoped that this technique will allow in vivo studies of carcinogen-induced DNA pathologies at physiological levels of carcinogen exposure. Similarly, the nucleoid technique will detect extremely low levels of DNA repair. For example, Yew and Johnson (1979b) used nucleoids, combined with the inhibition of the DNA strand-break-rejoining step of DNA excision repair, to detect the repair of the damage induced by 0.01joules/m2 UV in unstimulated lymphocytes but had to increase the UV exposure to 0.1 joules/m2 in order to detect damage by alkaline sucrose gradients. One advantage of such a technique is that it does not require hydroxyurea to inhibit scheduled DNA synthesis and thus avoids possible artifactual inhibition of unscheduled excision-repair DNA synthesis b y hydroxyurea (Collins and Johnson, 1979). C. POSSIBLECHROMATINORIGINSOF NUCLEOID DNA SUPERCOILING

DNA supercoiling primarily results from interactions between DNA and histone proteins (Crick and Klug, 1975; Sobell et al., 1976). The wrapping of approximately 140 base pairs (bp) of DNA in a nucleosomal particle (DNA plus an octomer of histones H2A, H2B, H3, and H4) can result in supercoiling of the DNA (Crick and Klug, 1975; Sobell et al., 1976; Felsenfeld, 1978; Champoux, 1978; Weintraub et al., 1976). The wrapping of DNA in a nucleosomal core particle introduces at least 1% DNA supercoil (McGhee and Felsenfeld, 1980). It is not clear whether the DNA supercoiling that results from the wrapping of DNA around nucleosomal core particles can vary and exert a regulatory influence. Such DNA supercoiling is bound in a nucleosome particle and therefore is constrained to a limited range of conformations (Champoux, 1978). However, if transcribing DNA re-

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gions are either not nucleosome associated (Foe, 1978; Scheer, 1978; Grainger and Ogle, 1978) or are in altered nucleosome conformations (Grainger and Ogle, 1978; Butler et aZ., 1978; Johnson et al., 1978, 1979), then organization of the nucleosomal induced DNA supercoiling may be a much more physiologically significant phenomenon. Furthermore, the conformational constraints placed upon nucleosomal induced DNA supercoiling will be at least partially relaxed during passage of the DNA replication fork (see below). The DNA of each nucleosome is wrapped around the histone octomer at least 1% times; thus, one would predict the introduction of 1% DNA supercoils per nucleosome. However, the average change in DNA supercoiling induced by the reconstitution of nucleosomes onto SV40 DNA is only 1% DNA supercoils induced per nucleosome (Shure and Vinograd, 1976). Similarly, Cook and Braze11 (1977) observed an interval of 90-180 bp between the center of HeLa superhelical coils: since there are 188 b p between HeLa nucleosomes (Compton et al., 1976), only 1 to lY2 DNA supercoils are observed per human nucleosome. Thus, it appears that the observed DNA supercoiling cannot b e solely and simply the result of DNA wrapping around histones to form nucleosomal core particles, since some process must reduce the theoretical average DNA supercoiling. One possible solution to this apparent conflict is if the nucleosomeassociated DNA varies by less than 5% in its twist when compared with internucleosomal linker region DNA (Prunell et al., 1979; Rhodes and Klug, 1980; Crick, 1976). This solution is possible since, if the DNA twist does vary in nucleosome-associated DNA, it will be observed as a change that will be interpreted as a change in DNA supercoiling. The quantity measured in electrophoresis of SV40 DNA or nucleoid sedimentation is a change in the topological linking number ( L ) ,defined as the sum of DNA twist ( T )and writhing ( W )where W represents conformational deformations including DNA supercoiling, and is usually negative (DNA negative superhelicity) (Crick, 1976; Fuller, 1975). In the nucleoid technique and in electrophoresis T is assumed to be held constant by ions, and thus dL = dW. When in vivo DNA supercoiling (-W) is made more negative (increased DNA supercoiling) by nucleosomal interactions, an in vivo increases in DNA twist will result in less of a decrease in the quantity, L , which is measured in vitro than would be predicted by the alteration in DNA supercoiling alone. Prunell et al. (1979) approached this problem by demonstrating that the periodicity of DNase I digestion of DNA is approximately 10.4 bp in both nucleosomal and linker-region DNA. If DNase I digestion is assumed to reflect DNA twist, then this suggests that DNA twist does not vary in nucleosomes. However, DNA twist

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may in fact vary in nucleosomes, but DNA-histone interactions may restrict the accessibility of DNA to DNase I digestion so that no difference in digestion pattern is visible (Prune11 et al., 1979; Rhodes and Klug, 1980; Girardet and Lawrence, 1979). Assuming that chromatin-associated DNA maintains a constant twist, Worcel et al. (1981) have proposed a model of nucleosome stacking that is mathematically consistent with both sets of conflicting data. The histone, H1, condensed 25-30 nm fiber of chromatin (Gorka and Lawrence, 1979; Matsumoto et aZ., 1980; McGhee and Felsenfeld, 1980) is proposed to consist of nucleosomes stacked with a specific arrangement of linker-region DNA such that the d L is approximately one per nucleosome. One prediction of this model is that adjacent nucleosomes are arranged in an opposing manner so as to minimize dL per nucleosome. This prediction appears to be consistent with Burgoyne and Skinner’s (1981) observation that the DNase I digestion pattern of chicken erthrocyte nuclei reflects a dinucleotide pattern where every other nucleosome is resistant to digestion. The nucleosome stacking pattern proposed by Worcel et aZ. would result in d L of one per nucleosome; however, there may be a dL of approximately 1% per nucleosome (Compton et al., 1976; Cook and Brazell, 1977; Shure and Vinograd, 1976; Lipetz, 1981). Thus, if the nucleosome stacking model is applicable, chromatin may be composed in a variety of nucleosome stacking patterns and DNA supercoiling could be both decreased and increased by varying the ratio of these types of stacking. Also theoretically possible are models in which L is altered either b y regions of positive DNA superhelicity or altered conformation to Z form DNA (Benham, 1980a); however, the transformation to Z form appears to require specific DNA sequences (Wang et al., 1979) and so is unlikely to be a generalized phenomenon. While a modified version of the nucleosomal stacking model of Worcel et d . (1981) is appealing, it is not yet possible unequivocally to determine which, if any, model is correct. However, in any of the above models, physiologically relevant alteration of nucleoid DNA supercoiling is a probe of chromatin structure, although the exact chromatin structure reflected is dependent upon which model(s) is correct. O F DECREASED NUCLEOID D. PHYSIOLOGICAL RELEVANCE DNA SUPERCOILING

One problem that has previously impeded the study of nucleoid DNA supercoiling has been that alterations of eukaryotic nucleoid DNA supercoiling do not necessarily indicate that correspondingly

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significant in vivo alterations of DNA supercoiling and chromatin structure have occurred. Decreased DNA supercoiling may be observed either in nucleoids with intact DNA molecules or in nucleoids with DNA molecules containing DNA strand breaks (Cook and Brazell, 1975; Lipetz, 1981). Nucleoids are prepared in high salt, which removes most chromosomal constraints, thereby allowing a single DNA strand break to relax the DNA supercoiling of an entire DNA domain (Cook and Brazell, 1975; Lipetz, 1981; Lipetz et al., 1982). In contrast, DNA supercoils are constrained in vivo by association with nucleosomal core particles and chromosomal proteins such that a DNA strand break does not appear to be able to relax DNA supercoiling (Sinden et al., 1980). Sinden et al. (1980)have attempted to observe the relaxation of linker region DNA after gamma irradiation. Their results indicate either that there is no relaxation of linker region DNA or that the length of DNA relaxed per gamma-induced DNA strand break is less than 5000 bp. [Interpretation of these experiments may be further complicated by the fact that gamma irradiation induces DNA-histone cross-links that may alter the relaxation characteristics of chromatin-associated DNA (Mee and Adelstein, 198l)l. It is possible to discriminate between decreases in nucleoid DNA supercoiling induced by DNA strand breaks (which probably do not represent large alterations in in vivo chromatin structure) and decreases in DNA supercoiling of intact DNA (which probably do represent large alterations ofin viuo chromatin structure). Nucleoid DNA is organized into domains whose D N A supercoiling is independently modulated in vitro (Cook and Brazell, 1975; Benjayati and Worcel, 1976; Lipetz, 1981). A DNA strand break will result in the loss of a single domain’s, and only that domain’s, DNA supercoiling. The average D N A supercoiling of the unaffected domains will remain unchanged. Thus, both unaltered nucleoids and nucleoids containing DNA strand breaks will exhibit minimum sedimentation at the same ethidium bromide (EB) concentration. In contrast, nucleoids with altered DNA supercoiling as a result of mechanisms involving intact DNA will exhibit minimal sedimentation at a different E B concentration than will control nucleoids. Verification of this analysis was demonstrated by the radical change in sedimentation pattern induced by 75 rad X ray [one strand break per 9 X lo9daltons DNA (Brash, 1979; Dean et al., 1969; Lipetz et al., 1982)l (Fig. 2). Figure 2 demonstrates that the EB concentration at which minimum nucleoid sedimentation is obtained does not change despite the fact that DNA strand breaks have decreased the average DNA supercoiling per DNA domain (including both broken and un-

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FIG.2. Nucleoid sedimentation in ethidium bromide (EB) gradients after 75 rad X ray. DNA strand breaks will relax nucleoid DNA supercoiling, but probably not in uiuo DNA supercoiling. Use of the nucleoid technique to measure DNA supercoiling requires that reduced nucleoid DNA supercoiling due to DNA strand breaks be differentiatable from altered DNA supercoiling with intact DNA, which probably represents altered in uiuo chromatin structure. FS1 cells were irradiated with 75 rad X ray while on ice and then converted to nucleoids and analyzed as per Lipetz et al. (1982) except that samples were centrifuged for 100 min at 10,000 rpm. The results indicate that in the presence of preexisting DNA strand breaks (one per 9 x lo8daltons of DNA), nucleoids continue to exhibit minimum nucleoid sedimentation at the same EB concentration as do nucleoids derived from cells without induced DNA strand breaks. Thus, a change in the EB concentration at which nucleoids derived from treated cells exhibit minimum sedimentation indicates that the DNA supercoiling has been altered in a way that probably reflects in uiuo chromatin structural alterations. Error bars indicate standard error of the mean.

broken domains). When decreased nucleoid DNA supercoiling represents a change in DNA conformation that will be reflected in in uivo chromatin structures, then the EB concentration at which minimum nucleoid sedimentation is observed will be decreased. Conversely, when DNA strand breaks have resulted in a decrease in the average DNA supercoiling per nucleoid domain, which probably is not indicative of large-scale in v i m alterations of chromatin structure, then the E B concentration at which minimum nucleoid sedimentation is achieved will remain the same so long as a majority of the domains are unaffected. It should be noted that the above discussion applies mainly to the DNA supercoiling associated with the 10 nm chromatin fiber; if stacking of nucleosomes into the 25-30 nm fiber also modulates DNA

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supercoiling, then it is relatively easy to construct models in which a linker-region DNA strand break alters the nucleosome stacking of a nucleosome oligomere. Such an alteration in nucleosome stacking would probably not be reflected in the experiments of Sinden et al. (1980). Thus, we are aware of models in which a DNA strand break might alter local regions of DNA supercoiling i n vivo; however, at the present time there is no evidence to support such a model.

E. NOVOBIOCINAND NALIDIXICACID AS

PROBES OF

DNA SUPERCOILING In prokaryotes, novobiocin and nalidixic acid have become accepted agents for decreasing DNA supercoiling (Cozzarelli, 1980). Because they also reduce eukaryotic DNA supercoiling, much effort has centered upon the metabolic alterations induced by these antibiotics in eukaryotes. Many eukaryotic topoisomerases have been isolated, and their modulation of chromatin structure may be the target of these antibiotics; however, aukaryotic topoisomerases are beyond the scope of this review. Novobiocin and nalidixic acid will be considered as preliminary model systems with which to study the significance of DNA supercoiling. In Section II1,C we shall propose that the carcinogenic promoter 12-O-tetradecanoyl-phorbol-13-acetate(TPA) is another such probe. Nalidixic acid and novobiocin have been repeatedly proposed as systems with which to study eukaryotic DNA supercoiling. Mattem and Painter (1979b) demonstrated that Chinese hamster ovary (CHO) cell nucleoid DNA supercoiling was decreased by novobiocin treatment. Mattern and Painter (1979a,b) also demonstrated that novobiocin treatment decreased initiation of scheduled replicative DNA synthesis and that similar inhibition could be induced by directly decreasing CHO DNA supercoiling with ethidium bromide. Since novobiocin also decreases prokaryotic DNA supercoiling by inhibiting the activity of subunit B of prokaryotic gyrase (topoisomerase 11) (Gellert et d.,1976a,b), Mattern and Painter (197913) suggested that such an enzyme might be present in eukaryotes. Collins and Johnson (1979) confirmed novobiocin’s activity and further demonstrated that the endonuclease recognition step of excision repair of UV-induced DNA damage was also inhibited b y novobiocin treatment. Lipetz et al. (1980a) and Mattern and Scudiero (1981)confirmed novobiocin inhibition of scheduled and repair DNA synthesis and demonstrated that similar inhibitions were induced by nalidixic acid. Since nalidixic acid also inhibits subunit A of prokaryotic gyrase (Sugino et al., 1977; Gel-

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lert et al., 1977), the total evidence appeared to be consistent with the existence of an eukaryotic topoisomerase analogous to gyrase. The significance of these demonstrations has been questioned, however, since direct efforts to detect gyrase-like activity in nuclear protein preparations have failed (Champoux, 1978). There appears to be little reason to require a gyrase, since nucleosomes are capable of inducing more eukaryotic DNA supercoiling than is observed in chromatin. Therefore, the problem would appear to be relaxation, not introduction, of eukaryotic DNA supercoiling. Finally, Edenberg (1980) demonstrated that while novobiocin did inhibit SV40 DNA supercoiling, coumermycin, another inhibitor of subunit B of prokaryotic gyrase (Gellert et aZ., 1976b), did not. This finding is inconsistent with the existence of a gyrase that would be truly analogous to the prokaryotic gyrase. Edenberg (1980) explained novobiocin inhibition of scheduled DNA synthesis by demonstrating and reviewing evidence that novobiocin inhibited the activity of eukaryotic DNA polymerases involved in scheduled DNA synthesis (Sung, 1974). Nalidixic acid also can inhibit the DNA polymerases involved in B

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FIG. 3. Nalidixic acid treatment reduces FS1 DNA supercoiling. FS1 cells were exposed to 7 hr of either 2000 pg/ml nalidixic acid (A) or 200 p g m l nalidixic acid (B). Both concentrations induce similar decreases in FS1 DNA supercoiling, and both decreases are physiologically relevant in that they are not due to DNA strand breaks. The difference in the sedimentation pattern of A and B results from different sedimentation ) that conditions; both experiments were via the protocols of Lipetz et al. ( 1 9 8 1 ~except samples in A were centrifuged for 4% hr at 5000 rpm in a Beckman SW27 rotor, and samples in B were centrifuged for 100 min at 10,000 rpm. Error bars indicate standard error of the mean.

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PHILIP D. LIPETZ ET AL.

scheduled DNA replication (Nakayama and Sugino, 1980; Poulson et al., 1974). Novobiocin inhibition of DNA supercoiling might also be explained as acting via inhibition of topoisomerase I (Nakayama and Sugino, 1980; Burrington and Morgan, 1978) resulting in decreased nucleosome formation (Germond et al., 1979; Ruiz-Carrillo et al., 1979; Nelson et al., 1979, 1981; Stein e t al., 1979). In contrast, nalidixic acid does not inhibit topoisomerase I (Nakayama and Sugino, 1980; Burrington and Morgan, 1978). As possible probes of the significance of eukaryotic DNA supercoiling, nalidixic activity was compared with novobiocin activity (Fig. 4). The results of Mattern and Painter (1979b) can be reinterpreted to FIG. 4. Nalidixic acid and novobiocin inhibition of scheduled and unscheduled DNA synthesis in normal human dermal fibroblasts. Scheduled DNA synthesis of FS1 cells was inhibited by both nalidixic acid (A) and novobiocin (B) addition at time zero. Twelve glass coverslips (11 x 22 mm) were placed in each Lab-Tek 100 x 15 mm plastic petri dish. Each coverslip was individually seeded with 5 x 103 FS1 cells by beading 250 p1 of monodispersed cell suspension onto the coverslip. The cell suspension was retained on the coverslip by its greater affinity for glass and the relative hydrophobic nature of the plastic. Cell growth does not extend either onto the petri dish or onto the reverse side of the coverslip. This procedure allows precise control of the number of cells on each coverslip. Cells were allowed to attach and acclimate for 24 hr in Eagle’s minimum essential medium (MEM) supplemented with 2 mM glutamine, 1 mM pyruvate, 100 U/ml penicillin, 100 pg/ml streptomycin, 100 pg/ml Fungizone, and 10% fetal bovine serum (Flow). The cultures were then washed with phosphate-buffered saline (PBS); 10 ml of new medium (containing 3% fetal bovine serum) were added to each plate, and the cultures were incubated for an additional 24 hr. The plates were then divided into groups for experimental protocols, and one plate from each group was W-irradiated as a control. Each group was treated with the proper concentration of nalidixic acid or novobiocin in Eagle’s MEM (3% fetal bovine serum) supplemented with 2 pCi/ml L3H]Tdr(25 Ci/mmol, Amersham). At each time point (0,1.5,3,6 hr) three coverslips were removed from each dish, rinsed with PBS, and fixed in Carnoy’s solution (ETOH and glacial acetic acid, 1:3). The coverslips were dried and placed in scintillation vials containing PC5 solution (New England Nuclear). Incorporation of radioactive label, [3H]Tdr,into FS1 was quantitated in a Beckman LS-8000 scintillation counter. Nalidixic acid (C) and novobiocin (D) inhibition of unscheduled DNA synthesis (presumptive excision DNA repair) was demonstrated via a modified version of the above protocol with the addition of either nalidixic acid or novobiocin immediately after UV irradiation. Scheduled DNA synthesis was inhibited by adding 3 mM hydroxyurea to the medium whenever 3% fetal bovine serum was utilized in the above protocol. FS1 not pretreated with hydroxyurea or UV irradiation are indicated as “no pretreatment” and demonstrate the inhibition of scheduled DNA synthesis by hydroxyurea. Such inhibition of scheduled DNA synthesis was also confirmed by autoradiography. Uptake of radiolabel after UV irradiation is considered to be proportional to repair DNA synthesis. Cells washed with PBS were exposed to 10 joules/m2 of UV radiation (primarily 254 nm) from a General Electric germicidal lamp. Dosimetry was determined using a Latejet meter.

A

10.000 NALIOIXIC AGIO FS I CELLS

1000

500 300

2OC I00 50

2c 10 o.uq/ml 201u)/ml mOpg/rnl 2000,q/ml

L

I

--------------

30

15

60

TREATMENT (HR)

B

10,000

W W I N FSI CELLS

1000 500

300

ax I00

5c

2C

7-----

IC

/" /"o,uq/ml 20,uq/ml 200,uq/ml 20001u)/ml

I

I 5

----_--------30

TREATMENT (HR)

FIGS.4A and B.

60

C

NALlDlXlC ACID FSI CELLS

.

I

I 5

30

60

TREATMENT IHR)

2 I

I .5

30 TREATMENT IHR)

FIGS.4C and D (see legend p. 180).

60

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show that novobiocin can induce physiologically relevant decreases of DNA supercoiling. After 6 hr of treatment with 2000 pg/ml novobiocin scheduled DNA synthesis was almost completely inhibited; 200 pg/ml induced approximately an 80% decrease; and 20 pg/ml, a 41% decrease (Fig. 4B). Unscheduled (excision repair) synthesis was decreased by approximately 98% by 6 hr of treatment with 2000 pg/ml novobiocin; 200 pg/ml induced approximately a 90% decrease; and 20 pg/ml induced approximately a 63% decrease (Fig. 4D). Nalidixic acid was examined (Lipetz et al., 1980a) to determine whether it could induce physiologically relevant decreases in DNA supercoiling in FS1 cells (normal human dermal fibroblasts). Nalidixic acid treatment (2000 and 200 pg/ml for 7 hr) decreases DNA supercoiling (Fig. 3). Treatment with 2000 or 200 pg/ml nalidixic acid results in minimum nucleoid sedimentation at 3-4 pglml EB, while minimum control sedimentation is 5 pg/ml. Clearly, these decreases in DNA supercoiling represent physiologically relevant mechanisms, not DNA strand breaks. Treatment with 20 pg/ml nalidixic acid does not result in a decrease in DNA supercoiling. In all reported FS1 DNA supercoiling experiments the cells were subjected to nalidixic acid exposure for 7 hr; 3-hr exposures altered nucleoid sedimentation but induced alterations that were not as clearly beyond the range of experimental error. This requirement for an extended exposure could reflect either low nalidixic acid activity or cell-cycle-dependent activity of the nalidixic acid target, which is discussed below. After 6 hr of treatment, 2000 pg/ml nalidixic acid induced approximately an 87% inhibition of FS1 scheduled DNA synthesis; 200 pg/ml induced an inhibition of approximately 26%; and 20 pg/ml induced approximately a 22% inhibition (Fig. 4A). It should be noted that while 2000 and 200 pg/ml nalidixic acid induced a similar response in decreasing DNA supercoiling, DNA synthesis was inhibited to different extents by these two concentrations. Thus, it appears that the inhibition of scheduled DNA synthesis b y nalidixic acid is a complex phenomenon involving more targets of inhibition than just DNA supercoiling. It is not known to what extent these results reflect direct inhibition of DNA polymerases. Unscheduled (excision repair) synthesis was decreased with 6 hr of treatment with 2000 pglml nalidixic acid to approximately 79% of the untreated value; 200 pg/ml induced a decrease of approximately 34%; and 20 pg/ml induced a decrease of approximately 26% (Fig. 4C). Nalidixic acid inhibition of DNA repair showed a definite dose-time response; at 1.5 hr of treatment neither 20 pg/ml nor 200 pg/m1 inhibited DNA repair; at 3 hr of treatment 20 puglml did not inhibit repair;

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and at 6 hr both doses inhibited DNA repair (Fig. 4C). The observation that inhibition of DNA repair and DNA supercoiling both require extended exposure for maximal inhibition is consistent with DNA supercoiling directly modulating DNA excision repair. Neither nalidixic acid nor novobiocin are known to inhibit excision-repair polymerases (Poulson et al., 1974; Sung, 1974; Edenberg, 1980). It should be noted that nalidixic acid exposure does not inhibit excision repair in Escherichia coli (Simon et al., 1974), and therefore it is unlikely that nalidixic acid is acting via inhibition of a topoisomerase truly analogous to prokaryotic gyrase. The observation that both 2000 and 200 pglml nalidixic acid induced similar decreases in DNA supercoiling may suggest that the long-exposure requirement is due to cell-cycle dependence rather than low nalidixic acid activity. We examined the activity of 2000 and 200 pglml nalidixic acid upon confluent, contact-inhibited FS1 cells that were not undergoing cellular division or DNA synthesis [Go cells (Pinon, 1978)l. In five trials, we were unable to detect any significant decrease in the supercoiling of nalidixic acid-treated cells resting in Go.These results are consistent with the target of nalidixic acid activity being cell-cycle dependent. The requirement for extended exposure in a population of dividing cells could reflect the fact that nucleoid gradients quantitate the average DNA supercoiling of all nucleoids. Hence, a sufficient proportion of treated cells must have entered a sensitive phase of the cell cycle in order that a decreased nucleoid migration can be manifest. The nalidixic acid target that modulates DNA supercoiling appears to require cell cycle progression. In DNA synthesis, nucleosome structure and stacking are restored shortly after DNA replication fork passage (DePamphilis and Wasserman, 1980). Interference with this process would result in altered DNA supercoiling. Histones H3 and H4 are transferred from preexisting chromatin to newly replicated chromatin (Jackson and Chalkley, 1981)and may act to introduce DNA supercoiling in the newly replicated DNA (Nelson et al., 1981; BinaStein and Simpson, 1977; Camerini-Otero e t al., 1976). Replication fork-induced DNA twisting creates regions of positive superhelicity (Champoux, 1978; Drlica et al., 1980). One of the functions of prokaryotic gyrase is to act locally to relax this replication fork-induced positive DNA superhelicity (Drlica et al., 1980). It is not known what eukaryotic topoisomerases perform this function. Both type I and type I1 eukaryotic topoisomerases can relax DNA positive superhelicity (Champoux, 1978; Hsieh and Brutlag, 1980; Liu et al., 1980). Failure

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to relieve local positive superhelical supercoiling could result in decreased nucleoid DNA supercoiling and may interfere with chromatin restoration after replication fork passage. If nalidixic acid and/or novobiocin act by inducing local regions of positive DNA superhelicity, then such alterations would probably be confined to a relatively small portion of the in vivo DNA domain and so probably could not serve as a probe of the generalized significance of DNA supercoiling. It is not clear what are the target(s) of nalidixic acid or novobiocin. Inhibition of scheduled DNA synthesis by these antibiotics is a complex process that does not clearly involve DNA supercoiling. There appears to be a stronger correlation between inhibition of DNA excision repair and inhibition of DNA supercoiling. This relationship is detailed in Section II1,D.

F. SIGNIFICANCE OF PROKARYOTIC DNA SUPERCOILING Since no significant body of evidence implicates or excludes DNA supercoiling as a primary regulator of eukaryotic DNA supercoiling, we are forced to rely on the prokaryotic literature in order to examine such a correlation. Prokaryotic DNA supercoiling is primarily regulated by topoisomerase I relaxation of DNA supercoiling and topoisomerase I1 (gyrase) introduction of DNA supercoiling (Cozzarelli, 1980). Although prokaryotic DNA supercoiling is partially stabilized b y histone-like proteins (Pettijohn and Pfenninger, 1980; RouviereYaniv and Gros, 1975; Varshavsky et al., 1977; Rouviere-Yaniv, 1977, 1979; Griffith, 1976), there are no known prokaryotic systems that stabilize and introduce DNA supercoiling in a manner truly analogous to eukaryotic chromosomal proteins. [For a review of the origins of prokaryotic DNA supercoiling, see Denhardt (1979) and Cozzarelli (1980).] Prokaryotic DNA supercoiling has been shown to regulate DNA metabolism and gene expression (Denhardt, 1979). Such demonstrations are possible because it is relatively easy to decrease prokaryotic DNA supercoiling. There are significant differences between the mechanisms of prokaryotic and eukaryotic gene regulation (Lewin, 1980; Marx, 1981). We will consider the prokaryotic literature regarding modulation of gene expression while bearing in mind the possible limited applicability of such results to eukaryotes. Examination of bacterial and viral DNAs of varying supercoiling has revealed that supercoiling may modulate (a) gene expression as measured by patterns of protein synthesis (Yang et al., 1979; Smith et al.,

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1978; DeWyngaert and Hinkle, 1979) and patterns of RNA synthesis (Smith et al., 1978; DeWyngaert and Hinkle, 1979; Botcham et al., 1973; Botchan, 1976); (b) RNA polymerase binding to DNA, both quantitatively and qualitatively (Richardson, 1975; Wang, 1974); (c) DNA replication (DeWyngaert and Hinkle, 1979; Itoh and Tomizawa, 1977; Crumplin and Smith, 1976; Marians et al., 1977; Staudenbauer, 1976; Pietsky et al., 1972); and (d) DNA recombination including recombinative integration of virus into host DNA (Holloman et al., 1975; Holloman and Radding, 1976; Radding, 1978; Abremski and Gottesman, 1979; Kikuchi and Nash, 1979). Direct evidence suggests that the expression of some prokaryotic and bacteriophage genes is regulated by DNA supercoiling. Decreasing DNA supercoiling inhibits the transcription of late, but not of early, genes in T7 bacteriophage (DeWyngaert and Hinkle, 1979). Decreasing DNA supercoiling alters patterns of protein synthesis from E . coli, ColEl plasmid, and phage genomes (Yang et al., 1979; Smith et al., 1978). Decreasing DNA supercoiling modulates the activity of lactose, maltose, and tryptophanase, but not threonine and trytophan in E . coli (Sanzey, 1979). There is a degree of correlation between those genes that can be modulated by decreased DNA supercoiling and sensitivity to catabolic repression (Sanzey, 1979; Shuman and Schwartz, 1975). Smith (1981) has reviewed evidence that supX mutations that alter gene expression of Salmonella may be topoisomerase I-difficient mutants and that such mutants may alter gene expression by increasing DNA supercoiling. DNA supercoiling may modulate prokaryotic and viral gene expression by altering the denaturation of promoter regions, thereby creating single-stranded regions that favor RNA polymerase binding (Botchan, 1976; Benham, 1979; Hsieh and Wang, 1975; Vollenweider et al., 1979). The initiator region of many promoters is A-T rich and thus more susceptible to denaturation than non-A-T-rich regions (Benham, 1979, 1980b; Botchan et al., 1973; Botchan, 1976; Vollenweider et al., 1978; Hossenlopp et al., 1974). Increased DNA supercoiling creates stress that denatures such regions (Botchan, 1976; Benham, 1979; Hsieh and Wang, 1975; Vollenweider et al., 1979; Brack et al., 1975; Delius et aZ., 1972; Beerman and Lebowitz, 1973; Dean and Lebowitz, 1971). It has been demonstrated that supercoiling stress controls the utilization of promoters for RNA polymerase binding (Levine and Rupp, 1978; Richardson, 1975; Wang, 1974). A comparison of the 429, A, M13, and SV40 viral partial denaturation maps and RNA polymerase binding sites indicates that A-T-rich sites coincide with some, but not

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all, of the in uitro polymerase binding sites (Sogo et al., 1979; Botchan, 1976; Vollenweider and Szybalsky, 1978; Dasgupta et al., 1977; Wasylyk et al., 1979). Denaturation maps of the 4x174 system can be similarly interpreted as predicting promoter regions that are not suggested b y gene mapping (Funnel1 and Inman, 1979); and RNA polymerase binding experiments appear to have supported these predictions (Rassert and Spencer, 1978). Thus, it appears plausible that DNA supercoiling may act differentially to control prokaryotic gene expression by altering RNA polymerase binding.

G . SIGNIFICANCE OF EUKARYOTIC DNA SUPERSTRUCTURE Cook (1973) gave momentum to the study of eukaryotic DNA superstructure with his proposal that DNA superstructure controlled differentiation. Although conceived before subchromosomal eukaryotic DNA superstructure had been elucidated, the model was a brilliant speculation. Akrigg and Cook (1980)have since shown that abnormally increasing HeLa nucleoid DNA supercoiling results in a dramatic increase in in uitro transcription by wheat germ RNA polymerase. However, other than in our demonstrations that TPA, a known modulator of eukaryotic gene expression, alters DNA superstructure, there is no direct evidence involving chromatin to support this hypothesis. Studies are underway in our laboratory to further examine Cook’s hypothesis. Two studies indicate that specific genes may have well defined locations with the loop of DNA represented by a DNA domain. Nucleoids are digested to various degrees with either nucleases or restriction endonucleases, and the DNA that remains attached to the nuclear matrix is separated from the digested fragments. Nuclear matrix attached DNA is transferred to a filter and hybridized against known probes. Nelkin et al. (1980) reported that SV40 genes are preferentially located near nuclear matrix attachment sites of SV40-transformed 3T3 cells. Also, Cook and Braze11 (1980) reported that a-,but not /3- or y-globin genes are preferentially located near the nuclear matrix attachment sites in HeLa. It should be cautioned that it has not yet been fully established that association of a gene with the nuclear matrix attachment sites indicates that it is preferentially associated with such sites; however, the observation that certain globin genes are not associated with the nuclear matrix tends to support such a hypothesis. As discussed by Nelkin et al. (1980), the association between the

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nuclear matrix and DNA domains may be different for transcribing and nontranscribing domains (Faiferman and Pogo, 1975).The nuclear matrix may provide support for transcriptional activities in a manner similar to what occurs in DNA replication in prokaryotes (Nelkin et al., 1980; Pardoll et al., 1980).Support for this hypothesis includes (a) the association of hnRNA and snRNA with the nuclear matrix (Miller et al., 1978a,b; Herman et al., 1978); (b) the specific binding of steroids to the nuclear matrix of target tissues (Barrack et al., 1977; Barrack and Coffey, 1980); (c) the preferential association of transcribed SV40 genes with the nuclear matrix of SV40-transformed 3T3 cells, while nontranscribed globin genes are not so associated (Nelkin et al., 1980); (d)alteration of DNA domain size in brain cells during mouse fetal and neonatal stages of development (P. Lipetz, 1981;unpublished observations); and (e)the alteration of lymphocyte DNA domain size under the stimulus of TPA, a known modifier of lymphocyte gene expression (Lipetz et al., 1981b) (see below). Transcriptionally active genomes are preferentially sensitive to DNase I digestion (Weintraub and Groudine, 1976; Garel e t al., 1977). Such sequences remain DNase I sensitive after transcription has been shut off, and DNase I sensitivity is independent of transcription rate (Weintraub and Groudine, 1976; Garel e t al., 1977; Younget al., 1978). DNase I sensitivity is increased when DNA supercoiling is increased (Campbell and Jackson, 1980). This may either imply increased DNA supercoiling in eukaryotic transcriptionally active regions, or else the increased DNase I sensitivity may reflect other structures. Weisbrod et al. (1980) suggest that HMG 14 and 17 (NHCP proteins) binding to chromatin may, in part, account for increased DNase I sensitivity. They hypothesize that such binding may alter basic nucleosome structure. Sandeen et al. (1980)have shown that the major sites of HMG 14 and 17 interaction is near the ends of the nucleosomal core DNA. Such interactions might alter DNA-histone interactions and thus might alter local supercoiling. While there exist conflicting reports as to whether histones and/or nucleosome conformation along the DNA is altered in transcriptionally active regions of the genome (Franke e t al., 1976; Johnson et al., 1978; Foe, 1978; Scheer, 1978; Grainger and Ogle, 1978; Butler et al., 1978), such alterations might also modify local supercoiling stress upon the DNA. These findings are compatible with, but fall considerably short of proving, the hypothesis that an alteration of higher order structures (including DNA superstructure) reflect a permissive condition in which other components (including protein binding) can modify the rate of transcription.

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Ill. Cancer and DNA Superstructure

A. CROWNGALLTUMOFUGENESIS The most straightforward correlation between altered DNA supercoiling and tumorigenesis is in the crown gall tumor system (Lipetz et al., 1981a). Crown galls are plant neoplasms arising from the integration of T DNA sequences from the Ti plasmid of Agrobacterium tumefaciens into dicotyledonous plant cell DNA (Watson et al., 1975; Thommashow et al., 1980; Chilton et al., 1977; Zambryski et al., 1980; Yadau et al., 1980; Willmitzer et al., 1980).The Ti plasmid system has been studied extensively since it is also a natural vector for introducing DNA sequences into plant cells (Klapwijki et al., 1978; Bomhoff et al., 1976; Montoya et al., 1977; Hernalsteens et al., 1980). There are obvious analogies between the integration of a cccDNA plasmid into a eukaryotic genome to induce neoplastic transformation and the integration of a cccDNA virus (such as SV40) into a eukaryotic genome to induce neoplastic transformation. However, unlike the viral systems, probes exist that will both increase and decrease the DNA supercoiling of the Ti plasmids and so allow the relationship between tumorigenesis and DNA supercoiling to be examined. Ti plasmid DNA supercoiling can be manipulated (Lipetz et al., 1981a). Novobiocin and nalidixic acid are antibiotics that decrease DNA supercoiling by inhibiting topoisomerase I1 introduction of DNA supercoiling (Cozzarelli, 1980; Gellert et al., 1976b, 1977; Sugino et al., 1977). Lipetz et al. (1980b, 1981) have demonstrated that under physiological cation conditions, physiological concentrations of polyamines (spermidine and spermine) inhibit the in vitro DNA superhelical relaxing activity of Micrococcus luteus. Similarly, spermidine inhibits the activity of purified A. tumefaciens and E . coli DNA superhelical relaxing enzymes. Spermidine also enhances in vitro M . luteus and E . coli topoisomerase I1 activity (Kung and Wang, 1977; Gellert et al., 1976a). Therefore, it is possible that spermidine may exert coordinate control over opposing enzyme activities to maximize prokaryotic DNA supercoiling. Alteration of Ti plasmid DNA supercoiling was verified by agarose gel electrophoresis. Assuming a constant molecular weight, the electrophoretic mobility of covalently closed circular (ccc) DNA in an agarose gel can be proportional (nonlinearly) to DNA supercoiling: the more negatively superhelical the molecule, the faster the migration (Shure and Vinograd, 1976; Keller, 1975) (see legend of Fig. 5). The

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1

0.I

1.0 10.0 SPERMIDINE (mMl NALlDlXlC ACID or NOVOBIOCIN bg/ml)

FIG. 5. Ti plasmid agarose gel electrophoretic mobility after treatment. Spermidine increased DNA supercoiling, and either nalidixic acid or novobiocin decreased Ti plasmid DNA supercoiling. Agrobacterium tumefaciens were cultured as per Favus et al. (1977) and incubated with spermidine, nalidixic acid, or novobiocin for 8 hr. Ti plasmids were extracted and subjected to electrophoresis in 0.8% agarose; the gels were ethidium bromide stained and photographed as per Birnboin and Doly (1979). The resulting photographs were scanned on an Ortec 4310 densitometer, and the distance migrated was measured. PM2 RFI bacteriophage controls were included in all runs to demonstrate that the Ti band had migrated proportionately to its molecular weight. Preincubation of PM2 RFI with spermidine did not alter migration. Molecules whose DNA supercoiling is above a certain range form a single fast migrating band during electrophoresis; the superhelicity of these molecules may be altered without affecting their electrophoretic migration (Shure and Vinograd, 1976: Keller, 1975). Ti plasmids exhibit a distribution about a migration value (not a sharp band) indicating that Ti plasmid negative superhelicity is within the range where electrophoretic mobility is nonlinearly proportional to DNA supercoiling. With covalently closed circular DNA molecules of less molecular weight, agarose gel electrophoresis of molecules within the superhelical range where electrophoresis is nonlinearly proportional to superhelicity should result in the appearance of distinct bands varying in superhelicity; this does not occur with Ti plasmids because the DNA molecule is large (90to 120 x 108 daltons) and possesses too much superhelicity for DNAs varying in linking number, L , by only one to form distinct bands in the narrow separation agarose gel electrophoresis induces in such large molecules. The error bars indicate SEM.

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O' 1

Y

Y

n

OJ

0.1

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PERMIDINE (mM) NALlDlXlC ACID or NOVOBIOCIN @g/ml)

EZG. 6. Rate of crown gall tumorigenesis after treatment. Spermidine pretreatment increased the rate of crown gall tumorigenesis, whereas either nalidixic acid or novobiovin decreased the formation of crown gall tumors. Spermidine- , nalidixic acid-, or novobiocin-treated cells were cultured on potato disks to determine the rate of tumor formation (Favus et al., 1977).All experimental points were the subject of at least two independent experiments of at least 15 disks, and the standard error of the mean of all points was less than 10%.

agarose gel electrophoretic mobility of selected linearized controls was examined to ensure constant molecular weight, therefore, migration is assumed to be proportional to DNA supercoiling. Figure 5 demonstrates that both novobiocin and nalidixic acid decrease the agarose gel migration rate of Ti plasmids isolated from B6 A . tumefaciens. When A . tumefaciens were incubated with spermidine the agarose gel migration rate of isolated Ti plasmid increased (Fig. 5 ) , indicating an increase in DNA supercoiling. Agrobacterium tumefaciens from the aforementioned cultures was tested for virulence on potato disks by the method of Favus et al. (1977) (Fig. 6). Cultures grown in novobiocin or nalidixic acid possess decreased tumorigenic capacity, whereas those grown in spermidine exhibit increased tumorigenic capacity (Fig. 6). The rate of tumor growth was constant in all experimental protocols, and the removal of antibiotic or spermidine before infecting potato disks with the bacteria did not alter results. Thus, it was a bacterial, not a plant, process that

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LJNEAR CORRELATION COEFFICIENTS" Treatment

r

Spermidine Nalidixic acid Novobiocin Combined

0.996 0.981 0.878 0.916

Significance (P)

<0.005 <0.01

<0.02

<0.0005

0 Linear correlation coefficients ( r ) between tumor formation and Ti plasmid electrophoretic mobility were performed for all experimental protocols including the appropriate control. The Student t test was performed to determine significance. Combined indicates that all experimental results and a single control were combined in a single analysis.

was modulated. Bacterial viability was not significantly affected by any of the pretreatments, and in all experiments concentrations of bacteria known to more than saturate the plant's ability to grow tumors were utilized (Anand and Herberlein, 1977);thus, bacterial cytotoxicity does not account for the results. A remarkable similarity exists between the agarose gel electrophoretic mobility of treated Ti plasmids (Fig. 5) and induced crown gall tumorigenesis (Fig. 6) (Lipetz et d., 1981a). These results can be analyzed to demonstrate a highly significant ( p < 0.0005) linear correlation between tumor formation and agarose gel electrophoretic mobility (see table). The least correlated value is for treatment with 10.0 pg/ml novobiocin, perhaps reflecting novobiocin inhibition of plant processes. Since all the experimental protocols can be combined to produce a single highly significant linear correlation coefficient, altered tumor formation results from the parameter represented by Ti electrophoretic mobility (DNA supercoiling), not from unrelated activities of the three treatments. The mechanism of T DNA recombination into plant cell DNA is unknown (Zambryski et al., 1980). Radding (1978) has proposed that increased DNA supercoiling enhances the integrative recombination of DNA into host DNA by creating conformational stress, thereby inducing denatured DNA regions where nucleation of homologous DNA can more easily occur. Since T DNA includes several long direct repeats as well as an inverted repeat (Zambryski et al., 1980), increased DNA supercoiling will tend to result in the formation of hairpin loops at the inverted repeat sequences (Woodworth-Gutai and Lebowitz,

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1976). These hairpin loops contain denatured sequences where recombination could easily occur. T DNA border sequences are easily denatured DNA sequences (80% A-T). These regions would be an attractive target for supercoiled modulated recombination between direct repeats to form the tandem repeats of T DNA that are observed in crown gall tumors (Zambryski et al., 1980). Altering Ti plasmid DNA supercoiling, as quantitated by agarose gel electrophoretic mobility, results in the modulation of tumorigenicity linearly proportional to the induced alteration of DNA electrophoretic mobility. Although our rationale suggests that recombinative integration of denatured Ti plasmid DNA sequences into plant cell DNA is modulated by the induced alterations of DNA supercoiling, our results do not eliminate alternative modes of activity, such as altered gene expression. These results suggest that the efficiency of host vectors in introducing foreign DNA sequences into target cell DNA may be enhanced by increasing the DNA supercoiling of the cccDNA vectors. At this time it is not clear to what degree these results apply to carcinogenic cccDNA viruses. B. CHRONICLYMPHOCYTIC LEUKEMIA Chronic lymphocytic leukemia (CLL) is a geriatric disease characterized by an accumulation of immature, probably nondividing, lymphocytes. The presence of IgM immunoglobins on the cell surface suggests that CLL lymphocytes are of B origin (Seligman et al., 1973). It has been suggested that CLL lymphocytes are arrested in a pre-S phase state (Lopez-Sandoval e t al., 1974); however, levels of [3H]thymidine uptake (Huang et al., 1972) can be interpreted as indicating a residual-amount of semiconservative DNA synthesis (Yew and Johnson, 1979a). The low CLL lymphocyte response to mitogens has led to the suggestion that they represent a clonal expansion of malignant B lymphoid cells arrested at a stage of development corresponding to that of small resting lymphocytes (Smith et al., 1972; Wybran et al., 1973; Monahan et al., 1975; Han and Dadey, 1979). In contrast to these conclusions, Fu et al. (1979) demonstrated two CLL patients with CLL cellular S-Ig idiotypically identical with serum monoclonal Ig components. They further demonstrated that a fraction of CLL cells could mature into monoclonal Ig-secreting cells by allogeneic helper T cells or Epstein-Barr virus (EBV). Robert (1979) similarly demonstrated that a subpopulation of CLL could be differentiated by B/T lymphocyte activators or EBV. Yew and Johnson (1979a) were the first to suggest that CLL lymphocytes may be characterized by increased DNA supercoiling. They re-

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ported that the nucleoid sedimentation rate of CLL lymphocytes was greater than that of normal lymphocytes, and that CLL lymphocytes exhibited a minimal sedimentation rate at 4-6 pg/ml E B while normals exhibited minimal sedimentation at 3-4 pg/ml. Our results confirm these observations. The CLL lymphocytes exhibited minimal sedimentation at 4-5 pg/ml EB (Fig. 8) and normals at 3 pg/ml (Figs. 7 and 8). There is a slight difference between the values obtained for normals in these two studies, since we consistently obtained the 3 pg/ml EB value for normal lymphocytes. Although we did not separate B and T lymphocytes, both types of lymphocytes exhibit identical sedimentation profiles in increasing amounts of EB (Yew and Johnson, 1978). Thus, a limited number of samples from two separate studies agree that CLL lymphocytes exhibit increased DNA supercoiling. The potent carcinogenic promoter 12-O-tetradecanoyl-phorbol-13acetate (TPA) (Boutwell, 1976) alters both the differentiation and proliferation of human lymphocytes. TPA inhibits terminal differentiation of mouse Friend erythroleukemia (Rovera et al., 1977; Yamasaki et aZ., 1977) or myeloid leukemia M 1 cells (Kasukabe et al., 1979). It also induces differentiation in other mouse erythroleukemia cells (Miao et al., 1978), mouse myeloid leukemia cells (Lotem and Sachs, 1979), human promyelocytic leukemia cells (Lotem and Sachs, 1979; Rovera

0 0

2

4

6

8

10

ETHIDIUM BROMIDE ( w / m l ) F I G . 7. DNA supercoiling of normal mixed human lymphocytes. Nucleoids were derived from normal mixed human lymphocytes. This is a typical nucleoid sedimentation pattern exhibited by normal mixed human dermal fibroblasts. Nucleoid and separation methods were as per Yew and Johnson (1979a) and Lipetzet al. (1981b, 1982). Error bars indicate standard error of the mean.

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et al., 1979, 1980; Vorbrodt et al., 1979), and human histiocytic lymphoma cells. TPA is also a lymphocytic mitogen (Touraine et al., 1977). This mitogenic activity is inhibited by retinol in both lymphocytes (Skinnider and Giesbrecht, 1979) and mouse skin, suggesting a common activity in these diverse cell types. The CLL lymphocytes can be differentiated by treatment with TPA (Totterman et al., 1980).TPA induces the appearance of lymphoblastoid and plasmactoid cells. TPA-treated CLL cells expressed increasing amounts of intracytoplasmic immunoglobins phenotypically identical to the S-Ig of control CLLs. Decreased monoclonal S-Ig density and DNA synthesis is also induced. TPA treatment induces ultrastructural maturation of CLL cells including increased mitochondria and endoplasmic reticulum as well as an increased cytoplasmic to nuclear ratio. There appears to be a maturation toward plasma cells or fibroblast-like structures.

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FIG.8. DNA supercoiling after 12-0-tetradecanoyl-phorbol-13-acetate (TPA) treatment of normal and chronic lymphocytic leukemia lymphocytes. The DNA supercoiling of normal lymphocytes (A) was increased, and the DNA supercoiling of chronic lymphocytic leukemia lymphocytes (B and C) was decreased by incubation in 1.6 x lo-' M TPA for 12 hours. Nucleoid and separation methods were those described by Yew and Johnson (1979a) and Lipetz et al. (1981b, 1982). Error bars indicate standard error of the mean.

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PHILIP D. LIPETZ ET AL. I00

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RELATIONSHIP OF DNA STRUCTURE TO CARCINOGENESIS

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Due to the central role of chromatin in gene expression, we hypothesized that DNA supercoiling might also be altered by TPA-induced differentiation. Figure 8 demonstrates that TPA reduces CLL lymphocyte DNA supercoiling. CLL lymphocytes exhibiting minimal nucleoid sedimentation at 4 pg/ml E B are reduced to minimal nucleoid M TPA treatment (Fig. sedimentation of 3 pg/ml by 12 hr of 1.6 X 8B). CLL lymphocytes exhibiting minimal untreated nucleoid sedimentation at 5 pg/ml are reduced to minimal nucleoid sedimentation at 3-4 pg/ml by similar treatment (Fig. 8C). Clearly both treated and untreated CLL sedimentation patterns fit our criteria for physiologically relevant alterations in DNA supercoiling. The observed pattern of increased CLL DNA supercoiling, and the ability of both TPA and chemotherapeutics to alter the pattern of DNA supercoiling, suggests that the chromatin structures inducing DNA supercoiling are responsive to the state of differentiation of the lymphocyte. Based upon prokaryotic models it would be tempting to speculate that such alterations in chromatin structure modulate gene expression and carcinogenic promotion; however, our knowledge of the mechanisms of eukaryotic gene expression is insufficient to withstand such a speculation. DNA-RNA hybridization studies are under way in our laboratory in an attempt to partially resolve this issue.

C. TPA ALTERATIONS OF DNA SUPERSTRUCTURE I N NORMAL HUMANCELLS After observing that TPA altered the DNA supercoiling of CLL cells, we wished to determine whether this is a general property of TPA activity. The DNA supercoiling of normal undifferentiated mixed lymphocytes is increased by TPA treatment (Fig. 8A). Since DNA supercoiling was measured after 12 hr of 1.6 x M TPA, it is unlikely that TPA-induced cell division is responsible for the observed effect. Similarly, TPA treatment (24 hr of 1.6 X M TPA) of normal human dermal fibroblasts results in a decrease in DNA supercoiling (Fig. 9). Thus, TPA can both increase and decrease DNA supercoiling. It is possible that TPA may tend to induce a specific chromatin conformation and thus will either increase or decrease DNA supercoiling depending on the preexisting conformation. TPA both differentiates and dedifferentiates cells; if such an activity is modulated by chromatin structure, then these opposing activities might induce opposing alterations in DNA supercoiling. Obviously more experimentation is needed to resolve this issue.

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FIG. 9. DNA supercoiling of 12-0-tetradecanoyl-phorbol-13-acetate (TPA)-treated normal human dermal fibroblasts. The DNA supercoiling of FS1 cells was decreased by 24 hr of treatment with 1.6 x lO-'M TPA. The pattern exhibited by untreated F S l cells is indicated in Fig. 3B. Untreated FS1 cells always exhibit minimum nucleoid sedimentation at 5 &ml EB whereas TPA-treated cells exhibit minimum nucleoid sedimentation at 3-4 &ml. The methods used are described by Lipetz et al. (1982),with centrifugation at 100 min and 10,000 rpm in a Beckman SW27 rotor. Error bars indicate standard error of the mean.

We also examined the effect of TPA upon normal human lymphocyte DNA domain size. The average DNA domain size of untreated lymphocytes was 8.4 x lo9 daltons of DNA. TPA treatment (1.6 x lo' M TPA for 12 hr) decreased the DNA domain size to 5.4 x lo9 daltons of DNA. If, as discussed earlier, some genes are preferentially located in specific regions of the DNA domains (near the nuclear matrix attachment site), and such attachment is a factor in controlling gene expression, then the alteration of DNA domain size may be a part of the mechanism by which TPA alters gene expression. Interpretation of DNA domain size experiments involving an alteration of DNA synthesis is difficult, since DNA domains may generate an additional membrane attachment site where DNA synthesis is performed, and thus transiently lower the size of the DNA domain. Since naladixic acid does not alter the DNA supercoiling of nondividing FSI cells, the effect of TPA on the supercoiling of nondividing FSI cells was examined. A 3 hr treatment (1.67 x lo-' M )resulted in a decrease of DNA supercoiling almost identical to that observed

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after 24 hr treatment of dividing FSI cells (Fig. 9). The minimum nucleoid sedimentation of nontreated nondividing FSI cells was obtained in the range of 5-6 pglml EB. Minimum nucleoid sedimentation of TPA treated nondividing FSI cells was obtained in the range of 3-4 pg/ml EB. Thus, unlike naladixic acid, TPA does not require cell division to alter DNA supercoiling. This suggests that the mechanism by which TPA alters supercoiling is different from that of naladixic acid and may relate to more generalized cellular control. These results are consistent with the observation that TPA can induce cellular differentiation without a round of scheduled DNA synthesis (Rovera et al., 1980). The TPA experimental series was originally designed to disprove the hypothesis that alterations of DNA supercoiling are important factors of cellular differentiation. If TPA had either failed to alter DNA supercoiling when it effected differentiation of CLL cells or such alterations of DNA supercoiling had required cell kinetics different from those required for cellular differentiation, then such an hypothesis would clearly be jeopardized. However, the fact that TPA induced alterations of DNA supercoiling are consistent with the differentiative phenomena so far examined does not establish that a functional correlation exists between altered DNA supercoiling and differentiation. Before these results can be interpreted as indicating a generalized mode of activity of carcinogenic promoters, phorbol esters of varying carcinogenic potency, nonphorbol ester carcinogenic promoters, and inhibitors of carcinogenic promotion should be examined in relation to supercoiling. D. CHROMATIN DAMAGEAND REPAIR The correlation between unrepaired DNA damage and tumorigenesis is well known (Brash and Hart, 1978) and will not be reviewed here. Herein we will consider only the possible relationships between DNA damage, DNA repair, chromatin damage, and chromatin repair. An impaired ability to repair damage to the DNA will obviously alter chromatin structure and hence DNA supercoiling. The strongest correlation between DNA supercoiling and impaired repair function appears to be with DNA excision repair. Collins and Johnson (1979) demonstrated that novobiocin acts to inhibit UV-induced DNA damage excision repair in HeLa and CHO cell cultures, and appears to act b y inhibiting the recognition of UV-induced DNA damage by endonucleases. Lipetz et d. (1980a) demonstrated that novobiocin and naladixic acid decreased UV-induced DNA damage excision repair in human dermal fibroblasts and human glia-like cells. Mattern and

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Scudiero (1981) demonstrated that nalidixic acid treatment results in the inhibition of the repair of DNA damage induced b y UV and N-methyl-N'-nitrosoguanidine. It can be demonstrated that novobiocin treatment following UV irradiation increases mutagenesis, and that this effect is due to unrepaired DNA damage, not to some other effect of novobiocin treatment. Paramecium macronuclear DNA supercoiling is decreased by exposure to 1 pg/ml novobiocin (Lipetz et al., 1981~). When novobiocin is administered before UV irradiation, the rate of induced mutagenesis is increased (Lipetz et al., 1981~).This activity is consistent with a novobiocin-induced decrease in repair of UV-induced DNA damage. However, when paramecia are exposed to photoreactivating light that results in the photoenzymic monomerization of the UV-induced cyclobutane pyrimidine dimers (Smith-Sonneborn, 1979), the rate of mutagenesis of novobiocin-treated cells returns to normal (SmithSonneborn and Lipetz, 1981). Thus, it appears that repair of cyclobutane pyrimidine dimers is inhibited by novobiocin-induced decreases in DNA supercoiling. Supercoiling stress may make DNA strand deformations easier for the repair endonucleases to recognize DNA damage. In a detailed analysis, Benham (1981) has proposed that relaxation of the DNA supercoiling stress may result in less of a strand deformation at the site of the UV-induced cyclobutane pyrimidine dimers, and hence lower its recognition by repair endonucleases. Thus, DNA repair processes should no longer be considered independent of chromatin structure and DNA supercoiling. It appears that repair of DNA strand breaks is not modulated by DNA supercoiling, since novobiocin does not inhibit repair of Xray-induced DNA damage (Collins and Johnson, 1979) and nalidixic acid does not inhibit the repair of methyl methylsulfonate-induced DNA strand breaks (Mattern and Scudiero, 1981). Unrepaired UV-induced damage to the secondary structure of the DNA will alter the twisting of the DNA about the duplex helix; and therefore, wiII result in altered DNA supercoiling (Benham, 1981; Woodworth-Geutai et al., 1977; Denhardt and Kato, 1973) and altered chromatin structure. Each unrepaired cyclobutane pyrimidine dimer alters DNA twisting by 5-6" (Denhardt and Kato, 1973). Psoralen-UV photoproducts unwind the DNA (Wiesehahn and Hearst, 1978). Carcinogens can also alter DNA supercoiling. It has been shown that the ability of benzo[a]pyrene (BaP) metabolites and Nacetoxy-2-actylaminofluorene to reduce DNA supercoiling correlates positively with each carcinogen's mutagenicity in the Ames assay (Drinkwater et al., 1978).This led Drinkwater et al. (1978) to propose that the ultimate carcinogenic metabolite of BaP, BaP diol epoxide

RELATIONSHIP OF DNA STRUCTURE TO CARCINOGENESIS

20 1

(Sims et al., 1974; Huberman et al., 1976; Newbold and Brookes, 1976; Wood et al., 1976; Yang et al., 1976; Kapitulnik et al., 1978), is intercalated as well as covalently bound to the DNA. The intercalating activity would reduce DNA supercoiling in a manner similar to that of EB. Hogan et al. (1981) appear to have overcome some earlier objections (Prusik et al., 1979; Geacintov et al., 1978) to the intercalative model of BaP diol epoxide interaction with DNA. Increasing preexisting DNA supercoiling appears to increase the magnitude of conformational deformations induced by BaP diol epoxide (Gamper et al., 1980). We have demonstrated that nucleoids from livers of rats treated with aflatoxin B, (AFB,) (Lipetz et al., 1982) (Fig. 10) and AFB,-treated human dermal fibroblasts resting in Go also exhibit decreased DNA supercoiling (Lipetz et al., 1981e). This suggests that the aflatoxin B,-DNA adduct (2,3-dihydro-2-( N7-guanyl)-3-hydroxyaflatoxin B1) (Croy et al., 1975) may also intercalate into DNA. It is also possible for strand deformations to decrease DNA supercoiling without intercalation. Such a mechanism of decreasing DNA supercoiling would be analogous to altered DNA supercoiling via cyclobutane pyrimidine dimer formation. AFBl treatment does not induce DNA strand breaks, thereby eliminating strand breaks as the cause for decreased supercoiling. Excision repair of damage to the DNA may alter DNA supercoiling and chromatin structure by either of two mechanisms: (a) when the damaged DNA sequence is excised from the DNA by repair enzymes, a DNA strand break is introduced and acts to reduce the DNA supercoiling as nucleosomes are dissociated for repair replication; or more speculatively, (b) excision repair processes may introduce conformational changes in nucleosome-associated DNA (Oleson et al., 1979) that could alter supercoiling. One of the targets of nalidixic acid and novobiocin might represent a system that repairs chromatin structure. Such a target could either be an inducible factor or could be constantly present but active only upon the proper chromatin substrate. Bodell and Cleaver (1981) have observed a transient alteration of chromatin conformation as a consequence of DNA repair. Observed alterations in nucleoid migration after excision repair require that a separate system exist to restore superstructure integrity; such a system is indicated by the data of Cook et al. (1978). However, the efficiency of structural chromatin repair is not known. For example, nucleoid migration has not been restored to normal 20.5 hr after the start of repair (Cook et al., 1978). Indirect evidence may suggest that the factors responsible for restoring chromatin conformation after DNA repair are decreased in the human genetic mutants Xeroderma pigmentosum, which are also known to

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manifest an increased rate of UV-induced tumorigenesis and decreased repair of UV-induced DNA damage (Cook et al., 1978). IV. Conclusion

The authors hope that DNA superstructure may eventually serve to reconcile two opposing sets of theories as to a critical mechanism of neoplastic transformation. One group argues that the transforming activity must be the result of altered informational flow from the DNA. The somatic mutation theory of carcinogenesis is the most popular of 14.00

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nucleoid sedimentation compared to the 0 pg/ml sedimentation rate (A) whereas it increases treated nucleoid sedimentation (B). This indicates that there was less preexisting DNA supercoiling in the nucleoids derived from treated cells and that 3 pg/ml EB both countered the previously existing DNA supercoiling and induced supercoiling in the opposite direction (positive DNA superhelicity), whereas 3 p d m l EB was only sufficient partially to counter the preexisting DNA supercoiling in the untreated controls. Protocols were those of Lipetz et 01. (1982). Female Fisher rats were maintained on a special lipotropic diet beginning at 28 days of age. On day 37 they were injected with 12 mdkg aflatoxin B,. Twenty-four hours after the injection, the experimental animals and matched controls (untreated sham controls) were sacrificed and the livers removed by dissection. The dissected organs were placed on ice in 15mM Tris. HCI, pH 6.9; 60 mM NaCl; 0.1 mM EDTA; 10 mM &mercaptoethanol, and 0.34 M sucrose. They were then minced, Dounce homogenized in 15 ml of buffer, and filtered through prewet sterile gauze. Nuclei were counted in a hemacytometer and diluted to 1 x 10' nuclei/ml. Fifty (5 x 105 nuclei) were gently layered onto the lysis layer of each nucleoid gradient (0 and 3 p d m l ethidium bromide). Similar results have been obtained with treatment at 1 mg/kg aflatoxin B, for 8 and 12 days (Lipetz et al., 1982, and unpublished results.) We would like to thank Drs. Su, Wani, Chang, and D'Ambrosio for furnishing the samples of treated rat liver.

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these theories. Proponents of such theories point to the success of the Ames (1976)mutagenesis assay and to demonstrations that unrepaired DNA damage can induce tumors (Hart and Setlow, 1974). Another group argues that membrane-associated events are critical to malignant transformation. They point to the failures of the Ames assay to predict correctly the carcinogenic potential based on the rate of induced mutations. Rubin (1980) has summarized this point of view. DNA superstructure could be responsive to both of these levels of control. We have already reviewed how mutagenic DNA damage can alter DNA superstructure. DNA superstructure may also be responsive to membrane-modulated signals, perhaps with polyamines as second messengers. Agrobacterium tumefaciens DNA supercoiling is responsive to polyamines. Similarly, the superhelical modulating ability of a Micrococcus luteus protein extract is modulated by physiological concentrations of polyamines under physiological ionic conditions (Lipetz et al., 1980b, 1981e). In eukaryotes, polyamines are synthesized in response to a wide variety of membrane-modulated signals (Tabor and Tabor, 1976). The observation that increased polyamine synthesis is the major correlate of TPA activity (Boutwell, 1976) is consistent with polyamine modulation of eukaryotic DNA superstructure. Polyamines may act to stabilize DNA domain structure (Flink and Pettijohn, 1975). It has also been demonstrated that rat ventral prostate DNA supercoiling relaxing activity is modulated b y androgens (Filipenko et al., 1981), again suggesting that eukaryotic DNA superstructure may be responsive to membrane-modulated stimuli.

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The critical unanswered question is: To what extent, if any, does DNA superstructure modulate eukaryotic gene expression? The results reviewed herein merely establish an interesting correlation between alterations in DNA superstructure and the processes associated with tumorigenesis. It is our working hypothesis that the nucleosomal level of DNA supercoiling may not be as significant a modulator as nucleosomal stacking, and that DNA domain structure may be the most significant modulator. Experiments are under way in our laboratory to attempt to resolve these important issues. It is our hope that the results reviewed herein will stimulate more research and perhaps lead to further correlations between DNA superstructure and carcinogenic transformation. ACKNOWLEDGMENTS The invaluable assistance of B. Muktananda is gratefully acknowledged. The efforts of S. Boyle, D. E. Brash, R.I. Chu, K. C. Ford, R.W. Hart, H. D. Jewett, L. B. Joseph, L. E. Lantry, M. P. Murphy, H. Newman, and J. Smith-Sonneborn in completing the experimental portions of this work are also gratefully acknowledged. Portions of this work were supported by USEPA No. CR 805337-03.

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NOTE ADDEDIN PROOF.We can now comment upon the relationship of DNA superstructure to the processes of carcinogenesis, carcinogenic promotion, and Z DNA. Although these topics have been the source of intense speculation within our laboratory, it was not felt that the previously published literature was sufficient to allow useful discussion of these topics. Alterations in eukaryotic nucleoid DNA supercoiling indicate perturbations in chromatin organization, and many models of carcinogenesis can involve such alterations. However, three recent observations may be worthy of future consideration in constructing such models. Moreau e t al. 11982,Nature (London) 295, 260-2621 recently observed that A-T-rich linkers define both the 3' and 5' ends of eukaryotic genes. Such regions are easily denatured. Increased DNA supercoiling, such as induced by TPA in human lymphocytes, increases denaturation and thus may alter gene expression. Lipetz et al. (in preparation) have observed that differentiation of rat neuroblastoma by sodium butyrate (KO and Keostner, 1980,J. Natl. Cancer Znst. 65, 10171021) is accompanied by increased nucleoid DNA supercoiling. Nordheim et al. [1981,Nature (London) 294,417-4221 have observed that Z DNA is present in Drosophila polytene chromosomes and suggest that Z DNA may act ,as a transcriptional control. The B to Z DNA transformation may occur as a result of the torsional stress induced by DNA supercoiling (Benham, 1980a). A recent review by Cairns [1981,Nature (London) 289, 353-3571 proposed that transpositions of DNA, rather than nonrecombinational DNA mutation, induces most human cancers. Klein [1981,Nature (London) 294, 313-3181 supported the transpositional hypothesis by reviewing evidence suggesting that B-lymphocyte-derived cancers derive from genetic transpositions. Klein also suggested that the translocation of DNA was into immunoglobulin regulatory regions of the genome. DNA supercoiling increases the rate of genetic recombination, and so increased DNA supercoiling might be favorable to increased translocation. However, eukaryotic DNA supercoiling results from nucleosome stacking and is most able to influence DNA recombination only when dissociated from chromatin proteins, such as during DNA replication. Therefore, an agent which would increase the frequency of carcinogenic transpositions by increasing DNA supercoiling should also induce DNA replication; carcinogenic promoters, TPA, also induce DNA synthesis. The structure of the eukaryotic genome appears to favor transposition events which could be modified by altered DNA supercoiling during DNA replication. The observation that eukaryotic genes are flanked by easily denatured A-T-rich regions suggests that when DNA supercoiling is increased recombination will tend to occur preferentially at either the 3' or 5' ends of specific genes. This may result in the transposition of intact genes. Klein has proposed that immunoglobulin genes may be the recipient site of carcinogenic transposition; it can be calculated that human immunoglobulin genes are similarly embedded in regions of relatively greater A-T richness and that immunoglobulin genes are also more A-T rich than are surrounding the exons. Thus, the proposed recipient area is also rich in easily denatured sites.

HUMAN B-CELL NEOPLASMS IN RELATION TO NORMAL B-CELL DIFFERENTIATION AND MATURATION PROCESSES

Tore Godal and Steinar Funderud Laboratory for Immunology. Department of Pathology and The Norwegian Cancer Society, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Oslo. Norway

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The B-Cell System . . . . . . . . . . . . . . . . . . . A. A Descriptive Framework . . . B. Immunoglobulin Gene Organization and Expression ......... ....... C. Repertoire Expression . . . . . . . . . . . . . . . . . . D. Surface Markers Related to BE . Regulation of B-Cell Function 111. B-Cell Neoplasms . . . . . . . . . . . . . . A. General Features and Their Exceptions .............................. B. B-Cell Neoplasia in Relation to B-Cell Development.. .. C. What Determines Clinical Prognosis? . . . . . . . . . . . . . . . . . . D . Subsets and Neoplasms in the B-Cell Compartment. An Attempt at a Synthesis . . . . . . . . . . . ............ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I . Introduction

Diagnostic systems in human oncology are based on the relationship between neoplastic cells and their normal counterparts. Within the immune system, up to the end of the 1960s this relationship was based on morphological criteria and serum immunoglobulin (Ig) analysis. During the last decade progress in immunology, at the basic level as well as at the clinical methodological level, has allowed reassessment to take place with regard to neoplasms associated with lymphoid tissues. Such studies have shown that among non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL) the great majority of neoplasms have B-cell characteristics. Acute lymphoblastic leukemia (ALL) appears in the great majority of cases to represent stem cell neoplasms of which less than 50%at present can be allocated into T or B cells. With regard to Hodgkin's disease, the pathogenetic processes remain largely unknown, but the balance of evidence suggests at present that the neoplastic cells are related to the phagocytic system. 21 1 ADVANCES IN CANCER RESEARCH, VOL.36

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006636-X

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While the subdivision of lymphocytes into T and B cells represented an important step forward in the classification of lymphoid neoplasias, it represented only the first step in uncovering the heterogeneity of lymphocytes and lymphoid neoplasias. The T-cell compartment with its three main subgroups of effector cells (helper, suppressor, and cytotoxic cells) can now be subdivided into a much larger number of phenotypically distinct subsets’ both in experimental animals (Cantor and Boyse, 1977) and in man (Reinherz and Schlossman, 1980). As will be discussed below, a similar level of heterogenity appears to exist in the B-cell compartment. A dissection of this complexity requires the study of functionally homogeneous, preferably monoclonal, populations. The easiest way to obtain such populations, especially in man, is from neoplasms. Just as myeloma proteins were essential to determine the structure of Igs, we believe that the study of monoclonal cell populations will be a fruitful avenue for basic immunological research. A major theme of this article will be to review the functional studies on human neoplasias that are now emerging. Since our own work is focused on NHL and, therefore, concerned with B-cell neoplasms, this review will be limited to the B-cell compartment. For those who want a definite outline of the B-cell compartment with a precise insertion of B-cell neoplasia according to the position of the normal counterparts, it is the wrong time for a review, because such powerful techniques as the hybridoma technology are in the process of being introduced into this area of research. The best we can hope for is consideration of an area of intense interplay between basic and applied research that is in rapid transition. II. The B-Cell System

A. A DESCRIPTIVE FRAMEWORK

The only known function of the B-cell compartment is the production of antibodies. This function also appears to be unique to the B-cell system. B cells may therefore be defined as cells that synthesize immunoglobulins (Ig).However, the B-cell compartment also includes B precursor cells committed to B-cell development,2 but not yet synthe-

’ We use the term “subset ofcells” solely to indicate a phenotypically distinct group of cells. We will here use the term differentiation for a bifurcation process giving rise to functionally distinct offspring. By maturation we understand phenotypic changes taking place along one lineage of B cells. Deoelopment is used as a noncommitted term covering differentiation and/or maturation.

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sizing intact Ig. The nomenclature here has remained confused (see Owen, 1980).We limit our discussion to cells synthesizing intracellular p heavy chains to be defined as pre-B cells, although studies on monoclonal antibodies may indicate that B-cell precursors also include other cells committed to the B-cell lineage (Stashenko et al., 1980a). The B-cell compartment occupies close to 50% of the immune system comprising between 10” and 10’’ lymphocytes. Its weight in man is a few hundred grams. The B-cell system may be described as four-dimensional. The first dimension represents the different antibody specificities in the system. They appear essentially unlimited. The second dimension may be defined as the differentiation and maturation pathways from stem cells to memory cells and Ig-secreting cells. During this process the variable part of the Ig molecule remains largely unchanged, while the constant part may vary by switching of the heavy chains (note the paradoxical terminology from a biological point of view). A third dimension of the system may be labeled the sociological dimension to indicate that the B-cell system is highly dependent on other cells, particularly T cells, and may develop differently in lymph nodes, spleen, and gutassociated tissues. The system does not remain constant, but changes with time because of antigens and turnover of cells. This is the fourth dimension of the system. Biological processes taking place in the B-cell compartment include a number of fascinating phenomena, including (a) generation of diversity; (b) allogeneic exclusion; (c) isotype light-chain restriction; (d) clonal “abortion”; (e) isotype coexpression and switch; (f) B-cell proliferation; (g) affinity maturation; (h) antibody production and secretion. In some of these areas, e.g., the generation of antibody diversity, spectacular progress has been made in recent years by DNA recombinant techniques. In others, e.g., B-cell triggering and the delineation of B-cell subsets, we have still not come to grips with the complexity of cell membrane receptors and their function (see Fig. 1). However, even in this area, information about the function of membrane Ig is well ahead of other receptors. Thus, in all the aspects to be discussed, emphasis will be on Ig-related phenomena. B.

IMMUNOGLOBULIN

GENEORGANIZATION AND EXPRESSION

1. Theories of Immunoglobulin Formation The ability of a human being or an animal to produce a large variety of antigen-specific antibodies is a remarkable biological phenomenon.

214

TORE CODAL AND STEINAH FUNDEHUD

'

Immunoglobulins MIogen Receptors-

93

Enzymesf

Fc Receptors

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Lyb Antigens

R G .1. An overall presentation of surface receptors of B cells. From Cooper (1979) and Levitt and Cooper (1980).

Since the molecular basis for the generation of this diversity has been largely clarified in recent years, it may be appropriate to review briefly the main theories offered to explain this variety. According to the selective theory of Ehrlich, antigens were bound to specific side chains on the surface of the cells, and specific cells were thereby stimulated to synthesize more side chains, which in the end appeared as antibodies in the serum (Ehrlich, 1900). Although the selective theory in principle has proved to be correct, it was gradually abandoned when the increasing number of immunogenic substances, including a large number of synthetic organic chemicals, suggested that the diversity of antigens was very large (>lo6 in the mouse). It seemed impossible that the corresponding antibodies could all preexist. Instead, an instructive or antigen-template theory was formulated in the 1930s (see Pauling, 1940). It was suggested that there was nonspecific y-globulin present that molded itself around the antigen and thus gave rise to a specific antibody. A central assumption in this theory is that the specificity of an antibody molecule is determined by the antigen, not the primary structure of the antibody molecule, as later proved to be the case (Haber, 1964). Another argument against the selective theory came with the demonstration that antibody molecules are newly synthesized in response to an antigen and are not derived from preexisting y-globulins. In 1955 Jerne proposed the first of what one may call the modem selective theories with his natural selection hypothesis. According to this hypothesis, antibody receptors will arise in absence of antigen. The role of the antigen is to select these receptors and thereby initiate antibody formation. Burnet, on the other hand, suggested that these preexisting receptors were associated with cells that underwent specific clonal proliferation on exposure to antigens (Burnet, 1959).

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215

The discovery that the specificity of antibodies was determined by the amino acid sequence raised the question of whether sufficient DNA was contained within the genome to encode antibodies for the almost infinite number of different antigens. The precise number of necessary specificities was not known, but a reasonable estimate was 1 to 10 x lo6in mammals. The supporters of the germ-line theory claimed that genes coding for these specificities, as a result of evolution, were all contained in each lymphocyte. Somatic mutational theories, on the other hand, proposed inheritance of only a few genes, which were then expanded by mutations during the differentiation of each individual cell. These two theories were seriously challenged by the “two genes-one polypeptide” theory introduced by Dreyer and Bennett in 1965. They proposed that two genes, one for the variable region and one for the constant region, were necessary for production of each chain of the antibody molecule. Furthermore, they suggested that there were many variable-region genes but only one copy of the constant-region gene. As we shall see from the discussion below, the Dreyer-Bennett model has in principle proved to be the correct one.

2. A Description of the Immunoglobulin Gene Complex As a result of the revolution in molecular biology that began in the early 1970s, new technology has been developed. Among these, cleaving of DNA with restriction endonucleases turned out to be a powerful tool as a first step in analyzing the Ig gene complex. In a second step, DNA fragments are separated and localized by special probes of radiolabeled cDNA (transcribed from Ig mRNA b y viral reverse transcriptase). These new techniques enabled Tonegawa and his colleagues to demonstrate that genes for the constant (C)and variable (V) region of a K light chain are located to different fragments in embryonic cells, whereas these genes are on the same fragment in plasma cells (Hozumi and Tonegawa, 1976). Later amino acid and DNA sequence studies have established the same pattern for the A light chain as well as for the various types of heavy chains (Brack et al., 1978; Early et al., 1979; Sakano et al., 1979a). During studies on the K light-chain gene segments, unexpected features were revealed. The germ-line V-region gene segments encoded only amino acids through position 95, whereas it was expected to do so through position 108 (Seidman et al., 1978; Tonegawa et al., 1978). Sequence and electron microscopic analyses have subsequently demonstrated that the remaining 13 amino acids are encoded by a segment J located 3.7 kb to the 5’ side of the C region (Seidman and Leder, 1978; Bernard et al., 1978; Brack et al., 1978).

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TORE GODAL AND STEINAH FUNDERUD

K chain locus

H chain locus

=. . ” ” -

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Apart from encoding the amino acids from 95 to 108, the J segment also specifies the site at which the V region is brought close to the C region to make an active gene. In the cases of h and K genomes, five different J genes are found in a cluster. Additional studies have indicated that there are several hundred different V genes in the germ line (Valbuena et d., 1978).In principle, all of them might join a J segment by somatic recombination. The V genes of A and heavy chains appear to be similar to K in this respect. The exact number of V genes is difficult to estimate, but genes in the order of 100-600 VX and 70-400 VHhave been suggested in the mouse genome (Adams, 1980). Hybridization experiments have revealed that V genes are arranged in tandem in subset clusters, and individual genes within one subset have similarities in the framework region (see Fig. 2). When a K light-chain V gene is brought into proximity to one of the J segments, this recombination is within the third hypervariable region (HV3) of the light chain. In the case of the heavy chain, the HV3 is encoded by an additional DNA segment, named D segment (D for diversity), situated between the V and J segments on the chromosome (Early et d . , 1980a). This D segment, together with a part of the J segment, comprises the total HV3 of the heavy chain. The economy of the Ig genes is striking. Only genes encoding variable regions are present in hundreds of different copies. The constant region, on the other hand, has only one C gene for the K light chain, and two C genes for the A light chain (mouse), while the heavy chain has eight closely linked C genes (C*, Cs, C y 3 ,C y l ,Cyzb,CyZa,C,, and C a ) . Each C gene is divided into several domains. A diagram of the current model of a light-chain and heavy-chain locus is given in Fig. 2. As shown in Fig. 2, the light-chain gene is made up of three sets of genes, while the heavy-chain gene has an additional D gene segment. The next question is then how these gene segments are put together to code for mRNA specifying the correct polypeptides.

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217

a . Somatic Recombination. In the case of the K light-chain genes the gene segments are combined through a site-specific somatic recombination event between one of several hundred germ-line V genes and one of five J segments (Max et al., 1979; Sakano et al., 1979b). The detailed mechanisms are not quite proved, but the finding that two short, conserved sequences 5’ to each germ-line J K and J H segment are inverse complements of sequences 3’ to germ-line VK and VH segments, makes it likely that V and J segments are freely recombining in presence of recombinases (Sakano et al., 1979b). In the case of the heavy chain VHgenes are created during B-cell differentiation by joining of VH,J H , and the additional D segment. The mechanism is most probably the same as that postulated for the light chain with an additional recombination event for the D segment (Early et al., 1980a). This prediction is strengthened by the finding that D segments in germ-line DNA are flanked by the same two sets of sequences as V and J segments (Sakano et al., 1981). b. RNA Splicing. The somatic recombinations within the V gene segment do not result in a fully continuous stretch of DNA carrying V and C genes in a linear array, but they are still separated by an intervening sequence of 2.4-3.7 kb, the length depending upon the number of J segments “trapped” between the V and C genes. Upon activation of the Ig gene a precursor RNA, including intervening sequences, is transcribed from DNA. The Ig mRNA, like every eukaryotic mRNA studied so far, is processed from this larger precursor mRNA by RNA splicing (Rabbitts, 1978). RNA splicing is possibly an obligatory step for the generation of a stable mRNA (Crick, 1979). The Ig genes, like all other eukaryotic genes, have the characteristic nucleotides 5’-GTintron-AG-3’ at junctures between introns and exons (Bernard et al., 1978; Tonegawa et al., 1978). These nucleotides most likely act as signals for RNA splicing and must be brought together for RNA splicing to occur. Another requirement for splicing to take place seems to be activation of J through V/J recombination (Perry et al., 1979). However, activation of J will not necessarily result in RNA splicing as observed by Max et al. (1979). Thus in the case of J3, J3 may be rearranged, but J3-encoded amino acids have not been found in light chains. The inactivation is caused by a single nucleotide change in its 3‘ border. 3. The Generation of Diversity The complete coding region for the antigen binding site is constructed by somatic recombination of one of 100-600 V segments at random with 1 of 4 J segments. If similar recombination events take place between V and J segments for both light and heavy chains,

218

TORE GODAL AND STEINAR FUNDERUD

AI

T

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I

I

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- T\ 23 - C/ I

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FIG.3. Inverted repeat stem structures formed between 5’4anking region of a J gene segment and 3’4anking region of a V gene segment (see text).

this diversity could alone account for the entire Ig repertoire in mammals. Several reports, however, have pointed to the fact that additional diversity is created through different mechanisms. The most frequent source of diversity is the recombination event between V and J segments. Adjacent to the 5’ of each J segment is a palindromic sequence of the heptamer CACTGTG. In addition, a T-rich nonamer sequence, GGTTTTTGT, is found 5’ to the palindrome. These conserved sequences are invertedly repeated in the 3’ of all embryonic V segments tested so far. As shown in Fig. 3, heptamers and nonamers are able to form a stemlike structure thought to be recognized by recombinases. By altering the frame of somatic recombination, both V, and V, will have amino acid substitution in the’HV3. The heavy chains have an additional source of variability by the D segment, which joined to a J segment constitutes most of the HV3 in

B-CELLLYMPHOMAS AND B-CELLDEVELOPMENT

2 19

these chains. A substantial increase in diversity of HV3 may also be created through recombination, as discussed above for the VIJ joining. The generation of the genetic repertoire of antibody diversity mainly appears to take place in cells not yet expressing any parts of the Ig gene complex at its surface or by secretion. Thus, the genetic repertoire appears to a large extent to be generated by antigen-independent mechanisms . However, there is growing evidence for additional diversification of the rearranged Ig gene by somatic mutation in the course of B-cell maturation. So far, the evidence is indirect because a B-cell line whose development can be followed in vitro does not exist, but the observation that the V gene exhibits more variations than can be explained b y the germ-line V-gene repertoire and the junctional diversity of V and J joining has led to this conclusion (Weigert and Riblet, 1976; Weigert et al., 1978). In accordance with this, Gearhartet al. (1981)observed that the V region from IgG antibodies is considerably more variable than those from their IgM counterparts. The variance was most pronounced in the hypervariable regions. The latter observation is explained by an idiotypic or antigen-driven “mutation”-like event with subsequent selection. IgA antibodies are also shown to be more diverse than their IgM counterparts. Thus, increased diversity seems to be associated with the class switch from IgM to other classes.

4. Heavy-Chain Class Switch Lymphocyte development involves the initial appearance of a L./ chain in the cytoplasm of a pre-B cell (Raff, 1976; Cooper et al., 1976) followed by the appearance of surface IgM (Kearney et al., 1977) and commonly IgD (Vitetta and Uhr, 1977). Upon stimulation the B lymphocyte can be induced to develop further to an IgG- or IgAproducing cell. The latter process involves secretion of antibodies that have almost the same specificity in the VHregion as IgM, but a different CH region. Such a shift in class expression is called a heavy-chain class switch. The switch is apparently accompanied by deletion of CH genes expressed earlier in ontogeny (Honjo and Kataoka, 1978; Davis e t al., 1980a). Based on the order of C H gene deletions in various murine myeloma cells, a heavy-chain gene order of 5’+, y3, y1, YZb, yza, a-3‘has been proposed by Honjo and Kataoka (1978), Coleclough et al. (1980), and Cory e t al. (1980). However, cloning experiments have in the meanwhile demonstrated that an E gene is 5’ to the (Y gene (Nishida et al., 1981).By analogy to the palindromes seen in the V gene region, flanking switch (S) sequences are also expected within the C gene region. Sequence examinations of switch regions have revealed their

220

TORE GODAL AND STEINAR FUNDEHUD

existence (Davis et al., 1980b; Kataoka et al., 1980), but they are clearly distinct from the sequences involved in the rearrangement of the V gene. There is, for instance, a lack of homology between the different S segments that make homologous recombination, as seen in V/J joining, an unlikely mechanism for class switching. Furthermore, Davis et al. (1980a) have found from sequencing studies on the S segments that each segment is made up of three distinct sites. In an event of class switching, it is postulated that special switching proteins recognize S sites, and thereby allow a recombination. In conclusion, it still remains unclear whether deletion in the C region occurs by looping out as demonstrated for V, D, and J rearrangement, or by other mechanisms, as for instance sister chromatid exchange, as proposed by Obata et al. (1981).The precise molecular mechanism of switching also remains unclear.

5 . Allelic and Isotype Exclusion In each individual antibody-producing cell only one of the pair of alleles at an Ig locus is expressed, whereas the other is phenotypically silent. This phenomenon is called allelic exclusion. In addition a choice is made between the two light-chain classes A and K-an isotypic exclusion. The question is: How does a given cell decide to express A or K rather than both, and only one K allele rather than both? The question is obviously easy to answer if one of the two light-chain (or heavy-chain) alleles remains in the germ-line configuration, because they will then not be expressed. Such cases have been observed (Seidman and Leder, 1978; Joho and Weissman, 1980).However, Alt e t al. (1980b) examined three long-established A-chain-producing cell lines and found that K genes had been rearranged in two of them with a possible loss in the third. From this they proposed that the control of allelic and isotypic exclusion is by the formation of functional genes. Similar results and conclusions were reached b y Coleclough et al. (1981)in a study on the organization of both light- and heavy-chain Ig genes in mouse splenic B cells. The question has also been pursued in man (Hieter et al., 1981), in whom the A gene contributes more to antibody diversity than in the mouse. In 10 B-lymphocyte lines producing A light chains, all genes for the K constant region had undergone rearrangement. I n contrast, genes for the A constant region remain in the germ-line configuration in all cells producing K light chains. These observations suggest that (a) there is a hierarchy of light-chain gene rearrangement beginning with K and proceeding to A; (b) Ig gene rearrangement is a probabilistic process that continues until a functional gene product is formed. A functional gene product

B-CELLLYMPHOMAS AND B-CELLDEVELOPMENT VH

Gene

-

CP1 CP2 CN3 C N 1 I 4 h UI

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-

1

U

Ps P

r

e

c

u

r

+

VH

sc , qI CM2 - c ,D 3 HC,L J

22 1

Pm

n

.

RNA

ps mRNA

c, I cp2 c? I?JI

I

I

I

CNL V A F A A

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pm m R N A

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FIG.4. Generation of secreted (s) and membrane (m) p mRNA by alternative modes of RNA splicing.

will then prevent further rearrangement. This mechanism can explain both allelic and isotype exclusion. 6. Membrane-Bound and Secreted ZgM Stimulation of a resting B lymphocyte by antigens leads to the development of a mature antibody-secreting plasma cell. During this transition, the cell is shifting the expression of surface IgM to production and secretion of pentameric secretory IgM. Several investigators have demonstrated that the p chains of membrane IgM (p,) and secreted IgM ( p J differ in structure (Melchers and Uhr, 1973; Vassalli et al., 1979). This seemed first to be a paradox because only a single copy of the C, gene exists per haploid genome (Cory and Adams, 1980; Coleclough et al., 1980).The paradox was resolved when it was demonstrated that the p m and p., are very similar throughout the VH,C,1, C,2, C,3, and C,4 domains (Alt et al., 1980a; Kehry et aZ., 1980), while the C terminus contains a segment of 41 and 20 residues, respectively, after the C,4 domain (Kehry et al., 1980; Rogers et al., 1980). Furthermore, it has been demonstrated that mRNA for p, and psis transcribed from the same p gene by alternative modes of RNA splicing, as shown in Fig. 4 (Early et al., 1980b; Rogers et al., 1980). 7. Simultaneous Expression of ZgM and ZgD A large proportion of B cells express both IgM, and IgD, (see Section 11,D). On a given cell these two surface Ig classes bear identical VH regions (Salsano et al., 1974; Goding and Layton, 1976; CoEman and Cohn, 1977).These data are difficult to fit in with the C, gene-dele-

222

TORE GODAL AND STEINAR FUNDERUD

tion model because they imply that a VH gene can be expressed with the C, gene without concomitant deletion of the C, gene. Two cloning studies (Liu et al., 1980; Moore et al., 1981) have investigated this apparent paradox. Both studies established that the 6 heavy-chain genes lie only 2.5 kb to the 3’ direction of the p domains, and that both p and 6 heavy-chain gene products are simultaneously associated with the same V region. In addition, no DNA rearrangement is found in the region between the C, and C, genes in cells that express IgM and IgD. This information and the fact that alternative RNA splicing or termination possibilities are utilized in the simultaneous expression of secreted and membrane-bound IgM, led both groups to suggest that p and 6 chains arise from a single transcript by alternate splicing of the transcript. However, such a transcript has so far not been detected. C. REPERTOIREEXPRESSION

As outlined above, present evidence suggests that the generation of diversity takes place mainly in receptor-negative B cells. Thus, the generation of diversity appears to be a stochastic process independent of antigen receptors. Nevertheless, the “prereceptor” repertoire (Klinman et al., 1980) is not identical to the repertoire as detected on receptor-positive B cells. The mechanisms that govern repertoire ex-. pression appear to be largely unknown, except for one type of immunological tolerance called “clonal abortion” (Nossal and Pike, 1975). This type of tolerance in B cells is intimately related to B-cell development during ontogeny as well as in adult subjects. The clonal abortion theory predicts that “lymphocytes at a particular stage of their differentiation at which some receptors for antigen have already appeared, can be permanently switched off or eliminated if they encounter antigen in appropriate concentration” (Nossal and Pike, 1975). A number of studies on B cells from fetus or newborns (Metcalf and Klinman, 1976; Cambier et al., 1976; Elson, 1977; Nossal et al., 1977; Teale and Mandel, 1980) or from adult bone marrow (Nossal and Pike, 1975) have provided evidence in favor of this concept. In addition, differences in susceptibility of mature and immature mouse B cells to anti-Ig. have been reported (Raff et al., 1975; Sidman and Unanue, 1975) that provide evidence that inactivation is mediated through sIg, as such antibodies do not affect pre-B cells (Cooper et al., 1980). A second mechanism that may also play an important role in shaping the post-receptor repertoire is the elimination of developing clones via anti-idiotypic or anti-allotypic immune responses (Pierce and Klinman, 1977). This network regulation may apparently take place at vari-

B-CELLLYMPHOMAS AND B-CELLDEVELOPMENT

223

ous stages of B-cell differentiation, but again early B cells seem particularly susceptible to suppression (or “tolerance”) induction (Strayer et al., 1975; Kaplan et al., 1978). D. SURFACE MARKERSRELATEDTO B-CELL DEVELOPMENT 1. Surface Zmmunoglobulin (sZg)

B cells are distinguished from pre-B cells by having sIg. This transition from pre-B cells to B cells is also closely associated with the commencement of light-chain synthesis (Siden et al., 1981). These observations, together with those showing defective light-chain synthesis in B cells (Bothwell e t al., 1981) and sequential rearrangement of light-chain genes (Alt et al., 1980b; Hieter et d., 1981), suggest that the cells remain in the pre-B-cell pool, allowing rearrangement of light-chain genes to take place until a light chain that can effectively combine with the p chain is formed. Only then, with the resultant complete IgM molecule, may IgM molecules be expressed on the cell surface. However, not all data are consistent with this hypothesis. Brouet e t al. (1979) and Vogler et al. (1981) have described cases of ALL with cells expressing p only on the surface, which they have suggested represent intermediate stages between pre-B and B cells. This is not a trivial point, because it would make it possible for the emerging B cell to become exposed to selective forces (either antigenic or idiotypic) before the structure of the light chain is determined. M’hether light chains are directly responsible for making Ig expression on the surface possible, remains unclear. Data from HLA A-B-C molecules suggest that &-microglobulin is required for expression of these molecules on the surface of cells (Krangel et al., 1979; Ploegh et al., 1979). However, as shown in Fig. 5, we have found a human lymphoma expressing y chains only without light chains, suggesting that light chains per se are not required for expression of Ig molecules on the surface of B cells (Coda1 et al., 1981b). As outlined in Section II,B,6, p, appears to be structurally different from p,, having an additional hydrophobic polypeptide segment at the C-terminal end. The precise differences in mouse p, as compared to p m consistent with biochemical data based on nucleic acid sequence studies have been reported by Kehry et al. (1980)and Rogers et al. (1980). Analogous differences appear to exist between y, and y s (Oi et al., 1980).Also 6, appears to contain a hydrophobic segment (Parkhouse et al., 1979). However, since each heavy chain appears to have its own

224

TORE GODAL AND STEINAH FUNDERUD

FIG. 5 . Two-parameter flow cytometric diagram of cells from biopsy 374/79 labeled with FITC-conjugated antibodies against A and K light chains, y heavy chains, and unstained control (c) as indicated. The origin is in the left-hand comer of the display of the 64 x 64 channel storage matrix of the multichannel analyzer. Fluorescence intensity per cell is measured along the x axis, which is parallel to the intensified line in each diagram. The fluorescence amplification factors were 2/3 for y, and 1 for the three other diagrams. Light-scatter intensity is measured along the y axis, and the vertical z axis represents the number of cells registered per channel. The intensified line (corresponding to channel y = 16) represents a threshold set on the light-scatter signal to prevent noise signals from fluorescent debris being registered. The figure shows a lymphoma expressing y chains only, without light chains at its surface. From Coda1 et al. (1981b).

DNA segment for the “membrane” polypeptides, the structure is likely to vary between the different Ig classes, and this may have functional implications.

2. Surface ZgD (sZgD)3 While the increased susceptibility to suppression of early B cells appears to be intimately related to the transition from pre-B cells to B “ T h e correct term for surface Ig (sIg) should be sIg,, e.g., sIgD,, to indicate its heavy-chain structure. However, since the great majority of data to be reviewed are based on surface analysis alone, we use here the term sIg.

B-CELLLYMPHOMAS AND B-CELLDEVELOPMENT

225

cells, i.e., the expression of Ig receptors, the phenotypic characteristics of these cells remain unclear. Since sIgD has been shown to be expressed after sIgM both during ontogeny (Vitetta et al., 1975) and during B-cell development in adult animals (Vitetta et al., 1976), Cambier et al. (1976) hypothesized that the acquisition of sIgD would terminate the suppression susceptible stage (Vitetta and Uhr, 1975). Evidence in favor of this hypothesis was presented by Cambier et al. (1977) by showing that the selective removal of sIgD, which is exquisitely susceptible to proteolysis by papain (Vitetta and Uhr, 1976), increased the susceptibility to tolerance of sIgM- + sIgD-positive cells. However, b y using the fluorescence-activated cell sorter (FACS) Layton et al. (1979) could not confirm these results and clearly demonstrated that sIgD-negative and sIgD-positive cells were equally susceptible to tolerance induction. Although sIgD appears after sIgM in differentiation, it usually also disappears earlier. Thus, while sIgM- + sIgD-positive cells are involved in primary immune responses (Black et al., 1978), sIgD is not present on most memory cells. Moreover, treatment of anti-6 antibodies (Dresser and Parkhouse, 1978)in uiuo prevents the generation of memory cells in primary responses, but not in secondary responses. Thus, sIgD appears to be present during a fairly limited period of B-cell development, but its precise function or functions remain(s) unknown. This will be discussed further in Section III,C,5, as studies on human nodular lymphomas suggest that the role of sIgD may be related to germinal center function.

3. Expression of Other Immunoglobulin Classes A characteristic feature of the immune response is that a shift from IgM production to the synthesis of IgG or IgA may take place. Such a switch has been observed in single clones of B cells (Anderson e t al., 1978; Wabl et al., 1978), showing that this is a maturation-associated event. Such a shift has also been observed with regard to membrane Ig, e.g., virgin B cells express sIgM and/or sIgM + sIgD, while memory cells may express sIgG or sIgA (Strober, 1976; Coffman and Cohn, 1977; Black et al., 1978). Whether a switch in Ig, (e.g., from IgM, + IgG,) is always accompanied by a switch in Ig, or vice versa, remains unclear, but since a considerable body of evidence suggests that this kind of switch is accompanied by a deletion of the p gene (see Section 11,B,4), it would appear likely that they switch simultaneously. While there is general agreement on the coexpression of sIgM and sIgD and switching, the appearance of sIgG and sIgA in relation to B-cell development and their coexpression with sIgM and sIgD remain a subject of discussion. Abney et al. (1978) have claimed, based

226

TORE GODAL AND STEINAR FUNDERUD

on observations in mice, that sIgA and sIgG may become coexpressed with sIgM prior to sIgD and that a major proportion of B cells that express sIgM and sIgD also express sIgG or sIgA. Such cells have also been described in man (Gathings et al., 1977) and have been clearly demonstrated in a case of B-cell lymphoma (Landaas et al., 1981) (Fig. 6), but their relationship to B-cell differentiation remains unclear.

4. Functional Roles of slg Since sIg spans the membrane, it meets the structural requirements for having the potential of transmitting signals across the cell membrane. Moreover, cross-linking of sIg at the surface has been shown to create binding to the cytoskeleton (Flanagan and Kock, 1978), which is required for redistribution, including capping, of sIg at the cell surface (see Schreiner and Unanue, 1976). However, whether sIg represents a functional receptor in the sense of being capable of transmitting signals across the cell membrane involved in activation of B cells, has remained a controversial issue and is still questioned (Wigzell and Binz, 1980). Coutinho and Moller (1974)and Moller (1975)have argued that B cells become activated by signals mediated b y other receptors and that sIg simply functions as a focusing mechanism for antigen to responding T cells, which may in turn deliver signals activating the B cells. While the Coutinho and Moller hypothesis may still be valid for certain subsets of B cells, there is now a large body of evidence, primarily based on the use of anti-Ig, that sIg ( p , 6, as well as y ) can initiate proliferative responses in mature, Cg-positive B cells (see Moller, 1980).This is more often acquired with insoluble rather than soluble anti-Ig (Parker, 1975; Purb and Vitetta, 1980; Henriksen et al., 1980). Anti-Ig in combination with T-cell factors can also initiate Ig, production (Parker et al., 1979). The chain of biochemical events elicited by cross-linking of sIg, which results in B-cell activation, remains unknown. However, studies on monoclonal B-cell populations suggest that the mitogenic effect is closely related to an early influx of potassium and is unrelated to capping (Heikkila et al., 1981). These studies suggest that sIg may activate two biochemically independent pathways-one leading to capping, and the other to B-cell proliferation. However, the capacity of anti-Ig to activate B cells depends on B-cell development. As described in Section II,C, immature, murine B cells readily become inactivated by anti-IgM, and activation by the same ligand has been achieved only in adult animals (Weiner et al., 1976; Sieckmann et al., 1978).

B-CELLLYMPHOMAS

AND

B-CELLDEVELOPMENT

227

FIG. 6. Two-parameter flow cytometric diagram of cells from biopsy 28/78 (2386/78) labeled with FITC-conjugated antibodies as indicated. The presentation is as outlined in the legend to Fig. 5. Diagrams of 6-, y-, and p-stained cells were registered with a fluorescence amplification factor of 2/3 relative to the amplification used for the unstained control and for K staining. For A staining a relative amplification factor of 1/5was used. The figure shows a lymphoma of A type, with distinct positive staining for p , 6, and y heavy chains. From Landaas et al. (1981).

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Thus, studies on the functional roles of sIg, which appears to be in advance of any other surface receptor on lymphocytes, have shown that a biochemically defined receptor (e.g., sIgM) may serve different functions dependent on the stage of development of the responding B cells. These findings show that the conditions for activation may differ from one B-cell subset to the other. Studies on highly homogeneous, preferably monoclonal populations, should therefore be particularly rewarding for a more detailed analysis of B-cell activation. Certain aspects of such studies can now be undertaken on neoplastic B cells (see Section III,A,2,b).

5. Complement Receptors ( C R s ) B cells have been shown to express two types of complement receptors, CR1and CR2,which appear to be structurally distinct and located on separate molecules within the lymphocyte membrane (Ross et al., 1973; Ross and Polley, 1975). While CRl can bind Cab, Cr, and Cs and is also found on primate erythrocytes, CR2 binds C3d and is found on B cells only (Ross, 1979). A third B-cell-associated C3 receptor has been reported on Raji cells (Okuda and Tachibana, 1980) apparently also distinct from the CR3 receptor binding C3bl present on monocytes and granulocytes (Ross, 1979). The structure of these receptors has been studied by several groups. Fearon (1979, 1980) has isolated and characterized CR, from human erythrocytes. He found that the receptor consists of a 205,000 molecular weight (MW) glycoprotein and has isolated a similar component from human granulocytes, monocytes, and B cells. Antibodies to the complement block CR1 receptor activity (Fearon, 1980). A single protein of similar physiochemical characteristics has also been isolated from human erythrocytes by Dobson et al. (1981). In contrast, Gerdes and Stein (1980, 1981) have reported the CRl receptor from human erythrocytes or B cells to consist of an 80,000 MW protein composed of several smaller polypeptide chains. Type CR2 has been isolated from Raji cells by Lambris et al. (1981) and found to consist of a 72,000 MW single polypeptide chain glycoprotein, whereas Gerdes and Stein (1981)reported CR, to have a similar structure to CR1. However, the last authors have not done studies to distinguish between CRl and CR2 either b y different cells or by specific antibodies to CR1 and CR2. The most common method so far for detection of CRs has been the use of complement-coated red cells. This assay gives precise information with regard to the proportion of receptor-positive cells with a sufficient density to be detected by the assay, but yields no information

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with regard to density. Slease et al. (1980) have approached this problem by using FITC-labeled CSb and analyzed fluorescence by flow cytofluorometry. Their data revealed large variations between different monoclonal B-cell populations with regard to CR1 receptor density. But since even cells with the lowest density (CLL cells) also were positive by EMC rosettes, their studies suggested that the rosette technique was as sensitive as the other method. However, these data need to be confirmed, as the conclusions critically depend on whether the EMC rosettes detected CRl only. They reported that all their CLLs were EMC positive. Their results are therefore at variance with those of Ross et al. (1973), who reported that the majority of CLLs express CR2 only. With the emergence of antibodies specific for CRI and CR2 receptors (Fearon, 1980; Lambris et al., 1981), studies on the precise distribution and density of CRs should be facilitated. For example Fearon (1980) has reported B cells to have 21,000 sites per cell for CRI with specific antibodies. Its positivity in the EMC3bassay may be due to release of endogenous P1H globulin and/or C3b inactivator, causing binding to CR2 via ECsbi. A close association between complement receptors, particularly CR2, and Epstein-Barr virus (EBV) receptors has been reported (Jondal et al., 1976; Yefenofet al., 1976; Yefenofand Klein, 1977; Klein et al., 1978). However, findings by Wells e t al. (1981) have indicated heterogeneities between CR expression and virus binding, suggesting that they are not identical moieties, although they belopg to the same receptor complex as determined by blocking and cocapping experiments. 6. Complement Receptors and B-Cell Diferentiation Complement receptors are not present on pre-B cells (Raff, 1976). During B-cell development in the bone marrow, CRs are acquired after sIgM and Fc receptors (Yang et al., 1978; Chan and Osmond, 1979). During ontogeny also, CRs are acquired after sIg and are not detected on B cells until 2 weeks after birth (Gelfand et al., 1974). Similar findings have been made after in vitro induction of receptors in mice (Hammerling e t al., 1976) and human bone marrow lymphocytes (Kagan e t al., 1979). Thus, CRs appear at about the same time as IgD (Vitetta et al., 1975; Sitia et al., 1979). Whether all B cells at one stage or the other during developmqt express CRs remains unclear. The findings of Yang et a2. (1978) that only about one-half of B-cell precursors homing to the spleen developed CRs may suggest that only a proportion of B cells develop CRs in this organ. However, just like IgD, CRs may also become lost during B-cell

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differentiation. This is clear from the fact that plasma cells lack CRs (Burns et al., 1979) and is also suggested by the findings of Mason (1976), who showed that sIgG-positive memory cells lacked CRs. The function of CRs remains unknown. Since the generation of memory cells is dependent on C3, C 3 may therefore be involved in B-cell activation, possibly by events taking place in germinal centers. The localization of antigen to germinal centers has been found to be a highly C3-dependent process (Klaus and Humphrey, 1977; Klaus, 1979).Complement receptors are likely to be involved in this focalization process (Klaus, 1979).

7. Other Surface Receptors Apart from receptors for the F c proportion of IgG (see Dickler, 1976) Fc receptors on human B cells for IgE (Gonzales-Molina and Spiegelberg, 1977) and IgM (Pichler and Knapp, 1977; Ferrarini et aZ., 1977) have been reported. Fcy receptors appear earlier during ontogeny than CRs (Hammerling et al., 1976; Yang et al., 1978), and apparently most, if not all, B cells develop such receptors (Yang et al., 1978). F c ~ receptors, however, are expressed b y only a proportion of B cells (Ferrarini et al., 1977). Moreover, and interestingly, Rudders et al. (1980) found that sIgG-positive B-cell lymphomas lacked this receptor. The biological function of Fc receptors on B cells is difficult to assess owing to their presence on both macrophages and T cells. Thus, not surprisingly, both enhancing and suppressive effects have been observed (see Morgan and Weigle, 1980; Nicholson and McDougal, 1981). The role of Fc receptors in B-cell development remains unknown. Other receptors that may be expressed on only a proportion of human B cells, and therefore may be related to B-cell development, are the mouse erythrocyte receptors (Stathopolos and Elliott, 1974), which may detect B cells in an early stage of differentiation (Kagan et aZ., 1979), and the insulin receptor, which is detected on activated lymphocytes (Helderman e t al., 1978). Differences in the density of this receptor have been reported between a B-cell line (Ramos) and EBV-transformed sublines (Spira et al., 1981). Another class of receptors of particular immunological interest are those that are known to interact with T cells. This includes the Ia determinants and receptors for T-cell factors. Ia determinants in mice and the related HLA-DR determinants in man have been found on all B cells with the possible exception of a small proportion of early sIgM-positive B cells (see Hammerlinget al., 1976; Mondet aZ., 1980). Pre-B cells in mice do not express Ia-antigens (Kearney et aZ., 1977)

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whereas human pre-B cells express HLA-DR antigens (Cooper and Lawton, 1979). E. REGULATION OF B-CELL FUNCTION Two major facts about the immune system have become apparent during the 1960s and 1970s. First, as initially demonstrated by Davies (1969), Claman and Chaperon (1969), and Miller and Mitchell (1969), T cells play a major role in the regulation of B-cell function. This regulation also involves suppressor effects (Gershon, 1974). Second, as initially described by Cantor and Boyse (1977), the T-cell compartment is very heterogeneous and comprises a large number of subsets, each with distinct differentiation markers and Ig functions. A similar level of heterogeneity may apparently also exist in the B-cell compartment (Godal et al., 1981b,c). These interactions may involve antigen-specific, antigen-nonspecific, or idiotype-specific events. Thus, the regulation of B-cell function represents a plethora of cell-tocell interactions and phenomena. It is not surprising, therefore, that our understanding of the regulation of the B-cell system, in spite of quite extensive research since 1960, remains fragmentary. The factors that govern B-cell development in the antigen-independent phase, including changes in the phenotypic pattern of receptors of early B cells developing to mature B cells, remain largely unknown. The possibility that idiotypes and antiidiotypes may play a role is considered in Section II,E,3. Considerably more information is available with regard to antigen-associated regulation of mature B cells. Experimental studies on B-cell function have to a large extent relied on Jerne’s plaque-forming assay and, therefore, the measurement of the net result of various events. However, it is severely limited in situations where proliferation takes place without antibody synthesis and secretion, or vice versa. Thus, from several points of view, it is important to consider such phenomena separately.

1. B-Cell Proliferation and Antibody Production Proliferation and generation of memory in the B-cell compartment seem to be only partly under T-cell control. For example, several investigators have reported development of B-cell memory in the T-cell deficient mice to T-dependent antigens (Roelants and Askonas, 1972; Davie and Paul, 1974; Diamantstein and Blitstein-Willinger, 1974). In in vitro systems, these antigen-triggered proliferating cells may be induced to Ig synthesis by T-cell replacing factor (TRF) (Askonas et

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al., 1974) or T cells (see Dutton, 1975).This led Dutton to suggest that T-cell activity is not critically required for the initiation of B-cell activation, but that T cells are required to turn on antibody synthesis and secretion. A macrophage-derived factor may be involved in the induction of proliferation under certain conditions (Hoffmann and Watson, 1979). The Dutton hypothesis has gained support from studies with anti-Ig. Thus, as described in Section II,D,4, both in experimental animals and man F(ab)z fragments of anti-Ig can induce proliferation in a mature subset of B cells carrying C3 receptors. This proliferation can take place in the absence of T cells and accessory cells (Sieckmann et al., 1978; Henriksen et al., 1980). At least in mice, Ig, production in anti-Ig activated B cells can b e initiated with T-cell factors (Parker et al., 1979). On the other hand, T cells can clearly also induce proliferation. In the mouse, such T-cell dependent B-cell proliferation has been analyzed in some detail by Tse e t al. (1981). An interesting feature that emerged from these studies was that, while Ir gene restricted at the B-cell level and induced by a specific antigen, the resulting B-cell proliferation was polyclonal.

2. Immunoglobulin Heavy-Chain Switch and Afinity Maturation In contrast to B-cell proliferation, Ig heavy-chain switch and affinity maturation appear to be under much tighter T-cell control (see Katz, 1977).These two phenomena appear to be associated, although affinity maturation can also take place in IgM immune responses, but this process is completed at an earlier stage than it is for the IgG class (Claflin et al., 1973). The precise role of T cells in these processes remains unclear. Whether antigen-specific as well as idiotype-specific T-cell help are involved, or whether these effects can be achieved with antigen nonspecific T-cell factors, is unknown to us. A major question that relates to both these phenomena is whether they are achieved only through selection in the pool of B cells precommitted from an early stage (pre-B-cell level) to a certain Ig class and affinity, or whether virgin B cells are pluripotent with regard to class and affinity change during maturation. Some studies have provided interesting data on both these aspects. In an elegant study Teale e t al. (1981) separated primary or “memory” B cells according to surface isotype and analyzed Ig production in the spleen focusing assay, which allows analysis of individual clones. Their studies provide strong evidence that mature B cells (primary as well as “memory”) have the potential of multiple Ig class production. This implies that environmental factors, including T cells, may play a

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predominant role with regard to determining Ig class. However, it does not exclude the possibility that switches may take place independently of T cells. The findings of Mongini et al. (1981)are relevant to this issue. Mongini et al. found that nude mice immunized with TNP-Ficoll, a T-independent antigen (TI-2), produced antibodies of different isotypes. The peak titers of these antibodies were of the following order: IgM > IgG3 > IgGl > IgG2b > IgG2a, which correlates directly with the 5‘ to 3’ heavy-chain constant-region gene order in the mouse. This finding may suggest that isotype switching in the absence of T cells is a probabilistic event directly related to the distance of a particular constant-region gene from the 5‘ end of the C, domains involved in gene switching (see Section II,B,5). However, when such mice were immunized in the presence of T cells, substantially higher levels of IgG2 antibody were produced, showing that T cells even in T-independent systems can promote class switching, as has also been reported in other systems (Brayley-Mullen, 1974). With regard to affinity maturation, analyses of the V region genes of IgM- as compared to IgA- or IgG-expressing cells have shown that more variations are found in VH gene segments in IgA- and IgG- as compared to IgM-producing cells (Gearhart et al., 1981).This intriguing finding suggests that affinity maturation not only is the result of a selection of B-cell clones committed at the pre-B-cell level to high affinity antibody production as suggested by the studies of Julius and Herzenberg ( 1974), but that genetic changes (“mutations”) may take place in the V region of mature B cells leading to antibodies of higher affinity.

3. Zdiotypes, Networks, and Suppression In the interaction between cells, soluble factors, and antibodies of the immune system, antigen obviously plays an essential role. However, as was first postulated by Jerne (1974), idiotypes may play important roles. As suitable test systems have become developed, idiotypes have in fact been shown to be capable of “mimicking” antigen in almost every aspect. This applies to post-receptor repertoire expression, as mentioned in Section II,C, generation of T-cell help (Gleason et al., 1981), and particularly suppression, where the documentation now is extensive (see below). As envisioned by Jerne (1974), idiotype anti-idiotype reactions put the immune system into networks of interlinked circuits by which an antigen or an idiotype not only will induce an immune response toward itself, but also elicit network perturbations. Whether idiotypic networks have distinct roles to play in B-cell development, particu-

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larly in the development of early B cells to virgin B cells, remains unclear. But the fact that both idiotypes (Bona et al., 1981) and antiidiotype antibodies (Hiernaux et al., 1981) may induce expression of silent clones clearly suggests this as a possibility. T-cell-mediated suppression of the B-cell system represents a very complex circuit involving different T cells and factors. These factors may carry anti-antigenic or anti-idiotypic determinants. Suppression may be specific or, under certain conditions, nonspecific (see Germain and Benacerraf, 1981). An essential consideration from our point of view is that T-suppressor cells have T-helper cells as target. Thus, according to Gershon (1980),there is no evidence that suppressor cells can act directly on B cells with the following exception. Lynch et al. (1979) have provided evidence that T-suppressor factors may prevent the secretion of Ig from myeloma cells. This finding again underlines the need for separate studies of different aspects of B-cell development. Ill. B-Cell Neoplasms

B-cell neoplasms represent a very heterogeneous group of diseases ranging from rapidly progressive ones such as ALL to very slowly progressing conditions found among CLL patients. It comprises stem cell neoplasms as well as cells in an end stage of differentiation (myelomas). However, in most of the conditions the precise relationship to normal B-cell development remains unclear. This is to a large extent due to a lack of knowledge about normal B-cell development. Moreover, precise prognostic factors are also lacking. Thus, both at the basic as well as at the clinical level much information is lacking. Nevertheless, most of the neoplasms have some features in common, such as monoclonality and maturation arrest. There are, however, some informative exceptions to these general features, which therefore require some discussion. A. GENERALFEATURES AND THEIREXCEPTIONS

1. Monoclonality To an immunologist diagnosing B-cell neoplasia expressing Ig, the single most important feature is light-chain isotype exclusion. Although this is not a formal proof of monoclonality, there is extensive evidence for monoclonality in B-cell tumors (see Fialkow, 1976). In an examination of more than 200 lymph node biopsies we have not yet

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seen a single case with a monoclonal B-cell staining pattern without histopathological or clinical evidence of malignant lymphoma. On the other hand, a lack of light-chain isotype exclusion may be found, but in such cases it is difficult to determine whether the cell suspension is representative of the neoplastic population. Using immunochemistry with reagents purified b y immunoadsorbents, a much lower occurrence of so-called bitypic cases of lymphoma has been found with the immunoperoxidase technique (Landaas et al., 1981) than was previously reported by Taylor (1978a). However, from a pathogenic point of view, monoclonality appears to be a fairly late event. Thus, there are several interesting conditions that suggest that the neoplastic process is initiated by a polyclonal phase followed by a selective process resulting in a monoclonal neoplastic population. Examples of these follow. a. Epstein-Burr Virus Znfection in Immunodeficient Subjects. Epstein-Barr virus, (EBV) can cause a spectrum of clinical manifestations in patients with immune defects against EBV (Purtilo, 1980). These include polyclonal B-cell lymphomas directly associated with primary infection with EBV (Robinson et al., 1980). b. Angioimmunoblastic Lymphadenopathy. This condition is characterized by hypergammaglobulinemia, fever, weight loss, occasionally a rash, and general lymphadenopathy. Histopathologically the condition is characterized by polyclonal immunoblastic and plasmacell proliferation and vascular proliferation (Rappaport and Moran, 1975). This condition is apparently triggered b y a hypersensitivity reaction (Schulz and Yunis, 1975) and may progress to malignant lymphoma (Lukes and Tindle, 1975). c. Autoimmune Conditions. Certain autoimmune conditions-most notably Sjogren’s syndrome, in which lymphoid cell infiltration of salivary and lacrimal glands is found-may progress to a more general involvement called “pseudolymphoma,” and terminate in malignant lymphoma (see Tala1 et al., 1980).This transition is often associated with a shift from polyclonal to monoclonal B-cell populations (Zulman et al., 1978). These clinical conditions, as well as other experimental data as outlined by Klein (1979), suggest that the pathogenesis of lymphomas is a multifactorial process that may often be initiated by a polyclonal B-cell proliferative stage, and that monoclonality represents a relatively late event, due to a selective process, in the case of EBV apparently mediated by immune-regulatory mechanisms, of a tumorigenic (“autonomous’’) clone of cells (Klein, 1979). Such cells often carry characteristic chromosomal changes (Klein, 1979).

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2. Maturation Arrest This phenomenon is quite striking in most cases of non-Hodgkin lymphomas and CLL. For example, in B-cell lymphomas expressing sIgM + sIgD the proportion of cells expressing sIgM is very similar to those expressing sIgD (Godal et al., 1981b), suggesting that both receptors are expressed simultaneously on the great majority of neoplastic B cells. On the other hand, there is clear evidence that the maturation arrest may be incomplete, and various maneuvers to release cells from maturation arrest in vitro have been reported. a. Zncomplete Maturation Arrest in Vivo. This is found in a proportion of B-cell lymphomas in which different stages of B cells from small lymphocytes to plasma cells may be found. Such cases belong to the lymphoplasmacytoid groups (see Lennert, 1978). Another case in point is diffuse lymphocytic lymphoma, in which Ig-containing immunoblastic cells may be found in so-called “maturation zones” contrasting the monotonous diffuse infiltration of small lymphocytes in such cases (Landaas et al., 1981).The presence of monoclonal Ig in a proportion of CLL with identical idiotype to that found on the CLL cells represents a third example (e.g., Fu et al., 1978). With regard to surface markers, the lymphoplasmacytoid group is heterogeneous (Godal et al., 1981b).This indicates that B-cell maturation toward plasma cells can take place from different subsets of B cells (see Section 111,D). b . In Vitro-Promoted Maturation and Diflerentiation. Maturation, and in some cases differentiation, of B-cell neoplastic populations in vitro has been reported. Based on the leads given by studies on murine erythroleukemia (see Marks and Riilcind, 1978), a large number of agents have been tried. Some of those found successful are described below. i. T-cell factors. Fu et al. (1978) have reported induction of plasma cells with allogeneic T cells in a high proportion of CLL cells synthesizing IgM of the same idiotype as sIgM present on CLL cells. DNA synthesis was not required for this induction. This maturation was found in two cases of CLL with monoclonal components in serum. T cells of these patients were found to be defective in providing help to normal B cells. ii. Phorbol ester and anti-immunoglobulin. Totterman et al. (1980, 1981a,b) have reported maturation toward plasma cells with cytoplasmic Ig synthesis in a high proportion of CLL cells by 12-O-tetradecanoyl phorbol-1Sacetate (TPA). Also in this case, the maturation was not associated with cell proliferation.

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Independently, we (Godal et al., 1981a,c) have found that TPA + anti-Ig can induce morphological alterations and proliferation in a proportion of B-cell lymphomas. In some of these, increased synthesis of intracellular Ig has also been detected. One case of sIgM + sIgD nodular lymphoma has been extensively studied. In this particular lymphoma, anti-IgD + TPA have been found to induce proliferation only, while anti-IgM + TPA induced both proliferation and Ig synthesis (Ruud et al., 1981).This case illustrates that differentiation (i.e., a bifurcation process) can be achieved in vitro. This case also suggests that sIgD may lead to a “readout” different from that of sIgM on the same cell. Some experimental studies are relevant to these findings. In a murine B-cell leukemia (BCL,), Isakson et al. (1981)have reported induction of proliferation with Sepharose-coupled anti-IgM or antiIgD. By adding T-cell factors, secretion of Ig could also be achieved with both anti-p and anti-6. Although their findings and ours are not strictly compatible since they did not need to use TPA, it is possible that the differences with regard to anti-8 may be due to different subsets. Thus, BCLl carries sIgM/sIgD in a high ratio and lacks CR receptors, whereas our particular lymphoma carries twice as much sIgD as sIgM and also carries CR. Both the findings of Totterman et ul. (1981a) and ours show that different subsets of B cells may be triggered to increased Ig synthesis and morphological transformation toward plasma cells, providing further support for our previous conclusion that plasma cell maturation may take place from different subsets of B cells. iii. Epstein-Burr virus (EBV)and other B-cell mitogens. EBV may be used to convert EBV-negative lymphoma cell lines such as Ramos. By comparing markers on the converted sublines as compared to the original Ramos, Spira et al. (1981) found that the converted sublines acquired sIgD. Transformation of CLL cells with EBV has been reported in a few cases, in which evidence of maturation also was found with regard to Ig synthesis and secretion (Hurley et al., 1978; Karande et al., 1980). Robert (1979) has reported both induction of DNA synthesis and Ig synthesis and secretion in CLL cells with mitogens, such as phytohemagglutinin, pokeweed mitogen, dextran sulfate, lipopolysaccharide, and anti-&-microglobulin and EBV. However, his findings with regard to proliferation are at variance with those of a number of other investigators, including our own (for references, see Godal et al., 1978), who have reported a lack of mitogen responsiveness in CLL cells. The only mitogen we found to which some CLL cells responded was the Ca2+ionophore A23187, suggesting together with other data

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that there is a cell membrane-associated block to mitogens in CLL cells. With regard to Robkrt’s report on Ig synthesis and secretion, the proportion of cells showing light-chain-specific plaques (reversed plaque assay), was in general small (<5% of the total number of CLL cells present). Nevertheless, light-chain responses corresponding to those of the CLL cells were striking in some cases after induction with E BV. c. Maturation Arrest and the Target of Transformation. At first sight the level of maturation arrest may be considered identical to the target cell of neoplastic transformation. However, there are clear examples of a dissociation between the two. First, as reported by LeBien et al. (1979), Vogler et al. (1979), and Greaves et al. (1979a), Philadelphia chromosome-positive pre-B cells in blastic crisis of chronic myelocytic leukemia (CML) provide strong evidence together with other data (Fialkow, 1976) that the target of transformation in this disease is a multipotential stem cell. Evidence for alterations in the stem-cell population has also been found in multiple myeloma. Kubagawa et al. (1979) studied pre-B cells and B cells in two patients with anti-idiotypic antibodies. They found that idiotype-specific B cells were expanded, expressing p , 6, y, as well as (Y heavy-chain isotypes, although the myeloma was of IgA origin. Moreover, a significant increase in pre-B cells carrying the same idiotype was also found. These data suggest that B cells as well as pre-B cells belonging to the same clone as the myeloma cells may be greatly expanded in multiple myeloma. Similar changes in peripheral blood B cells have been observed by others (Van Acker et aZ., 1979; Petterson et al., 1980; Schedel et al., 1980). The most likely explanation for these findings is that the neoplastic transformation in multiple myeloma involves stem cells. Since neoplastic transformation is usually a multistage process, some of the events may only have “hit” the stem cells. Alternatively, the expansion of the clone may be a result of idiotype-specific network perturbations. However, we consider this possibility unlikely, since pre-B cells do not express sIg. The mechanism behind maturation arrest remains unknown. It may be due to intrinsic disturbances of the gene regulation of the neoplastic clone, but could also be due to lack of “help.” The study of Fu et al. (1978) (see Section III,A,2,b) is interesting, as their cases demonstrate that “environmental factors” may play an important role in some cases of maturation arrest. The increasing number of methods by which neoplastic B cells may be “rescued” from maturation arrest in vitro provides further evidence that maturation arrest is not solely due to an

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intrinsic defect of the neoplastic clone, but that it may result from a “stalemate” in the interaction of environmental factors and the neoplastic clone. B. B-CELL NEOPLASIAI N RELATIONTO B-CELL DEVELOPMENT

1. Neoplasms in Pre-B Cells Apart from blast crisis of CML referred to above, neoplasms with pre-B-cell phenotypic characteristics (cytoplasmic p heavy chains) are found in a proportion of ALLs. This entity was first described b y Vogler et al. (1978). It has been found to occur in approximately 20% of ALLs, but they do not appear to have clinical and hematological features distinct from other ALLs (see Humphrey et al., 1980). With regard to surface and other markers, pre-B-cell ALLs usually express CALL antigen and HLA-DR determinants and contain terminal deoxynucleotidyltransferase (Greaves et al., 1979b), a phenotype also found among normal pre-B cells (Janossy et al., 1979). On the other hand, among the four cases of Vogler et al. (1978),two were reported to express Fc receptors and one Cs receptors, which are not detected on normal pre-B cells. Cell lines with pre-B-cell characteristics have also been established (Hunvitz et al., 1979; Minowada et al., 1979).

2 . Chronic Lymphocytic Leukemia ( C L L ) .Does It Include Neoplasms of Early B Cells? Chronic lymphocytic leukemia represents a very heterogeneous group of mainly B-cell neoplasms. To an extent, the diagnosis is arbitrary, depending on whether the diagnosis is based on the examination of peripheral blood or a lymph node biopsy. Since in our experience any histological type of lymphoma, including those very close to plasma cell maturation as well as germinal center cell tumors, may give rise to CLL-type leukemia, CLL must be very heterogeneous with regard to B-cell developmental stages with a considerable overlap to non-Hodgkin lymphomas (NHL). Efforts have been made to distinguish CLL from leukemia arising secondarily to lymphoma because the latter has a somewhat poorer prognosis (Zacharski and Linman, 1969). Among the parameters used are capping of sIg, the mouse erythrocyte rosette test, and surface concentration of sIg. Cohen (1978) claimed that capping of sIg occurs in NHL with secondary leukemia, but not in CLL. We have confirmed his findings to the extent that typical CLL cases lack capping (Godal et al., 1978) and that leukemia occurring after the presentation often

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MARKERSIN CHRONIC LYMPHOCYTIC LEUKEMIA

IMMUNOLOGICAL SURFACE

sIgM sIgM sIgM sIgM

Markers

No.

+ sIgD + CRs" + sIgD + CRs

2 3 1 8 14

Total No. examined CRs,

complement receptors.

shows capping, but the distinction is not absolute. Moreover, since a considerable proportion of NHL do not cap sIgM (Godal et al., 1981b) in the initial tests on cell suspensions from biopsies, it is difficult to understand how this parameter could be absolute. The same applies to surface concentration of Ig, another difference proposed to distinguish between these conditions (Aisenberg and Wilkes, 1976), but this parameter is also highly heterogeneous in biopsy material from NHL (Godal et al., 1981b). However, the mouse erythrocyte rosette test is of interest both because it appears to discriminate quite well between CLL and non-CLL neoplasms (Catovsky et al., 1976; Forbes et al., 1978; Burns and Cawley, 1980), and because it appears to be a marker of early B cells (see Section II,D,7). These data therefore suggest that a considerable proportion of CLLs are derived from early B cells. Other data support this hypothesis. In our series of 14 CLLs, the distribution of surface markers was as shown in the table. This pattern is compatible with a derivation from early B cells with a sequence of surface marker expression of sIgM + sIgM + sIgD + sIgM + sIgD + CR. Whether the altered cell membrane functions (Godal et al., 1981b) and asynchronous synthesis of light chains (Maino et al., 1977; Gordon et al., 1978) also reflect normal features of certain subsets of early human B cells remains unknown. In conclusion, CLL represents an ill-defined and heterogeneous group, most of which are derived from B cells. Many CLLs have phenotypic markers compatible with a derivation from early B cells.

3. Neoplasia in Mature B Cells This group is found in NHL and multiple myeloma. Since multiple myeloma, a neoplasia of plasma cells, represents terminally differentiated B cells, this group is not of particular interest from the perspective of our discussion here and will therefore not be considered further.

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Since the pioneering work on Burkitt lymphoma cells (Klein e t al., 1968), numerous investigations during the 1970s have shown that the great majority (80% or more) of NHL, including “reticulosarcomas,” are derived from B cells (Aisenberg and Bloch, 1972; Taylor, 1974; Garvin et al., 1976; Levy et al., 1977; Payne et al., 1977; Belpomme et al., 1978; Daveyet al., 1978; Lennert, 1978; Lukes et al., 1978; Pinkus and Said, 1978; Stein et al., 1978; Warnke et al., 1978, 1980; Habeshaw e t al., 1979). For review, see Stein (1978) and Mann et al. (1979). The methods used in such studies have been mainly the peroxidase anti-peroxidase method (PAP) on tissue sections or immunofluorescence on cells in suspension. Both methods have distinct advantages and disadvantages. The PAP method can be carried out on routinely fixed tissues and allows direct correlation with ordinary light microscope examinations, and therefore also allows the Ig location to be related directly to neoplastic cells. The main limitation of the method is that fixation to a variable degree may denature or covert the Igs in the tissues, reducing their specificity and sensitivity. In addition, PAP when used on routinely fixed sections detects cytoplasmic Ig, but not surface Ig (Taylor, 1978a). By immunofluorescent staining of intact cells in suspension, on the other hand, surface Ig is detected without interference from fixation procedures providing a higher sensitivity and specificity. For adequate analysis of lymph node biopsies both methods are required (Landaas et al., 1981).The application of monoclonal antibodies to immunohistochemistry is likely to provide a new powerful tool to such investigations. Non-Hodgkin lymphomas can be divided into a number of different histological groups. The nomenclature of the histopathological classification remains a subject of discussion, but there is general agreement that some B-cell lymphomas are derived from germinal center cells, whereas others, such as immunoblastic lymphomas and lymphomas with plasmacytoid (lymphoplasmacytoid) features, appear to be at a maturation stage close to plasma cells with high intracellular concentrations of Ig as demonstrated b y immunohistochemistry (Taylor, 1978b). These observations suggest that B-cell lymphomas are derived from different stages of B-cell maturation. By using multiple immunological parameters, we have tried to obtain more detailed information on this point (Godal e t al., 1981b). Our findings can be summarized as follows. Based on sIg isotypes and their concentrations as determined by flow cytofluorometry, capping, and CRs, NHL may be subdivided into a large number of distinct immunological subsets. The majority of histological subgroups comprised more than one immunological subgroup. These data suggest that there is extensive heterogeneity within

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the B-cell compartment. This was most striking for lymphomas derived from germinal centers. 4. Germinal-Center Cell Lymphomas and Germinal-Center Function Germinal centers are populated mainly by B cells (Parrot and deSousa, 1971; Durkin et al., 1972; Howard et al., 1972), but contain a few T cells (Poppema et al., 1981).Moreover, nodular or diffuse lymphomas of germinal-center cell morphology are B cells (see Stein, 1978). A high turnover of cells is found in germinal centers, and a considerable body of evidence suggests that they are associated with the generation of memory cells (see Thorbecke et al., 1974; White, 1975).Moreover, the localization of immune complexes within lymphoid follicles is a C3-dependent process (Papamichail et al., 1975; White et al., 1975), and C3 is required for generation of memory cells (Klaus and Humphrey, 1977; Klaus, 1978). Since sIgD becomes lost during the generation of memory cells (see Section II,D,2), it is interesting to note the immunological subtypes detected in follicular lymphomas (Godal et al., 1981b). They were ( a ) sIgM + sIgD + C3; (b) sIgM + C3; ( c ) sIgM + sIgD; ( d ) sIgM; ( e ) sIgG. The relative concentration of sIgD was found to range from strongly positive to barely detectable by flow cytofluorometry. These findings suggest that IgD becomes lost during maturatioddifferentiation processes taking place in germinal centers; and that switching from IgM to IgG also may take place in germinal centers, and this process is associated with the loss of CRs. The possibility that IgD plays an important role in events taking place in germinal centers has been entertained (Godal et al., 1981b). C. WHATDETERMINESCLINICALPROGNOSIS? The clinical course of B-cell neoplasia is very heterogeneous. There are not only characteristic differences in the overall clinical behavior of ALL, CLL, and NHL, but also within each of these groups, especially in NHL. Histopathological classification systems in NHL have clearly demonstrated significantly longer survival in some groups than in others (Rappaport, 1966; Math6 et al., 1976; Dorfman, 1977; Lukes and Collins, 1977; Lennert, 1978). More recently, immunological methods have been applied to the same problem. Data have been presented with regard to B-, T-, and ‘‘0”-cell lymphomas (Brouet et al., 1975; Bloomfield et al., 1976) showing that B-cell lymphomas have a better prognosis than 0- and T-cell lymphomas, but these findings are of

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limited value, as T- and 0 - cell lymphomas most often are found in histopathological groups known to have a poor prognosis. Classification related to these markers, therefore, adds little to the information obtained by histopathology. We are not aware of data correlating immunological B-cell subtyping, i.e., Ig isotype, CRs, etc. One often encounters the view that prognosis is closely related to differentiation; i.e., poorly differentiated tumors have a poor prognosis, and well differentiated tumors have a good prognosis (e.g., Rappaport’s nomenclature). However, in B-cell neoplasms there does not seem to be such a relationship. For example, among ALL the neoplasms with B-cell characteristics seem to have a poorer prognosis than pre-B-cell ALL (Humphrey et al., 1980). Neoplasms in early B cells, which appear to be found in the CLL group (see Section III,B,2), certainly have a much better prognosis than immunoblastic B-cell lymphomas that consist of mature Ig-producing B cells. On the other hand, some studies suggest that cell proliferation is related to prognosis. Several studies (Cooper et al., 1968; Sandritter and Grimm, 1977; Silvestrini et al., 1977; Scarfee and Crowther, 1978; Braylan et al., 1978; Diamond and Braylan, 1980; Kvaldy et al., 1981a) have reported a correlation between tumors of unfavorable histology and a high proliferative activity as determined by autoradiography, flow cytofluorometry, and/or thymidine incorporation in uitro. Simonsson and Nilsson (1980) have made similar findings within the CLL group. The extensive heterogeneity found within several groups (e.g., nodular lymphomas) (Kvaldy et al., 1981a) makes it important to follow up each patient to establish to what extent analysis of cell kinetics may play a role in predicting the prognosis in individual patients. Because proliferating lymphocytes morphologically have the appearance of “blasts,” it is easy to understand that a confusion between differentiation and proliferation has occurred. Since many features of neoplastic B cells reflect that of their normal counterparts, we consider it likely that the proliferative profile also to a large extent reflects the proliferative activity characteristic of normal counterparts. Since proliferation of normal B cells to a large extent is controlled by other cells, especially T cells, it seems likely that proliferation and prognosis in malignant lymphomas would also be modulated by the T-cell compartment. Perturbations in the composition of T cells have been reported in the peripheral blood of patients with CLL (Kay et aZ., 1979; Hattori et al., 1980; Lauria et al., 1980; Platsoucas et al., 1980; Catovsky et al., 1981) and in PBL from children with NHL (Beck et al., 1980). In these studies an increased proportion of T-suppressor cells, as determined by Fcy-receptors on T cells, was found.

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Kvaldy et al. (1981b) have used monoclonal antibodies to define T-cell subsets in cell suspensions of B-cell lymphomas. Also by this method aberrations in T-cell subsets were detected. Most often a relative increase in T-suppressor/cytotoxic (T8’) cells was found. Moreover, in such cases a high proportion of T8’ cells expressed HLA-DR antigen, indicating that they were activated. The relationship of these findings to the prognosis of the patients remains to be determined, but suggests that “network” perturbations take place in B-cell neoplasms. If the T8+ cells release nonspecific factors, they could also modify the prognosis by promoting immunodeficiency, which is a factor of importance for prognosis in patients with B-cell neoplasms (see Wintrobe, 1975).Treatment will modify the “natural” prognosis of lymphomas, and this change may not necessarily relate to the “natural” prognosis of the disease; e.g., Burkitt’s lymphomas have a poor prognosis if untreated, but respond well to treatment. Indicators of prognosis therefore have to be reassessed as new therapies are introduced. D. SUBSETS AND NEOPLASMS IN THE B-CELL COMPARTMENT. AN ATTEMPTA T A SYNTHESIS

A considerable body of evidence has been provided showing the existence of functional B-cell subsets in the mouse. Thus, B cells responding to thymus-dependent antigens appear to be distinct from B cells responding to T-independent antigens (Playfair and Purves, 1971; Jennings and Rittenberg, 1976; Quintans and Cosenza, 1976). Lewis et al. (1976) found that B cells responding in a T-cell-dependent fashion expressed C 3 receptors, while those responding to T-independent (TI) antigens lacked such receptors. Moreover, responses to T I antigens can be further divided into two sets by so-called TI-1 and TI-2 antigens. The CBNN mouse strain carries a sex-linked B-cell defect manifested by a failure to respond to TI-2 antigens such as DNP conjugates of Ficoll or dextran, while responding normally to TI-1 antigens such as DNP-LPS or DNP-Brucella abortus (Mosier e t al., 1977a,b). Similar differences are found in neonatal normal animals, but not in adult animals. The activation of B cells by TI-2 antigens is associated with a surface determinant recognized by anti-Lyb-5 serum. The Lyb-5 alloantigen is present on 50-60% of B cells (Subbarao et al., 1979).Cyclosporin has been found to block responses to TI-2, but not to TI-1 (Kunkl and Klaus, 1980),and TI-2-responding B cells carry a low density of surface Ia (Greenstein et al., 1981). However, whether this functional heterogeneity represents different lineages of B cells or maturationassociated differences along one lineage only, remains unclear.

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Also in man, evidence in favor of multiple, functionally distinct subsets is accumulating. For example, we have found that human B cells responding to insoluble F(ab’)* are CR-bearing B cells, whereas B cells responding to other B-cell mitogens, such as pokeweed mitogen (PWM) and Sepharose protein A, lack C 3 receptors (Henriksen et aZ., 1981). Ault and Towle (1981) have reported that PWM selectively stimulates a minor subset of B cells carrying IgG. To what extent the selective activation of B cells in the case of T-cell-dependent mitogens may be determined b y T cells, remains unclear (Nishikawa et aZ., 1979). Thus, both in mouse and man there is a considerable body of evidence favoring distinct functional subsets in the B-cell compartment. However, whether this heterogeneity involves separate lineages of B cells remains at present unknown. It is likely that hybridoma antibodies will make important contributions on this point. So far, hybridoma antibodies to a common B-cell antigen (Brooks et al., 1980; Stashenko et aZ., 1980b) and to a subfraction of B cells and a proportion of neoplastic B cells have been reported (Brooks et al., 1981; Nadler et al., 1981). Another antibody prepared against a pre-B-cell line was found to react with most B cells, CLL, and NHL, but not with myelomas (Abramson et aZ., 1981). However, this antibody was not specific to cells of the B-cell lineage, as it also reacted with the erythroleukemia K562.A similar antibody crossreacting with neuroblastoma and fetal brain has also been reported (Greaves et ul., 1980). In our attempt to relate the extensive phenotypic heterogeneity uncovered by studies on neoplastic populations, we will primarily base our discussion on the assumption that the B-cell compartment consists of one lineage, since there is no definite information to the contrary. The attempt is shown in Fig. 7. This model represents an elaboration of earlier-presented models of B-cell differentiation (e.g., Parkhouse and Cooper, 1977; Vitetta and Uhr, 1977; Preud’homme et al., 1977) by trying to incorporate the role of germinal centers into the scheme. The model is based on the basic hypothesis that B cells during most stages of development have multiple choices with regard to further development, i.e., the potential for diflerentiation at most stages. This hypothesis is mainly based on the evidence for Ig, production and possibly plasma cell development from phenotypically different subsets of B cells, which has been elaborated previously. The evidence in favor of this hypothesis we consider to be strong. Other evidence relating to this hypothesis includes our studies on follicular lymphomas, which suggest that B cells in germinal centers selectively may lose sIgD or CR and sIgD during processes leading to IgM + CR memory

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Iymmste bulwmla

Re-8-cell

cells

FIG.7. A hypothetical scheme of B-cell neoplasias related to normal B-cell development. For explanations, see text.

cells and cells (“memory” cells?) expressing sIgG or sIgM only (see Section 111,B74).T h e evidence on this point remains circumstantial. When drawn in this way, there is a striking symmetry in the system as shown by the following pathway: sIgM + sIgM

+ sIgD + sIgM + sIgD + CR + sIgM + sIgD + sIgM

or as an alternative pathway: sIgM+ sIgM

+ CR+

sIgM

+ CR + sIgD+

sIgM

+ CR+

sIgM

If confirmed by other markers as well, this symmetry could make it possible for B cells to make “shortcuts” in secondary responses. This could be one important mechanism with regard to affinity maturation (see Section II,E,2) and could be an explanation for studies of Herzenberg et al. (1980) that showed that sIgD-positive cells carrying immunological memory retained lower affinity than IgD-negative mem-

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ory cells. The apparent symmetry also makes it more difficult to sort out B-cell subsets based on surface markers alone. The overall distribution of the neoplastic counterparts is shown on the left-hand side of Fig. 7. They are linked together by a narrow band on the left side to show the existing overlaps between the groups with regard to their relationship to B-cell maturation. The different categories of non-Hodgkin lymphomas as based on the Kiel terminology (Lennert, 1978) are indicated in parentheses. Their positions are derived from surface marker studies published elsewhere (Godal et al., 1981b,c).

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EVOLUTION IN THE TREATMENT STRATEGY OF HODGK IN’S DISEASE

Gianni Bonadonna and Armando Santoro Dlvision of Medical Oncology. National Tumor Institute. Milan, Italy

I . Introduction ........................... 11. Radiation Therapy: From t A. First Strategic Concepts .......................... B. Concept of Cure ...................................... C. Definitive Treatment . 111. Chemotherapy: From Sing A. Historical Developmen B. The Era of Combination Chemotherapy .............................. IV. New Treatment Strategies .........................

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A. Age, Histology, and Symptoms C. Limited Extranodal Disease

............................... ................................

..........................

B. Morbidity from Radiotherapy

References

......................

284

. . . . . . . . . . . . . . . 290

I. Introduction

Treatment of Hodgkin’s disease has continuously improved, particularly during the 1960s and 1970s, and today about 70% of all patients with this type of lymphoma can be offered a chance of cure. Although its fundamental nature, etiology, and pathogenesis remain to be further elucidated, Hodgkin’s disease represents a remarkable example in the history of clinical oncology of how progress in clinical and laboratory research has been successfully translated into effective management programs (62, 65, 67). The continuing progressive improvement in prognosis has been based initially on meticulous clinicopathological observations and subsequently on results derived from sound prospective trials. Thus, Hodgkin’s disease long 257 Copyright

0 1982 by Academic Press, Inc.

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ago became an important human model for the management of other human neoplasms (26). Since the treatment strategy applied to Hodgkin’s disease has evolved through important concepts and technical events (Table I), a critical review of the major steps leading to present achievements should be useful to outline the potential new advances aimed at the total conquest of this disease.

II. Radiation Therapy: From the Kilovoltage to the Megavoltage Era

A. FIRSTSTRATEGICCONCEPTS Historically, the first therapeutic attempts to treat Hodgkin’s disease with X-ray therapy were performed by Pusey in 1902 and Senn in 1903 (64). However, modern radiotherapy for Hodgkin’s disease really began with the Swiss radiation therapist R. Gilbert, who published from 1925 to 1939 (41,42).He was in fact the first radiation therapist to emphasize the fundamental principle of treatment, i.e., the destruction of “all granulomatous lesions” in the first course of irradiation. Gilbert also stressed the formulation of a systematic plan of irradiation in each case after careful clinical and radiological evaluation of all detectable sites of involvement. Based on clinical observations about “recurrence developing in the immediate vicinity of a field too narrowly treated” (42), he advocated the concentration of the therapeutic effort first on lymph node-bearing regions clinically involved by the disease and then extending the field of treatment to encompass the apparently uninvolved regions “which experience shows are more frequently invaded by the process.” By utilizing the strategy of segmental irradiation, Gilbert and Babaiantz (43) were able to report the first patients with prolonged survival: 4.3 years for the entire group and 6.5 years for the living patients. B. CONCEPT OF CURE Although other radiation therapists, such as Ratkoczy and Desjardins

(64), adopted treatment plans similar to those advocated by Gilbert, and Craft in 1940 published (24) the first reasonably convincing evidence that the survival of patients with Hodgkin’s disease was significantly prolonged by X-ray therapy, widespread interest in the curative potentialities of radiation therapy arose only after the two classical papers of V. Peters in 1950 (82) and Peters and Middlemiss in 1958 (84). The initial publications of Peters, who with Gordon Richards

TABLE I CHRONOLOGICAL FLOWOF MAJOR CONCEPTS AND EVENTSINFLUENCING THE EVOLUTION OF HODGKIN’SDISEASE 1925- 1939

Gilbert

1950-1958

Peters

1952 1963 1963-1964 1962-1965

Kinmonth Easson and Russel Lukes Kaplan

1965 1965 1966 1969 1970-1980 1971 1973 1968- 1981 1973- 1974

Rosenberg and Kaplan Lacher and Durant Frei Kaplan and Glatstein De Vita De Vita Young Rosenberg Bonadonna

THE

TREATMENT OF

Concept of destruction of all lesions in the first course of radiotherapy; segmental irradiation to encompass suspected microscopic disease Improved 5- and 10-year survival by prophylactic irradiation of adjacent lymphoid areas; first 3-stage clinical classification Lower extremity lymphangiography Concept of cure by radiotherapy Relationship of histologic features to clinical stages and prognosis Development of wide-field technique with irradiation in continuity of multiple node chains (mantle, inverted Y, and total lymphoid radiotherapy); identification of tumoricidal dose levels Evidence for an orderly progression in the spread Increased complete remission rate by vinblastine plus chlorambucil compared to single agents Efficacy of a cyclical four-drug combination (MOMP) Staging laparotomy and further studies on the pattern of anatomic distribution Concept of high cure rate by MOPP chemotherapy Staging laparoscopy No real advantage of maintenance chemotherapy in pathologic complete responders Trials with combined radiotherapy and chemotherapy, especially MOPP Development of non-cross-resistant chemotherapy (ABVD) and of alternating regimens (MOPP/ABVD)

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GIANNI RONADONNA AND ARMAND0 SANTORO

used from 1928 to 1953a treatment plan very similar to that of Gilbert, were important from many points of view. First, she reported treatment results according to a three-stage clinical classification, which also included the presence or the absence of systemic symptoms. By doing this, she started the new era of rational emphasis on diagnostic evaluation and treatment reporting based on the anatomic extent of involvement. The survival results of the Toronto series (51% at 5 years and 25% at 10 years for all stages) included impressive figures for stage I (88% at 5 years) and stage I1 disease (72% at 5 years). The doses employed ranged from 18 to 50 Gy, depending on the site and extent of lymphoma. Most important, the prolonged survival was ascribed to the extent of radiotherapy, which included in many patients “prophylactic” irradiation of adjacent lymph node-bearing regions clinically uninvolved, with doses ranging from 4 to 8 Gy. By extending the period of observation (84), Peters observed no substantial decrement of survival after the tenth year, and concluded that if a patient with Hodgkin’s disease survives 10 years without recurrence the risk of new disease manifestations during the subsequent two decades is very limited. In a subsequent publication (83) she noticed that the long-term survival was not significantly related to the initial site of presentation but rather was influenced by the presence or the absence of systemic symptoms, age below or over 40 years, and sex. Thus, the work of the Toronto group with “complementary” irradiation represented the first systematic application of the principles advocated by Gilbert and a treatment plan based on technical factors as well as on natural spread of the disease. The statistically valid evidence that patients with Hodgkin’s disease are potentially curable with kilovoltage irradiation, if they have localized lymphadenopathy at the time of treatment and if they are treated over an adequate volume of tissue and an adequate level of dosage, was further defined by Easson and Russel in 1963 (34). On the basis of retrospective analysis of 698 patients with localized adenopathy treated at the Christie Hospital in Manchester from 1934 to 1959, they reported a 5-year survival rate ranging from 53.5% to 56.5%, a 10-year survival of 44.1-43.2%, and a 15-year survival of 38.9% for patients treated from 1934 to 1949. No attempt was made “prophylactically” to irradiate the clinically uninvolved contiguous node groups. The irradiation coverage included from 5 or 7 cm beyond the uppermost and lowermost of palpable lymph nodes in the chain, and the level of dosage employed was between 27.5 and 25 Gy to field sizes from 20 to 30 cm long (33).In reporting the results, Easson and Russel adopted the method of age-corrected survival rates, and their

TREATMENT EVOLUTION I N HODGKIN’S DISEASE

26 1

definition of cure was as follows: “We can speak of cure when in time, probably a decade or so after treatment, there remains a group of disease-free survivors whose progressive death rate from all causes is similar to that of a normal population of the same sex and age constitution” (34).This concept as well as the accompanying survival results made a substantial contribution to subsequent treatment strategy with radiotherapy. C. DEFINITIVETREATMENT Between the end of the 1950s and the beginning of the 1960s two events, megavoltage irradiation and lymphography, made a major impact on the evolution of treatment for Hodgkin’s disease. The highenergy beams generated by new megavoltage devices induced some research centers in England and the United States to adapt “supervoltage” irradiation to medical radiotherapeutic use (52, 61, 63, 64). Cobalt teletherapy units as well as new electronic devices such as the betatron and the linear accelerator provided apparatus capable of yielding beams of very high energy while operating at quite nominal voltages. The physical advantages of megavoltage equipment have greatly increased the versatility and precision of modem radiation therapy and have opened the way to entirely new treatment approaches for Hodgkin’s disease and other malignant lymphomas. By utilizing since 1956 the Stanford 5 MV linear accelerator, Kaplan was able to introduce the wide-field technique of radiotherapy for stage I and I1 Hodgkin’s disease (58),and this represented the major event in the development of the more successful radiotherapy techniques of today. The studies of the Stanford group, by identifying the tumoricidal dose levels (59,64),established one of the milestones for definitive treatment and provided confidence in the capacity of radiotherapists to eradicate tumors in irradiated areas that were involved by Hodgkin’s disease. Kaplan’s strategy was aimed from the beginning (58,61,68)at treating multiple lymph node chains in continuity with as few fields as possible. The development of optimal field size and shape led to the classical “mantle” and the “inverted Y” fields for the irradiation of all the major lymph node chains above and below the diaphragm, respectively; hence the concept of total lymphoid (TLI) or total nodal irradiation (TNI) when both fields were utilized (45, 61, 64). The technique to permit the delivery of tumoricidal doses to virtually all lymphoid structures in the body was tested at Stanford through a prospective trial in which patients with clinical stage I11 were randomized to receive either low-dose palliative irradiation or high-dose total lymphoid

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radiotherapy. To the pleasant surprise of the investigators, total lymphoid megavoltage irradiation proved to be remarkably well tolerated and not as dangerous as feared (65). Subsequent studies have further defined the optimal selection of dose levels and the dose fractionation patterns (64). The second event that influenced the survival results through improved treatment selection was the adoption by several specialized centers of lower extremity lymphography first developed by Kinmonth in 1952 (70). This new diagnostic method, by revealing that many patients had unsuspected lymph node involvement in the retroperitoneal space, increased tremendously the knowledge as to the extent of lymphoma at diagnosis and allowed one to determine the orderly progression in the spread of Hodgkin’s disease (4,64, 93).The information gained by the systematic use of lymphography allowed the extension of wide-field radiotherapy to patients with stage I11 lymphoma and considerably improved the follow-up of the disease in areas previously obscured b y inadequate diagnostic methods. The new ideas about the nature, epidemiology, etiology, modes of spread, and treatment have been thoroughly discussed by people working in many different disciplines in a series of small international meetings held in Paris (106),Rye (107),London, Ann Arbor (108), and Palo Alto (55).These meetings have served to concentrate effort, disseminate information, agree on classification and staging, secure uniformity of histological reporting, and emphasize the importance of prospective clinical trials. In particular, the pattern of anatomic distribution and the knowledge that in the great majority of patients Hodgkin’s disease spreads nonrandomly and predictably via lymphatic channels to contiguous lymph node chains and other lymphatic structures were further improved by the introduction of staging laparotomy in 1969 (46, 66). Surgical staging, which became rapidly popular among research centers, documented that a considerable fraction of patients (20-30% in clinical stage 1-11, 60-80% in clinical stage 111) had microscopic disease within abdominal structures. Therefore, staging laparotomy further improved the selection of candidate patients for radical radiotherapy (17,66,92). Thus, at the beginning of the 1970s sufficient information had accumulated on patterns of spread of Hodgkin’s disease as well as main prognostic factors and, more important, on the cure rate with megavoltage radiotherapy (55, 64). In adequately staged patients, the 5-year relapse-free survival (RFS) rose to 70-80% for stage 1-11 disease and to about 50% for stage IIIA. Moreover, a careful analysis of patterns of treatment failure after radical radiotherapy indicated that about 90% of all primary relapses

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occurred within the first 3 years from completion of irradiation and rapidly diminished to approach the zero baseline by the fourth or the fifth year (58,60,76,118).This observation substantially improved the concept of cure as previously defined by Easson and Russel (34).

111. Chemotherapy: From Single Agents to Multiple Drug Treatment

A. HISTORICAL DEVELOPMENTS The activity of nitrogen mustard in Hodgkin’s disease was discovered in 1940 (44),and the initial series of clinical studies attempting to delineate the proper use of this compound in the treatment of various neoplastic diseases culminated in the report of C. P. Rhoads in 1946 (91). During the 1950s other alkylating agents, namely chlorambucil and cyclophosphamide, as well as corticosteroids and some of the antimetabolites were found to be useful in inducing a temporary response in advanced Hodgkin’s disease (29). The decade of 1960s was probably the most important for the development of medical treatment of malignant lymphomas. First of all, numerous classes of drugs were identified for effectiveness against Hodgkfn’s disease: the vinca alkaloids, procarbazine, adriamycin, and bleomycin. The clinical observation that all these compounds were not individually cross-resistant to each other led to their use in sequence. In fact, the principle governing the clinical use of chemotherapeutic agents was based on the administration of sequential single drugs, often through initial maximum tolerated doses, until relapse or excessive toxicity occurred. Treatment was usually started by using one of the alkylating agents (nitrogen mustard, cyclophosphamide). Once the initial response was obtained, maintenance treatment with an oral alkylating agent (chlorambucil, cyclophosphamide) was applied to keep the patient in either complete or partial remission with minimal bone marrow toxicity. If and when the lymphoma became refractory to the alkylating agents, one of the vinca alkaloids (usually vinblastine) was tried next, and then procarbazine if symptoms and signs persisted or recurred. When all these agents were given an adequate trial and chemotherapy was still indicated because of further progression, one of the new antibiotics (adriamycin or bleomycin) was administered. Bleomycin was usually selected instead of adriamycin in the presence of concomitant myelosuppression. Only in the 1970s were the nitrosourea derivatives (BCNU, CCNU, streptozotocin), the epipodophyllotoxin derivatives (VP-16,VM-26),and dacarbazine (DTIC)tested

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in patients refractory to conventional agents. In general, with singleagent chemotherapy complete remission occurred in about 20% and the duration of response rarely exceeded 6 months. However, with the sequential approach more than half of the patients were maintained in partial (PR) or complete remission (CR) for many months and occasionally for a few years. The overall median survival was in the range of 24 months. Survival was longer in patients responding to at least two drugs (40-42 months) compared to those responding less or not at all (9-18 months) (10, 49). Advances in the drug treatment for Hodgkin’s disease followed the clinical application of some of the principles advocated b y Skipper et al. (29, 104) after studying drug effects in the transplantable L1210 leukemia system. As early as 1963, Frei, influenced by the activity of drug combinations in acute lymphoblastic leukemia, began studies with combination regimens also in Hodgkin’s disease (123). In 1966, Frei, De Vita, Moxley, and Carbone published the results of a fourdrug combination (MOMP: cyclophosphamide, vincristine, methotrexate, and prednisone) (38). This group then substituted mechlorethamine for cyclophosphamide, and procarbazine for methobexate, respectively. This became the famous MOPP regimen, and the formal detailed report of De Vitaet al. in 1970 (30),showing a high incidence of prolonged CR, represented the first glimmer that Hodgkin’s disease might, in fact, be curable by chemical agents. Prior to this report, Lacher and Durant (72) showed in 1965 a 62% CR rate with polychemotherapy (vinblastine plus chlorambucil) as compared to 30% for single agents.

B. THEERA

OF

COMBINATION CHEMOTHERAPY

There is general agreement that MOPP chemotherapy represented a breakthrough in the treatment of advanced (stages I11 and IV) Hodgkin’s disease. The importance of MOPP as a successful example of optimum combination of active drugs without severe overlapping acute toxicity went far beyond the treatment of Hodgkin’s disease. In fact, the modern approach of intermittent (cyclical) combination Chemotherapy with full or nearly full dose regimen, as well as the concept of planned dose attenuation schedules in the presence of various types and degrees of toxicity and of average percentage of the projected total dose administered for each drug, were all developed during the initial trial with MOPP (30). The strategic hypothesis underlying the design of the MOPP regimen was based on the general concepts that the curative potential of

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265

chemotherapy developed in rodents with L1210 leukemia (fractional cell-kill hypothesis, dose-response effect, inverse relations hip between number of tumor cells and cure rate) ought to be operative also in human tumors for which sufficient partially effective drugs were available. Therefore, considering that combination chemotherapy had been shown in rodents to slow or prevent the development of drug resistance, the NCI group attempted for the first time to cure Hodgkin’s disease, not merely to palliate with drugs. This explains the administration of full doses for each of the four drugs, the use of cyclical rather than continuous schedule, and the relatively long duration of therapy (26).The clinical results were impressive (27, 30, 31). In 198 patients with advanced stages treated from 1964 to 1975, 80% attained CRYa fourfold increase over that achievable with single agents. Furthermore, 68% of patients who attained CR were continuously progression-free 5 years from the end of all treatment. Since only four relapses were documented beyond 4 years from the end of MOPP, the RFS for patients at risk 10 years was 63.4%.The contrast to the results using single agents in the past trials was quite striking, since less than 10%of patients treated with a single agent survived 5 years and even fewer survived free of tumor (10, 21, 26, 49). The experience with MOPP has also provided important general information that should be considered in the current treatment of Hodgkin’s disease as well as in the design of future strategies (27, 30,

31). 1. Although the median number of cycles needed to achieve CR was three, there should not be a fixed period of induction therapy. Patients should receive, at minimum, 6 monthly cycles of chemotherapy at maximally tolerated doses and, if clinical complete remission is confirmed by pathologic restaging, 2 more cycles of chemotherapy should be given as consolidation treatment. In a few patients, especially with nodular sclerosis histology, as many as 12 cycles are required before the patient can be designated as a true complete res pon der. 2. Once CR has been achieved, there is no real advantage in prolonging treatment, either with the same combination or with single agents (121);thus, the achievement of the status of true CR is the most important factor affecting prognosis. 3. In untreated patients, the only characteristic significantly associated with a greater probability of attaining CR is the absence of systemic symptoms (“A”: loo%,“B”: 78%). 4. Prior chemotherapy compromises the capacity of patients with Hodgkin’s disease to attain CR, whereas prior radiotherapy does not

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TABLE I1 COMPLETE REMISSION (CR) AFTER MOPP OR MOPP-LIKECOMBINATIONS“ Acronym

Drugsb

Evaluable patients

Percentage of CR

MOPP MVPP COPP CVPP ChlVPP BOPP BCOP BCVP MABOP B-MOPP BCVPP

HNZ, VCR, PCZ, PRD HNZ, VLB, PCZ, PRD CTX, VCR, PCZ, PRD CTX, VLB, PCZ, PRD Chl, VLB, PCZ, PRD BCNU, VCR, PCZ, PRD BCNU, CTX, VCR, PRD BCNU, CTX, VLB, PRD HNZ, ADM, BLM, VCR, PRD BLM, HNI, VCR, PCZ, PRD BCNU, CTX, VLB, PCZ, PRD

1189 227 102 114 118 114 31 25 56 156 115

68 69 66 64 69 67 35 52 63 82 68

Modified from Bonadonna and Santoro (7). HNZ,Mechlorethamine; VCR, vincristine; PCZ, procarbazine; PRD, prednisone; VLB, vinblastine; CTX, cyclophosphamide; Chl, chlorambucil; BCNU, carmustine; ADM, adriamycin; BLM, bleomycin.

compromise the ability of patients who relapsed to respond to combination chemotherapy (16). 5. Patients with nodular sclerosis histology have a significantly shorter 10-year RFS (53%) compared to patients with other histological variants. 6. Patients with “B” symptoms have a significantly lower 10-year RFS (53%) than those without symptoms (94%). The results with MOPP chemotherapy in advanced Hodgkin’s disease, though with comparatively fewer follow-up periods, were confirmed by a number of research centers throughout the world. It is important to emphasize that none of the three-, four-, and five-drug regimens designed subsequently by deletion (COP), substitution (MVPP, COPP, CVPP, BCOP, BCVP, MABOP), or addition (MABOP) of various components of MOPP regimen could be considered significantly superior to MOPP in terms of incidence and duration of CR (7, 10, 49) (Table 11). Only recently, a few regimens (ChlVPP, BOPP) were reported to be less toxic than MOPP while the cure rate has remained essentially unchanged (69, 80). Besides the high incidence of durable CR, the most important results affecting the evolution of treatment strategy concerned the time and the pattern of relapse. Similarly to what has been observed after treatment with radical megavoltage radiotherapy (64, 118),about 90% of patients showing relapse did

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267

so within the first 2-3 years from completion of chemotherapy, thus suggesting that the likelihood of being cured by drugs becomes very

high after a RFS exceeding 2 years. Analysis of the specific patterns and sites of relapse (39, 122) indicated that patients relapsed primarily (92%) in sites of previous lymphoma, particularly in nodal sites (75%); the sites most frequently involved at relapse were the central nodal areas as well as areas of previous bulky disease. Furthermore, the probability of relapse increases with increasing stage, and presence of nodular sclerosis histology, and systemic symptoms. The aforementioned observations led to subsequent studies utilizing chemotherapy followed by radiotherapy. IV. New Treatment Strategies

A. COMBINEDMODALITYTREATMENT Even in theoretically optimal conditions provided by surgical staging procedures, both megavoltage irradiation and MOPP or MOPPlike chemotherapy, when given alone, have reached a plateau in their capability to cure early and advanced stages of Hodgkin’s disease. For this reason, during the past decade radiotherapy and chemotherapy have been sequentially combined in an attempt to improve the cure rate in several prognostic subgroups. In fact, the failure of optimal radiotherapy to obtain a long-term RFS in 25-60% of patients with Hodgkin’s disease spread either above or below the diaphragm and/or with systemic “B” symptoms is most likely due to the presence of occult foci of lymphoma cells beyond the field of irradiation. Likewise, in patients with bulky lymphoma, especially in central nodal areas, the cytoreductive effect of chemotherapy is often inadequate and treatment could be supplemented with irradiation to achieve complete cell eradication (11, 88, 89). Although several attempts were made in this direction as early as 1950 (10, 65), we shall limit our discussion to the results derived from some important prospective controlled trials. Probably the first important randomized trial was that started by the EORTC group in 1964. In clinical stages I and 11, radiotherapy (mantle field or inverted-Y technique) followed by prolonged vinblastine chemotherapy was compared to radiotherapy alone. The 10-year results (81, 114) showed that the proportion of relapse-free patients was significantly higher among those who received chemotherapy. When the data on the MOPP prcgram became available, the Stanford group led by S. Rosenberg (94, 95, 96) utilized the information to integrate

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MOPP chemotherapy with radiotherapy. Completed with detailed staging b y laparotomy, the Stanford program included a brilliant series of randomized clinical experiments. The use of MOPP (or MOP with prednisone withheld if patients had received mediastinal irradiation) as an adjuvant after irradiation was the major question tested in studies from 1968 to 1974 for patients with pathologic stage (PS) I, 11, and I11 disease, including those with limited extranodal lymphoma, defined as the “E” lesion in the Ann Arbor classification (108).A pilot study on the use of irradiation as an adjuvant to MOPP for patients with PS IV lymphoma was initiated during the same period as well. In 1974, based on the preliminary results of these combined modality studies, new randomized protocols were initiated. Patient eligibility, diagnostic study, and protocol design were detailed in several publications (94, 95), and the major end points consisted in freedom from progression (FFP) and total survival. The most important updated results (96) are summarized in Table 111. By combining more study groups, the 12-year actuarial analysis indicated a highly significant improvement in F F P in the adjuvant MOP(P) group and a probable survival advantage. The lack of significant difference in survival in the Stanford study has been attributed to the success of “salvage” chemotherapy with MOPP after relapse in patients who failed to respond to radiotherapy alone. The advantage in FFP after adjuvant chemotherapy was most significant for asymptomatic “A” patients (irradiation 68%, irradiation MOPP 81%, p = 0.002), especially those with PS I11 lymphoma. Patients with PS IIIB did poorly with TLI and hepatic irradiation alone (FFP 9%), as compared to the combined modality approach (FFP 52%, p = 0.04). In the pilot study of 33 patients with PS IV disease, testing combined modality therapy, there was no benefit of adjuvant radiotherapy after MOPP chemotherapy. In PS IA-IIA Hodgkin’s disease, the 6-year results demonstrated that adjuvant MOP(P) can replace extended field irradiation to clinically uninvolved sites, without compromise of excellent FFP and actuarial survival. Alternating chemotherapy (MOPP or PAVe) and radiotherapy for PS IIIB demonstrated considerable improvement in F F P and survival, as compared retrospectively to previous management programs of TLI alone or TLI followed by MOP(P) chemotherapy. The overall results for all patients treated on protocol studies have improved since 1968. In fact, in 147 patients with stages 1-111 treated from 1962 to 1968 with primary radiotherapy, the 10-year FFP was 48% and survival 58%. In 363 patients (all stages) treated from 1968 to 1974, the corresponding findings were 64% and 75%, respectively, and the 6-year results of 235 patients (all stages) treated from 1974 to 1980 were 77% for F F P and 86% for survival (96).

+

TABLE 111 SUMMARY OF

MOST IMPORTANT

Pathologic stages IB-IIB-IIIA-IIIB IIIsA-IIIsBc

STANFORD RANDOMIZED TRIALSWITH RADIOTHERAPY 2

FFPb Treatment TLI

R

R

TLI

-+

6 MOP(P)

IIIB'

0.09 (Gehan) 0.04 (Cox) 78

82

91 0.48 (Gehan)

I F + 6 MOP(P)

Alternating MOP(P) or PAVe and TLI

P

63

76

/ \

(%)

0.001 (Gehan) 0.002 (Cox) STLI

IA-IIA

CHEMOTHERAPY

Survival P

61

/ \

(%)

MOPP

0.62 (Gehan)

82

97

78

88

TLI, Total lymphoid irradiation; STLI, subtotal lymphoid irradiation; IF, involved-field irradiation; PAVe, procarbazine, alkeran, and vinblastine. FFP, Freedom &om progression. ' Patients also received radiotherapy to the entire liver.

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Subsequent to the initiation of combined treatments at Stanford, the efficacy of adjuvant chemotherapy in Hodgkin’s disease confined to lymph nodes was tested by a number of research groups, and we shall summarize the results of trials that had adequate follow-up. From 1970 to 1974, the Baltimore Cancer Research Program (119) treated 87 patients with PS IA-IIA-IIB-IIIA after laparotomy who were assigned at random to receive either extended field radiotherapy (subtotal or total nodal irradiation) alone or radiotherapy followed b y six cycles of MOPP. The median follow-up time from the end of all treatments was in excess of 69 months. Patients with stages I and I1 had a 31%relapse rate with radiotherapy alone compared to a 6% relapse rate with combined modality treatment ( p = 0.04). The difference in total survival was not significant ( p = 0.4). In a limited number of patients with stages IIIA and IIIEA (total 28 cases), both remission duration ( p = 0.01) and survival ( p = 0.02) at 78 months from the end of all therapy were significantly in favor of combined therapy. Although 83% of the patients failing on radiotherapy alone were initially salvaged with chemotherapy after their first relapse, 42% ultimately died of Hodgkin’s disease and only 25% remained in continuous CR 3 years after first relapse. A similar study was carried out b y the Southwest Oncology Group in 230 patients (22, 23). Patients with PS IA-IB-IIAIIB after laparotomy were randomly allocated from 1972 to 1978 to either subtotal nodal radiotherapy or involved-field radiotherapy followed b y six cycles of MOPP. Patients with pelvic adenopathy received total nodal irradiation. Patients (all stages) treated by involved-field radiotherapy plus MOPP had a significantly better FFP than those treated by extended-field radiotherapy ( p = 0.04). The FFP advantage for the combined modality was seen only among the patients with stages IB and IIB disease ( p = 0.02) but not with stages IA and IIA lymphoma ( p = 0.4).There was, however, no survival advantage in either the I and IIA or I and IIB groups. The opposite sequence, i.e., combined modality treatment with chemotherapy first to be followed by radiotherapy was performed by some research groups with the rationale that (a) chemotherapy can produce a prompt control of all sites of the disease, and possibly eradication of disseminated microfoci beyond the fields of irradiation; (b) hematosuppression recovers more quickly after intensive cyclical chemotherapy than after total or subtotal nodal irradiation; (c) a partial response after chemotherapy can more easily become complete after subsequent irradiation, rather than vice versa; (d) in patients with either complete or partial response following chemotherapy, the volume of irradiation (and even the dose in complete responders) can be

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considerably reduced (11, 89). The first prospective study utilizing induction chemotherapy followed by low-dose chemotherapy was initiated by Prosnitz et al. (88, 89) at Yale, and their initial findings were updated (35).In 124 patients (53 untreated and 61 with relapses after radical radiotherapy) with stages 11, 111, and IV, the investigators first administered three cycles of M W P P (mechlorethamine, vincristine, vinblastine, procarbazine, and prednisone) during a 6-month period. Patients achieving CR then received megavoltage radiation therapy (15-25 Gy) to all areas known to be involved with disease prior to the initiation of chemotherapy, including extranodal sites, but not the bone marrow. In particular, patients with hepatic involvement received whole-liver irradiation at rates of 1.5 Gy per treatment with a total dosage not exceeding 20 Gy. Similar fractions and dosages were used for lung irradiation, but here treatment portals were individualized and did not necessarily include bilateral whole-lung irradiation. After completion of radiation therapy, two additional cycles of chemotherapy were given, and the total treatment time ranged from 14 to 18 months. After combined therapy, 102 of 124 patients (84%)have entered CR and 92 remained in CR with a median follow-up time of 5 years. The cumulative survival rate for all 124 patients was 80% at 5 years, and the RFS rate was 74%.The 5-year survival rate was affected by age (>40 years 45%; 5 4 0 years 89%) and extent of extranodal involvement (multiple extranodal sites 48%, single extranodal sites 81%).In 1973, a similar strategic approach was initiated in Milan (9, 12) in 76 consecutive patients either previously untreated (55cases) or with nodal and/or extranodal relapses following radical radiotherapy (21 cases). Pathologic stages included IIB-IIIB-111s and IV Hodgkin’s disease. Induction chemotherapy comprised at random six cycles of either MOPP or ABVD. Complete plus partial responders were then irradiated with a technique similar to that described by Prosnitz, but the tissue doses were higher compared to those delivered b y the Yale group (nodal sites, 30-35 Gy; extranodal sites, 20-25 Gy). At the completion of combined modality therapy, CR was 71% in the MOPP group and 80%in the ABVD group, and its incidence was high regardless of nodal or extranodal disease, prior or not prior irradiation, age below or over 40 years, histological subgroups, “A” or “B” symptoms (9). Furthermore, the comparative analysis failed to identify a significant difference between MOPP and ABVD. The 4-year RFS from end of radiotherapy was 84% for MOPP patients and 91% for ABVD patients, and the 5-year survival rate was 88% and 90%,respectively. No patient showed relapse after 26 months from the end of the treatment program, and in both groups the duration of CR was not unfavorably

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influenced by the presence of age over 40 years, systemic symptoms, extranodal disease (single or multiple sites), and nodular sclerosis histology. The Memorial Hospital group (105) has designed a rather complex combined treatment program alternating monthly MOPP and ABVD chemotherapy followed by low-dose radiotherapy (20 Gy in 2 weeks) to areas of initial bulky disease in untreated patients. In 84 evaluable patients, CR rates were 80% for previously untreated, 65% for prior irradiation or minimal chemotherapy treated, and 50% for heavily pretreated patients. Among 49 previously untreated patients there were no primary treatment failures, and the estimated 2-year relapse rate for the CR group was 9%. Besides the usefulness of low-dose adjuvant radiotherapy to initial sites of bulky lymphoma, the results were attributable to the effect of the two non-cross-resistant drug combinations. The research group in Milan started in 1974 a second study with combined modality therapy, and the 4-year results were reported by Santoro et al. (100).The main reason for this prospective randomized trial was to compare iatrogenic morbidity, especially sterility and carcinogenesis, between MOPP and ABVD chemotherapy when combined with radiotherapy. All 153 patients with PS IIB and I11 (A + B) enrolled so far in the study, were started with three cycles of chemotherapy (either MOPP or ABVD) and, after subtotal or total nodal irradiation (35 Gy to involved lymphoid areas and 30 Gy to adjacent areas), treatment was completed with three additional cycles of either chemotherapy. The total incidence of CR was significantly higher in the ABVD group (94%) compared to the MOPP group (79%, p < 0.01), and the difference was particularly evident in subgroups with “B” symptoms (73% vs 93%, p = 0.01) and nodular sclerosis histology (79% vs 94%, p < 0.03). The 4-year results suggested an important trend in both RFS (77% vs 90%) and total survival (78% vs 86%) favoring ABVD. However, this drug regimen combined with radiotherapy resulted so far significantly superior to MOPP plus irradiation only in the RFS of patients with PS IIIA ( p = 0.02). From 1971to 1975 the Southwest Oncology Group (22, 23) treated a total of 143 patients with PS IIB and I11 (A + B) who were started on MOPP Chemotherapy (less than three cycles, 6; three cycles, 72; four cycles, 62; more than four cycles, 6) followed by total nodal radiotherapy. The 8-year actuarial results showed a FFP of about 65% and a total survival of about 85%, with no significant differences with respect to stage subgroups. I n two other prospective studies entering a limited number of patients, MOPP chemotherapy preceded total nodal

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radiotherapy either in patients with nodal (71) or extranodal (96) disease, but the results were definitely less successful. The details are known at present only for the NCI study (71) where the treatment program was discontinued because of excessive toxicity. This was probably due to prolonged MOPP chemotherapy before irradiation as well as to high-dose radiotherapy. In conclusion, the strategy utilizing combined modality therapy has not yet evolved into a standard approach for given subgroups with Hodgkin’s disease. Furthermore, available results are not always easily comparable, particularly because the various investigators reported their findings at different follow-up periods and often grouped together various prognostic subsets. However, all investigators have clearly shown that the RFS (or FFP) was significantly improved over optimal radiotherapy alone. The improvement in total survival was not always clearly demonstrable in patients with nodal extent, whereas in patients with extranodal lymphoma the survival rate was increased after chemotherapy followed by low- or intermediate-dose radiotherapy (9, 35, 105) compared to chemotherapy alone (27, 29). Also, there was preliminary evidence of a superiority in the RFS when chemotherapy preceded radiotherapy (9,35, 105) or was alternated with radiotherapy (96) compared to irradiation followed by adjuvant MOPP (96, 100). Within this context, ABVD (100) or MVVPP (35) appeared to be superior to MOPP. Probably, as reported by the Southwest Oncology Group (23)as well as by a French Group (2, log), we could reduce the number of chemotherapy cycles without compromising the results since the combination of chemotherapy plus radiotherapy carries a high risk of second neoplasms when treatment includes alkylating agents and/or procarbazine (3, 14, 15, 19-21,112,115,116). In view of this considerable risk, the regimen of choice could be ABVD (8). The indications for low-dose radiotherapy and chemotherapy in children with Hodgkin’s disease will be mentioned during the discussion of late-treatment morbidity. REGIMENS B. ALTERNATINGNON-CROSS-RESISTANT The experience with MOPP chemotherapy (27, 31) has indicated that about 20% of all patients with advanced Hodgkin’s disease do not attain CR and usually succumb rapidly to their disease. Furthermore, an additional 40% of patients who do achieve CR ultimately relapse, and they are not easily salvaged with other forms of treatment. The explanation for the failure to attain CR and for the early relapse is attributable to primary drug resistance, and the explanation for recur-

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rent disease after prolonged CR suggests that the initial induction therapy was probably stopped too soon. In fact, the NCI group (36) provided evidence that in relapsing patients retreatment with MOPP yielded a second CR in a total of 59% and the likelihood of attaining a second long complete remission was significantly affected by the duration of the first CR (<12 months 29%, >12 months 93%).As suggested by the mathematical model of Goldie and Coldman (48) based on current somatic mutation theory, stable genetic alterations arise in tumor cells and result in phenotypic changes in drug sensitivity. As the tumor size increases, the probability of resistant clones increases and therefore, for reasons quite independent of growth kinetics, we would expect increasing resistance to chemotherapy with increasing tumor size, as commonly occurs in patients with clinically advanced cancer. In responsive tumors, such as malignant lymphomas, the proportion of drug-sensitive cells will almost always be much greater than the resistant fraction. For this reason, the tumor can be expected to exhibit temporary measurable remission to chemotherapy (e.g., MOPP in Hodgkin’s disease) until the size of the resistant fraction approximates that of sensitive fraction. As further emphasized by Skipper (103), a large fluctuation in the proportion and absolute number of drug-resistant tumor cells, a necessary consequence of the mutation theory, probably exists in comparably staged individuals with a given tumor such as Hodgkin’s disease. In the attempt to eradicate the mix of MOPP-sensitive and -resistant neoplastic cells, the research group in Milan has designed the ABVD chemotherapy (12) and mounted a number of prospective trials to prove its effectiveness as a non-cross-resistant regimen to MOPP chemotherapy (8, 9, 97-99). The most recent results (99) of this strategic plan have first confirmed the initial findings (12, 97); i.e., ABVD can induce a high incidence of CR in MOPP-resistant patients. In 54 consecutive patients defined as resistant to MOPP chemotherapy because of either progressive disease during primary MOPP or relapse within the first 12 months after achievement of complete response, CR was obtained in 59%.The likelihood of achieving CR was significantly influenced b y the absence of extranodal disease (nodal 74%,extranodal 44%, p = 0.03) and of systemic symptoms (“A” 82%, “B” 49%, p = 0.02). Futhermore, CR was achieved regardless of whether patients during primary MOPP chemotherapy were classified as failures (52%), partial responders (67%),or complete responders (68%).There was also no statistical evidence that age (< or 240 years), sex, histologic characteristics, and prior irradiation affected the probability of attaining CR with ABVD. The 5-year RFS was 37% (“A” 44%, “B” 25%), and the median survival for all patients was 27 months (complete

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responders >60 months, partial responders 12 months, failures 9 months). The opposite sequence, i.e. the administration of MOPP in 16 ABVD-resistant patients, yielded CR in 25% and PR in 12.5%.The median duration of complete remission was 6.5 months and the median survival of complete responders was in excess of 20 months. The conclusion was that ABVD should be considered as an effective salvage regimen and, when utilized as first treatment in MOPP-resistant patients, is probably able to cure about one-third of complete responders. The important problem of MOPP-salvage therapy was also considered by other investigators, who have utilized various forms of chemotherapy as summarized in Table IV. Contrary to the moderate acute toxicity and lack of delayed morbidity observed with ABVD chemotherapy (8, 99), most MOPP-salvage programs often involved severe, and even fatal, toxicity due to prolonged myelosuppression, renal tubular dysfunction secondary to streptozotocin, and even cardiomyopathy following high single doses (60 mg/mz) of adriamycin, such as in the B-CAVe regimen. Therefore, some of the drug regimens listed in Table IV cannot always be recommended for general use. The alternating MOPP-ABVD program was started in 1974 to maximize the incidence of CR in untreated patients and to minimize relapses. The therapeutic strategy involving the cyclical administration of non-cross-resistant chemotherapeutic regimens seems rational because it offers the possibility of reducing failure to achieve CR with MOPP alone and preventing or delaying relapses due to overgrowth of singly-, doubly-, or multidrug-resistant phenotypes to MOPP. Based on the clinical observation that most patients resistant to MOPP showed progressive disease by the third cycle (26), MOPP was alternated monthly with ABVD. From 1974 to 1980, 75 patients with initial stage IV Hodgkin’s disease (55 cases) or extranodal relapse following primary irradiation (20 cases) were randomly allocated to receive either 12 cycles of MOPP or MOPP monthly alternated with ABVD for a total of 12 cycles. In complete responders after 12 cycles of either chemotherapy, no additional treatment was given, whereas in partial responders drug administration was continued at maximum tolerated doses until either CR or progressive disease occurred. The 5-year results (8, 98) are summarized in Table V. The incidence of CR was significantly superior after alternating chemotherapy (92%) compared to MOPP alone (71%).Even more important, cyclical switches of chemotherapy were able to prevent in all patients progressive lymphoma during the period of drug therapy. The cyclical administration of drug regimens was superior to MOPP alone in all prognostic subgroups. Thirty-seven percent in the MOPP group remained progression-free at 5 years compared to 70% of those given MOPP plus ABVD; and the median RFS

TABLE IV MOST COMMON REGIMENSUTILIZED IN MOPP FAILURES" Response' Acronym

Drugsb

Evaluable patients

ABVD ABDIC B-DOPA B-CAVe BVDS SCAB CVB

ADM, BLM, VLB, DTIC ADM, BLM, PRD, DTIC, CCNU BLM, DTIC, VCR, PRD, ADM BLM, CCNU, ADM, VLB BLM, VLB, ADM, STZ STZ, CCNU, ADM, BLM CCNU, VLB, BLM

54 29 15 22 10 17 39

CR

(a)

CR

+ PR

59 34.5

72 82.5

60

80

50

77

30 35 26

50

59 84.5

Median duration of CR (months)

17 28' 14+ 35+ ?

Median survival of CR, (months)

60+ 28+ ?

24 26+

8+

16'

4,5+

4,5+

Modified from Santoro et nl. (99). ADM, Adriamycin; BLM, bleomycin; VLB, vinblastine; DTIC, dacarbazine; PRD, prednisone; CCNU, lomustine; STZ, streptozotocin. 'CR,Complete remission; PR, partial remission. (I

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TABLE V ESSENTIALRESULTSIN STAGE IV HODGKIN’SDISEASE TREATED WlTH MOPP OR MOPP ALTERNATEDWITH ABVD MOPP, 38 patients

Complete response Partial response Disease progression Five-year results Progression Free Relapse-Free Survival Survival N E D “ Overall survival

MOPP + ABVD, 37 patients

P value

(%I

(a)

71 5 24

92 8 0

0.02

37 47 54 61

70 77 84 83

0.0001 0.01 0.005 0.10

~

-

~~

N ED , No evidence of disease.

was 20 months compared to >31 months, respectively. Also the 5-year survival of patients with no evidence of disease was significantly in favor of those treated with alternating chemotherapy (84%) compared to MOPP alone (54%). Because of the limited number of patients at risk for 5 years, the difference in total survival was not significant. It is important to emphasize that in both treatment groups the survival curves leveled off after the second year from starting treatment. The conclusion from this study was that the cyclical delivery of two noncross-resistant combinations offers a promise for higher CR rate, longer duration of RFS, and possibly higher cure rate of advanced Hodgkin’s disease compared to the continuous administration of MOPP alone. A prospective randomized study similar to that carried out in Milan is in progress at the NCI (26). It employs alternating cycles of MOPP with cycles of a combination of streptozotocin, CCNU, adriamycin, and bleomycin (SCAB). Only a limited number of patients with stages 11, 111, and IV have so far been entered into the trial, and therefore the results are premature (120). V. Prognostic Factors Influencing Current Strategy

A. AGE, HISTOLOGY, A N D SYMPTOMS Although impressive advances have been made in the treatment of Hodgkin’s disease, in some instances prognosis remains bleak. Improvement could possibly reside in the accurate identification of pre-

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treatment variables affecting the therapeutic response. A number of factors including age, sex, histopathologic type, anatomical extent of disease, presence or absence of systemic symptoms, treatment modality, and response to therapy are strictly correlated to the prognosis of Hodgkin’s disease. It is noteworthy that a more effective prognostic index could be constructed by considering multiple prognostic factors simultaneously (64).In fact, frequently the association of more prognostic factors in the same group of patients reduces the value of each one separately. It is also important to emphasize that modern aggressive treatment with cyclical chemotherapy or chemotherapy plus radiotherapy, has a tendency to blur the influence of classical prognostic factors such as sex, age, histologic type, and pathologic stage. The overall survival in the pediatric age group is now slightly superior to that of young adults, and in general the prognosis for patients older than 40 years remains less favorable than that for younger patients (64).At present, however, there are no treatment approaches that specifically take into consideration the age factor for adults with Hodgkin’s disease. On the contrary, as will be discussed later, a modification in the treatment strategy is being applied to children under the age of 10 to minimize morbidity from radiation therapy. Results with MOPP chemotherapy (31) and combined treatment modality identified age as an important prognostic factor. However, the observation made by the Yale group (35)that chemotherapy plus low-dose radiotherapy produced the best results in patients under 40 years of age was not confirmed by the Milan group (9) utilizing a similar strategic approach. In patients subjected to modern megavoltage irradiation, the prognosis of mixed cellularity, once almost as poor as that of lymphocyte depletion, has markedly improved, with an RFS that plateaued at 50% at about 10 years. When the analysis was further subdivided according to stage, there was no longer any significant difference in either RFS or survival between mixed cellularity and nodular sclerosing lymphoma in patients with stage I, 11, and I11 disease (64).Thus, at present, in patients who are candidates for primary irradiation there is no particular strategic approach based on histology. In patients subjected to MOPP chemotherapy (31), nodular sclerosing Hodgkin’s disease had significantly shorter RFS than mixed cellularity or lymphocytedepleted lymphoma. Since there is preliminary evidence that such a difference was not observed after ABVD alone or combined with radiotherapy or alternated with MOPP (9,98),future trials should also consider the use of this new combination regimen in the presence of nodular sclerosis histology.

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TABLE VI PROGNOSTIC FACTORS IN HODGKIN’S DISEASE

Factor Age greater or lesser than 40 years Histologic subgroup, particularly nodular sclerosis Disease extent at diagnosis (stage) Systemic “B” symptoms Extensive disease: mediastinal adenopathy :thoracic ratio > 0.33; abdominal adenopathy > 5 cm; multiple extranodal “E” sites; extensive spleen involvement; multiple visceral involvement Prior chemotherapy Complete pathologic remission Relapse-free survival > 2-3 years

Clinical relevance

++

++

++ +++ +++

+++ +++ +++

The presence of systemic symptoms remains strictly correlated with an unfavorable prognosis (1, 64) in patients treated with MOPP chemotherapy, either alone or combined with irradiation. Thus, systemic symptoms represent the most important single prognostic factor in practically each subset of patients. However, some results with ABVD combined with radiotherapy (9, 100) failed to indicate a difference in the CR rate, RFS, and total survival between patients with “A” and “B” symptoms. Therefore, also in this prognostic subgroup ABVD chemotherapy should be considered in the design of future strategies. Studies (96) have identified several new unfavorable prognostic factors that seem to acquire a growing importance in treatment planning. These are (a) bulky disease either in the mediastinum (masdthoracic ratio >0.33) or in the abdomen (mass > cm 5; extensive spleen involvement >5 nodules); and (b) multiple extranodal (E) involvement (Table VI). B. MEDIASTINALBULKYDISEASE The presence of large mediastinal involvement (greater than onethird of the chest diameter) in patients with pathologically staged supradiaphragmatic Hodgkin’s disease has been associated with an increased risk of relapse as compared to patients with lesser or no mediastinal disease. About 50-74% of such patients initially treated with radiation therapy alone relapse compared to 5-27% with small mediastinal masses (40,53,73,76,77,79,87,111, 117).The majority of

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relapses are intrathoracic, as recurrences within the treated volume, recurrences at the margin of the treated field, and diffuse pulmonary relapses. On this clear evidence, several institutions are evaluating the role of chemotherapy (MOPP) associated with radiotherapy. The preliminary results obtained with chemotherapy plus radiotherapy strongly suggest the use of a combined modality approach for this subset with poor prognosis. Another controversial point is the sequence of administration of chemotherapy and radiotherapy. The increased risk of complications, especially pericarditis and pneumonitis, because of the large radiation fields needed if radiotherapy is initially utilized, suggests that chemotherapy should precede irradiation to secure bulk reduction and to facilitate subsequent radical radiotherapy (79, 100, 117).The feasibility and utility of low-dose lung irradiation as an initial part of the supradiaphragmatic field was examined by Lee et al. (74).Fifteen patients were treated with 10-20 Gy to the lung as part of the extended-field radiotherapy, and results were compared with those in 20 similar patients who were treated only with total nodal radiotherapy. With a minimum follow-up of 24 months, only 13% of patients who received lung irradiation have recurred (only one in the lung) compared to 79% who were treated without lung irradiation, nine of whom relapsed in the intrathoracic region. Thus, lung irradiation appears feasible and should be further evaluated with adjuvant chemotherapy in the treatment strategy of patients with Hodgkin’s disease and large mediastinal masses. C. LIMITEDEXTRANODAL DISEASE

The Ann Arbor classification (108) assumes that the prognosis in patients with localized extranodal (E) disease is similar to that of comparable patients with the same stage disease without extranodal spread. The introduction of the “E” subgrouping has had an important influence on subsequent therapeutic approaches employed by many centers for these patients. The implication has been that patients with E stage could be treated adequately with similar radiation techniques used for comparable patients with nodal disease (108). Confusion may arise, however, with respect to the E classification (86).The excessive use of the E stage sometimes may have led to conceptual errors in the management of patients. In fact, there is always some subjective element in deciding whether a patient should be placed in stage IV or E category. Following Musshoffs observation (108), E designation should be restricted to patients whose disease appears to be curable

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28 1

with radiotherapy alone. Levi and Wiernik (75) in reporting the results of a comparative analysis of patients with stage IIA-IIIA and IIEAIIIEA treated concurrently with extended field irradiation alone or limited-field irradiation followed by MOPP, found a wide difference in the 5-year relapse rate for patients treated with radiotherapy alone (stage IIA-IIIA, 29%;IIEA-IIIEA, 82%).By contrast, in patients receiving adjuvant MOPP after irradiation the relapse rate was 6% and 14%, respectively. Most relapses were observed within the lung parenchyma. Statistical analysis revealed a significantly shorter remission duration and survival for patients with E stage after irradiation alone, but there was no difference between the two treatment groups treated with RT plus MOPP. The authors concluded that the use of extendedfield irradiation alone has been inadequate for patients with E Hodgkin’s disease and suggested a combined approach for these patients. The Stanford group (113) analyzed patients with E disease and failed to confirm the results of Levi and Wiernik. The difference in results obtained by the two groups could be related in part to different patient selection, and maybe Levi and Wiernik classified as E stage some patients who actually had stage IV disease (86).Nevertheless, the optimal treatment strategy for stage E Hodgkin’s disease remains controversial, and definitive conclusions cannot as yet be drawn.

D. STAGEIIIA The optimal treatment strategy for PS IIIA with or without splenic involvement remains controversial. Total nodal irradiation has resulted in a definite improvement in both RFS and survival compared to less aggressive forms of radiotherapy (64).Nevertheless, a significant proportion, ranging from 30 to 65% of patients treated with aggressive irradiation alone, continue to relapse, especially in extranodal sites (47,90).Mauch et al. (78) utilizing total nodal irradiation alone in stage IIIA reported RFS and overall survival rates that were inferior to those obtained in stage IIIB with the same irradiation followed b y MOPP, and they suggested the use of a combined therapy program in stage IIIA. In addition, as mentioned before, the updated results from Stanford (96) confirmed a significant improvement in RFS and probably also in total survival for patients receiving total lymphoid radiotherapy plus six cycles of MOP(P) compared to those treated with irradiation alone. However, some of the treatment complications, such as sterility and second neoplasms, following combined treatment may suggest caution before adopting intensive radiotherapy followed b y

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adjuvant MOPP for all patients with stage IIIA. The use of primary irradiation alone may also be justified because of the high salvage rate (about 50%) with MOPP chemotherapy administered at the time of first relapse after radiotherapy. However, the observation of Canellos et al. (15) and Valagussaet al. (115,116),that patients receiving MOPP chemotherapy as salvage treatment for relapse after extensive radiotherapy have a considerably higher risk of developing second acute leukemia compared to patients receiving extensive radiotherapy and MOPP as first-line treatment, would contraindicate the aforementioned strategy. The use of MOPP chemotherapy alone in the treatment of IIIA has also been investigated. However, with the exception of the NCI study (31), the results of chemotherapy alone seem to be inferior to those obtained with radiotherapy alone (13). New information concerning optimal treatment strategy has been provided by research in staging. In fact, specific patterns of intraabdominal involvement were reported to correlate with response to therapy, patterns of relapse and survival. As reported by the Chicago group (25, 50), detailed surgical staging allowed subdivision of patients with PS I11 into two “anatomic substages”: PS 1111(involvement limited to those lymphatic structures in the upper abdomen that accompany the celiac-axis group of arteries, i.e., spleen, splenic hilar nodes, celiac nodes, and/or portal nodes); and PS IIIz (involvement of lower abdominal nodes, i.e., para-aortic, iliac, or mesenteric nodes with or without involvement of nodes belonging to 1111groups). The 5-year RFS and total survival rates after total nodal irradiation were definitely higher for substage 111, than for substage II12. The findings were improved by the addition of chemotherapy only in PS IIL, not in 1111. The large number of PS IIIA patients studied gave strong support to the contention that the anatomic substage is the major prognostic indicator for patients in PS IIIA. However, the different prognosis of patients with PS 1111 vs 1112 was not confirmed b y the Stanford series (54). Analysis of a large number of potential prognostic factors among patients with PS IIIA treated at Stanford showed that the most important adverse prognostic factors were extensive splenic involvement (five or more nodules visible on the cut section of the spleen), bulky abdominal masses ( 2 5 cm), and presence of five or more sites of involvement. Thus, PS IIIA Hodgkin’s disease appears to include several groups with different prognostic factors. Probably, the optimal management remains to be further elucidated and alternating chemotherapy with non-cross-resistant regimens in conjunction with moderate doses of radiotherapy appears to be worth testing.

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VI. Morbidity Influencing Current Strategy

A. MORBIDITYFROM SURGICAL STAGING During the 1960s and 1970s, various efforts were made to stage Hodgkin’s disease properly. The major intent was to make proper decisions regarding therapy by improving the selection between patients suitable for curative radiotherapy and those who are candidates for a systemic treatment program with or without radiotherapy. Over the years, lymphography, needle marrow biopsy, laparotomy, and laparoscopy have become important steps in the clinical accuracy of staging both in adults and children. The systemic use of available procedures to determine the extent of disease led first to the Rye (1965)and then to the Ann Arbor (1970) international staging classification for Hodgkin’s disease (107, 108). A major advance was the introduction (46, 66) of staging laparotomy, which has provided an unparalleled contribution in staging accuracy and knowledge of the natural history of disease. Today, the role of laparotomy is under critical reevaluation as a routine staging procedure. It should be performed only if management decisions depend on the identification of occult abdominal lymphoma, particularly of a positive spleen. Since a combined treatment approach is presently being applied for many patients with intermediate stages of Hodgkin’s disease, laparotomy is becoming less important as a routine staging procedure. To detect patients with stage IV disease in the liver, laparoscopy with multiple hepatic biopsies can substitute for laparotomy in the large majority of patients (5, 6, 28). Staging laparotomy remains at present a necessary procedure in clinical stage IA and IIA with no bulky mediastinal mass, as the 5- and 10-year survival rates of patients without occult disease below the diaphragm and treated with subtotal or total nodal irradiation alone approach 90%. As in adults, staging laparotomy in children with Hodgkin’s disease modifies the clinical stage in a considerable number of patients, particularly because in the pediatric age group there is a high incidence of occult splenic involvement (64).Complications from surgical staging are not high, but they are definitely less after laparoscopy compared to laparotomy. In the sequential laparoscopy-laparotomy study carried out in Milan on 146 consecutive patients (6), laparoscopy was associated with less morbidity (3%)compared to staging laparotomy (35%).The morbidity from laparotomy is of particular importance in children under 5 years of age because of the specific hazard of overwhelming postsplenectomy infection (57). This complication as well

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as the need of a combined modality approach in children to decrease radiation morbidity, has led the large majority of pediatric oncologists to abandon routine staging laparotomy in children, particularly in those under the age of 5. Current results with combined modality therapy in children not subjected to laparotomy has yielded a 5-year survival in the range of 90% (56). B. MORBIDITYFROM RADIOTHERAPY

The most important complications from radical radiation therapy (18, 110) that can influence the current treatment strategy in adult patients with Hodgkin’s disease are (a) radiation pneumonitis and fibrosis; (b)radiation pericarditis and carditis; (c) radiation nephritis; (d) radiation myelitis; (e) prolonged myelosuppression; (fj sterility. The aforementioned complications are primarily due to high-dose, large treatment volume techniques, and they are more likely to be expressed with the increased number of long survivors. Therefore, prevention is the best treatment and may be accomplished by appropriate shielding of vital organs and/or by keeping the dose below the risk threshold. I n particular, the incidence of symptomatic radiation pneumonitis (1530%) can be decreased in patients with massive mediastinal and/or hilar involvement by starting treatment with one to three courses of combination chemotherapy, which can substantially reduce the irradiated volume, and, in complete responders, also the dose of radiotherapy. In patients with symptomatic pneumonitis, steroid therapy is indicated. The radiation-induced heart disease, particularly pericarditis (13-15%), depends on the total dose, dose fraction, relative weighting of the anterior to posterior, volume of the heart included, and whether one or two fields are treated each day (110).Since it has been suggested that a lower dose, in the order of 30-35 Gy, should suffice for the eradication of microscopic disease in apparently uninvolved lymph node chains (64), a decreased morbidity and risk of late complications should be observed by delivering the aforementioned dosage to the mediastinal area in the absence of radiological or radioisotopic signs of Hodgkin’s disease. It should be recalled that acute steroid withdrawal, either during mantle irradiation or during the first and fourth courses of MOPP, may activate occult radiationinduced heart disease (64). Although after the administration of ABVD in previously irradiated patients, no episodes of symptomatic heart disease were documented (8), it is important to remember that there is at least an additive, if not synergistic, effect between conventional

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doses of radiotherapy and cumulative doses of adriamycin exceeding 400 mg/m’. To limit radiation nephritis, appropriate shielding of a portion of the kidneys and keeping the dose to both organs below 24 Gy, particularly in the presence of negative lymphography and laparotomy, usually minimizes or prevents renal damage. Similar technical precautions are applied to limit radiation myelitis and sterility in man (110). Attempts to preserve ovarian function were initiated at Stanford in 1964 (64) through oophoropexy at the time of staging laparotomy. The procedure gained popularity, since many patients have been noted to retain menstrual ability and became pregnant with the delivery of normal children. The irradiation of pelvic nodes is often followed by prolonged myelosuppression, especially thrombocytopenia, and this side effect markedly increases in elderly patients as well as in those subjected to combined chemotherapy and radiotherapy. For this reason, many radiation therapists altered the treatment strategy in patients with pathologic supradiaphragmatic IA and IIA after the report of Goodman et al. (51). These investigators noticed that, utilizing subtotal nodal rather than total nodal irradiation, the failure rate in pelvic lymph nodes was less than 10%. Subtotal nodal irradiation is also often applied in a combined modality setting when only the para-aortic nodes appear to b e involved b y Hodgkin’s disease. In children, extensive high-dose radiation therapy has produced a number of delayed complications, namely growth retardation, particularly in the shoulders and clavicles following mantle field irradiation. Growth retardation is manifested primarily as decreased sitting height and is most severe in children treated when less than 6 years of age or when they are 12-13 years of age (64, 85). To overcome this type of morbidity, reduction in field extension as well as in the dose and combining low-dose radiotherapy with adjuvant chemotherapy (MOPP or ABVD) has represented the most important evolution in the treatment strategy for young children. Current results (32, 37, 56, 64) have indicated that in both clinical and pathologic stages 1-111 the relapse rate was low with bimodal treatment and was not associated to important acute and delayed side effects from radiotherapy and chemotherapy. c . LATE COMPLICATIONS FROM CHEMOTHERAPY The toxic effects of chemotherapy that are relevant to the treatment strategy of Hodgkin’s disease are as follows: (a) cardiotoxicity; (b) lung fibrosis; (c) infertility; (d) second neoplasms. Cardiotoxicity may be

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secondary to adriamycin therapy, and the risk becomes very high when

the cumulative dose exceeds 550 mg/mz. Patients with prior radiotherapy to the mediastinurn may manifest congestive heart failure after cumulative doses higher than 400-450 mg/mz. Therefore, all adriamycin-containing regimens should be administered with caution. The Milan experience with ABVD has been so far successful also from the toxicologic point of view, for no patient out of the 400 treated so far with this regimen has exhibited signs and symptoms compatible with cardiomyopathy. It should be pointed out that in all studies the cumulative dose of adriamycin has almost never exceeded 350 mg/m' (8). However, a longer follow-up analysis is required to define better the risk of heart damage following the administration of adriamycin with and without irradiation. Pulmonary toxicity may be observed after treatment with bleomycin and BCNU or CCNU. Also from this point of view, ABVD appears to be a safe combination (8) provided the total dose of bleomycin does not exceed 200-250 mg/m' and its administration is withheld in patients with chronic lung disease or overt pulmonary postirradiation fibrosis. Male infertility and second neoplasms are of particular concern, especially because many patients with Hodgkin's disease are young and the potential for cure is high. The NCI group (101, 102) has noted a high incidence of male infertility following MOPP chemotherapy, and this has been attributed to the toxic effect on spermatogenesis by alkylating agents and procarbazine. However, in 25-40% of patients treated with MOPP or MOPP-like combinations, spermatogenesis may return after about 2 years from completion of treatment. The Milan group (8, 100) has reported that azoospermia occurred in 100% of patients treated with MOPP and in only 15%of patients given ABVD. A similar difference was also noticed in the comparative incidence of prolonged amenorrhea. These observations are important, also in that the therapeutic activity of ABVD appears to be equivalent to that of MOPP, at least in the randomized studies so far carried out in Milan (8). Thus, treatment strategy for young patients who desire to have children could be modified by selecting ABVD rather than MOPP. Another alternative could be a more careful analysis of toxicological effects of MOPP alternated with ABVD (98). By administering MOPP every 2 months, and therefore reducing the total dose of mechlorethamine and procarbazine, it is conceivable that their action on reproductive organs will be decreased. Second neoplasms, particularly acute nonlymphocytic leukemia and non-Hodgkin's lymphomas, are now being reported with more frequency than in the past in patients subjected to intensive prolonged chemotherapy with MOPP or

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CASE

SERIES

TABLE VII SHOWING THE RISK OF ACUTE LEUKEMIA AFTER DIFFERENT, TYPESOF THERAPY FOR HODGKIN'S DISEASE

Author Baccarani et al. (3)

Coleman et al. (20)

Valagussa et ul. (1 15)

a

Treatment

Patients

Risk (%)

Time (years)

Radiotherapy Chemotherapy" Combined" Radiotherapy Chemotherapy" Radiotherapy-gold t chemotherapy" Combined" Adjuvant Salvage Radiotherapy Cornbined" Adjuvant Salvage Combined Radiotherapy + ABVD

117 152 344 417 37

0 2.0 2.04 0 17.6

7 7 7 9 9

65

7.7

9

453 124 272

5.1 2.8 0

9 9 10

430 176

1.5 6.1

10 10

0

10

84

All regimens including alkylating agents and/or procarbazine.

MOPP-derived regimens (3, 14, 15, 19-21, 112, 115, 116). Within 10 years from diagnosis of Hodgkin's disease, the risk was considerable for patients treated with combined extensive radiotherapy -chemotherapy and was even higher, at least in some published series (15,115, 116), after salvage chemotherapy (Table VII). In contrast, the updated results with ABVD (115) confirmed that this regimen, either when administered alone or when combined with extensive irradiation, appeared to be devoid of carcinogenic activity. Also this observation may change the future therapeutic strategy, and the practical considerations on this subject are identical to those made about infertility. VII. Conclusions: Toward the Total Conquest of Hodgkin's Disease

The evolution of treatment strategy for Hodgkin's disease indicates that improvement in results is being achieved in all stages at major research centers. Current achievements are based on solid advances and represent the results of brilliant leadership in the rational development of therapeutic programs. The fact that today no stage of the disease is beyond cure after the initial treatment program, and even

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after recurrence following primary irradiation, represents a dramatic improvement in the overall prognosis of a disease that only about 20 years ago was considered to be almost universally fatal. Future strategies should refine available treatment programs, because given subgroups with unfavorable prognostic signs will probably require a more aggressive approach whereas in other subgroups the risk of treatment complications should be minimized. First of all, since the reason why some patients with widespread lymphoma are not cured by drugs is due to primary cell resistance, more attention should be given to the potential of alternating treatments. At present the only approach that we can see to increase the cure rate of advanced Hodgkin’s disease is by cyclical (or sequential) use of non-cross-resistant combinations in the manner now being investigated in the MOPP-ABVD (8,98) and the MOPP-SCAB (26, 120) protocols. By this we do not mean that these particular combinations, or the ratios of doses in them, or the timing of the treatment in each cycle is the optimum, but the effort to minimize failures due to the overgrowth of MOPP-resistant tumor cells seems a step in the right direction. Thus, in the presence of stages IIIB and IV or at relapse following primary irradiation, MOPP monthly alternated with ABVD (minimum six cycles to achieve CR and then two additional cycles as consolidation therapy) appears to be the treatment of choice. Further to improve present results, the research group in Milan is now exploring the therapeutic effect of three non-cross-resistant combinations, i.e ., MOPP, ABVD, and C E P (CCNU, VP-16, and prednimustine). In fact, in a pilot study being carried out in patients resistant to both MOPP and ABVD, CEP was able to induce objective tumor response in about 60% and CR in about 40% at the expense of minimal toxicity. If ongoing trials continue to show an advantage of cyclical delivery of two or three non-cross-resistant combination of drugs, then the Hodgkin’s disease model will have taught us another important lesson, one that should be taken into consideration in future design of treatment for other neoplastic diseases that are highly responsive, moderately responsive, and not so responsive to available chemotherapy (103).The unanswered question in stages IIIB and IV is how to use irradiation, and potentially improve the results, without compromising the chemotherapy program in these patients. In other words, the relative merits of a single drug combination plus low-dose radiotherapy versus multiple non-cross-resistant combinations with and without irradiation remains to be clearly demonstrated. The greatest disagreement in terms of optimal treatment strategy concerns stage IIIA as to whether irradiation should be the primary

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treatment, or whether chemotherapy should be added to total nodal irradiation, or even whether combination chemotherapy should be used without irradiation. Considering the cure rate after primary radiation therapy (64) and the most consistent finding of a survival benefit for combined modality therapy for patients with PS IIIA (96, loo), we believe the new clinical studies should evaluate combined therapy utilizing non-cross-resistant combinations. Treatment should start with chemotherapy and should be alternated with irradiation. Most probably, by making drug therapy more effective than MOPP, also the survival benefit could become more evident than was observed in the past experience. The same strategic approach should be explored further in patients with PS IIA-IIB with massive mediastinal involvement and with lymphocytic depletion histology, as well as in young children, to limit excessive morbidity from high-dose radiotherapy. As far as PS IA and IIA is concerned, surgical staging with laparotomy followed by extensive-field radiotherapy (probably subtotal nodal irradiation) represents the established treatment method. In fact, this strategic approach is followed by a high cure rate after about 2 months of treatment and a moderate morbidity if treatment is carried out b y experienced radiation therapists. Furthermore, most patients showing relapse can now be saved with effective chemotherapy. Nearly one-third of the patients with Hodgkin’s disease die without evidence of lymphoma at autopsy. Infection, mainly bacterial, remains the most common cause of death, but a significant number of patients die of complications of therapy, both benign and malignant, including patients with hematologic or de novo lymphoid neoplasms. Therefore, new treatment programs should give more attention to the costs and morbidity of primary and salvage therapies. Most of the problems, particularly those related to prolonged aggressive chemotherapy (change in physical appearance, fear of treatment programs, loss of libido, dislocation from home, etc.) are difficult to express as actuarial curves and p values, but should become important considerations in the design and selection of new treatment programs (96). In conclusion, the total conquest of Hodgkin’s disease does not appear to be a too distant goal. To achieve this goal new treatment studies are needed for high-risk groups as well as more consideration to overt and relatively occult treatment morbidity. To accomplish this program, patients with Hodgkin’s disease should continue to b e referred to major cancer research centers where efforts in accurate diagnosis, proper staging, discipline of controlled trials, and identification of complications will remain the essential ingredients of treatment approach. The laudable goal of total conquest of Hodgkin’s disease

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will probably not be represented b y the delivery of combination chemotherapy to all patients in private offices or in community medical centers. Rather, it will be the judicious balance of refined diagnostic, predictive, and therapeutic methods for the various prognostic subsets.

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EPSTEIN-BARR VIRUS ANTIGENS-A CHALLENGE TO MODERN BlOCH EM I STRY

David A. Thorley-Lawson,l Clark M. Edson, and Kathi Geilinger' Sidney Farber Cancer Institute. Boston, Massachusetts

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. What Is Known about the Antigens of Epstein-Barn Virus? B. What Questions Remain To Be Answered about the Antigens o Epstein-Barr Virus? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Transformation Antigens . ........... A. Epstein-Barr Virus Nuclear Antigen (EBNA) . . . . . . . . . . . . . . . . . . . . . . . . . Surface of EBV-Transformed Cells (LYDMA) 111. Early Antigens

.. ... ... .. ... ...... ... . .. . .. ... . ... ......... ... .... . .....

A. Intracellular Early Antigens.. . . . B. Early Membrane Antigens ........... ........... IV. Late Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction B. Membrane Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . A. Molecular Biology of the Antigens and the Polypeptides . . . . . . . . . . . . . . . B. Immune Responses to the Viral Antigens.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . ............ ..............

295 295 297 298 298 304 309 309 318 319 319 32 1 335 336 336 339 342

I. Introduction

A. W H A T IS KNOWN ABOUT T H E ANTIGENS OF EPSTEIN-BARR VIRUS?2

Epstein-Barr virus (EBV) has two fascinating features in its biology. The first is its ability to transform human B lymphocytes in uitro (W. Henle et al., 1967; Pope et al., 1969; Miller et al., 1969; Gerber et al., 1969).The ability of EBV to convert small, normal, resting B lymphocytes into exponentially proliferating, transformed lymphoblasts is one ofthe most dramatic events that may be witnessed in tissue culture. I Present address: Department of Pathology, and Department of Medicine, Division of Geographic Medicine, Tufts University Medical School, Boston, Massachusetts. For a comprehensive overview of Epstein-Barr virus, see Epstein and Achong (1979).

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The second is its association with human disease, in particular malignant disease. The central question of importance for EBV studies is derived from the synthesis of these two observations. To what extent does the ability of the virus to transform human lymphocytes play a critical role in human disease? Is EBV truly a human tumor virus? The weight of the evidence indicates that this transformation is either malignant or premalignant (Shope et ul., 1973; Giovanella et ul., 1979). This leads to the questions of what is the mechanism of transformation; what is the nature of the immune responses that control transformation in viuo; and what is different about these processes in individuals who succumb to the EBV-associated diseases-nasopharyngeal carcinoma (NPC) (Old et al., 1966; W. Henle e t al., 1970b),Burkitt’s lymphoma (BL) (Levy and Henle, 1966; G. Henle e t al., 1969; de-Tht5 et al., 1979), and fatal infectious mononucleosis (fatal I M ) (Bar et al., 1974; Purtillo et al., 1975; Virelizier et al., 1978; Crawford et al., 1979; Robinson e t al., 1980b)? The virus was discovered originally as a typical herpesvirus in cultured tissue of BL biopsies (Epstein et al., 1964) and was shown to be distinct from other human herpesviruses on a serological basis (G. Henle and Henle, 1966a,b). Until recently, the biology of EBV has been studied primarily in terms of serologically defined antigens that can be grouped into three classes depending on the stage of the virus lytic cycle.

1. Transformation Antigens EBV-transformed lymphocytes have the capacity to grow indefinitely in culture and possess three markers for the presence of EBV: (a) multiple copies of the viral genome (zur Hausen and SchulteHolthausen, 1970; zur Hausen et al., 1970; Nonoyama and Pagano, 1971, 1973); (b) expression in their nucleus of a characteristic, serologically defined antigen termed the EB nuclear antigen (EBNA) (Reedman and Klein, 1973); (c) expression, on the plasma membranes, of antigens that elicit cellular immune responses, described under the general title of lymphocyte-determined membrane antigens (LYDMA) (Svedmyr and Jondal, 1975; Misko et al., 1980; Thorley-Lawson, 1981). The transformation antigens are expressed in all EBV-infected cells so far studied. 2. Early Antigens At any one time, a small number of EBV-transformed cells will spontaneously enter a viral lytic cycle. The absolute number of these cells in a culture depends on several factors, such as (a) the cell type [cell lines derived by in vitro transformation or spontaneous outgrowth

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from the peripheral blood of IM patients have a low number, <1%, perhaps as low as 0.1-0.01% (Sugden et al., 1979), whereas cell lines derived from BL or marmoset tumors tend to have a higher level, in the range of 1-lo%]; (b) the tissue culture conditions [starvation tends to increase the percentage (Magrath et al., 1980)l; (c) the addition of inducers [phorbol esters (zur Hausen et al., 1978),n-butyrate (Luka et al., 1979)l.The entrance of a cell into the lytic cycle is indicated by the presence of early antigens (EA) (W. Henle et al., 1970a), so called because they are not sensitive to DNA synthesis inhibitors (Gergely et al., 1971b). These probably include two intracellular forms, termed diffuse (EA-D) and restricted (EA-R) (G. Henle et al., 1971a), and a membrane form (E-MA) (Ernberg et al., 1974; Sairenji et al., 1977). It is generally accepted that a cell that expresses early antigens is doomed to complete the lytic cycle, release infectious virus, and die (Gergely et al., 1971a).

3. Lute Antigens When a cell containing the early antigens begins to replicate the viral DNA, it synthesizes a number of late antigens, so called because their expression is blocked by inhibitors of DNA synthesis. There are two serologically defined groups of late antigens-plasma membrane (Klein et al., 1966; Ernberg et al., 1974; Sairenji et al., 1977) and cytoplasm associated (G. Henle and Henle, 196613). These antigens share some determinants with structural components of the virion. The membrane antigens (MA), expressed on the plasma membrane of cells replicating the virus, are cross-reactive with components of the viral envelope (Sugawara and Osato, 1970; Silvestre et al., 1971; ThorleyLawson, 1979a) and include components able to generate virusneutralizing antibodies (Pearson et al., 1970; De Schryveret al., 1974; Qualtiere and Pearson, 1979; Thorley-Lawson, 1979b). It is generally accepted that the cytoplasmic antigen cross-reacts with capsid components (W. Henle et al., 1966; Mayyasi et al., 1967; Silvestre et ul., 1971), and these antigens are termed viral capsid antigens (VCA). In fact, the classical cytoplasmic “VCA” stain is due to ii mixture of intracellular membrane and capsid antigens (Thorley-Lawson and Geilinger, 1980) (see Section IV,B,2).

B . WHAT QUESTIONSREMAINTO BE ANSWERED ANTIGENS OF EPSTEIN-BARRVIRUS?

ABOUT THE

Both the biology and immunology of Epstein-Barr virus had been mapped out in a preliminary fashion on the basis of immunologically defined antigens, whose role in the biology of EBV was unknown and

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whose significance in the normal immune response, which controls EBV infection, was undetermined. At this stage, a number of obvious questions arose about the function and significance of these molecules.

1. EBNNLYDMA: What role do these molecules play in the process of transformation and in the maintenance of the transformed state? 2. EBNNLYDMA: Are these antigens, which are detected in vitro, also present in infected cells in uiuo? 3. LYDMA: Are the same antigens recognized by mechanisms of cellular immunity defined both in vivo and in vitro? Can isolated LYDMA be used to generate cellular immunity in vivo against EBVinfected and -transformed cells? 4. EA: What roles do these molecules play in initiating the lytic cycle and viral DNA replication? 5. EA: Does the role of anti-EA antibodies in predicting the course of diseases have biological significance, and can isolated EA molecules be used to develop a more effective test for these antibodies? 6. EMA: Does immunity against this antigen allow cells replicating the virus to be destroyed before infectious virus is produced? 7. MA: How many components are there, and are they the same on the plasma membrane of producer cells and the viral envelope? 8. MA: Which components stimulate neutralizing antibodies, and do such antibodies play a role in the control of infection? Could a vaccine made of such components be effective in preventing infection? 9. MA: Which of these components on the viral envelope is necessary for interaction with the putative EBV receptor on the B lymphocyte? 10. VCA: How many components are there in the viral capsid, and how are they assembled?

The substance of this review will be concerned with the answers to these questions, to the extent that they are known, and a discussion of possible ways to approach the unanswered questions. II. Transformation Antigens

A. EPSTEIN-BARRVIRUS NUCLEAR ANTIGEN(EBNA) 1. lntroduction

The EBNA was first discovered in the nucleus of EBV-positive cell lines (Reedman and Klein, 1973) and BL biopsy cells (Reedman et al., 1974). Since then, it has been found in all cell lines tested that also

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contain copies of the viral genome. This includes cell lines derived from BL tumor cells or from the peripheral blood lymphocytes of IM patients or by in vitro transformation of normal lymphocytes. These studies have been extended to show that as many as 1-2% of the lymphocytes in the peripheral blood of acute-phase I M patients contain EBNA (Klein et al., 1976; Lenoir et al., 1978; Katsuki et al., 1979; Robinson et al., 1980a). Most critically, EBNA has also been demonstrated in the tumor cells of all NPC biopsies that contain viral genomes (Klein et d., 1974; Huang et al., 1974; Andersson-Anvret et

al., 1977). In the case ofin vitro transformation, EBNA may b e detected within a few hours after infection (Aya and Osato, 1974; Yata et al., 1975; Einhorn and Ernberg, 1978; Takada and Osato, 1979), well before the first cell division, which occurs perhaps 30-40 hr post infection. Its synthesis, as might be expected, is not affected by the presence of inhibitors of DNA synthesis (Thorley-Lawson and Strominger, 1978; Takada and Osato, 1979). Several lines of evidence suggest that EBNA may have some unusual antigenic features, in particular, that it may be a weak antigen.

1. A triple sandwich fluorescence technique is required to detect EBNA (Reedman and Klein, 1973). Human EBV-positive serum is used as the first step followed by complement and fluoroscein isothiocyanate (FITC) anti-complement antibody. This method allows great sensitivity, but it is also technically difficult and not always successful, even in experienced hands. 2. Anti-EBNA antibodies do not arise until weeks or months after recovery from IM (G. Henle et al., 1974), whereas antibodies to EA, VCA, and MA all appear during the acute phase of the disease (W. Henle et al., 1974; W. Henle and Henle, 1975). This, despite the fact that EBNA-positive cells are present during acute I M and that EBNA presumably is being released from destroyed cells. 3. In certain cases of immunodeficiency, no EBNA antibodies are made, despite clear evidence for the establishment of EBV infection on the basis of antibodies to other viral antigens. For example, boys with the X-linked proliferative syndrome (XLP), who are immunoincompetent to EBV infection, occassionally make anti-VCA antibodies but never make substantial levels of anti-EBNA antibodies or occasionally have low titers (Sakamoto et al., 1980). By comparison, normal seropositive individuals have both anti-EBNA and anti-VCA antibodies. A similar observation is seen upon infection of certain new world monkeys with EBV. Several species readily establish long-term infection, associated with a stable anti-VCA titer, but never produce

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detectable anti-EBNA antibodies (G. Miller et al., 1977). This is true even for cottontop marmosets that can develop tumors upon EBV infection (Shope et al., 1973) and where the tumor cells can be shown to contain EBNA. Another form of nuclear antigen, termed rheumatoid arthritis nuclear antigen (RANA), has been associated with EBV. This antigen is found only in EBV-infected cells. Antibodies to this antigen are found at a higher level in patients with rheumatoid arthritis (Alspaugh et al., 1978), although anti-EBNA antibodies are also elevated in these individuals (Catalan0 et al., 1979). For this reason the association of antiRANA antibodies with rheumatoid arthritis remains unsubstantiated. RANA may be distinguished from EBNA because (a) EBNA is detected b y anti-complement and RANA by anti-immunoglobulin indirect immunofluorescence tests; (b) RANA is expressed in the nucleus and, to some extent, the cytoplasm, whereas E BNA is exclusively expressed in the nucleus; (c) RANA immunofluorescence has a much more speckled appearance than EBNA; (d)RANA is expressed primarily in the GI phase, whereas EBNA is primarily expressed in the S phase of the cell cycle (Slovin et al., 1980). At present it is not yet clear if EBNA and RANA are different or the same antigen recognized by different tests.

2. Biochemical Characteristics of EBNA Most of the studies on the characteristics of EBNA have been performed on crude or partially purified material, assaying for EBNA on the basis of its ability to fix complement. What follows is an attempt to summarize, in a consistent fashion, the observations of several groups. a . Sensitivity of the Antigenic Activity of EBNA to Proteases. It has been demonstrated, both in histochemical studies (Ohno et al., 1977a) and by loss of complement-fixing activity from partially purified EBNA preparations (Baron and Strominger, 1978; Klein et al., 1979), that the antigenic activity of EBNA may be destroyed with trypsin. One unconfirmed report has suggested that EBNA may also be partially DNase sensitive (Baron and Strominger, 1978). Sensitivity of the antigen to mild denaturing conditions, such as urea and nonionic detergents, has also been reported (Luka et al., 1977). b. Heat Stubility of the Antigenic Activity. The activity is remarkably stable to heat treatment. Crude material has been shown to be stable to 56°C for 30 min (Ohno et al., 1977a),65°C for 10 min (Lukaet al., 1980), 80°C for 10 min (Matsuo et al., 1977), and, in one report, 100°C for 30 min (Baron and Strominger, 1978). The purified antigen

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reflects the same property, having been shown to be stable at 80°C for 10 min (Klein et al., 1979). c . EBNA Is a DNA-Binding Protein. EBNA fluorescence is found associated with the chromosomes in the nucleus of EBVinfected cells (Reedman and Klein, 1973). It does not appear to be localized on a particular chromosome or region of the chromosomes, suggesting that it is not specifically associated with the viral DNA. Crude EBNA, extracted from cells, will bind to the DNA in acid-fixed nuclei of any cell (AFNB assay), and this preparation provides an effective reproducible means of demonstrating EBNA fluorescence (Ohno et al., 1977b). Inhibition of AFNB has been used as an assay for purifying EBNA (Luka et al., 1978, 1980). Last, several investigators have demonstrated the ability of EBNA to bind to DNA-cellulose columns, and this property has been used for affinity purification of EBNA (Lenoir et al., 1977; Luka et al., 1977; Matsuo et al., 1978; Baron and Strominger, 1978). It is reported that EBNA binds preferentially to double-stranded DNA, compared to single-stranded DNA (Luka et al.,

1977). d . Purification and Molecular Weight of EBNA. The most detailed and elegant study to date on the nature of EBNA is the reported purification of EBNA to homogeneity (Luka et al., 1980). The purification procedure used was a modification of a previous method published by this group and makes use of the known properties of EBNA, notably its heat stability and its affinity for double-stranded DNA. Thus, purified material was obtained by treatment at 65°C for 10 min, followed b y DNA-cellulose chromatography and a final step of hydroxyapatite chromatography. In this study EBNA activity was found associated with a 48,000 MW protein that copurified with a 53,000 MW protein that was unrelated by peptide mapping and could also b e purified from EBV-negative lymphoblastoid cell lines. Confirmation of these results was obtained by studies that revealed a 53,000 and 48,000 MW polypeptide after immunoprecipitation with an EBNA-positive serum (Fig. 1). The undenatured antigen migrates with an approximate molecular weight of 200,000 on Sephacryl S-200 (Luka et al., 1978,1980). Thus, the 53,000 and 48,000 MW polypeptides are probably associated, in situ, with each other in a tetramer. Interestingly, the separated 53,000 and 48,000 MW proteins can also form tetramers with themselves, indicating that the native tetramer may be in multiple forms, containing various proportions of the 53,000 and 48,000 MW components. The 48,000 MW protein carries the EBNA antigenic specificities, as it gives a positive result in the AFNB assay and successfully inhibits the complement-fixation assay. To date, this rep-

FIG.1. Identification of “C-labeled Epstein-Barr virus nuclear antigen (EBNA) and 53,000 M W protein by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, and subsequent fluorography. RAJI, RAMOS, and BJAB cells were labeled separately by growth in a 14C-labeledamino acid mixture. Extracts were treated with anti-EBNA-positive or -negative sera as described in the text. Lanes 1-3: RAJI cell extract treated with three different anti-EBNA-positive sera; lanes 4-6: RAJI cell extract treated with anti-EBNA-negative sera; lanes 7 and 8: RAMOS cell extract treated with anti-EBNA-positive and -negative sera; lanes 9 and 10: BJAB cell extract treated with anti-EBNA-positive and -negative sera. Reprinted from Luka et al. (1980) by courtesy of the author and publisher.

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resents the only report of successful purification and immunoprecipitation of EBNA despite considerable efforts from other groups. However, the isolated material has the properties of EBNA agreed on by most investigators. Thus, other workers agree that the molecular weight of EBNA, under nondenaturing conditions, is approximately 200,000, with reports of 180,000 on Sephadex G-200 or sucrose density gradients (Lenoir et al., 1976) and 220,000-240,000 on Sephadex G-200 (Matsuo et al., 1978). Furthermore, Hentzen et a2. (1980) have reported that the antigenic activity of EBNA is present on a 50,000 MW component, derived from the 180,000 MW native form b y denaturation, and that this monomer will re-form the 180,000 MW tetramer upon removal of the denaturing conditions.

3. Functions of E B N A The few studies to date on the function of EBNA rely heavily on analogies to the T antigens of the papova- and adenoviruses. The T antigens are thought to play a role in the establishment of transformation by initiating DNA synthesis. This has been demonstrated directly by microinjection of purified T antigen into the nuclei of TC7 monkey cells (Tijan et al., 1978). Because of the ubiquity of EBNA among EBV-transformed cells and its appearance before the induction of cellular DNA replication during in uitro transformation, EBNA is clearly a target for manifest speculation. It could play a role in the initiation and/or maintenance of transformation and the suppression of gene functions, required for the production of virus (stably transformed cells do not usually produce virus and when they do, they die). The possibility that EBNA is an artifact, i.e., a cellular antigen,,that occurs because of EBV infection, but is not involved in any virus transforming function, is, however, a hypothesis with equal validity, if less exciting, and in the absence of experimental data to the contrary, should be given equal status in speculative writing. It is an absolute prerequisite to demonstrate that EBNA is virally encoded before statements about its role in transformation can have any meaning. Some progress has been made toward answering this question if it can be assumed that the 48,000 MW polypeptide is EBNA. Only about five distinct EBV-specified RNA species have so far been detected in transformed cells (Rymo, 1979; van Santen et al., 1981). If in vitro translation of these RNAs produces a polypeptide with a molecular weight of 48,000 that is identical by peptide mapping or by immunoprecipitation to the putative purified EBNA, then the foundation for future studies will be firmly established. As the next step, it will be important to attempt to induce EBNA in cells by microinjection of the appropriate restriction enzyme fragment of viral

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DNA and to demonstrate the stimulation of DNA synthesis in cells by microinjection of purified E BNA. Preliminary reports have already been presented indicating that both approaches may be successful (Graessmann et al., 1980; Klein et d., 1979). Last, it would be very useful to have available a monospecific antiEBNA antiserum or monoclonal antibody. No such antibody has yet been produced, despite the availability of partially purified and purified EBNA and repeated attempts to develop such antibody reagents. This failure may represent the result of the apparently weak antigenicity of EBNA as discussed in Section II,A,l. Attempts to immunize animals with EBNA should take this into account; in particular, repeated immunizations over a long period of time may be necessary (e.g., it takes many months postinfection to develop anti-EBNA antibodies). Furthermore, EBNA may not be immunogenic at all in certain species (e.g., cottontop marmosets never develop anti-EBNA antibodies). In order to obtain monoclonal antibodies against ENBA, it may be necessary to await the arrival of an effective human myeloma cell line for hybridoma production and to fuse this with spleen cells from a patient chronically infected with EBV. B. LYMPHOCYTE-DETERMINED MEMBRANEANTIGENS-ANTIGENS O N THE SURFACE OF EBV-TRANSFORMED CELLS (LYDMA)

1. Introduction Evidence has been presented indicating that there may be EBVassociated antigens on the surface of transformed lymphocytes. As these antigens are detected by various forms of cell-mediated immunity, they are referred to as lymphocyte-determined membrane antigens. Four kinds of cell-mediated immunity have been demonstrated against EBV-infected cells. Whether these different forms of cellmediated immunity are directed against the same or different EBVassociated antigens is not known at this time. First, it was shown that there are cytotoxic T lymphocytes in the peripheral blood that will lyse a broad spectrum of EBV-infected cells including cell lines derived from BL biopsies, from the peripheral lymphocytes of acute IM patients, and by in uitro transformation (Svedmyr and Jondal, 1975). These cytotoxic T cells had specificity for EBV, as none of the EBVnegative cell lines tested could be lysed, but showed no preference for targets with matched major histocompatibility complex (MHC) antigens (i.e.,no MHC restriction) (Lipinski et al., 1979). Recent evidence indicates, however, that this form of cytotoxicity is due to natural killer cells rather than specific T cells (G. Klein, personal communication).

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Second, it has been demonstrated that plasma membranes from EBV positive but not negative cells will inhibit the migration of leukocytes from EBV seropositive, but not seronegative individuals (Szigeti et al.,

1981). The other two kinds of cell-mediated immunity have been demonstrated as a consequence of the observation, made by several investigators, that T cells will prevent the outgrowth of newly EBV-infected autologous B lymphocytes (Thorley-Lawson et al., 1977a; ThorleyLawson, 1980; Moss et al., 1978,1979; Rickinson et al., 1979; Schooley et al., 1981).A typical experiment is shown in Fig. 2. When newly isolated B cells are infected with EBV, they proliferate exponentially, and this can be demonstrated b y measuring [3H]thymidine incorporation. When the experiment is repeated in the presence of autologous T cells, the initial number of B cells transformed is drastically reduced, as may be seen by the level of proliferation at day 2. The number of

FIG. 2. The proliferation of human B cells after Epstein-Barr virus infection in the presence and in the absence of autologous T cells (Thorley-Lawson et al., 1977b). Note that the initial level of proliferation is lower in the presence of T cells, owing to interferon-mediated “suppression” of transformation, and that the proliferation is again reduced 7-10 days postinfection owing to cytotoxic T-cell-mediated “regression.” Reprinted from Advances i n Comparative Leukemia Research, Vol. 8,1977 by courtesy of the editor and publisher.

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transformed and proliferating B cells is again reduced approximately 7-10 days postinfection. The early event has been termed “suppression” and is probably mediated b y leukocyte interferon (ThorleyLawson, 1981), which will prevent the transformation and proliferation of infected B cells, but will not prevent proliferation once transformation is established. The later event has been termed “regression” and appears to be mediated by cytotoxic T cells that are specific for EBVinfected cells and show MHC preference in that they will lyse autologous EBV-infected B cells more effectively than allogeneic cells (Misko et al., 1980). Only T cells from seropositive individuals will perform regression (Moss et al., 1978). A similar kind of cytotoxic cell may be generated by repeatedly stimulating peripheral T cells with the autologous EBV-transformed B-cell line (Sugamura and Hinuma,

1980). The other type of virus-specific molecule that might be found in the plasma membrane of transformed cells may be hypothesized by extension of the analogy between EBNA and the nuclear T antigen of, for example, polyoma virus. It is now thought that the gene product required for maintaining the transformed state in polyoma-infected cells is not the nuclear antigen, but a plasma membrane-associated transformation antigen (middle T) derived by splicing of the gene coding for the nuclear T antigen (Benjamin et al., 1979). Thus, it is conceivable that EBV may also code for a plasma membrane-associated transformation antigen. No evidence for such a molecule has been demonstrated.

2. Biochemical Characteristics of LYDMAs To date, nothing has been published about the biochemical characteristics of any EBV-associated antigens that may be expressed on the plasma membrane of all EBV-transformed cells. The first step in isolating such molecules would be to develop specific antisera or monoclonal antibodies. Attempts to detect such antibodies in the sera of infected individuals have been unsuccessful. Similarly, attempts to produce such antibodies b y immunizing experimental animals with EBV-infected cells and absorbing with EBV-negative cells have not worked. These negative results lead to the conclusion that these molecules must be so weakly antigenic that they do not generate a substantial antibody response although they may be detected by the more sensitive cellular responses. If an antibody response is being made in an immunized animal, it will be so weak as to be lost upon repeated absorption of the antiserum. Using this as a theoretical basis, we have approached the problem of developing antibodies against EBVassociated antigens on the plasma membrane of transformed cell lines

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(Thorley-Lawson et al., 1981; Thorley-Lawson and Geilinger, 1981). Purified plasma membranes were solubilized in detergent, and the major antigenic molecules, the HLA-A, B, C and DR products were removed by passage over columns of monoclonal antibodies with specificity for those molecules. The material that passed through these columns was used to immunize mice and hybridomas produced. Of the resulting hybridomas, only 3 of 72 reacted with EBV-transformed cells, reflecting the prior removal of MHC antigens. One of these 3 hybridomas (17D6) reacted preferentially with EBV-transformed lymphocytes. In binding assays, the cloned antibody reacted strongly with all 8 EBV-transformed cell lines tested and only weakly with a variety of EBV-negative cell lines, including B, T, null, myeloid, and erythropoietic leukemia cell lines. Peripheral B and T cells, with or without mitogenic transformation, reacted weakly with the antibody. However, EBV transformation of the B cells made them highly reactive with the antibody. The EBV-positive cell lines derived by spontaneous outgrowth from the peripheral blood of IM patients or EBVpositive individuals, and by cocultivation of peripheral lymphocytes with NPC biopsies, were all strongly reactive with the 17D6 antibody. EBV-positive BL cell lines also reacted weakly, but specifically, as shown by analysis with the cytofluorograph. Thus, 17D6 appears to define an antigen preferentially expressed on EBV-infected cells. This antigen has an apparent molecular weight of 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation from an EBV-transformed cell line. When RAMOS, an EBV-negative cell line lacking the 45,000 MW protein, was infected with EBV it expressed the 45,000 MW polypeptide as shown in Fig. 3. The antigen recognized by the 17D6 antibody is also present on 1-2% of cells [equivalent to the percentage of EBNApositive cells (Robinson et al., 1980a)l in the peripheral blood of acute-phase IM patients, but not of normal individuals (ThorleyLawson et al., 1981). By cytofluorography these are in the blast cell population in agreement with previous studies indicating that the EBV-infected cells in the peripheral blood of IM patients are EBNApositive, blast-transformed lymphocytes (Robinson et al., 1980a). Whether this 45,000 MW protein is responsible for generating cellmediated immunity in z)iz?o, i.e., is truly a LYDMA, remains to be tested.

3. Function of LYDMAs There are two distinct functions possible for LYDMAs. One, already defined, is the stimulation of cell-mediated immunity against EBVtransformed cells, the other could be the maintenance of the trans-

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FIG. 3. Radioimmunoprecipitation with the 17D6 antibody on lWI- and lactoperoxidase-labeled RAMOS and EBV-infected RAMOS (EBR) cells. Note the specifically precipitated EBV-associated 45,000 MW polypeptide from EBR. Lanes: A, EBVinfected RAMOS + 17D6; B, EBV-infected RAMOS + control; C, RAMOS + 17D6; D, RAMOS + control.

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formed state. An antigen that stimulates cellular immunity does not have to be virally encoded, only presented at the cell surface in such a way that it be seen as foreign by the immune response. This could occur through structural alteration of cell surface antigens, unmasking of buried antigens, or expression of new antigens, such as oncofetal antigens. In order to test whether an antigen is the target of a cellmediated immune response, it will be necessary to demonstrate that an antibody against this antigen will block the response. It will also be important to isolate the antigen and attempt to use it as a target for cell-mediated immunity when reconstituted in liposomes (Almeida et al., 1975; Manesis et al., 1979; Six et al., 1980) or in detergent-free protein micelles (Helenius and von Bonsdorff, 1976; Simons et al., 1978; Morein et al., 1979). Last, attempts should be made to stimulate cell-mediated immunity against EBV-transformed cells in uiuo with such reconstituted materials. The corollary to this will be to study the expression of such antigens on infected cells in the peripheral blood of acute IM patients, patients with chronic or fatal EBV-associated proliferative diseases, and on the tumor cells of BL and NPC patients. Such studies should allow us to define the normal cellular immune response during IM and what is wrong with cell-mediated immunity in the EBV-associated neoplasms. Finally, the production of immunologically active liposomes or protein micelles could prove effective in stimulating the immune response to EBV-transformed cells in such patients. An EBV antigen that is able to maintain the transformed state, like middle T of polyoma, will presumably have to be encoded by the viral DNA, specifically by a gene coding for one of the mRNAs present in EBV-transformed cells. Continuing the analogy with middle T, one function that could readily be tested with such a molecule would be whether it is phosphorylated and possesses kinase activity (Smith et al., 1979; Eckhart et al., 1979; Schafiausen and Benjamin, 1979).

Ill. Early Antigens

A. INTRACELLULAR EARLYANTIGENS

1. Introduction Early antigens (EA) were discovered as a consequence of a discrepancy found in the number of cells that would stain in a virus-producer cell line when EBV-positive sera from different sources were used (W.

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Henle et al., 1970a). In particular, a larger number of cells were stained when sera from BL, NPC, or acute-phase IM patients were compared to sera from normal healthy seropositive individuals. This serological definition of two types of antigens was given a biological basis when it was noted that the expression of the antigen recognized b y all positive sera was sensitive to inhibitors of DNA synthesis (Gergely et al., 1971b).The new antigen, recognized by BL, NPC, and acute IM sera, was insensitive and, therefore, termed early antigen (EA). Thus, one may obtain EA-, VCA+ (healthy seropositive individual) and EA+, VCA+ (BL, NPC, or acute IM patient) human sera, but not EA+, VCA- sera. More detailed studies have shown that EA can be resolved into two components on a serological basis ( G . Henle et al., 1971a). Sera from acute-phase IM and NPC patients generally give a stain that is diffuse over the entire cell and is resistant to methanol fixation. This antigen has been termed the diffuse form of EA, or EA-D. Sera from BL patients, on the other hand, tend to give a stain that is localized in the cytoplasm and is methanol sensitive. This antigen has been termed the restricted form of EA, or EA-R. The association of antibodies to the R and D forms with different disease states is not a hard and fast rule, but holds in general. Thus, it is unusual, for example, for IM patients to develop anti-EA-R antibodies (Horwitz et al., 1975).The observation that anti-EA antibodies are present only in the tumor-associated disease states and in the acute phase of IM has proved to be very useful in diagnostic studies. Thus, the presence of anti-VCA (IgM and IgG) and transient anti-EA-D antibodies with the absence of anti-EBNA antibodies (Section II,A,l) is serodiagnostic of a primary EBV infection; with or without clinical symptoms of IM (W. Henle et al., 1974; W. Henle and Henle, 1975). Declining levels of anti-EA and VCA (IgM)antibodies are indicative ofthe convalescent phase of IM. Stable levels of anti-VCA (IgG) and EBNA antibodies in the absence of anti-EA antibodies are indicative of an established latent infection. The reappearance of anti-EA antibodies is generally thought to be indicative of reactivation of this latent infection. This can occur as a consequence of immunosuppression caused by neoplasms such as Hodgkins lymphoma (Johansson et al., 1970) or nonmalignant diseases such as sarcoidosis and systemic lupus erythematosus (W. Henle and Henle, 1973) or can be induced by immunosuppressive therapy, for example, in kidney transplant patients (Cheeseman et al., 1980).Similarly, the persistence of anti-EA antibodies, in some individuals, is considered indicative of an ongoing active infection that may nevertheless be subclinical. Such persistent anti-EA antibodies can occur in

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individuals who are genetically impaired in their response to EBV, such as in the XLP syndrome (Sakamoto et al., 1980).Antibodies to the R and D forms of EA have also been extremely useful in diagnostic studies with both BL and NPC. In general, individuals with EBVassociated BL show unusually high antibody titers to EA-R (G. Henle et al., 1971a; W. Henle and Henle, 1977), often higher than anti-VCA titers. On the other hand, antibodies to the EA-D component are, by comparison, low or absent. The level of antibodies against EA-R may also be used, to some extent, to predict the subsequent course of the disease (G. Henle et al., 1971b; W. Henle et al., 1973a,b; Nkrumah et al., 1976). Thus, a drop in the titer (for example, due to treatment) is indicative of a good prognosis, whereas an increase in titer often predicts a fatal relapse. With NPC the predominant anti-EA antibody is against the D component, although antibodies against the R component are also frequently found (G. Henle et al., 1971a; W. Henle et al., 1973b). As with BL, the antibody titers to all antigens, including EA, increase with the tumor burden (Ho, 1971; G. Henle et al., 1974). However, patients who respond well to treatment and who go into long-term remission show a steady decline in antibodies to EA-D, often to an undetectable level. A plateauing or rise in titers is indicative, in some cases, of subsequent relapse or metastases. The study of antibody profiles to the EA components, together with those against VCA (see Section IV,A) have, thus, been extremely useful in serodiagnostic studies on EBV and its relationship to human disease. However, nothing is known, to date, about the functions of the serologically defined antigens in the molecular biology of EBV and whether these viral functions in any way relate to the serodiagnostic correlations discussed above. For example, two intriguing questions are: (a)Why are anti-EA antibodies present only transiently during IM, whereas anti-VCA antibodies persist? Does this mean that a different kind of productive infection is occurring (qualitatively rather than quantitatively) during the acute phase, compared to the convalescent and healthy phases? (b) Does the predominance of the anti-EA-R antibodies in BL, but EA-D in IM and NPC, relate to a different kind of infection process in these diseases? For example, is it related to the site of the primary center of replicative infection, which is probably the lymphocytes of the BL tumor, but the epithelial cells in acute IM and NPC?

2. Biochemical Characterization of EAs Several laboratories have attempted to identify the early antigen polypeptides using a variety of systems (Bayliss and Nonoyama, 1978;

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Kallin et al., 1979; Mueller-Lantzsch et al., 1979; Bodemer et al., 1980; Feighny et al., 1980a, 1981; Kawanishi et al., 1981; Bayliss and Wolf, 1981). Most of these studies have made use of the observation that the P3HR-1 strain of virus will superinfect RAJI cells, causing a large percentage of those cells to express both R and D forms of the early antigen (W. Henle et al., 1970a).The development of late antigens can be prevented by superinfecting in the presence of inhibitors of DNA synthesis, such as phosphonoacetic acid (PAA) [virus specific (Summers and Klein, 1976)] and arabinoside C (Ara C) (Gergely et al., 1971b). Although this is the best system to date for obtaining a high percentage of EA-positive cells, it also has severe drawbacks. Most notably, superinfection is not a property of wild-type virus, but it is a unique property of the P3HR-1 laboratory strain. Furthermore, it is known that superinfection is primarily an abortive lytic process with accumulation of defective viral DNA (Lee et al., 1977), and thus, minor polypeptides may also accumulate in deceptively large amounts. Despite this, superinfection is the best choice for initial experiments, although it is important to try to extend findings to more typical systems, particularly the EBV-producing cells, without chemical inducers where possible. In many cases such complete studies may prove to be extremely difficult because of the small number of EA-positive cells in these cultures. Most studies have used EA+ human sera to immunoprecipitate proteins from superinfected RAJI cells metabolically labeled with [35S]methionine in the presence of PAA. A variety of other systems have also been used, including direct analysis of superinfected RAJI cells over time with and without immunoprecipitation, and immunoprecipitation from virus-producing cell lines induced by a variety of chemicals, such as n-butyrate, IUdR, and 12-0-tetradecanoy1phorbo1-13-acetate (TPA), in the presence of various inhibitors of DNA synthesis. A large number of human sera from different sources (IM, BL, NPC, seropositive, and seronegative healthy individuals) have been used in the immunoprecipitation experiments. The results of studies conducted by six different groups are presented in Table I. The main conclusion that may be drawn from these studies is that early antigens are too complex to be satisfactorily studied with reagents of such unreliable specificity as human sera. One may find polypeptides of almost any molecular weight between 16,000 and 165,000, and the variability in gel systems makes comparison difficult. Nevertheless, careful analysis of the data reveals some striking similarities in the results, and the following general conclusions may be

SUMMARY OF

EARLYANTIGEN

TABLE I DEFINEDBY DIFFERENT INVESTIGATORS'

COMPONENTS

~~

Investigators Bayliss and Nonoyama (1978) Bayliss and Wolf, 1981 Bodemer et al. (1980) Feighny et al. (1%) Feighny el al. (1981) Kallin et ol. (1979) Kawanishi et al. (1981)d

Mueller-Lantzsch et ol. (1979, 1980)

Source of antigen'

DNA inhibitofl

Immunoprecipitation performed

Polypephdes (MW x 1 0 . ' ) -

138

-

110

90

80

70

165 155

-

100 100 100 103

-

-

-

138

130 125/122 125 134

-

-

155 140 140 152

90

79

65

51

3 0 37 33

+

-

140

125

120

90

85

80

66

50

40

+

-

120

85

-

-

-

-

3 5 -

-

s-RAJI

PAA, Ara C

s-RAJI s-RAJI

PAA ACV.PAA,AraC ACV,PAA,AraC Ara C

+

+

PM

AnC

+ s-RAJI BP3HR-1 S-FL4Jl I-P3HR-1 B-P3HR-1 S-NC37 T-P3HR-I T-B95-8

}

~~

-

+

-

-

-

7

-

63

5

45

35

-

5 5 4 5 3 0 6 2 6 8 3 0 -

38

31

-

-

-

2 6 2 6 31

-

-

35/30

17

-

18/16

Comparative alignments were made on the hasis of relative intensity and mobility on SDS-PAGE. BP3HR-I. n-butyrate-induced P3HR-1; I-P3HR-1, IUdR-induced P3HR-1; T-P3HR-l, TPA-induced P3HR-1; T-B95-8, TPA-induced 8958. TPA, 12-0-tetradecanoyl~rbol-lsacetate. PAA, Phosphonoacetic acid; ACV, acyclovir: Ara C, anbinoside C. Some minor components in the 50,000 to 70,000 MW region described in this study are not included. a

'S-RAIL Superinfected RAJl; S-NC37, superinfected NC37;

3 14

D A V I D A. THORLEY-LAWSON E T AL.

drawn on the basis of the relative intensity and mobility of the polypeptides seen on SDS-PAGE in the various studies.

1. Five major polypeptides are reproducibly seen with approximate molecular weights (k 10%)of 145,000, 125,000, 95,000, 35,000, and 30,000. 2. In addition, at least 8 other major components are seen with variable frequency. In particular, polypeptides of approximate molecular weights (k10%)of 90,000, 80,000, 60,000, 50,000, 34,000, and 17,000 are prominent. 3. There is coordinate synthesis of the EBV-associated polypeptides somewhat analogous to that found with herpes simplex (Honess and Roizman, 1974). There is an initial synthesis of some early polypeptides. Termination of their synthesis occurs concomittant with synthesis of a different and later occurring group of early polypeptides followed by the synthesis of late, i.e., post DNA synthesis polypeptides (Bayliss and Wolf, 1981, Feighny et al., 1981). 4. Early polypeptides are probably synthesized that are not recognized by human sera, and there are early polypeptides that are recognized by both EA', VCA' and EA-, VCA- sera. 5. Not all human sera recognize the same components; for example, BL and IM sera from patients at different disease stages tend to precipitate different polypeptides. 6. The 145,000 and 125,000 MW proteins are present in both cytoplasmic and nuclear subcellular fractions, whereas the 95,000,35,000, and 30,000 MW proteins are detected only in the cytoplasmic fraction (Feighny et al., 1980a; Kawanishi et al., 1981). The subcellular locations of the other components have not been consistently assigned. Thus, it is premature to conclude which components comprise the EA-D and -R components. It may be speculated, however, that the 145,000 and 125,000 MW polypeptides may be part of the EA-D (cytoplasmic and nuclear) and the 95,000, 35,000, and 30,000 MW polypeptides may be part of EA-R (cytoplasm only). It should be stressed, however, that in all the studies where immunoprecipitation was performed correlation was made on the basis of direct and indirect immunofluorescence and immunoprecipitation with protein A-binding antibodies from [35S]methionine-labeledcells. Clearly, at the sensitive level of analysis afforded by immunoprecipitation, some polypeptides will be missed either because they are poor in methionine or because antibodies against them do not bind protein A. Now that the preliminary work has been completed, indicating that early polypeptides may be identified by immunoprecipitation, there is

EPSTEIN-BARR VIRUS ANTIGENS

315

a need for polypeptide-specific antibodies, so that the components may be purified and analyzed individually in detail (see Section V,A). In particular, from the clinical point of view, it would seem necessary to begin developing experimental approaches designed specifically to identify which polypeptides comprise the R and D components. For this purpose, monoclonal antibodies again seem indispensable. It should be possible to develop immunization protocols that favor the production of hybridomas secreting anti-EA antibodies and screen such hybridomas by fluorescence tests on RAJI cells, superinfected in the presence of PAA and fixed with and without methanol. Apart from these studies very little work has been done on the biochemical characteristics of EA, and only preliminary reports of purification procedures have been presented. I n one report, it was suggested that nonionic detergents were required for complete solubilization of the EA-D component (Ogburn and Zajac, 1977), implying that it may be membrane associated. The solubilized antigen would bind to DEAE-cellulose and eluted with low salt. In another report, EA activity was shown to migrate with a molecular weight of 150,000 in gel filtration experiments (Lenoir et al., 1977). Similar results have been published by a third group (Veltri et al., 1976; Wainwright and Veltri, 1977). Whether this is equivalent to the 145,000 MW protein seen by most investigators in their immunoprecipitates remains to be seen. What this study suggests, however, is that the EA seen b y immunofluorescence is not a massive complex of all the early proteins, but may consist of only a few of the proteins seen by immunoprecipitation. The resolution of this point will have to await clear-cut experiments showing that immunofluorescence may be blocked with purified polypeptides.

3. Functions of Early Antigens It is thought that cells that express EAs are committed to die and usually replicate viral DNA and release infectious virus (Gergely et al., 1971a). The biological roles for EAs would seem, therefore, most likely to be involved in the activation of the viral genome in the stably transformed cell, perhaps, with concomitant shutdown of host-cell functions. Although this shutdown happens in the superinfected RAJI system, little is known about what occurs in the virus-producer cell lines, which presumably represent the most normal mechanism of virus production. It may also be speculated that early antigens could be involved in the replication of viral DNA and even be involved in

316

DAVID A. THORLEY-LAWSON E T AL.

the initial assembly of the virions themselves. For these reasons, it seems reasonable to look for activities associated with activation, replication, and packaging of viral DNA. a. DNA-Binding Proteins. One study has looked at the ability of early polypeptides to bind to DNA-cellulose and at least four such proteins have been identified with molecular weights of 152,000, 134,000, 55,000, and 51,000. These proteins were shown to be EBVspecific by immunoprecipitations with EA+ sera, and three of them correspond to polypeptides previously defined as early antigens by this group. The DNA-binding proteins include DNA polymerase and DNase activity. However, no specific correlation was shown between these activities and the immunoprecipitated polypeptides (Roubal et al., 1981). b. DNA Polymerase. Various kinds of EBV-associated DNA polymerase have been identified. Four groups have independently presented evidence for an EBV-associated DNA polymerase in EBVproducing lymphoblastoid cell lines. The activity may be detected in producer cell lines such as P3HR-1 and B95-8 (Goodman et al., 1978), P3HR-1 cells induced with TPA (Datta et al., 1980) or IUdR (Feighny et al., 1980b), superinfected RAJI cells (Ooka et al., 1979), and IUdRinduced D98/HR-1 hybrid cells (R. Miller et al., 1977). The criterion for EBV specificity is that the activity is not detected in either EBVnegative or EBV-positive nonproducer cell lines. The increase in polymerase activity upon induction or superinfection follows temporally the rise in the number of EA-positive cells (Ooka et al., 1979; Feighny et at., 1980b). Despite the variety of systems used, the polymerase activities described by all groups, except Goodman et al. (1978), are remarkably similar. Thus, the enzyme has many properties in common with the better-characterized virus-specific DNA polymerase of other herpesviruses. The enzyme is insensitive to 50-100 mM (NHJ2S04, and stimulation of enzyme activity by this salt treatment varying between two- and sixfold has been reported. The enzyme is relatively insensitive to PAA compared to the host cell polymerases, although the sensitivity is close to that of the a polymerase. The viral polymerase has also been shown by one group (Feighny et al., 1980b; Datta et al., 1980) to be more sensitive to acyclovir than the cellular polymerase. The polymerase does not show reverse transcriptase or terminal transferase activity and performs well with synthetic template primers such as poly(dA)-oligo(dT),,, with a sharp pH optimum, reported as 7.5 or 8.5. Several hundredfold partially purified material has been obtained using various combinations of gel filtration, DNA-cellulose, DEAE-cellulose, and phosphocel-

EPSTEIN-BARR VIRUS ANTIGENS

317

lulose chromatography, the latter proving to be particularly effective. However, no pure enzyme is yet available. Goodman et al. (1978) have reported two distinct polymerase activities, one from EBV-producing cells and one that copurified with virions. The cell-associated polymerase has properties somewhat like those described b y the other authors, but sufficiently at variance to suggest that the enzyme may have been isolated in an altered form. In particular, the enzyme was not especially sensitive to PAA and did not perform well with the poly(dA)-oligo(dT)lotemplate, and, although the activity was resistant to 50 mM (NH&S04, no activation was seen. The virion-associated activity appears to be completely different, behaving more effectively as an endonuclease than a DNA polymerase and having many characteristics of a DNA repair enzyme (Clough and McMahon, 1981).The authors suggest that this enzyme could play an important role in EBV biology, as virion DNA is known to be multiply nicked and would need a repair activity to be completed, upon release from the virion, so as to be available for replication and transcription. This enzyme can also be detected transiently in newly superinfected RAJI cells. c . DNase. An EBV-specific DNase has been detected in superinfected RAJI cells, IUdR-induced D98/HR-1 hybrid cells (Cheng et al., 1980a), and EBV-producing lymphoblastoid cell lines (Clough, 1979). The specificity of the enzyme was shown by its unique electrophoretic mobility and, most interestingly, in one study by its ability to be specifically inhibited by one EBV-positive serum. In a more extended study (Cheng et al., 1980b), it was demonstrated that anti-DNase antibody activity was present in most EBV seropositive individuals, particularly in NPC patients (46 of 49). No correlation was found between the anti-DNase antibody titers and the titers of antibody to the EA-D and EA-R components or any other viral antigens. d . Other Enzymes. Other enzyme activities associated with EBV include an endonuclease in virus-producing cells (Clough, 1980) a ribonucleotide reductase in superinfected RAJI and IUdR-treated D98/HR-1 cells (Henry et al., 1978), and thymidine kinase in superinfected RAJI and EBV-producing cells (Chen et al., 1978). More recent evidence, however, from the same laboratory suggests that there is no detectable thymidine kinase (A. Datta and J. Pagano, personal communication). However, another laboratory reports independent corroboration of the existence of an EBV-associated thymidine kinase (W. Clough, personal communication). The final judgment as to the existence of this enzyme will remain moot until more experiments have been performed.

318

DAVID A. THORLEY-LAWSON E T AL.

B. EARLYMEMBRANEANTIGENS

1. Introduction Early membrane antigen (EMA) has been detected on the plasma membrane of BL biopsies and EBV-producing cell lines (Klein et al., 1966; Yata and Klein, 1969; Yata et al., 1970). It is not always clear in many early studies whether LMAs or EMAs were being studied, as the LMAs constitute the majority of membrane antigens on superinfected RAJI cells and EBV-producing cell lines (see Section IV,B,2). It is, nevertheless, clear that antigens may be detected by fluorescence on superinfected cells and EBV-producer cells after Ara C or PAA treatment. This residual activity is antigenically distinct from the LMA as shown b y absorption studies (Ernberg et al., 1974; Sairenji et al.,

1977). 2. Biochemical Characteristics of EMA There is some evidence from immunoprecipitation studies to suggest the existence of at least one EMA-associated polypeptide. Thus, Mueller-Lantzsch et al. (1980) have reported that the plasma membranes of superinfected NC-37 cells contain an Ara C-insensitive component of 90,000 MW, which can be iodinated by lactoperoxidase. This polypeptide is probably the same as a 90,000 MW polypeptide detected by Kallin et al. (1979) and a 95,000 MW PAA-resistant component recognized by a monoclonal antibody developed b y our group. The specificity of the monoclonal antibody for EBV could be shown by immunofluorescence and by immunoelectron microscopy using peroxidase-coupled rabbit anti-mouse Ig as the second antibody. The most intense stain is seen on cells that contain internal virions. The 95,000 MW component copurifies with virions and may be immunoprecipitated from iodinated detergent extracts of the virus (ThorleyLawson, 1982).

3. Function of EMA The EMA component that we and others have defined (see preceding section) is probably a structural component of the virion. It is not clear what other functions it may perform. However, it could be very useful to the host as a marker on cells in the early stages of the lytic cycle. Thus, a cytotoxic immune response to such an antigen, mediated by antibody and complement or antibody-dependent cellular cytotoxicity (ADCC), would destroy the cell before it could make and release infectious virus. It is known that anti-MA antibodies will medi-

EPSTEIN-BARR VIRUS ANTIGENS

319

ate ADCC against superinfected RAJI cells (Pearson and Orr, 1976); however, it is not clear as yet whether EMA or LMA is the target antigen. Although such a response would not destroy transformed cells, it could certainly play a role in reducing or preventing the release and spread of infectious virus. Early MA could also serve as a target for natural killer (NK) cells. It has been demonstrated (Blazer et al., 1980) that NK cells will kill superinfected RAJI and n-butyrate-induced P3HR-1 cells. The antigen recognized by the NK cells is not inhibited by Ara C and must, therefore, be an early membrane antigen component. The biochemical characteristics of the antigens recognized by ADCC and NK cells are as yet unknown.

IV. Late Antigens

A. INTRODUCTION Late antigens are defined as being sensitive to inhibitors of DNA synthesis, including the general inhibitor Ara C and the virus-specific inhibitor PAA (Summers and Klein, 1976). These antigens include both viral capsid (W. Henle et al., 1966; Mayyasi et al., 1967; Silvestre e t al., 1971) and viral envelope (membrane) antigens (Sugawara and Osato, 1970; Silvestre et al., 1971) and may also include nonstructural polypeptides of the virus. Studies with monoclonal antibodies against EBV structural components have revealed that the original techniques used to describe these antigens may be misleading (Thorley-Lawson and Geilinger, 1980). The cytoplasmic stain obtained with EBV-positive sera on EBV-producing cells (G. Henle and Henle, 1966a,b) is the classic test for the presence of viral capsid antigen (VCA). However, both viral envelope (membrane antigens) and capsid (capsid antigen) components are present in the cytoplasm of the cells (see Section IV7B72).This is particularly important, because almost all of the seroepidemiology and serodiagnosis of E BV infection involving the study of antibodies to late antigens is by this staining technique. Workers within and outside of EBV research should b e made aware that in such studies the anti-VCA antibody titer does not differentiate between antibodies to viral capsid components, envelope (membrane) components present in the cytoplasm and on the cytoplasmic membranes, early antigens that may also be expressed late, and nonstructural late polypeptides. The definition of the exact polypeptide target

320

D A V I D A. THORLEY-LAWSON ET AL.

of an antibody detected in serum is crucial for the correct evaluation of the role of antibody responses in the control of EBV infection, a cornerstone to the rationale for developing a subunit EBV vaccine. This is because antibodies to true capsid components are probably irrelevant in controlling infectious virus, but antibodies to envelope (i-e.,membrane antigen) components may prevent the spread of virus and thus prevent the infection of a vaccinated individual. In the discussion below, therefore, references to antibody titers against VCA should be read as the staining of the cytoplasm of EBV-producing cells, not as being indicative of anti-capsid activity per se. The study of antibody responses to late antigens, together with EBNA and EA, has proved to be extremely useful in investigations of the seroepidemiology and serodiagnosis of EBV-associated diseases. The original observation that sera from EBV-infected individuals would stain the cytoplasm of BL-derived cell lines (G. Henle and Henle, 1966a,b) and that there was no serological cross-reactivity with other herpesviruses was the first evidence indicating that EBV was a distinct virus. It was subsequently shown that the VCA, defined by fluorescence, was only expressed in B cells actively replicating EBV as defined b y the presence of intracellular virions (zur Hausen et al., 1967; Epstein and Achong, 1968). Using the classic fluorescence test for VCA it has been possible to detect anti-EBV antibodies and, therefore, infection in virtually every individual in every geographic location studied (Levy and Henle, 1966; Tischendorf et al., 1970; Black et al., 1970; Lang et aZ., 1977). However, the age of infection was found to vary considerably, 3 years of age in primitive or overcrowded societies, but delayed until adolescence or early adult ages in the higher socioeconomic groups of Western societies (G. Henle et aZ., 1968; Porter et al., 1969; Hinuma et al., 1969; Niederman et al., 1970; G. Henle and Henle, 1970). By following the anti-VCA antibody titers in patients, convincing evidence was obtained for a causative role for EBV in I M (G. Henle et al., 1968).In a prospective study of students at Yale University, all pre-IM sera were found to have no antibodies against VCA, whereas consecutive acute-phase and convalescent sera all showed persistent antibodies to VCA (Niederman et al., 1968). The presence of transient IgM and long-term IgG antibodies to VCA, combined with transient anti-EA-D (Section III,A,l), is now taken to be indicative of a current primary EBV infection (W. Henle et al., 1974; W. Henle and Henle, 1975). In the case of Burkitt’s lymphoma, individuals with BL have geometric mean antibody titers to VCA, 8- 10 times higher than those of control sera (Levy and Henle, 1966; G. Henle et al., 1969; Kafuko et

EPSTEIN-BARR VIRUS ANTIGENS

32 1

al., 1972). More recently, in a prospective study in Uganda, it was shown that individuals who later develop BL have unusually elevated antibody titers to VCA, months and even years before onset of clinically detected tumors (de-Th&et al., 1979). With nasopharyngeal carcinoma the picture is similar; that is, individuals with the tumor have geometric mean antibody titers 8-10 times higher than normal controls (W. Henle et al., 1973b; de-The et al., 1975; G. Henle and Henle, 1976). One unique feature of NPC is the presence of IgA antibodies to VCA (G. Henle and Henle, 1976). The detection of such antibodies has been useful as a diagnostic tool for the discovery of otherwise undetected tumors (Li et al., 1980).

B. MEMBRANEANTIGENS 1. Introduction As discussed in Section III,B,l, it is not always clear to what extent

early studies on membrane antigen were of the early (EMA) or late (LMA) forms. Membrane antigen was first discovered in freshly cultured BL biopsy material (Klein et al., 1966) and is expressed on the surface of EBV-producing cells in cultured cell lines. Generally, a low percentage of the cells in such cultures are producing EBV. Suggestive evidence has been developed that membrane antigens are also expressed on the viral envelope and include the molecules responsible for generating virus-neutralizing antibodies. Thus it was demonstrated by immunoferritin labeling that MA+ sera would stain the envelope of virions (Silvestre et al., 1971, 1974) and that there was a correlation between antibody titers to MA and neutralizing antibody titers (Pearson et al., 1970; DeSchryveret al., 1974). In further serological studies, it was suggested that MA had at least three distinct antigenic specificities independent of the LMA and EMA divisions (Svedmyr et al., 1970). Last, it has been demonstrated that antibodies to MA are capable of mediating ADCC against MA-positive superinfected RAJI cells (Pearson and Orr, 1976).Although the role of such an activity in vivo is unclear, it was possible to demonstrate that, in patients with NPC and BL who responded well to therapy and survived more than 2 years, the geometric mean titer of ADCC antibody was significantly higher than in individuals who died within 2 years of therapy (Pearson et al., 1978, 1979). Also indicative of a role for antiMA antibodies in the control of EBV-associated tumors, is the finding that anti-MA antibodies often drop suddenly several months prior to fatal relapse in BL (Gunven et al., 1974). These observations, together

322

DAVID A. THORLEY-LAWSON ET AL.

with the ability of viral envelope antigens to stimulate neutralizing antibodies, make the study of membrane antigens critical to our understanding of the immunological control of EBV infection, including the possibility of developing a subunit vaccine. In addition, the existence of EBV-specific MAS provides a system for beginning the study of the structure and assembly of the virion.

2. Biochemical Characteristics of MA Several early attempts to isolate and characterize the components of the MA complex were unsuccessful, leading some authors to speculate that the antigen was due to capsid antigen on the cell surface (Boone et al., 1973; Dolken and Klein, 1977).Our approach to the isolation and identification of MA was to produce a high-titered rabbit antiserum against purified virions (Thorley-Lawson, 1979a). The antiserum was made specific for late antigens by absorption with PAA-treated cells of the same type as those used to produce the virus (B95-8). This serum had the following properties.

1. It neutralized EBV infection in uitro. 2. The neutralizing antibodies could b e absorbed by EBVproducing, but not b y nonproducing, cell lines, demonstrating that the molecules responsible for generating neutralizing antibodies were expressed on the plasma membranes of EBV-producing cell lines. 3. It gave membrane immunofluorescence (MIF) with a small population of cells in EBV-producing cultures. Separation of the MIFpositive cells revealed that they were the ones actively replicating virus, as judged by the VCA stain using a human serum. 4. The membrane antigens on intact cells could be identified by means of a quantitative two-step binding radioimmunoassay using '251-labeledprotein A as the second step. Inhibition of this assay could be used as a quantitative measure for the isolation of membrane antigens. Using the protein A assay it was possible to demonstrate, for the first time, the presence of EBV-associated membrane antigens on highly purified plasma membranes (Thorley-Lawson and Edson, 1979).The membranes were made by N2 cavitation of EBV-producing lymphoblastoid cell lines, followed by differential centrifugation to obtain crude plasma membranes and sucrose density centrifugation to produce purified plasma membranes. Using the assay, it was possible to follow the purification of the membrane antigen through a series of steps by which (a) the antigenic activity could be effectively solubilized by a range of detergents, including n-octyl glucoside, Triton

EPSTEIN-BARR VIRUS ANTIGENS

323

X-100, and sodium deoxycholate; (b) the antigenic activity would bind specifically to ricin and lentil lectin affinity columns. Thus it was possible to obtain a partially purified detergentsolubilized extract of plasma membrane antigens from EBV-producing cell lines. Most critically, this material possessed antigenic activity, as it was capable of generating high-titered neutralizing antibodies when injected in uivo (Thorley-Lawson, 1979b). Pearson and Qualtiere (1978) had previously been able to show that antigenically active MA could be solubilized from intact cells by papain digestion. Analysis by immunoprecipitation of detergent-solubilized material from [35S]methionine-labeled, purified plasma membranes revealed that the membrane antigen consisted of four polypeptides with molecular weights of 350,000,220,000, 140,000, and 85,000 (Fig. 4). These polypeptides were precipitated from all of 14 EBVproducing cell lines tested (Thorley-Lawson and Edson, 1979; Edson and Thorley-Lawson, 1981) and from superinfected RAJI cells. Their production could be increased by treatment of the cells with TPA and n-butyrate (P3HR-1 only) and was decreased by treatment with PAA with concomitant changes in the number of virus-producing cells. These polypeptides could not be precipitated from a wide range of EBV-positive nonproducer cell lines or EBV-negative cell lines. Thus, they are EBV-specific late polypeptides. Using three independent methods, it was possible to demonstrate that the 350,000,220,000, and 85,000 MW polypeptides, but not the 140,000 MW polypeptide, were glycoproteins (Edson and Thorley-Lawson, 1981)because (a) the 350,000,220,000, and 85,000, but not the 140,000, MW proteins bound to ricin and lentil lectin affinity columns; (b) the 350,000,220,000, and 85,000, but not the 140,000, MW proteins could be labeled metabolically with [3H]glucosamine and [3H]mannose; (c) the 350,000, 220,000, and 85,000, but not the 140,000, MW proteins were sensitive to tunicamycin, an inhibitor ofN-asparagine-linked glycosylation (Kuo and Lampen, 1974; Schwarz et al., 1976). For these reasons we have adopted the terminology gp350, gp220, p140, and gp85 to describe the four major components of the EBV-MA. In subcellular fractionation studies it was possible to demonstrate that all the components of the membrane antigen are present on the membranes throughout the cell, with the possible exception of the gp85 polypeptide, which is poorly expressed in the nucleus (Edson and Thorley-Lawson, 1981).As expected, none of the glycoproteins are found in the cytosol, although the p140 is found there. Interestingly, although p140 is found both in membrane-bound and free forms in the cell, it is the membrane antigen component requiring the most strin-

324

DAVID A. THORLEY-LAWSON ET AL.

FIG.4. Immunoprecipitation (4% SDS-PAGE) of Triton X-100-soluble Epstein-Barr (EBV) antigens from ["Slmethionine-labeled M-ABA (marmoset) and J-ABA (human) cells transformed with the same EBV isolate. The samples were run on the same gel and are shown side by side for clarity. These cells were induced by 12-0-tetradecanoylphorbol-13-acetate. M-ABA cells plus (A) preimmune or (B) immune serum; J-ABA cells plus (C) preimmune or (D) immune serum. Reprinted from Edson and Thorley-Lawson (1981) by courtesy of the publishers.

EPSTEIN-BARR VIRUS ANTIGENS

325

gent extraction procedures, high concentrations of sodium deoxycholate at room temperature for several hours, to be completely solubilized, although partial extraction may be obtained with milder procedures (D. Shalloway and D. A. Thorley-Lawson, unpublished observation). Last, we have been able to demonstrate that these four proteins are the major constituents of [3sS]methionine-labeled virions, purified according to the method described by Dolyniuck et al. (1976a,b). They are virtually the only detectable proteins solubilized from such virions with detergents and may be immunoprecipitated by the rabbit anti-EBV serum (Edson and Thorley-Lawson, 1981). Thus, in summary, we may conclude that gp350, gp220, p140, and, p85 are truly membrane components in the plasma membrane of EBV-producing cells and the envelopes of virions because (a) they copurify with plasma membranes and viral envelopes; (b) they require detergents to be solubilized from both plasma membranes and virions; (c) at least three of the proteins are glycoproteins; (d) at least three of the proteins (the glycoproteins) may be labeled in intact cells by lactoperoxidase and "'I (D. A. Thorley-Lawson, unpublished observations). In addition to the four major components, we have also detected several other components, including a glycoprotein of molecular weight 115,000 (gp115) in producer cells (Fig. 4) and a component of molecular weight 56,000 (p56), which is most readily detected in virions, Furthermore, we have detected minor glycoprotein components with molecular weights in the range of 120,000-190,000 that are precursors of the two large glycoproteins (Edson and Thorley-Lawson, 1982).A typical immunoprecipitation pattern from whole cell lysates is presented in Fig. 4, and a summary of the components that we have defined and of their properties is presented in Table 11. We have performed parallel studies using [3H]lysine to investigate the possibility that there may be components present in our preparations that would not be detected because they lack, or are poor in, methionine. Essentially the same results were obtained as with [35S]methionine-no new polypeptides were detected. Although no other laboratories have attempted the isolation of membrane antigens, several have performed radioimmunoprecipitation studies using human anti-MA sera. They have used a variety of systems including superinfected RAJI and NC37 cells (Qualtiere and Pearson, 1979; Strnad et al., 1979; Mueller-Lantzsch et al., 1980), TPA-induced EBV-producer cells (Strnad et al., 1979; MuellerLantzsch et al., 1980; North et al., 1980; Qualtiere and Pearson, 1980), n-butyrate-treated PSHR-1 (Kallin et al., 1979) and isolated virions

TABLE I1 PROPERTIES OF THE [35S]METHIONINE-LABELEDPROTEINS IMMUNOPRECIPITATED

BY THE RABBIT

ANTI-EBV SERUM

ProMWa

posed nomenclature*

350,000 gp350 220,000

160,OOO

gp220 p160

140,000 p140 115,000 gp115

85,000 56,000

gp85 (g)p56

Distribution Physical properties‘ Contains N- and 0-linked carbohydrate, soluble in TX Same as gp350 Unglycosylated, poorly labeled by r3H]lysine Unglycosylated, soluble in DOC Contains N-linked carbohydrate, soluble in TX Same as a115 Soluble in TX; probably not glycosylated

In cellsd

In virus

Comments

PM, ER, M, N

Envelope

Slightly lower MW in human cells

PM, ER, M, N

Envelope Capsid

Same as gp350 Appears to occur in TX-soluble (“immature”) and refractory (“mature”) forms Relatively insoluble in TX Not always seen in cells; not detected in virus Poorly represented in nuclear fraction Not always seen in cells

CS, N (plus membranes?) PM, ER, M, N, CS ?

Envelope ?

PM, ER, N, (N?) ?

Envelope Envelope

The molecular weights given are for the marmoset cell lines B95-8, M-ABA, and W-91. In human producer cell lines, there are only two differences: gp350 and gp220 are gp320 and gp200, respectively. * gp, Glycoprotein; p, protein. ‘TX, Triton X-100; DOC, deoxycholate. PM, Plasma membrane; ER, endoplasmic reticulum; M, mitochondria; N, nucleus; CS, cytoplasmic supernatant.

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(Mueller-Lantzsch et al., 1980; North et aZ., 1980).The experimenters have used cell surface labeling procedures such as lactoperoxidasecatalyzed iodination to label protein (Qualtiere and Pearson, 1979; Mueller-Lantzsch et al., 1980; North et ol., 1980) and galactose oxidase-catalyzed tritiation to label glycoproteins (Strnad et aZ., 1979; Qualtiere and Pearson, 1980). Glycoproteins have also been identified by means of metabolic incorporation of radioactive sugars (Kallin et al., 1979; Qualtiere and Pearson, 1980; Hoffman et al., 1980). Others have looked at the polypeptides in the envelopes of virions purified by density gradient centrifugation (Dolyniuck et al., 1976a,b; MuellerLantzsch et al., 1980) or by affinity-binding to RAJI cells followed by labeling with radioactive iodine (North et al., 1980). Given the wide variety of labeling techniques used, all of which are to some degree selective, and the large number of different human sera, it is not surprising that the results from different groups are somewhat at variance. Further confusion is added by the lack of adequate molecular weight markers in the 200,000-400,000 range, leading to inaccuracy in estimating molecular weights in this region. Failure to use appropriate gel systems (5% acrylamide or less) and molecular weight markers (i.e., myosin, 200,000; fibronectin, 220,000; and thyroglobulin large subunit, 330,000) results in a drastic underestimation of the size of the large glycoproteins. Thus, the largest glycoprotein (gp350) has a mobility on SDS-PAGE equal to or slightly slower than the large subunit of thyroglobulin, giving a molecular weight of about 350,000. The second largest glycoprotein (gp220) migrates more slowly than myosin and about at the rate of fibronectin, giving a molecular weight of 220,000. The confusion is further compounded by the observation that gp350 and gp220 have different sizes when synthesized in marmoset compared to human cells (North et ul., 1980; Edson and ThorleyLawson, 1981) (Fig. 4). The molecular weights, described above, are for proteins synthesized in marmoset cells. However, gp350 and gp220 have molecular weights approximately 10% lower when synthesized in human cells. When these factors are taken into account, the results obtained by the various groups are quite consistent with each other and with our findings as summarized in Table 111. Recently, a general nomenclature was agreed upon by most investigators working on EBVLMAs as follows: gp350/300, largest glycoprotein; gp250/200, next largest glycoprotein; p140, unglycosylated protein; gp85, smallest glycoprotein. The format 350/300 and 250/200 was used because of the variability in the molecular weights of the components depending on the cell type used, as discussed above. For the identification of precursor polypeptides, the nomenclature,

TABLE I11 SUMMARY OF LATE MEMBRANEANTICEN COMPONENTS DEFINEDBY DIFFERENTINVESTIGATORS

State of glycosylation; MW x Investigators

Labeling procedure“

Thorley-Lawson and Edson ( 1979)d See text Edson and Thorley-Lawson (1981)d Qualtiere and Pearson (1979, 1980) “2jIlLPO L3H]GLcNAc r3H]BH Mueller-Lantzsch et al. (1980) [L*jI]LPO (1981)’ Kallin et al. (1979) Stmad et al. (1979) S h a d et al. (198l)e North et al. (1980)d Hoffman et nl. (1980)’ Dolyniuk et al. (1976a)’

Cell type

Marmoset Human Marmoset Human Human

[3H]GLcNAc Human L3H]BH Marmoset Human [‘2jI]LPO Marmoset Human [3H]GLcNAc Marmoset Cold Marmoset

Proposed nomenclature

Isolates tested 3 11 B95-8 P3HR-1 S-RAJI S-NC37 P3HR-1 Virions B-P3HR-l T-B95-8 S-RAJI 2 2 B95-8 B95-8

gP

gP

P

gP

gP

350 320 320

220 200

140 140

115 115

85 85 90

300 -

250 250

140

130

-

90 -

340 275 236 236 340 320 250 290

240 236 2 12 212 270 240

140 158

95

90

-

-

-

-

-

-

85 85

275

152

-

90

140

-

85

’

-

350/300 250/200

I, LPO, Lactoperoxidase; GLcNAc, N-acetylglucosamine; BH, sodium borohydride. For abbreviations see Table I, footnote b. Precursor of larger glycoproteins. Rabbit anti-EBV serum used in addition to human sera. Monoclonal antibody used only. No immunoprecipitation performed.

-

(gPY

-

-

160

-

-

-

P

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for example, gp190 pre gp350/300 (see below) was suggested. Hopefully, as other components are better defined they will be designated in a similar fashion. We have studied a large number (14) of independent EBV isolates and have found no consistent or significant variations in the patterns of the MA polypeptides, with two exceptions. First, as mentioned above, gp350/300 and gp250/200 have slightly different molecular weights when synthesized in marmoset compared to human cells. This difference is best illustrated using the human J-ABA and marmoset M-ABA pair of cell lines (Fig. 4), which were derived at the same time with the same EBV isolate. The second case is the B95-8 cell line, which expresses very low levels of gp250/200 (Thorley-Lawson and Edson, 1979; Qualtiere and Pearson, 1980). B95-8, is therefore, a variant in that it is the only virus strain that we have tested with a defective glycoprotein expression. Marmoset and human cell lines transformed in oitro with B95-8 virus are also atypical in their expression of gp250/200 (Edson and Thorley-Lawson, 1981) indicating that the defect is in the viral DNA itself, not in the host cells. Interestingly, this defect must have been acquired by the virus during the establishment of the B95-8 cell line, as the parental isolate, the 833L cell line, is normal. This genetic defect in glycoprotein expression may be related to the observation that B95-8 lacks a small portion of its genome (Raab-Traub et al., 1980). However, the atypical glycoprotein expression is not related to the origin of the B95-8 virus (i.e., I M ) nor to its transforming abilities, as has been suggested (Qualtiere and Pearson, 1980). Although no functional aberrance has been associated with B95-8, the presence of two already characterized defects indicates that care should be taken in drawing general conclusions on the basis of studies with this isolate. Two independent studies, one involving cross-absorption of human sera (Franklin et al., 1981) and one using monoclonal antibodies (Mueller-Lantzsch et al., 1981), suggest that there is minor antigenic variation between the two large glycoproteins of different strains of EBV. However, an insufficient number of strains have been analyzed to conclude whether these differences are significant for the biology or epidemiology of EBV infection. Because of the number of polypeptides in the MA, it would be difficult to isolate each component by standard biochemical techniques. We decided, therefore, to separate them by producing monoclonal antibodies against each component. a. gp35Ol300 and g p 2 5 0 / 2 0 0 . We have derived a series of monoclonal antibodies against EB virions. Of those that will immunoprecipi-

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tate polypeptides, most recognize the two large glycoproteins gp3501 300 ahd gp250/200 (Thorley-Lawson and Geilinger, 1980). Although these hybridomas have been repeatedly subcloned, they will nevertheless precipitate both polypeptides, even in the presence of SDS , which separates the two polypeptides (D. A. Thorley-Lawson, unpublished observation). This means that gp350/300 and gp250/200 share antigenic determinants. Because of the observed cross-reactivity of gp350/300 and gp250/200, we have attempted to discover if they derive from a common precursor (Edson and Thorley-Lawson, 1982). In experiments with cells pulse-labeled for 15 min with [35S]methionine we can identify a 190,000 MW precursor for gp350/300 and a 160,000 MW precursor for gp250/200, which chase into their respective final forms. We can identify these precursors with a specific serum (see below) and can assign them to either gp350/300 or gp250/200 using the B95-8 cell line, that expresses gp350/300, but virtually no gp250/200 (see above). There is no suggestion in these studies that gp250/200 is a precursor of gp350/300 or that they share a common precursor. The antigenic cross-reactivity of these molecules implies that they are structurally related. They may have arisen through a gene duplication or an RNA splicing event. Alternatively, the antigenic specificites may be shared by the unusual carbohydrate structure that both possess (see below). Such conclusions will be resolved by detailed peptide analyses of gp350/300 and gp250/200. A duplication event could explain why B95-8 can function in an apparently normal way with virtually no gp250/200. The carbohydrate present on glycoproteins pulse-labeled for 15 min is of the N-asparagine-linked high-mannose form and can be removed (with the exception of the proximal N-asparagine-linked N-acetylglucosamine) by digestion with endo-8-N-acetylglucosaminidase H (endo H), leaving the precursor polypeptide backbone intact (Robbins et al., 1977). Such studies reveal the polypeptide sizes for gp350/300 and gp250/200 to be 160,000 and 130,000 MW, respectively. This is a startling finding, suggesting that about half of the apparent molecular weight of these glycoproteins is due to carbohydrate. More intriguing still, the molecular weights of these polypeptides in the presence of tunicamycin, which inhibits N-linked, but not 0-linked, glycosylation (Kuo and Lampen, 1974; Schwarz et al., 1976), are 300,000 and 180,000 MW, respectively. This means that there is a discrepancy of 140,000 and 50,000 MW, respectively, between the unglycosylated forms (160,000 and 130,000 MW) and the forms lacking N-linked sugar (300,000 and 180,000 MW), implying that a large amount of 0-linked sugar is present. These results

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TABLE IV MOLECULARWEIGHTAND PROPERTIES OF

THE VARIOUS STAGESOF GLYCOSYLATION OF gp350/300 AND gp250/200

Form Mature Mature without N-linked sugar Immature Immature without sugar

How obtained

sugar linkages

Overnight labeled with [35S]methionine 0 + N 0 Overnight label with [33S]methionine plus tunicamycin Pulse (15 min) label with [3JS]methionine Pulse (15 min) label with [3sSJmethionine followed by endo H treatment

MW x 350/300 300

250/200 180

N

190

160

-

160

130

are summarized in Table IV. The presence of such large amounts of both 0and N-linked carbohydrate on a polypeptide may be unique in herpesviruses and rare among glycoproteins in general. One crucial observation made with the monoclonal antibodies to the two large glycoproteins is that they will give both membrane immunofluorescence on whole cells and a classical VCA-like stain in the cytoplasm of acetone-fixed virus-producing cells (Thorley-Lawson and Geilinger, 1980).This means that classical VCA stain has no value as a test for serum antibodies against viral capsid components if those sera also contain anti-MA antibodies. One of the monoclonal antibodies ( C l ) against the two large glycoproteins will neutralize the virus in vitro (Thorley-Lawson and Geilinger, 1980) and a similar antibody has been described b y Hoffman et al. (1980). We have confirmed the presence of these glycoproteins on the virion b y performing immunoelectron microscopy on E BV-producing cells using the C1 antibody and peroxidase-coupled rabbit anti-mouse Ig (Fig. 5) (Thorley-Lawson and Poodry, 1982). In this experiment C1 antibody stained both the plasma membrane of EBV-producing cells and the envelope of virions. The stain was specific because every cell observed with internal virions stained with the antibody and every cell without internal virions did not stain. No single exception was seen. These studies show in the most convincing way, short of genetic analysis, that gp350/300 and gp250/200 are EBVspecific structural components of the viral envelope, which are also expressed independently (i.e., not in virions) on the plasma membrane of EBV-producing cells. In addition, gp350/300 and gp250/200 are

FIG.5. (A) Electron micrographs of a producer cell in which the gp350/300gp250/200 antigen is revealed using the monoclonal antibody C1 followed by a peroxidaseconjugated rabbit anti-mouse Ig. The cell in the upper right portion of the figure has virus particles in its nucleus and a dense peroxidase reaction product over its surface, whereas the nonproducer cells to the left and lower right have no indication of a positive reaction. The magnification bar is 2 pm. The inset shows a virion and a portion of plasma membrane from a different cell. Both the viral envelope and cell membrane are stained by the immunochemical reaction. The magnification bar in the inset is 0.5 pm. (B) Electron micrograph of an EB virion on the surface of protein A Sepharose beads coated sequentially with rabbit anti-mouse Ig, C1 monoclonal antibody, and EB virions. The bead is represented by the darker region in the lower part of the electron micrograph. Virions were found sparsely but regularly around the perimeter of the C1 beads because the electron micrographs were of ultrathin sections. No virions were found on beads with control antibodies or on C1 beads incubated with virions in the presence of 0.5% Triton X-100or sodium deoxycholate. The magnification bar is 0.5 pm. From ThorleyLawson and Poodry, 1982.

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capable of generating virus-neutralizing antibodies in vivo (i.e., in mice). To purify the molecules, crude plasma membranes are extracted with detergent, and the solubilized glycoproteins are purified b y ricin lectin affinity chromatography. The resulting glycoproteins are then passed sequentially through columns of Sepharose 4B and normal mouse Ig coupled to Sepharose 4B (preclearing steps) and finally over a column of C1 antibody coupled to Sepharose 4B. The material bound to the C1 column is eluted and consists of pure gp3501300 and gp250/ 200. A more crude, but quicker, preparation may be obtained by omitting the preclearing columns. The purified material is antigenically active as it may be immunoprecipitated, and, most important, upon injection into rabbits, it will generate a high-titered virus-neutralizing antibody that specifically immunoprecipitates only gp350/300 and gp250/200. Last, we have used the purified glycoproteins, either in solution or, more effectively, coupled to Sepharose 4B, to absorb human sera. In this manner it is possible to demonstrate that when the anti-gp350/300 and gp250/200 antibodies are absorbed from human sera most of the virus-neutralizing antibodies are also absorbed. This indicates that the two large glycoproteins can generate virusneutralizing antibodies in vivo, both in animals and humans, and is the main component responsible for generating virus-neutralizing antibodies during infection. A summary of these findings is presented schematically in Fig. 6. b. p 1 4 0 . There is some evidence to suggest that p140 may not be exposed on the outer surface of the plasma membrane. First, it is not labeled by lactoperoxidase and 1251,and second, monoclonal antibodies against p140 do not give detectable membrane fluorescence (Mueller-Lantzsch et aZ., 1981; D. A. Thorley-Lawson, unpublished observation). c. gp85. This glycoprotein may also play a role in virus neutralization as anti-gp85 and monoclonal antibodies of the right class will neutralize the virus in the presence, but not absence, of complement (Stmad et aZ., 1981).This would suggest neutralization of the virion by disruption mediated by complement rather than the blocking mechanism which occurs with antibodies against gp350/300 and gp250/200 (see below). 3. Function of Membrane Antigens

The major function that could be ascribed to membrane antigens on the envelope of the virus would be a role in the binding and adsorption of the virion to the target cell. There is good evidence for a specific EBV receptor on B lymphocytes (Jondal and Klein, 1973; Greaves et

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DAVID A. THORLEY-LAWSON E T AL.

FIG.6. Schematic representation summarizing our findings on gp350 and gp220 and their role in stimulating neutralizing antibodies. Epstein-Barr virus (EBV) infection of humans results in the generation of virus-neutralizing antibodies. Production of monoclonal antibodies after immunizing mice with purified EBV allowed the isolation of a monoclonal anti-EBV antibody that would neutralize the virus in uitro. This antibody could then be used to isolate the appropriate molecules (gp350/220) in a form that would both generate neutralizing antibodies in uiuo and absorb the neutralizing antibodies from the sera of EBV-infected humans.

al., 1975), the only cell type that may be infected with EBV both in vitro (Schneider and zur Hausen, 1975; Yata et al., 1975; Menezes et al., 1976; Thorley-Lawson and Strominger, 1978) and in vivo (Moore and Minowada, 1973; Huber et al., 1976). It seems reasonable to assume that it is the virus that has adapted itself to infect B lymphocytes rather than the other way around, and thus it is more appropriate to say that EBV has a receptor for B cells. This receptor is presumably in the viral envelope. Given that gp350/300 and gp250/200 are the major components of the viral envelope and that they contain polysaccharide structures that must protrude from the surface of the envelope, it is likely, purely in stereochemical terms alone, that these molecules make first contact with the target cell and, thus, bind to the EBV receptor. In this context, it is noteworthy that MA-positive, but not MA-negative, cells will rosette with EBV receptor-positive cells (Jondal and Klein, 1973). Very recently, it has been shown that purified gp350/300 and gp250/200, but not other viral components, will bind to the EBV receptor on B cells. Specific antibodies against these glycoproteins will inhibit viral binding, confirming a role for these glycoproteins as the viral receptor for B cells (Wells et al., 1982).

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It is also of interest that the p140 may be on the inner surface of the membrane. As EBV buds through membranes and acquires its envelope, it must identify specific membrane components. For example, it acquires MA components, but not MHC products (Silvestre et al., 1974). It is conceivable, therefore, that the viral capsid binds to and buds through specific regions of membrane designated by a primer protein of some kind. Such a protein would have to be on the inner surface of the membrane, as has been described for the M protein of vesicular stomatitis virus (Knipe et al., 1977), and p140 would be a good candidate for such a protein with EBV. Other functions that could be associated with membrane antigens are the stimulation of immune responses mediated by antibody, notably neutralizing antibodies and ADCC. Although our studies indicate that the primary components responsible for stimulating neutralizing antibodies are the two large glycoproteins gp350/300 and gp250/200, it is likely that antibodies against other components, particularly gp85, could also neutralize the virus. Nothing is yet known about the target for the antibody that mediates ADCC, although gp350/300 and gp250/200 must be the prime candidates because of their highly immunogenic nature. However, other components that stimulate antibody production could also be involved. C. VIRALCAPSIDANTIGENS 1. Zntroduction The detection of antigens on viral capsids (VCA) was shown unequivocally by electron microscopic studies demonstrating that human sera would stain naked viral capsids and that discordent MA-, VCA+ sera stained only naked capsids, not enveloped capsids (Silvestre et al., 1971). Such sera will also stain the cytoplasm, but not membranes, of virus-producing cells to give the classic VCA stain that has been so useful in the study of EBV (for discussion of problems of confusion in the VCA stain technique between true VCA and cytoplasmic MA, see Sections IV,A and IV,B,2). 2. Biochemical Characteristics of Viral Capsid Antigens Dolyniuck et al. (1976a,b) have described a procedure for the isolation of EB virions and have shown 7 polypeptides that are resistant and 9 partially resistant to detergent extraction (removal of envelope). Of these, the major resistant component has a molecular weight of 160,000. Mueller-Lantzsch et al. (1979) have detected a major component of 150,000 in detergent-extracted virions and have shown that a

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similar protein may be immunoprecipitated from [35S]methioninelabeled cells with VCA+ sera. In our experiments, we have also detected a 160,000 MW protein (Edson and Thorley-Lawson, 1981) that is the major polypeptide left after detergent extraction of virions purified by the method of Dolyniuck et al. (1976a,b). The same polypeptide can be precipitated from the cytoplasm and nucleus, but not intracellular or plasma membranes, of virus-producing cells (Fig. 4). It seems reasonable to conclude, therefore, that p160 is the major component of the viral capsid and is synthesized in the cytoplasm in a soluble form and then transported to the nucleus, where it condenses into capsids and may no longer be solubilized by detergents. 3. Functions of Viral Capsid Antigens No functions have been demonstrated for EB viral capsid antigens, although presumably they must play a role in packaging of viral DNA and possibly in attachment to membranes during budding. V. Conclusions

A. MOLECULAR BIOLOGYOF THE ANTIGENS AND THE POLYPEPTIDES

Epstein-Barr virus has 85 to 90 X lo6 MW of unique DNA (Dambaugh et al., 1980)and therefore has the capacity to code for at least 50 polypeptides. Herpes simplex virus, which has a similar genomic size, is thought to code for between 50 and 200 distinct polypeptides (Honess and Roizman, 1973; Haarr and Marsden, 1981). As yet no single polypeptide has been shown definitively to be coded for by EBV DNA. However, a number of polypeptides, summarized in Table V, have been shown, to a greater or lesser degree of certainty, to be found associated with EBV-infected cells. The maximum number to be so identified is about 20-25. Additional polypeptides can be assumed on the basis of already known information. There are enough mRNAs in transformed cells to account for at least 5 more transformation proteins (Rymo, 1979; van Santen et al., 1981).From the work of Dolyniuck et al. (1976a,b) on purified virions, there may be as many as 25 more structural polypeptides in the envelope, tegument, and capsid of virions, although these are probably overestimates because minor protein bands, which could easily be contaminants, were also counted. The remaining possible 50- 100 polypeptides are presumably nonstructural and could be synthesized either early, late, or both early and late. Immunochemical studies have provided a breakthrough in the study

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TABLE V SUMMARY OF EBV-ASSOCIATED POLYPEPTIDES AND ANTIGENS SO FARIDENTIFIED

Antigen Transformation Early antigens (EA)

Early plasma membrane antigen Plasma membrane and viral envelope antigens Viral capsid antigens (VCA)

Number of polypeptides known 1 1 10- 15

1 6

1

Molecular weight ( X 10-3 10%)

Antigenic association"

48 45 1451125 95/35/30 1601130190/80160150/17 95

EBNA LYDMA EA-D EA-R Not assigned EMA

3501300, 2501200 140 85 115156 160

VCAb/MAINA VCAb VCAblMA Not assigned VCA

With the exception of the 48,000 MW EBNA and the gp350/300 and gp2501200 MA components, no direct evidence of association has been given. EBNA, EBV nuclear antigen; LYDMA, lymphocyte-determined membrane antigen (MA); EA-D, EA-R, diffuse and restricted forms of EA; EMA, early MA; NA, antigens that stimulate neutralizing antibodies. bThese are membrane components that give a specific VCA-like strain as defined by monoclonal antibodies.

of EBV antigens and their constituent polypeptides. The use of human sera, particularly discordant ones, has provided evidence for the existence of the 20-25 EBV-associated polypeptides. The limitations of these antibodies, however, are clearly seen in studies on the MAS where various groups have reported from 2 to 7 components. The situation becomes even more chaotic with the EAs, reports varying between 5 and 15 components with ranges of molecular weights such that, unlike the MA studies, it becomes difficult to draw general conclusions. The best characterized antigens and polypeptides so far are the 48,000 MW EBNA polypeptide and the gp350/300 and gp250/200 components of MA. This is because EBNA appears to be simple, consisting of a single polypeptide. In the case of gp350/300 and gp250/200 the task was made possible by the production of monoclonal antibodies. As an overall strategy for defining EBV specificity and function for the various antigens, it would obviously be simplest if a permissive in vitro system were available so that genetic manipulation could be

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DAVID A . THORLEY-LAWSON ET AL.

used to study these questions. This has been very useful, for example, in the study of herpes simplex virus. Unfortunately, no such system is available as yet for EBV. In the absence of such an approach, it seems reasonable to conclude that a more laborious method is necessary. To begin with, it will be necessary to produce monospecific antibodies to each antigen; that is, monoclonal antibodies. Attempts to correlate observations made with an antibody using various techniques such as immunofluorescence, immunoelectron microscopy, and immunoprecipitation can have only limited value with polyspecific antibodies. Thus, for example, one can never be sure that the same molecules are being immunoprecipitated (requiring protein A-binding antibodies) as are recognized in a direct or indirect immunofluorescence test. For this reason, a defined serum such as a rabbit heteroantiserum against a limited number of antigens is more desirable than a human serum with antibodies against many antigens. However, none of these has the unique power of unequivocal certainty that is given by the monoclonal reagent. The next step will then b e to isolate the molecules. For this, the power of monoclonal antibodies as immunoaffinity reagents is unequaled. Having isolated the antigen and developed a monoclonal antibody reagent, it will be possible to discover whether the molecule corresponds to antigens previously defined by immunofluorescence with human sera. It is not necessary a priori to isolate only molecules that are recognized by human sera, as any molecule that may be virus-specific is intrinsically interesting. Nevertheless, for certain studies it is important to know which molecules are recognized by human sera. Thus, for example, if an attempt were being made to study the role of neutralizing antibodies in prevention of EBV infection and tumorigenesis, it would be necessary to show that isolated envelope components used in the study were the ones recognized by human neutralizing antibodies. Similarly, if EAs were being isolated to develop some form of assay to screen human sera, it would be necessary to establish that the antigens were of the kind already known to b e important, notably the D and R components of EA. The next stage in the study of such molecules will be to define their virus specificity. Although a number of experimental methods have been devised, as discussed above, to provide circumstantial evidence for the specificity of virus-associated antigens, there is only one proof, and that is to show that the polypeptides are coded for by the viral genome. The entire viral genome has been cloned (Dambaugh et al., 1980), and attempts have been made to identify and isolate virusspecific mRNAs (Rymo, 1979; van Santen et al., 1981). The next step

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will be to translate these mRNAs in uitro and identify the translation products b y immunoprecipitation with polypeptide-specific antibodies. This method will allow genetic mapping to b e performed with EBV in the absence of a traditional genetic system. The last stage in these studies would be to define the role of the isolated polypeptides and antigens in the biology of EBV. In some cases these studies will be relatively easy. Thus, for those molecules that stimulate the immune response, it should be possible to demonstrate the appropriate activity with isolated molecules in the proper form (e.g., reconstituted in liposomes or protein micelles). Similarly, it should be possible to demonstrate the binding of appropriate envelope components to the viral receptor on B cells. To understand the role of transformation proteins and nonstructural early and late proteins in the induction and maintenance of transformation and the lytic cycle, respectively, will be much more difficult and will probably rely on repeating studies already established with more manageable and amenable tumor virus systems. B. IMMUNE RESPONSESTO THE VIRAL ANTIGENS Besides the molecular biological aspects of EB antigens, there is the major question of their role in the immune response and the control of EBV infection. There is no doubt that the major interest in EBV derives from its association with human disease, particularly malignant disease. Only a masochist would chose EBV as a model system for molecular biology studies. The interest in EBV and disease means that a considerable effort has and will be directed toward defining the immune responses to EBV and what is defective in individuals who succumb to the EBV-associated diseases BL, NPC, and chronic or fatal IM. This may be studied both from the point of view of the immune responses and the antigens that stimulate those responses. Antigens may be either essential viral components that are incidentally antigenic or cellular components that act as beacons directing the immune response to the area of infection. It is conceivable that there are immune responses directed against every part of the viral cycle, as illustrated in Fig. 7. Virus-neutralizing antibodies may intercede against the spread of free infectious virus. Infected cells may be prevented from transforming by interferon. The infected-transformed cell can then either proliferate, in which case it would be the target for specific cytotoxic T cells, or it could enter the lytic phase. Cells in the early phase of virus production could be destroyed by NK cells directed against EMAs. Cells in both the early and late phase could be

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DAVID A. THOHLEY-LAWSON E T AL.

FIG. 7. Schematic representation summarizing the immune responses, defined in oitro, against Epstein-Barr virus (EBV) and EBV-infected cells. LMA, Late membrane antigen (MA); EMA, early MA; ADCC, antibody-dependent cellular cytotoxicity; NK, natural killer cell; C’, complement.

destroyed by means of antibody to EMAs and LMAs mediated by ADCC or complement. Such activities could prevent the production and release of intact, enveloped, infectious, virus particles, particularly when directed against EMAs. We do not know at present what role these different responses play in uiuo. Protocols are at present underway involving the treatment of immunosuppressed kidney transplant patients (at risk to infection with several herpesviruses, including EBV) with interferon (Cheeseman et al., 1980) and boys at risk to the EBV-associated XLP syndrome with immune y-globulin (D. Purtillo, personal communication). In the interferon study, it is too soon to know whether any effect can be demonstrated. In the XLP study, the boys do well, but it is unclear whether this is due to overall protection (the patients are aggamaglobulinemic) or due to specific protection against EBV. Nevertheless, we are now in a situation to begin studying the role of various antigens in preventive immunity. We already have purified gp350/300 and gp250/200 which are capable of stimulating neutralizing antibodies. Thus, we can study the role of neutralizing antibodies in preventing the establishment of infection in the animal model, namely, the cottontop marmoset, which is suscepti-

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ble to EBV infection and tumorigenesis (Shope et al., 1973). Opinions appear to be divided as to the appropriateness of such studies in marmosets. The argument for these experiments can best be made by answering the major points made against them. 1. The cottontop marmoset is not an appropriate animal model. There is some validity to this argument. These animals appear to be somewhat impaired in their immune responsiveness, possibly because they are always chimeric twins (Harvey et al., 1974). However, this fact makes these animals even more appropriate as a model system, because it is now accepted that fatal EBV infections and tumorigenesis only occur in immunologically impaired individuals. 2. The lymphoma that develops in the marmoset is not like BL, but more like the lymphoma that occurs in fatal IM, for example. This view is based primarily on the observation that marmoset tumors lack the characteristic chromosome abnormalities that occur in the BL tumor (Manolov and Manolova, 1972; Jarvis et al., 1974; Zechet al., 1976) and on the suggestion that the marmoset tumor, like fatal and normal IM (Glade and Chessin, 1968; B k h e t et al., 1974), is polyclonal in origin, whereas BL is monoclonal (Fialkow et al., 1970, 1973). Although no consistent chromosome abnormality has been reported in marmoset tumors, there is one report claiming that the marmoset tumor may be monoclonal, polyclonality occurring because of multiple sites of infection (Rabin e t al., 1977). Little or no work has been done with marmosets using the natural route of infection, which is presumed to be oral. In another respect the marmoset tumor is more like the BL in that the cells in culture are high-level virus producers (Shope et al., 1973; Miller and Lipman, 1973), unlike the cell lines derived from IM, which are much lower level or nonproducers (Sugden e t al., 1979). At present the issue is not settled, but, for the initial experiments, unimportant, because the development of any EBV-positive tumor is merely one indicator for the establishment of infection, and the first question will simply be: Can immunization prevent the establishment of infection? 3. The notion that neutralizing antibodies can prevent EBV tumorigenesis is too simplistic. This may be so, but it requires that an experiment be performed to find out. It may be that more sophisticated approaches will be required involving the isolation of more MA components (both early and late) and the presentation of the antigen in a more effective way, in liposomes or protein micelles, for example. It will also be desirable to isolate components of LYDMA and reconstitute them in a form that will stimulate cell-mediated immunity. These

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studies can be developed only in parallel with an increased understanding of the role of these antigens during in vivo infection of both humans and marmosets and are not feasible at present. Another exciting, but real, possibility will be the availability of cheap, virus-free antigens derived from bacteria containing cloned EBV DNA. The first step will be the isolation of virus-specific mRNAs followed by the cloning of cDNA and finally expression of those cDNAs in the bacteria. Such experiments have already been successfully performed with other systems and should be feasible for EBV within the next few years. The study of viral subunit vaccination in primates should be regarded as a first step in developing our understanding of the interaction of EBV with the immune response during infection, which may eventually lead to preventive measures in man. Such studies should not be presented as one experiment in monkeys followed by use in humans, both for the sake of the integrity of the members of the scientific community and so as not to raise false hopes in the general population, particularly among the families of children suffering from EBVassociated diseases.

ACKNOWLEDGMENTS We would like to thank Joyce Culgin for typing the manuscript. This work was supported by NIH Grants Nos. 1R01-A15310 and -CA28737.

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A Acute lymphocytic leukemia (ALL) characteristics of, 211 chromosome markers in, 126-132 clinical prognosis of, 242-244 Acute myeloblastic leukemia (AML), chromosome abnormalities in, 121-

122 Acute nonlymphocytic leukemia (ANLL) chromosome abnormalities in, 116-118 in adults, 118-122 in children, 123-124 prognostic significance, 124-125 as second neoplasm, 125-126 Acute-phase reactant proteins (APRPs), as tumor markers, 22 Acute promyelocytic leukemia (APL), chromosome abnormalities in, 122 S-Adenosyl-L-methionine decarboxylase inhibition of, 62 pharmacological, 81-84 Allelic exclusion, definition of, 220 Amniotic fluid, polyamines in, normal levels, 50 Angioinnnunoblastic lymphadenopathy lymphoma derived from, 235 Antigens of Epstein-Barr virus, 295-348 Autoimmune conditions, lymphomas in,

235

function of, regulation, 231-234 functional subsets of, 244-247 generation of diversity in, 222-223 idiotypes, networks, and suppression of, 233-234 immunoglobulins on, 223-228 functions of, 226-228 heavy-chain switch in, 232-233 neoplasms, 211,234-247 B-cell development and, 239-242 clinical prognosis, 242-244 hypothesis for, 246 maturation arrest in, 236-239 in mature cells, 240-242 monoclonality of, 234-235 pre-cells, neoplasia in, 239 proliferation of, 231-232 surface receptors on, 214, 223-231 system of, 212-234 description of, 212-213 Bile, polyamines in, normal levels, 50 Blood of normal and cancer patients, polyamines in, 38-48 Bone marrow polyamine levels in, 47 in cancer patients, 48-49 Bone neoplasias, urinary polyamines in,

34 Breast cancers, urinary polyamines in, 34 Burkitt’s lymphoma, polyamines in, 26-

27

B

C

B cell(s), 21 1-255 antibody production by, 231-232 in chronic lymphocytic leukemia, 239240 complement receptors on, 228-230 Fc receptors on, 230-231

Carcinogenesis, DNA structure and, 165-

210 Cell cultures (neoplastic) growth rate and cell-cycle phases, 11-

12 349

350

INDEX

microenvironmental factors in, 2-1 1 polyamines in, 2-20 Central nervous system (CNS) neoplasias, polyamines from, 34, 49-50 Cerebrospinal fluid polyamines in in cancer patients, 49-53 normal levels, 50 Chromatin damage and repair of, tumorigenesis and, 199-203 as possible origin of nucleoid DNA SLIpercoiling, 173-175 Chromosome abnormalities in acute lymphocytic leukemia, 126132 in acute nonlymphocytic leukemia, 116-126 in chronic myelogenous leukemia, 107116 in malignant hematologic diseases, 103-148 methods for study of, 105-107 in polycythemia Vera, 132-139 as tumor markers, 22 Chronic lymphocytic leukemia (CLL) as B-cell neoplasm, 239-240 characteristics of, 211 clinical prognosis of, 242-244 DNA supercoiling in, 193 maturation arrest in, 236 Chronic myelogenous leukemia (CML) acute phase of, 111-114 chromosome abnormalities in, 107-1 16 clinical corroborations, 113- 114 chronic phase of, 107-111 Clinical oncology, “markers” used in, 2223 Complement receptors, on B cells, 228230 Cyclic nucleotides polyamines in tumors and, 12-18 as tumor markers, 22

D Diamine oxidase (DAO), 56-62 in human tumors, 57-60 Digestive system neoplasias, urinary polyamines in, 33

DNA in chronic lymphocytic leukemia, 193197 of eukaryotes, significance of superstructure of, 187-193 focus-induced, from transformed cells, 156-157 of nucleoids, 167-169 chromatin origins, 173-175 strand breaks, 169-173 supercoiling decrease, 175-178 structure carcinogenesis and, 165-210 TPA alterations of, 197-199 supercoiling of, novobiocin and nalidixic acid as probes of, 178-185 DNA-binding proteins, of EBV early antigens, 316 DNA polymerase, of EBV early antigens, 316-317 DNase, in EBV early antigens, 317 Duodenal fluid, polyamines in, normal levels, 50

t Early antigens of Epstein-Barr virus, 296-297,309-319 Early membrane antigens of Epstein-Barr virus, 318-319 Endocrine system neoplasias, urinary polyamines in, 34 Enzymes, as tumor markers, 22 Epstein-Barr virus (EBV) effect on immunodeficient patients, 235 transformation of CLL cells by, 237238 Epstein-Barr virus (EBV) antigens, 295348 early antigens, 296-287, 309-319 biochemical characteristics, 31 1-315 functions, 315-317 intracellular, 309-31 1 membrane type, 3 18-319 late antigens, 297, 319-336 membrane type, 318-319,321-335 transformation antigens, 296, 298-304 viral capsid type, 335-336 Erythrocytes, polyamine levels in, 46

351

INDEX

F

I

Fc receptors, on B cells, 230-231 Fetal antigens, as tumor markers, 23 Fibronectin, polyamines in cells and, 20

Immunoglobulin( s) on B-cell surfaces, 223-228 formation theories for, 213-215 gene organization and expression for, 213-222 description of, 215-217 generation of diversity in, 217-219 as tumor markers, 23 Immunoglobulin D on B-cell surface, 224-225 simultaneous expression with IgM, 221-222 Immunoglobulin M membrane-bound and secreted, 221 simultaneous expression with IgD, 221-222 Isozymes, as tumor markers, 22

G Genes allelic and isotype exclusion in, 22022 1 for cancer, see Oncogenes Germinal-center cell lymphomas, characteristics of, 242

H Heat, as cancer treatment, 8-9 Hematologic diseases (malignant) chromosome abnormalities in, 103-148 implications of, 139-142 Hematopoietic system neoplasias, urinary polyamines in, 33 Histaminase, see Diainine oxidase Hodgkin’s disease, 257-293 alternating non-cross-resistant regimens of, 273-277 chemotherapy of, 263-267 combination type, 264-267 historical aspects, 263-264 late complications from, 285-287 combined modality treatment of, 267273 major concepts of therapy of, 259 morbidity influencing current strategy, 283-287 new treatment strategies, 267-277 pathogenesis of, 21 1 prognostic factors affecting, 277-282 age, histology, and symptoms, 277279 liniited extranodal disease, 280-281 mediastinal bulky disease, 279-280 stage IIIA, 281-282 radiotherapy of, 258-263 morbidity from, 284-285 surgical staging of, morbidity from, 283-284 total conquest of, goals for, 287-290 Hormones, as tumor markers, 22 Human cancer, polyamines in, 20-56

L Late antigens of Epstein-Barr virus, 297, 319-336 membrane type, 321-335 viral capsid type, 335-336 Leukemias, chromosome abnormalities in, 139-142 Leukocytes, polyamine levels in, 47 Lymphocytes development of, 212, 219 heavy-chain class switch in, 219-220 Lymphomas, B-cell type, 211-255

M Malignolipin, in human neoplasms, 27 Mammalian tumors, polyamines in, 1-102 Membrane antigens (MA) of Epstein-Barr virus, 321-335 biochemical characteristics of, 322-333 function of, 333-335 3-Methylcholanthrene, cells transformed by, oncogenes in, 155-156 5’-Methylthioadenosine (MTA), polyaniine synthesis and, 20

N Nalidixic acid, in studies of DNA supercoiling, 178-185

352

INDEX

Neoplasms, B-cell derived, 211, 234-247 Novobiocin, in studies of DNA supercoiling, 178-185 Nucleoids DNA superstructure of, 167-169 strand breaks, 169-173

0 Oncogenes activation of, 159-160 proteins encoded by, 161-162 multiplicity of, 157-158 retrovirus-associated, 150-153 role in carcinogenesis, 160-161 of spontaneous and chemically induced tumors, 149-163 in transformed cells, 153-155 Ornithine decarboxylase (ODC) inhibition of, 62-67 pharmacological, 68-81 in neoplastic cell cultures, 2-8, 11-18

as tumor markers, 23 in urines normal and cancer patients,

27-38 Polyamine biosynthetic decarboxylases (PBD) as human neoplasm markers, 24 degree of malignancy and, 53-55 induction of, cyclic nucleotides and,

12-18 Polycythemia Vera, abnormal chromosomes in, 132-139 Putrescine in blood, normal levels, 40-41, 46-47 effects on neoplastic cells, 19 in human neoplasias, 25 in urine cancer patients, 32-38 normal, 28-30

R Radiotherapy of Hodgkin’s disease, 258-

263

P Philadelphia chromosome in acute lymphocytic leukemia, 131-

132

Reproductive system neoplasias, urinary polyamines in, 33 Respiratory system neoplasias, urinary polyamines in, 33 Retroviruses, oncogenes associated with,

150-153

as leukemia marker, 114-116 Phorbol ester, effect on CLL cells, 236-

237

S

Plasma, polyamines in, in cancer patients,

42-43 Platelets, polyamine levels in, 47 Polyamines biosynthesis of, physiological inhibitors, 63-68 cyclic nucleotides and, 12-18 in human oncology, 20-56 in mammalian tumors, 1-102 metabolic conjugation of, 55-56 in normal and cancer patients blood, 38-48 blood cells, 46-47 urine, 27-38 miscellaneous effects of, 18-20 in neoplastic cell cultures, 2-20 role in cell-killing effects, 9-10

Saliva, polyamines in, normal levels, 50 Serum, polyamines in, in cancer patients,

43 Skin neoplasias, urinary polyamines in, 34 Spermidine biosynthesis of, inhibitors, 84-85 in blood, normal levels, 40-41, 46-47 effects on neoplastic cells, 19 in human neoplasias, 25-26 in urine cancer patients, 32-38 normal, 28-30 Spermine biosynthesis of, inhibitors, 84-85 in blood, normal levels, 40-41, 46-47 effects on neoplastic cells, 18-20

353

INDEX

in human neoplasias, 24-25 in urine cancer patients, 32-38 normal, 28-30 Sterols, as tunior markers, 23 Sweat, polyamines in, normal levels, 50

Tumors diamine oxidase in experimental 60-62 human, 57-60 oncogenes of, 149-163 polyamines in, 1-102

T U

T cells allogeneic, plasma cell induction by,

236 differentiation into, 212 TPA, effects on DNA superstructure, 197-

Urinary system polyamines in, Urine, of normal polyamines in,

neoplasias,

urinary

33 and cancer patients,

27-38

199 Transformation antigens of Epstein-Barr virus, 296 lymphocyte-determined membrane antigens, 304-309 biochemical characteristics, 306-307 function, 307-309 nuclear antigens, 298-304 biochemical characteristics, 300-303 functions, 303-304

v Vaccinia virus, effect on neoplastic cells,

18 Viral capsid antigens of Epstein-Barr virus, 335-336 Viruses, nononcogenic, effects on neoplastic cells, 18

This Page Intentionally Left Blank

CONTENTS OF PREVIOUS VOLUMES

Volume 1

Carcinogenesis and Tumor Pathogenesis

Electronic Configuration and Carcinogenesis

Ionizing Radiations and Cancer

1. Bcronhluni Airstin M . Bruijs

C. A . Coulson

Survival and Preservation of Tumors in the Frozen State

Epidermal Carcinogenesis E . V . Coudry The Milk Agent in the Origin of Mammary Tumors in Mice L . Dmochoi\,ski Hormonal Aspects of Experimental Tu morigenesi s

Jartics Craigio

Energy and Nitrogen Metabolism in Cancer Leonard D. Fcnningc,r and G. Burroughs Mider

T . U . Gardner

Properties of the Agent of Rous No. 1 Sarcoma R. J . C . Harris

Applications of Radioisotopes to Studies of Carcinogenesis and Tumor Metabolism Charles Heidelbergtv

The Carcinogenic Aminoazo Dyes James A . Millpr and Elizahcth C. Mi1Ic.r

Some Aspects of the Clinical Use of Nitrogen Mustards Calvin T. K l o p p and Jcannc C . Bor1,rnuri Genetic Studies in Experimental Cancer L. w. Lull, The Role of Viruses in the Production of Cancer C . Oberling and M . G u i ~ i n Experimental Cancer Chemotherapy C . Chester S10c.A AUTHOR INDEX-SUBJECT INDEX

The Chemistry of Cytotoxic Alkylating Agents M . C . J . Ross Nutrition in Relation to Cancer Albert Tannenbauni and Hc,rbl,rt Silverst one

Plasma Proteins in Cancer Richard J . Winder AUTHOR INDEX-SUBJECT INDEX

Volume 3 Etiology of Lung Cancer Ric,harcl DCJII The Experimental Development and Metabolism of Thyroid Gland Tumors Harold P. Morris

Volume 2 The Reactions of Carcinogens with Macromolecules

Electronic Structure and Carcinogenic Activity and Aromatic Molecules: New Developments A . Pullman and B. Pullmuti Some Aspects of Carcinogenesis P. Rondoni

Peter Alexander

Chemical Constitution and Carcinogenic Activity G. M . Badger

355

Pulmonary Tumors in Experimental Animals Michurl B. Shirnkin

356

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues S i d n q W~iiihouso A U T H O R IN D E X - S U BJ ECT I N D E X

Volume 4 Advances in Chemotherapy of Cancer i n Man Sidiioy Furher. Rudolj Toch, Ethiwtl Manning S m r s . atid Doriultl Pinlpl The Use of Myleran and Similar Agents in Chronic Leukemias D. A . G . Galton The Employment of Methods of Inhibition Analysis in the Normal and TumorBearing Mammalian Organism Abruhani Goldin Some Recent Work on Tumor Immunity P. A . Coriv Inductive Tissue Interaction in Development Clifford Crobsti,iti Lipids in Cancer Franc.i,s L . Ha\vn and W . R . Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A . Liicussagne. N . P . Buu Hoi. R . Duudi)l, atid F. Zujilola The Hormonal Genesis of Mammary Cancer 0. MUhlbocA A U T H O R IN D E X - S U BJECT I N D E X

Volume 5 Tumor- Host Relations R . W . Bcgg Primary Carcinoma of the Liver

Char/i,s Bc,ri?iari Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal P. N . Canipbcll

The Newer Concept of Cancer Toxin Wnro NaAuharu and Fut?iiX(~Fukiroh Chemically Induced Tumors of Fowls P. R . P r r r c ~ ~ A Anemia in Cancer Vinc,i,nt E. Prici, und Robvrt E. Grc,cnJic,ld Specific Tumor Antigens L. A . Lilbi,r Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizabeth K . Weishurgi,r und John H . Wi,isburgc,r A U T H O R I N D E X - SU BJ ECT I N D E X

Volume 6 Blood Enzymes in Cancer and Other Diseases Oscar Bodunsky The Plant Tumor Problem Arniin C . Brauri and Hiwry N . Wood Cancer Chemotherapy by Perfusion Oscar Crw1.h. Jr. and Ediiw-cl T . KriJtni’ntz Viral Etiology of Mouse Leukemia Ludii.ic.k Cross Radiation Chimeras P. C. Koller. A . J . S. Daries. arid Sheila M . A . Douk Etiology and Pathogenesis of Mouse Leukemia J . F. A . P. Mi1lc.r Antagonists of Purine and Pyrimidine Metabolites and of Folic Acid G. M. Tininiis Behavior of Liver Enzymes in Hepatocarcinogenesis Grorgi, Wrbor AUTHOR INDEX-SUBJECT INDEX

Volume 7 Avian Virus Growths and Their Etiologic Agents J . W . Beard

CONTENTS OF PREVIOUS VOLUMES

357

Mechanisms of Resistance to Anticancer 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 Bro+t*nand lshbel M . Tough Ethionine Carcinogenesis Emmanuel Farber Atmospheric Factors in Pathogenesis of Lung Cancer Paul Kotin and Hans L . Falk Progress with Some Tumor Viruses of Chickens and Mammals: The Problem of Passenger Viruses G . Ncgroni

The Relation of the Immune Reaction to Cancer Louis v. Caso Amino Acid Transport in Tumor Cells R. M . Johnstoni~and P . G . Scholejidd Studies on the Development, Biochemistry. and Biology of Experimental Hepatomas Harold P . Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes, and Bone Marrow Cells 1. F . Seitz

AUTHOR INDEX-SUBJECT INDEX

Carcinogens, Enzyme Induction, and Gene Action H . V . Gelboin I n Vitro Studies on Protein Synthesis by Malignant Cells A . Clark Grijjiii The Enzymatic Pattern c f Neoplastic Tissue W . EugiJnc, Knox Carcinogenic Nitroso Compounds P . N. Magee arid J . M . Bariic,.s The Sulfhydryl Group and Carcinogenesis J . S. Harrington The Treatment of Plasma Cell Myeloma DaiiiiJl E . Bc,rgsagc)l. K . M . Grijjith. A . Haut. arid W . J . Stuchley. Jr.

Volume 8 The Structure of Tumor Viruses and Its Bearing on Their Relation to Viruses in General A . F . Ho,vatson Nuclear Proteins of Neoplastic Cells Harris Busch and William J . Strele Nucleolar Chromosomes: Structures, Interactions, and Perspectives M . J. Kopac and Gladys M . Mazeyko Carcinogenesis Related to Foods Contaminated by Processing and Fungal Metabolites H . F . Kraybill and M . B . Shimkin Experimental Tobacco Carcinogenesis Ernest L. Wynder and Dietrich Hoffman AUTHOR INDEX-SUBJECT INDEX

Volume 9 Urinary Enzymes and Their Diagnostic Value in Human Cancer Richard Stambaugh and Sidney Weinhouse

AUTHOR INDEX-SUBJECT INDEX

Volume 10

AUTHOR INDEX-SUBJECT INDEX

Volume 11 The Carcinogenic Action and Metabolism of Urethran and N-Hydroxyurethran Sidn1.v S . Mirvish Runting Syndromes. Autoimmunity, and Neoplasia D . Keast Viral-lnduced Enzymes and the Problem of Viral Oncogenesis Saul Kit

358

CONTENTS OF PREVIOUS VOLUMES

The Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology WilliCJiIl Regrlson Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Joseph C . A r c w uritl Mary F . Argus AUTHOR INDEX-SUBJECT INDEX CUMULATIVE INDEX

Volume 12 Antigens Induced by the Mouse Leukemia Viruses G. Pustc,rnuL Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses G . 1. Dc,ic~hinuii Replication of Oncogenic Viruses in VirusInduced Tumor Cells-Their Persistence and Interaction with Other Viruses H . Hariujrisu Cellular Immunity against Tumor Antigens Karl EriX H~~llstri~rri and Iiigi,prcl HcNstroni Perspectives in the Epidemiology of Leukemia I n i n g L . Kessler arid Abraham M . Lilirnfrld AUTHOR INDEX-SUBJECT INDEX

Volume 13 The Role of lmmunoblasts in Host Resistance and lmmunotherapy of Primary Sarcomata P . Alc,xaiidiv and J . G . Hall Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Os\t~oldJarrcjtt

The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Hoirn Ginsburg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gajanari V . Sherbet The Characteristics of Animal Cells Transformed in Vitro l a t i Macphrrsoii Role of Cell Association in Virus Infection and Virus Rescue J . Svohodrl and 1. HloianoA Cancer of the Urinary Tract D. B . CI(iysoii and E . H . Cooper Aspects of the EB Virus M . A . Epsti,iii AUTHOR INDEX-SUBJECT INDEX

Volume 14 Active lmmunotherapy Goorgos Math6 The Investigation of Oncogenic Viral Genomes in Transformed Cells by Nucleic Acid Hybridization Eriii>st Winocour Viral Genome and Oncogenic Transformation: Nuclear and Plasma Membrane Events Goorgc, M o y r Passive lmmunotherapy of Leukemia and Other Cancer Roland Motto Humoral Regulators in the Development and Progression of Leukemia Doiiuld Metcalf Complement and Tumor Immunology Kusuva Nishioka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors C . 1. Ahelcv Low Dose Radiation Cancers in Man A1ic.t Steitwrt AUTHOR INDEX-SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 15 Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J . S.B ~ i t ~Sl.. S . T(,v(,thiu. ~ i i .dI . L . Moll1iC.X

Nasopharyngeal Carcinoma (NPC) J. H . C. H o Transcriptional Regulation in Eukaryotic Cells A . J . MncGillii~ruy.J . Purr/. u i i d G . T h r d full

Atypical Transfer RNA's and Their Origin in Neoplastic Cells Eriiost Borcl. uiitl Sylriii J . Korr Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females Philip J . FiulAoii. Electron Spin Resonance Studies of Carcinogenesis Huroltl U . Sii'urt; Some Biochemical Aspects of the Relationship between the Tumor and the Host V . S. Shupot Nuclear Proteins and the Cell Cycle G o r y Stc,iti crnd Rc>tiutri Buscrgu AUTHOR INDEX-SUBJECT INDEX

359

I ,3-Bis(2-Chloroethyl)- I -Nitrosourea (BCNU) and Other Nitrosoureas in Cancer Treatment: A Review Stopheii K . Curtor. Fraiil. M . Schubrl. J r . , Luiiwric,e 15. Broder. uiid Thor?ius P . Johiistoii AUTHOR INDEX-SUBJECT I N D E X

Volume 17 Polysaccharides in Cancer: Glycoproteins and Glycolipids VIjtri N . Niguiti uriil Atitotiio C u t i t e m Some Aspects of the Epidemiology and Etiology of Esophageal Cancer with Particular Emphasis on the Transkei. South Africa Gpruld P . Wurwick uiid Johii S.Huringtoir

Genetic Control of Murine Viral Leukemogenesis FruiiX Lilly u i i t l Thcoilori~Piric,us Marek's Disease: A Neoplastic Disease of Chickens Caused by a Herpesvirus K . Noioriuii Mutation and Human Cancer Aijir(4 G . Kiiudsoii. J r . Mammary Neoplasia in Mice S . Nuiitli u i i d Churlcs M . M&ruth AUTHOR INDEX-SUBJECT INDEX

Volume 16 Polysaccharides in Cancer Vijui N . N i g u t ~ icrirtl Aiitoiri(i Caiitc,rri Antitumor Effects of Interferon l o i i Grc,ssor Transformation by Polyoma Virus and Simian Virus 40 Jot, Scri?ibrooX Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing? Sir A/oxuiiclc,r Hucltloii, The Expression of Normal Histocompatibility Antigens in Tumor Cells AIcriu Lriigeroi,ri

Volume 18 Immunological Aspects of Chemical Carcinogenesi s R . W . Baldii~iii lsozymes and Cancer Fuiiiiy Schupiru Physiological and Biochemical Reviews of Sex Differences and Carcinogenesis with Particular Reference to the Liver Yer Chu Toh Immunodeficiency and Cancer Johir H . Kersc,.v. Biwtricr D . Spoc1or. uiid Rohort A . Good

360

CONTENTS OF PREVIOUS VOLUMES

Recent Observations Related to the Chemotherapy and Immunology of Gestational Choriocarcinoma K. D. Bagshave Glycolipids of Tumor Cell Membrane Sen-itiroh Hakomori

Chemical Oncogenesis in Culture Charles Heidelberger AUTHOR INDEX-SUBJECT INDEX

Principles of Immunological Tolerance and Immunocyte Receptor Blockade G. J . V . Nossal The Role of Macrophages in Defense against Neoplastic Disease Michael H. L P and ~ E. Frc.dcric,k Whetl O Ck

Epoxides i n Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis

P. Sims

and P. L. Groviv

Virion and Tumor Cell Antigens of C-Type RNA Tumor Viruses Htjinz Bouer

Volume 19 Comparative Aspects of Mammary Tumors J . M . Hamilton The Cellular and Molecular Biology of RNA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses, and Their Relatives Honsard M . Temin Cancer, Differentiation, and Embryonic Antigens: Some Central Problems J . H . Coggin, Jr. and N. G . Anderson Simian Herpesviruses and Neoplasia Fredrich W. Deinhardt, L a n w n w A . Falk, and Lauren G . Wolfr

Cell-Mediated Immunity to Tumor Cells Ronald B. Herberman Herpesviruses and Cancer Fred Rapp

Cyclic AMP and the Transformation of Fibroblast s I r a Pastan and Georgc

S.Johnson

Tumor Angiogenesis Judah Folkman SUBJECT INDEX

Volume 20 Tumor Cell Surfaces: General Alterations Detected by Agglutinins Annette M. C . Rapin and Max M . Burger

Addendum to "Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing?" Sir Alexandi>r Haddoit, SUBJECT INDEX

Volume 21 Lung Tumors in Mice: Application to Carcinogenesis Bioassay Michaid B . Shimkin and Gary D . Stotwr Cell Death in Normal and Malignant Tissues E. H. Coopor. A . J . Bi@i)rd. and T . E . Konny

The Histocompatibility-Linked Immune Response Genes Baruj B e n a c ~ w aand j David H . K a t z Horizontally and Vertically Transmitted Oncornaviruses of Cats M . Esscx Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms K w f A . RajJiwy. Jr. Selection of Biochemically Variant, in Some Cases Mutant, Mammalian Cells in Culture G . B . Cli~mc~nts The Role of DNA Repair and Somatic Mutation in Carcinogenesis James E . Trosko and Ernest H. Y . Chu SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

36 1

Volume 22

Volume 24

Renal Carcinogenesis

The Murine Sarcoma Virus-Induced Tumor: Exception or General Model in Tumor Immunology?

J . M . Hamilton

Toxicity of Antineoplastic Agents in Man: Chromosomal Aberrations, Antifertility Effects, Congenital Malformations, and Carcinogenic Potential Susan M . Sieber and Ric,hard H . Adam-

son Interrelationships among RNA Tumor Viruses and Host Cells Raymond V . Gilden Proteolytic Enzymes, Cell Surface Changes, and Viral Transformation Richard Roblin. lih-Nan Chou, and Paul H . Black

Immunodepression and Malignancy Osias Slutman SUBJECT INDEX

Volume 23 The Genetic Aspects of Human Cancer W. E . Heston The Structure and Function of Intercellular Junctions in Cancer Ronald S . Weinstein. Frederic h B . M i d .

J . P. Lerty and J . C. Leclrrc

Organization of the Genomes of Polyoma Virus and SV40 Mihi. Fried and B i w r l y E . Grifjrin P,-Microglobulin and the Major Histocompatibility Complex Per A . Peterson. Lars Rash. und Lors Osibcvg

Chromosomal Abnormalities and Their Specificity in Human Neoplasms: An Assessment of Recent Observations by Banding Techniques Joachim Mark Temperature-Sensitive Mutations in Animal Cells Claudio Basilicr) Current Concepts of the Biology of Human Cutaneous Malignant Melanoma Wallace H. Clark. J r . , Michurl J . Masiratigc~lo.Ann M . Ainsiiwrth. David Brrd. Robert E. Belli,r+ and Eivlina A . Brrriardirio SUBJECT INDEX

and Joseph Ahoy

Genetics of Adenoviruses Harold S. Ginsberg and C. S. H . Young

Molecular Biology of the Carcinogen, 4-Nitroquinoline I-Oxide Minaho Nagao and Tahashi Suginrura

Epstein-Barr Virus and Nonhuman Primates: Natural and Experimental Infection A . Franh, W . A . Andiman. and C . Mil1i.r Tumor Progression and Homeostasis Richmond T . Prehn Genetic Transformation of Animal Cells with Viral DNA or RNA Tumor Viruses Miroslav H i l l and Jaria Hillo\u SUBJECT INDEX

Volume 25 Biological Activity of Tumor Virus DNA F . L . Graham Malignancy and Transformation: Expression in Somatic Cell Hybrids and Variants Harrvy L . Ozer and Krishria K . Jha Tumor-Bound Immunoglobulins: Irr Siiu Expressions of Humoral Immunity Isaac P. Wiiz

The Ah Locus and the Metabolism of Chemical Carcinogens and Other Foreign Compounds Snorri S . Thorgrirssoti and Dani~l W. Nobrrr

362

CONTENTS OF PREVIOUS VOLUMES

Formation and Metabolism of Alkylated Nucleosides: Possible Role in Carcinogenesis by Nitroso Compounds and Alkylating Agents Anthony E . P r g g Immunosuppression and the Role of Suppressive Factors in Cancer Isao Kanio and Horman Fricvlman Passive lmmunotherapy of Cancer in Animals and Man Sti,b~,n A . Rosc,nbi,rg and William D. Torry SUBJECT INDEX

Volume 26

The Choice of Animal Tumors for Experimental Studies of Cancer Therapy Harold B. Hrn’itt Mass Spectrometry in Cancer Research Johii Rohoz Marrow Transplantation in the Treatment of Acute Leukemia E . Donna// Thomas. C . Dean Buchier. Alexander Fcfor. Paul E . Neiinan. and R a i w r Storb Susceptibility of Human Population Groups to Colon Cancer Martin Lipkin Natural Cell-Mediated Immunity Ronald B. Hcrhcwnan and Howard T . HoldPn SUBJECT INDEX

The Epidemiology of Large-Bowel Cancer P d a y o Correa and William Haenszc4 Interaction between Viral and Genetic Factors in Murine Mammary Cancer Volume 28 J . Hilgers and P. Bentvi>lzi,n Cancer: Somatic-Genetic Considerations Inhibitors of Chemical Carcinogenesis Lee W . Wattenhijrg F. M . Burnot Latent Characteristics of Selected Herpes- Tumors Arising in Organ Transplant Recipviruses ients Jack G. Stc.\vns Israc4 Pc~nn Antitumor Activity of Coryi?c~hac.li~riurnStructure and Morphogenesis of Type-C Retroviruses porvum LuIa Milas arid Martin T . Scott R ~ i i a l dC. Montdaro and Dan; P. BolSUBJECT INDEX

Volume 27 Translational Products of Type-C RNA Tumor Viruses John R . Stc,phenson. Sushilkurnar G. Devare. and Fred H . Ri>ynolds.Jr. Quantitative Theories of Oncogenesis Alice S . Whitternore Gestational Trophoblastic Disease: Origin of Choriocarcinoma, Invasive Mole and Choriocarcinoma Associated with Hydatidiform Mole, and Some Immunologic Aspects J . I . Briwvr. E. E. Torok. B. D. Kahan, C . R. Stanhopt. and B. Halpi)rn

( J ~ I W S ~

BCG in Tumor lmmunotherapy Robert W . Balduin and Malcolm V . Pillll?? The Biology of Cancer lnvasion and Metastasis Isaiah J. Fidler. Douglas M. Gerst1.n. and Ian R . Hart Bovine Leukemia Virus Involvement in Enzootic Bovine Leukosis A . Burny, F. B1.x. H . Chontrc.niic,. Y. CIeutcv. D. Dekc~gi4.J . Ghysdael. R . Kottmann. M . Leclrrcy. J . Li>uni,n. M . Mammc,rickx, and D. Portrtrlli, Molecular Mechanisms of Steroid Hormone Action Stephen J . Higgins and Ulrich Gchring SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 29 Influence of the Major Histocompatibility Complex on T-cell Activation J . F. A . P. Miller Suppressor Cells: Permitters and Promoters of Malignancy? David N a m Retrodifferentiation and the Fetal Patterns of Gene Expression in Cancer Jost? Uriel The Role of Glutathione and Glutathione S-Transferases in the Metabolism of Chemical Carcinogens and Other Electrophilic Agents L. F. Chasseaud a-Fetoprotein in Cancer and Fetal Development Erkki Ruoslahti and Markku Seppiiki Mammary Tumor Viruses Dan H . Moore, Carole A. Long, Akhil B . Vaidya, Joel B. Shefield, Arnold S . Dion, and Etienne 1'. Lasfargues Role of Selenium in the Chemoprevention of Cancer A . Clark Grifin SUBJECT INDEX

Volume 30

363

The Molecular Biology of Lymphotropic Herpesviruses Bill Sugden, Christopher R. Kintner, und Willie Mark Viral Xenogenization o f Intact Tumor Cells Hiroshi Kohayashi Virus Augmentation of the Antigenicity of Tumor Cell Extracts Faye C . Austin and Charles W. Boone INDEX

Volume 31 The Epidemiology of Leukemia Michael Alder.son The Role of the Major Histocompatibility Gene Complex in Murine Cytotoxic T Cell Responses Hermann Wagner, Klaus Pfizenrnaier, und Martin Rollinghofl The Sequential Analysis of Cancer Development Emmanuel Furber and Ross Camerson Genetic Control of Natural Cytotoxicity and Hybrid Resistance Edward A. Clark and Richard C . Harmon Development of Human Breast Cancer Sefton R . Wellings INDEX

Acute Phase Reactant Proteins in Cancer E. H. Cooper and Joan Stone Induction of Leukemia in Mice by Irradiation and Radiation Leukemia Virus Variants Nechama Haran-Ghera and Alpha Peled On the Multiform Relationships between the Tumor and the Host V. S. Shapot Role of Hydrazine in Carcinogenesis Joseph Bald Experimental Intestinal Cancer Research with Special Reference to Human Pathology Kazymir M. Pozharisski, Alexei J . Likhavchev, Valeri F. Klimasheuski, and Jacob D. Shaposhnikov

Volume 32 Tumor Promoters and the Mechanism of Tumor Promotion Lrilu L)icrmotid, Thomur G . O'Hrieii, ~ n &'illiam d M. Rmird Shedding from the Cell Surface of Normal and Cancer Cells Pnzri H . m t c k Tumor Antigens on Neoplasms Induced by Chemical Carcinogens and by DNA- and RNA-Containing Viruses: Properties of the Solubilized Antigens Lloyd \V. Law, A4ichael J . Rogers, und Ettore Appellrr Nutrition and Its Relationship to Cancer Rundaru S . Reddy, Leonard A . Cohen,

364

CONTENTS OF PREVIOUS VOLUMES G. David McCoy, Peter Hill, John H . Weisburger, and Ernst L. Wynder

INDEX

Volume 33 The Cultivation of Animal Cells in the Chemostat: Application to the Study of Tumor Cell Multiplication Michael G. Tovey Ectopic Hormone Production Viewed as an Abnormality in Regulation of Gene Expression Hiroo Imura The Role of Viruses in Human Tumors Harald zur Hausen The Oncogenic Function of Mammalian Sarcoma Viruses Poul Andersson Recent Progress in Research on Esophageal Cancer in China Li Mingxin ( L i Min-Hsin), Li Ping, and Li Baorong (Li Pao-Jung) Mass Transport in Tumors: Characterization and Applications to Chemotherapy Rakesh K. Jain, Jonas M. Weissbrod, andJames Wei INDEX

Volume 34 The Transformation of Cell Growth and Transmogrification of DNA Synthesis by Simian Virus 40 Robert G. Martin Immunologic Mechanisms in UV Radiation Carcinogenesis Margaret L. Kripke The Tumor Dormant State E. Frederick Wheelock, Kent J . Weinhold, and Judith Levich

Marker Chromosome 14q+ in Human Cancer and Leukemia Felix Mitelman Structural Diversity among Retroviral Gene Products: A Molecular Approach to the Study of Biological Function through Structural Variability James W. Gautsch, John H. Elder, Fred C. Jensen, and Richard A . Lerner Teratocarcinomas and Other Neoplasms as Developmental Defects in Gene Expression Beatrice Mintz and Roger A . Fleischman Immune Deficiency Predisposing to Epstein-Barr Virus-Induced Lymphoproliferative Diseases: The X-Linked Lymphoproliferative Syndrome as a Model David T. Purtilo INDEX

Volume 35 Polyorna T Antigens Walter Eckhart Transformation Induced by Herpes Simplex V i s : A Potentially Novel Type of V i s -Cell Interaction Berge Hampar Arachidonic Acid Transformation and Tumor Production Lawrence Levine The Shope Papilloma-Carcinoma Complex of Rabbits: A Model System of Neoplastic Progression and Spontaneous Regression John W: Kreider and Gerald L. Bartlett Regulation of SV40 Gene Expression Adolf Graessman, Monika Graessmann, and Christian Mueller Polyamines in Mammalian Tumors, Part I Giuseppe Scalabrino and Maria E. Ferioli Criteria for Analyzing Interactions between Biologically Active Agents Morris C . Berenbaum INDEX