Advances in Cancer Research, Volume 23

ADVANCES IN CANCER RESEARCH VOLUME 23

Contributors to This Volume

Joseph Alroy

Frederick 6. Merk

W. A. Andiman

G. Miller

A. Frank

Minako Nagao

Harold S. Ginsberg

Richmond T. Prehn

W. E. Heston

Takashi Sugimura

Miroslav Hill

Ronald S. Weinstein

lana Hillova

C. S. H. Young

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnrtitutet Stockholm, Sweden

SIDNEY WEINHOUSE Fels Research Institute Temple University Medical School Philadelphia, Pennsylvania

Consulting Editor

ALEXANDER HADDOW Chester Beatty Research Institute Institute of Cancer Research Royal Cancer Hospital London, England

Volume

23 - 7976

ACADEMIC PRESS

New York

A Subsidiary of Harcourt Brace Jovanovich, Publishers

San Francisco

London

COPYRIGHT 1976, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED O R TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road. London NW1

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 52-13360 ISBN 0-12-006623-8 PRINTED IN

THE UNITED STATES OF AMERICA

CONTENTS CONTRIBUTORS

TO

VOLUME 23

.

.

.

.

.

.

.

.

.

.

.

ix

. .

. .

. . . .

1 3

The Genetic Aspects of Human Cancer

.

W E. HESTQN

I. Introduction . . I1. Cancer of the Breast . I11. Leukemia . . . IV. Colorectal Cancer . V. Gastric Cancer . . VI. Retinoblastoma . . VII. Xeroderma Pigmentosum VIII . Albinism . . . IX . Lung Cancer . . References . . .

. . . . . . . . . .

. . . . . . . . . .

. .

. . . . . . . .

. .

. . . . . . . .

. .

. . . . . . . .

. .

. . . . . . . .

. .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

7 10 12 13

15 16 17 19

The Structure and Function of Intercellular Junctions in Cancer

RONALDS . WEINSTEIN.FREDERICK B. MERK.AND JOSEPHALROY

I. Introduction . . . . . . . . . . I1. Membrane Ultrastructure . . . . . . . . 111. Cell Junction Classification . . . . . . . . . . IV. Occurrence of Cell Junctions in Tumors . V. Cell-to-Cell Communication and Growth Control . . . VI . Cell Junctions in Embryonic Development . VII . Cell Junctions and the Biological Behavior of Tumor Cells VIII. Intercellular Adhesion in Tumors . . . . . . IX . Transepithelial Permeability and Malignant Transformation . . . . . . . . . . X. Summary . References . . . . . . . . . . .

. .

.

. . . . . . . . . . .

.

.

. . . . . . . . .

. . . . . . . . .

.

.

.

.

23 26 32 43 61 70 71 75 77 77 79

Genetics of Adenoviruses

HAROLD S. GINSBERGAND C. S. H . YOUNG I. Introduction . . . . I1. Isolation of Adenovirus Mutants HI. Genetic Characterization . .

.

.

.

.

.

.

.

. . . . . . . . . . . . . . . . . . V

91 101 105

vi

CONTENTS

IV . Phenotypes of Adenovinis Mutants . . . . V. Summing Up . . . . . . References .

.

.

. .

.

. .

. .

.

. .

.

. .

.

. .

.

. .

.

. .

116 123 126

Molecular Biology of the Carcinogen. 4-Nitroquinoline 1 -Oxide

MINAKONAG.40

AND

TAKASHI SUGIMURA

I. Introduction . . . . . . . . . . . . . I1. Mutagenic Activity of 4-Nitroquinoline 1-Oxide on Organisms . . . 111. Chromosome Aberrations . . . . . . . . . . . IV . Repair of 4-Nitroquinoline 1-Oxide-Damaged DNA . . . . . V . Interaction of 4-Nitroquinoline 1-Oxide and Its Derivatives with Nucleic . . . . . . . . . . . . . . Acids . 1’1. Interaction of 4-Nitroquinoline 1-Oxide and Its Derivatives with Protein VII . Recent Information on Carcinogenesis by 4-Nitroquinoline 1-Oxide . . VIII . 4-Nitroquinoline 1-Oxide and Microbial Screening Method for Carcinogen IX . Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . . .

132 133 140 143

151 156

157 163 163 164

Epstein-Barr Virus and Nonhuman Primates: Natural and Experimental Infection

A. FRASK.W . A . ASDIMAN.

AND

C . MILLER

I. Introduction . . . . . . . . . . I1 . EBV Reactive Antibodies in Nonhuman Primates . . 111. Lymphoblastoid Cell Lines ( L C L ) from Nonhuman Primates IV . Experimental Infection of Nonhuman Primates with EBV . V . Summary and Conclusions . . . . . . . . . . . . . . . . . References .

.

.

.

. .

. .

. .

. .

. .

177 186 . 197 . 1%

. .

.

.

171

. 172

Tumor Progression and Homeostasis

RICHMOXDT. PREHN I. Introdrrction . . . . . . . . I1. Initiation: The First Step in Tumor Progression . I11. The Subsequent Steps in Tumor Progression . IV . Immunity as a Homeostatic Mechanism . . V . Concluding Remarks . . . . . . References . . . . . . . . .

. .

. .

. .

. .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

203 204 . 213 . 218 . 233 . 233

Genetic Transformation of Animal Cells with Viral DNA of RNA Tumor Viruses

Mmosmv HILL AKD I . Introduction I1. Endogeneous

.

.

Viruses

. .

. .

. .

JAXA

. .

. .

HILLOVA

. .

. .

. .

. .

. .

. .

237 239

vii

CONTENTS

I11. Virus-Specific DNA in Virus-Infected and Uninfected Cells IV. Infectivity of the Viral DNA . . . . . . . V . Sizing the RNA Genome in Virus Particles . . . . VI Search for Transforming Genetic Material . . . . . VII. Conclusions . . . . . . . . . . References . . . . . . . . . . .

.

SUBJECTINDEX . CONTENTS OF

.

.

.

. PREVIOUS VOLUMES.

. .

. . .

. .

. . . . . . . .

. . . . .

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

243 246 271 274 287 289 299 304

This Page Intentionally Left Blank

CONTRIBUTORS

TO VOLUME 23

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ALROY, * Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts (23)

JOSEPH

W. A. ANDIMAN,Departments of Pediatrics and Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut ( 171) A. FRANK,Departments of Pediatrics and Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut ( 171 )

HAROLD S . GINSBERG, Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York (91)

W . E. HESTON,Laboratory of Biology, National Cancer Institute, National lnstitutes of Health, Bethesda, Maryland ( 1 ) MIROSLAVHILL, Department of Cellular and Mokculur Biology and Equipe de Recherche No. 148 du C.N.R.S., lnstitute of Cancerology and Immunogenetics, Villejuif, France (237) JANA HILLOVA, Department of Cellular and Molecular Biology and Equipe de Recherche No. 148 du C.N.R.S., lnstitute of Cancerology and lmmunogenetics, Villejuif, France (237) FREDERICK B. MERK,Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts ( 23) G. MILLER,Departments of Pediatrics and Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut ( 171 ) MINAKO NAGAO, National Cancer Center Research Institute, Chuo-ku, and lnstitute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan (131) RICHMONDT. PREHN,The lnstitute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pennsylvania ( 203 )

’Present address: Rush Medical College and Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois. ix

X

CONTRIBUTORS

TAKASHI SUGIMURA, Nationul Cancer Center Research Institute, Chuo-ku, and Institute of Medical Science, University of Tokyo, Minuto-ku, Tokyo, Japan ( 131)

RONALD S . WEINSTEIN,'Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts ( 2 3 ) C . S . H. YOUNG, Department of Microbiology, College of Physicians and Surgeons, Columbia University, New Ymk, New York (91)

Present address: Rush Medical College and Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois.

THE GENETIC ASPECTS OF HUMAN CANCER W. E. Heston Loboratory at Biology, National Cancer Institute. National Institutes of Health, Bethesda, Maryland

I. Introduction . . . . . . . 11. Cancer of the Breast . . . . . 111. Leukemia . . . . . . . . A. Studies on Inheritance . . . . B. Chromosomal Aberrations and Leukemia IV. Colorectal Cancer . . . . . . V. Gastric Cancer. . . . . . . VI. Retinoblastoma . . . . . . . VII. Xeroderma Pigmentosum . . . . V I E Albinism . . . . . . . . . . . . . . IX. Lung Cancer . References

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

. . .

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

.

.

. . .

.

.

. . .

.

.

.

.

. . . . . .

. . . . . .

.

.

.

.

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

1 3

7 7 9

10 12 13 15 16 17 19

1. Introduction

This chapter is concerned with the genetic transmission of factors influencing the occurrence of cancer in man. It is not a complete review of the subject. Such a review would require a large volume with half a volume of references. Cancers that have received greater emphasis in this area of investigation are discussed, and an assessment is made of the kinds of evidence in support of genetic influences. Selected references are given. An effort was made not to slight early investigations as is often done in reviews today. There was a peak of emphasis on the genetics of human cancer during the 1940s and early 1950s, stimulated in large part in the United States by a very informal Conference on Parental Influence in Relation to the Incidence of Human Cancer conceived and organized by Dr. C. C. Little and held as a part of the Fifteenth Anniversary Celebration of the Roscoe B. Jackson Memorial Laboratory at Bar Harbor, Maine, in 1944. It was concluded at this conference that evidence at that time on parental factors influencing occurrence of cancer in experimental animals, particularly mice, together with information already known regarding genetic influences in respect to cancer in man, made it imperative that geneticists extend their knowledge on the genetic influences in cancer in man, and place such knowledge in proper balance 1

2

W. E. HESTON

with the increase in information on the chemical and physical agents and other nongenetic factors. Several large studies were initiated soon thereafter. One on breast cancer was developed by Dr. Madge T. Macklin at Ohio State University. Another also on breast cancer was developed by Dr. C. P. Oliver at the Dight Institute of the University of Minnesota. Dr. Oliver later transferred to the University of Texas, where he developed a program in the same area; the Dight Institute program was carried on principally by Dr. Elving Anderson. At the same time a very significant program, principally on the genetics of gastrointestinal cancer, was being developed at the University of Utah by Drs. F. E. Stephens, Eldon J. Gardner, and Charles M. Woolf. This group had the advantages of access to the large polygamous families of the Mormons and the extensive family records in the Church's Genealogical Library in Salt Lake City. Another program deserving special mention was the one in Denmark involving the University Institute of Human Genetics under the direction of Dr. Tage Kemp, the Danish Cancer Registry under the direction of Dr. Johannes Clemrnenson, and the University Institute of Pathologic Anatomy directed by Dr. Julius Engelbreth-Holm. Extensive studies on genetics of leukemia and of breast cancer were carried out there. Productive programs were carried out elsewhere by other investigators, one of whom was Dr. R. P. Martynova in U.S.S.R. She deserves special mention not only because of the early work she did on the genetics of breast cancer in women, but also for her signal role in keeping the genetics of cancer alive in the U.S.S.R. during the Lysenko era. These programs, which utilized for the most part comparison of incidence of cancer in relatives of cancer probands with that in relatives of control probands, established that genetic factors were iiivolved in many kinds of cancer in man and could be demonstrated provided enough data were collected. The studies also defined some of the limitations of this approach. These studies are reviewed because of their own merit and so that today we may distinguish between new discoveries and extension or confirmation of observations made at that time. The concept of human cancer as a somatic mutation disease does not receive special emphasis here, but not because somatic mutation is not involved. It surely is, as some form of change in the genetic material of the cell, but the subject has recently been covered thoroughly bv Knudson (1975). Furtheimore, no attempt has been made to give a complete review of cytogenetics and cancer. This subject has been covered thoroughly in a recent volume edited by German (1974). Some attempt is made to forecast what future investigations might

GENETIC A S P E W OF HUMAN CANCER

3

reveal from what is being discovered in experimental animals. Of principal interest here is that many kinds of cancer in experimental animals are now known to be induced by viruses, and that many of these viruses are endogenous. These are of particular concern to the geneticist because of evidence that they are transmitted vertically as a part of the host genome, much as genes are transmitted. If viruses are involved in the induction of cancer in man, and they most surely will be shown to be involved, the geneticist must take a very active role in their discovery. It. Cancer of the Breast

Studies to date indicate an inherited influence in the etiology of breast cancer that is especially prominent in the case of premenopausal cancer. From a large proband study of cancer in Holland, Wassink (1935) observed that when the proband had cancer of the breast there was a significant increase of cancer among female relatives owing to an increase in the homologous form of cancer. This was followed by Martynova’s (1937) rather extensive study in the U.S.S.R. From her data on 201 breast cancer family histories, she concluded that hereditary factors play a definite role in predisposition to cancer of the breast in women. She also concluded that predisposition to cancer of the breast is in some way connected with predisposition to cancer in general. Jacobsen (1946), working at the University Institute of Human Genetics in Copenhagen and in collaboration with the Danish Cancer Registry, compared relatives of 200 breast cancer probands with like relatives of 200 controls and found what he termed an indubitable excess incidence of breast cancer among the female relatives of the breast cancer probands with exception of grandmothers. He interpreted these results as indicating that the hereditary predisposition was a major factor in the development of breast cancer. It was noted that the curve of the age distribution at the first manifestation of the disease in the 200 breast cancer probands had two peaks, at ages 45-49 and 60-64. It was further observed that probands with stronger evidence of hereditary iduence were in the early age group. This is comparable with the difference between pre- and postmenopausal breast cancer described recently by Anderson ( 1972). In 1955, Woolf reported on his study of breast cancer in the Utah population. He,selected 216 patients who had died from breast cancer as probands and collected cancer data on their relatives. The number of deaths from cancer in these relatives was compared with an expected number based on proportionate mortality rates from the general popula-

4

W. E. HESTON

tion. Also, by the sequential analysis method he compared the. frequency of cancer in the families of the breast cancer probands with the frequency in a control sample. In addition to data on mothers and sisters of the probands, he also collected data on fathers and 'brothers since he was interested in whether his data would confirm Martynova and others who considered that susceptibility to cancer of the breast was only one manifestation of susceptibility to cancer in general or confirm Penrose et al. (1948) and others who considered the predisposition to be organ specific. Female relatives of the breast cancer probands had a higher incidence of cancer of the breast than the female relatives of the control probands and higher than expected from the general population, confirming that there was an inherited predisposition. The fact, however, that the frequency of other types of cancer in all four groups of relatives g a s no greater than in the control sample indicated that the inherited predisposition was organ specific. Later studies of Penrose et al. (1948), Anderson et aZ. (1958), Oliver ( 1959), and Macklin ( 1959) have been published with similar conclusions. Since Macklin's study was probably the most comprehensive, it will be discussed here. She collected complete data on mothers, grandmothers, aunts, and sisters of three groups of probands: ( 1 ) women with diagnosed breast cancer, ( 2 ) women with some cancer other than of the breast, and (3) women who had had no known cancer. Breast cancer occurred 2 or 3 times as frequently among relatives of the breast cancer probands as would have been expected from mortality rates or proportionate death rates. Among relatives of the probands with some other cancer, breast cancer occurred even slightly less frequently than expected, especially on mortality rates, thus failing to support any possible genetic relationship between breast cancer and other types. There was no difference between the frequency of breast cancer among relatives of the probands with no cancer and what would have been expected on mortality rates or proportionate death rates. From these data, Macklin concluded that there was some factor or factors that caused the relatives of breast cancer patients to have significantly more breast cancer than would have been expected if they had experienced the same risk as the population with which they were compared. She pointed out that these factors might be in the environment or the genes or both. Macklin compared paternal aunts of the breast cancer probands with their maternal aunts and found no difference in frequency of breast cancer. She suggested that this would tend to rule out environmental factors and indicates the action of genetic factors, since it would be

GENETIC ASPECTS OF HUMAN CANCER

5

unusual to find the same environmental factors influencing the fathers’ mothers and sisters in the same way they influenced the mothers’ mothers and sisters. Comparison of paternal and maternal grandmothers of the breast cancer probands was of particular importance in view of the maternally transmitted mammary tumor virus (MTV) of the mouse. If there were a milk-transmitted breast cancer virus in women as in mice, one could expect a higher frequency of breast cancer in the maternal grandmothers than in the paternal grandmothers. From the fact that there was no difference between the two groups of grandmothers, she concluded that, if there is a milk-borne virus for breast cancer in women, it must be ubiquitous and some other agent is the deciding factor. These are very significant observations in relation to our present knowledge of transmission of mammary tumors in mice (for review, see Heston, 1973). While Macklin did not rule out the possibility of a breast cancer virus transmitted from parents to offspring, if such a virus exists in women the best experimentaI model is probably not the strain C3H model, where the virus (MTV) is primarily transmitted through the milk. The best model may be the strain C3HfB model where the virus, in this case nodule-inducing virus (NIV), is transmitted through the male as readily as through the female and is not transmitted through the milk; or the strain GR model, where the virus is transmitted through the male as readily as through the female but can also be transmitted through the milk. Present efforts are directed toward trying to find a virus in the milk of women, but this seems to be primarily because the milk is a convenient place to look for it, It must be pointed out that some suggestive, although far from conclusive, evidence for the presence of virus has been found. However, if any breast cancer virus in women is like NIV, it would not be expected to be in the milk, at least in detectable amounts. There is considerable evidence that the mammary cancer virus in mice is transmitted as a provirus, i.e., that the viral information is integrated in the genome of the mouse. It may prove even to be transmitted as a dominant gene ( Bentvelzen, 1972). Information thus far from studies of human breast cancer would suggest that if there is a breast cancer virus, it too is probably transmitted genetically and thus the virologists are going to need the assistance of geneticists in discovering it. Again one is reminded that geneticists made the original discovery in respect to the MTV in mice (Jackson Laboratory Staff, 1933; Korteweg, 1934). In relation to some of the earlier observations of Jacobsen (1946) referred to above, Anderson (1972) separated his breast cancer probands

6

W. E. HESTON

into premenopausal and postmenopausal cases. Breast cancer was increased about 3-fold in the relatives of the premenopausal group, but was not increased in the relatives of the postmenopausal group. He also noted a 5-fold increase in relatives of bilateral breast cancer patients; and in relatives of patients with both premenopausal and bilateral cancer the rate was increased 9-fold. He concluded that genetic factors must play a more important role in patients with early onset of multiple disease than in patients with late onset of a single tumor. The same situation could be expected if a vertically transmitted virus were a factor in inducing breast cancer. Anderson's observations are in line with those made in the mouse, where a strong genetic component or a strong viral factor, or both, results in early mammary tumors, the females often having multiple mammary tumors. It is thus in these patients with premenopausal and bilateral cancers that one would expect to have the greatest chance of demonstrating any breast cancer virus. The possibility of a genetic relationship between breast cancer and other forms of cancer suggested by the early works of Martynova (1937) and others is finding support in certain family studies reported recently. Li and Fraumeni (1969) described four families showing a concordance of soft tissue sarcomas, leukemia, breast cancer, and an apparent excess of multiple primary malignant neoplasms. Later Lynch et a2. (1973~) reported a study of 34 families in which two or more first- or seconddegree relatives had breast cancer. Of these 34 families, 11 had firstor second-degree relatives with associated soft tissue sarcomas, leukemia, or brain tumors, or combinations of these malignant neoplasms. In another study of 22 families, Lynch et al. (1973b) observed an association of gastrointestinal and breast cancer. Through three generations of two families reported by Lynch et al. (1974), there was an apparent association between breast -and ovarian cancer. One might expect the association between breast and ovarian cancer to be caused by hormonal factors. The administration of estrogen results in neoplasms in several organs of the endocrine and reproductive systems in mice. Woolley et al. (1952) induced adrenal, pituitary, and mammary gland neoplasms in certain hvbrid mice by the hormonal imbalance resulting from early castration. *In the absence of administered hormonal factors, one might expect such endocrine influences to be basically genetic. Such associations of different forms of cancer presumably also could result from viral or genetic factors. A vertically transmitted virus with the oncogenic capacity of the polyoma virus (Stewart et al., 1958) could result in such combinations. On the other hand, a single gene, like the Av or AZ'V genes of the mouse that increase the occurrence of hepa-

GENETIC ASPECTS OF HUMAN CANCER

7

tomas, mammary tumors, pulmonary tumors, and leukemias (Heston and Vlahakis, 1968), if present in human beings could also result in such associations. 111. Leukemia

A. STUDIESON INHERITANCE One of the most extensive proband studies on the genetics of leukemia was carried out in Denmark by Videbaek (1947). Data were collected on 209 leukemia probands and their 4041 relatives and on 200 sound control probands and their 3641 relatives with good agreement between the age distribution of the two groups of relatives. Videbaek reported among the relatives of the leukemia patients an excessive incidence of cancer, but this was due to high incidence of all forms of the disease. In the families of the 209 leukemia patient probands, however, 17 had at least one other verified case of leukemia whereas in the families of the 200 control probands there was only one case of leukemia. Thus, there was significantly more leukemia among the relatives of the leukemia probands than among the relatives of the controls. From analysis of these 17 families and others from the literature making a total of 39, it was concluded that genetic factors had a role in the occurrence of leukemia, but the mode of inheritance could not be determined. It appeared that genes were controlling a predisposition to the disease, leaving open the possibility of the additional influence of chemical or physical carcinogens or viruses. There was no evidence of sex limitation or sex linkage and no evidence of extrachromosomal or maternal inheritance, as had been shown by that time for mammary cancer in mice. Genetically, leukemia appeared as an entity with the various types occurring among the relatives of the probands. Chronic lymphogenous leukemia, and probably also acute leukemia and chronic myelogenous leukemia, tended to occur earlier in the familial cases than in the sporadic ones. These observations of Videbaek, with the exception of the increase in cancer in general in families of leukemia probands, might be expected from what we have observed of the occurrence of leukemia in certain inbred strains of mice and from the results of the classic studies of Cole and Furth (1941) and MacDowell and co-workers (1945). They demonstrated that genetic factors were involved in mouse leukemia, and these appeared to be multiple. Although Videbaek's observations were not confirmed by a more recent study of leukemia in man by Steinberg (1960), the excess of leukemia in sibships has been confirmed

8

W. E. HESTON

for childhood leukemia by Stewart (1961) and Miller (1963). The frequency is about four times normal expectation. A classical approach to the ident&cation of genetic factors is the comparison of concordance in identical or monozygous twins with that in fraternal or dizygous twins. Since nongenetic factors would be about as nearly alike in the dizygous twins as in the monozygous twins and since the latter would be identical genetically except for mutations occurring after the splitting of the zygote, a greater concordance would be expected between the monozygous pairs if genetic factors were involved. MacMahon and Levy (1964) have reported a concordance rate of about 25%for childhood leukemia among monozygous twins while in the literature only three concordant sets were described as dizygotic and none was well documented. If a twin child falls ill with leukemia the monozygous mate has one chance in four or five of also developing the disease and usually within weeks or a few months. Although the data from twins are strong evidence for genetic factors in childhood leukemia, Clarkson and Boyse (1971) have suggested as an alternative explanation the possibility that high concordance in the monozygous twins may be due to fusion of placentas with common circulation permitting the formation of hematopoietic chimerism. They are suggesting that the neoplastic change may occur before birth and that many cases of concordance may represent only one occurrence of leukemia, not two. They further point out that whether or not this is the case might be shown through cytogenetic studies. This evidence for inherited influences, especially in childhood leukemia, is in line with the high incidence and early appearance of leukemia in certain inbred strains of mice where the genetic influence is strong, but it is also generally true in mice where a potent leukemia virus is involved. The significant demonstration by Gross (1951) of a vertically transmitted leukemia virus in the mouse eventually led to the concept of genetically transmitted C-type leukemia viruses put forth by Huebner and his associates (Huebner and Todaro, 1969) as their oncogene theory. This postulates, like the provirus theory of the transmission of the B-type mammary tumor virus, that the C-type oncogenic RNA viral information is transmitted as DNA in the host genome. Actual identificaton and location of the locus or loci has come forth from works of others, particularly Rowe and associates (1972). From rapidly accumulating information on leukemia viruses in mice and other experimental animals, it appears likely that a leukemia virus will eventually be found in man. If so, it probably also will be genetically transmitted, and thus here geneticists working in collaboration with virologists will again be able

GENETIC ASPECTS OF HUMAN CANCER

9

to make a real contribution to out understanding of the transmission of the disease.

B. CHROMOSOMAL ABERRATIONS AND LEUKEMIA In the 1930s, Dr. Warren H. Lewis was observing that neoplastic cells had more chromosomal morphologic irregularities than did normal cells. He asked the basic question whether these changes were the cause of the neoplasia, the result of the neoplastic change, or a manifestation of a basic factor causing both the neoplasia and the chromosoma1 changes. We still do not have the final answer to his question although certain chromosomal traits are found to be of value in diagnosis and in determining cancer risks. Dr. Lewis’ basic observations stimulated a vast number of karyotypic studies of tumors of all kinds, which for the next two decades were relatively unproductive. The changes did not appear to have much uniformity in their patterns of manifestation or in their causation. The picture changed in 1960 when Nowell and Hungerford reported that a minute chromosome replaced one of the smallest autosomes in cells of seven patients with chronic granulocytic leukemia which they had studied. This minute chromosome was not seen in cells of four cases of acute granulocytic leukemia in adults or of six cases of acute leukemia in children. There were no other frequent or regular chromosomal changes in the cells of the chronic granulocytic leukemia patients, and all patients had many cells with a normal karyotype. Thus, a chromosomal marker for chronic granulocytic leukemia, that was to be confirmed many times, had been identified. This minute chromosome is now commonly referred to as the Philadelphia chromosome (Phl) (Sandberg et al., 1964). This observation of a definite chromosomal change associated with a particular kind of neoplasm has given a renewed stimulus to cytogenetic studies of neoplasia, particularly the leukemias and other reticulum cell neoplasms. The observation that Bloom’s syndrome is associated with increased susceptibility to acute leukemia is of particular interest to the geneticist because the syndrome itself is inherited through an autosomal recessive gene (Sawitsky et al., 1966). The syndrome is characterized by photosensitive telangiectasia of the face. Data indicate that one of eight persons with the syndrome will develop leukemia during the first 30 years of life. It is thought that the causation of leukemia is related to the observation of excessive chromosomal breakage and rearrangement in cultured cells from patients with the syndrome. Similarly, the recessively inherited Fanconi’s aplastic anemia ( Bloom

10

W. E. HESTON

et al., 1966) and ataxia-telangiectasia (Hecht et al., 1966) are also associated with an abnormal number of chromosomal aberrations, and both diseases also appear to be associated with an increased risk for leukemia and other neoplasms. Children with Down’s trisomy syndrome, mongolism, have an increased incidence of leukemia (Jackson et al., 1968). They have an extra chromosome number 21 of the G group of autosomes. Males are invariably infertile, but female mongols have been reported with offspring. The female mongol produces two kinds of gametes, one with 24 chromosomes which when fertilized by a normal sperm results in a mongol, and the other with 23 chromosomes which when fertilized produces a normal zygote (see Penrose and Smith, 1966). To this list could be added numerous less well defined chromosomal aberrations that appear to be associated with increased leukemia. It appears from these observations that there is an association between unbalanced karyotypes and neoplasia, particularly leukemia. It may be that such cells are more susceptible to oncogenic viruses. Todaro and co-workers (1966; Todaro and Martin, 1967) have shown that cultured fibroblasts from patients with Fanconi’s anemia or mongolism show increased malignant transformation when exposed to SV40. Hirschhorn and Block-Shtacher (1970) have evidence that these cells are also more susceptible to malignant transformation when treated with some of the chemical carcinogens. Hirschhorn ( 1970) concludes that these chromosomal aberrations lead to a selective advantage causing the increase in cell number called leukemia and he postulates that the advantage may be due in part to membrane changes leading to alterations in pseudoimmunological recognition mechanisms. How far have we come since the 1930s in answering Dr. Lewis’ questions on the relationship of these chromosomal aberrations to the neoplastic process? We have made progress and have opened up new areas for research, but we do not have the final answers. In the meantime we have discovered some markers to aid in prediction and diagnosis. IV. Colorectal Cancer

While there is abundant evidence that colorectal cancer may be familial, the primary evidence of genetic factors is found in the adenocarcinoma of the colon arising from inherited polyposis. It is well established that in individuals with this condition some of the polyps progress into carcinomas. Dukes (1952) gave an extensive review of the subject, and a recent review has been published by Burdette (1970). The classical condition is multiple polyposis of the colon first shown by Cockayne (1927) to be due to a single dominant gene. This has

GENETIC ASPECTS OF HUMAN CANCER

11

been confirmed by data from many more recent family pedigrees. Reed and Nee1 (1955) in a study of multiple polyposis of the colon estimated that in the state of Michigan, the minimum frequency at birth of individuals with the dominant gene for the trait is about 1 in 8300. Relative fitness of individuals with the gene derived from relative reproductive span was estimated to be 0.78. From these estimates of frequency and relative fitness and with consideration of known biases involved, the mutation rate of the multiple polyposis dominant gene was estimated to be within the range 1 to 3 x 105/geneper generation. A second polyposis important in the genesis of colorectal carcinoma is that described by Gardner (1955, 1962) and now usually referred to as the Gardner syndrome. Here the gastrointestinal polyps occur in association with osteomas, fibromas, and sebaceous cysts. Since these associated lesions occur earlier than the intestinal polyps or the resultant carcinomas, they are of diagnostic value. Analysis of the original kindred brought to the attention of Gardner and Stephens (1950) because it contained 9 cases of cancer of the digestive tract, 8 of which were colorectal, indicated that like multiple polyposis, the Gardner syndrome is inherited as a single dominant gene. Starting with probands obtained from death certificates of the state of Utah and family histories from the records of the Latter-Day Saints Genealogical Society in Salt Lake City, Woolf (1958) carried out a study to test the hypothesis that a genetic component, independent of the gene associated with multiple polyposis, existed for colorectal cancer. Individuals selected as probands were white persons born or raised in Utah with diagnosed carcinoma of the large intestine. Individuals with any indication that multiple polyposis had been present were excluded. In the study involving 242 families, he observed death certificates of 145 fathers, 142 mothers, 309 brothers, and 167 sisters of the probands or a total of 763. Of these, 26 had cancer of the large intestine as compared with 8 among the relatives of well-matched controls. Furthermore, there was a significant increase in all four classes of relatives over controls. There was no difference between relatives of the intestinal cancer probands and controls in respect to cancer at other sites. Woolf concluded that his results were compatible with the hypothesis of a genetic component, but he pointed out that his study, like so many comparable studies for other types of human cancer, did not distinguish between genetic and familial factors. Macklin (1960) studied families of 167 patients with gastric cancer and 145 patients with cancer of the large intestine where inherited multiple polyposis was lacking. Her aim was to see whether there was evidence for a genetic basis for these two forms of cancer, and if so was

12

W. E. HESTON

there evidence of any genetic factors in common with the two types. Gastric cancer was found more frequently among relatives of gastric cancer probands than in the general population, but cancer of the large intestine was not. Likewise, cancer of the large intestine was found more frequently among relatives of the probands with cancer of the large intestine than in the general population, but gastric cancer was not. This indicated that both types had familial factors favoring induction that did not influence the development of the other type. The fact that husbands and wives were not affected more often than expected on the basis of random distribution whereas parent and child and two siblings were, indicated that the familial factors probably were genetic. No simple genetic pattern for inheritance was revealed in the families with gastric cancer or those with cancer of the large intestine where there was no multiple polyposis. She concluded that the genetics of both was probably polygenic in contrast with the single dominant gene inheritance of multiple polyposis. From a genetic study of colon cancer based upon cancer in probands and first-degree relatives with matched controls, Lynch et al. (1973a) have reported site-specific colon cancer, breast cancer, and multiple primary malignant neoplasms occurring in significant excess among firstdegree relatives of the colon cancer probands compared with the controls. This suggests a common familial etiologic factor, which they suggest may be a gene or genes or a familial occurring oncogenic virus. While this is an interesting observation, it is not supported by the previous genetic studies of cancer of the colon by Woolf or those by Macklin. Furthermore, these observations do not find general support in the work on genetics of cancer or of viral oncogenesis in experimental cancer. The only virus definitely shown to induce mammary cancer is the B-type RNA mouse mammary tumor virus with its variations, and this induces only mammary cancer and only in mice. The other known oncogenic C-type RNA viruses induce only leukemias and sarcomas but have not been shown to induce carcinomas. The results observed by Lynch et al. (1973a) would require some unusual virus like the so-called polyoma virus that is a DNA virus ( Stewart et al., 1958). V. Gastric Cancer

A number of investigators have searched for evidence of a genetic factor in gastric cancer (Videbaek and Mosbeck, 1954; Hagy, 1954; Woolf, 1955, 1956; Macklin, 1960). The approach has been the usual one of comparison of gastric cancer in relatives of gastric cancer probands with that in comparable relatives of controls. In general there has been found to be more gastric cancer in the relatives of the probands

GENETIC ASPECTS OF HUMAN CANCER

13

than in those of the controls, although in some studies the difference has been barely significant. Woolf conducted an extensive study from the Utah families including 173 fathers, 168 mothers, 390 brothers, and 260 sisters of gastric cancer probands and a like number of controls. More gastric cancer occurred in each group of relatives of probands than in the comparable groups of control relatives, the total numbers being 66 compared with 32. That the tendency was site-specific was indicated by the fact that deaths due to other types of cancer in the families of the probands totaled 94 compared with 82 in the controls. The difference between these two incidences was not considered significant. Here again he indicated that it is difficult to tell whether the factor is genetic or familial. Interesting associations between gastric cancer and other nonneoplastic diseases have been revealed. Mosbeck (1953) compared the incidence of gastric cancer among relatives of 234 patients with pernicious anemia with that in relatives of 226 control patients and also with that in the general population of Denmark, Incidence of gastric cancer was significantly greater in the relatives of the pernicious anemia patients than in either of the other groups, but there was no difference in incidence of cancer at other sites. Achlorhydria has also been shown to be associated with gastric cancer (Comfort et al., 1947), and Videbaek and Mosbeck (1954) have suggested that the genetic factor is that causing achlorhydria, which in turn predisposes to both pernicious anemia and gastric cancer. The incidence of gastric cancer seems also to be higher in people of blood group A than in those of the other groups (Aird and Bentall, 1953). A number of genes that control other traits in the mouse also increase or decrease the occurrence of various kinds of cancer, but these genes usually have an effect on growth of the animal and those that increase growth increase occurrence of cancer while those that decrease growth decrease occurrence of cancer ( Heston et al., 1952, Heston and Vlahakis, 1961). These genes, however, have not as yet been associated with gastric cancer in the mouse. The histocompatibility H-2 locus of the mouse has been associated with mammary tumors (Miihlbock and Dux, 1974) and with leukemia of the mouse (Lilly and Pincus, 1973), but in each case the effect of H-2 is on the respective oncogenic virus. VI. Retinoblastoma

Retinoblastoma is a highly malignant tumor of the eye that usually occurs in children from a few months to 4 years of age. In the past the disease was almost invariably fatal, and at present it is considered to be responsible for about 1%of all deaths from cancer in early childhood

14

W. E. HESTON

and 5%of all blindness in children (Smith and Sorsby, 1958). In early publications a few cases were reported, however, of individuals who survived the disease and transmitted it to one or more of their offspring. With recent advances in therapy, the number of such cases is increasing. Through the years retinoblastoma frequently has been cited as an example of an inherited neoplasm in man, and it was generally considered to be due to a single dominant gene (see Neel and Falls, 1951). Neel and Falls made a complete survey of the state of Michigan and found 49 sibships with one or more children with retinoblastoma born to normal parents. On the assumption that these were due to a dominant mutation involving a single locus and with the total number of births in the state during that period, they calculated the mutation rate per gene to be 2.3 x This was considerably higher than the rate of 1.4 x which they cited as having been determined by Philip and Sorsby from material from London and which suggested to them that the rate might be different in different geographical areas. Recent work indicates that in considering the genetics of the disease, the bilateral cases must be separated from the unilateral cases. Sorsby (1972) has reported from his own records and from the literature 19 survivors of sporadic bilateral retinoblastoma with a total of 39 offspring of whom 17 were affected in both eyes and 3 in one eye. This confirms the single dominant gene inheritance of the bilateral disease. It has even been determined through cvtogenetic studies that this locus is on the long arm of a D chromosome (see Orye et al., 1971). The much more common unilateral cases apparently are not transmissible to the offspring escept for 5-10% that appear to be due to the same inherited dominant gene but are unilateral because of reduced expression. The major portion of the unilateral cases, if genetic, would be due to a somatic mutation presumably at the same locus. These are factors to be considered in genetic counseling which is becoming more urgent as chemotherapy and other fonns of treatment become more successful. For the bilateral cases one would expect 50% of offspring to have the trait. One would expect the majority of the unilateral cases not to be transmitted. Difficulty will arise in identifying the 5 1 0 %phenotypically unilateral cases that in fact represent incomplete expression of the germinal mutation for bilateral retinoblastoma. These individuals, if thev could be identified, could be advised that they also could expect to transmit the disease to 50% of their offspring. Sorsby (1972) suggests that there is a possibility that these few hereditary unilateral cases may be identified histologically. Cumings and Sorsby ( 1944) observed that unilateral neoplasms originated exclusively from the outer nuclear laver of the retina whereas the bilateral ones had

GENETIC ASPECTS OF HUMAN CANCER

15

a more diffuse origin. It remains to be seen whether the hereditary unilateral disease also has a diffuse origin, VII. Xeroderrna Pigmentosurn

Xeroderma pigmentosum is a hereditary disease found in one person in approximately 250,000 of the general population. Its occurrence is worldwide in all races. Most patients with the disease have an acute sensitivity to sunlight manifested early in life. Freckles develop on sunexposed areas where the skin becomes dry and scaly, hence the name “dry pigmented skin.” Later neoplasms of the skin appear. These are usually basal cell carcinomas and less frequently squamous cell carcinomas. In patients in which early neoplasms receive effective treatment, up to 50% may later develop malignant melanoma. Abnormalities of other organ systems, including the nervous system, are often associated with the cutaneous lesions. By analyzing family histories in which xeroderma pigmentosum occurred, Cockayne (1933) cdncluded that inheritance was by a single autosomal recessive gene, a conclusion supported by additional family histories published by Macklin ( 1936). Haldane’s analysis ( 1936) indicated to him that the true pattern of inheritance was incomplete sex-linkage, the gene being borne in that particular region of the sex chromosome common to X and Y. Later, in a critical review of autosomal and partial sex-linkage in man, Morton (1957) showed that the inheritance could not be partial sex-linkage and presented evidence. that the earlier tests had confounded sex-bias with partial sex-linkage. Since heterozygotes show the freckling and since homogygotes seldom reproduce, the trait sometimes has been referred to as semidominant sublethal. Recent work of El-Hefnawi et al. (1965) indicates that the locus for xeroderma pigmentosum is linked with the genes for the ABO blood types with a recombination frequency of about 18%.This would refute the claim that it was on the sex chromosome and reestablish it as autosomal. There was, however, an unusually strong nonrandom association between the disease and blood group 0. Eighty percent of the patients in this study were 0 whereas on a random basis one would expect only about 35%.This suggested to the authors some selective mechanism favoring survival of xeroderma pigmentosum homozygotes who are type 0 as compared with those who are A, B, or AB. A possible lead to the mechanism of gene action in xeroderma pigmentosum carcinogenesis appeared in a signal discovery of Cleaver (1968). His observation was that whereas normal skin fibroblasts in culture can repair ultraviolet radiation damage to D N A by inserting new bases into DNA, in fibroblasts with the xeroderma pigmentosum

16

W. E. HESTON

gene such repair replication of DNA is either absent or much reduced. He concluded that this faulty DNA repair must be related to carcinogenesis. Soon thereafter, Epstein and co-workers (1970) showed that this defect in DNA synthesis occurred in the epidermal cells in vivo of patients with the disease. They postulated that since defects in DNA repair result in increased mutation rate induced by ultraviolet light in bacteria, a similar increase in mutation rate in the skin of these patients after exposure to the sun might cause the subsequent neoplasms of the skin. This has been followed by extensive biochemical studies of the disorder not only in fibroblasts of xeroderma pigmentosum patients, but also in their lymphocytes, where ultraviolet-stimulated thymidine incorporation has been investigated (see Robbins et al., 1974). Cell fusion studies have been carried out that suggested heterogeneity of the genetic lesion. Fibroblasts from certain pairs of patients when fused show complementation overcoming the defect in DNA repair in each member of the pair. Yet all these studies have not actually linked the faulty DNA repair to carcinogenesis in these patients. In fact, studies by Robbins and Burk (1973) of one xeroderma pigmentosum patient whose cells failed to show faulty DNA repair have suggested that some mechanism other than enhancement of UV carcinogenesis by defective DNA repair may be responsible for the skin tumor carcinogenesis in this patient and possibly all patients with xeroderma pigmentosum. Despite the apparently normal DNA repair in the fibroblasts and lymphocytes, this patient developed numerous cutaneous neoplasms. The ,possibility that the xeroderma pigmentosum gene is increasing carcinogenesis by increasing oncogenic virus-induced transformation in cells of patients with the disease, has been suggested. Fibroblasts from patients with Down’s syndrome and Fanconi’s anemia, both of which are associatcd with a high incidence of neoplasia, show a marked increase in susceptibility of SV40-induced transformation as noted in the above section on leukemia. In a studv by Key and Todaro (1974) neither xeroderma pigmentosum fibroblasts, which show n o m d susceptibility to SV40 transformation, nor normal skin fibroblasts showed any increase in susceptibility to the virus-induced transformation following exposure to UV radiation. Their results, however, are contrary to some preliminary reports referred to by these authors. VIII. Albinism

In certain aspects albinism in man can be compared with xeroderma pigmentosum. Albinism has even been more clearly shown to be due

GENETIC ASPECJX OF HUMAN CANCER

17

to a simpIe MendeIian recessive gene than has xeroderma pigmentosum, and in this respect it is like albinism in other species of mammals. However, whereas albinism is one of a series of alleles in other species, such as the mouse, definite evidence for other alleles has not been found for man. Albinism occurs much more frequently than xeroderma pigmentosum: the incidence in Europeans is estimated as about one in 10,000, and in the Navajo Indians, it appears twice in 10,000. It has been studied extensively by Keeler (1963, 1964) among the Cuna Indians of the San Blas islands in the Caribbean. Here is found the highest incidence in the world, now being about 45 per 10,000. Albinos have snow-white hair and a great reduction of pigmentation in the eyes and skin. This causes them to be sensitive to the ultraviolet rays of the sun, but in the temperate zone they get along comparatively well by wearing dark glasses and protective clothing and they seldom develop skin cancer. In the tropics, however, where the Cuna Indians are, the intense sun produces a premalignant actinic keratosis in practically all albino children by two or three years of age. Frequently, the terminal episode is metastatic carcinoma. All skin cancer in the Cuna albinos is squamous cell carcinoma, in contrast with the xeroderma pigmentosum patients, in which the basal cell carcinoma is the usual cutaneous neoplasm. The abnormal DNA repair of UV damage of the cells of xeroderma pigmentosum cited in the previous section raises the question whether the cells of albinos are similarly affected. The carcinogenic action of the UV rays in the albino individuals, however, may not be unlike that in the usual Caucasians in whom excessive sunlight] such as is experienced in the Southwestern states, induces neoplasms of the skin. In the albinos the UV rays are probably more effective because of lack of pigmentation, just as Hereford cattle are more susceptible to sunlightinduced cancer eye than other breeds of cattle with pigmentation around the eyes (see Anderson, 1959). However, this still does not explain the mechanism by which UV rays induce neoplasms. Gene mutation is undoubtedly involved. IX. lung Cancer

One of the neoplasms in experimental animals for which genetic factors have been clearly shown is the lung tumor in the mouse. As early as 1926, Lynch published data showing an hereditary influence. Later, in a rather extensive study of both induced and spontaneous lung tumors, Heston (1942a,b) showed that the tumors resulted from multiple genetic and nongenetic factors with additive effects, the tumor

18

W. E. HESTON

appearing when the total effect passed a certain threshold. Thus, a carcinogen did not wipe out the genetic effect, but its influence was added to the genetic influence. For years geneticists avoided any genetic study of lung cancer in man, pointing out that its incidence was so low that a genetic study would be difficult and also that the disease in man was not comparable with the inherited disease in mice. While lung tumors in mice are alveolar in origin, most lung cancer in man is bronchogenic. With the great increase in lung cancer due to cigarette smoking and probably other factors, such as fumes from industry and automobiles, a genetic study became feasible. In 1963, Tokuhata and Lilienfeld reported on a comparison of relatives of 270 lung cancer probands with race-sex-age-residence matched controls. They observed a significant excess in lung cancer mortality among the proband relatives that could not be accoiinted for by age, sex, generation, and cigarette-smoking factors. The effect of familial factors among nonsmokers was similar in both men and women. The smoking factor appeared to be greater than the familial factor among men, but among women the familial factor appeared to be greater than the smoking factor. Their conclusion was that genetic factors may play a role in the etiology of lung cancer together with such environmental factors as cigarette smoking-a conclusion that might have been predicted from the experimental studies. Questions arose as to the possible relationship between the genetic susceptibility of the individual to lung cancer and his smoking habits. Several investigators in arguing against any causal relationship between cigarette smoking and lung cancer suggested that cigarette smoking and lung cancer might have a common genetic basis. Fisher (1958) concluded from his observations on smoking habits among twins that such was the case. To obtain further information on this question, Tokuhata (1963) obtained lifetime smoking experience data on his 270 lung cancer probands and their relatives and on the 270 controls and their relatives. He found evidence for a familial predisposition of the individual to the smoking habit, which he observed both in relation to and independentlv of the familial history of lung cancer. He found that lung cancer aggregates in families independentlv of smoking habit and that smokers aggregate in families independently of lung cancer. From this he concluded that the host susceptibilitv to cigarette smoking and the like susceptibility to cancer of the lung, both of which appeared to have a genetic basis, did not share a common genetic basis. Thus far, no virus has been shown to be causally related to lung tumors in mice, so one might not expect to find a virus in the etiology of cancer of this organ in man. If a lung cancer virus does occur in

GENETIC ASPECTS OF HUMAN CANCER

19

man, however, it may well be related to carcinogens that are involved. It may even be activated by them. It will certainly be controlled by the genetics of the individual. Therefore, the most likely individual in which to look for such a virus probably is the younger lung cancer patient with a strong familial factor and with a strong carcinogenic factor, such as cigarette smoking.

REFERENCES Aird, I., and Bentall, H. D. (1953). Brit. Med. J . 1,799-803. Anderson, D. E. ( 1959). In “Genetics and Cancer” (Staff of M.D. Anderson Hospital and Tumor Inst., eds.), pp. 364-374. Univ. of Texas Press, Austin. Anderson, D. E. (1972). J. Nut. Cancer Inst. 48, 1029-1034. Anderson, V. E., Goodman, H. O., and Reed, S. C. (1958). “Variables Related to Human Breast Cancer,” Univ. of Minnesota Press, Minneapolis. Bentvelzen, P. ( 1972). In “RNA Viruses and Host Genome in Oncogenesis” (P. Emmelot and P. Bentvelzen, eds.), pp. 309-337. North-Holland Publ., Amsterdam. Bloom, G. E., Warner, S., Gerald, P. S., and Diamond, L. K. (1966). N . Engl. J , Med. 274, 8-14. Burdette, W. J. ( 1970). In “Carcinoma of the Colon and Antecedent Epithelium” ( W. Burdette, ed. ) pp. 78-97. Thomas, Springfield, Illinois. Clarkson, B. D., and Boyse, E. A. (1971). Lancet 1, 699-701. Cleaver, J. E. ( 1968). Nature (London) 218,652-656. Cockayne, E. A. (1927). Cancer Reo. 2,337447. Cockayne, E. A. (1933). “Inherited Abnormalities of the Skin and its Appendages.” Oxford Univ. Press, London and New York. Cole, R. K., and Furth, J. ( 1941). Cancer Res. 1, 957-965. Comfort, M. W., Kelsey, M. P., and Berkson, J. ( 1947). J . Nut. Cancer Inst. 7, 367-373. Cumings, J. N., and Sorsby, A. (1944). Brit. 1. Ophthalmol. 28, 533-537. Dukes, C. E. ( 1952). Ann. Eugen., London 17, 1-29. El-Hefnawi, H. S., Smith, S. M., and Penrose, L. S. (1965). Ann. Hum. Genet. 28, 273-290. Epstein, J. H., Fukuyama, K., Reed, W. B., and Epstein, W. L. (1970). Science 168, 1477-1478. Fisher, R. A. ( 1958). Nature (London) 182, 108. Gardner, E. J. ( 1955). In “Novant’anni della Leggi Mendelione” (L. Gedda, ed.), pp. 321329. Istituto Gregorio Mendel, Rome. Gardner, E. J. (1962). Amer. J . Hum. Genet. 14, 376-389. Gardner, E. J., and Stephens, F. E. (1950). Amer. J. Hum. Genet. 2, 4148. German, J. ( 1974). “Chromosomes and Cancer.” Wiley, New York. Gross, L. ( 1951). Proc. SOC. Exp. Bid. Med. 78,342348. Hagy, G. W. (1954). Amer. J. Hum. Genet. 6,434-447. Haldane, J. B. S. (1936). Ann. Eugen. 7 , 2 8 5 7 . Hecht, F., Koler, R. D., Rigas, D. A., Dahnke, G. S., Case, M. P., Tisdale, V., and Miller, R. W. (1966). Lancet 2, 1193. Heston, W. E. (1942a). 1. Nut. Cancer Inst. 3,69-78. Heston, W. E. (1942b). J . Nut. Cancer Imt. 3, 79-82.

20

W. E. HESTON

Heston, W. E. ( 1973). Methods Cancer Res. 7, 115-129. Heston, W. E., and Vlahakis, G. (1961). J. Not. Cancer Inst. 26, 969-983. Heston, W. E., and Vlahakis, G. (1968). J. Nat. Cancer Inst. 40, 1161-1166. Heston, W. E., Deringer, M. K., Hughes, I. R., and Cornfield, J. (1952). J. Nut. Cancer Inst. 12, 1141-1157. Hirschhorn, K. (1970). Proc. Not. Cancer Conf. 6th, 1968 pp. 107-112. Hirschhorn, K., and Bloch-Shtacher, N. (1970). Proc. 23rd Annu. Symp. Fundam. Cancer Res. I969 pp. 191-204. Huebner, R. J., and Todaro, G. (1969). Proc. Not. Acad. Sci. US.64, 1087-1094. Jackson, E. W., Turner, J . H., Klauber, M. R., and Norris, F. D. (1968). J. Chronic Dis. 21, 247-253. Jackson Laboratory Staff. ( 1933). Science 78, 465-466. Jacobsen, 0. (1946). “Heredity in Breast Cancer,” Vol. 11. Lewis, London. Keeler, C. E. (1963). Nut. Cancer Inst., Monogr. 10,349359. Keeler, C . E. ( 1964). J. Hered. 55, 115-120. Key, D. J., and Todaro, G. J. (1974). J . Znoest. Dennutol. 62,7-10. Knudson, A. G. (1975). In “Cancer: A Comprehensive Treatise 1,” (F. F. Bedker, ed.), pp. 59-74. Plenum, New York. Korteweg, R. ( 1934). Ned. Tiidschr. Geneesk. 78, 240-245. Li, R. P., and Fraummi, J. F. ( 1969). Ann. Intern. Med. 71,747-752. Lilly, F., and Pincus, T. (1973). Adoan. Cancer Res. 17,231-277. Lynch, C. J. ( 1926). J . Exp. Med. 43,339-355. Lynch, H. T., Guirgis, H., Swartz, M., Lynch, J,, Krush, A. J., and Kaplan, A. R. ( 1973a). Arch. Surg. (Chicago) 106, 66-75. Lynch, H. T., Krush, A. J., and Guirgis, H. (1973b). Amer. J. Gastroentefol. 59, 31-40. Lynch, H. T., Krush, A. J., Harlan, W. L., and Sharp, E. A. ( 1 9 7 3 ~ ) .Amer. Surg. 39, 199-206. Lynch, H. T., Guirgis, H. A., Albert, S., Brennan, M., Lynch, J., Kraft, C., Pocekay, D., Vaughns, C., and Kaplan, A. (1974). Surg., Gynecol. Obstet. 138,717-724. MacDowell, E. C., Potter, J. S., and Taylor, M. J. (1945). Cancer Res. 5, 65-83. Macklin, M. T. (1936). Arch. Dermatol. Syph. 34, 656-675. Macklin, M. T. (1959). I.Nut. Cancer Inst. 22,937-952. Macklin, M. T. (1960). J. Nut. Cancer Znst. 24,551571. MacMahon, B., and Levy, M. A. (1964). N . Engl. J . Med. 270, 1082-1085. Martynova, R. P. ( 1937). Amer. J . Cancer 29,530-540. Miller, R. W. (1963). N. EngZ. J . Med. 268,393-401. Morton, N. E. ( 1957). Amer. J . Hum. Genet.9,55-75. Mosbeck, J. (1953). O p . Dom. Biol. Hered. Hum. 34, 108. Muhlbock, O., and D u x , A. ( 1974). J . Nut. Cancer Inst. 53,993-996. Ned, J. V., and Falls, H. F. ( 1951). Science 114, 419-422. Nowell, P. C., and Hungerford, D. A. (1960). Science 132, 1497. Oliver, C. P. (1959). In “Genetics and Cancer” (Staff of M.D. Anderson Hospital and Tumor Inst., eds.), pp. 427-438. Univ. of Texas Press, Austin. Orye, E., Delbeke, M. J., and Vandenabeele, B. ( 1971). Lancet 2, 1376. Penrose, L. S., and Smith, G. F. (1966). “Down’s Anomaly.” Little, Brown, Boston, Massachusetts. Penrose, L. S., Mackenzie, H. S., and Kam, M. N. (1948). Ann. Eugen., London 14, 234-266. Reed, T. E., and Neel, J. V. ( 1955). Amer. J. Hum. Genet. 7, 236-263.

GENETIC ASPECIS OF HUMAN CANCER

21

Robbins, J. H., and Burk, P.G. ( 1973). Cancer Res. 33, 929-935. Robbins, J. H., Kraemer, K. H., Lukner, M. A., Festoff, B. W., and Coon, H. G . (1974). Ann. Intern. Med. 80,221-248. Rowe, W. P., Hartiey, J. W., and Bremmar, T. (1972). Science 178, 860-862. Sandberg, A. A., Ishihara, T., Kikuchi, Y.,and Crosswhite, L. H. (1964). Ann. N.Y. ACUU!. Sci. 113,663-716. Sawitsky, A., Bloom, D., and German, J. (1966). Ann. Intern. Med. 65, 487495. Smith, S. M., and Sorsby, A. (1958). Ann. Hum. Genet. 23,50. Sorsby, A. (1972). Brit. Med. J. 2,580583. Steinberg, A. G. (1960). Cancer 13, 985-989. Stewart, A. ( 1961). Brit. Med. J. 1,452-460. Stewart, S. E., Eddy, B. E., and Borgese, N. (1958). J . Nut. Cancer Inst. 20, 1223-1243. Todaro, G. J., and Martin, G. M. (1967). Proc. SOC.Ezp. Bid. Med. 124,1232-1236. Todaro, G. J., Green, H., and Swift, M. R. (1966). Science 153,1252-1254. Tokuhata, G. K. (1963). J. Nut. Cancer Inst. 31, 1153-1171. Tokuhata, G. K., and Lilienfeld, A. M. (1963). J. Nut. Cancer Inst. 30, 289412. Videbaek, A. ( 1947). “Heredity in Human Leukemia and its Relation to Cancer.” Lewis, London. Videbaek, A., and Mosbeck, J. (1954). Acta Med. Scand. 149,137-159. Wassink, W. F. ( 1935). Genetica 17, 103-144. Woolf, C. M. (1955). “Investigations on Genetic Aspects of Carcinoma of the Stomach and Breast,” Pub. Health, Vol. 11, pp. 265-350. Univ. of California Press, Berkeley. Woolf, C. M. (1956). Amer. J. Hum. Genet. 8, 102-109. Woolf, C. M. (1958). Amer. J . Hum. Genet. 1 0 , 4 2 4 7 . Woolley, G. W., Dickie, M. M., and Little, C. C. (1952). Cancer Res. 13, 231-245.

This Page Intentionally Left Blank

THE STRUCTURE AND FUNCTION OF INTERCELLULAR JUNCTIONS IN CANCER

.

Ronald S Weinstein. * Frederick

B . Merk.

and Joseph Alroy’

Departments of Pathology. Tufts University School of Medicine. Boston. Massachusetts and Rush Medical College. Chicago. Illinois

I. Introduction . . . . . . . . . . I1. Membrane Ultrastructure . . . . . . . A . Principal Electron Microscopy Techniques . . . B. General (Nonjunctional) Plasma Membranes . . . I11. Cell Junction Classification . . . . . . . A . Occludentes Junctions . . . . . . . B. Gap (“Nexus”) Junctions . . . . . . . C . Adherentes Junctions . . . . . . . . D. Junctional Complexes . . . . . . . . E. Miscellaneous Junctions . . . . . . . N . Occurrence of Cell Junctions in Tumors . . . . A. Solid Tumors (and Nonmalignant Growth Disorders) . B. Tissue Culture . . . . . . . . . V. Cell-to-Cell Communication and Growth Control . . . A. Introduction . . . . . . . . . . B. Intimate Communication at Gap Junctions . . . C . Control of Gap Junction Permeability . . . . D. Metabolic Coupling at Gap Junctions . . . . E. Coupling between Tumor Cells . . . . . . F. Genetic Correlations . . . . . . . . VI. Cell Junctions in Embryonic Development . . . . VII . Cell Junctions and the Biological Behavior of Tumor Cells . A Contact Inhibition of Movement (Locomotion) . . B. Postconfluence Inhibition of Growth (Cell Division) . C. Invasion and Metastases . . . . . . . VIII . Intercellular Adhesion in Tumors . . . . . . A Adhesion at Cell Junctions . . . . . . . B. Tumor Dissemination . . . . . . . . IX. Transepithelial Permeability and Malignant Transformation X . Summary . . . . . . . . . . . References . . . . . . . . . . .

.

.

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

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

. .

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

23 26 26 30 32 32 35 39 42 43 43 43 60 61 61 62 64 66 67 69 70 71 71 74 74 75 75 77 77 77 79

I. Introduction

One of the fundamental problems in cancer research is to determine the cell products coded for by the genes of neoplastic transformation * Present address: Rush Medical College and Rush.Presbyterian.St . Luke’s Medi-

cal Center. Chicago. Illinois.

23

24

RONALD S . WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

and to identify the product(s) that accounts for the autonomous and destructive growth that is characteristic of malignant tumors. The cell surface membrane, which in large measure is a product of gene expression, probably plays a crucial role in the control of normal growth (Pardee, 1961; Pardee et al., 1974; Rapin and Burger, 1974). Malignant tumors are characterized by abnormalities in the regulation of their growth and by their invasiveness. Changes in the cell surface membrane may be related either indirectly or directly to these properties of tumors. A broad spectrum of structural, compositional, and functional membrane alterations appear in the course of malignant transformation, and it is becoming increasingly evident that individual instances of malignant transformation may involve a multiplicity of these membrane changes (Pollack and Hough, 1974; Nachbar et al., 1974). This multiplicity substantially complicates the task of correlating a single membrane change with any specific aspect of biological behavior. Cell junctions, a clearly recognizable set of membrane components, are frequently altered in the course of malignant transformation. These alterations may account for some significant properties of malignant cells. At present, research on the normal function and disposition of cell junctions and on the relationships of junctional changes to malignancy is still at a data-gathering stage. In our opinion, it is premature to attempt a theoretical consolidation of the tumor cell-junction field. Instead, our purpose in this review will be to summarize available data on junctions, and to outline and examine some of the hypotheses that have been used to interpret these data. Cell junctions are defined as structurally specialized domains that are formed at regions of contact between two cells and to which both cells contribute a part. A number of different types of cell junctions have been identified: adherens, occludens, “gap,” septate, etc. It is standard practice to consider these various cell junctions together since they are all components of the cell surface membrane, provide a structural means for cell-cell interactions, and enable cells to form compex multicellular structures. Cell junctions are customarily subclassified on the basis of their ultrastructure (Farquhar and Palade, 1963; Brightman and Palay, 1963; Weinstein and McNutt, 1972), as will be discussed in Section 111. Cell junctions perform the following functions: 1. They provide strong structural links between cells. The cells in turn form mechanically coherent tissues. 2. They serve as conduits through adjoining membranes of cell pairs. By this mechanism, junctions mediate direct communication between cells by allowing the transfer of ions and small molecules from cell to cell without leakage into the extracellular space.

INTERCELLULAR JUNCI’IONS IN CANCER

25

3. They seal cells together into a coherent tissue that can act as a highly selective barrier to diffusion. 4. They mediate the unidirectional propagation of electrochemical impulses from one cell to another. Some of these functions are the responsibility of a combination of specific types of cell junctions. There is a considerable body of evidence to support the notion that cell junctions are particularly important with respect to the cancer problem. First, many studies show that cell junctions contain structural components that are probably gene products, and that junction formation is genetically controlled (Campbell and Campbell, 1971; Azarnia et al., 1974). Second, all categories of cell junctions contribute to cell-to-cell adhesion ( McNutt and Weinstein, 1973), a property that is often altered in malignant tumors (Coman, 1944, 1961). Third, cell junctions may be involved in growth regulation (Loewenstein and Penn, 1967; Loewenstein, 1968a,b), and alterations in junctions could conceivably account for some of the growth abnormalities in tumors (Jamakosmanovic and Loewenstein, 1968; Loewenstein, 1974). Last, one type of junction, the occludens (i.e., “tight”) junction, modulates transepithelial permeability. Structural and functional changes in occludens junctions induced by exposing tissues to any one of a number of noxious stimuli could increase the flow of certain carcinogens, promoters, or cofactors to target cells within an epithelium. To date there is no irrefutable proof that cell junctions or, for that matter, any cell membrane components are causally related to malignant behavior. However, comparison of some of the properties of normal and malignant tissues inevitably focuses attention on cell junctions as possible candidates for an important role in malignancy since cell junctions provide a primary mechanism for various cell-cell interactions in tissues. In this review, we shall consider the ultrastructure, biochemistry and function of cell junctions in both normal and malignant tissues, emphasizing information that may be particularly pertinent to the cancer problem. Recent advances have been made in the development of methodology for isolating, purifying, and characterizing cell junctions. This work is of vital importance and is relevant to the cancer effort since it generates fundamental information about the molecular organization of cell junctions and also provides a framework for mechanistic studies on cell junction formation in tumors. Therefore, we will survey significant advances in cell junction physical-chemical characterization. We will also discuss the frequency of occurrence and potential significance of junctional abnormalities in tumors in relation to tumor biological behavior. At the outset we shall briefly outline data relating to the biochemical

26

ROSALD S. WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

ultrastructure of cell junctions, since progress recently has been made in this important area and many nonmembranologists working in the cancer field may be unacquainted with the electron microscopy literature. The reader is referred to several comprehensive reviews for more detailed discussions of cell junctions, their ultrastructure and function ( McNutt and Weinstein, 1973; Satir and Gilula, 1973; DeHaan-Sachs, 1973; Staehelin, 1974; Overton, 1974 ).

It. Membrane Ultrastructure

A. PRINCIPAL ELECTRON MICROSCOPY TECHNIQUES The majority of reports dealing with cell junctions in tumors are electron microscopy studies. Three principal electron microscopy preparative procedures, thin sectioning, negative staining, and freeze fracturing, are commonly used to study the ultrastructure of the general plasma membrane and cell junctions. 1. Thin-Section Electron Microscopy For thin sectioning, tissue blocks are first stabilized by various chemical fixatives, dehydrated, and then embedded in plastic. A two-dimensional image representing a single plane within the specimen is obtained by examining stained sections of embedded material with a transmission electron microscope. Thin sections of most biological materials in an unstained state are virtually electron lucent. To enhance image contrast, specimens are exposed to heavy-metal ions (“electron dense” stains). The heavy-metal ions bind to cell structures that have an affinity for the stains. Most of the image contrast visible in the transmission electron microscope is due to electron scattering caused by the heavy-metal stains. Thin-section electron microscopy of the general plasma membrane of many cells reveals a characteristic triple-layered structure at the cell periphery. Membranes can be resolved into two 2.5-3.5 nm electronopaque lines separated by an electron-lucent space that varies in thickness from 3.5 to 7.5 nm, depending on the cell type, This trilaminar structure is generally referred to as the “unit” membrane, a term introduced by Robertson to emphasize that all three layers in the 7-15 nm structure are components of a single membrane, and also to suggest that membranes of diverse biological systems share this structure ( Robertson, 1959, 1964 ). Many investigators have attempted to deduce information about the molecular architecture of membranes from the unit-membrane image. However, it is now generally acknowledged that

INTERCELLULAR JUNCTIONS IN CANCER

27

the triple-layered image is essentially an artifact of electron microscopy preparative techniques and that it cannot serve as a basis for a precise interpretation of membrane structure at the molecular level (Korn, 1966; Stoeckenius and Engelman, 1969). The “unit” membrane image is a useful indicator of the location of the main permeability barrier at the cell surface, and it may give a rough approximation of the thickness of the diffusion barrier ( McNutt and Weinstein, 1973). In thin sections, the membrane components of cell junctions of all types appear as triple-layered unit membranes. Many recent studies employing the freeze-fracture technique show substantial heterogeneity in the central plane of junctional membranes. The discrepancy between these observations on freeze-fractured junctions and thin-section data may be attributed to section thickness, and to the disorganization and/or removal of membrane components during fixation, dehydration, and the embedding processes (Singer, 1962; Korn and Weisman, 1966; Lenard and Singer, 1968; Moretz et al., 1969).

2. Negative Staining The negative staining technique is useful for studying selected aspects of membrane fine structure. For negative staining, membranes are isolated and purified, washed, and then dried onto thin support films in the presence of a highly soluble heavy-metal salt, such as phosphotungstic acid. During the final phase of drying, the heavy-metal salt precipitates as extremely fine, clectron dense crystals. The appearance of negatively stained membrane structures is related to the distribution of the small stain crystals at the membrane. In general, membrane components which are impermeable to the salt appear electron lucent and stand out in negative contrast to adjacent permeable regions that contain stain. Negative staining is particularly useful for visualizing membrane components that protrude from the membrane surface but may reveal other components as well. The interpretation of negative stain data is frequently hampered by a lack of knowledge about the degree of penetration of the stain into the membrane. Since some globular structures are seen exclusively in an en face view in negative stain preparations the exact location of these components within the membrane can be difficult to resolve. Interpretation of negative stained preparations is further complicated by artifacts that may be introduced during isolation of cell membranes and the subsequent dehydration that occurs while labile membranes are exposed to increasing concentrations of the heavy metal salt during the staining procedure itself. The negative staining technique has demonstrated novel components in general plasma mem-

28

RONALD S.

WEINSTEIN, FREDERICK B.

MERK, AND JOSEPH ALROY

branes (Haggis, 1969), as well as a number of structural elements in cell junctions (Benedetti and Emmelot, 1968a,b; Goodenough and Revel, 1970).

3. Freeze-Fracturing The freeze-fracture technique circumvents some of the technical limitations of thin-sectioning and negative staining ( Bullivant, 1970). It is particularly useful as a tool for probing biological membrane ultrastructure since it provides a unique view of the internal organization of membranes. In freeze-fracturing, biological specimens are rapidly frozen and then cleaved at low temperatures (-loo0 to -196°C). Replicas of the fracture faces are prepared in oucuo by deposition of heavy metal (e.g., platinum) at an oblique angle onto the frozen and freshly cleaved surface of the specimen (Moor, 1966). The result is a coherent metal film that is reasonably representative of the ultrastructural topography at the fracture face. After casting of the replica the original specimen is first digested and then removed by washing. The replica is examined in a conventional transmission electron microscope ( Moor, 1966; Bullivant, 1973). An ancillary step variously referred to as “freeze-etching,” “heat etching,” or “deep etching” can be added to the freeze-fracture procedure. This step is equivalent to freeze-drying. It is used to sublime water-ice away from membranes and to demonstrate the true outer surfaces of membranes at the specimen-vacuum interface. In general, freeze-fracturing splits open membranes along planes of low mechanical resistance within their hydrophobic interior ( Branton, 1966; Deamer and Branton, 1967). The cleaving process generates novel fracture faces that represent aspects of the internal structure of the membrane ( Branton, 1969; Wehrli et ul., 1970; Weinstein et a?.,1970a; Chalcroft and Bullivant, 1970) at a resolution close to the molecular level. McNutt and Weinstein (1970) introduced several terms to describe membrane interior faces and the natural surfaces that are revealed by freeze-fracturing and freeze-etching. This nomenclature has emerged as the popular convention for referring to membrane fracture faces and heat-etched surfaces ( McNutt and Weinstein, 1973; Staehelin, 1974; Gilula, 1974; Bullivant, 1974), although at the time of this writing, a new nomenclature for freeze-fractured membranes is being devised ( D. Branton, personal communication ).I According to McNutt and Weinstein (1970), the fracturing process divides the cell membrane into two lamel‘ A new nomenclature for freeze-etching has now been published (Branton et al., 1975). According to this system: Face-A becomes Face-PF; Face-B becomes FaceEF; Face C becomes Surface-€%; Face-D becomes Surface-ES; Lamella-1 becomes the P-half membrane: and Lamella-2 becomes the E-half membrane.

29

INTERCELLULAR JUNCXIONS IN CANCER

lae: Lamella-1 ( LM-1 ), the juxtacytoplasmic membrane IamelIa that is in contact with the cell interior; and Lamella-2 (LM-2), which is in contact with the extracellular compartment ( Fig. 1).Two novel fracture faces ( A and B ) are generated by membrane fracturing, and the natural surfaces or faces ( C and D ) are revealed exclusively by freezeetching. Fracture face A is the fractured surface of the inner lamella LM-1 and fracture face B is the fractured surface of the outer lamella, LM-2. The natural surfaces of the membrane are: face-C, the inner (juxtacytoplasmic) surface, and face-D, the outer surface of the membrane which abuts the extracellular milieu. Fracture faces A and B of metabolically active nonjunctional plasma membranes bear a population of small particles called membrane-associated particles or intramembrane particles (MAP or IMP). As a rule,

--

Lm, Lmp Lmz Lm, -41

Face C -

-Face A -Face

B

-Face

c

Face D

f l t

PCP Intercellular PCP compartment

I

Intercellular compartment

FIG.1. These diagrams illustrate the relative positions of membrane lamellae and surfaces that are demonstrated at gap (nexus) cell junctions by the freeze-fracture technique. Left: An uncleaved junction in cross section. When each junctional membrane is fractured along the potential cleavage plane (PCP), which lies within the interior of the membrane, it is split into two lamellae, LMI and LM,. As seen on the right, fracture faces A and B are generated by the fracturing process. Reprinted from McNutt and Weinstein (1970), with permission of the Rockefeller University Press.

30

RONALD S. WEINSTEIN, FREDERICK B. MEW, AND JOSEPH ALROY

metabolically active enzyme-rich membranes contain more of these particles than relatively inert membranes, such as myelin (Branton and Deamer, 1972). It has been proposed on the basis of a considerable body of indirect evidence that smooth areas of fracture faces correspond to lipid bilayer regions of membranes and that the MAP correspond to proteins intercalated within the bilayer (Branton, 1969; Hong and Hubbell, 1972; Singer and Nicolson, 1972).

B. GENERAL ( NONJUNCXONAL) PLASMA MEMBRANE Cell junctions are structurally distinct domains embedded within the general plasma membrane. However, they share certain ultrastructural characteristics with the general plasma membrane ( McNutt and Weinstein, 1973). This is predictable, since junctions are formed by the stepwise modification of the general plasma membrane (Overton, 1962; Campbell and Campbell, 1971; Krawczyk and Wilgram, 1973; Johnson et al., 1974). Since cell junctions are specializations within the general plasma membrane and are structurally related to it, a brief description of general plasma membrane architecture will serve as a useful point of departure for our discussion of cell junctions. Many contemporary ideas about the general plasma membrane are summarized in the fluid-mosaic membrane model (Fig. 2) of Singer and Nicolson (1972). This model depicts the major membrane components, lipids, proteins, and oligosaccharides (not shown), in a lowest free-energy configuration. It incorporates one of the central themes of the earlier Danielli-Davson-Robertson membrane models by placing a lipid bilayer within the membrane. The hydrophobic groups of amphipathic lipids (mainly phospholipid) are sequestered within the interior of the bilayer while hydrophilic groups reside at exterior (natural) surfaces, where they can interact strongly with the aqueous phase. Singer (1971) introduced two terms to describe proteins associated with the membrane lipid bilayer : integral proteins and peripheral proteins. Integral proteins are amphipathic, having an ionic exterior segment in contact with water at the membrane surface and a hydrophobic segment intercalated within the membrane lipid matrix. Presumably, the extent to which an integral protein penetrates the membrane is determined by thc amino acid sequence and covalent structure of the protein molecule as well as by its interactions with the surrounding microenvironment. Some but probably not all integral proteins extend through the entire thickness of the lipid bilayer within the membrane (Weinstein and McNutt, 1970b; Marchesi et al., 1972; Steck, 1974). Peripheral proteins associate with the membrane surface by electrostatic interactions and other weak bonds. Removal of integral proteins from membranes by

INTERCELLULAR JUNCTIONS IN CANCER

31

FIG.2. Fluid-mosaic membrane model. The bulk of the phospholipids are organized in a discontinuous lipid bilayer (solid circles represent polar head groups; and wavy lines, their fatty acid chains). Integral proteins are embedded in the lipid bilayer but can protrude from it. Peripheral proteins may bind to phospholipid polar headgroups or to the membrane via protein-protein interactions. The arrow shows the position of a natural cleavage plane within the membrane. Adapted from Singer and Nicolson ( 1972 ) .

experimental manipulation requires drastic treatment with chemicals, such as detergents and organic solvents, whereas peripheral proteins can usually be dissociated from membranes by the addition of chelating agents or other mild treatments. On the basis of thermodynamic considerations, Singer and Nicolson ( 1972) suggested that, under physiological conditions, membrane lipids and some integral proteins may be in a fluid state although there probably are constraints on the lateral mobility of at least some integral proteins (Nicolson, 1973; Elgsaeter and Branton, 1974; Peters et al., 1974; Marikovsky et al., 1976). Examples of integral proteins (Bretscher, 1972; Steck et al., 1971; Marchesi et al., 1972) and peripheral proteins (Marchesi and Steers, 1968) have been purified from red cell ghosts. Membrane oligosaccharide residues are present at the exterior surface of the plasma membrane as constitutents of proteoglycans and glycolipids ( Winder, 1970). General plasma membranes, and presumably junctional membranes, are constantly in a state of flux owing to synthetic and degradative processes (Gurd and Evans, 1973). As recently reviewed by Rapin and Burger (1974), the composition and molecular architecture of the general plasma membrane varies with mitotic cycle, level of differentiation, cell microenvironment, metabolic state, and other factors. These variables

32

RONALD S. WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

must be considered in efforts to interpret quantitative membrane changes in pathological states. 111. Cell Junction Classification

Cell junctions can be grouped into two major categories (Weinstein and McNutt, 1972). The first category includes junctions at which surface membranes of neighboring cells come into direct contact (e.g., occludentes junctions, “gap” or nexus junctions, and probably septate desmosomes). These junctions can be further subclassilied according to differences in the components within the interior of the junctional membranes. The second general category includes junctions where the surface membranes of adjacent cells are separated by a 15-35-nm interspace (e.g., adherentes junctions). This interspace typically contains electron-dense proteinaceous material. Junctions in this category can be subclassified on the basis of the morphology of cytoplasmic and extracellular matefial associated with the membrane at the junction. Cell junctions in both of these general categories are further subclassified on the basis of their overall shape and size. Latin terms, introduced into the cell junction literature by Farquhar and Palade, are used to describe the shape and total area of membrane-to-membrane contact at junctions. The term “zonula” (plural, zonulae) is used to describe junctions that extend as a belt around the entire cell. “Fascia” (plural, fasciae) is used to describe a junction that forms an extensive sheetlike area of attachment that does not completely encircle the cell. A junction that is a single spot or disk-shaped area of attachment is termed a “macula” ( Farquhar and Palade, 1963) . Cell junctions in normal tissues and in tumors are generally indistinguishable at the ultrastructural level although they may differ with respect to overall size, distribution at the cell surface, level of development, and numerical density ( McNutt and Weinstein, 1969; Martinez-Palomo, 1970a; Wiernik et al., 1973). Because of the apparent similarities of junctions in normal cells and cancer cells, their ultrastructure, biochemistry, and function will be discussed together. In view of available information on the many compositional and structural alterations in the general plasma membrane that occur during malignant transformation, it seems reasonable to anticipate that biochemical differences will be found for tumor cell junctions as well. However, the development of cell junction isolation methods is in its infancy (Benedetti and Emmelot, 1968a; Goodenough, 1974; Skerrow and Matolsty, 1974a), and, to date, available isolation methods have not been used to compare normal junctions with tumor cell junctions.

INTERCELLULAR JUNCITONS IN CANCER

33

A. OCCLUDENTES JUNCTIONS

Occludentes junctions occur in many epithelia and endothelia. 1. Ultrastructure

Unit membranes of adjacent cells come into immediate contact at zonulae, fasciae, and maculae occludentes junctions, and the outer leaflets of the membranes appear to “fuse,” thereby eliminating the extracellular space ( Figs. 3 and 7). Freeze-fracture replicas of occludentes junctions reveal fibrils 6-8 nm in diameter within the interiors of the junctional membranes (Weinstein et d.,1970b; Chalcroft and Bullivant, 1970) that correspond in their distribution to the lines of union of the membranes (Chalcroft and Bdlivant, 1970; Staehelin, 1973). An occludens junction is the only type of cell junction known to contain long intramembrane fibrils. Single intramembrane fibrils have been further resolved into two 3-4-nm filaments which lie side-by-side in the plane of the membrane ( McNutt and Weinstein, 1973). In many epithelia (including some endothelia), the occludens junction forms a belt that completely encircles the perimeter of the cell and is therefore called a zonula occludens (Farquhar and Palade, 1963). The occludentes junctions of some tissues are discontinuous and are thus designated maculae or fasciae occludentes. Comparative ultrastructural studies on unfixed and aldehyde-fixed tissues provide indirect evidence that the intramembrane fibrils at occludentes junctions may be integral membrane proteins. In unfixed tissue, membrane splitting by freeze-fracturing breaks the fibrils into a series of short segments that can be attached to either fracture face A or B. After prefixation with glutaraldehyde, a bifunctional reagent that cross-links membrane proteins at amino, imino, and guanidino groups, individual fibrils resist fragmentation during freeze-fracturing and selectively remain attached to fracture face A. This strengthening of their structure by glutaraldehyde fixation supports the notion that the fibrils contain proteins (Weinstein et al., 1970b; McNutt and Weinstein, 1973; Staehelin, 1973). 2. Isolation

Occludentes junctions have not been isolated. 3. Functions The primary function of the zonulae occludentes is to control transepithelial permeability. A secondary function is to contribute to cell-cell adhesion. Because the junction occludes the interspace between adjacent cells, it forms a seal between them preventing “bypass diffusion.” Zonulae

34

RONALD S. WEINSTEIN, FREDERICK B. B R K , AND JOSEPH ALROY

. A OCCLUDENS

A

ADHERENS

.A

ADHERENS

J l1 NCTI ON

FIG.3. Highly schematic representation of the intercellular junctions between two simple columnar epithelial cells. Surface “A” is a representation of freeze-fractured plasma membrane A-face. The general ( nonjunctional plasma membrane bears a sparse population of membrane-associated particles ( MAP) which may represent integral membrane proteins. The A-face fine structure of four types of intercellular junctions are sho\vn. (See text for details.) Plane X represents the thin section appearance of the same types of cell junctions. Adapted from Weinstein and McNutt (1972).

occludentes form a physical barrier between the lumen of cavitary organs and the adluminal compartments. In some tissues this barrier may be totally impermeable to molecules, small ions, and even water whereas in other tissues zonulae occludentes are relatively leaky. The extent of occludens junction formation (Farquhar and Palade, 1963; Friend and Gilula, 1972b) is tissue specific, and junction ultrastructure correlates well with transepithelial bypass diffusion in some tissues (Claude and

INTERCELLULAR JUNCTIONS IN CANCER

35

Goodenough, 1973) but not others ( Martincz-Palomo and Erlij, 1975). Zonulae occludentes junctions, together with mcrnbrane pumps, enable sheets of cells (e.g., intestinal epithcliurn) to create and maintain steep chemical and clcctrical gradients between compartments in various organs. Maculae and fasciae occludentes are usually found in endothelia. They morphologically resemble zonulae occludentes ( Simionescu et d.,1975; Staehelin, 1975), but form an incomplete network at the perimeter of cells (Karnovsky, 1967; Weinstein and McNutt, 1970a). The discontinuities in maculae or fasciae occludentes junctions provide shunt pathways for bypass diffusion of molecules and ions around endothelial cells (Karnovsky, 1967; Fromter and Diamond, 1972; Whitternburg and RawIins, 1971; Diamond, 1974; Simionescu et uZ., 1975). Experimentally induced changes (see Section I X ) , in the configuration and distribution of occludens junction structural elements (Wade and Kamovsky, 1974) can have a profound influence on the overall permeability across epithelium (Wade et al., 1973).

B. GAP (“NEXUS”)JUNCTIONS Gap junctions occur in many normal tissues and in tissue culture systems. They may join homotypic, heterotypic (Michalke and Loewenstein, 1971), or heterologous cells in coculture (Stoker, 1967). In these settings their junction ultrastructure is the same (Johnson et al., 1973). They also occur in benign and malignant tumors (see Section I V ) . 1. Uttrastructure A number of studies provide detailed information on gap junction fine structure (Robertson, 1963; Revel and Kamovsky, 1967; Benedetti and Emmelot, 1968a,b; McNutt and Weinstein, 1970; Goodenough and Revcl, 1970, 1971; Chalcroft and Bullivant, 1970; Goodenough and Stoeckenius, 1972; Merk et al., 1973; Peracchia, 1973a,b). Mch’utt and Weinstein ( 1970) have attempted to summarize the detailed ultrastructurar data obtained with several electron microscopy techniques in the form of a model. The data upon which the model was originally based have been reviewed in detail ( McNutt and Weinstein, 1973) and rccent studjes provide additional support for the model (Goodenough and Gilula, 1974). For our present purpose it shall suffice to briefly mention selccted aspects of gap junction ultrastructure and survey the essential features of the McNutt-Weinstein model. The reader is referred to recent reiriews for a complete discussion of the gap junction ultrastructure literature ( McNutt and Weinstein, 1973; Staehelin, 1974; Gilula, 1974). In conventional thin sections, the gap junction bears a superficial re-

36

RONALD S .

WEINSTEIN,

FREDERICK B. MEW, AND JOSEPH ALROY

semblance to the occludens junction since both appear as regions where the plasma membranes of neighboring cells come into intimate apposition. However, there are differences. The occludens junction has an overaIl thickness of less than twice that of the general plasma membrane whereas the gap junction has a thickness of more than twice that of the general plasma membrane ( Weinstein and McNutt, 1972). The increased thickness of the gap junction is due to the presence of a minute 2-nm “gap” between the membrane outler leaflets (Revel and Karnovsky, 1967). Special tracer studies show that the so-called “gap” is actually spanned by membrane components ( McNutt and Weinstein, 1973) which provide the structural basis of membrane-to-membrane contact. Freeze-fracturing demonstrates that the internal organization of gap junctional membranes is different from that of the general plasma membrane in that it contains ordered globular subunits (Chalcroft and Bullivant, 1970; Goodenough and Revel, 1970; McNutt and Weinstein, 1970). In the McNutt-Weinstein model, the gap junction is pictured as a bipartite interlocking array of subunits which receives equal structural contributions from each partner of the cell pair (Fig. 4 ) . As seen by freeze-fracturing, gap-junction membranes contain particles 4-5 nm in diameter on fracture face A. These particles often appear in a hexagonal array with a center-to-center spacing of approximately 9-10 nm. A 2.0-2.5 nm central depression is frequently observed at the apex of the particles. McNutt and Weinstein suggest that this central depression may represent a segment of a hydrophilic channel which is thought to extend through the gap junction ( McNutt and Weinstein, 1970; Johnson et al., 1974). Face B of gap junction membranes displays a polygonal arrangement of pits which house the face-A particles prior to membrane fracturing. In some tissues the particles are attached to the B face and the pits are located on the A face (see Staehelin, 1974, for a discussion of additional nomenclature). Face D of the outer lamella (LM-2) of the junctional membrane is organized as foot processes that bridge the extracellulax space and join foot processes projecting from the surface of the neighboring cell. A network of channels, which are an extension of the extracellular compartment, course around the foot processes ( Payton et al., 1969; McNutt and Weinstein, 19’70).When sectioned transversely (in the plane of the membrane) the foot processes are circular, approximately 7 nm in diameter, and have essentially the same center-to-center spacing as the intramembrane particles in freeze-fracture replicas. Morphological variants of the typical gap junction have been described (Staehelin, 1973; Peracchia, 1973a,b; Gilula, 1972; Satir and Gilula, 1973; Raviola and Gilula, 1973; Albertini and Anderson, 1974; Kogon and Pappas, 1975).

’

INTERCELLULAR JUNCTIONS IN CANCER

37

FIG.4. The McNutt-Weinstein Model: An interpretation of the dtrastructure of the Hap junction. (Left) The junctional membranes (Ma and M,) are shown pulled back to demonstrate the appearance of foot processes that normally project from each membrane and meet in the extracellular space at the midline. (Right) Aspects of the gap junction are shown as they appear in freeze-fracture replicas (see text for details). Intramembrane particles, which partially ( ? or completely) span the junctional membranes are present on the A face. The inset illustrates an interpretation of the position of hydrophilic channels within the gap junction. These channels may provide the structural basis for low-resistance electrotonic coupling of neighboring cells. The model is not drawn to exact scale. Reprinted from Mch’utt and Weinstein (1970), with permission of the Rockefeller University Press.

38

RONALD S .

WEINSTJ3N, FREDERICK

B. MERK, AND JOSEPH ALROY

2. Zsolation and Characterization Gap junctions are remarkably resistant to chemical and mechanical stress (Berry and Friend, 1969), a characteristic that is exploited in gap junction isolation methods ( Benedetti and Emmelot, 1968a,b; Goodenough, 1974). Benedetti and Emmelot (1968a) were the first to successfully concentrate gap junctions. They isolated liver plasma membranes according to the method of Neville (1!360), and then, by means of a brief treatment with Ahe detergent deoxycholate, preferentially solubilized the general plasma membrane while leaving the gap junctions intact. These isolated junctions were useful for ultrastructural studies, although they were contaminated with amorphous nonjunctional material which limited their usefulness for biochemical analysis. Other isolation procedures have been described by ,Evans and Gurd ( 1972), Zampighi and Robertson (1973), and Dunia et al. ( 1974). In 1972, Goodenough and Stoeckenius developed a method for preparing mouse hepatocyte gap junctions in a more highly purified form. They treated cell membranes with collagenase and then selectively solubilized the nonjunctional membrane with the detergent Sarkosyl NK-97 and ultrasonification. They separated the gap junctions from the amorphous nonjunctional debris by sucrose gradient ultracentrifugation. Goodenough and Stoeckenius produced only small amounts of purified gap junctions by this method, but their yield was adequate for a preliminary analysis of gap-junction biochemistry and structure. Thin-layer chromatography of the gap-junction fractions revealed one major phosopholipid and some neutral lipids. Low-angle X-ray diffraction of both wet and dry isolated gap junctions showed reflections which index on an 8.6 nm center-to-center hexagonal lattice, corresponding to the center-to-center spacing of intramembrane gap junction particles. Protein analyses with polyacrylamide-gel electrophoresis of sodium dodecyl sulfate ( SDS) solubilized gap junctions showed one major and two minor protein bands ( Goodenough and Stoeckenius, 1972). Recently, Goodenough ( 1974) modified the Goodenough-Stoeckenius method and achieved yields of up to milligram quantities. Goodenough's procedure employs continuous-flow centrifugation and a discontinuous sucrose gradient in a zonal rotor. The fraction of purified gap junctions is solubilized in SDS and then analyzed for protein content in electrophoretographs. Analysis of the gap-junction fraction shows a rather simple protein profile as compared to that of the hepatocyte nonjunctional membrane. There are two major protein peaks, one at 34,000 daltons and the second at 18,000 daltons plus two minor bands at 10,000 daltons. After exposure to disulfide reducing agents, most of the protein migrates as a doublet to the 10,000-dalton position. On the basis of these findings, Goodenough suggests that gap-junction proteins are composed of two

INTERCELLULAR JUSCTIONS IN CANCER

39

small polypeptides with molecular weights of about 10,000 daltons and that the polypcptides arc linked covalently either by interpeptide or intrapeptide disulfide bonds. The names “Connexin-A” and “-B” have been proposed for thc two 10,000-dalton components ( Goodenough, 1974). These peptides may conncct adjacent cells at gap junctions (Goodenough, 1974) although probably not by covalent bonds, since the two junctional membranes can be separated by immersion of tissue in a hypertonic disaccharide solution that “unzips” the junction in the central plane (Goodenough and Gilula, 1974). Dunia et d.( 1974) succecded in isolating lens-gap junctions and also found a 34,000-dalton major protein. It is noteworthy that the existence of the 18,000- and 10,000-dalton proteins in gap junctions in the native state is open to question because of the possibility that these low-molecular-weight polypeptides may represent proteolytic breakdown products of a large-molecular-weight subunit. Gilula ( 1974) examined additional properties of isolated gap junctions and found an absence of both negatively charged surface groups and concanavalin A binding sites. No endogenous enzyme activity has been consistently associated with gap junctions.

3. Functions Gap junctions mediate ionic metabolic coupling between cells by providing conduits across cell membranes. By definition, cell pairs are “ionically” ( electrotonically ) coupled when they freely exchange ions along concentration gradients without leakage into the extracellular compartment. They are “metabolically coupled” when nutrients or intermediate metabolites of low molecular weight are exchanged by diffusion. Gap junctions also contribute to strong intercellular adhesion ( Muir, 1967; Goodenough and Stoeckenius, 1972).

C. ADHERENTESJUNCTIONS In this section, we shall focus our attention on the macula adhercns, since this is the most frequently encountered type of adherens junction and it has received particular attention in cancer research. The ultrastructure of fasciae and zonulae adherentes is summarized elsewhere (McNutt and Weinstein, 1973). Maculae adherentes occur in nearly all epithelia. As in the case of gap junctions, maculae adherentes may join either homotypic or heterotypic cells (Hay, 1961; Overton, 1974, 1975). 1. Ultrastructure of the Macula Adherens ( D e m o s o m e ) In transverse sections, the membrancs of thc fully developed macula adhcrens junction are parallcl to one another and are separated by a

40

RONALD S. WEINSTEIN, FREDERICK B. M E R K , AND JOSEPH A L R O Y

2535-nm interspace (Fig. 5 ) . Within the extracellular zone of the junction there is a condensation of proteinaceous material that sometimes forms a central dense stratum in a plane equidistant from, but parallel to, the junctional membranes. A dense fibrillar plaque is located within the cytoplasm of each cell subjacent to the junctional membrane (Fawcett, 1961; Farquhar and Palade, 1963). These plaques are attachment sites for bundles of cytoplasmic tonofilaments. The tonofilaments do not terminate in the dense plaques, but instead loop through them and then back into the cytoplasmic matrix (Kelly, 1966). Freeze-fracturing reveals a nonpolygonal arrangement of closely packed granules and short

FIG.5. hpdculae adherentes junctions ( desniosomes ) in Fischer rat urinary bladder.

This micrograph shows an area of squamous cell differentiation in a transitional cell carcinoma induced with the carcinogen N-[4- ( S-nitro-2-furyl)-2-thiazolyl]formamide. At each inacula adherens, cytoplasmic tonofilaments converge on the dense plaques ( D P ) of the junction. A gap junction ( C J ) is illustrated. B. U. Pauli, R. S. Weinstein, and S. M. Cohen, unpublished micrograph. ~ 7 0 , 0 0 0 .

INTERCELLULAR JUNCI’IONS I N CANCER

41

filaments approximately 8-10 nm in rcplica diameter within the interior of the adherens junction membranes ( McNutt et al., 1971; Breathnach et al., 1972). The granules and filaments may represent segments of filaments arising out of the dense fibrillar plaques ( McNutt and Weinstein, 1973). The filaments may intercalate into the hydrophobic interior of the membranes. Junctions resembling one half of a macula adherens lie along the basal surface of many epithelia where they attach epithelial cells to the connective tissue substratum. These junctions are called “hemidesmosomes” ( Kelly, 1966) although there are structural differences between these junctions and intraepithelial desmosomes (Kelly, 1966; Hay and Revel, 1!369). Another type of adherens junction resembles the hemidesmosome in that it is an asymmetric adherens junction. Such junctions are intraepithelial, unlike true hemidesmosomes, and may represent imperfectly formed maculae adherentes junctions (Weinstein et d.,1974). We propose the name “ d a adherens imperfecta” (or “imperfect desmosome”) for this junction. The macula adherens imperfecta (see Fig. 7 in Weinstein et al., 1974) has a fully developed dense plaque subjacent to the junctional membrane of one cell but none in its neighbor, unlike asymmetric adherentes junctiofis in embryonic tissues, which have a well developed dense plaque in one cell and a poorly developed plaque in the neighboring cell (Hay, 1961) . Tonafilaments, when present, loop through the single dense plaque of a macula adherens imperfecta, and the extracellular condensation is moderately well developed. Macula adherens imperfecta could arise through one of several mechanisms: (1) in uiuo shearing of defective symmetrical adherentcs junctions; ( 2 ) incomplete formation of adhcrentes junctions due to the failure of one cell to assemble its contribution to the junction; or ( 3 ) as part of a cell-cell dissociationreassociation cycle (Wcinstein et al., 1974). Two other forms of the adhcrcns junction, namely zonula and fascia adherens, are usually included for convenience in the general adhercns category although their ultrastructure is significantly different from that of the macula adherens ( McNutt and Weinstein, 1973). These junctions have not been investigated with respect to cancer. Histochemical and cytochemical studies have dcmonstrated that the cytoplasmic dense plaques and the intercellular condensations of desmosomes are susceptible to proteolysis (Overton, 1968; Douglas et al., 1970; Borysenko and Revel, 1973). The junctional interspace is rich in carbohydrate residues (Kelly, 1966; Luft, 1971; Rambourg, 1969), and these may be components of membrane glycoprotein ( Staehelin, 1974). Selec-

42

RONALD

s. WEINSTEIN,

FREDERICK B. MERK,AND JOSEPH ALROY

tive removal of divalent cations with the chelator EDTA diminishes intercellular zone material and results in a widening of the intercellular space (Muir, 1967; Overton, 1968). The EDTA experiment may indicate that some maculae adherentes are adhesive structures which operate by calcium bridging (Sedar and Forte, 1964). Borysenko and Revel ( 1973) recently demonstrated that maculae adherentes from different normal tissues have varying sensitivities to proteolysis, chelators, and detergents, suggesting that adherentes junctions can have different chemical compositions. This heterogeneity suggests that intercellular adhesion at adherentes junctions in different tissues may be accomplished by different mechanisms. 2. Isolation and Chracterization Skerrow and Matoltsy ( 1974a,b) have isolated maculae adherentes junctions from cow snout epidermis at a level of purity suitable for biochemical analysis. Their purification method utilizes selective solubilization by citric acid-sodium-citrate buffer, pH 2.6, and discontinuous sucrose density gradient centrifugation. The isolated maculae adherentes contain protein, carbohydrate, and lipid at a ratio of 76:17:10. The protein is rich in nonpolar amino acid residues. Gel electrophoresis of junctional proteins demonstrates 24 bands with mobilities corresponding to a molecular weight range of 15,OOo-230,000. Two proteins, with molecular weights of 210,000 and 230,000, comprise 28% of the desmosome weight. Skerrow and Matoltsy suggest that the high-molecular-weight proteins may derive from the dense fibrillar plaque. Two other bands are periodic acid-Schiff positive and constitute 23%by weight of the macula adherens protein. These bands may derive from the carbohydrate-rich material at the junction interspace ( Skerrow and Matoltsy, 1974b). 3. Function

Adherentes junctions play an important role in cell-to-cell adhesion. They and their associated tonofilaments are elements in a cytoskeletal system that may mediate a complex form of cooperation between individual cells for the regulation of mechanical properties of the tissue as a whole.

D.

JUNCTIONAL

COMPLEXES

At regions of cell-to-cell apposition, several types of cell junctions can occur in groups called junctional complexes. A classic example is the “terminal bar” region of intestine columnar epithelium ( Farquhar and Palade, 1!363), in which a series of junctions whose order is invari-

LVTERCELLULAR JUNCTIONS IK CANCER

43

able appears at the apical margin of the lateral surfaces of columnar epithelial cells (Fig. 3 ) . A zonula occludens is the apical junction of this complex, and this is closely followed by a zonula adherens and then by a macula adherens. In other tissues, several types of junctions may occur in close juxtaposition but not arranged into an identifiable pattern ( McNutt and Weinstein, 1973). Unusual combinations of junctions are sometimes observed in tumors. For example, Ahoy and Weinstein (1976) studied the ultrastructure of an adenoacanthoma arising spontaneously in canine mammary gland. An adenoacanthoma is characterized by bidirectional differentiation; i.e., cells within the tumor have structural characteristics of both columnar and squamous epithelial cells. Carcinoma cell membranes in adenoacanthomas have prominent occludentes junctions that resemble those of nonneoplastic columnar cells. Interspersed among the intramembrane fibrils and grooves of the occludentes junctions are fully developed maculae adherentes (Fig. 7B), which resemble the adherentes junctions of normal squamous cells. Thus, the columnar and squamous cell phenotypes are both represented within a single region of the membrane.

E. MISCELLANEOUS JUNCTIONS Several additional types of Cell junctions have been characterized (Lasansky, 1969) including septate desmosomcs (Wood, 1959; Locke, 1965; Gilula et al., 1970), septatelike contacts (Friend and Gilula, 1972a), etc. These and other unusual junctions (Breton-Gorius et d., 1975) are either restricted in their occurrence to one or two organs or are rarely encountered in mammals. Since these junctions have not been related to the cancer problem, we will not consider them further. IV. Occurrence of Cell Junctions in Tumors

A. SOLIDTUMORS(AND NONMALIGSANT GROWTHDISORDERS) The descriptive literature regarding tumor ultrastructure is voluminous. Surgical pathologists recognize that cell junctions can be useful

for classifying certain tumors which present difficult diagnostic problems, and therefore cell junctions are mentioned in many pathology papers. Unfortunately, only a few of the many papers on tumor ultrastructure contain quantitative information on specific types of cell junctions. 1. Descriptions of Cell ]unctions in Tumors We have surveyed the ultrastructure literature and in Table I have listed representative reports that describe cell junctions in many types

I I)ESCHIPTIONS OF CF:LL JUNCTIONS I N TUMOHW-~ TABLE Cell junction

Organ Adrenal

Bone

Breast

Tumor (species)

Terminal barc

Occludens

“Gap”

Adherens

+

+

Kovaca el at. (1974) Sharmaand Hashimoto (1972) Brown el al. (1972) Steiner el al. (1973) Hou-Jensen el al. (1972) Steiner et al. (1972) Gonealez-Licea el al. (1907) Martinez-Palomo (1970b) Tobon and Price (1972)

+, HI)

Tobon and Price (1972)

Adenoma (human) Carcinoma (rat)

+

Pheochromocytoma (human) Chondrosarcoma (human) Ewing’s sarcoma (human) Giant cell tumor (human) Osteogenic sarcoma (human)

+ + + +

Adenocarcinoma (mice) Carcinoma, lobular, in s i t u , epithelium (human) Carcinoma, lobutar, in situ, myoepithelium (human) Carcinoma, mucinous (human) Carcinoma, scirrhous (human) Carcinoma, tubular (human) Cystosarcoma phylloides (human) Fibroadenosis (human) Mixed tumor myoepithelium (canine)

1

0

+

1

1

1

1

f

Reference

+ +

+ 1

Ahmed (1974) Ahmed (1974) Erlandson and Carstens (1 972) Toker (1968) Barton (1964) von Bombard and von Sandersleben (1973)

m

+, HI) +

Mixed tumor myoepithelium (canine) Sclerosing adenosis (human) Carotid body Craniopharyngioma Ear Epididymis Eye

Adenoma (human) Human Ceruminous gland adenocarcinoma (human) Adenomatoid tumor (human) Corneal “preinvasive” cancer (human) Retinoblastoma (human) Retinoblastoma-like tumor (rat)

Fallopian tube Hypopharynx Intestine

Kidney

Larynx Liver

Adenofibroma (human) Squamous cell carcinoma (human) Adcnocarcinoma (human) Papilloma (human) Transitional cloacogenic carcinoma (human) Villous adenoma (human) Congenital mesoblastic nephroma (human) Mesenchymal renal tumor (human) Renal cell carcinoma (clear cell) (human) Renal cell carcinoma (granular cell) (human) Wilms’s tumor (human) Papillomatosie (human) Adenoma, liver cell (human) Hepatoma (human)

+, I n )

+

+

+

+

+

1, HD

+

+

1

+ +

+ 1

+

+

+ + + + + + + 1

+ or 1 +, HD + +

Pulley (1973) Wellings and Roberts (1963)

Welsh el al. (1972) Ghatak et ol. (1971) Welti et al. (1972) Mackay et al. (1971) Tripathi and Garner ( 1972)

Ts’o el al. (1969) Kobayashi and Mukai (1974)

Kanbour el al. (1973) Rangan (1972) Imai and Stein (1963) Fisher and Sharkey (1962)

Fisher (1969) Ioachim el ol. (1974) Fu and K a y (1973) Favara et al. (1968) Tannenbaum (1971) Tannenbaum (1971) Tannenbaum (1971) Svoboda el al. (1963) K a y and Schatzki (1971)

Wills (1968) (Continued)

$

TABLE I (Continued) Cell junction

Organ

Tumor (species)

Terminal barc

m 0

z

Occludens

‘‘Gap” Adherens

Reference

E

r 8

Liver (Cont’d)

Hepatoma BH3 (mouse) Hepatoma BC3 (mouse) Hepatoma BRL (mouse) Hepatoma BNL (mouse) Hepatoma H-31780 (rat) Hepatoma, Morris 9121 (rat) Hepatoma, Novikoff (rat)

Lung

1

Hepatoma (human)

Hepatoma, Yoshida ascit,es, AH602 (rat,) Hepatoma, Yoshida ascites, AH7974 (rat) Hepatoma, Yoshida ascites, AH130 (rat,) Hepatoma, Yoshida ascites, AH13 (rat) Bile duct adenocarcinoma (rat) Acinic cell tumor (human) Adenomatosis (sheep) Carcinoid, bronchial (human) Carcinoma, anaplastic (hamster) Carcinoma, bronchoalveolar (human) Carcinoma, oat cell (human) Carcinoma, squamous cell (human)

+

+ 1 + 0

+

+ 1

1 1

+ 1 1

+ +

+ + 1

1

0

+ +

+

+ 0 + 0

n

+ + +

Ma and Blackburn (1973) Malick (1972) Malick (1972) Malick (1972) Malick (1972) Martlnez-Palomo (1970b) Urban el al. (1972) Babai and Tremblay (1972) Hoshino (1963) Hoshino (1963) Hoshino (1963) Hoshino (1963) Ma and Webber (1966) Fechner et al. (1972) Perk et at. (1971) Hage (1973) Harris et al. (1973) Kuhn (1972) Bensch et al. (1968) Lupulescu and Boyd (1972)

M

1

9 q

8

1 m

z

“E

1 8 !

w

4

E

2

Mouth

Nasopharynx Nervous System

Ovary

+

Adenomatoid tumor (human) Ameloblastoma (human) Calcifying epithelial odontogcnic tumor of Pindborg (human) Carcinoma, spindle-cell (human) Granular cell tumor (human) Carcinoma (human) Astrocytoma (human) Astrocytoma, glioblastoma multiformis (human) Choroid plexus papilloma (human) Ependymoma (human) Germinoma, in tracranial (seminoma) (human) Medulloblastoma (human) Medulloepithelioma (rat, mouse, hamster) Meningioma (human) Adenomatoid tumor (human) Brenner tumor (human) Cystadenocarcinoma (human) Cystadenofibroma (monkey) Cystadenoma (human) Clear cell tumor (human) Dysgerminoma (human) Gonadoblastoma (human) Granulosa cell tumor (human) Granulosa theca cell tumor (human) Sertoli-Leydig cell tumor (human) Surface papilloma (monkey)

+, HI)

+

+, HD 0

+ + or 1 + 1

+ +

+

1

+ + + + + +

+

+ + + +

+ +

+ + 1 + +

+

+ + 1 +

1

0

Taxy et al. (1974) -Minter and McGinnis (1972) Anderson el al. (1969) Leifer el al. (1974a) K a y el al. (1971) Lin et al. (1969) Rubinstein el al. (1974) Tani et al. (1973) Carter et al. (1972) WolR et al. (1972) Tani el al. (1974) Rubinstein el al. (1974) Mukai et al. (1974) Popoff el al. (1974) Ferencsy et al. (1972) Bransilver el al. (1974) Gondos (1971) Amin et al. (1974) Gondos (1971) Salazar et al. (197.4) Lynn el al. (1967) AMackayel al. (1974) Hamlett el al. (1971) Bransilver el al. (1974) Murad et a / . (1973) Amin el al. (1974)

13F:

5 z

2

$

2

v)

2

2

3z

.h

(Continued)

=j

b b 06

TABLE I (Continued) Cell junction

Y

Organ Pancreas

Parathyroid Pineal Prostate Salivary glands

Skin

Tumor (species) Acinar cell carcinoma (human) Alpha cell tumor (human) Beta cell tumor (human) Carcinoma, infantile type (human) Giant cell tumor (human) Adenoma (human) Carcinoma, acinar (human) Large cell tumor (human) Carcinoma (human) Acinar cell tumor (human) Mixed tumor, chondroid pattern (human) Mixed tumor, (epithelial, myoepithelial, and myxoid patterns) (human) Myoepithelioma (human) Sebaceous carcinoma (human) Basal cell carcinoma (human) Bowen disease (human) Keratoacanthoma (human) Melanoma (human) Pilomatrixoma (human) Squamous cell carcinoma (rat) Squamous cell carcinoma (human)

Terminal barc

Occludens

“Gap”

Adherens

Reference

+ + + +

Burns et al. (1974) Goldenbcrg et al. (1969) Bencosme et al. (1963) Frable el al. (1971) Rosai (1968) Marshall et al. (1967) Echevarria (1967) Ramscy (1965) Mao et al. (1966) Erlandson and Tandler (1972) Welsh and Mcyer (1968) Welsh and Meyer (1968)

I! YJ 5:

+

+

+

0

+ or 1 + 0

+

+ + 1

1, HL)

0

1

+, HD + + 1 1

Leifer et al. (1974b) Akhtar el al. (1973) Flaxman (197’2) Yeh (1973) Fisher et al. (1972) Mishima (1967) McGavron (1965) Martinez-Palomo (1970b) Fisher el al. (1972)

3

“g

i F! w

li

”

1

LI

8

u

3:

b

Soft tissues

+

Squamous cell carcinoma (human) Alveolar soft part sarcoma (human) Fibromyxosarcoma (human) Fibroxanthoma, malignant (human) Hemangioendothelial sarcoma (human) Hemangiopericytoma (human) Histiocytosis (human) Lciomyoblastoma (human) Leiomyosarcoma (human) Lymphoangiosarcoma (human) Mesothelioma (human) Mesothelioma (rat)

+ 1

+ 1 0

Ithabdomyoma (human) Sarcoma, clear cell (human) Sarcoma, epithelioid (human)

Stomach Testes

1

+ +

Adenocarcinoma (human) Embryonal carcinoma (human) Leydig cell tumor (rat) Leydig cell tumor (rat) Seminoma (human)

+

Seminoma, spermatocytic (human)

0

0 0

Sarcoma, Moloney virus (rat) Sarcoma, synovial (human) Sarcoma 180 (Crocker’s tumor) (mouse) Sclerosing hemangioma (human)

Scminoma (human)

+ + +

+

+

+,HD

+ + + +

+ or 1 +

T

+ +

+ + +

Hashirrioto el al. (1973) Welsh et a/. (1972) Leak et al. (1967) Merkow el al. (1971) Steiner and Dorfrnan (1972) Battifora (1973) Hou-Jensen el al. (1973) Cornog (1969) Wang et al. (1974) Merrick et al. (1971) Ferenczy et ul. (1972) Shin and Firminger (1973) Battifora et al. (1969) Kubo (1969) Fisher and Horvat (1972) Stanton el ul. (1970) Kubo (1974) Zuckerberg (1973) Hill and Eggleston (1972) Ming et ul. (1967) Pierce (1966) Roth el al. (1970) lteddy et al. (1973) Holstein and Korner (1974) J. Ahoy et al. (unpublished) Rosai el al. (1969)

(Continued)

TABLE I (Continued) Cell junction

Organ Testes (Cont’d) Thymus Thyroid

Ureter Urinary bladder

Uterus

Tumor (species) Sertoli cell adenoma (human) Teratocarcinoma (mouse) Yeminoma, thymic (human) Thymoma (human) Adenoma, follicular (human) Carcinoma, follicular (human) Carcinoma, papillary (human) Hurthle cell tumor (human) -Medullary carcinoma (human) AMedullarycarcinoma (human) Ultimobranchial adenoma (bovine) Transitional cell carcinoma (human) Transitional cell carcinoma, Grade I (human) Transitional cell carcinoma, Grade 11-111 (human) Squamous cell carcinoma (human) Carcinoma, adenosquamous (human) Clear cell adenocarcinoma (human) Papillary adenocarcinoma (human) Squamous cell carcinoma, cervix, in situ (human)

Terminal barc

Oceludens

‘‘Gap’’ Adherens

f

+

+ 1 +

+ + + +

+ + + 1 + +

1 +or1

1 or 0

+ +

+ t

1 T

+ +

f, H D

+

1 1

11

1

lleferen ce Able and Lee (1969) Pierce el al. (1967) Levine (1973) Leviqe (1973) Lupulescu and Boyd (1972) Tonietti el al. (1967) Tonietti el al. (1967) Feldman et al. (1972) Horvath el al. (1972) Bordi et al. (1972) Black el al. (1973) Flaks et al. (1970) R. S. Weinstein et al. (unpublished) It. S. Weinstein el al. (unpublished) R. S. Weinstein et al. (unpublished) Aikawa and Ng (1973) Rorat et al. (1974) Hameed and Morgan (1972) McNutt et al. (1971)

m

Squamous cell carcinoma, cervix, (human) Sauamous cell carcinoma, corpus (rat) Vagina

Clear cell carcinoma (human)

11 or 0

1

McNutt et al. (1971)

+, HI) +

Baba and von Haam (1971)

Silverberg and DeGiorgi (1972)

+

= junctions are present at a numerical frequency similar to t h a t in the tissue of Semiquantitation of intercellular junctions; tumor origin; 0 = junctions are absent; f = junctions are increased in number; 1 = junctions are moderately reduced in number; 11 = junctions are markedly reduced in number; HD = hemideamosomes are present. * Blank spaces do not indicate t h a t specific types of junctions are necessarily absent; rather they indicate t h a t the junction has not been described in relevant papers. c Terminal bar refers to the junctional complex at the luminal border in many cuboidal and columnar epithelia. Usually, the terminal bar region contains three types of junctions: zonuls occludens, zonula adherens, and macula adherens. In many papers, the terminal bar is mentioned, b u t individual component junctions are not identified.

1 C

$

0

Z

v)

2!

9

E

52

HONALD

s. WEINSTEIN,

FREDERICK B. MEXK, AND JOSEPH ALROY

of solid tumors. It is noteworthy that many of the cited reports omit from their descriptions some types of junctions. There are a number of reasons for this. Certain types of junctions, particularly gap junctions and tight junctions, are difficult to identify in surgical or autopsy material, even under the best preparatory conditions. Positive identification of gap junctions requires the use of special specimen preparation techniques that are generally not used in surgical pathology laboratories. Also, until recently, the classification of junctions was a matter of controversy. The earlier tumor ultrastructure literature contains many inaccurate descriptions of cell junctions. We can draw several conclusions about the occurrence of cell junctions in tumors from the data in Table I. These data show that the various types of cell junctions are present in many types of epithelial and mesenchymal tumors although the occurrence of these junctions often is less frequent than in normal tissue. The type of cell junction most frequently reported in solid tumors is the macula adherens (desmosome). This may be related more to the relative ease with which desmosomes are visualized in the electron microscope than to their relative abundance or importance. 2. Qtiuntitative Studies of Junctions in Solid Tumors

Systematic quantitative studies of cell junctions have been performed for two solid tumor systems, squamous cell carcinomas of human cervical epithelium ( McNutt and Weinstein, 1969; McNutt et al., 1971; Wiernik et al., 1973) and uroepithelial tumors (i.e., transitional cell carcinomas) arising in human urinary bladder ( Weinstein et al., 1974). a. Gay ]unctions. blcNutt et al. (1971) compared the population frequency of gap junctions in normal human cervical squamous epithelium, squamous metaplasia and dysplasia (two reversible nonmalignant states), carcinoma in situ, and invasive squamous cell carcinoma. Human uterine cervical epithelium is particularly well suited for quantitative studies of gap junction frequencies, since gap junctions are remarkably abundant in normal cervix ( McNutt and Weinstein, 1969). In addition, the neoplastic state carcinoma in situ, which arises spontaneously in cervical cpithclium, can serve as a positive control in studies correlating ultrastructure with biological behavior, since carcinoma in situ is a well recognized preinvasive malignant state which can persist in the cervix for years before the appearance of stromal invasion (Richart and Barron, 1969). Structural changes arising during the protracted in situ stage may be either early events in a series that leads to invasiveness or are unrelated to invasiveness. In either case, the in situ carcinoma is a control that is clearly noninvasive.

53

INTEHCELLULAR JUNCTIONS IN CANCER

The intermediate cells of normal cervical epithelium have up to 225

gnp junctions per cell (Table 11). In contrast, invasivc squamous cell carcinomas in uterinc cervix (Fig. 6 ) arc markedly deficient in gap junctions ( McNutt and Weinstein, 1969). In relatively well differentiated areas of human tumors, up to four gap junctions pcr cell arc present, but in poorly differentiated areas nonc are found. This variance in the distribution between well differcntiatcd and poorly differentiated areas may help to explain thc variations in electrotonic coupling that are found in different regions within solid tumors (Sheridan, 1970). Very fcw gap junctions arc prescnt in carcinoma in situ of the cervix (Table 11). This allows us to infer that there is a poor temporal correlation betwecn the development of severe gap-junction deficiencies and tumor invasion, although the possibility remains that the loss of gap junctions is one of several prerequisites required for stromal invasion. In reversible nonmalignant states, including squamous metaplasia and moderate dysplasia, there are statistically significant decreases in gapjunction frequency although junctions remain plentiful ( McNutt et a]., 1971). 11. Maculae Adherentes ( Desmosomes) . Numbers of maculae adherentes (desmosomes) have been estirnatcd for many types of tumors (Table I ) and have been precisely quantitatcd for two solid tumors, human cervical and urinary bladder carcinomas. McNutt and Weinstein

Basal zone

State Normal squsmous cpithelium Sorinal squamous cpithcliuni Squnmous mctaplasia l)ysplssia, mild to moderate Ilysplasia, severe carcinoma in situ Invssivc carcinoma

I

53 f 26.‘ 11 f 4 d 10 f 1‘ 11 5‘

*

Intermediate zone 187 f 40 -

+

60 8 72 f 31

2 f 1“ 0 . :P

6

a Adapted from h l c S u t t el nl. (1971). The ENF was estimated hy dividing the n u m h r of nexuses per section (n) by thc product of thc number of rell profiles pcr section (c) niultiplicd by the fraction of cell surface included per section (f),where:

j = section thickncss;/average cell diameter

* Value

and ESF = n / ( c f )

? one standard deviation. Basal zone, consisting of npproximalely thrce cell layem. d Bmal laycr, consisting of the single layer of cells adjacent to tho basement membfanc.

54

RONALD S. WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

INTERCELLULAR JUNCXIONS IN CANCER

55

(19sS) were the first to observe that maculae adherentes are less frequent in invasive cervical squamous cell carcinomas than in normal cervix. In a more detailed study, Wiernik and his associates found a small yet statistically significant decrease in the total number of maculae adherentes per cell with malignant transformation. Their data also show that maculae adherentes in cervical invasive carcinomas are smaller than those in normal cervix. They found no significant difference in the number of maculae adherentes per unit length of membrane (Wiernik et al., 1973). Comparable studies of maculae adherentes have not yet been reported for preinvasive cervical carcinoma, although it is known that their numbers are reduced in Bowen’s disease, a form of preinvasive intraepidermal squamous cell carcinoma of the skin (Yeh, 1973; Yeh et al., 1974). Recently, studies of the occurrence of maculae adherentes in tumors have been extended to include transitional cell carcinomas arising in human bladder uroepithelium (Fulker et al., 1971; Weinstein et al., 1974; Ahoy et al., 1976). These studies provide some of the strongest circumstantial evidence for an association between abnormalities in maculae adherentes and malignant behavior. Relatively few maculae adherentes are present in normal human bladder uroepithelium (Richter and Moize, 1963; Battifora et aZ., 1965). In 1971, Fulker and FIG 6. Electron micrographs of cell junctions in normal and malignant epithelium of the human uterine cervix. ( A ) Normal cervical epithelium. Intermediate layer cells are connected to each other by gap junctions (nexuses ) and desmosomes. At gap junctions, the cell membranes are very closely apposed. At desmosomes, the neighboring cell membranes are attached by filamentous extracellular material, often showing a distinct central stratum (S).~37,420. ( B ) Normal cervical epithelium. Cap junction ultrastructure as demonstrated by lanthanum tracer impregnation. Four cell processes (PI-,) are attached at gap junctions. Electron-opaque lanthanum hydroxide fills thin channels of extracellular space around subunits (foot processes) which join the junctional membranes. Gap junctions, viewed en fuce, appear as a closely packed array of subunits outlined with lanthanum. They have a 9-10-nm center-to-center spacing. In cross section, this subunit pattern is obbcured and the so-called “gap” appears as a dark central 7-nm line ( S ). XSil20. Inset is a detailed en face view of a subunit array. Individual subunits (arrow) are 7 nm in diameter. x 120,960. (C)Squamous carcinoma of the cervix. Processes of adjacent cells are attached by desmosomes, but gap junctions are infrequent. Some tumor desmosomes lack a centml dense stratum. ~38,800. ( D ) Squamous carcinoma of the cervix. A rare gap junction connects two tumor cells. This junction has been infiltrated with lanthanum, revealing in cross section the characteristic five-layered appearance of a lanthanum-stained gap junction ( X ) and in en face sections a closely packed subunit array. ~62,640. A-D: From McNutt and Weinstein (1989);copyright 1969, American Association for the Advancement of Science.

56

RONALD

s. WEINSTEIN,

FREDERICK B. MERK,AND JOSEPH ALROY

his associates reported that the numbers of maculae adherentes often increase above normal in low-grade papillary transitional cell carcinomas and decrease below normal in moderate and high-grade tumors (Fulker et al., 1971; Cooper, 1972). These observations have since been confirmed ( Weinstein et al., 1974). Since low-grade papillary transitional cell carcinomas are infrequently invasive, unlike higher grade bladder tumors, an obvious question is whether loss of junctions facilitates invasion in higher-grade tumors. This question can be tested since low-grade bladder tumors occasionally invade stroma, and since a considerable percentage of higher-grade tumors are either noninvasive or pass through a preinvasive stage in the course of development. These variants have been used in a preliminary study to determine whether the number of maculae adherentes in bladder carcinomas show a stronger correlation with tumor grade per se or with invasive behavior. We recently quantitated maculae adherentes in low-grade human papillary transitional cell carcinomas which had invaded stroma and muscle, and in several higher grade tumors which were noninvasive at the time of surgical removal. Junctions were decreased in frequency in the low-grade invasive tumors and increased in frequency in the noninvasive higher-grade tumors. It would therefore seem that maculae adherentes frequency in bladder tumors is indeed more strongly correlated with invasive behavior than with tumor grade (Alroy et aE., 1976). If these observations are confirmed for a larger series of biopsies, they could have a considerable impact on diagnostic pathology since numbers of demosomes in transitional cell carcinomas may turn out to be a useful prognosticator of tumor behavior. Hruban et aZ. (1972) quantitated maculae adherentes in 35 different transpIantabIe Moms hepatomas which exhibited a broad spectrum of growth rates. Their data show no correlation between maculae adherentes frequency and growth rate. c. Occludentes Junctions. Quantitation of zonulae occludentes junctions requires more than simple counting of junctional sites. Since many normal epithelial cells typically have a solitary zonula occludens junction, quantitation is generally accomplished by counting the number of parallel intramembrane fibrils within the junctional zone (Claude and Goodenough, 1973). Although this measurement correlates well with junctional sealing capacity, simple counting of occludentes intramembrane fibrils in tumors can be meaningless since the numbers of fibrils are frequently extremely variable along the surfaces of individual tumors cells. Attenuation or loss of zonulae occludentes is a common occurrence in anaplastic carcinomas (see Table 11; also Martinez-Palomo, 1970b;

INTERCXLLULAR JUNCTIONS IN CANCER

57

Weinstein et al., 1974). These changes were systematically examined in spontaneous transitional-cell carcinomas arising in human urinary bladder. As is found in normal bladder uroepitheliums, in low-grade papillary tumors, zonulae occludentes are confined to the apical regions of the lateral surfaces of superficial tumor cells (Fig. 7A). In nonneoplastic states, the junctions contain four or five parallel intramembrane fibrils, but in low-grade tumors they are focally reduced to the width of a single intramembrane fibril. In invasive tumors, occludentes junctions are discontinuous (i.e., they become maculae occludentes ) and are markedly attenuated. They are found at all surfaces of individual superficial tumor cells as well as at the surfaces of cells deep within the tumors (R. S. Weinstein, F. B. Merk, and J. Alroy, unpublished observations). The presence of occludentes junctions on cells deep within tumors may be a manifestation of loss of cell polarity which is commonly observed in anaplastic tumors. The finding suggests that invasive tumor cells may be capable of retrograde migration. Loss of occludentes junctions also results in a decrease in intercellular adhesion, an increase in leakiness across the epithelia, and possibly enhances the loss of cellular polarity. The first two functional changes are discussed elsewhere (Sections VIII, A and IX). Differentiated epithelial cells are highly polarized with respect to their biochemical surface topography. For example, in renal proximal tubules, leucine aminopeptidase activity is located exclusively at the luminal border and Na-K-MgATPase is located exclusively at the basal border of the epithelial cells. In normal tissues and low-grade tumors, the intramembrane fibrils of zonula occludens may serve as a physical barrier to the lateral diffusion of individual membrane components in the plane of the membrane and, by this mechanism, keep membrane components segregated along specific membrane surfaces. Disruption zonulae occludentes may permit membrane components to migrate over the entire surface of the cell. This would result in a haphazard arrangement of mobiIe membrane components at the cell surface (Porter et al., 1974) and thus deprive the cell of its capacity to perform specialized functions. Other mechanisms are also contributors to the control of cell surface topography ( NicoIson, 1973; Elgsaeter and Branton, 1974). 3. Cell ]unctions between Blood Cells Normal erythrocytes, granulocytes, and small lymphocytes do not form morphologically detectable cell junctions. Small lymphocytes can be induced by immunologically specific as well as nonspecific stimulants to shift from a resting state to an activated state with high metabolic activity. In the activated state, lymphocytes apparently are able to form

58

RONALD S. WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

INTERCELLULAR JUNCXIONS IN CANCER

59

low-resistance junctions. This was shown by Hulser and Peters (1972), who incubated bovine lymphocytes with the nonspecific stimulant phytohemagglutinin ( PHA ) and successfully demonstrated electrotonic coupling within 1 minute of initiation of stimulation. Also, Sellin et al. ( 1971, 1974) demonstrated fluorescein dye transfer from anti-immunoglobulin-stimulated lymphocytes to unstimulated lymphocytes and macrophages. R. S. Weinstein and M. Stadecker (unpublished observations) were unable to demonstrate any type of intercellular junctions between PHA-stimulated lymphocytes by either freeze-fracture or thinsection electron microscopy. Their finding is curious since electrotonic coupling and gap junctions coexist in all other cell systems that are capable of ionic communication, with the obvious exception of cell pairs joined by intercellular bridges (Woodruff and Telfer, 1973). Possible explanations for this discrepancy are that: the junctions in stimulated lymphocytes form transiently and may not be preserved by electron microscopy methods; or that gap junction subunits are widely dispersed over the lymphocyte cell surface and therefore are not identifiable as components of gap junctions since they fail to organize into the characteristic geometric array of the gap junction. It is also noteworthy that Cox et a2. (1974) were unable to demonstrate metabolic coupling between lymphocytes in continuous culture. There is evidence that unusual cell junctions may be present in some lymphomas (Behrens et al., 1974) and leukemias (Sane1 and Serpick, 1970) and in bone marrow of some patients with ineffective erythropoiesis ( Breton-Gorius et al., 1975).

4. Cell Junctions in Tumor Metastases The relationship between the occurrence of cell junctions and the ability of tumors to metastasize has not yet been systematically explored. FIG.7. Freeze-fractured occludentes junctions in tumors. ( A ) A zonula occludens (A face) is present at the boundary between the lateral and luminal surfaces of a cell in a Grade 1 noninvasive papillary transitional cell carcinoma in human urinary bladder. Microvilli (MV) are prominent at the luminal surface. x57,000. From Weinstein et al. (1974). ( B ) Unusual junctional complex, found in an adenoacanthoma which arose spontaneously in canine mammary gland. Intramembrane fibrils and grooves which are characteristics of occludentes junctions, partially encircle maculae adherentes ( MA). ~71,000.From Alroy and Weinstein ( 1976). ( C ) An occludens junction at fracture face B of an H-35hepatoma cell in culture. At this fracture face, the junction appears as an extensive network of grooves. Frequently, membrane-associated particles which presumably have been fractured away from the complimentary A-face membrane are present in these furrows. ~70,000. By courtesy of Mr. M. Porvamik.

60

RONALD

s. WEINSTEIN, FREDERICK B.

MEFX, AND JOSEPH ALROY

It is likely that studies on this topic will be complicated because of the presence of phenotypically different cells within tumors ( Bennington, 1969; Harris et al., 1970; Pierce and Wallace, 1971). An obvious approach is to determine whether the tumor cells with a capacity to metastasize have junctions by examining tumor cells at metastatic tumor sites. Data on this topic are fragmentary and inconclusive, although cell junctions are observed in metastatic tumors (Gondos, 1969; Tarin, 1970; Letourneau et al., 1975).

5 . Cell Junctions in Nonmalignant Growth Disorders The occurrence of cell junctions in nonneoplastic disease states is a topic of considerable interest, since many of the changes in tumor junctions may represent nonspecific reactions. Cell junctions are described in a few papers on tissue ultrastructure in nonmalignant growth states (Pitelka et al., 1973; Vogel and Narasimnan, 1974; Tice et al., 1975). For example, several studies have examined junctions in epithelial metaplasia. Metaplasia is a nonneoplastic, reversible form of abnomial tissue regeneration in which one adult cell type is replaced by another adult cell type. Harris et al. (1973) studied maculae adherentes in chemically induded squamous metaplasia of the tracheobronchial epithelium. They found that the maculae adherentes in metaplastic tissue are more fully developed than in normal respiratory epithelium. This finding is expected since maculae adherentes are more fully developed in normal squamous epithelium than in pseudostratified respiratory epithelium. Recently, Prutkin ( 1975) found that topical applications of vitamin A-acid to tumors, e.g., keratoacanthomas, arising in rabbit ear skin epithelium and to normal skin epithelium produced mucous metaplasia. Gap junctions form early in the process of mucous metaplasia both in tumors and in normal epithelium. Junctions have been examined in other growth states, such as hyperplasia, hypertrophy, and atrophy (Pitelka et al., 1973). R. TISSUE CULTURE Occludentcs junctions ( Xlartinez-Palonio, 19703; Lavin and Koss, 1971), gap junctions (Pinto da Silva and Gilula, 1972), and adherentes junctions (McNutt et al,, 1973) are present in some culture systems. Gap junctions occur in many fibroblastoid cell lines (Pinto da Silva and Gilula, 1972; Revel et al., 1971; OLague and Dalen, 1974), but, according to Hiisler, they are less frequently encountered in epithelioid lines of cultured cells (Hiilser and Demsey, 1973; Hulser and Webb, 1973) . I n general, positive correlation cannot be demonstrated between

INTERCELLULAR JUNCTIONS IN CANCER

61

tumorigenicity of cultured cells and the presence or the absence of gap junctions ( Hiilser and Webb, 1973). Cultured cells often manifest different junctional properties from the corresponding cells in uiuo; quite frequently, junctions are more conspicuous in culture systems. For example, malignant hepatocytes isolated from Novikoff hepatoma readily form low-resistance junctions ( Borek et al., 1969) although most of the hepatocytes in the tumor of origin are electrically uncoupled ( Loewenstein and Kanno, 1967). Cells in ethylnitrosourea-induced malignant neurinomas are uncoupled, but cell lines derived from the tumors are coupled. Occludentes junctions may be relatively inconspicuous in solid tumors but flourish in comparable culture systems (Lavin and KOSS,1971) (Fig. 7C). There are several possible explanations for the differences between the occurrence of junctions in vivo and in uitro. First, while several cell phenotypes may coexist in solid tumors, there may be preferential growth of one cell phenotype (i.e., junction formers) in culture ( Azarnia and Loewenstein, 1971). Another possibility is that factors in the cellular microenvironment may inhibit cell junction formation in uiuo. This mechanism could account for the finding that morphologically normal liver cells growing in close proximity to cancer cells display a marked reduction in low-resistance electrotonic coupling ( Loewenstein and Kanno, 1967). Alternatively, factors in the culture media (e.g., proteolytic enzymes activated by plasminogen activator) may stimulate junction proliferation in tissue cultures.

V. Cell-to-Cell Communication and Growth Control

A. INTRODUCTION The growth of constituent tissues is precisely balanced and coordinated in normal animal organs. In most adult tissues, the rate of new cell production offsets the rate of cell loss enabling the tissue mass to remain essentially constant (Castor, 1968; Vasiliev et al., 1969). Cell growth and differentiation in complex tissues has been extensively studied. While a number of theories have been advanced to explain the modus operundi of homeostatic mechanisms for the control of growth (Bullough, 1965; Loewenstein, 1968a; Roth, 1973), the identity and nature of regulatory systems that influence growth in embryonic and adult tissue remains a matter for speculation. Nonetheless, our knowledge of the general features of control systems lends support to the popular notion that cell junctions play a significant role in the regulation of growth. All

62

RONALD S. WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

control systems consist ofdhree cardinal elements. These are: (1) the control signals, such as chemical mediators; ( 2 ) a conduit for the conveyance of signals; and (3) a signal receptor (and effector). Theoretically, defects in any of these elements could account for the loss of growth control that may be characteristic of malignancies. It is reasonable to expect that different etiologies of cancer might disrupt any one of these three elements and still produce an identical perturbation of growth control ( Loewenstein, 1974). Changes in cell junctions associated with neoplastic transformation may constitute a breakdown in one element of the growth control system, the conduit for the conveyance of signals. In mammals there are three levels of cell-to-cell communication by means of transfer of substances. The first two are long-range communication, which involves the exchange of diffusible regulatory substances, such as blood-borne tropic hormones, over long distances, and shortrange communication, which involves the transfer of signal molecules, such as embryonic inducer substances, between cells that are close to each other but not intimately joined. Both of these require that the signal molecules move through the intercellular space. The third, intimate communication, requires actual contact between cells for the passage of information. This is accomplished either by allosteric mechanisms involving interaction of membrane components (Roth, 1973) or by the direct exchange of ions and molecules either through permeable intercellular junctions (Loewenstein, 1968a,b, 1973) or, far less commonly, through true cytoplasmic bridges (Fawcett et al., 1959; Zamboni and Gondos, 1968; Woodruff and Telfer, 1973). Whereas the general plasma membrane may participate in long-range and short-range communication, intimate communication by the direct transfer of substances is mediated by cell junctions or bridges.

H.

INTIMATE COMAIUNICATION AT

GAP JUNCTIONS

Pioneer studies demonstrating the existence of intimate communication via the selective transfer of substances (Furshpan and Potter, 1959; Loewenstein and Kanno, 1964; Loewenstein, 1966) were conducted by inserting micropipettes into neighboring cells, passing a current through them, and then measuring the resting potential between the micropipettes. These studies showed low-resistance electrotonic coupling between manv types of cells (Furshpan and Potter, 1959; Loewenstein and Kanno, 1967) indicating that contact areas of cell membranes do not impede ionic flow from cell to cell. This type of ion transfer is related to the presence of gap junctions (Barr et aZ., 1965; Dreifus et al., 1966; Gilula et al., 1972) .

INTERCELLULAR JUNCTIONS IN CANCER

63

Low-resistance junctions are found in both excitable tissues ( Furshpan and Potter, 1959; Woodbury and Crill, 1961; Potter et al., 1966) and nonexcitable tissues ( Loewenstein et al., 1965; Loewenstein, 1966). Their occurrence in tissues that do not propagate action potentials raised the possibility that these junctions serve functions in addition to ionic coupling of cells. In addition to inorganic ions, small organic ions and molecules pass freely from cell to cell at gap junctions. This was first shown by Loewenstein and Kanno (1964), who followed the transfer of the organic dye fluorescein (MW 342) between cells. They injected fluorescein into Drosophila salivary gland cells with micropipettes and observed that the probe molecules diffuse throughout the cytoplasm of the injected cell and then pass with apparent ease into neighboring cells. This observation raised the possibility that biologically active molecules such as nucleoside triphosphates ( MW 482523 ) or cyclic 3’,5’-adenosine monophosphate ( CAMP; MW 349) may pass from cell to cell at gap junctions (Sheridan, 1971; Merk et al., 1972). Since small organic molecules pass freely through gap junctions, it is important to establish the maximum sizes of molecules that are transferred via this route. Gap junction channels have been probed with tracer molecules of graded size (Kanno and Loewenstein, 1966; Reese et al., 1971). In 1966, Kanno and Loewenstein reported the largest molecule capable of passing by diffusion from cell to cell in salivary gland epithelia is bovine serum albumin (BSA), which has a molecular weight of 69,000 and an equivalent hydrodynamic radius of 3.6 nm. With a micropipette they injected radioactively labeled BSA into cells and then examined neighboring cells for the presence of radioactivity. Although the radioactive label was present in neighboring cells, no effort was made to recover and analyze it, leaving the possibility that the label was attached to split products rather than to intact BSA. Loewenstein (1966) also claimed that BSA, conjugated with the fluorescent label fluorescein-isothiocianate, can diffuse through cell junctions, but here again injected BSA was not recovered. Reese et al. (1971) used peroxidase to probe gap junction channels in the lateral giant axon fiber of the crayfish. Peroxidases can be visualized by histochemical methods at the ultrastructural level. In order to monitor the spread of tracer molecules in the light microscope, they added fluorescein to the peroxidase solution prior to injection into the segmental axons. In the presence of electrical coupling and fluorescein dye transfer, horseradish peroxidase ( MW 40,000) failed to transfer to neighboring axons. A low-molecularweight microperoxidase ( MW 1800), however, succeeded in passing from cell to cell. Reese and his associates interpreted the results as

64

RONALD S .

WEINSTEIN, FREDERICK B. MERK, A N D

JOSEPH ALROY

showing that the hydrophilic channels traversing gap junctions can accommodate molecules with molecular weights of up to at least 1800. A criticism of their experimental design has been raised by McNutt and Weinstein (1973), who point out that microperoxidase may not be immobilized during fixation with glutaraldehyde and so may pass through low-resistance passageways after they are altered by fixation for electron microscopy. Thus, the upper size limit for molecules that can pass through junctional channels remains an open question, but it is probably around 1000 daltons ( Staehelin, 1974). Gap junctions in many different tissues are indistinguishable from one another in the electron microscope although there is compelling evidence that they may have different sieving properties. This evidence comes from studies on embryonic tissues. Certain embryonic cells that are electrotonically coupled, presumably at gap junctions, do not transfer fluorescein in detectable quantities (Slack and Palmer, 1969; Sheridan, 1971; Bennett et al., 1972). Further, cells in Xenopus laevis embryos are electrotonically coupled, but fluorescein dye does not pass between them during early developmental stages. However, at later stages of development both electrotonic coupling and dye transfer are observed. These data suggest that the hydrodynamic pore diameter of gap junction channels may not be fixed and may be modulated by an as yet unidentified control mechanism. Alternatively, gap junctions may represent a family of structures that differ with respect to fixed pore size (Bennett et al., 1972).

C . CONTROL OF GAP JUNCTION PERMEABILITY Intracellular calcium ( Ca?+), adenosine triphosphate ( ATP ), cyclic adenosine monophosphate (CAMP), and electrochemical potentials across the plasma membrane each seem to play a significant part in the regulation of gap junction permeability in normal intact tissues.

1. Intracellular Calcium and Adenosine Triphosphate Gap junctional permeability requires a cytoplasmic concentration of A4 (Loewenstein, 1967; Loewenstein et free Ca2+ below 10-5 to al., 1967; Payton et al., 1969; Rose and Loewenstein, 1971). Permeability falls markedly when free Ca?+ is injected into a cell through a micropipette, enters the cell from the extracellular compartment via a tear in the cell membrane, or diffuses into the cell sap from damaged mitochondria (Loewenstein, 1967; Politoff et al., 1969; Rose and Loewenstein, 1975a,b). When intracellular Ca2+levels equilibrate to levels of Ca2+typically present in extracellular fluids (i.e., lo-?to 1 0 - ~M ) junctional perme-

INTERCELLULAR JUNCTIONS IN CANCER

65

ability falls and becomes essentially the same as that of the general plasma membrane ( Oliveira-Castro and Loewenstein, 1971). Junctional permeability can be restored to normal by reestablishing low cytoplasmic free Ca2+levels (Rose and Loewenstein, 1975a). Maintenance of a low intracellular level of free Caz+ requires the adequate function of ATP-driven membrane pumps. Predictably, inhibition of ATP synthesis or excess ATP utilization raises intracellular Ca2+ levels and depresses junctional permeability (Politoff et al., 1969). The uncoupling of the gap junction by an elevation of intracellular Ca2+ may represent a significant early pathophysiologic response to cell injury since an influx of extracellular Caz+ into the cytoplasm of an injured cell will effectively insulate that cell from its noninjured neighbors ( Loewenstein and Penn, 1967; Loewenstein, 1972). Extracellular levels of Ca2+and Mg2+also influence junctional permeability by regulating the tightness of the junctional seals that insulate junctional conduits from the extracellular compartment ( Rose and Loewenstein, 1971). 2. 3s-Cyclic Adenosine Monophosphate (CAMP) cAMP is an intermediate or “second messenger” in the action of a number of hormones ( Robison et al., 1971). This nucleotide is of particular interest in light of the theme of this review because it probably is involved in the regulation of cell growth in normal cells and is capable of restoring contact inhibition of cell proliferation to transformed CUItured cells (Burk, 1968; Hsie and Puck, 1971; Otten et al., 1971; Sheppard, 1971; Burger et al., 1972; Seifert and Paul, 1972; Smets, 1972; Willingham et al., 1972; Tee1 and Hall, 1973). Whereas low intracellular levels of cAMP are present in actively growing cell cultures, high concentrations inhibit cell proliferation ( Sutherland, 1970; Frank, 1972; Froehlich and Rachmeler, 1972; Seifert and Paul, 1972). There is evidence that intimate communication, by the transfer of information carrying molecules at gap junctions, is a CAMP-mediated phenomenon (Hax et al., 1974a,b). Studies on salivary glands of the larvae of Drosophila hy&i in which intracellular cAMP levels are elevated by incubation of the gland in a medium containing either dibutyryl-CAMP, theophylline, or ecdysterone have shown that increases in intracellular cAMP are accompanied by increases in gap junctional permeability and decreases in the permeability of the general plasma membrane (Hax et al., 1974a,b).

3. Electrochemical Potential across the General Plasma Membrane Permeability of gap junctions is influenced by the potential across the general ( nonjunctional ) membrane. Evidence for a relationship be-

66

RONALD s. WEINSTEIN, FREDERICK B. MEW, AND JOSEPH ALROY

tlveen general plasma-membrane potential and gap-junction permeabdity comes from several observations. A depolarizing current uncouples lowresistance junctions in glandular epithelium (Socolar and Politoff, 1971), and junctional permeability can be restored by a repolarizing current under certain experimental conditions (Rose, 1971; Rose and Loewenstein, 1971) . Changes in junctional permeability that accompany changes i n membrane potential are probably due in part to intracellular Ca2+, since depolarization of the membrane produces an increase in Ca2+permeability and Ca2+enters the cell. 4. Suminary Based upon these observations and theoretical considerations, Hax and his associates have proposed that CAMPlevels may play a significant role in the molecular regulation of coupling phenomena (Hax et al., 1974a,b) . They suggest that hormonal activation of the adenylate-cyclase system, which results in an increase in intracellular cAMP and a concomitant decrease in ATP (Robison et al., 1971), may increase junctional permeability and thus enhance the flow of low-molecular-weight informational molecules from cell to cell. Conversely, decreased intracellular concentrations of cAMP would impede the transfer of substances at low-resistance junctions. According to their scheme, cAMP and intracellular Ca’+ may be components of a feedback system that regulates cell to cell communication. It is known that low intracellular concentrations of Ca?+ are responsible for a high level of adenylate-cyclase activity, which in turn results in an increased synthesis of CAMP (Bradham et al., 1970). Conversely, increased levels of intracellular Ca2+ inhibit adenylate-cyclase activation ( Bar and Hechter, 1969; Rasmussen, 1970; Bradham et al., 1970; Rubin et al., 1972). Therefore, cAMP and intracellular free Ca’+ appear to be mutually involved in a negative feedback control of their respective concentrations, although the precise mechanisms by which this is accomplished remain to be elucidated. Hax et al. (1974a) postulate that the same control system might regulate intracellular communication at gap junctions. They suggest that an elevated cAMP concentration may initiate the conversion of an inactive precursor of membrane-bound phospholipase A into its active form. A resulting increase of lysophospholipid of the junctional membrane may enhance intercellular communication ( Hax et al., 1974a).

D. METABOLICCOUPLING AT GAP JUNCTIONS Metabolic coupling (also called “metabolic cooperation”) of cells (see Section III,B ) has been demonstrated in several tissue-culture systems. Subak-Sharpe and his associates first demonstrated metabolic coupling

INTERCELLULAR JUNCTIONS I N CANCER

67

by showing that a defective phenotype of certain mutant cells in tissue culture can be corrected by intimate contact with normal cells (Burk et al., 1968; Subak-Sharpe et al., 1969). They found that mutant Chinese hamster fibroblasts, deficient in the enzyme inosinic guanylic pyrophosphorylase ( IPP- ) , are incapable of incorporating exogenous hypoxanthine into their nucleic acids when they grow alone, but that they do incorporate hypoxanthine when grown in contact with a wild-type cell (IPP'). The most probable explanation of these observations is that a low-molecular-weight molecule, such as a nucleotide or a nucleotide derivative, is transferred from the wild-type cell to the mutant cell, thus bypassing the enzyme block in the IPP- mutant (Subak-Sharpe et al., 1969; SubakSharpe, 1969). Another possible, but less likely, explanation is that the substance that passes to the mutant cell and endows it with the ability to incorporate [ 3H]hypoxanthine i s episomal DNA, informational RNA, or a regulatory substance that endows the mutant with the ability to synthesize or activate a functional inosinic pyrophosphorylase (Cox et al., 1970; Pitts, 1971). Gilula et al. (1972) examined the relationship between metabolic and ionic coupling and gap junctions. They studied several types of cocultured cells in various combinations in order to determine whether cell-tocell transfer of molecules could be correlated with the presence or the absence of gap junctions. Their test system included three cell lines: Don hamster fibroblasts, a normal cell type, which can incorporate exogenous [3H]hypoxanthine into nucleic acids; and two mutant lines, DA cells and A9 cells, which are both IPP- and therefore cannot incorporate [3H]hypoxanthine when cultured alone. The Don and DA cells are gapjunction formers, whereas the A9 cells are not. In these experiments Don:Don cell combinations formed gap junctions and were both ionically and metabolically coupled. A9:A9 combinations did not form gap junctions and were uncoupled. Don :DA cells formed gap junctions and were ionically and metabolically coupled. These observations elegantly demonstrate that ionic and metabolic coupling may occur if gap junctions are present but not in their absence (Gilula et al., 1972).

E. COUPLING BETWEEN TUMORCELLS Loewenstein and his colleagues provided the first evidence of a defect in low-resistance coupling in solid malignant tumors. They made electrical measurements on carcinomas in liver (Loewenstein and Kanno, 1967), thyroid (Jamakosmanovib and Loewenstein, 1968), and stomach (Kanno and Matsui, 1968) and found an absence of electrotonic coupling. Based on these observations and theoretical considerations, Loewenstein proposed that a genetically determined interruption of junc-

68

RONALD S. WEINSTEIN, FREDERICK B. MEXK, AND JOSEPH ALROY

INTERCELLULAR JUNCrIONS IN CANCER

69

tional communication may be one of many cazrses of cancerous growth (Loewenstein, 196813). Later, Sheridan ( 1970) succeeded in demonstrating electrotonic coupling between some tumor cells within Novikoff hepatomas, an observation consistent with Loewenstein’s prediction of coupling in some tumors of other etiologies (Loewenstein, 1974) as well as with other interpretations ( Sheridan, 1970; McNutt et al., 1971). Loewenstein and his co-workers also studied metabolic coupling in malignant cells and found a correlation between defective cell-to-cell transfer of [3H]hypoxanthine-derived material, and a lack of electrotonic coupling and the capability to transfer dye. They loaded three lines of cancer cells, two derived from the Morris 5123 hepatoma and one from an X-irradiated embryonic hamster cell, with [3H]hypoxanthine, and showed the absence of transfer of the labeled nucleotide to heterotypic IPP- mutant cells in coculture. These tumor lines also lack junctions that are permeable to either inorganic ions or fluorescein (Azarnia et al., 1972). F. GENETICCORRELATIONS In an elegant study, Azarnia and Loewenstein demonstrated a genetic correlation between the simultaneous occurrence of gap junctions, ionic coupling and contact ( density-dependent ) inhibition of growth ( Azarnia and Loewenstein, 1973; Azarnia et al., 1974). Further, they found evidence that the reinstatement of contact inhibition of growth by cell hybridization is accompanied by a parallel correction of gap junction effects. Cultured human Lesch-Nyhan fibroblast cells have gap junctions, are electrotonically coupled, and fluorescein freely diffuses from one cell to another (Fig. 8). They show contact inhibition of growth. CulFIG. 8. Demonstration of a correlation between the occurrence of gap junctions and cell-to-cell coupling in a human cell/mouse cell hybrid system. Left column: Figures a-d show the morphology of four cultured cell lines: ( a ) Lesch-Nyhan human parent cells; ( b ) mouse parent cells CI-1D derivative of an L-cell mouse line); ( c ) early hybrids between human cells and mouse cells; ( d ) revertant hybrids after loss of about 30 out of 46 human chromosomes. Right column: For a cell pair (cell I and 11) in each culture, current ( i = 1 x A) was injected into cell I and, after a 100-msec delay, another current was injected into cell 11. Resulting changes in membrane voltage ( V ) were measured simultaneously in the two cells with intracellular microelectrodes and displayed on an oscilloscope (inset). Simultaneously, fluorescein was injected into cell I, and the fluorescence was photographed in a dark field after a 5-minute interval. The relatively large V, in ( a ) and ( c ) show that there is cell-to-cell passage of small ions (the carriers of i ) . Fluorescein spread in ( a ) and ( c ) demonstrates cell-to-cell passage of small organic molecules. Azarnia et aZ. (1974), by permission.

70

RONALD s. WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

tured mouse Cl-1D cells have a coupling frequency of zero, will not exchange fluorescein with neighboring C1-1D cells, lack gap junctions, and are not contact inhibited. Hybrids were prepared from Lesch-Nyhan and Cl-1D cells bv fusion with inactivated Sendai virus (Harris 1970). The resulting hybrid cells contain a nearly complete complement of chromosomes, are contact inhibited, have gap junctions, and are coupled with neighboring hybrid cells. This shows that the capacity to form cell associations necessary for coupling is expressed in hybrid membrane. Azarnia and Loewenstein (1973) have also shown that as hybrid cells lose human chromosomes with serial passage, clones appear among the segregants which have reverted to the noncoupling and junction-deficient trait of the mouse parent cells. Azarnia et al. (1974) postulate that the human cells may contribute a genetic factor to the hybrids that corrects the junctional deficiency of the mouse cells. The factor could be a junctional component or, alternatively, a component of the general plasma membrane which may be essential for cell-cell recognition or junction assembly. VI. Cell Junctions in Embryonic Developmeni

If gap junctions are essential for normal growth control processes, we would expect cell junctions to appear early in phylogeny and in embryogenesis of higher organisms (Trelstad et a?.1966, , 1967; Loewenstein, 1968a; Sheridan, 1970; Pappas et al., 1971; Bennett, 1973; Ducibella et al., 1975; Fisher and Linberg, 1975). Cell junctions have now been observed during cmbryogenesis in many systems. For example, blastomeric cells of Triturus pyrrhogaster (It0 and Loewenstein, 1969) and newt embryo cells at the morula stage of development are joined by gap junctions (It0 and Hori, 1966). Potter and his associates (1966) demonstrated junctional communication between dissimilar groups of cells during the later stages of squid embryo development, and, recently, the formation of gap junctions during amphibian neurulation has been described ( Decker and Friend, 1974). Adherens and occludens junctions also have been described in embryonic and fetal development (Hay and Revel, 1969; Revel et aL, 1973). The formation and proliferation of cell junctions in embryos cannot be considered synonymous with progressive differentiation since selective disappearance of junctions is often coincident with maturation. In the newt Xenopus 2aeui.s the eye becomes irreversibly polarized at developmental stage 32, after which it is insensitive to stimuli from tissue surrounding the eye (Hunt and Jacobson, 1972). Gap junctions are present throughout the embryonic neural retina and pigment epithelium up to

INTERCELLULAR JUNCXIONS I N CANCER

71

stage 31/32. At stage 32, gap junctions disappear from the central portion of the retina and from the pigment epithelium. This event appears to be correlated with the time of retinal specification (Dixon and CronlyDillon, 1973,1974).

VII. Cell Junctions and the Biological Behavior of Tumor Cells

A. CONTACT INHIBITION OF MOVEMENT( LDCOMOTION) Normal adult epithelial cells and some nonepithelial cells have a latent but restrained locomotor capacity ( Abercrombie and Heaysman, 1954; Oldfield, 1963). These locomotor restraints are diminished in the course of malignant transformation ( Abercrombie et al., 1957; Temin and Rubin, 1958; Abercrombie and Ambrose, 1962). In general, mobile nonneoplastic epithelial cells become quiescent late in embryonic development. However, they retain the ability to resume locomotion when tissues are damaged or when cells are excised and transferred to a tissue culture environment. Normal cells at saturation density in confluent culture exhibit locomotor restraints that may employ the same growth control mechanisms which are operative in intact tissues. Locomotor restraint manifests itself in culture systems by the distinct tendency of cells to uniformly occupy space on the culture substratum and to spread out as monolayers. This growth pattern was first examined in detail by Abercrombie and his associates. They observed that cell locomotion appears to be inhibited when cells collide with their neighbors (Abercrombie and Ambrose, 1958). The overall result is that cells spread out evenly and tend not to overlap ( Abercrombie and Heaysman, 1952, 1954; Abercrombie et al., 1957). The phenomenon of directional inhibition of locomotion in culture has been called “contact inhibition of movement” ( Abercrombie and Heaysman, 1954; Abercrombie, 1970). Many strains of malignant cells in culture are relatively insensitive to contact inhibition. However, this characteristic cannot be considered an invariant property of all malignant cells. It has been suggested that the loss of contact inhibition observed in vitro can be compared with loss of control over cellular movement, thought to exist in solid tumors ( Abercrombie and Ambrose, 1962). This line of reasoning das been extended to compare loss of contact inhibition with tumor invasiveness in vivo, a notion unsupported by current evidence. Several theories attempt to identify a single mechanism of contact inhibition of movement. However, there is a growing awareness that it is probably a multifactorial process (Martz and Steinberg, 1972; Stein-

72

RONALD

s. WEINSTEIN,

FREDERICKB. MF.RK, AND JOSEPH ALROY

berg, 1973). The three unitary theories that have received widest attention are: (1) the mutual adhesion theory; ( 2 ) the differential adhesion theory; and (3) the locomotory paralysis theory. Two of these theories, the mutual adhesion and the locomotory paralysis theory, could implicate cell junctions in the process of contact inhibition of movement.

1. Mutual Adhesion Theory The mutual adhesion theory suggests that contact inhibition results from the formation of strong and long-lasting adhesions between cell pairs. The observation that untransformed 3T3 fibroblasts form adhesions that last three times longer than adhesions between simian virus (SV40) transformed 3T3 cells has been interpreted as strong evidence for this theory (Gail and Boone, 1971). According to the mutual adhesion theory, the reduced susceptibility of transformed cells to contact inhibition is a reflection of the relatively short time that they are immobilized by their adhesions. Cell-to-cell adhesion in these culture systems is partially mediated by cell junctions. McNutt et al. (1973) found that untransformed 3T3 cells are joined together by many adherentes junctions, whereas junctions are less prominent in SV40-transformed 3T3 cells, an observation that may explain the differences in adhesion observed by others (Gail and Boone, 1971). The mutual adhesion theory fails to explain how cell-cell overlapping is prevented by contact inhibition. 2. Differential Adhesion Theary The differential adhesion theory proposes that contact-inhibited cells have a greater affinity for their glass or plastic supporting substratum than for one another. Contact-inhibited cultured cells, according to some experimental data, may adhere more strongly to the substratum than to each other, which would explain their tendency to form a monolayer (Abercrombie, 1961; Carter, 1967). Overlapping of cells would be minimized, since overlapping would require the loss of strong cell-substratum adhesions and the retention of weaker cell-to-cell adhesion. However, Harris (1973) has challenged the central assumption of the differential adhesion theory. He has made the observation that many contact-inhibited cells form coherent sheets and that considerable portions of these cell sheets often pull away spontaneously from the substratum. This would require a relatively greater strength of intercellular adhesiveness than would be present at the substratum front (Coman, 1961; Harris, 1973).

3. Locomotoy Paralysis Theoy According to the locomotory paralysis theory, cell locomotor mechanisms are paralyzed by a signal that emanates from the point of contact

INTERCELLULAR JUNCTIONS I N CANCER

73

with neighboring cells ( Abercrombie, 1961) . This theory attempts to account for the events that accompany contact inhibition of movement, but the theory cannot be evaluated in a detailed fashion at the molecular level because of broad gaps in our understanding of mechanisms by which cells propel themselves. Some proponents have speculated that contact inhibition by locomotory paralysis might be mediated by a chemical agent, perhaps in the form of a diffusible substance that is transferred from cell to cell at gap junctions. This idea was particularly attractive to cancer biologists, since many tumors are deficient in gap junctions (see Sections I V and V,E). Recent experimental evidence does not support the notion that molecular transfer from cell to cell at gap junctions can account for contact inhibition of movement. Ultrastructural and electrical studies reveal that gap junctions are absent at the initiation of contact inhibition in some culture systems (Flaman et al., 1969; Heaysman and Pegrum, 1973a,b; Goshima, 1969). On the other hand, some sarcoma cell lines, which are contact inhibited, are electrotonically coupled ( Furshpan and Potter, 1%8) and have gap junctions (Pinto da Silva and Gilula, 1972). Also, [3H]hypoxanthine-loaded Lesch-Nyhan skin fibroblasts can transfer nucleotides or their derivatives to either contacted-inhibited mouse 3T3 cells or contact-uninhibited transformed 3T3 cells with equal efficiency (Cox et al., 1974) showing that contact inhibition and metabolic coupling are unrelated phenomena. Thus, there is no correlation between the presence of functional gap junctions and contact inhibition of movement. Kolodny speculates that cell-to-cell transfer of macromolecules, such as RNA (Kolodny, 1971) is involved in contact inhibition of movement ( Kolodny, 1974). Exchange of large molecules would presumably occur at the general plasma membrane, since there is no evidence that these macromolecules are transferred through cell junctions.

4. Reevaluation of the Concept of Contact Inhibition Movement Steinberg and his associates have challenged the concept of contact inhibition of movement. They used a Nomarski optical system to prepare time-lapse films of 3T3 mouse cells in confluent cultures and found that so-called contact-inhibited 3T3 cells are capable of translocational mobility along the substratum and, in fact, move readily with respect to their neighbors, although they are restrained from moving over the surface of one another (Martz and Steinberg, 1972; Steinberg, 1973). Wiseman and Steinberg (1973) extended these observations to solid tissues. They monitored the movements of single cells within three-dimensional tissue masses under conditions where contact inhibition of movement would be expected to be present. They seeded radiolabeled embryonic cells onto the surface of embryonic heart and liver tissue

74

RONALD S .

WEINSTEIN, FREDERICK B. MERK, AND

JOSEPH ALROY

fragments (or aggregate$) and followed the positions of the seeded cells by autoradiography. They were able to demonstrate that single cells can penetrate solid tissues and can migrate for considerable distances within the tissue masses. These observations are inconsistent with the idea that cell-to-cell contact entirely curtails cell movement.

B. POSTCONFLUENCE INHIBITIONOF GROWTH(CELLDIVISION) In many nonmalignant culture systems, extensive cellular contact switches off net RNA and protein synthesis, and blocks the synthesis of new DNA (Stoker, 1967). This phenomenon has been called variously “contact inhibition of growth,” “density-dependent inhibition,” “contact regulation of cell division,” and “postconfluence inhibition of cell division‘‘ ( Martz and Steinberg, 1972). Contact inhibition of movement and postconfluence inhibition of cell division are operationally distinct and may be the manifestations of different control mechanisms (MacieiraCoelho, 1967; Stoker, 1967). However, postconfluence inhibition of cell division, like contact inhibition of movement, tends to bc markedly reduced in cultured tumor cells. Kinetic studies relating cell contact to inhibition of cell division suggest that the two processes are unrelated. Martz and Steinberg (1972) examined 3T3 cells at confluence and found that inhibition of cell division appears one cell generation after cells come into initial contact. From this they concluded that factors in addition to simple cell-to-cell “contact” must contribute to inhibition. They proposed several alternative mechanisms to explain the phenomenon. One of the mechanisms may implicate gap junctions. They suggested that areas of membrane contact may not become effective inhibitors of division until the contacts have “matured.” Stated another way, time may be required for fully functional cell junctions to form at the cell surface. Rates of formation for junctions are consistent with this idea since junctions take minutes or hours to become coupled (It0 et aZ., 1974a,b; Johnson et al., 1974). On the other hand, many cells that remain in contact probably never become uncoupled, even during division. Electrotonic coupling ( O’Lague et al., 1970) and gap junctions (Merk and McNutt, 1972) persist between mitotic and interphase cells, indicating that intercellular communication probably is not related to inhibition of division. C. INVASION AND METASTASES

Cell junctions are discussed in relation to tumor invasion in Sections 1\7,A,2,b; IV,A,4; and VIII,B. Several additional points about the invasion

INTERCXUULAR JUNCXIONS IN CANCER

75

phenomenon and its relation to growth control should be underscored for purposes of clarity. First, the control of growth (i.e., DNA synthesis) and the phenomenon of invasion are different processes and may well be unrelated to one another. Growth control is compromised in both benign and malignant tumors, but invasiveness is associated exclusively with cancer. Because of an abnormality of growth control, a benign tumor may grow expansively and may achieve enormous size, but the tumor will not invade the connective tissue stroma. On the other hand, in some malignan t tumors cell proliferation is reasonably well controlled, as evidenced by the tumors’ small size and low mitotic index, and yet such tumors may readily invade stroma and even metastasize widely throughout the body. Therefore, invasion and metastases, the aspects of tumor behavior which most frequently threaten life, are not the direct sequelae of loss growth control but result from other tumor cell properties and host factors. VIII. Intercellular Adhesion in Tumors

A. ADHESIONAT CELLJUNCTIONS A relatively low strength of adhesion between tumor cells may contribute to the biological behavior of malignancies (Coman, 1944, 1961) and may explain certain patterns of tumor growth (Steinberg, 1963). Intercellular adhesion in normal tissues as well as tumors is clearly a multifactorial process (Curtis, 1973). All classes of cell junctions, plus general plasma membrane components ( e.g., gIycoproteins, glycolipids, etc. ), microexudates, and divalent ions may all contribute to intercellular adhesion. Many methods have been developed to quantitate the strength of cell-to-cell adhesion in vitro (Berwick and Coman, 1962; Roth and Weston, 1967; Orr and Roseman, 1969; Curtis, 1970; Steinberg, 1970; Armstrong, 1971; Roth et al., 1971; Walther et al., 1973), but it is doubtful that each method measures the same molecular event (Walther et al., 1973). None of these quantitative methods have been used to estimate the relative contributions of each type of cell junction or other membrane components to adhesiveness. Since cell junctions form rapidly after cells come into contact (It0 and Loewenstein, 1969; Flaxman et al., 1969; Johnson et al., 1974; Ito et al., 1974a,b), it is likely that they are major contributors to cell adhesion as measured by these methods. Several electron microscopy studies provide qualitative information on the relative contributions of various plasma membrane components to adhesiveness under highly artificial conditions, including extracellular Caz+depletion (Sedar and Forte, 1964; Muir, 1967) or incubation of

76

RONALD S. WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

tissue in a markedly hyperosomotic medium ( B a n et al., 1965, 1968; Goodenough and Gilula, 1974). In general, these studies suggest that cell-to-cell adhesion per unit area of surface membrane is considerably greater at cell junctional sites than in regions of nonjunctional close apposition of neighboring cells. Intercellular adhesion may be decreased in some solid tumors. This concept gained early support from the work of Coman and his associates, who attempted to compare the cell-to-cell adhesion of different tissues (Coman, 1944; McCutcheon et al., 1948; Coman and Anderson, 1955; Berwick and Coman, 1962). They devised a micromanipulation method to pull cells apart, using a calibrated microneedle. When pulling away an impaled cell, the degree of bend in the microneedle was used as an index of strength of adhesion (Coman, 1944, 1961). They found that carcinoma cells are less adherent than normal epithelial cells. This conclusion is consistent with the clinical observation that carcinoma cells exfoliate more readily than normal cells. However, many factors, including the absence of strongly interadherent superficial cells and increases in cell turnover rates, probably contribute to cell shedding from tumors. The reduction of intercellular adhesion described by Coman may arise at an early stage of malignant transformation (Coman, 1960). Analysis and comparison of intercellular adhesion within both normal stratified epithelium and tumors is a complex problem because of differences in intercellular adhesion at different levels in tissues. Heterogeneity of adhesiveness within epithelia is suggested by ultrastructural data. Basal cells in uterine cervical epithelium (McNutt et al., 1971) and in urinary bladder uroepithelium (B. U. Pauli and R. S. Weinstein, unpublished data) have fewer cell junctions than intermediate and superficial cells, suggesting that cell interadhesion may increase with maturation in these tissues. Further, a junctional complex (e.g., the terminal bar of columnar epithelia), consisting of several types of cell junctions in close juxtaposition along the membrane, forms near the apex of superficial cells in many normal epithelia and provides a very strong zone of intercellular adhesion. Junctional complexes are attenuated or absent in many malignancies ( Fulker et al., 1971; Weinstein et aE., 1974). These and other variations in ultrastructure should be considered when attempting to compare cell-to-cell adhesion in normal tissue with adhesion in tumors. Since typical tumor cells are usually incompletely differentiated, the only valid comparisons would be between strengths of adhesion between tumor cells and adhesion between cells from a level in normal epithelium at a comparable stage of differentiation. These comparisons would be exceedingly difficult to make in soiid tissues for technical reasons, including the position of comparable cell layers deep within normal epithelium.

INTERCELLULAR JUNCTIONS I N CANCER

77

B. TUMOR DISSEMINATION Since the turn of the century, it has been suspected that cancer cells may be disseminated into the general circulation by surgical trauma ( Tyzzer, 1913). Mechanical manipulation of malignant tumors during surgery presumably dislodges weakly adhering tumor cells. This idea provided the rationale for the development of the “no touch isolation” procedure for resecting colonic cancers, as described by Turnbull et al. (1967). They found that ligation of efferent tumor blood vessels prior to handling of the tumors improved survival rates. IX. Transepithelial Permeability and Malignant Transformation

Zonula occludens junctions (tight junctions) form a permeability seal between epithelial cells (see Section 111,A). Leakiness of zonula occludens junctions is increased by many factors, including electrical shock (Hirano et uZ., 1970), surgical trauma (Rhodes and Karnovsky, 1971), elevation of intravascular hydrostatic pressure (Pietra et d.,1969), immersion in hypertonic solutions (Erlij and Martinez-Palomo, 1972), inhalation of cigarette smoke (Simani et al., 1974), and exposure to chemical carcinogens, such as dibutylnitrosamine or methylnitrosourea ( Hicks et al., 1974). These and other noxous stimuli, which compromise the normal protective function of epithelia, may increase the exposure of basal cell layers to water-soluble carcinogens (Simani et al., 1974) and by this mechanism exert a cocarcinogenic effect. Attenuation of occludentes junctions ( Martinez-Palomo, 1970a; Fulker et al., 1971) (see Table I ) and a concomitant increase in transepithelial permeability (Hicks et aZ., 1974) is a common occurrence in carcinomas. The extent of junctional attenuation appears to correlate well with the level of tumor anaplasia (Weinstein et aZ., 1974), at least in some tumor systems. X. Summary

Intercellular junctions (cell junctions) are a set of structurally complex membrane components that are incorporated into the general plasma membrane at sites of close cell-to-cell apposition. The functions of some types of cell junctions are reasonably well understood. For instance, we now know that all types of junctions contribute to intercellular adhesion and that the zonula occludens endows epithelia with a sealing capacity against bypass diffusion. However, the primary function of some types of junctions, such as gap junctions, remains obscure; this is unfortunate since a considerable body of information on the occurrence, bio-

78

RONALD

s. WEINSTEIN, FREDERICK B. MERK, AND

JOSEPH ALROY

chemical ultrastructure, and physical properties of junctions suggests that they probably do play a central role in important biological phenomena. The pathology literature contains many references to cell junctions in benign and malignant solid tumors. Quantitative evaluations of the occurrence of junctions are subjective in most of these reports, although there are a few reports in which the data were obtained by quantitative electron microscopy techniques. Included in this review is a collation of much information on the occurrence of cell junctions in different types of tumors (Table I). These data show that junctional deficiencies are common in tumors, but they fail to demonstrate any consistent patterns of junctional deficiencies. This may be due to the imprecision of the data more than anything else. In spite of numerous reports to the contrary, there is neither concrete nor compelling circumstantial evidence which supports the popular notion that junctional defects contribute to those properties which are the hallmarks of malignant growth, namely, invasiveness and the ability to metastasize. In our introductory remarks, we noted that a fundamental problem in cancer research is to identify the cell products that are coded for by the genes of neoplastic transformation and to determine how these products can account for the biology of tumors. It is therefore germane to ask whether genes of neoplastic transformation may be related to qualitative and quantitative abnormalities in intercellular junctions, which are observed in some tumors. At the present time, we are unable to provide a definitive answer to this question. However, there are a number of different mechanisms by which gene products could influence intercellular junction formation and function. The most obvious mechanism involves the abnormal synthesis and/or assembly of junctional components. Evaluation of these possibilities must await the successful isolation of specific junctional components and comparative studies of these components in normal tissues and in tumors. Methodology for the isolation of junctional components is rapidly evolving. Another mechanism affectingjunctions probably involves alterations in the general ( nonjunctional) plasma membrane. As discussed in Section II,B, most cell junctions are formed by the structural modification of the general plasma membrane in a stepwise fashion. In tumors, abnormal gene products may modify the general plasma membrane and thus deprive the cell of normal junction assembly sites. This could account for quantitative changes in junctions as well as abnormalities in their biochemical ultrastructure and function. Such a mechanism might account for the linked changes in the occurrence of several types of junctions, as has been observed in several tumor systems (Weinstein et id.,1974). Alternatively, genetic modifications of cell surface components involved in cell-to-cell

INTERCELLULAR JUNCTIONS IN CANCER

79

recognition, either by modification of elements of the cellular machinery responsible for recognition or by masking of recognition sites, could interfere with the formation of junctions. Conversely, unmasking of sites may enhance junction formation. Thus, there are a number of ways in which genes of neoplastic transformation may influence intercellular junction structure and function. Additional research will be required to define the contribution of tumor gene products to the pathogenesis of junctional abnormalities in tumors and to elucidate the roles, if any, of these cell membrane defects in malignant growth.

ACKNOWLEDGMENTS We are grateful to Mrs. Marianne Alroy and Mr. Steven Halpern for editing the manuscript and Ms. Shirley Hunter and Ms. Renee Slack for typing the manuscript. This study was supported by National Cancer Institute Grants CA-14447 and CA16377 from the National Institutes of Health, United States Public Health Service.

REFERENCES Abercrombie, M. ( 1961). Exp. Cell Res., Suppl. 8, 188. Abercrombie, M. (1970). In Vitro 6, 128. Abercrombie, M., and Ambrose, E. J. (1958). Exp. Cell Res. 15, 332. Abercrombie, M., and Ambrose, E. J. (1962). Cancer Res. 22, 525. Abercrombie, M., and Heaysman, J. E. M. ( 1952). Exp. Cell Res. 5, 11. Abercrombie, M., and Heaysman, J. E. M. (1954). Exp. Cell Res. 6, 293. Abercrombie, M., Heaysman, J. E. M., and Karthauser, H. M. (1957). Exp. CeZl. Res. 13, 276. Able, M. E., and Lee, J. C. (1969). Cancer 23,481. Ahmed, A. (1974). J. Pathol. 112, 177. Aikawa, M., and Ng, A. B. P. (1973). Cancer 31,385. Akhtar, M., Gosalbez, T. G., and Brody, H. (1973). Arch Pathol. 96, 161. Albertini, D. F., and Anderson, E. (1974). J. Cell Biol. 63,234. Alroy, J., and Weinstein, R. S. ( 1976). J. Natl. Cancer Inst. In press. Alroy, J., Weinstein, R. S., and Pauli, B. U. (1976). Am. J. Path. In press. Amin, H. K., Ferenczy, A., and Richart, R. M. (1974). J. Comp. Pdhol. 84, 161. Anderson, H. C., Byunghoon, K., and Minkowitz, S. (1969). Cancer 24, 585. Armstrong, P. B. ( 1971). Wilhelm Roux' Arch. Entwicklungsmech. Organismen 168, 125. Azarnia, R., and Loewenstein, W. R. ( 1971). J. Membrane Biol. 6, 368. Azamia, R., and Loewenstein, W. R. (1973). Nature (London) 241, 455. Azamia, R., Michalke, W., and Loewenstein, W. R. (1972). J. Membrane Biol. 10, 247. Azarnia, R., Larsen, W. J., and Loewenstein, W. R. (1974). Proc. Nut. Acud. Sci. U.S. 71, 880. Baba, N., and von Haam, E. ( 1971). J. Nut. Cancer Inst. 47,675. Babai, F., and Tremblay, G. ( 1972). Cancer Res. 32,2765. Bar, H. P., and Hechter, 0. (1969). Biochem. Biophys. Res. Commun. 35, 681. Ban, L., Dewey, M. M., and Berger, W. (1965). 1. Gen. Physiol. 48, 797.

80

RONALD s. WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

Barr, L., Berger, W., and Dewey, M. hl. (1968). J. Gen. Physiol. 51, 347. Barton, A. A. ( 1964). Brit. J. Cancer 18,682. Battifora. H. A. (1973). Cancer 31, 1418. Battifora, H. A., Eisenstein, R., Sky-Peck, H. H., and McDonald, J. H. (1965). J. Urol. 93, 217. Battifora, H. A., Eisenstein, R., and Schild, J. A. (1969). Cancer 23, 183. Behrens, U. J., Mashbum, L. T., Stevens, J., Hollander, V. P., and Lampen, N. ( 1974). Cancer Res. 34, 2926. Bencosnie, S. A., Allen, R. A., and Latta, H. (1963). Amer. J . Pathol. 42, 1. Benedetti, E. L., and Emmelot, P. (1968a). J. Cell Biol. 38, 15. Benedetti, E. L., and Emmelot, P. (1968b). In “The Membranes” (A. J. Dalton and F. Haguenau, eds.), pp. 51, 52, and 56. Academic Press, New York. Bennett, M. V. L. (1973). Fed. Proc., Fed. Amer. SOC. Exp. Bwl. 32, 65. Bennett, M. V. L., Spira, M. E., and Pappas, G. D. (1972). Deuelop. B i d . 29, 419. Bennington, J. L. (1969). Cancer Res. 29, 1082. Bensch, K. G., Conin, B., Pariente, R., and Spencer, H. (1968). Cancer 22, 1163. Berry, h4. N., and Friend, D. S. ( 1969). J. Cell Biol. 43, 506. Berwick, L., and Coman, D. R. ( 1962). Cancer Res. 22, 982. Black, H. E., Capen, C. C., and Young, D. M. ( 1973). Cancer 32, 865. Bordi, C., Anversa, P., and Vitali-Massa, L. ( 1972). Virchows Arch., A 357, 145. Borek, C., Higashino, S., and bewenstein, W. R. (1969). J. Membrane Bipl. 1, 274. Borysenko, J . Z., and Revel, J. P. (1973). Amer. J. Anat. 137, 403. Bradham, L. S., Holt, D. A,, and Sims, M. (1970). Biochim. Biophys. A d a 201, 250. Bransilver, B. R., Ferenczy, A., and Richart, R. M. (1974). Arch. Pathol. 98, 76. Branton, D. ( 1966). Proc. Nut. Acad. Sci. U.S. 55, 1048. Branton, D. (1969). Annu. Reu. Plorlt Physiol. 20,209. Branton, D., and Deamer, D. W. (1972). In “Membrane Structure,” p. 41. SpringerVerlag, Berlin and New York. Branton, D., Bullivant, S., Gilula, N. B., Karnovsky, M. J., Moor, H., Miihlethaler, K., Northcote, D. H., Packer, L., Satir, B., Satir, P., Speth, V., Staehelin, L. A., and Weinstein, R. S. (1975). Science 190, 54. Breathnach, A. S., Stolinski, G., and Gross, M. ( 1972). Micron 3,287. Breton-Gorius, J., Flandrin, G., Daniel, M. T., Chevalier, J., Lebeau, M., and Sanel, F. T. ( 1975). Virchows Arch B. Cell Path. 18, 165. Bretscher, M. S. ( 1972). Nature (London),New Biol. 236, 11. Brightman, M. W., and Palay, S. L. (1963). J. Cell Biol. 19, 415. Brown, W. J., Barajas, L., Waisman, J., and DeQuattro, V. (1972). Cancer 29, 744. Bullivant, S. ( 1970). In “Biological Techniques in Electron Microscopy” (D. F. Parsons, ed. ), p. 101. Academic Press, New York. Bullivant, S. ( 1973 ). In “Advanced Techniques in Biological Electron Microscopy” (J. K. Koehler, ed.), p. 67. Springer-Verlag, Berlin and New York. Bullivant, S. (1974). Phil. Trans. Roy. SOC.London, Set. B 268,5. Bullough, W. S. ( 1965). Cancer Res. 25,1683. Burger, M. M., Bombik, B. M., Breckenridge, B. McL., and Sheppard, J. R. (1972). Nature (London),New Biol. 239, 161. Biirk, R. R. (1968). Nature (London) 219, 1272.

INTERCELLULAR JUNCTIONS IN CANCER

81

Burk, R. R., Pitts, J. D., and Subak-Sharpe, J. H. (1968). Exp. Cell Res. 53,297. Burns, W. A., Matthews, M. J., Hamosh, M., van der Weide, G., Blum, R., and Johnson, F. B. (1974). Cancer 33,1002. Campbell, R. D., and Campbell, J. H. (1971). In “Origin and Continuity of Cell Organelles” (J. Reinert and H. Ursprung, eds.), p. 261. Springer-Verlag, Berlin and New York. Carter, L. P., Beggs, J., and Waggoner, J. D. ( 1972). Cancer 30, 1130. Carter, S. B. ( 1967). Nature (London) 213, 256. Castor, L. N. (1968). J. Cell. Physiol. 72, 161. Chalcroft, J. P., and Bullivant, S. (1970). J. Cell Biol. 47, 49. Claude, P., and Goodenough, D. A. (1973). J. Cell Biol. 58, 390. Coman, D. R. ( 1944). Cancer Res. 4, 625. Coman, D. R. ( 1960). Cancer Res. 20, 1202. Coman, D. R. ( 1961). Cancer Res. 21, 1436. Coman, D. R., and Anderson, T. F. (1955). Cancer Res. 15,541. Cooper, E. H. (1972). Ann. Roy. Coll. Surg. Engl. 51, 1. Cornog, J. L. (1969). Arch. Pathol. 87, 404. Cox, R. P., Krauss, M. R., Balis, M. E., and Dancis, J. (1970). Proc. Nut. Acad. Sci. US.67, 1573. Cox, R. P., Krauss, M. R., Balis, M. E., and Dancis, J. (1974). In “Cell Communication” (R. P. Cox, ed.), p. 67. Wiley, New York. Crick, F. ( 1970). Nature (London) 225,420. Curtis, A. S. G. (1970). J. Embryol. Exp. Morphol. 23,253. Curtis, A. S. G. (1973). Progr. Biophys. Mol. Biol. 27, 315. Deamer, D. W., and Branton, D. (1967). Science 158,655. Decker, R. S., and Friend, D. S. ( 1974). J. Cell Biol. 62, 32. DeHaan, R. L., and Sachs, H. G. (1973). Curr. Top. Deuelop. Biol. 8, 193. Diamond, J. M. (1974). Fed. Proc., Fed. Amer. SOC. Exp. Biol. 33, 2220. Dixon, J. S., and Cronly-Dillon, J. R. (1973). J. Embryol. Erp. Morphol. 28, 659. Dixon, J. S., and Crody-Dillon, J. R. (1974). Nature (London) 251, 505. Douglas, W. H. J., Ripley, R. C., and Ellis, R. A. ( 1970). J. Cell Biol. 44, 211. Dreifuss, J. J., Girardier, L., and Forssmann, W. G. (1966). PfEzregers Arch. Gesamte Physiol. Menschen Tiere 292, 13. Ducibella, T., Albertini, D. F., Anderson, E., and Biggers, J. D. (1975). Deuelop. Biol. 45, 231. Dunia, I., Sen Ghosh, C., Benedetti, E. L., Zweers, A., and Bolemendal, H. (1974). FEBS (Fed. Eur. Brochem. SOC.) Lett. 45,139. Echevarria, R. A. ( 1967). Cancer 20,563. Elgsaeter, A., and Branton, D. (1974). J . Cell Biol. 63, 1018. Erlandson, R. A., and Carstens, P. H. B. ( 1972). Cancer 29,987. Erlandson, R. A., and Tandler, B. (1972). Arch. Pathol. 93, 130. Erlij, D., and Martinez-Palomo, A. ( 1972). J. Membrane Biol. 9, 229. Evans, W. H., and Curd, J. H. (1972). Biochem. J. 138,1972. Farquhar, M. G., and Palade, G. E. (1963). 1. Cell Biol. 17, 375. Favara, B. E., Johnson, W., and Ito, J. ( 1968). Cancer 22, 845. Fawcett, D. W. (1961). Exp. Cell Res., Suppl. 8, 174. Fawcett, D. W., Slautterbach, D. L., and Ito, S. (1959). J. Biophys. Biochem. Cytol. 5, 453. Fechner, R. E., Bentinck, B. R., and Askew, B., Jr. (1972). Cancer 29,501. Feldman, P. S., Horvath, E., and Kovacs, K. (1972). Cancer 30, 1279.

82

RONALD S. WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

Ferenczy, A., Fenoglio, J., and Richart, R. M. ( 1972). Cancer 30,244. Fisher, E. R. (1969). Cancer 24, 312. Fisher, E. R., and Horvat, B. (1972). Cancer 30, 1074. Fisher, E. R., and Sharkey, D. A. (1962). Cancer 15, 160. Fisher, E. R., McCoy, M. M., XI, and Wechsler, H. L. (1972). Cancer 29, 1387. Fisher, S. K., and Linberg, K. A. ( 1975). J. Ultmstruct. Res. 51,69. Flaks, B., Cooper, E. H., and Knowles, J. C. (1970). Eur. I. Cancer 6, 145. Flaxman, B. A. (1972). Cancer Res. 32,462. Flaxman, B. A., Revel, J, P., and Hay, E. D. ( 1969). Exp. Cell Res. 58,438. Frable, W. J., Still, W. J. S., and Kay, S. ( 1971). Cancer 27, 667. Frank, W. (1972). Exp. CeU Res. 71, 238. Friend, D. S., and Cilula, N. B. (1972a).J. Cell Biol. 53, 148. Friend, D. S., and Gilula, N. B. ( 1972b). J. Cell Biol. 53, 758. Froehlich, J. E., and Rachnieler, M. (1972). J. Cell Biol. 55, 19. Fromter, E., and Diamond, J. (1972). Nature (London), New Biol. 235, 9. Fu, Y. S., and Kay, S. ( 1973). Arch. Pathol. 96, 66. Fulker, M. J., Cooper, E. H., and Tanaka, T. ( 1971). Cancer 27, 71. Furshpan, E. J., and Potter, D. D. ( 1959). J. Physiol. (London) 145, 289. Furshpan, E. J.. and Potter, D. D. (1968). Curr. Top. Deoelop. Biol. 3, 95. Gail, M. H., and Boone, C. W. ( 1971). Exp. Cell Res. 65,221. Chatak, N. R., Hirano, A., and Zimmernian, H. M. (1971). Cancer 27, 1465. Gihla, N. B. (1972). J. Ultrastruct. Res. 38, 215. Gilula, N. B. (1974). In “Cell Communication” ( R . P. Cox, ed.), pp. 1-29. Wiley, New York. Gilula, N. B., Branton, D., and Satir, P. (1970). Proc. Not. Acad. Sci. 67, 213. Gilula, N. B., Reeves, 0. R., and Steinbach, A. ( 1972). Nature (London) 235,262. Goldenberg, V. E., Goldenberg, N. S., and Benditt, E. P. (1969). Cancer 24, 236. Condos, B. (1969). Cancer 24, 954. Condos, B. ( 1971).Cancer 27, 1455. Gonzales-Licea, A., Yardiey, J. H., and Hartmann, W. H. (1967). Cancer 20, 1234. Goodenough, D. A. ( 1974). J. Cell Biol. 61,557. Goodenough, D. A., and Cilula, N. B. ( 1974). J, Cell Biol. 61, 575. Goodenough, D. A., and Revel, J. P. (1970). 1. Cell Biol. 45,272. Goodenough, D. A., and Revel, J. P. (1971). 1.Cell Biol. 50,81. Goodenough, D. A., and Stoeckenius, W. ( 1972). J. Cell Biol. 54,646. Coshima, K. (1969).Exp. Cell Res. 58,420. Curd, J. W., and Evans, W. H. ( 1973). Eur. 3. Biochem. 36,273. Hage, E. (1973). Virchows Arch., A 361,121. Haggis, G . H. (1969). Biochim. Biophys. Acta 193, 237. Hameed, K., and Morgan, D. A. (1972). Cancer 29, 1326. Hamlett, J. D., Aparicio, S. R., and Lumsden, C. E. ( 1971). J. Pathol. 105, 111. Harris, A. ( 1973). Develop. Biol. 35, 97. Harris, C. C., Kaufman, D. G., Spron, M. B., and Staffiotti, U. (1973). Cancer Chemother. Rep., Part c 4,43. Harris, H. (1970). In “Cell Fusion,” p. 1. Harvard Univ. Press, Cambridge, Massachusetts. Harris, J. W., Meyskens, F., and Patt, H. M. (1970). Cancer Res. 30, 1937. Hashimoto, K., Yamanishi, Y., Maeyens, E., Dabbous, M. K., and Kanzaki, T. (1973). Cancer Res. 33, 2790.

INTERCELLULAR JUNCTIONS IN CANCER

83

Hax, W. M. A,, van Venrooij, G. E. P. M., and Vossenberg, J. B. J. (1974a). 1. Membrane Bid. 19, 253. Hax, W. M. A., Demel, R. A., Spies, F., Vossenberg, J. B. J., and Linnemans, W. A. M. (1974b). Exp. Cell Res. 89, 311. Hay, E. D. ( 1961). J . Biophys. Biochem. Cytol. 10,457. Hay, E. D., and Revel, J. P. (1969). Monogr. Deuelop. B i d . I, 1. Heaysman, J. E. M., and Pegrum, S. M. (1973a). E r p . Cell Res. 78, 71. Heaysman, J. E. M., and Pegrum, S. M. ( 1973b). Exp. Cell Res. 7,479. Hicks, R. M., Ketterer, B., and Warren, R. C. (1974). Phil. Tram. Roy. SOC. London, Ser B 268, 23. Hill, G. S., and Eggleston, J. C. (1972). Cancer 30, 1092. Hirano, A., Becker, N. H., and Zimmerman, H. M. (1970). J. Neurol. Sci. 10, 205. Holstein, A. F., and Korner, F. ( 1974). Virchows Arch., A 363,97. Hong, K., and Hubbell, W. L. (1972). Proc. Nut. Acad. Sci. U.S.A. 69, 2617. Horvath, E., Kavacs, K., and Ross, R. C. (1972). Virchows Arch., A 356, 281. Hoshino, M. ( 1963). Cancer Res. 23,209. Hou-Jensen, K., Priori, E., and Dmochowski, L. (1972). Cancer 29, 280. Hou-Jensen, K., Rawlinson, D. G., and Hendrickson, M. (1973). Cancer 32, 809. Hruban, Z., Mochizuki, Y., Slesers, A., and Morris, H. P. (1972). Cancer Res. 32, 853. Hsie, A. W., and Puck, T. T. (1971). Proc. Nut. Acad. Sci. U.S. 68, 358. Hiilser, D. F., and Demsey, A. ( 1973). Z . Natulforsch. C . 28, 603. Hulser, D. F., and Peters, J. H. ( 1972). E r p . Cell Res. 74, 319. Hulser, D. F., and Webb, D. J. (1973). E r p . Cell Res. 80,210. Hunt, R. K., and Jacobson, M. ( 1972). Proc. Nat. Acad. Sci. US.69,2860. Imai, H., and Stein, A. A. ( 1963). Gastroenterology 44, 410. Ioachim, N. J., Delaney, W. E., and Madrazo, A. (1974’). Cancer 34, 586. Ito, S., and Hori, N. (1966). 1. Gen. Physiol. 49, 1019. Ito, S., and Loewenstein, W. R. (1969). Develop. B i d . 19, 228. Ito, S., Sato, E., and Loewenstein, W. R. (1974a). J. Membrane B i d . 19,305. Ito, S . , Sato, E., and Loewenstein, W. R. (197413). J. Membrane Biol. 19, 339. Jamakosmanovih, A., and Loewenstein, W. R. ( 1968). J. Cell Biol. 38, 556. Johnson, R., Herman, W. S., and Preus, D. M. ( 1973). J. Ultrastruct. Res. 43,298. Johnson, R., Hammer, M., Sheridan, J., and Revel, J. P. (1974). Proc. N d . Acad. Sci. US. 71, 4536. Kanbour, A. I., Burgess, F., and Salazar, H. ( 1973). Cancer 31, 1433. Kanno, Y., and Loewenstein, W. R. (1966). Nature (London) 212, 629. Kanno, Y.,and Matsui, Y. (1968). Nature (London) 218, 755. Karnovsky, M. J. (1967). J. Cell Biol. 35, 213. Kay, S., and Schatzki, P. F. ( 1971). Cancer 28,755. Kay, S., Elzay, R. P., and Willson, M. A. ( 1971). Cancer 27,674. Kelly, D. E. ( 1966). J . Cell Biol. 28, 51. Kobayashi, S., and Mukai, N. (1974). Cancer Res. 34, 1646. Kogon, M., and Pappas, G. D. ( 1975). J . Cell Biol. 66, 671. Kolodny, G. M. ( 1971). E r p . Cell Res. 65,313. Kolodny, G. M. (1974). I n “Cell Communication” (R. P. Cox, ed.), p. 97. Wiley, New York. Korn, E. D. (1966). Biuchim. Biophys. Acta 116,317. Korn, E. D., and Weisman, R. A. (1966). Biochim. Biophys. Acta 116, 309.

84

RONALD S . WEINSTEIN, FREDERICK B. MERK, AND JOSEPH ALROY

Kovacs, K., Horvath, E., Delarue, N. C., and Laidlaw, J. C. (1974).Horn. Metab. Res. 5, 47. Krawnyk, W. S., and Wilgram, G. F. (1973). 1. Ultrustruct. Res. 45, 93. Kubo, T. ( 1969).Cancer 24,948. Kubo, T.(1974).Acta Pathol. lap. 24,163. Kuhn, C. (1972).Cancer 30,1107. Lasansky, A. ( 1969).I . Cell Biol. 40,577. Lavin, P., and Koss, L. G. ( 1971). I . Nut. Cancer Inst. 46, 597. Leak, L. V., Caulfield, J. B., Burke, J. F., and McKhann, C. F. (1967). Cancer Res. 27, 261. Leifer, C., Miller, A. S., Putong, P. B., and Min, B. H. ( 1974a).Cancer 34,597. Leifer, C., Miller, A. S., Putong, P. B., and Harwick, R. D. (1974b).Arch. Pathol. 98, 312. Lenard, J., and Singer, S. J. (1968).1. Cell Biol. 37, 117. Letoumeau, R. J., Li, J. J., Rosen, S., and Villee, C. A. (1975). Cancer Res. 35, 6. Levine, G . D. (1973).Cancer 31,729. Lin, H.S., Lin, C. S., Yeh, S., and Tu, S.-M. ( 1969).Cancer 23,390. Locke, M. (1965).J . Cell Biol. 25, 166. Loewenstein, W.R. (1966).Ann. N.Y. A d . Sci. 137,441. Loewenstein, W.R. ( 1967). J. Colloid. Interface Sci. 25, 34. Loewenstein, W. R. ( 1968a).Deuelop. B i d . , Suppl. 19,20. Loewenstein, W. R. (1968b).Perspect. B w l . Med. 11, 260. Loewenstein, W. R. (1972).Arch. Intern. Med. 129,299. Loewenstein, W. R. (1973). Fed. Proc., Fed. Amer. SOC. Exp. Biol. 32, 60. Loewenstein, W. R. ( 1974). In “Membrane Transformation in Neoplasia” (J. Schultz and R. E. Block, eds. ), p. 103.Academic Press, New York. Loewenstein, W. R., and Kanno, Y. ( 1964).1. CeU Biol. 22,565. Loewenstein, W.R., and Kanno, Y. ( 1967). 1. Cell B i d . 33, 225. Loewenstein, W.R.,and Penn, R. D. (1967).I . Cell Biol. 33,235. Loewenstein, W. R., Socolar, S. J., Higashino, S., Kanno, Y., and Davidson, N. ( 1965). Science 149, 295. Loewenstein, W. R., Nakas, M., and Socolar, S. J. (1967).1. Gen. Physiol. 50, 1865. Luft, J.H. (1971).Anat. Rec. 171,347. Lupulescu, A., and Boyd, C. B. ( 1972).Cancer 29, 1530. Lynn, J. A., Varon, H. H., Kingsley, W. B., and Martin, J. H. ( 1967). Amer. J. Pathol. 51, 639. Ma, M. H., and Blackburn, C. R. B. (1973).Cancer Res. 33, 1766. Ma, M. H., and Webber, A. J. (1966).Cancer Res. 26,935. McCutcheon, M., Coman, D. R., and Moore, F. B. (1948). Cancer 1, 460. McGavron, M.H. ( 1965).Cancer 18, 1445. Macieira-Coelho, A. (1967).Exp. Cell Res. 47, 193. Mackay, A. M., Pettigrew, N., Symington, T., and Neville, A. M. (1974). Cancer 34, 1108. Macka!., B., Bennington, J. L., and Skoglund, R. W. ( 1971).Cancer 27, 109. McNutt, N. S., and Weinstein, R. S. (1969).Science 165,597. McNutt, N. S., and Weinstein, R. S. (1970).1. Cell Biol. 47,666. McNutt, N. S., and Weinstein, R. S. (1973). Progr. Biophys. Mol. Biol. 26, 45. McNutt, N. S., Hershberg, R. A., and Weinstein, R. S. (1971).J . Cell Biol. 51, 805.

INTERCELLULAR JUNCTIONS IN CANCER

85

McNutt, N. S., Culp, L. A., and Black, P. H. (1973). 1. Cell Biol. 56,412. Malick, L. E. ( 1972). J. Nut. Cancer Inst. 49, 1039. Mao, P., Nakao, K., and Angrist, A. (1966). Cancer Res. 26,955. Marchesi, V. T., and Steers, E., Jr. (1968). Science 159, 203. Marchesi, V. T., Tillack, T. W., and Jackson, R. L. (1972). Proc. Nut. Acad. Sci. U S . 69, 1445. Marikovsky, Y., Brown, C. S., Weinstein, R. S., and Wortis, H. H. (1976). Exp. Cell Res. In press. Marshall, R. B., Roberts, D. K., and Turner, R. A. (1967). Cancer 20, 512. Martinez-Palomo, A. (1970a). In Vitro 6, 15. Martinez-Palomo, A. (1970b). Lab. Invest. 22,605. Martinez-Palomo, A. (1971). Pathobiol. Annu. I, 261. Martinez-Palomo, A., and Erlij, D. (1975). Proc. Nut. Acad. Sci., U S . 72, 4487. Martz, E., and Steinberg, M. S. (1972). J. Cell Physiol. 79, 180. Merk, F. B., and McNutt, N. S. ( 1972). J. Cell Biol. 55,511. Merk, F. B., Botticelli, C. R., and Albright, J. T. ( 1972). Endocrinology 90, 992. Merk, F. B., Albright, J. T., and Botticelli, C. R. (1973). Anut. Rec. 175, 107. Merkow, L. P., Frich, J. C., Slifkin, M., Kyreages, G., and Pardo, M. (1971). Cancer 28, 372. Merrick, T. A., Erlandson, R. A., and Hajdu, S. I. ( 1971). Arch. Pathol. 91,365. Michalke, W., and Loewenstein, W. R. (1971). Nature (London) 232, 121. Mincer, H. H., and McGinnis, J. P. (1972). Cancer 30, 1036. Ming, S. C., Coldman, H., and Freiman, D. G. (1967). Cancer 20, 1418. Mishima, Y. ( 1967). Cancer 20,632. Moor, H. (1966). Int. Reu. Exp. Pathol. 5, 179. Moretz, R. C., Akers, C. K., and Parsons, D. F. (1969). Biochim. Biophys. Actu 193, 1. Muir, A. R. (1967). J . Anat. 101,239. Mukai, N., Kobayashi, S., and Oguri, M. (1974). Acta Neuropathol. 28, 293. Murad, T. M., Mancini, R., and George, J. (1973). Cancer 31, 1440. Nachbar, M. S., Oppenheim, J. D., and A d , F. (1974). Amer. J. Med. Sci. 268, 122. Neville, D. M., Jr. (1960). J. Biophys. Biochem. Cytol. 8,413. Nicolson, G. L. (1973). J. Supramol. Struct. 1,410. OLague, P., and Dalen, H. ( 1974). Exp. Cell Res. 86,374. O’Lague, P., Dalen, H., Rubin, H., and Tobias, C. (1970). Science 170, 464. Oldfield, F. E. ( 1963). Exp. Cell Res. 30, 125. Oliveira-Castro, G. M., and Loewenstein, W. R. (1971). J . Membrane B i d . 5, 51. Orr, C. W., and Roseman, S. ( 1969). J . Membrane Biol. 1, 109. Otten, J., Johnson, G. S., and Pastan, I. (1971). Biochem. Biophys. Res. Commun. 44, 1192. Overton, J. (1962). Deuelop. Biol. 4,532. Overton, J. (1968). J. Exp. Zool. 168, 203. Overton, J. (1974). Progr. Surface Membrane Sci. 8, 161. Overton, J., and Kapmarski, R. (1975). J. Exp. 2001.192, 33. Pappas, G. D., Asada, Y.,and Bennett, M. V. L. ( 1971). J. Cell Biol. 49, 173. Pardee, A. B. (1964). Nut. Cancer Inst., Monogr. 14, 7. Pardee, A. B., Jimenez de Asua, L., and Rozengurt, E. (1974). In “Control of Proliferation in Animal Cells” (B. Clarkson and R. Baserga, eds. ), p. 547. Cold Spring Harbor Lab., Cold Spring Harbor, New York.

86

RONALD S . WEINSTEXN, FREDERICK B. MERK, AND JOSEPH ALROY

Payton, B. W., Bennett, M. V. L., and Pappas, G. D. (1969). Science 166,1641. Peracchia, C. (1973a). J. Cell Biol. 57, 54. Peracchia, C. ( 197313). J . Cell Biol. 57, 66. Perk, K., Hod, I., and Nobel, T. A. (1971). 1. Not. Cancer Inst. 46, 525. Peters, R., Peters, J., Tews, K. H., and Bahr, W. (1974). Biochim. Biophys. Acta 367, 282. Pierce, G. B., Jr. (1966). Cancer 19, 1963. Pierce, G. B., Jr., and Wallace, C. ( 1971). Cancer Res. 31, 127. Pierce, G. B., Jr., Stevens, L. C., and Nakane, P. K. (1967). J. Nut. Cancer Inst. 39, 755. Pietra, G. G., Szidon, J. P., Leventhal, M. M., and Fishman, A. P. (1969). Science 166, 1643. Pinto da Silva, P., and Gilula, N. B. (1972). Exp. CeU Res. 71,393. Pitelka, D. R., Hamamoto, S. T., Duafala, J. G., and Nemanic, M. K. (1973). J. Cell Biol. 56, 797. Pitts, J. D. ( 1971). Growth Contr. Cell Cult., Ciba Found. Symp., 1970 p. 89. Politoff, A. L., Socolar, S. J., and Loewenstein, W. R. (1969). J. Gen. Physiol. 53, 498. Pollack, R. E., and Hough, P. V. C. ( 1974). Annu. Reu. Med. 25, 431. Popoff, N. A., Malinin, T. I., and Rosomoff, H. L. (1974). Cancer 34, 1187. Porter, K. R., Fonte, V., and Weiss, G. ( 1974). Cancer Res. 34, 1385. Potter, D. D., Furshpan, E. J., and Lennox, E. S. (1966). Proc. Nut. Acad. Sci. U S . 55, 328. Prutkin, L. (1975). Cancer Res. 35, 364. Pulley, L. T. (1973).Amer. J. Vet.Res. 34, 1513. Rambourg, A. ( 1969). 1. Microsc. (Paris) 8,325. Ramsey, H. J. (1965). Cancer 18, 1014. Rangan, S. R. S. ( 1972). Cancer 29, 117. Rapin, A. M. C., and Burger, M. M. ( 1974). Aduan. Cancer Res. 20, 1. Rasmussen, H. (1970). Science 170,404. Raviola, E., and Gilula, N. B. (1973). Proc. Not. Acad. Sci. U.S. 70, 1677. Reddy, J., Svoboda, D., Azamoff, D., and Dawar, R. (1973). J. Nut. Cancer Inst. 51, 891. Reese, T. S., Bennett, M. V. L., and Feder, N. ( 1971). Anat. Rec. 169, 409. Revel, J. P., and Karnovsky, M. J. ( 1967). J. Cell Biol. 33, C7. Revel, J. P., Yee, A. G., and Hudspeht, A. J. (1971). Proc. Nut. Acad. Sci. US. 68, 2924. Revel, J. P., Yip, P., and Chang, L. L. ( 1973). Deuelop. Biol35,302. Rhodes, R. S., and Karnovsky, M. J. ( 1971). Lab. Inoest. 25, 220. Richart, R. M., and Barron, B. A. ( 1969), Amer. J. Obstet. Gynecol. 105,386. Richter, W. R., and Moize, S. M. (1963). J. Ultrastruct. Res. 9, 1. Robertson, J. D. (1959). Biochem. SOC. Symp. 16,3. Robertson, J. D. ( 1963). J . Cell Biol. 19,201. Robertson, J. D. ( 1961). In “Cellular Membranes in Development” ( M . Locke, ed.), p. 1. Academic Press, New York. Robison, G. A., Butcher, R. W., and Sutherland, E. W. (1971). “Cyclic AMP.” Academic Press, New York. Rorat, E., Ferenczy, A., and Richart, R. M. (1974). Cancer 33, 880. Rosai, J. (1968). Cancer 22, 333. Rosai, J., Khodadoust, K., and Saber, I. (1969). Cancer 24, 103.

INTERCELLULAR JUNCXIONS IN CANCER

87

Rose, B. (1970). Science 169,607. Rose, B. (1971). J. Membrane Biol. 5, 1. Rose, B., and Loewenstein, W. R. (1971). J. Membrane Biol. 5,20. Rose, B., and Loewenstein, W. R. (1975a). Nature 254,250. Rose, B., and Loewenstein, W. R. (1975b). Science 190, 1204. Roth, L. M., Spurlock, B. O., Sternberg, W. H., and Rice, B. F. (1970). Amer. 1. Pathol. 60, 137. Roth, S. (1973). Quart. Reo. B i d . 48, 541. Roth, S., and Weston, J. A. (1967). Proc. Nut. Acad. Sci. US. 58, 974. Roth, S., McGuire, E. J., and Roseman, S. ( 1971). J. Cell Biol. 51, 525. Rubin, R. P., Carchman, R. A., and Jaanus, S. D. (1972). Nature (London), New Biol. 240, 150. Rubinstein, L. J., Herman, M. M., and Hanbery, J. W. (1974). Cancer 33, 675. Salazar, H., Merkow, L. P., Walter, W. S., and Pardo, M. (1974). Obstet. Gynecol. 44, 551. Sanel, F. T., and Serpick, A. A. (1970). Science 168, 1458. Satir, P., and Gilula, N. B. ( 1973). Annu. Reu. Entoml. 18, 143. Sedar, A. W., and Forte, J. G. (1964). J. Cell Biol. 22, 173. Seifert, W., and Paul, D. (1972). Nature (London),New Biol. 240, 281. Sellin, D., Wallach, D. F. H., and Fischer, H. (1971). Eur. 1. Immunol. 1, 453. Sellin, D., Wallach, D. F. H., Weltzien, H. U., Resch, K., Sprenger, E., and Fischer, H. (1974). Eur. J. Immunol. 4, 189. Sharrna, R. K., and Hashimoto, K. ( 1972). Cancer Res. 32,666. Sheppard, J. R. ( 1971). Proc. Nut. Acad. Sci. U.S. 68, 1316. Sheridan, J. D. (1970). J. Cell Biol. 45,91. Sheridan, J. D. ( 1971). J. Cell Biol. 50, 795. Shin, M. L., and Firminger, H. I. ( 1973). Amer. J. Pathol. 70,291. Silverberg, S . G., and DeGiorgi, L. S. (1972). Cancer 29, 1680. Simani, A. S., Inoue, S., and Hogg, J. C. (1974). Lab. Invest. 31,75. Simionescu, M., Simionescu, N., and Palade, G. E. (1975). J . Cell Biol. 67, 863. Singer, S. J. (1962). Aduan. Protein Chem. 17, 1. Singer, S. J. (1971). In “Structure and Function of Biological Membranes” (L.I. RotMeld, ed.), p. 145. Academic Press, New York. Singer, S. J., and Nicolson, G. L. (1972). Science 175,720. Skerrow, C . J., and Matoltsy, A. G. ( 1974a).J. Cell Biol. 63,515. Skerrow, C. J., and Matoltsy, A. G. (1974b). J. Cell Biol. 63,524. Slack, C., and Palmer, J. F. ( 1969). Exp. Cell Res. 55,416. Smets, L. A. ( 1972). Nature (London), New Biol. 239, 123. Socolar, S. J., and Politoff, A. L. ( 1971).Science 172,492. Staehelin, L. A. (1973). J . Cell Sci. 13, 763. Staehelin, L. A. (1974). Int. Reu. Cytol. 39, 191. Staehelin, L. A. (1975). J. Cell S c i . 18,545. Stanton, M. F., Ting, R. C., and Miller, E. (1970). J. Nut. Cancer Inst. 45, 195. Steck, T. L. ( 1974). J. Cell Biol. 62, 1. Steck, T. L., Fairbanks, G., and Wallach, D. F. H. (1971). Biochemistry 10,2617. Steinberg, M. S. (1963). Science 137, 762. Steinberg;M. S. (1970). J. Ezp. ZOO^. 173, 395. Steinberg, M. S. (1973). Locomotion Tissue Cells, Ciba Found. Symp. p. 33. Steiner, G. C., and Dodman, H. D. (1972). Cancer 29, 122. Steiner, G. C., Ghosh, L., and Dorfman, H. D. (1972). Hum. Pathol. 3, 569.

88

ROSALD

s. WEINSTEIN,

FREDERICK B. MERK, AND JOSEPH ALROY

Steiner, G. C . , hfirra, J. M., and Bullough, P. G. (1973). Cancer 32, 926. Stoeckenius, W., and Engelman, D. M. (1969). J . Cell Bwl. 42,613. Stoker, hf. (1967). Curr. Top. Develop. BioZ. 2, 107. Subak-Sharpe, H. (1969). Homeostatic Regul., Ciba Found. S y m p . Subak-Sharpe H., Biirk, R. R., and Pitts J. D. (1969). J. Cell Sci. 4, 353. Sutherland, E. W. (1970). J. Amer. Med. Ass. 214, 1218. Svoboda, D. J., Kirchner, F. R., and Proud, G. 0. (1963). Cancer Res. 23, 1084. Tani, E., Nishiura, M., and Higashi, N. (1973). A d a Neuroputhol. 26, 127. Tani, E., Ikeda, K., Kudo, S., Yamagata, S., Nishiura, M., and Higashi, N. (1974). Acta Neuropathol. 27, 139. Tannenbaum, M. ( 1971). Pathobiol. Annu. p. 249. Tarin, D. (1970). J. Invest. Dermutol. 55, 26. Taxy, J. B., Battifora, H., and Oyasu. R. (1974). Cancer 34, 306. Tee], R. W., and Hall, R. G. ( 1973). Exp. Cell Res. 76,390. Temin, H. hl., and Rubin, H. (1958). Virology 6, 66a. Tice, L. W., Wollman, S. H., and Cartier, R. C. ( 1975). J . Cell Biol. 66, 657. Tobon, K , and Price, H. M. ( 1972). Cancer 30, 1082. Toker, C. (1968). Cancer 21,1171. Tonietti, G . , Baschieri, L., and Salabe, G. (1967). Arch. PuthoE. 84, 601. Trelstad, R. L., Revel, J. P., and Hay, E. D. (1966). J. Cell Biol. 31, C6. Trelstad, R. L., Hay, E. D., and Revel, J. P. (1967). Develop. Biol. 16, 78. Tripathi, R. C., and Gamer, A. ( 1972). Cancer Res. 32,90. Ts’o, M. 0. M., Fine, B. S., and Zimmerman, L. E. ( 1969). Arch. Pathol. 88, 664. Turnbull, R. B., Kyle, K., Watson, F. R., and Spratt, J. (1967). Ann. Surg. 166, 420. Tyzzer, E. E. (1913). J. Med. Res. 28, 1913. Urban, J., Kartenbeck, J., Zimber, P., Timko, J., Lesch, R., and Schreiber, G. (1972). Cancer Res. 32, 1971. Vasiliev, J. XI., Gelfand, I. M., Domnina, L. V., and Rappoport, R. I. (1969). Exp. Cell Res. 54, 83. Vogel, A., and Narasimnan, S. ( 1974). Dermutologica 148,201. von Bomhard, D., and Sandersleben, von J. (1973). Virchows Arch. A. 359, 87. Wade, 1. B., and Karnovsky, M. J. ( 1974). 3. Cell Biol.62, 344. Wade, J. B., Revel, J. P., and DiScala, V. A. ( 1973). Amer. J . Physiol. 224,407. Walther, B. T., Oehman, R., and Roseman, S. (1973). Proc. Nut. Acad. Sci. US. 70, 1569. Wang, X. S., Seeniayer, T. A., Ahmed, M. N., and Morin, J. (1974). Arch. Pathol. 98, 100. Wehrli, E., Muhlethaler, K., and Moor, H. (1970). Exp. Cell Res. 59, 336. Weinstein, R. S. ( 1974). In “The Red Blood Cell” ( D. M. Surgenor, ed.)., 2nd ed., Vol. 1, p. 213. Academic Press, New York. Weinstein, R. S., and McNutt, N. S. (197Oa). In “Microcirculation, Pedusion and Transplantation of Organs” (T. J. Malinin, B. S. Linn, A. B. Callahan, and W. D. Warren, eds.) p. 23. Academic Press, New York. Weinstein, R. S., and McNutt, N. S. (1970b). Semin. Hematol. 7,259. Weinstein, R. S., and McNutt, N. S. (1972). N . Engl. J. Med. 286, 521. Weinstein, R. S., Clowes, A. W., and McNutt, N. S. (1970a). P r w . SOC. E x p . Biol. Med. 134, 1195. Weinstein, R. S., McNutt, N. S., Nielsen, S. L., and Pinn, V. W. (1970b). Proc. 28th Meet. Electron Microsc. SOC. Amer. p. 108.

INTERCELLULAR JUNCI’IONS IN CANCER

89

Weinstein, R. S., Zel, G., and Merk, F. B. (1974). In “Membrane Transformation in Neoplasia” (J. Schultz and R. E. Block, eds.), p. 127. Academic Press, New York. Wellings, S. R., and Roberts, P. (1963). J. Nut. Cancer Inst. 30,269. Welsh, R. A., and Meyer, A. T. (1968). Arch. Pathol. 85, 433. Welsh, R. A., Bray, D. M., 111, Shipkey, F. H., and Meyer, A. T. (1972). Cancer 29, 191. Welti, C. V., Pardo, V., Millard, M., and Gerston, K. (1972). Cancer 29, 1169. Whittemburg, G., and Rawlins, F. A. ( 1971). Pjluegers Arch. Gesamte Physiol. Menschen Tiere 330, 302. Wiernik, G., Bradbury, S., Plaut, M., Cowdell, H., and Williams, E. A. (1973). Brit. J. Cancer 28, 488. Willingham, M. C., Johnson, G. S., and Pastan, I. (1972). Biochem. Biophys. Res. Commun. 48,743. Wills, E . J. (1968). Cancer 22, 1046. Winzler, R. J. (1970). Int. Reu. Cytol. 29, 77. Wiseman, L. L., and Steinberg, M. S. (1973). Erp. Cell Res. 79,468. Wolff, M., Santiago, H., and Duby, M. M. (1972). Cancer 30, 1046. Wood, R. L. (1959). J . Biophys. Biochem. Cytol. 67, 213. Woodbury, J. W., and Crill, W. E. (1961). In “Nervous Inhibition” ( E . Florey, ed.), p. 124. Pergamon, Oxford. Woodruff, R. I., and Telfer, W. H. (1973). J. Cell B i d . 58, 172. Yeh, S. ( 1973). Hum. Pathd. 4,469. Yeh, S., Chen, H. C., How, S. W., and Deng, C. S. (1974). J. Nat. Cancer Inst. 53, 31. Zamboni, L., and Gondos, B. (1968). J. Cell Biol. 36,276. Zampighi, G., and Robertson, J. D. (1973). J. Cell Biol. 56,92. Zuckerberg, C. ( 1973). Cancer Res. 33, 2278.

This Page Intentionally Left Blank

GENETICS OF ADENOVIRUSES

Harold S. Ginsberg and C. S.

H. Young

Department of Microbiology, Collegd of Physicians and Surgeons, Columbia University, New York, New York

I. Introduction . . . . . . . . A. The Virion . . . . . . . . B. Viral Replication . . . . . . . 11. Isolation of Adenovirus Mutants . . . . A. Types of Mutants . . . . . . B. Mutagenic Procedures . . . . . . C. Selection . . . . . . . . D. The Problem of Multiple Mutation . . . 111. Genetic Characterization . . . . . . A. Complementation . . . . . . . B. Recombination Tests . . . . . . C. Correlation between Genetic and Physical Maps D. Heterotypic Complementation . . . . IV. Phenotypes of Adenovirus Mutants A. Characterization . . . . . . . B. Functional Studies Using Adenovirus Mutants . . . . . . . . V. Summing Up References

.

.

.

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. .

. .

. .

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

. . . . . . . . . . .

. . .

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

. 9 1 92 95 .lo1 .lo1 .lo3 .lo4 .lo5 .lo5 .lo5 .lo9 . 113 . 115 . 116 .116 . 118 .123 .126

. .

1. Introduction

Adenoviruses infect many species of warm-blooded animals and effect varied clinical reactions including acute disease, latent infection, and the induction of malignancy. Similarly, in cell culture, the response to infection is varied, ranging from extensive cytopathic effects to cellular transformation. The establishment of a nuclear infection triggers this array of host responses, and offers an unusual potential to investigate the regulation of replication and transcription of DNA and the translation of mRNAs in eukaryotic cells. Studies with bacteriophage, however, vividly exposed the problem that biochemical techniques alone were inadequate to obtain detailed data on the controls governing many biosynthetic reactions. Selected conditionally lethal and deletion mutants were essential for the studies that yielded exquisite evidence on the mechanisms regulating bacteriophage replication. In recognition of the critical requirement of appropriate mutants, several laboratories ( see Table I ) have selected conditionally lethal temperature-sensitive mutants of adenoviruses, predominantly types 5 and 12, to study the genetic 91

92

HAROLD S. GINSBERG AND C. S. H. YOUNG

mechanisms of a DNA-containing virus and to investigate the reactions that control adenovirus replication. Since mammalian cells are so complex and contain of the order of 10‘ genes, a direct investigation of the molecular events regulating biosynthetic reactions and cell division is fundamentallv impossible at this time. Studies with a smaller genome, which contains a maximum of 50 genes and replicates in the nucleus of a eukaryotic cell, offers a simpler model that should yield evidence germane to the molecular biology of mammalian cells as well as to viral biosynthesis. The objectives of this article are to review the studies that described the mutagenesis and selection of adenovirus mutants, their use in formal genetic studies, their phenotypes, and their utility in experiments for understanding the regulation of adenovirus replication and cellular transformation. A number of reviews have described different aspects of adenovirus structure and replication ( Ginsberg, 1969; Schlesinger, 1969; Green, 1970; Philipson and Lindberg, 1974; Norrby, 1968; Philipson and Petterson, 1973), but to familiarize the nonspecialist with the data pertinent to this review, a brief summary of the essential evidence will be presented. A. THE VIRION The viral particle is an icosahedron with a mean diameter of 70-80 nm. Contrary to the initial prediction of Crick and Watson (1956), the isometric virus is not composed of identical repeating subunits, but rather the capsid is made of three unique major multimeric proteins and several minor proteins ( Ginsberg, 1969; Schlesinger, 1969; Green, 1970; Philipson and Lindberg, 1974; Norrby, 1968; Philipson and Pettersson, 1973; Maize1 et al., 1968a,b). It has been possible to describe the architecture of the virion and the topography of its components owing to the early demonstration that the virion can be dissociated into intact native subunits, that the capsid components are identical with the great excess ( approximately 10-fold) of unassembled “soluble antigens” present in infected cell extracts, and that the capsid proteins have the unusual feature of being soluble in aqueous media while remaining in their native functional forms (Wilcox, et d.,1963; Valentine and Pereira, 1965). The capsid contains 252 capsomers (Fig. 1 ) of which the faces and edges of the 20 equilateral triangles are comprised of 240 hexons, so termed because each has six neighbors (Ginsberg et d.,1966). At each of the 12 vertices of the icosahedron, the axis of 5-fold symmetry, is a penton (Valentine and Pereira, 1965), which consists of a base, the vertex capsomer, and a fiber. Three minor proteins of less than

93

GENETICS OF ADENOVIRUSES

MOLECULAR WEIGHT

HEXONASSOCI4TID PROTEIN

1 PENTON(

Fiber

H

PI

2S.SK

,\

FIG.1. Diagrammatic representation of a type 5 adenovirus particle partially disrupted to demonstrate the viral capsid and nucleoprotein core. A drawing of a representative pattern of the denatured virion proteins electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel shows the relative sizes of the polypeptide chains. The nomenclature follows that of Maizel et al. (1968a) and Everitt et d. (1973). The molecular weights of the major type 5 virion polypeptide chains are presented, since most are significantly different from the values given for the comparable proteins of type 2 adenovirus (R. Kauffman, unpublished data).

25,000 daltons are reported to be associated with the hexons that make up the faces of the triangles, and one minor protein is said to be structurally related to the hexons surrounding the pentons, the so-called peripentonal hexons (Maizel et al., 1968a,b; Everitt et al., 1973). The minor proteins have been isolated in relatively constant amounts and may serve to assemble and stabilize the capsid, but until chemical evidence is presented showing that each is indeed a unique protein, the possibility must also be entertained that some are degradation products of one or more of the major virion proteins. Internally, there are two core proteins, closely associated with the viral DNA (Laver, 1970; Prage and Pettersson, 1971; Russell et aZ., 1971). The hexon, which induces the production of type-specific neutralizing antibodies ( Wilcox and Ginsberg, 1963a; Kjellkn and Pereira, 1968; Kasel et aZ., 1964) as well as broad cross-reacting antibodies which immunologically identify the adenoviruses as a group (Wilcox and Ginsberg, 1961; Pereira, 1960), is composed of three identical polypeptides of 100,000-120,000 daltons each, depending upon the type (Maizel et al., 1968a,b; Franklin et al., 1971; Cornick et al., 1973; Stinski and Ginsberg, 1975). The fiber varies morphologically in length in different types (Wil-

94

HAROLD S. GINSBERG AND C . S. H. YOUNG

cox et al., 1963; Valentine and Pereira, 1965; Norrby, 1968, 1969) and therefore probably in molecular weight. The types 2 and 5 fibers have molecular weights of 183,000 (Dorsett and Ginsberg, 1975) to 200,000 (Sundquist et al., 1973b) and consist of three polypeptide chains of 60,000 to 65,000 daltons each (Dorsett and Ginsberg, 1975; Sundquist d al., 1973b). The fiber, which is glycosylated (Ishibashi and Maizel, 1974b) and phosphorylated (Russell et al., 1972b), is either composed of two polypeptide chains which are chemically identical and one which is unique or three chemically different chains (Dorsett and Ginsberg, 1975). The penton base is relatively unstable and highly sensitive to proteolytic enzymes ( Pereira, 1958; Everett and Ginsberg, 1958), and it therefore has not yet been well characterized except to note that it consists of several polypeptide chains ( 4 or 5) of around 70,000-80,000 daltons (Maizel et al., 1968a,b). The fiber is associated with the base through noncovalent bonds, which are disrupted by 2.5 M guanidine HCl (Norrby and Skaaret, 1967), 8%pyridine (Pettersson and Hoglund, 1969), or 33%formamide ( Neurath et al., 1968). The viral genome is a linear, double-stranded DNA molecule of 20 to 25 X lo6 daltons (Green et al., 1967; van der Eb et al.,1969), depending upon the immunological type (there are 31 human, 23 simian, 8 avian, 6 bovine, 4 porcine, 2 canine, and 1 murine types reported). It is striking that the viral DNA is not terminally redundant nor circularly permuted, but, like the adeno-associated viral DNA (Koczot et al., 1973), each strand of the molecule contains an inverted terminal repetition (Garon et al., 1972; Wolfson and Dressler, 1972). Hence, when the viral DNA is denatured at a limited concentration, both strands can form single-stranded circles through hydrogen bonds between the complementary ends (Garon et al., 1972; Wolfson and Dressler, 1972). However, the function of this novel DNA structure is unclear. It has also been reported that a small protein molecule associated with the viral DNA maintains the genome in a circular form in the virion and when artifically released from it (Robinson et al., 1973). A similar circular genome has not been detected, however, after intracellular uncoating of the virion (Robinson et al., 1973) or during viral DNA replication (Sussenbach et al., 1972; Pettersson, 1973). Just as adenoviruses have been divided into subgroups according to biological properties, such as oncogenicity or hemagglutination ( Huebner et al., 1965; Rosen, 1960), the viral DNAs of different types may be similarly classified according to base composition (Piiia and Green, 1965) and DNA-DNA hybridization (Green, 1970; Garon et al., 1973). Thus adenoviruses have been arranged into three subgroups (A, B, and C ) according to their oncogenic potential in newborn hamsters (Huebner

95

GENETICS OF ADENOVIRUSES

et al., 1965). The viruses fall into similar subgroups when divided according to G C content and nucleotide sequence homology. The G C content of subgroup A, which consists of the most oncogenic adenoviruses (types 12, 18, and 31) is 48-49%; subgroup B, which contains weakly oncogenic viruses (e.g., types 3, 7, and 21) has a G C content of 49-52%;and subgroup C, which consists of viruses that are essentially nononcogenic (e.g., types 1, 2, and 5 ) has a G C content of'55-602 (Green, 1970; Piiia and Green, 1965). It is also striking that, between members of a subgroup, the nucleotide sequences are up to 95%homologous, whereas DNA-DNA hybridizations between selected members of different subgroups show only about 10%homology (Green, 1970; Garon et al., 1973 ) . Electron microscopic examination of heteroduplexes formed between DN As from different subgroup viruses reveal considerable heterology (Garon et al., 1973) while DNAs from viruses belonging to the same subgroup (types 1, 2, and 5 ) show almost complete homology (Garon et al., 1973; Bartok et al., 1974) except for two discrete regions of nonhomology representing a total of 16% of the genome (Bartok et al., 1974).

+

+

+

+

B. VIRALREPLICATION Production of infectious virions follows from an ordered series of reactions which is initiated by association of a viral particle with a susceptible cell and culminated by the assembly of virions from its subunits. Replication of types 2 and 5 adenoviruses has been most extensively studied because the yield is great (about lo4 PFU/cell) and the virion, as well as most of the components, can be easily purified and analyzed. Accordingly, the molecular biology of adenovirus infection is known in greatest detail with these types, and the description of adenovirus replication that follows will summarize the investigations with types 2 and 5 viruses. A single cycle of type 5 adenovirus multiplication with the accompanying biosynthetic events are diagrammatically summarized in Fig. 2. The temporal characteristics and viral yield vary, however, with different types of adenoviruses: for example, the eclipse period for type 12 adenovirus at 36OC is 16-18 hours (Gilead and Ginsberg, 1965), and biosynthesis of the viral DNA cannot be detected until 1 2 1 5 hours (Piiia and Green, 1969) after infection, whereas DNA replication of types 2 and 5 begins 6-7 hours after infection and their eclipse periods terminate 13-14 hours after infection (Pifia and Green, 1969; Ginsberg et al., 1967). The fiber is the virion's organ of attachment to receptor sites of susceptible cells (Levine and Ginsberg, 1967; Philipson et al., 1 W ) . The nature of the specific receptors, however, has not yet been identified. Attached

96

HAROLD S. GINSBERG AND C. S. H. YOUNG

lo’[ HOST PROTEINS

2 10 5

...-

- -- -

LATE PROTEINS L A T E / mRNAs V I R A L ~ D N A .__. .. .. -EARLY /PROTEINS.. . .-CLASS11 EARLY/m RNAs I EARLY/?RNAS CLASS .. -

___

J

,

___ I

virions enter susceptible cells by phagocytosis ( Dales, 1962; Chardonnet and Dales, 1970) or direct penetration of the plasma membrane (Morgan et cd., 1969). Whether either entry mechanism is preferred is still unclear, but even after phagocytosis the virions must directly penetrate the membrane of the phagocytic vacuole to gain access to the cytoplasm. Once within the cytoplasm, viral uncoating (i.e., viral eclipse) is rapidly initiated with disengagement of the pentons (Sussenbach, 1967), which is soon followed by dissociation of the capsid and release of the nucleoprotein core ( Sussenbach, 1967; Lawrence and Ginsberg, 1967; LonbergHolm and Philipson, 1969). Neither synthesis of proteins nor replication of nucleic acids is required for these initial uncoating reactions (Lawrence and Ginsberg, 1967; Lonberg-Holm and Philipson, 1969), which uncover the viral DNA so that it is susceptible to DNase although still associated with about 20% of the viral proteins, the core proteins (Lawrence and Ginsberg, 1967; Lonberg-Holm and Philipson, 1969). The partially uncoated virus appears to be transported in microtubules to nuclear pores (Chardonnet and Dales, 1970), where further dissociation of DNA from the internal proteins occurs at the nuclear membrane (Morgan et al., 1969) or in the nucleus (Lonberg-Holm and Philipson, 1969). But the extent and the mechanisms of the terminal uncoating reaction have not been determined. The description of viral penetration and uncoating presented carries with it a caveat. The ratio of total viral particles to infectious virions (i.e., plaque formers) varies from 10 to several hundred, according to the viral species (type), and all the experiments described employed

GENETICS OF ADENOVlRUSES

97

high multiplicities of infection. Consequently, the sequence of events that was observed, which was followed by biochemical analysis of isotope-labeled virus and electron microscopic observations, involved the large population of viral particles in each cell, but the virion or virions that established the infectious process may not necessarily have followed the same sequence.

1. Transcription Shortly after the viral DNA reaches the nucleus, 2-3 hours after infection, transcription begins (Thomas and Green, 1969; Lucas and Ginsberg, 1971; Parsons and Green, 1971). Since protein synthesis is not required (Parsons and Green, 1971) and the virion does not contain its own RNA polymerase, transcription apparently involves a host enzyme. This has been shown to be a-amanitin sensitive and therefore probably corresponds to the DNA-dependent RNA polymerase I1 (Ledinko, 1971; Price and Penman, 1972). Prior to viral DNA replication only a portion of the genome is transcribed, yielding early mRNAs (Thomas and Green, 1969; Parsons and Green, 1971; Lucas and Ginsberg, 1971; Tibbetts et al., 1974). However, the extent of the genome copied into early transcripts is still uncertain, with reports varying from 1&20% (Thomas and Green, 1969; Lucas and Ginsberg, 1971) to 4050%(Tibbetts et al., 1974). It is particularly important to note that the genes for early transcripts are not limited to a single region but are distributed throughout the genome (Craig et al., 1975). After the initiation of DNA replication, ‘‘late’’ transcripts appear (Bello and Ginsberg, 1969; Green et al., 1970; Lucas and Ginsberg, 1971 ) , and apparently the transcription of about 50% of the early mRNAs, the so-called Class I early mRNAs (Lucas and Ginsberg, 1971; Craig et al., 1975) ceases, although there is also not complete agreement on these findings (Thomas and Green, 1969; Tibbetts et al., 1974). The reactions that regulate the limited transcription of the parental viral genome, the onset of late transcription, and the switching of transcription from one strand to the other during late transcription (Tibbetts et al., 1974; Fujinaga and Green, 1970; Tibbetts and Pettersson, 1974) are still unknown. Indeed, there are several reactions by which the original transcripts are modified to produce functional mRNAs, and their mechanisms and control are also uncertain: ( 1 ) the primary transcripts are considerably larger than the polysomal mRNAs (McGuire et al., 1972; Wall et al., 1972), which are aIso heterogeneous in size, varying from 9 S to 27 S (Parsons and Green, 1971; Lindberg et al., 1972; Philipson et al., 1973; Craig et al., 1975); ( 2 ) only 70-804: of the RNA transcribed is processed and transported into the cytoplasm (McGuire et al., 1972; Lucas and Ginsberg, 1972); and ( 3 ) most if

98

HAROLD S. GINSBERG AND C. S. H. YOUNG

not all viral mRNAs are polyadenylated in the nucleus after transcription (Philipson et al., 1971). It is noteworthy that the heterogeneity and processing of adenovirus-specific RNAs resemble the characteristics of the uninfected host cell’s heterogeneous ( H n ) RNAs, and hence investigation of transcription of adenovirus mRNAs offers another convenient probe for study of the molecular biology of eukaryotic cells. 2. DNA Replication In productive infections, under conditions of multiple infection of each cell, replication of viral D N A begins about 6 hours after infection, reaching a maximum about 18 hours after viral attachment (Pifia and Green, 1969; Ginsberg et al., 1967). Because viral DNA has a higher G C content than that of the host, the two species can be effectively separated (Piiia and Green, 1969; Ginsberg et al., 1967). Accordingly, it is noted that synthesis of host DNA begins to decline about 6 hours after infection, when replication of viral DNA commences, and production of host DNA is effectively blocked by 12 hours (Ginsberg et al., 1967). But prior synthesis of either host or viral DNA is not essential for the inhibition of host DNA (Ginsberg et al., 1967). Like replication of most linear DNA molecules, little is known about the precise mechanism of duplicating adenovirus DNA. Although protein synthesis is required for the initiation of viral DNA replication (Wilcox and Ginsberg, 1963b), there is no evidence to indicate whether the polymerase is host or viral coded. Continued protein production, however, is not required after replication of viral DNA is established (Horwitz et al., 1973). Van der Vleit and Levine (1973) have identsed in type .j-infected cell extracts proteins of 72,000 and 48,000 daltons which bind preferentially to single-stranded DNA ( a so-called DNAbinding protein), similar to the T4 phage gene-32 protein ( Alberts and Frey, 1970). Peptide maps of the two suggest, however, that the 48,000-dalton polypeptide is a proteolytic breakdown product of the 72,000-dalton protein ( A. J. Levine, personal communication ) . Whether both proteins are functional is uncertain. Like T4 gene-32 protein, the DNA-binding protein in adenovirus-infected cells appears to be a viral gene product present in large numbers of copies per cells and essential for DNA replication (Alberts and Frey, 1970). Type 12 infected cells contain similar proteins of 60,000 and 48,000 daltons (Rosenwirth et QZ., 1975). Present in virus-infected cells are replicative forms of viral DNA, which sediment more rapidly and are denser than mature virion DNA (Belfett and Younghusband, 1972; van der Vleit and Sussenbach, 1972; van der

+

GENETICS OF ADENOVIRUSES

99

Eb, 1973; Pettersson, 1973). These replicative intermediates can also be isolated by the so-called “M-band” ( Sarkosyl) technique ( Pearson and Hanawalt, 1971; Shiroki et al., 1974; Yamashita and Green, 1974). In each instance, the replicative intermediate appears to consist of double-stranded DNA with single-stranded branches ( Bellett and Younghusband, 1972; van der Vleit and Sussenbach, 1972; van der Eb, 1973; Pettersson, 1973). Studying viral DNA replication in nuclei from infected cells, Sussenbach and colleagues have presented data which indicate that the DNA is replicated semiconservatively but asymmetrically, that the strand which has greater buoyant density in alkaline CsCl is initially replicated, displacing the complementary strand, and that the lighter strand is then copied in discontinuous segments that are subsequently joined (Sussenbach et al., 1972, 1973; Ellens et al., 1974). It should be stressed that although each viral DNA strand has the structure to generate a single-stranded circle (i.e., the strands have inverted terminal repetitious ends, as described above ) , such single-strand “circles” have not been observed in the replication complexes (Pettersson, 1973; Ellens et al., 1974).

3. Protein Synthesis Like host nuclear proteins, adenovirus proteins, whose transcripts are made in the nucleus, are produced in the cytoplasm and rapidly transported into the nucleus for assembly (Velicer and Ginsberg, 1968, 1970). Evidence cIearIy demonstrates that synthesis of early proteins precedes and is mandatory for replication of viral DNA (Wilcox and Ginsberg, 1963b; Horwitz et al., 1973). Thus far, only the T (Pope and Rowe, 1964; Gilead and Ginsberg, 1965) and P (Russell and Knight, 1967) antigens and the DNA-binding protein (van der Vleit and Levine, 1973; Rosenwirth et al., 1975) have been identified as probable early viral gene products. Translation of late proteins, which primarily consist of virion components, occurs on cytoplasmic polyribosomes of 180-200 S (Velicer and Ginsberg, 1968; Thomas and Green, 1966) and requires 1-2 minutes (Velicer and Ginsberg, 1970; White et al., 19sS). Kinetic data suggest that, after completion and release from polyribosomes, the nascent polypeptide chains are rapidly transported into the nucleus, where they are assembled into immunologically reactive multimeric capsid proteins ( Velicer and Ginsberg, 1970). However, cytoplasmic assembly of newly made protomers into capsid proteins can occur, as will be noted below with certain temperature-sensitive mutants (Kauffman and Ginsberg, 1975). Indeed, nascent polypeptides made in uitro can even self-assemble into capsid proteins in the cell-free reaction mixture ( Wilhelm and Ginsberg, 1972).

100

HAROLD S. GINSBERG AND C. S. H. YOUNG

Hexon protein, the major viral protein, is the first capsid protein to appear in the cell and is made in greatest amounts. All the capsid proteins, however, are produced in relative abundance since only about 10-20% of each of the proteins synthesized are assembled into virions ( Green, 1962; Wilcox and Ginsberg, 1 9 6 3 ~ ) .The excess viral proteins (often termed “soluble antigens”), along with similar amounts of excessive viral DNA, are the constituents of the characteristic intranuclear inclusion bodies seen in adenovirus-infected cells (Boyer et al., 1957, 1959; Morgan et al.,1960). 4. Assembly

It was generally accepted until several years ago that the capsid of isometric viruses self-assembled around previously formed viral nucleoprotein cores. Evidence has accumulated, however, showing that an RNA-deficient poliovirus procapsid may be initially assembled as the precursor of the infectious virion (Maizel et al., 1967; Phillips et aZ., 1968). With insertion of the viral RNA a procapsid protein is processed to form the stable capsid (Jacobson and Baltimore, 1968). Incomplete particles of SV40 have also been described as putative precursors of intact virions (Ozer, 1972; Ozer and Tegtmeyer, 1972). King and his collaborators studying the assembly of the SaZmonelZu typhimurium phage P22 reported convincing evidence that formation of a prohead is an initial phase of virion assembly. The prohead is a shell consisting of the capsid protein and a “scaffolding protein,” which exits from the precursor shell in concert with encapsulation of viral DNA (King and Casjens, 1974). The P22 proheads are similar to the T4 tau particles, which are precursors to T4 heads (Kellenberger et aZ., 1968; Simon, 1972). Data have been recently presented reporting that incomplete adenovirus particles (i.e., empty capsids) are also assembled as precursors into which viral DNA is inserted (Sundquist et al., 1973a; Ishibashi and Maizel, 1974a). With final assembly, two or three precursor viral proteins appear to be processed by cleavage and at least one disappears from the putative precursor particle (Sundquist et al., 1973a; Ishibashi and Maizel, 1974a; Anderson et al., 1973). Evidence obtained from the experiments summarized above, as well as from studies of abortive infections produced by elevated temperatures ( Okubo and Raskas, 1971), arginine deprivation ( Rouse and Schlesinger, 1972; Everitt et al., 1971), and temperature-sensitive mutants (Williams et al., 1974; Ensinger and Ginsberg, 1972), imply that assembly is controlled at several junctures during viral replication: (1) transport of nascent polypeptide chains or capsomers; ( 2 ) assembly of protomers

GENETICS OF ADENOVIRUSES

101

into capsomers; ( 3 ) formation of capsids; and (4)incorporation of viral nucleoprotein cores into virions.

II. Isolation of Adenovirus Mutants

A. TYPESOF MUTANTS In recent years, a number of investigators have applied to adenoviruses the techniques developed with bacteriophages in the hope of obtaining various kinds of mutants, with which to explore the interactions between virus and cell in both permissive and nonpermissive infections. The techniques and mutants obtained have also been employed to develop a genetic system. The next two sections will be concerned with the isolation and preliminary genetic characterization of adenovirus mutants. Theoretically, the types of mutants that may be sought include: 1. Plaque morphology mutants 2. Host range mutants-both those with an increased and those with a restricted host-cell range compared to that of the wild type 3. Conditional lethal mutants: At present this category is confined, in animal viruses, to temperature-restricted mutants, in particular, the so-called temperature-sensitive ( t s ) mutants that fail to replicate at high temperature while doing so at the lower. The other major class of conditional lethals, i.e., suppressor-sensitive mutants, which are suppressed by host cells containing UAG, UAA, or UGA suppressors, awaits the development of the appropriate cells and test systems 4. Drug-resistant mutants-those resistant to drugs that act specifically on viral replication 5. Virion structural mutants: These might include alterations to the stability or resistance of the virion to in uitro treatments, including heat or antiserum inactivation. Also, virions with altered buoyant density may be selected. Table I lists the publications dealing with the isolation of adenovirus mutants. It can be seen that all categories except ( 4 ) have been represented, but that ts mutants have been most actively searched for. The reasons for this are clear. Any gene that codes for a protein product is expected to be able to mutate to a t s form, if the temperature range over which the protein is active is mutable. Thus, t s mutants should represent lesions in a variety of functions essential to the replicative process. Furthermore, they are useful for several technical reasons: (1) the phenotype may be directly compared with wild type at both the restrictive and permissive temperatures and the functional lesion peculiar

T.4BLE I PIJHI.lChTION8 I)KSCRIBINO

Virus and mutant type H2 1s

H5 ts

H5

IS

Mutagenu

NN(; FIN02 NHzOH HNOz BrdUrd NHzOH HNO2 NNG NHzOH NNG BrdUrd NNG

H31 t s A1 Is H12 cyt Ad 2+ ND1 host range H 5 host range I15 heat stable

Killing or yield depression -10-4 -10-3 lo-' 10-5 10-2

gq 0.5 -

NHzOH Spontaneous

0.63 10-'-10- 3 10-*-10-3 10-2-10-3 10-~-10-3 5 x lo-' 10-1 10-1-10-4 8 x 10-4 -

TJV

10-'-10-~

uv

€I12 Is

ISOLATION O F

NH20H BrdUrd NNG HNO? NNG

uv

10-4

Spontaneous

-

ADKNOVIINJS SZUTANTS

Frequency of mutant (1s plaques/total) 7/172 5/170 14/146 8/95 2/355

References BBgin and Weber (1975) Williams el al. (1971) Ensinger and Ginsberg (1972)

0.01-0.10% 8/372 2/317 3/165 10/1440 45/700 2413370 2/260 171250

27/238d 5-&fold increase over spontaneous background 3/2-50 7/372 Only one mutant isolated

8

I"

Takahashi (1972) Rubinstein and Ginsberg (1974)* Lundholm and Doerfler (1971) Shiroki et al. (1972)

Ledinko (1974) Suzuki et al. (1972) Ishibashi. (1971) Takemori el al. (1968) Grodzicker el al. (1974s) Takahashi (1972) Young and Williams (197.5)

BrdUrd, bromodeoxyuridine ; NNG, N-methyl-N'-nitro-N-nitrosoguanidine;UV, ultraviolet irradiation. error, the wildtype virus used was type 5, rather than type 12 adenovirus as reported. I n this and the following table, the mutants isolated and characterized have been included as type 5 ts mutants. survivors. Range varied from 1/85 for -10-1 survivors to 7/144 for 2 X lo-' survivors to 3/94 for d ts mutants isolated from stocks which had been inactivated t.o give only from 0.5 to 3.0 % Is. a

* Owing to an unfortunate

E

8

E R

GENElTa OF ADENOVIRUSES

103

to the mutant may be identified; ( 2 ) the time and mode of action of the function may be identified by appropriate temperature-shift experiments; (3) they are eminently suitable for the performance of those genetic tests that depend on the ability to select a particular phenotype or phenotypic interaction.

PROCEDURES B. MUTAGENIC During serial passage of a virus, diverse spontaneous mutations will accumulate, giving rise to a stock of virus that is genetically heterogeneous, Thus most investigators have cloned their viral preparations by a number of plaque purifications before proceeding to isolate mutants from them, Since the starting material is thus relatively homogeneous and since there are frequently no selective methods to isolate particular classes of mutants (see below), a variety of mutagenic treatments have been used to enhance the frequency of mutants within the population (Table I). The mutagens have been of the kind that will predominantly induce “mis-sense” base-pair changes. This type of mutation is considered to be essential for the lesion that leads, for example, to a t s phenotype. The treatments fall into two experimental categories: ( 1 ) those used in uitro which are directed against the virion DNA (nitrous acid, hydroxylamine, UV irradiation) and ( 2 ) those used in the infected cell during viral replication ( nitrosoguanidine, bromodeoxyuridine) , I n uitm methods would be expected to induce lesions of the mismatched basepair type ( m / + ) , and it is perhaps surprising that many plaques isolated directly after such treatments have proved to be mutant (see, for example, Williams et al., 1971; Ledinko, 1974; BBgin and Weber, 1975) since it would be expected that an m/+ virus would segregate mlm and progeny during growth of the plaque. These mixed plaques might easily be scored as wild type under many of the screening procedures to be described below. The errors associated with treatment directed against virus replicating in uiuo arise from the fact that mutations occurring early will be replicated, and “separate” mutants isolated from the treated stock may prove to be siblings. This could lead to false conclusions concerning the mutation frequency of certain gene functions. The problem may be avoided by isolating single mutants from a number of separately treated stocks, and in general this has been the method employed. However, even where this precaution has not been taken, separate mutants isolated from one stock or mutagenized virus have often proved to be different. Thus Lundholm and Doerfler (1971) isolated mutants from a treated stock of type 12 adenovirus, several of which were different as judged by their temperature-sensitive phenotype.

+/+

104

HAROLD S . GINSBERG AND C . S . H. YOUNG

The frequencies of mutants induced by the various mutagens are listed in Table I. In no case, is there a clear indication of the optimal dose of mutagen or length of mutagenic treatment in induction of mutation; although Suzuki et aZ. (1972) have published a table showing the frequency of ts mutants after various doses of UV irradiation, the numbers of mutants obtained were limited by the difficulty of screening many hundreds of plaque isolates, and thus the dose versus mutant induction response was hard to assess accurately. C. SELECTION

All the phenotypic changes being looked for are the result of so-called “forward mutations, i.e., mutations from wild to mutant phenotype. In one case, the cytocidal mutants of adenovirus type 12 (Takemori et al., 1968), the search involved visual inspection of plaques; in another, the isolation of a heat-stable mutant of type 5 adenovirus followed three rounds of selection by heat inactivation (Young and Williams, 1975). More frequently, however, the change involves the loss of some function, e.g., the ability to grow on a certain host cell or to replicate at a particular temperature. As yet there have been no published reports of efforts to select adenovirus mutants on the basis of their inability to grow under restrictive conditions, while the wild type grows and incorporates some toxic substance such as a base analog or an amino acid analog. This procedure, which should lead to the selective destruction of wildtype virus in a mixed population, would be expected to enrich the nongrowing mutant phenotype. Thus, in the absence of selective methods, most mutant hunts have been random in the sense that plaques arising from viral stocks have been individually checked for the mutant characteristic. Naturally, this is a time- and material-consuming procedure. In the case of the ts mutants isolated by Williams et at. (1971), well separated plaques which had been grown at 31OC were picked and checked by plaquing at the restrictive and permissive temperatures. Plaques were tentatively classified as ts if they gave 50-fold less progeny plaques at the restrictive temperature compared with the permissive temperature. Essentially similar procedures were carried out by Ishibashi ( 1971) and Grodzicker et al. (1974a) in their searches for ts mutants of CELO adenovirus and of the nondefective SV40 adenovinis type 2 hvbrid AdPND1, respectively. Ensinger and Ginsberg ( 1972) employed a two-step, semiselective method to help speed the isolation procedure. Mutagenized virus was plated and incubated at the permissive temperature until small plaques appeared. Then, the plaques were marked and the plates were shifted

GENETICS OF ADENOVIRUSES

105

to the nonpermissive temperature. Plaques that failed to enlarge were picked and tested by infecting cells at a low multiplicity of infection to screen for inability to produce cytopathic effect (CPE) at the high temperature. Suzuki and Shimojo ( 1971), Shiroki et al. (1972), Takahashi ( 1972), and Ledinko (1974) screened plaque isolates directly by checking for CPE at the high temperature, while Lundholm and Doerfler (1971) picked plaques but transferred samples by sterile toothpick to plates already overlaid with agar, thus confining CPE to a small region and allowing more than one isolate to be screened per plate. BCgin and Weber (1975) have used the plaque enlargement and CPE methods and also have screened plaque isolates for the ability to make “inclusion bodies” at the high temperature. All these methods of selection tend to underestimate the frequency of ts mutants in the population either because ‘‘leaky’’ mutants will give CPE or because plaque enlargement may occur by cytocidal effects of defective virus or of viral proteins.

D. THE PROBLEM OF MULTIPLEMUTATION In all the cases where mutagenesis has been used to enhance the frequency of the mutant type being sought, those mutants which have been isolated will inevitably have more mutational lesions than the one that will give rise to the desired phenotype. No attempt has been made to determine whether or not all the phenotypic characteristics of a particular mutant are pleiotropic effects of one mutation or are caused by multiple mutations, by, for example, checking wild-type revertants or recombinants to see whether phenotypic characteristics are separable. Another potentially serious source of confusion, which was first pointed out for adenoviruses by Ishibashi (1971) and by Williams and Ustacelebi (1971b), lies in the isolation of mutants whose phenotype is caused by multiple mutations of the same class. For example, in a population where 10%of the survivors, following mutagenic treatment, are ts, 10% of the ts mutants are expected to arise from double mutations. This situation often can be revealed by complementation and recombination analysis which will be described below. 111. Genetic Characterization

A. COMPLEMENTATION The complementation test is designed to determine whether or not two mutants have mutations lying in the same or in different genes. Complementation tests of adenovirus ts mutants have been performed

106

HAROLD S . GINSBERG AND C . S . H. YOUNG

by infecting cells with a mixture of the pairs of mutants to be tested and with the mutants individually. The cells are then incubated at the restrictive temperature, and the yields from the mixed and single infections are compared by titration at the permissive temperature. To ensure that any increase is not caused by wild-type revertants or recombinants occurring in the mixed infections it is necessary to plaque the yields at the nonpermissive temperature as well, Various arbitrary criteria have been adopted for identifying positive complementation. In the case of the Ads t s mutants isolated by Williams et al. (1971), an increase of 10-fold in the yield of the mixed infection compared with either of the single infection controls was taken as being positive (Williams and Ustacelebi, 1971a), while in the tests of Ensinger and Ginsberg (1972) a continual increase with time in the ratio of mixed to single infection yields was evidence for complementation. In general, complementation has been found to be an efficient process both in terms of the increase in the yield in the mixed infection compared with singles and also in the absolute amount of virus produced, which may be as high as 5M (Ledinko, 1974) of the comparable yield of wild type grown at the restrictive temperature. It should be pointed out ( a ) that the efficiency of complementation is easy to determine if the t s mutants used are not leaky or do not have high levels of reversion and ( b ) that if complementation is low it is essential to determine whether or not the increase has been caused solely by wild-type virus arising in the mixed infection (see above). Table I1 summarizes the results published so far. It is important to point out the information that these results imply as well as the experimental and theoretical limitations that apply to them. 1. The results of Williams et al. (1974) can be interpreted to indicate that as many as 16 different genes have been identified, i.e., from onethird to one-half of the coding capacity of which a double-stranded DNA of 23 x lo6 MW is capable. Their results illustrate two points. ( a ) The accumulation of mutations in certain complementation groups implies that the mutagenic methods used are going to reveal further groups slowly. The increase from 22 mutants in 14 groups (Russell et al., 1972a), to 51 mutants in 16 groups (Williams et al., 1974) clearly indicates this point. Thus without rapid screening methods for complementation, as have been developed for SV40 (Chou and Martin, 1974), the task becomes progressively more laborious as each new ts mutant must be tested against all preexisting complementation groups. ( b ) The numbers of complementation groups may exceed the number of gene functions that have t s mutations located in them. Thus, in the complementation tests summarized by Williams et al. (1974), certain mutants

TABLE I1 COMPLEMENTATION GROUPSA N D THEIRPHENOTYPIC DEFECTS Complementation tests Virus

No. of No. of mutants groups

Mutants with single phenotypic defects” DNA Synthesis “Hexon”

“Hexon transport”

Fiber

15

13

0

0

0

Ad5

51

120

l(3)

I (24)C

l ( 7 ) ~ 3(3, 2, 1) 0

15

6

3

3

34

13

10 12

6 8 -

Ad12

Ad31 CELO

-

0

“Assembly”

3(1, 1, l ) b 8(6 X 1; 2 X 2)

Ad2

0

0

Penton base

0

4(3, 2, 1, 1)

1(1)

References BBgin and Weber (1975), Weber et al. (1975) Russell et al. (1972a, 74), Williams et al. (1974), Wilkie et al. (1973) Ensinger and Ginsberg (1972), Ginsberg et al. (1974a) Rubinstein and Ginsbergd (1974) Shiroki et al. (1972), Shiroki and Shimojo (1974) Ledinko (1974) Suauki et al. (1972) Ishibashi (1971)

a Some complementation groups have more than one phenotypic defect and are not included in this table (with the exception of DNA synthesis mutants, which fail to synthesize hexon, fiber, and penton base). Not all complementation groups have been examined phenotypically. b Numbers in parentheses refer to the numbers of mutants in each complementation group; e.g., 2(1, 1) indicates two complementation groups with one mutant each and (6 X l) indicates six groups with one mutant each. Not all mutants in each group have been examined phenotypically. c The publication by Williams et al. (1974) classified these mutants as hexon: 2(1, 1) and hexon transport: 4(20, 7, 1, 1). The reassignment is based on preliminary complementation data, as explained in the text. See also footnote in Section II1,A. d See footnote a in Table I. 8 Complementation was not performed with the CELO t s mutants.

8

3

8 mG

2

z

m

v)

108

HAROLD S. GINSBERG AND C. S. H. YOUNG

defective in hexon antigen or in the transport of hexon from cytoplasm to nucleus gave low but positive values for complementation among themselves. It was suggested (Williams et al., 1974) that this might be accounted for by intracistronic complementation ( Fincham, 1966) between hexon polypeptides with defects located at different positions that might yield a partially active multimeric hexon capsomer. In this context it is important to distinguish between two types of hexon transport mutants-that which involves a separate gene function and that which involves the failure of the hexon polypeptide to assume the correct conformation for transport at the restrictive temperature. In complementation tests carried out by R. S. Kauffman (unpublished data) at a higher restrictive temperature (39.5”C compared with 38.S°C), several of the Williams ts mutants have been classified using Ad5 ts mutants whose phenotype has been pinpointed as being in either hexon polypeptide or in the separate function of hexon transport (Kauffman and Ginsberg, 1975). Preliminary data suggest that at least two of the Williams compiementation groups, one of which was previously classified as “hexon transport” (Russell et al., 1972a; Williams et al., 1974), fall into the hexon polypeptide class, not into the transport function class. One of the complementation groups previously described by these authors as “hexon transport” does fall into the transport function class ( R . S. Kauffman, unpublished data). Using these data and extrapolating that all the low complementating groups fall into the single hexon gene, an alternative classification of the Williams hexon and hexon transport mutants is included in Table 11.‘ 2. Some of the ts mutants that have been isolated fail to complement mutants from more than one group (e.g., in Suzuki et al., 1972). This strongly suggests that they are multiple mutants. If the mutant fails to adsorb, penetrate, or uncoat at the restrictive temperature, it should fail to complement with any other complementation group. In contrast to SV40, where such mutants have been found (Robb and Martin, 1972; Chou and Martin, 1974), there are as yet, no examples of such a class in adenoviruses. It can be stated, by contrast, that mutants that do complement all other groups are not doubles involving any other group 40 far isolated. 3. The availability of more than one mutant within a particular comRecent data describing the physical map and the transcriptional program of the genome suggest, however, that the assignment of mutations to proteins may be at variance with the interpretation based solely on phenotypic expression. Since the writing of this review, the interpretation based on the physical experiments places the hexon gene at the site previously referred (Fig. 3 ) to hexon transport and the mutants phenotypically defective in hexon antigen in the region of the lOOK protein.

GENETICS OF ADENOVIRUSES

109

plementation group has been useful in checking that the physiological defect found in one mutant of the group at the restrictive temperature is common to the group and is not peculiar to that mutant (Ensinger and Ginsberg, 1972; Russell et a,?., 1972a; Shiroki et al., 1972). This approach may be used instead of comparing the mutant with a ts+ revertant. In some cases, such revertants are difficult to obtain owing to the low reversion frequencies and to the cytotoxicity of the t s viral population on plaquing at high concentration during selection for ts' viruses. Despite the limitations to complementation analysis outlined above, the technique is useful in restricting the numbers of mutants that are to be examined physiologically, and it gives indications of the number of genes that may be easily identified by ts mutations. Furthermore, complementation tests have predictive value in the recombination and mapping analyses to be described below, in that mutants in the same complementation group are expected to map closely together. The use of heterotypic complementation tests will also be discussed.

B. RECOMBINATIONTESTS Recombination between adenovirus genomes has been observed using ts mutants (Williams and Ustacelebi, 1971a; Ensinger and Ginsberg,

1972; Ledinko, 1974; BBgin and Weber, 1975), cyt-kb mutants which may be considered a type of host-range mutant (Takemori, 1972), and a heat-stable mutant (Young and Williams, 1975). With the first two classes of mutants, the ability to select the recombinant wild type from a predominantly mutant population greatly facilitated the demonstration of genetic exchange. The phenomenon of recombination has been or may be exploited in a number of ways: (1) to arrange mutants in order, by constructing a genetic map based on the recombination frequencies (r.f.) that occur between them, the greater the r.f., the greater the genetic distance (Williams et al., 1974; M. J. Ensinger, R. S . Kauffman, and H. Ginsberg, unpublished); ( 2 ) to establish whether or not mutants in the same complementation group were identical, as for example the demonstration by Takemori (1972) that many cyt-kb mutants were separable by recombination; ( 3 ) to utilize recombination, as mentioned earlier, to investigate whether or not all phenotypic characteristics of a particular mutant are caused by a single base change, i.e., are pleiotropic expressions of a single mutational lesion; and ( 4 ) to construct new genotypes, as in the case of the transfer of a heat-stable marker from the t s mutant in which it was isolated to wild type and thence to other t s mutants (Young and Williams, 1975).

110

HAROLD S. CINSBERG AND C . S. H. YOUNG

Recombination tests using t s mutants are normally arranged as follows. Cells are infected with both parental strains of virus and parallel cultures of cells are infected with each virus alone. The infected cells are incubated for several days at the permissive temperature, and the yields are titrated at both the restrictive and the permissive temperatures, to select for ts+ rcombinants and to measure total viral yields, respectively. The recombination frequency, r.f., expressed as a percentage has commonly been calculated as: (titer at the restrictive temperature) / (titer at the permissive temperature) x 2 x 100. In general the frequency of revertants i n the single parent controls has been so low as to make a correction for reversion in thc mixed infection unnecessary. The factor of 2 in the expressip allows for the presence of the unmonitored double ts recombinant class which is assumed to arise at equal frequency to the ts' class. This has never been established with adenoviruses as it is a formidable technical task to screen t s offspring for double ts genotype by complementation analysis. Limited data from crosses using a heatsiable ts mutant, ts-hs, and wild type, ts+-hst, suggest that the two recombinant types, heat-stable wild type, ts+-hs, and the ts mutant ts-11s' occur at frequencies that are not grossly dissimilar (Young and Williams, 197.5). In the absence of evidence of the reciprocal class occurring in ts x ts crosses, some investigators have preferred to use r.f. as expressing only the frequency of ts+ (Ledinko, 1974). Some technical points should be noted. ( 1 ) The r.f. values for a particular cross vary over a considerable range (Williams and Ustacelebi, 1971a; Williams et al., 1974), and thus to obtain a reliable statistic, several separate tests should be run. ( 2 ) It is important to establish that the plaques appearing at the restrictive temperature are genuinely ts+ and are not caused by complementation of different t s mutants on the assay plate. This has been tested by picking plaques and checking that the progeny have a wild-type genotype, i.e., yield the same number of plaques at both restrictive and permissive temperatures (Williams and Ustacelebi, 1971a; Ensinger and Ginsberg, 1972; Ledinko, 1974; Begin and Weber, 1975). Although one study demonstrated that complementation on the plate may be a serious disturbance to r.f. values (Ensinger and Ginsberg, 1972), this has not been reported by others. ( 3 ) The mixed infections should be incubated sufficiently long to permit the exponential phase of viral replication to be completed, since the r.f. increases with viral increase (Williams et d., 1974). With adenoviruses, despite the detection and biochemical and biological characterization of defective viruses with deleted genomes ( Mak, 1971; Burlingham et al., 1974), no attempt has been made to clone such deletions and use them for genetic purposes. The ordering of genes

GENETIC3 O F ADENOVIRUSES

111

with deletion mutants would seem to be technically feasible. Similarly, although some mutants have been isolated that would be excellent third markers in ts x ts crosses, no advantage has been taken of them. In particular the cgt mutants of Ad12 (Takemori et uZ., 1968) and the host-range markers of Ad5 (Takahashi, 1972) would be eminently suitable since the yield from three-factor crosses could be tested directly by selection to screen both for ts+ and for the third marker. The only investigation to date which has involved the use of a third marker is that using a heat-stable mutant of Ad5. The data were limited by the since the necessity of picking ts+ plaques and checking for h and h+, yield showed phenlotypic mixing for the heat-stable phenotype (Young and Williams, 1975). In view of the lack of more precise mapping procedures, the data from two-factor ts X ts crosses have had to be used to generate a genetic map. To date the only published orders or maps for adenoviruses are those for human adenoviruses type 2 (Bkgin and Weber, 1975) and type 5 (Williams et al., 1974). The former is based upon data using twelve mutants from twelve complementation groups and yields a relatively unambiguous order. It should be pointed out that very high values for r.f. were obtained for some crosses by these investigators; e.g., values as high as 38%were obtained for the percentage of ts+ among the progeny. This is formally equivalent to the statement that the mutants are unlinked. The significance of the map positions of the twelve mutants in the crosses must await the discovery of their phenotypic characteristics, an investigation that is under way ( Weber et al., 1975). The type 5 adenovirus map of Williams et al. (1974) is reproduced in Fig. 3, top line. This map is a compilation of data obtained over a number of years and represents a “best fit” for the r.f. values. The additivity of r.f. is good in many instances, an observation that lends support to the view that most, if not all, of the ts mutants used were single mutations, but there are a number of anomalies, as the authors pointed out. Whether or not these anomalies are significant in terms of the molecular organization of the chromosome, or are related to specific marker effects, remains to be determined. For comparison, the map of Ad5, which has been constructed recently by M. J. Ensinger, R. S. Kauffman, and H. S. Ginsberg, (unpublished) using t s mutants which have been independently isolated and characterized, is shown in Fig. 3, line 2. Several features of the two maps are worth emphasizing. ( 1 ) Where direct cqmparisons can be made, the order of markers of known phenotypes and in corresponding complementation groups are identical. In many instances, the genetic distances (r.f.’s) are similar. ( 2 ) In both maps, it is apparent that there is no absolute segregation of early and

HAROLD S . GINSBERG AND C. S . H. YOUNG

112

I

420 19

---

--+ 49

125

ASSEMBLY

\/

? 1,8

36

!.

59

I

i

I I

I

24

56

ASSEMBLY

I

I I I

,-

I;

HEXON TRANsPoRT I I

!

j

i I

I

’,

ASSEMBLY

I

-- -

149

:

15

30

1

__

‘ 1

j ‘.,I

I

FlBEp 1.2,3 I

I

I I

I

I/

I I

125 122

147

I38

;

II

II

HFXON

I I

---

115

47

142 56

04_18

I

I I

I

?

- --

5

I

II

DNA SYNTHESIS

9

I

110 10 l cr u

I

I I

DNA SYNTHESIS II

65

I

I I

I

22

1:!!~3

w

~

FIG.3. The genetic map of type 5 adenovims based upon two-factor recombination frequencies between pairs of t s mutants. Adapted from Williams et al. (1974), upper line, and M. J. Ensinger, R. S. K a u h a n , and H. S. Ginsberg, (unpublished), lower line. Symbols:

49

=+a

I

122 115

1 . 5.6

I

FI+R I

-

mutant position on map; except for the “hexon” gene, each mutant represents one complementation group alleles of the “hexon” gene (see footnote in Section II1,A) recombination frequency between two mutants mutant phenotype; vertical dashed lines indicate that complementation tests have been performed between mutants isolated in the laboratories of J. F. Williams and H. S. Ginsberg, and the mutants have been assigned to particular complementation groups.

late functions. Both DNA synthesis genes, which are early acting, are bracketed by late functions. (3) The maps demonstrate that the two functions related to hexon phenotype are separated by as many as three other genes. In this context, it is worth pointing out that the five complementation groups that map closely together and are variously de-

GENETICS OF ADENOVIRUSES

113

scribed as hexon-antigen negative and hexon-transport defective (mutants 1, 3, 4, 17, and 20) probably consist of mutants that can undergo intracistronic complementation. ( 4 ) Although the data are not shown on the maps, alleles within the same complementation group map closely together and at approximately the same distance from alleles in other genes. This is to be expected if the r.f.'s reflect physical genome distances. C. CORRELATION BETWEEN GENETIC AND PHYSICAL MAPS

The value of genetic mapping rests on the assumption that the genetic order corresponds to the physical order of genes in the genome. The proof of this assumption in other organisms encourages one to believe that it will be true for adenoviruses, but it must be established, if genetic mapping is to be a useful predictive tool. The physical structures of the DNAs of many serotypes of adenovirus have been investigated using site-specific restriction endonucleases obtained from a variety of microorganisms. The characteristic fragments that are produced may be used to construct a physical map (Mulder et al., 1974) against which genetic and biochemical markers may be aligned. With the development of hybridization techniques for selecting messenger RNAs that bind to specific fragments, and in uitm translation systems for such exogenous messengers, it is possible to map early and late transcripts and to locate genes for specific viral polypeptides (Lewis et al., 1975). Thus genetic and physical maps may be compared to correlate the position of the fragment that codes for a particular viral polypeptide and the map position of the gene that appears to control this polypeptide. The pitfall in this approach is that t s mutations may not lie in the structural gene for the p~lypeptidewhose activity is altered. For example, the hexon may be altered in quantity or the processing of its mRNA or a protein precursor may be altered by a mutation lying outside its structural gene. To avoid this danger, methods must be devised that directly correlate the t s mutant with the physical genome. The use of ts mutants in different adenovirus serotypes, from which different restriction endonuclease fragments may be generated, allows the selection and physical description of intertypic ts+ recombinants. This approach has been developed by Grodzicker et d. (1974b) and Williams et al. (1975a); it will be described in some detail because it is a novel example of the power of a combined genetic and physical investigation and because it has led to the conclusion that the recombination map for type 5 adenovirus, despite its being based on somewhat ambiguous data, is a faithful representation of the physical structure underlying it. Ad2'NDl is a nondefective hybrid between adenovirus type 2 and

114

HAROLD S. CINSBERG AND C. S . H. YOUNG

simian virus 40 (SV40), which is capable of plaquing with equal efficiency on both human and monkey cells (Lewis et al., 1969) and contains 17% of the SV40 genome located 15%from the right-hand end of the adenovirus genome ( Kelly and Lewis, 1973). Temperature-sensitive and host-range mutants of this virus are now available (Grodzicker et d., 1974a,b). Types 2 and 5 adenovirus belong in the same subgroup of adenovirus serotypes, which are classified as nononcogenic and show considerable DNA base homology (Garon et al., 1973; Bartok et al., 1974). Probably because of this close relatedness, the ts mutants from Ad 5 and Ad2'NDl can complement each other and recombine with frequencies close to those obtained in homologous crosses with corresponding homotypic mutants. The ts' recombinants from heterologous crosses can be checked for their ability to plaque on monkey cells, the SV40 fragment acting as a host range third marker; more important, since the restriction fragment patterns of Ad5 and Ad2'NDl are distinct, the genetic contribution of both parents to the recombinant may be determined. Thus, when a crossover from one parent to another occurs, the event is often marked by an alteration in the fragment pattern. When several ts' recombinants from a particular cross are compared in this way, those crossovers that are seen to be present in all of them are taken to be necessary for production of the recombinant. The position of these invariant crossovers set limits on the positions of the ts markers that enter the cross. It is important to note that (1) some restriction endonucleases may not yield distinctive patterns of fragments from certain areas of the molecule, in which case alternative enzymes must be used, and ( 2 ) double crossovers within the limits of the heterologous endonucleolytic cleavages will not be observed, which will lead to ambiguity unless other recombinants from the cross are available for comparison. Using this technique, it has been possible to locate the positions of three mutants of Ad5 and one of Ad2'NDl in the right-hand end of the genetic map. The orders and distances of the physical and genetic maps corresponded remarkably well ( Grodzicker et al., 1974b; Williams et al., 1975a). Recent data suggest that this correspondence holds for several more markers examined, including the DNA synthesis negative mutants H5ts36 and H5ts125 (J. Sambrook, H. S. Ginsberg, and J. F. Williams, personal communication). Some further extensions of this heterotypic analysis should be mentioned. First, the recombinants may be tested with antisera directed against specific polypeptides from each of the two viruses. Thus, it has proved to be possible to locate the fiber and hexon using recombinants that are expectcd to have crossover points between them and thus to have the fiber of one serotype and the hexon of the other (V. Mautner

GENETICS O F ADENOVlRUSES

115

and J. F. Williams, personal communication). Second, polypeptides from recombinants may be compared with those of either parent on sodium dodecyl sulfate ( SDS ) -polyacrylamide geIs; where polypeptide differences between serotypes can be detected, the genes may be mapped. It should be noted that restriction endonuclease analysis of recombinants may be used also to examine the frequency and nature of genetic exchange in animal viruses. D. HETEROTYPIC COMPLEMENTATION As has been mentioned in Section I, adenoviruses fall into three groups, depending on their oncogenic potential. Heterotypic complementation analysis provides a method to study the functional differences between them to determine which replicative functions and which structures can be substituted by one serotype for another. In adenoviruses, it is apparent that complementation between a serotype from the weakly oncogenic group (e.g., Ad2 or Ad5), and from the highly oncogenic group (e.g., Ad12 or Ad31), would be worth examining since DNA hybridization (Green, 1970), heteroduplex studies (Garon et d.,1973) and partial denaturation studies (Doerfler and Kleinschmidt, 1970; Doerfler et aZ., 1972) have shown these viruses to have different DNA structures. Heterotypic complementation between Ad5 and Ad12 has been examined by Williams et a2. (1975b). The complementation tests involved the use of a strain of Ad12 (1131) which failed to produce plaques on HeLa cells, although capable of infecting and producing cell-associated virus. The complementation tests therefore were of the form Ad12 wild type x Ad5 ts, and the yields were plated on HeLa celIs at the permissive temperature. Eight complementation groups from Ad5 could be complemented but seven other groups could not. Phenotypic mixing, as measured by the ability of neutralizing antisera against Ad5 and Ad12 to inactivate the yield, was observed in some positive complementation tests but not in others (Williams et al., 197%). It should be emphasized that it has not been possible to demonstrate recombination between type 5 and type 12 adenoviruses (Williams et al., 1975b), in contrast with the ability of Ad5 and Ad2'NDl to recombine freely. In bacteriophage, a correlation has been drawn between the homology of the DNA between different members of a related series and their ability to recombine [see, for example, the T3, T7, $11 series studied by Hyman et aZ. (1973)]. Heterotypic complementation within oncogenic classes to classify mutants has been alluded to in Section III,C. Shiroki and Shimojo (1974) have also used this method to classify ts mutants of Ad12 with respect to a ts mutant of Ad31 known to be defective in viral DNA synthesis.

116

HAROLD S . CINSBERG AND C. S . H. YOUNG

IV.

Phenotypes of Adenovirus Mutants

A. CHARACTERIZATION 1. Temperature-Sensitive Mutants As many as 22 virus-induced, possibly virus-coded, proteins have been detected in extracts of adenovirus-infected cells (Maizel et al., 1968a,b; Anderson et al., 1973; Everitt et al., 1973) (Fig. 1).Several are precursors of virion proteins (Sundquist et al., 1973a; Anderson et al., 1973; Ishibashi and Maizel, 1974a) (e.g., pVI and pVII), and several minor components have not yet been shown to be unique polypeptides. Hence, 12-15 putative primary gene products have been identified (Maizel et al., 1968a,b; Everitt et al., 1973; Anderson et al., 1973) although potentially the adenovirus genome could code for as many as 50-60 proteins with the average size of 25,000 daltons. Of the known viral gene products only a few appear to be altered in the large number of adenovirus temperature-sensitive mutants that have now been characterized (Table 11). Perhaps this can be attributed to the fact that the precise function and immunological properties of the native proteins have been identified only for the major viral proteins, and these are the viral products that appear to be represented in most of the phenotypes thus far described. Thus, the best characterized protein phenotypes listed in Table I1 represent lesions in the most easily studied and assayed capsid proteins, the hexon and fiber proteins. In contrast, so-called “assembly mutants” constitute a large proportion of the ts mutants classified, but whose defects have not yet been identified. These mutants are termed “assembly” mutants because at the nonpermissive temperature all identifiable proteins are synthesized and all the proteins that can be assayed are immunologically functional (Ginsberg, 1969; Schlesinger, 1969; Green, 1970; Philipson and Lindberg, 1974; Norrby, 1968; Philipson and Pettersson, 1973). These mutants may represent defects in one of the minor virion components or in a nonvirion protein that is essential for modulating the assembly process. If the “assembly” mutants represent a melange of mutations in a number of genes coding for nonvirion proteins, such as the 100 K, 50 K, 27 K, 26 K, or virion proteins XIII-XI1 ( Fig. l ) , a comparable number of complementation groups should have been identified. Instead, a maximum of five “assembly” complementation groups for type 5 ts mutants have been described (until mutants isolated in different laboratories are compared, the precise number of complementation groups remains uncertain). Two putative assembly mutants belonging to different complementa-

GENETICS OF ADENOVIRUSES

117

tion groups, also fail to induce interferon in chick embryo fibroblasts ( CEF) at the nonpermissive temperature (Ustacelebi and Williams, 1972). Since interferon is not a virus-coded product, nor is its induction viral specific, one simple interpretation of this observation is that the uncoating of these two mutants does not proceed to completion in CEF at the nonpermissive temperature because structural components of the capsid are altered. The observation that, although the mutants complement in the infectious cycle in HeLa cells, they failed to complement in the induction of interferon on CEF (Ustacelebi and Williams, 1972) is consistent with this interpretation, as is the finding that the induction of interferon was thermosensitive only during the first 6 hours after infection but the ts lesion blocked the production of virus in HeLa cells at late times during infection (Ustacelebi, 1973). In addition, both mutants were found to be considerably more thermolabile than wild type in uitro (Ustacelebi, 1973; Young and Williams, 1975), which is not unexpected for ts mutations in capsid structural components (Fenner, 1969). Thermolability of some ts mutants has also been observed in type 12 adenovirus (Shiroki et al., 1972) and in type 2 (Weber et al., 1975). During viral replication, adenovirus DNA can be separated from host cell DNA by virtue of its higher G C content (Piiia and Green, 1969; Ginsberg et al., 1967). Thus it has been a relatively simple task to screen ts mutants for the ability to synthesize DNA at the restrictive temperature. So far, mutants with DNA synthesis defects have been isolated in type 5 (Ensinger and Ginsberg, 1972; Wilkie et al., 1973), type 12 (Ledinko, 1974; Rubenstein and Ginsberg, 1974; Shiroki aad Shimojo, 1974), type 31 (Suzuki et al., 1972), and CELO (Ishibashi, 1971). Several of these mutants have also been examined for alteration in the frequency of transformation of rat or hamster embryo cells; these investigations will be discussed later.

+

2. Plaque Morphology and Host-Range Mutants Takemori et al. (1968) isolated a large number of mutants of adenovirus type 12 which gave plaques that were larger and clearer than those given by wild-type virus on early-passage human embryo kidney (HEK) cells. These mutants were discovered to be of low tumorigenicity in newborn hamsters and failed to transform newborn hamster kidney cells in oitro. Some of the mutants, (“cyt-kb”),failed to propagate in one line of KB cells (KB-1) while others, (“cyt-kb”’),multiplied in them; both types propagated in KB-2 cells (Takemori et al., 1969). Although there is evidence that the product of the cyt gene is diffusible

118

HAROLD S. GINSBERG AND C. S. H. YOUNG

(see Section IV,B,4 on transformation and tumorigenesis), as yet it is not known what viral product is involved. Host-range mutants of human adenovirus type 5 have been isolated (Takahashi, 1972; T. J. Harrison and J. F. Williams, personal communication) that are capable of growing on human cells but unable to do so on hamster cells. As yet no specific lesion has been identified, but on SDS-polyacrylamide gels, it is clear that all virus-infected, cell-specific proteins are made, but in reduced quantity (T. J. Harrison and J. F. Williams, personal communication ) . As mentioned previously, the nondefective, adeno-SV40 hybrid AdB'NDl, is capable of growing on both human and monkey cell lines (Lewis et al., 1969). It was thought that host-range mutants that fail to grow on monkey cells would be mutated in the integrated SV40 fragment, which presumably promotes adenovirus lytic cycle functions in the normally semipermissive monkey cells. Accordingly, Grodzicker et al. (1974a) selected such mutants and examined the polypeptides made in both human ( HeLa) and monkey cells (CVI clone of AGMK) infected with Ad2"Dl wild type, a host-range mutant, and Ad2. In the HeLa cell line, a protein product of 30,000 daltons, which is characteristic of Ad2'NDl infection, was absent in both Ad2 and host-range mutant infected cells. In CV1 cells, the characteristic underproduction of many adenovirus late proteins was observed in both Ad2 and hostrange mutant infections. The authors also examined the production of the perinuclear SV40 U antigen and found that in permissive cells infected with the host-range mutant the appearance of maximal detectable antigen was delayed, whereas in CV1 cells, very few nuclei ever displayed antigen. Whether or not the host-range mutation lies in the structural gene for the 30,000 dalton protein or in a viral gene that induces a host-cell protein is not clear. It should be noted that host-range mutants can be absolutely defective if the function(s) that are mutable are required only for replication of the virus in the restrictive host. This is perhaps unlikely in the hamster/ human Ad5 mutants but is a distinct possibility in the Ad2'NDl hostrange mutants. Absolute defectives are of considerable interest, since, as a class, they include deletion mutants that may be used for mapping purposes and, where they delete part of a gene, may be used to identify specific gene products.

B. FUNCTIONAL STUDIESUSINGADENOVIRUS MUTANTS Since, during replication, conditionally lethal temperature-sensitive mutants can be shifted conveniently from conditions that permit full

GENETICS OF ADENOVIRUSES

119

expression of the viral genome to nonpermissive conditions that do not allow expression of a defective gene, the gene product affected can be identified and its functional role in viral synthesis can be explored. This approach to the study of viral replication and its regulation has had noteworthy success with bacterial viruses and offers similar promise for unraveling the intricacies of adenovirus synthesis. Studies taking advantage of adenovirus ts mutants are in effect just beginning, and significant progress has been made only investigating three central areas in viral replication: DNA synthesis (Suzuki and Shimojo, 1974; Shiroki and Shimojo, 1974; van der Vliet et al., 1975; Levine et al., 1974), transcription (Carter and Ginsberg, 1975), and transport of viral proteins (Kauffman and Ginsberg, 1975). In addition, both ts and absolute mutants are being employed to explore the mechanisms underlying the establishment and maintenance of adenovirus transformation ( Ledinko, 1974; Ginsberg et al., 1974a,b; Williams et al., 1974; Rubenstein and Ginsberg, 1974; Takemori et aZ., 1968; Takahashi et aZ., 1974). 1. Viral DNA Synthesis

Relatively few mutants that cannot replicate their DNA under nonpermissive conditions have been reported. But the mutants available thus far encompass two complementation groups for type 5 virus (Ginsberg et al., 1974a,b; Williams et aZ., 1974) and three unique complementation groups for type 12 virus (Shiroki and Shimojo, 1974). It is striking that all the mutants isolated appear to be defective in initiation (Shiroki and Shimojo, 1974; Ginsberg et al., 1974a,b). The methods employed, however, do not distinguish between a mutant that cannot initiate replication and one that can elongate but not engage in a final reaction that may be required to terminate the synthesis of a complementary strand in order to initiate a new round. Thus, at least three virus-coded gene products appear to be essential for adenovirus DNA replication, and each is probabIy required for a reaction in chain initiation. One virus-specific protein, which binds preferentially to single-stranded DNA (van der Vliet and Levine, 1973), in a similar manner to the T4 gene-32 protein ( Alberts and Frey, 1970), has been shown to be defective in H5ts125 (van der Vliet et al., 1975) and H12ts275 ( Rosenwirth et al., 1975). Moreover, the so-called DNA-binding protein made in H5tsl25-infected cells is degraded at 39.5"C and dissociates from single-stranded DNA at lower temperatures than the wild-type protein (van der Vliet et al., 1975). Although the function of the DNAbinding protein is uncertain, since the adenovirus replicating form contains extensive single-stranded regions ( Sussenbach et al., 1972, 1973; Ellens et al., 1974; Pettersson, 1973), the binding protein could serve

120

HAROLD S . GINSBERG AND C. S . H. YOUNG

to maintain the single strands and thus permit effective copying of the displaced strand (van der Vliet et al., 1975). 2. Transcription of the Viral Genome Evidence obtained using pyrimidine analogs to inhibit DNA replication showed that only early mRNAs could be transcribed, from the infecting parental genome, and that late transcripts could be made only after DNA synthesis was begun (Bello and Ginsberg, 1969; Lucas and Ginsberg, 1971). These conclusions were subject to criticism, however, since it was not possible to demonstrate unambiguously that the chemicals employed ( i.e., 5-fluoro-2-deoxyridine, and arabinosylcytosine), did not effect any other intracellular biosynthetic reactions. With the availability of appropriate mutants, it became possible to investigate more rigorously the relationship between viral DNA replication and viral transcription. Carter and Ginsberg (1975) used two DNA-minus mutants, H5ts125 and H5ts149, to infect KB cells at nonpermissive temperature (41°C) and employed hybridization techniques to measure DNA replication and RNA transcription. The data obtained confirmed the earlier studies that no late transcripts appeared if onset of DNA replication was not permitted, and that under the restrictive conditions all early mRNAs Classes I and I1 (Lucas and Ginsberg, 1971) were transcribed. It was further shown that both Class I and I1 mRNAs continued to be transcribed as long as 15 hours after infection at 41"C, indicating that the shutoff of Class I mRNA was the work of a late gene function. It is also striking to note that although the onset of DNA replication is essential for the switch to transcription of late messages, the continuous replication of viral DNA is not required for late transcripts to be made: i.e., when cells were infected with H5ts125 or H5ts149 at 32°C for 25 hours and then shifted to 41"C, the rate of DNA synthesis decreased rapidly for H5ts125 and slowly for H5ts149; but the rate of viral RNA synthesis after the shift up did not change for 3 to 4 hours for ts 125 and even continued to increase for ts 149; and within the limits of the hybridization-competition techniques employed, the data indicated that the mRNAs made consisted of all the late sequences and Class I1 early RNAs.

3. Transport of the Hexon Protein As noted earlier, adenovirus proteins are synthesized on cytoplasmic polyribosomes and rapidly transported into the nucleus (Velicer and Ginsberg, 1968, 1970), where about 10%are assembled into virions (Wilcox and Ginsberg, 1963~).The mechanism of protein transport has been difficult to investigate owing to the marked leakiness of the infected

GENETICS OF ADENOVIRUSES

121

nucleus for viral proteins (Velicer and Ginsberg, 1968), although it was considered likely that host-cell functions played a major role. The discovery of ts mutants in which one or more capsid proteins were made but accumulated in the cytoplasm rather than moving into the nucleus under nonpermissive conditions ( Ishibashi, 1970, 1971; Shiroki et al., 1972; Russell et al., 1972a; Ginsberg et al., 1974a,b), suggested that these may offer an opportunity to study the process of protein transport. The mutants of one complementation group, whose hexon proteins specifically cannot move into the nucleus (Russell et al., 1972a; Ginsberg et al., 1974a,b; Kauffman and Ginsberg, 1975), appear to be of particular value for this purpose. The hexon, like the other capsid proteins, is immunologically active and folds into its native, multimeric structure, but only the hexon is not transported (Kauffman and Ginsberg, 1975). These transport mutants are clearly distinct from the hexon antigen minus mutants, since genetic recombination analysis shows that at least one other gene separates their respective gene loci. Biochemical, immunological, and physical studies of H5ts147 indicate that unaffected hexons are assembled and accumulate in the cytoplasm at 39.5"C; and that upon shift-down to 32"C, the preformed, cytoplasmic hexons can be transported into the nuclei and assembled into virions if protein synthesis is permitted (Kauffman and Ginsberg, 1975). Preliminary data suggest that the precursor (pVI) to protein VI (Anderson et al., 1973), a putative hexon-associated protein (Everitt et al., 1973), is defective in H5ts147 replication at 39.5"C (Kauffman and Ginsberg, 1975). The precise function of the small (27,OOO daltons) pVI protein is still unknown. 4. Transformation Conditionally lethal mutants offer the hope that specific gene product( s ) that effect cellular transformation can be defined, provided that these products are essential not only for lytic growth, but also for transformation. This optimistic note appears to have some validity since specific temperature-sensitive mutants of Rous sarcoma virus ( Tooze, 1973) and SV40 (Martin and Chou, 1975; Tegtmeyer, 1975; Brugge and Butel, 1975; Osborn and Weber, 1975) either cannot transform or cannot maintain the transformed state at the nonpermissive condition. The results of transformation studies with adenovirus ts mutants do not clearly identify a specific gene function to be directly concerned with transformation. Data do suggest, however, that at least one viral protein serves in the regulation of transformation. Some, but not all, DNA minus mutants [H5ts125 (Ginsberg et al., 1974a,b), H12ts307 (Ginsberg et al., 1974a,b), and H12ts401 (Ledinko, 1974)l transform two to eight times more rat

122

HAROLD S. GINSBERG AND C. S . H. YOUNG

or hamster embryo cells than does wild-type 5 or 12 virus. In sharp contrast, H5ts149 (Ginsberg et al., 1974a) and H12ts406 (Ledinko, 1974), which are also DNA-minus mutants in different complementation groups from H5ts125 and H12ts401, respectively, transform at the same frequency as wild-type viruses. H5ts36 and H5ts37 (Wilkie et al., 1973) however, which belong to the same complementation group as H5ts149 (Williams et d.,1974; Ginsberg et al., 1974a,b) transform rat embryo cells at a 10- to 20-fold lower frequency than wild type at the nonpermissive temperature, although cells transformed by the mutants at 32.5"C do not lose their transformed characteristics at 38.5"C (Williams et d.,1974). (One clone of such cells, however, has the curious property of being unable to grow at 38.5"C although it still maintains its transformed morphology). The failure of the cells of all such clones to revert to a normal morphology on shift-up to the nonpermissive temperature, suggests that H5ts36 and 37 are defective in the initiation, rather than in the maintenance, of transformation (Williams et al., 1974). Temperature-shift experiments, during the establishment of transformation, suggest that the temperature-sensitive step occurs before 48 hours growth at 32.5"C (Williams et al., 1974). Type 5 adenovirus ts mutants have also been used to transform hamster embryo cells, which are normally permissive for Ad5 (Williams, 1973). By performing the transformation at 38.5"C, t s mutants were unable to enter the lytic cycle and transformed clones were obtained. The clones were found to be highly oncogenic in newborn hamsters (Williams, 1973), and, furthermore, sera taken from such animals were found by indirect immunofluorescence techniques to react with the transformed cell nuclei (Williams et al., 1974). Another approach to restricting the usual lytic cycle of type 5 adenovirus infection in hamster cells, has been taken by Takahashi et al. (1974) and T. J. Harrison and J. F. Williams ( personal communication ) . They have demonstrated that hostrange mutants that fail to replicate in hamster cells nevertheless can transform them. It is noteworthy that the portion of the genome that contains the H5ts125 gene (J. Sambrook and H. S. Ginsberg, unpublished), which codes for a DNA-binding protein (van der Vliet et al., 1975),and perhaps that which contains the H5ts36 gene (J. Sambrook and J. F. Williams, unpublished) are not present in that minimum segment of the viral genome that has been detected in adenovirus-transformed cells ( Gallimore et al., 1974). Thus, as little as 14%of the distal end of the Eco.R1 restriction endonuclease generated A fragment (Mulder et al., 1974) appears to be necessary to maintain the transformed state (Gallimore et al., 1974; Sambrook et al., 1974). Whether under conditions of viral infection,

GENETICS OF ADENOVIRUSES

123

all or only a small portion of the genome is initially integrated to induce cell transformation is unknown, but transformation can be effected by only a fragment of the viral DNA representing as little as 5%of the viral genome and consists of only a small piece, which is present in the large Eco.R, “A” fragment (Graham et al., 1974). The finding that H5ts125, as well as related type 12 DNA-minus ts mutants, transforms at an increased frequency suggests that the DNA-binding protein may normally play a role that modulates the viral genome-cell interaction to reduce the opportunity for transformation, perhaps for DNA integration. These high-efficiency transforming mutants do clearly demonstrate that transformation is not dependent on replication of the viral DNA. The use of nonconditionally lethal mutants of defined genotype to study transformation has so far been restricted to the cyt mutants of human adenovirus type 12 (Takemori et al., 1968).These mutants, which gave larger and clearer plaques on early-passage HEK cells than did the parental wild-type strain, were found to have lost the ability to transform hamster cells in z)itro and to cause tumors in newborn hamsters. The mutants cooperated with low tumorigenic cyt+ field strains of Ad12 and the weakly tumorigenic viruses Ad3 and Ad7, to produce high levels of tumorigenicity, which suggested that the gene product was diffusible (Takemori et al., 1968). The cyt mutants have also been examined for the percentage of defective particles which they and the parental strain generated, to determine whether there was a positive correlation between the frequency of transformation and the proportion of defectives (Ezoe and Mak, 1974). No such correlation could be found. This does not rule out the possibility, however, that a specific class of defectives is the agent of transformation. Such a demonstration may have to await the development of methods for cloning virus of known defective constitution in ways analogous to those devised for SV40, in which specific deletion mutants were complemented by specific ts mutants at the restrictive temperature (Brockman and Nathans, 1974). It should be pointed out that all mutants that have been examined for transforming abilities were selected, in the first instance, for alterations in the lytic cycle and only secondarily for changes in transformation. This method of screening precludes mutants that are deficient only in transformation, if indeed such exist. V. Summing Up

Beauty may be attributed to a perfect object, but observation of nature’s imperfections has often revealed the elegance of structure and function awarded to earthly creatures. Thus, the study of inheritable

I!%

HAROLD S . CINSBERG AND C.

S. H. YOUNG

defects noted in a vast variety of plants and animals has uncovered rules and mechanisms of genetic interactions as well as processes regulating differentiation, morphogenesis, and biosynthetic events. Viruses, bacterial and animal, have been similarly studied and shown to follow the same genetic game rules as plants and animals-perhaps the most telling point that viruses may be considered to be organisms in that perennial argument as to whether viruses are living. Adenovirus genetics has been reviewed in this paper; as predicted, these viruses participate in the same genetic interactions as other organisms, and their genetic imperfections ( i.e., mutations ) are elucidating viral structure and reactions regulating viral function. It is striking that although the adenovirus genome can potentially code for fifty or more proteins, unique mutations have been detected in only ten to twelve genes. And of the number of conditionally lethal, temperature-sensitive mutants isolated, there is considerable clustering of two phenotypes, hexon and “assembly” mutants. Hexon mutants segregate into a single complementation group in which there is some evidence for intracistronic complementation; “assembly” mutants probably comprise four or five nonoverlapping complementation groups involving several gene functions essential to the regulation of virion morphogenesis. It is not clear, however, why the number of unique mutants detected falls so far short of the genome’s coding potential. Nevertheless, the mutants isolated follow predictive intergenic reactions; and despite the relative imprecision of two factorial crosses, a reproducible linear map has been constructed (Williams et al., 1974; M. J. Ensinger, R. S. Kauffman, and H. S. Ginsberg, unpublished). Indeed, the recombination maps produced (Williams et al., 1974; M. J. Ensinger, R. S . Kauffman, and H. S. Ginsberg, unpublished) correspond remarkably well with two independent techniques of physical mapping ( Grodzicker et al., 1974b; Lewis et al., 1975). Viral genetics, in addition to furnishing means for investigating viral structure and function, has predictive values. For example, Hirst noted the relatively high frequency of recombination between influenza virus mutants and suggested from these findings that the viral genome may be fragmented (Hirst, 1962). No such dramatic prognostication can be made from evidence accumulated with adenovirus mutants. The intergenic reactions demonstrated, however, do permit certain hypotheses that explain the evolutionary development of numerous adenovirus types in many animal species, and the viruses’ potential for incorporating viral genetic materials into host genomes to produce cell transformation, and tumors, and possibly latent viral infections. As noted earlier ts mutants have proved to be of greatest value for

GENETIa OF ADENOVIRUSES

125

investigating the structure and function of adenovirus proteins. Thus, the grouping of hexon mutants in a single cistron confirms chemical and physical evidence that the hexon is formed from the assembly of three identical polypeptide chains (Franklin et al., 1971; Cornick et al., 1973; Stinski and Ginsberg, 1975). And the demonstration, using genetic techniques, that a mutant defective in the transport of hexon protein into the nucleus is distinct from the hexon antigen minus mutants, revealed evidence that synthesis, assembly, and transport of hexon capsomers require more than a single gene product (Kauffman and Ginsberg, 1975). Furthermore, the detection of three nonoverlapping complementation groups representing three genes essential for production of a functional fiber (Russell et al., 1972a) implies that the fiber consists of three rather than two unique species of polypeptides (Dorsett and Ginsberg, 1975). Studies using adenovirus ts mutants for discovering the viral proteins involved and their function( s ) in regulating viral biosynthesis are still in their infancy. Nevertheless, types 5 and 12 mutants have revealed a minimum of three viral proteins essential for initiation of DNA replication (Shiroki and Shimojo, 1974; Ginsberg et al., 1974a,b). But only one gene product has yet been identified, a DNA-binding protein which prefers single-stranded DNA (van der Vliet and Levine, 1973; van der Vliet et d.,1975; Rosenwirth et al., 1975). Two of these mutants, H5ts125 and H5ts149, have also been valuable for studying regulation of transcription (Carter and Ginsberg, 1975). Using these mutants, it has been possible to confirm the earlier finding (Bello and Ginsberg, 1969) that late transcripts can be made only after replication of viral DNA has begun (Carter and Ginsberg, 1975). The reduction in transcription of early class I mRNAs also requires viral DNA synthesis. Despite these demands, continued viral DNA replication is not essential for transcription of late genes, since change to the nonpermissive temperature and the consequent rapid cessation of viral DNA synthesis does not alter the rate or the quality of late transcription (Carter and Ginsberg, 1975). The ts mutants unable to replicate DNA at the restrictive temperature have also proved to be valuable for investigating cell transformation; it was found that H5ts125 ( Ginsberg et al., 1974a,b) H12ts307 (Rubenstein and Ginsberg, 1974), and H12ts401 ( Ledinko, 1974) transform significantly more cells than wild-type viruses or other ts mutants. H5ts125 has been most thoroughly characterized and, as noted above, is defective in a virus-specific, single-stranded DNA-binding protein ( van der Vliet et al., 1975). It is striking that the portion of the viral genome that codes for the ts 125 gene product (Fig. 3), which is approximately 0.65 physical map unit from the left-hand end of the genome (J. Sam-

126

HAROLD S. GINSBERG AND C. S . H. YOUNG

brook and H. S. Ginsberg, unpublished data), is rarely integrated in adenovirus-transformed cells ( Gallimore et al., 1974; Sambrook et al., 1974). The significance of these findings is emphasized by the fact that three independently isolated mutants present this same phenomenon of increased transforming capacity, whereas three other DNA-minus mutants, belonging to another complementation groups, H5ts36 and 37 (Williams et al., 1974) and H5ts149 (Ginsberg et al., 1974a), show either decreased or normal transformation frequency. It appears inescapable that the adenovirus DNA-binding protein, whatever its function is, plays a regulatory role in transformation as well as DNA replication. The beginnings of adenovirus genetics recounted in this review invite continued optimism, since the data thus far obtained, as predicted, not only reveal new mechanisms controlling viral replication, but also offer greater understanding of genetic interactions of DNA-containing animal viruses.

REFERENCES Alberts, B., and Frey, L. (1970). Nature (London) 227, 1313-1318. Anderson, C. W., Baum, P. R., and Cesteland, R. F. (1973). J. Virol. 12,241-252. Bartok, K., Garon, C. F., Berry, K. W., Fraser, M. J., and Rose, J. A. (1974). J. Mol. Biol. 87, 437-449. BCgin, M., and Weber, J. (1975). J. Virol. 15, 1-7. Bellett, A. J. D., and Younghusband, H. B. (1972). J. Mol. Bid. 72,691-709. Bello, L. J., and Ginsberg, H. S. ( 1969). J . Virol. 3, 106-113. Boyer, G. S., Leuchtenberger, C., and Ginsberg, H. S. (1957). J. Exp. Med. 105, 195-216. Boyer, G. S., Denny, F. W., Jr., and Ginsberg, H. S. (1959). J. Exp. Med. 110, 827-844. Brockman, W. W., and Nathans, D. (1974). Proc. Nut. Acad. Sci. U.S. 71, 942-946. Brugge, J. S., and Butel, J. S. (1975). J. Virol. 15, 619-635. Burlingham, B. T., Brown, D. T., and Doerller, W. (1974). Virology 60,419-430. Carter, T. H., and Ginsberg, H. S. ( 1975). J . Virol. (in press). Chardonnet, Y.,and Dales, S. ( 1970). Virology 40, 462-477. Chou, J. T., and Martin, R. G. (1974). J . Virol. 13,1101-1109. Cornick, G., Sigler, P. B., and Ginsberg, H. S. (1973). J. Mol. Biol. 73, 533-537. Craig, E. A., Zimmer, S., and Raskas, H. J. (1975). J . Virol. 15, 1202-1213. Crick, F. H. C., and Watson, J. D. (1956). Nature (London) 177, 473475. Dales, S. (1962). J. Cell Biol. 13, 303-322. Doerfler, W., and Kleinschmidt, A. D. (1970). J . Mol. B i d . 5 , 5 7 9 5 9 3 . Doerfler, W., Hellman, W., and Kleinschmidt, A. D. ( 1972). Virology 47, 507-512. Dorsett, P. H., and Ginsberg, H. S. ( 1975). J. Virol. 15,208-216. Ellens, D. J., Sussenbach, J. S., and Jansz, H. S. (1974). Virology 61, 427-442. Ensinger, M. J., and Ginsberg, H. S. ( 1972). J. Virol. 10,328-339. Everett, S. F., and Ginsberg, H. S. (1958). Virology 6,770-771. Everitt, E., Sundquist, B., and Philipson, L. (1971). J. Virol. 8, 742-753. Everitt, E., Sundquist, B., Pettersson, U., and Philipson, L. (1973). Virology 52, 130-147.

GENETIG OF ADENOVIRUSES

127

Ezoe, H., and Mak, S. (1974). J . Virol. 14, 733-739. Fenner, F. (1969). Curr. Top. Microbiol. Immunol. 48, 1-28. Fincham, J. R. S. ( 1966). “Genetic Complementation.” Benjamin, New York. Franklin, R. M., Pettersson, U., Akervall, K., Strandberg, B., and Philipson, L. ( 1971). J. Mol. Biol. 57, 383395. Fujinaga, K., and Green, M. (1970). Pruc. Nut. A c d . Sci. U.S.65,375482. Gallimore, P. H., Sharp, P. A., and Sambrook, J. (1974). J. Mol. Biol. 89, 49-72. Garon, C. F., Berry, K., and Rose, J. (1972). Proc. Nut. A c d . Sci. US. 69, 2391-2395. Garon, C. F., Berry, K. W., Hierholzer, J. C., and Rose, J. A. ( 1973). Virology 54, 414426. Gilead, Z., and Ginsberg, H. S. (1965). 1. Bucteriol. 90,120-125. Ginsberg, H. S. (1969). In “The Biochemistry of Viruses” (H. B. Levy, ed.), pp. 329-359. Dekker, New York. Ginsberg, H. S., Pereira, H. G., Valentine, R. C., and Wilcox, W. C. (1966). Virology 28, 782-783. Ginsberg, H. S., Bello, L. J., and Levine, A. J. (1967). In ‘The Molecular Biology of Viruses” (S. J. Colter and W. Paranchych, eds.), pp. 547-572. Academic Press, New York. Ginsberg, H. S., Ensinger, M. J., Kauffman, R. S., Mayer, A. J., and Lundholni, U. ( 1974a). C d d Spring Hurbor Symp. Quunt. Biol. 39,419-426. Ginsberg, H. S., Ensinger, M. J., Rubinstein, F. E., and Kauffman, R. S. (1974b). In “Viruses, Evolution and Cancer” (E. Kurstak and K. Maramorosch, eds. ), pp 167-181. Academic Press, New York. Graham, F. L., van der Eb, A. J., and Heijneker, H. L. ( 1974). Nature (London) 251, 687-691. Green, M. (1962). Cold Spring Harbor Symp. Quunt. Biol. 27, 219-235. Green, M. ( 1970). Annu. Rev. Biochem. 39,701-756. Green, M., Piiia, M., Kimes, R., Wensink, P. C., MacHottie, L. A., and Thomas, C. A., Jr. (1967). Proc. Nut. Acad. S c i . U.S. 57, 13021309, Green, M., Parsons, J. T., Piiia, M., Fujinaga, K., Caffier, H., and Landgraf-Leurs, I. (1970). Cold Spring Hurbor Symp. Quunt. Biol. 35,803-818. Grodzicker, T., Anderson, C., Sharp, P. A., and Sambrook, J. (1974a). J. Virol. 13, 1237-1244. Grodzicker, T., Williams, J., Sharp, P., and Sambrook, J. (1974b). Cold Spn’ng Hurbor Symp. Quunt. Biol. 39, 439-446. Hirst, G. K. (1962). Cold Spring Hurbor Symp. Qmnt. Biol. 27,303-309. Horwitz, M. S., Brayton, C., and Baum, S. G. (1973). J. Virol. 11, 544-551. Huebner, R. J., Casey, M. J., Chanock, R. M., and Schell, K. (1965). Proc. Nut. Acud. Sci. US.54, 381388. Hyman, R. W., Brunovskis, I., and Summers, W. C. (1973). J. Mol. Biol. 77, 189-196. Ishibashi, M. (1970). Proc. Nut. Acud. Sci. US.65, 304409. Ishibashi, M. ( 1971 ). Virology 45, 42-52. Ishibashi, M., and Maizel, J. V., Jr. ( 1974a). Virology 57,409-424. Ishibashi, M., and Maizel, J. V., Jr. (197413). Virology 58,345-361. Jacobson, M. F., and Baltimore, D. (1968). J. Mol. Biol. 33, 369-378. Kasel, J. A., Huber, M., Loda, F., Banks, P. A., and Knight, V. (1964). Prac. SOC. Erp. Biol. Med. 117, 186190. Kauffman, R. S., and Ginsberg, H. S. ( 1975). J . viTO1. (in press). Kellenberger, E., Eiserling, F., and Boy de la Tour, E. (1968). J. Ultrastruct. Res. 21, 335-360.

128

HAROLD S. CIh’SBERG AND C. S. H. YOUNG

Kelly, T. J., Jr., and Lewis, A. M., Jr. (1973). J. Virol. 12, 643-652. King, J., and Casjens, S. ( 1974). Nature (London) 251, 112-119. Kjellhn, L. and Pereira, H. C. ( 1968). J. Gen. Virol. 2, 177-185. Koczot, F. J,, Carter, B. J., Garon, C. F., and Rose, J. A. (1973). Proc. Nat. Acad. Sci. U.S. 70, 215-219. Laver, W. G. ( 1970). Virology 41, 488-500. Lawrence, W. C., and Ginsberg, H. S. ( 1967). J. Virol. 1,851-867. Ledinko, N. (1971). Nature (London), New Biol. 233, 247-248. Ledinko, N. (1974). J. Virol. 14, 457-468. Levine, A. J., and Ginsberg, H. S. ( 1967). I . ViroI. 1, 747-757. Levine, A. J., van der Vliet, P. C., Rosenwirth, B., Rabek, J., Frenkel, G., and Ensinger, hl. (1974). Cold Spring Harbor Symp. Quant. Biol. 39, 559-566. Lewis, A. M., Jr., Levin, M. J., Wiese, W. H., Crumpacker, C. S., and Henry, P. H. (1969). Proc. Nat. Acad. Sci. U.S.63, 1128-1135. Lewis, J. B., Atkins, J. F., Anderson, C. W., Baum, P. R., and Gesteland, R. F. ( 1975). Proc. Nat. Acad. Sd. US.72, 1344-1348. Lindberg, U., Persson, T.,and Philipson, L. (1972). I. Virol. 10, 909-919. Lonberg-Holm, K., and Philipson, L. ( 1969). J. Virol. 4,323338. Lucas, J. J., and Ginsberg, H. S. ( 1971). J. ViroZ. 8,203-213. Lucas, J. J., and Ginsberg, H. S. ( 1972). J . Virol. 10, 1109-1117. Lundholm, U., and Doerfler, W. (1971). Virology 45, 827-829. McGuire, P. M., Swart, C., and Hodge, L. D. (1972). Proc. Nut. Acad. Sci. U.S. 69, 1578-1582. hfaizei, J. V., Jr., Philiips, B. A., and Summers, D. F. ( 1967). Virology 32, 692-699. and Scharff, h4. D. ( 1968a). Virology 36, 115-125. Maizel, J, V., Jr., White, D. 0.. Maizel, J. V., Jr., White, D. O., and Scharff, hl. (1968b). Virology 36, 126-136. Mak, S. ( 1971). J. V i d 7, 426433. Martin, R. G., and Chou, J. Y. (1975). I . Virol. 15,599-612. Morgan, C., Godman, G. C., Breitenfeld, P. M., and Rose, H. M. (1960). J. E r p . Med. 112,373-382. Morgan, C., Rosenkranz, H. S., and Mednis, B. (1969). J. Virol. 4, 777-796. Mdder, C., Arrand, J. R., Delius, H., Keller, W., Pettersson, U., Roberts, R. J., and Sharp, P. A. ( 1974). Cold Spring Harbor Symp. Quant. Biol. 39, 397-400. Seurath, A. R., Rubin, B. A., and Stasny, J. T. (1968). 1. Virol. 2, 1086-1095. Norrby, E. (1968). Curr. T o p . Microbiol. lmmunol. 43, 1-43. Norrby, E. (1969). J. Gen. Virol. 5, 221-236. Norrby, E., and Skaaret, P. ( 1967). Virology 32, 489502. Okubo, C. K., and Raskas, H. J. ( 1971 ). Virology 46, 175-182. Osborn, hl., and Weber, K. (1975). 1. Virol. 15, 636-644. Ozer, H. L. (1972). 1. Virol. 9,41-51. Ozer, H.L., and Tegtmeyer, P. (1972). 1. Virol. 9 , 5 2 4 0 . Parsons, J. T., and Green, M. (1971). Virology 45, 154-162. Pearson, G . D., and Hanawalt, P. C. ( 1971). J. Mol. Biol. 62, 65-80. Pereira, H. G. (1958). Virology 6, 601-611. Pereira, H. C. ( 1960). Nature (London) 186, 571-572. Pettersson, U. (1973). 1. Mol. Biol. 81,521527. Pettersson, U.,and Hoglund, S. ( 1969). Virdogy 39,90-106. Philipson, L., and Lindberg, U. (1974). Compr. Virol. 3, 143-227. Philipson, L.,and Pettersson, U. (1973). Progr. Exp. Tumor Res. 18, 155. Philipson, L., Lonberg-Holm, K., and Pettersson, U. (1968). J. Virol. 2, 1064-1075.

GENETICS OF ADENOVIRUSES

129

Philipson, L., Wall, R.,Glickman, G., and Darnell, J. E. (1971). Proc. Nut. Acad. Sci. US.68, 2806-2809. Philipson, L., Lindberg, U., Person, T., and Vennstrom, B. (1973). Adoan. Biosci. 11, 167-183. Phillips, B. A., Summers, D. F., and Maizel, J. V., Jr. (1968). Virology 35, 216226. Piiia, M., and Green, M. (1965). Proc. Nut. Acad. Sci. US. 54, 547-551. Piiia, M., and Green, M. (1969). Virology 38, 573-586. Pope, J. H., and Rowe, W. P. (1964). J. Erp. Med. 120,577-588. Prage, L., and Pettersson, U. ( 1971) . Virology 45, 364-373. Price, R.,and Penman, S. (1972). J .ViroZ. 9, 621-626. Robb, J. A., and Martin, R. G. ( 1972). 1. Virol. 9,956-968. Robinson, A. J., Younghusband, H. B., and Bellett, A. J. D. (1973). Virology 56, 54-69. Rosen, L. ( 1960). Amer. J. H y g . 71, 120-128. Rosenwirth, B., Shiroki, K., Levine, A. J., and Shimojo, H. (1975). Submitted for publication. Rouse, H. C., and Schlesinger, R. W. ( 1972). Virology 48,463-471. Rubenstein, F . E., and Ginsberg, H. S. (1974). Interoirology 3, 170-174. Russell, W. C., and Knight, B. ( 1967). J. Gen. Virol. 1, 523-528. Russell, W. C., McIntosh, K., and Skehel, J. J. (1971). J. Gen. Virol. 11, 35-46. Russell, W. C., Newman, C., and Williams, J. F. (1972a). J. Gen. Virol. 17, 265-279. Russell, W. C., Skehel, J. J., Machado, R., and Pereira, H. G. (1972b). Virology 50, 931-934. Russell, W. C., Skehel, J. J., and Williams, J. F. (1974). J. Gen. Virol. 24, 247-259. Sambrook, J., Botchan, M., Gallimore, P., Ozanne, B., Pettersson, U., Williams, J., and Sharp, P. (1974). Cold Spring Harbor Symp. Quunt. Bwl. 39, 615-682. Schlesinger, R. W. (1969). Aduan. Virus Res. 14, 1-61. Shiroki, K., and Shimojo, H. (1974). Virology 61, 474-485. Shiroki, K., Irisawa, J., and Shimojo, H. (1972). Virology 49, 1-11. Shiroki, K., Shimojo, H., and Yamaguchi, K. (1974). Virology 60,192-199. Simon, L. (1972). Proc. Nut. Acad. Sci. US.69, 907-911. Stinski, M. F., and Ginsberg, H. S. (1975). J. Virol. 15, 898-90s. Sundquist, B., Everitt, E., Philipson, L., and Haglund, S. (1973a). J. Virol. 11, 449-459. Sundquist, B., Pettersson, U., Thelander, L., and Philipson, L. ( 1973b). Virology 51, 252-256. Sussenbach, J. S. (1967). Virology 33, 567-574. Sussenbach, J. S., van der Vliet, P. C., Ellens, D. J., and Jansz, H. S. (1972). Nature (London),New Biol. 239, 47-49. Sussenbach, J. S., Ellens, D. J., and Jansz, H. S. (1973). J. Virol. 12, 1131-1138. Suzuki, E., and Shimojo, H. (1971). Virology 43, 488-494. Suzuki, E., and Shimojo, H. (1974). J . Virol. 13, 538-540. Suzuki, E., Shimojo, H., and Moritsugu, Y. (1972). Virology 49, 426-438. Takahashi, M. ( 1972). Virology 49, 815-817. Takahashi, M., Minekawa, Y., and Yamanishi, K. (1974). Virology 57,300-303. Takemori, N . (1972). Virology 47, 157-167. Takemori, N., Riggs, J. L., and Aldrich, C. (1968). Virology 36, 575-586. Takemori, N., Riggs, J. L., and Aldrich, C. D. (1969).Virdogy 38, 8-15. Tegtmeyer, P. (1975). J. Virol. 15, 613-618. Thomas, D. C., and Green, M. ( 1966). Proc. Not. Acad. Sci. U.S. 56,243-246.

130

HAROLD S. CINSBERG AND C. S. H. YOUNG

Thomas, D. C., and Green, M. (1969). Virology 39, 205-210. Tibbetts, C., and Pettersson, U. (1974). J. Mof. Biol. 88, 767-784. Tibbetts, C., Pettersson, U., Johansson, K., and Philipson, L. (.1974). J. Virol. 13, 370-377. Tooze, J., ed. (1973). “The Molecular Biology of Tumour Viruses.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Ustacelebi, S. (1973). Ph.D. Thesis, University of Glasgow, Scotland. Ustacelebi, S., and Williams, J. F. (1972). Nature (London) 235, 5253. Valentine, R. C., and Pereira, H. G. (1965). J. Mol. Biol. 13, 13-20. van der Eb, A. J. (1973). Virology 51, 11-23. van der Eb, A. J., van Kesteren, L. W., and van Bruggen, E. F. J. (1969). Biochim. Biophys. Acta 182, 530-541. van der Vliet, P. C., and Levine, A. J. (1973). Nature (London), New Biol. 246, 170-174. van der Vliet, P. C., and Sussenbach, J. S. (1972). Etrr. J. Biochem. 30, 584-592. van der Vliet, P. C., Levine, A. J., Ensinger, M. J., and Ginsberg, H. S . (1975). 1. Virol. 15, 348-354. L’elicer, L., and Ginsberg, H. S. (1968). Proc. Nut. Acad. Sci. U S . 61, 1264-1271. Velicer, L., and Ginsberg, H. S. ( 1970). J. Virol. 5, 338-352. Wall, R., Philipson, L., and Darnell, J. E. ( 1972). Virology 50,2734. Weber, J., BCgin, M., and Khittoo, G. (1975).J. Virol. 15, 1049-1056. Scharff, M. D., and Maize], J. \’., Jr. ( 1969). Virology 38, 395-406. White, D. 0.. Wilcox, W. C., and Ginsberg, H. S. ( 1961). Proc. Nut. Acad. Sci. U.S. 47, 512-526. Wilcox, W. C., and Ginsberg, H. S. (1963a). Proc. SOC. Exp. Biol. Med. 114,3742. Wilcox, W. C., and Ginsberg, H. S. ( 1963b). Virology 20,269-280. Wilcox, W. C., and Ginsberg, H. S. ( 1 9 6 3 ~ ) J. . Erp. Med. 118, 295-306. Wilcox, W. C., Ginsberg, H. S., and Anderson, T. F. (1963). J. Exp. Med. 118, 307-3 14. Wilhelm, J. M., and Ginsberg, H. S. ( 1972). J. Virol. 9,973-980. Wilkie, N. M., Ustacelebi, S., and Williams, J. F. ( 1973). Virology 51, 499-503. Williams, J. F. ( 1973). Nature (London) 243, 162-163. Williams, J. F., and Ustacelebi, S. (1971a). J. Gen. Virol. 13, 345-348. Williams, J. F., and Ustacelebi, S. (1971b). Strategy Viral Genome, Ciba Found. Symp. pp. 275-290. Williams, J. F., Gharpure, M., Ustacelebi, S., and McDonald, S. (1971). J. Gen. Virol. 11, 95-101. Williams, J. F., Young, C. S. H., and Austin, P. E. (1974). Cold Spring Harbor Symp. Quant. Biol. 39, 427-437. Williams, J. F., Grodzicker, T., Sharp, P., and Sambrook, J. (1975a). Cell 4, 113-119. Williams, J. F., Young, C. S., and Austin, P. E. (1975b). J. Virol. 15, 675-678. Wolfson, J., and Dressler, D. (1972). Proc. Nat. Acad. Sci. U S . 69, 30543057. Yamashita, T., and Green, M. (1974). 1. Virol. 14, 412-420. Young, C. S. H., and Williams, J. F. (1975).J. Virol. 15, 1168-1175.

MOLECULAR BIOLOGY OF THE CARCINOGEN. 4-NITROQUINOLINE 1 -OXIDE Minako Nagao and Takashi Sugirnura

.

National Cancer Center Research Institute. Chu0.b. and Institute of Medical Science University of Tokyo. Minato.ku. Tokyo. Japan

I . Introduction . . . . . . . . . . . . . I1. Mutagenic Activity of 4-Nitroquinoline I-Oxide on Organisms . . . A . Historical Aspects of Mutations by 4-Nitroquinoline 1-Oxide . B. Base-Pair Change Mutations . . . . . . . . . . . . . . . . . . . C. Frameshift Mutations D . Deletion Mutations . . . . . . . . . . . E . Mitotic Gene Conversions . . . . . . . . . . F. Loss of the Cytoplasmic p Factor in Yeast . . . . . . G. Mutagenic Activities of 4-Nitroquinoline 1-Oxide and Related . . . . . . . . . . . . Compounds H. Phage Induction from Lysogenic Bacteria . . . . . . I11. Chromosome Aberrations . . . . . . . . . . A . Chromosome Aberrations in Cells with Normal Repair Function . . B. Chromosome Aberrations in a Repair-Deficient Strain of Human Cells . . . . . . . C. Endoreduplication of Chromosomes . IV. Repair of 4-Nitroquinoline 1-Oxide-Damaged DNA . . . . . A . Repair in Bacteria . . . . . . . . . . . B . Repair in Yeast . . . . . . . . . . . . C. Repair in Bacteriophages . . . . . . . . . . D. Repair in Plant Cells . . . . . . . . . . . E. Repair in Mammalian Cells . . . . . . . . . V. Interaction of 4-Nitroquinoline I-Oxide and Its Derivatives with Nucleic . . . . . . . . . . . . . . Acids . A . In Viuo Formation of Quinoline-Base Adducts . . . . . B . Enzymic Activation Steps Required for Modification of DNA by . . . . . . . . . 4-Nitroquinoline I-Oxide . C. Biological Activity of Modified DNA . . . . . . . D. Chemical Structure of Quinoline-Purine Adducts . . . . . E. Chemical Interaction of 4-Nitroquinoline 1-Oxide and Its Deriva. . . . . . . . . . . tives with DNA . VI . Interaction of 4-Nitroquinoline 1-Oxide and Its Derivatives with Protein VII. Recent Information on Carcinogenesis by 4-Nitroquinoline 1-Oxide . . A . I n Viuo Carcinogenesis . . . . . . . . . . B . Immunity . . . . . . . . . . . . . C. I n Vitro Carcinogenesis by, and Effect of, 4-Nitroquinoline 1-Oxide on in Viuo Viral Carcinogenesis . . . . . . . . D. Decarcinogenesis by 4-Nitroquinoline 1-Oxide . . . . . E. Other Biologic Effects of 4-Nitroquinoline I-Oxide . . . .

.

131

132 133 133 133 136 136 137 137

138 138 140 140 140 141 143 143 145 146 146 146

151 151 152 152

153 155 156 157 157 160 160 162 162

132

MINAKO NAGAO AND TAKASHl SUGIMUFlA

VIII. 4-Nitroquinoline l-Oxide and Microbial Screening Method for Carcinogen 163 IX. Conclusions . . . . . . . . . . . . . 163 References . . . . . . . . . . . . . 164

I. Introduction

Studies on chemical carcinogenesis have three main purposes. One is to elucidate carcinogenic mechanisms, another is to prevent possible carcinogenic hazard due to chemicals in our environment, and the third is to provide clinical model systems in experimental animals. The carcinogenicity of 4-nitroquinoline 1-oxide (4NQO) was first reported by Nakahara et al. (1957). However, the mutagenicity of 4NQ0 was reported even earlier, in 1955, by Okabayashi. Mutagenicity by a carcinogen was observed, among others, by Latarjet et al. (1949). Recent studies on chemical carcinogens and mutagens have shown that with a few exceptions carcinogens are mutagenic and mutagens are carcinogenic. 4NQ0 is an example of a compound that has long been known to be both carcinogenic and mutagenic. Many geneticists have used 4NQ0 in experiments on biological materials ranging from bacteriophages to mammals. In many studies, 4NQ0 has also been used as a positive control substance in mutagenic and carcinogenic experiments. 4NQ0 has been used in studies on cultured cells in vitro as well as on in vivo carcinogenesis. Very interesting results have been reported on its induction of certain types of gastric carcinomas, pancreatic tumors, and lung tumors that mimic cases of human tumors. The molecular mechanisms of the interaction of metabolites of 4NQ0 with nucleic acids have been fairly well clarified. Moreover genetic studies on the effect of 4NQ0 are more advanced than those on many other carcinogens. Owing to the relation between carcinogens and mutagens, it seems worthwhile to review recent advances in studies on 4NQO. A monographic review of studies on this compound entitled “Chemistry and Biological Actions of 4-Nitroquinoline l-Oxide” by Endo et al. (1971) has been published. Therefore, the present review principally aims to cover more recent reports on this compound. One advantage of using 4NQ0 as a carcinogen is that metabolic activation systems for it are commonly present in microbial and mammalian cells. Thus it differs from most typical carcinogens, like acetylaminofluorene, 3’-methyl-Ndimethyl-4-aminoazobenzene, and many aromatic hydrocarbons, which are metabolically activated only by mammalian microsomal enzymes, but not by any microbial enzyme. This has made possible many experiments using 4NQ0 on microbes. Only a few other compounds like 4NQ0

MOLECULAR BIOLOGY OF THE CARCINOGEN,

4NQO

133

are known. These include nitrofuran derivatives, which are now under intensive study. We hope that this review on 4NQ0 will be of general use for studies on chemical carcinogenesis. II. Mutagenic Activity of 4-Nitroquinoline 1 -Oxide on Organisms

A. HISTORICAL ASPEW

OF

MUTATIONSBY 4-NITROQUINOLINE 1-OXIDE

Okabayashi was the first to observe that 4NQ0 had biological activity, finding first antifungal activity ( Okabayashi, 1953) and subsequently mutagenic activities on fungi ( Okabayashi, 1955). 4NQ0 was found to cause mutation with regard to the pattern of colony growth (Okabayashi, 1955) and to induce biochemical mutants (Yamagata et al., 1956) of Aspergillus niger. It also caused reversion of amino acid-requiring auxotrophs of Streptomyces griseo@vus to their prototrophs ( Mashima and Ikeda, 1958). Okabayashi and Yoshimoto (1962) proposed that microorganisms, such as Escherichiu coli, Candidu utilis, Aspergillus niger, Brevibacterium liquefaciens, and Pseudomonas aeruginosa, reduced 4NQ0 metabolically to 4-hydroxyaminoquinoline l-oxide (4HAQO) and that the latter was further reduced to 4-aminoquinoline l-oxide (4AQO). Treatment of Aspergillus niger with the metabolic intermediate 4HAQO also induced morphological mutants and nutrient-requiring auxotrophic mutants ( Okabayashi et al., 1964). Subsequent analyses of the mutagenic actions of 4NQ0 on various kinds of microbes and their mutants have provided a clearer understanding of the mechanisms of the mutagenic actions of 4NQO.

B. BASE-PAIR CHANGE MUTATIONS Molecular analyses of mutagenesis induced by 4NQ0 were initiated

by Ishizawa and Endo (1970) using three types of rII mutants of coliphage T4. After treatment of Escherichiu coli infected with T4 phage mutants with 4NQ0, they demonstrated reversion patterns of rII mutants, as indicated in Table I. In vivo treatment of T4 wild type phage with 4NQ0 induced remarkable GC + AT transitions, but did not induce AT + GC transitions or cause reversion of frameshift mutants induced by proflavine. I n Vivo treatment of T4 phage with 4NQ0 preferentially yielded rII mutants having AT base pair at mutant sites, and none of them reverted on treatment with 4NQ0 or proflavine (Ishizawa and Endo, 1971). Ishizawa and Endo (1972) also reported the induction of amber suppressor mutants by 4NQ0 and 4HAQO in Escherichia coli B9601 (trpBa,, his,,). These

134

MINAKO NAGAO AND TAKASHI SUGIMURA

TABLE I REVERSION OF THREE TYPESOF rII MUTANTS BY 4NQ0° ~~

~

~~

Reversion index in units, 10-6

rIT mutant

Probable alteration at mutation site

Without 4NQOb (Io)

With 4NQ0 (30 pg/ml) (1)

GC GC GC GC AT

0.08 1.0 0.05 0.16 0.01 0.04 0.2 0.07 0.02 0.37

25.2 292 21.5 33.2 0.06 0.18 0.15 0.2 0.03 0.92

AT AT AT Frameshif t Frameshift a

b

Factor of increase (I/ZO) 316 292 430 207 6

4.5 0.8 2.9 1.5 2.5

Data from Ishizawa and Endo (1970). 4-NQO, 4-nitroquinoline I-oxide.

prototrophic reversions were mainly based on GC + TA transversion (SupD). In vitro treatment of T4 phage with 4NQ0 did not induce any mutation, and metabolic activation of 4 N Q 0 seemed to be essential for its mutagenic activity ( Ishizawa and Endo, 1967). Recently, extensive studies on the mutagenic specificity of 4NQ0 were carried out by Sherman and collaborators. They used various kinds of structural gene mutants of iso-l-cytochrome c of Saccharomyces cereuisiae (Prakash et al., 1974; Prakash and Sherman, 1974). The use of these mutants allowed differentiation of intragenic reversion from extragenic reversion, since intragenic revertants produced large colonies on lactate agar plates, whereas extragenic revertants ( suppressor revertants) produced small colonies. These results are summarized in Table 11. It can be seen from these results that there are three distinct responses to the mutagenic action of 4NQO: high-frequency reversion, low-frequency reversion, and no reversion. The cycl-131 strain, which is an initiator tester strain with valine codon GUG mutation from the initiation codon was reverted with high frequency by GC + AT transition. Three different types of mutants showed a low-frequency response. Type 1 included the initiator mutants, cycl-51, cycl-100, and cycl-181, in which the responses all involved the mutant leucine codon UUG or CUG at the initiation codon. Type 2 was also an initiator mutant cycl-133 in which the

TABLE I1 CODONA N D BASE-PAIRCHANGES ASSOCIATED WITH REVERSION OF cycl TESTER STRAINSA N D THEIRREVERSION FREQUENCIES’S* Reversion frequency mRNA change Initiation mutant

Base-pair change G+ -A C T

GUG-+ AUG

T

MU-

4NQ0

Re-

tant

(1 pg/ml)

sponse

811

High

~ycl-131

A

U

cycl-51

C

C

81 102 CYC~-181 55

Low

cycl-133

67

LOW

‘cycl-13 cycl-74 cycl-85 ,cycl-163

1 0

{cycl-~oo

or

UG -+ AUG

A

6’5; G+ -T -

AGG ---t AUG

C

A

T A

G C

-4-

or C - + -G G C or A+ -G T C

U AUC -+ AUG A

Ochre mutant

A

T

T+A

and A G

amino acid UAA +

T-+ c

codons

and A C

T’G

1 1 0 0

No

0

5 4

All the above, plus G T

Amber mutant

amino acid

E-+X

codons

and G + -C C G

UAG --+

Frameshift mutant

cycl-2 cycl-9 cycl-45 cycl-72 cycl-94 cycl-140 cycl-156

No

2 16

UA ?-+ deletion, etc. AAAA + - base, etc.

LA deletion, AT etc. AAAA + - base TTTT pair, etc. ~

AA TT

+

+

base pair, etc.

CYC~-179 92

cyc 1-31

0

CYC~-183 16 ~y~l-239

LOW

No

1

Data from Prakash et al. (1974) and Prakaah and Shermann (1974). Revertants per lo7 survivors after treatment with 4-nitroquinoline 1-oxide (4NQ0).

136

MINAKO NAGAO AND TAKASHI SUGIMURA

response involved arginine codon AGG mutated from the initiation codon. Type 3 was an amber mutant which had an UAG codon and might be reverted by any kind of base change. GC +JTA transversion was considered to be the molecular mechanism of these low-frequency responses. Mutants showing no reversion included initiator mutants, which contained the isoleucine codon, AUU, AUC, or AUA, replacing the initiator codon, and the ochre (UAA) mutant. Three types of frameshift mutants containing an AT base pair also showed no reversion. Thus a G - C base pair at the mutation site is a common character of all strains which showed efficient reversion on treatment with 4NQO. Studies using mutated codons with completely known nucleotide sequences clearly supported this conclusion. It could not be decided from these experiments whether 4 N Q 0 induced GC + CG transversion, but it certainly induced GC + TA transversion. Other mutagens besides 4NQ0 also preferentially induce GC + AT transition. These are ethylmethane sulfonate, diethyl sulfate, N-methyl-N’-nitro-N-nitrosoguanidine(MNNG), nitrosoimidazolidone, nitrous acid, and p-propiolactone ( Prakash and Sherman, 1973). However, these chemicals did not produce significant GC transversion. Thus 4 N Q 0 is a unique mutagen in this respect.

C. FRAMESHIFT MUTATIONS

4NQ0 did not induce a frameshift revertant of Saccharomyces cerevisiae, as previously mentioned (Table 11). However, Hartman et al.

(1971) and Ames et al. (1973) demonstrated that it induced frameshift mutations in Salmonella typhimurium. Three histidine requiring frameshift tester strains were examined: TA1536 with an unknown lesion, 1 base pair insertion and TA1538 with TA1537 with :ggEgr :$$?Egg:+ 2. Of these, only TA1538 was reverted by 4NQO. TA1538 also showed a high rate of reversion with 2-nitrosofluorene (Ames et al., 1973). The failure of 4NQ0 to induce reversed frameshift mutants of Saccharonyces cerevisiae could be due to the different base compositions of the frameshift mutants that were examined.

+

D. DELETION MUTATIONS

4NQ0 induces deletion mutations, as does UV-light. Yamamoto and Ishii (1974) examined deletions of chromosomes of Eschen’chia coli including the colB (sensitivity to colicin B ) , tonB (receptor for phage T1) and trp genes. Deletion from the coZB to tonB locus was “short” and deletion from the colB to trp locus was “long.” When E. coli B

MOLECULAR BIOLOGY OF THE CARCINOGEN,

4NQO

137

( H / r 30 argFam) and its repair-deficient derivatives, uurA- (with a defect in excision repair), poEA- (with a defect in DNA polymerase I ) , and recA- (with a defect in the recoinbinational mechanism) were treated with 4NQ0, the induced deletion mutants were “short” type, as on UV treatment. On the other hand, in spontaneous deletions the numbers of “short” and “long” deletions were similar. Deletion mutation of the uurA- strain was about 30 times higher than that of the parental strain. Like UV-light 4NQ0 did not induce deletion mutation of the recA- strain. Thus the modes of action of 4NQ0 and UV-light are quite similar.

E. Mrromc GENECONVEFSIONS In S . cerevisiae, mitotic gene conversions are induced by various agents including UV light (Roman and Jacob, 1957), alkylating agents (Zimmermann, 1971) , and acridine ( Fahring, 1970). Zimmermann and Schwaier (1967) reported that nitrite, MNNG, and other alkylating agents induced 103-foldmore mitotic gene conversions than reverse mutations. The D,, D4, and D, strains, which were established by Zimmermann, have frequently been used in gene conversion studies. Fahring (1973) reported that 4NQ0 induced gene conversion in high frequency in the D4 strain, which is diploid and heteroallelic at the a&, and frp5 loci. After treatment with 2 p M 4NQ0 for 4 hours, 45 convertants at each of these loci were found per 5 X los survivors. The D3 strain is heteroallelic at the a&, locus, and it also showed mitotic gene conversions on treatment with 4NQ0 (V. Simmon, Stanford Research Institute, personal communication). The genetic mechanism of mitotic gene conversion was relatively well elucidated by Mortimer and Manney (1971), but the molecular events involved are not completely understood. It is generally accepted that mitotic gene conversion is an indicator of repair of damaged DNA.

F. Loss

OF THE

CYTOPLASMIC p FACTOR IN YEAST

The formation of the respiratory system of yeast is controlled by both a nuclear gene and a cytoplasmic self-duplicating genetic factor p. A respiration-deficient ( RD ) mutant could be induced by various chemical and physical agents. Agents like acriflavine (Slonimski and Ephrussi, 1949), ethidium bromide (Slonimski et al., 1968), dimethyl sulfoxide (Yee et al., 1972), pinacyanol (Sugimura et al., 1969), and 5-fluorouraciI (Moustacchi and Marcovich, 1963) were found to induce the cytoplasmic

138

MIN'AKO NAGAO AND TAKASHI SUGIMURA

RD mutant with extremely high efficiency, especially from growing yeast. Nitrous acid induced exclusively the nuclear RD mutant (Schwaier et al., 1968) , and UV light, nitrosoimidazolidone, and nitrosomethylurethane produced both nuclear and cytoplasmic RD mutants. According to Mifuchi et aL ( 1963), RD mutants from a diploid strain of S. cereuisiae, Hansen 0209,induced by 4NQ0 consisted of cytoplasmic and/or nuclear RDs. These nuclear RD mutants showed the spectra of various types of cytochromes (Morita and Mifuchi, 1970). However, Nagai (1969) reported that 4NQ0 specifically induced cytoplasmic RD mutants with high efficiency. Epstein and St. Pierre (1969) also reported that 4NQ0 induced RD mutants of S. cerevisiae, but they did not identify these as cytoplasmic or nuclear. In synchronized yeast cultures, the frequency of induction of RD mutants by 4NQ0 was closely correlated with mitochondriogenesis (Morita and Mifuchi, 1974). A cytoplasmic RD, the N-1 mutant, which was derived from the Hansen 0209 strain by 4NQ0, showed deletion of mitochondria1 DNA (Morita and Mifuchi, 1974).

Epstein and St. Pierre (1969) investigated the induction of RD mutants of S. cerevisiae by compounds related to 4NQO and 4-nitropyridine l-oxide (4NPO). They found that the concentrations of chemicals that were required to induce RD mutants were low with carcinogenic compounds but high with noncarcinogenic compounds. Thus there seems to be a correlation between mutagenicity and carcinogenicity. Many substances showed simultaneous mutagenic, carcinogenic, and photodynamic activities as well as activities for free-radical production and induction of phages from lysogenic bacteria (Table 111).

H. PHAGEINDUCTIONFROM LYSOGENIC BACTERIA Phage induction by a carcinogen was first observed by Lwoff ( 1953). Endo et al. reported that 4NQ0 induced the lysogenic h phage of E. coEi ( 1963).Treatment of lysogenic Salmonella typhirnurium with 4NQ0 or 4HAQO resulted in the induction of phage P22 (N. Yamamoto et al., 1970). The induction of phage by these compounds was far greater from hcr- lysogen than from the wild-type lysogen, and no phage-induction from the rec- lysogen was observed. This phage-induction pattern of 4NQ0 was also very similar to that of UV light (Yamamoto, 1969).

TABLE I11 4-NITROQUINOLINE I-OXIDE (4NQO) DERIVATIVES: MUTAGENICITY O N Saccharomyces cerevisiae; CARCINOGENICITY, PHOTODYNAMIC ACTIVITY,FREE-RADICAL FORMATION, A N D PHAGE INDUCTION FROM LYSOGENIC BACTERIA"

Compound*

Conc. producing 50% growth inhibition (GO) (pglml)

Conc. causing double the spontaneous mutation rate (2SMR) Gg/ml)

Mutagenic potency (2SMR-1)

4NQ0 8-CHr4NQO 7-CH3-4N QO 2-CHs4NQ0 6-CHr4NQO 7-C1-4NQ0 4NQ 4HAQO.HCl 7-CI-4HAQO.HC1 4 HAPO.HC1 3-CH3-4NQ0 4NP0 3NQ0 5NQ0 6-NOr4HAQO.HCI

0.07 0.06 0.12 0.07 0.23 0.48 14 26 > 100 > 100 > 100 > 100 > 100 > 100 >200

0.06 0.10 0.13 0.27 0.31 1.5 9.0 26 > 100 > 100 > 100 > 100 >100 > 100 > 100

16.7 10.0 7.69 3.70 3.23 0.67 0.11 0.04 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.005

x Carcinogenicity

+ + ++ ++ ++ + + +d

-

+

Photodynamic activity 11.1 2.4 7.1 2.9 1.3 1.1 1.7 < 0.1

< <

0.1

0.1

< 0.1 < 0.1 < 0.1 < <

0.1 0.1

Freeradical formation

+ + ++ + +-

NDc

+-

+-

Phase induction

+++

+++

+++ ++ ++ +++ ++ -

Data from Epstein and St. Pierre (1969). 3-, 4, 5NQ0, 3-, 4,5-nitroquinoline 1-oxide; 4HAQ0, ehydroxyaminoquinoline 1-oxide; 4-HAPO, 4hydroxyaminopyridine 1-oxide; 4-NPO, Pnitropyridine 1-oxide. c ND, no data available. d Modified by recent data of Kawaroe (personal communication).

ti m

g 8

U

.e

8 j!M

2m

!! z

0

"g e ro 0

a

+

8

140

MINAKO NAGAO AND TAKASHI SUGIMURA

111. Chromosome Aberrations

A. CHROMOSOME ABERRATIONSIN CELLS WITH NORMAL REPAIRFUNCI~ON Chromosome aberrations induced in uiuo by 4NQ0 was first reported by Yoshida et d. (1965). They injected 4NQ0 intraperitoneally into Wistar strain rats bearing Yoshida ascites sarcoma cells and found that the mitotic index of the cells decreased from 2.7 for controls to 0.3 after treatment. They classified the chromosome aberrations induced by 4NQ0 into three types: abnormal elongation of chromosomes, chromatid breaks, and chromatid exchanges. They observed abnormal elongation of chromosomes within 3 hours after the injection of 4NQ0, and chromatid breaks mainly from 4 to 12 hours after the injection. The frequency of chromatid breaks increased in proportion to the amount mole to mole. They of 4NQ0 injected in the range of 2 X observed chromatid exchanges 12-48 hours after the injection. Kurita et al. (1965) observed that the distribution of chromatid breaks among cells was not random. Chromosome aberrations of Yoshida sarcoma cells induced in uitro by 4NQ0 was also reported by Isaka (1970). Treatment with 4NQ0 also induced chromosome aberrations in cultured Syrian hamster cells (Stich and San, 1970), but no significant difference was noticed in the rates of induction of chromosome aberrations in growing and resting cells. Frequencies of chromatid breaks and chromatid exchanges induced by carcinogenic derivatives of 4NQ0 were higher than those by noncarcinogenic derivatives with cultured Yoshida sarcoma cells ( Isaka, 1975).

ABERRATIONSIN B. CHROMOSOME OF HUMAN CELLS

A

REPAIR-DEFICIENT STRAIN

Xeroderma pigmentosum (XP) is a hereditary disease associated with high UV sensitivity and a high incidence of skin cancer. Cultured cells from patients with severe manifestation of XP showed a markedly reduced capacity to repair UV-induced DNA lesions (Cleaver, 1968), but could correct DNA damage induced by X-rays (Cleaver, 1969). XP cells were suggested to be deficient in a specific endonuclease involved in DNA repair (Cleaver, 1971 ), or postreplication repair of DNA (Lehmann et al., 1975), and five complementation groups have been reported (Bootsma et al., 1975; Kraemer et al., 1975; Robbins et al., 1974). Fibroblast cells from a case of XP, which possessed only 20%of the repair capacity of control fibroblasts, were very sensitive to induction

MOLECULAR BIOLOGY OF THE CARCINOGEN,

4NQO

141

4NQO CONCENTRATION ( M 1

FIG. 1. Frequency of metaphase plates with chromosome aberrations of cultured XP cells ( 0 )and cells from normal person ( 0) exposed for 90 minutes to various concentrations of 4-nitroquinoline l-oxide (4NQO). From Stich et al. ( 1973).

of chromosome aberrations by 4NQ0 (Fig. 1 ) (Stich et al., 1973). The highest frequency of chromosome aberration in XP cells was observed 24 hours after treatment with 1 x lo-' M 4NQ0 for 90 minutes. In contrast there was no difference between chromosome aberrations of XP cells and of control cells to MNNG. The ratios of chromatid exchanges to chromatid breaks with and without 4NQ0 were also almost the same in control and XP cells. OF CHROMOSOMES C. ENDOREDWLICATION

4NQ0 induced diplochromosomes in Chinese hamster cells ( Sutou, 1973) in addition to chromosome aberrations (Fig. 2 ) . Diplochromosomes could be formed by endoreduplication, a process in which additional chromosome reproduction is concealed as normal chromosome reproduction. Diplochromosomes have been observed in various kinds of cells including mouse ascites tumor cells ( Levan and Hauschka, 1953), cultured human euploid fibroblasts ( Schwarzacher and Schnedl, 1965), human tumor cells (Ising and Levan, 1957), and hamster cells in primary culture (Schmid, 1966). Endoreduplication is a consequence of disturbed chromosome reproduction, Thus 4NQ0 seems to be able to induce disturbance of chromosome reproduction. The effective dose of 4NQ0 for this action on Chinese hamster cells was 0.5 pglml, and its effective dose range was narrow. The highest number of diplochromosomes was observed 27 hours after 4NQ0 treatment. In the diplochromosomes induced by 4NQ0 the predominant aberrations were gaps and breaks of chromatid type (Fig. 2 ) . Sutou (1973) suggested that the interaction of a 4NQ0 metabolite with proteins in the structure of chromosomes

142

MINAJLO NACAO AND TAKASHI SUGIMURA

FIG.2. Induction of endoreduplication by 4-nitroquinoline 1-oxide (4NQO). Chinese hamster cells were treated with 0.5 pg of 4NQ0 per milliliter for 4 hours. Chromosome specimens were prepared 18 hours after the treatment. ( a ) Ordinary metaphase chromosome with aberrations. ( b ) Diplochromosomes caused by endoreduplication with aberrations. Kindly prepared by Sutou.

MOLECULAR BIOLOGY OF THE

CARCINOGEN, 4NQO

143

might be important in their formation and that the resulting disturbance of chromosome reproduction might lead to formation of diplochromosomes. IV. Repair of 4-Nitroquinoline 1 -0xide-Damaged DNA

Like most other chemical carcinogens (Ames et al., 1973) 4NQ0 causes DNA damage, as described later. Most cells have the capacity to repair DNA damaged by UV light or chemical agents. Three types of repair processes to eliminate modified DNA are known. (1) Photoreactivation. The photoreactivating enzyme acts on UV-induced pyrimidine dimers of DNA under illumination at 300-600 nm, its e6ciency being maximal at around 400 nm. This process has been detected in various species (Cook and McGrath, 1967; Sutherland, 1974; Sutherland et al., 1974). ( 2) Excision-repair. This repair process involves many enzymatic steps. The damaged part of the DNA strand is first excised by endonuclease and exonuclease. The resulting gap is then filled by the process of repair replication involving DNA polymerase and ligase. This process has been shown to be error free (Witkin, 1969a). (3) Postreplication repair. In E . coli, this process is thought to involve recombination repair. Semiconservative DNA synthesis proceeds with gaps facing the sites of damage of the parental strands of DNA. These gaps are filled by recombinational exchange ( Rupp et al., 1971). This recombinational process is error prone (Bridges, 1969; Witkin, 1969b). Similar processes have been reported in Chinese hamster cells and mouse lymphoma cells by Cleaver and Thomas (1969) and Rupp et al. ( 1970). However, Lehman (1972) proposed that postreplication repair in mouse lymphoma cells does not involve recombinational exchange. At present, the postreplicational repair process has not been elucidated precisely in mammalian cells and even in E . c o k Quite recently, the induction of postreplication repair enzyme( s ) after UV irradiation was reported ( Witkin, 1974; Gudas and Pardee, 1975; Sedgwick, 1975). Excision repair and postreplication repair are termed dark repair. Kondo (1974b) proposed the use of the term tolerance repair instead of postreplication repair, and Radman (1975) proposed the term SOS repair. Recent advance on DNA repair mechanism was reviewed by Kondo ( 1975). DNA repair processes play important roles in mutation and possibly also in carcinogenesis (Trosko and Chu, 1975).

A. REPAIRIN BACTERIA In Bacillus subtilis hcr (defective in host-cell reactivation, that is, in endonuclease for pyrimidine dimer induced by UV light) or rec-

144

MINAKO NACAO AND TAKASHI SUCIMURA

( defective in the recombination process) mutants were more sensitive to the lethal effects of 4NQ0 and UV light than were the wild strains (Felkner and Kadlubar, 1968; Tanooka and Takahashi, 1972; Kada et al., 1972). From extensive studies, Kondo and his co-workers (1970) concluded that the molecular mechanisms of base-change mutagenesis and the phage-inducing ability of 4NQ0 in E . coli are very similar to those of the effects of UV light. Experiments using various kinds of repair-deficient mutants including, inc- (uurA-, urvB-; defective in endonuclease for pyrimidine dimer), polA- (defective in DNA polymerase I ) and recA-, showed that the mechanism of repair of 4NQO-damaged DNA was similar to that for repair of UV-damaged DNA. The recAmutant of E . coli was more sensitive to the lethal action of 4NQ0, but the frequency of mutation in survivors was very low after treatment with UV light or 4NQO. On the other hand, the inc- mutant was extremely sensitive to the lethal effect of 4NQ0, and the frequency of mutation in survivors was very high. This indicates that excision repair plays a major role in rescue from the lethal action of 4NQ0, while mutation induced by 4NQ0 results from recombinational repair processes-that is, complicated recA gene-dependent factors result in formation of mutated base sequences from 4NQO-induced lesions of DNA (Kondo, 1973). One hundred pyrimidine dimers per genome of the E . coli uvrA- strain resulted in 37% survival and two hundred 4NQOpurine adducts had an equivalent effect ( Ikenaga et al., 1975a). Kondo et al. (1970) also reported that photoreactivation treatment of E . coli did not reduce the lethal or mutagenic effects of 4NQ0, whereas the same photoreactivation treatment markedly reduced the lethal and mutagenic effects of UV irradiation. N. Yamamoto et al. (1970) isolated 5 mutants of S . typhimudum that were sensitive to 4NQ0 by treatment with MNNG. Four of them were hcr mutants, and one was a rec- mutant. They were all sensitive to UV light. Five mutants that were sensitive to UV light were also obtained, and these were all hcr mutants. Four of them were 4NQOsensitive and one was resistant to 4NQO. The resistant one was a double mutant of hcr- with deficiency in the enzyme converting 4NQO to an active intermediate. More recently, a UV-sensitive mutant, uurl2 of Salmonella abony, which showed the normal response to 4NQ0 was isolated by Skavronskaya et al. (1973). This mutant had normal photoreactivating ability. Mutant uurl2 had a different mutation site from uurB2, and the affected gene was required for repair of UV-damaged DNA, but not for repair of 4NQO-damaged DNA. The step of enzymic excision and its genetic control are not well understood at present (Skavronskaya et al., 1974).

MOLECULAR BIOLOGY OF THE CARCINOGEN, ~ N Q O

145

It seems probable that the mechanism of this step is more complicated than is now generally thought. Micrococcus radiodurans is very resistant to the lethal effect of UVradiation. For example, the l / e value (i.e., the dose causing an average of one lethal event per bacterium) of the wild type of M. radiodurans is 1890 ergs/mm2, whereas the value for E . coli B/r (the most UV-resistant strain of E. coli) is 470 ergs/mmz (Witkin, 1966). The extreme radiation resistance of M. rudiodurans is due to its very efficient dark repair of UV-induced damage, which is known to involve both excision and recombination mechanisms. The recombination repair of damaged DNA seems to be important in radiation,resistance. A temperature-sensitive mutant with regard to rcombination capacity was also obtained. It was extremely UV resistant at the permissive temperature, but UV sensitive at the restrictive temperature, and it was found that its genetic recombination ability decreased at the restrictive temperature ( Moseley et al., 1972). Sweet and Moseley (1974) reported that 14,850 ergs/mm2 of UV light which reduced its survival to 5%, caused no detectable increase in the frequency of mutants, Exposure of M. rudiodurans wild type to 50 pg of 4NQ0 per milliliter for 4 hours had no effect on either the viability or the production of mutants. Exposure of E. coli B/r 2/1 to a similar concentration of 4NQ0 resulted in only 0.1%viability with production of many mutants. Treatment of M. radiodurans with 100 pg/ml of MNN'G for 40 minutes resulted in 70%survival and increase in the mutation frequency to 7.5 times the rate of spontaneous mutation, but UV irradiation that resulted in 70%survival had no effect on the production of mutants. Sweet and Moseley (1974) suggested that these phenomena were due to an unusual accuracy of recombination repair of UV-damaged DNA in M. radwdurans, unlike the case in E . coli.

B.

REPAIR IN YEAST

In the eukaryotic cells of S. cerevisiae, repair mechanisms are more complicated than in E . coli (Nakai and Mortimer, 1969; Kobalizoba, 1973). UV-sensitive mutants, UV," (radl-1) and UV," (rad2-1), have been isolated which are defective in error-free excision repair mechanisms (Nakai and Matsumoto, 1967; Hunnable and Cox, 1971). The rudl gene is concerned with repair of damaged nuclear DNA and the rud2 gene with repair of damaged nuclear and mitochondria1 DNA (Moustacchi, 1971; Bucking-Throm et al., 1973). Twenty-one cistrons were found to be related to the process of repair of damaged DNA in yeast. Nagao and Sugimura (1972) found that UV,* and UV," mutants were also very sensitive to the lethal effects of 4NQ0, 4HAQ0, 3-methyl-4NP0, and

146

MINAKO NAGAO AND TAKASHI SUGIMURA

2,3-dimethyl-4NPO. A double mutant UVISXx,"was more sensitive to 4NQ0 than the single mutant ( Koske and Stich, 1973).

C. REPAIRIN BACTERIOPHAGES T4 Phage is known to be more resistant to UV irradiation than T2 and T6 phages. Harm (1963) showed that this resistance was due to the u gene of T4 phage and that this gene was not present in T2 and T6 phages. The u gcne codes cndonuclease, which specifically excises pyrimidine dimers produced by UV irradiation (Sekiguchi et al., 1970; Freidberg and King, 1971; Freidberg and Clayton, 1972). Infection of UV-irradiated E. coli Bs-l with UV-inactivated phage T ~ u rescued + a certain fraction of the host cells. However, u gene-mediated rescue was not observed after damage by 4NQ0 or other chemicals. Purified T4 endonuclease degraded UV-irradiated DNA in uitro but did not attack DNA modified by 4NQ0, mitomycin c, or nitrogen mustard ( Freidberg, 1972). D. REPAIR IX PLANTCELLS No repair replication was detected in Vicia faba after treatment with 4NQ0 ( Wolff and Cleaver, 1973). Moreover, exposure of V. faba root tips to 4NQ0 (5 x 1@+it$) solution for 1 hour did not induce repair replication, while 4NQ0 decreased 3H-labeled bromodeoxyuridine incorporation into DNA in semiconservative replication indicating uptake of 4NQ0 into cells. In Chlamydomonas ( Swinton and Hanawalt, 1973), Haplopappus, and Nicotiana (Trosko and Mansour, 1968), excision repair of UV damage was not detected. Thus it seems possible that plants in general may lack this excision repair process. The photoreactivation system was demonstrated in Haplopappus, h7icotiana, and other higher plants ( Trosko and Mansour, 1968). In the alga Eudrina elegans, excision-repair synthesis did not function on 4NQO-damaged DNA, but the error-prone postreplication repair system did (Kemp and Malloy, 1973). This organism also has a photorepair system (Kemp, 1972).

E. REPAIR IN

MAMMALIAN

CELLS

1. Excision Repair In many types of mammalian cells, DNA repair synthesis, namely, unscheduled DNA synthesis, occurs after exposure of cells to UV light

MOLECULAR BIOLOGY OF THE

CARCINOGEN,

~NQO

147

(Rasmussen and Painter, 1966; Regan et al., 1968; Evans and Norman, 1968) or chemical agents (Roberts et al., 1968; Hahn et al., 1968). Tada et al. (1970) reported that ascites hepatoma AH130 cells from rats injected with 4HAQO intraperitoneally contained DNA with a lower molecular weight under denatured conditions. In the native state, the DNAs from treated and untreated cells had similar molecular weights of 8 X lo6 and 11x loG,respectively, while in the single-stranded form the molecular weights of DNAs from treated and untreated cells were 1.4 X lo6 and 5.1 x loG,respectively. This change might be produced by a repair process, although the possibility that the breaks were induced chemically by 4HAQ0, as described later, could not be completely ruled out. Horikawa et al. (1970) reported that 4NQ0 treatment of cultured Ehrlich ascites cells induced single-strand scissions of cellular DNA and concomitant DNA repair synthesis. The DNA in cells pretreated with 4NQ0 decreased in size, but it returned to the same size as that in untreated cells on incubation without 4NQ0 for 3050 minutes. This indicates that, in Ehrlich ascites cells, DNA modified by 4NQ0 is mended by an excision repair process. Double-strand scissions of DNA resulting in change in the size of DNA to 130 S were found in cultured mouse fibroblast cells L-P3 (substrain of L-929) and mouse mammary tumor cells, FM3A, after treatment with M and M 4NQ0, respectively (Andoh and Ide, 1972). Double-strand breaks of DNA were repaired on further incubation of treated cells at 37°C without 4NQ0, and repair was complete in 24 hours (Ide and Andoh, 1972). Stich and San (1970) reported that Syrian hamster cells in the GI phase showed DNA repair synthesis after exposure to 4NQO. Almost all the cells incorporated [3H]-labeled thymidine (TdR) into the acidinsoluble fraction after exposure to 4 X M 4NQ0 for 1.5 hours. The capacity for DNA repair synthesis following 4NQ0 treatment appears to be a common feature of many types of mammalian cells, as shown in Table IV. Stich et al. (1971) investigated the induction of unscheduled DNA synthesis in Syrian hamster cells after exposure to carcinogenic and noncarcinogenic derivatives of 4NQO. A good correlation was obtained between carcinogenicity and the capacity to induce DNA repair synthesis. On incubation with [3H]TdR, the average number of grains per nucleus depended on the time and the concentration of 4NQ0 to which the cells were exposed. In the case of human lymphoM 4NQ0 was sufficient to induce DNA repair synthesis cytes, 5 X M 4NQ0 caused maximal [3H]TdR incorporation (Jacobs et and aZ., 1972a). In phytohemagglutinin-M stimulated lymphocytes, DNA

MINAICO NAGAO AND TAKASHI SUGIMURA

148

TABLE IV [ a H ] T (TdR) ~ ~ INTO ~ ~ NUCLEI ~ ~O F ~HAMSTER ~ AND HUMANCELLS ARRESTED BY ARGINIXE DEPRIVATION A N D T R E A T E D WITH 4NQ0“ ~NCORPORATI~N OF

Labeled nuclei (%) Cells Syrian hamster Fibroblasts (young) Fibroblasts (aged) Myoblasts Heart cells Fortner tumor cells B H K 2 l cells Human embryo Fibroblasts (diploid) Fibroblasts (aged) Fibroblasts (triploid’) Fihroblasts (abnormale) Fibroblasts (infected’)

Grain countsd

Control*

4NQOC

Control

4NQ0

0.9 0.0 0.5 0,o 1.6 1.1

100 96 100 100 100 100

100 112 101 90 84 98

0.1 0.0 0.2 0.6 0.6

100 94 100 100 98

115 122 183 109 84

Data from Stich and San (1970). Cell cultures exposed to a [SH]TdRpulse of 1.5 hours, but not to 4NQ0. Cell cultures exposed simultaneously for 1.5 hours to 4 X 1 0 P M 4NQ0 and to [aH]TdR. d Average number of grains per diploid nucleus of embryonal Syrian hamster M, 1.5 hours) was taken as 100%. The few fibroblasts exposed to 4 N Q 0 (4 X heavily labeled nuclei seen in the S phase were excluded from the counts. e Fibroblast cultures from spontaneously aborted embryos with abnormal karyotypes. The last sample in the table consisted of cells shedding rubella virus. a

repair synthesis was also induced by 4NQ0 (Jacobs et al., 1972a). Like UV irradiation, 4NQ0 induced DNA repair synthesis in nuclei in the GI, G2,and S phases and also in metaphase chromosomes (Stich and San, 1970). Recently, the very interesting phenomenon of a “refractive period” was reported by Warren and Stich (1975). Cultured human fibroblasts were first treated with 5 x M 4NQ0 for 60 minutes. Then, after intervals of 0.5-12 hours, they were reexposed to 4NQ0 at the same concentration for 60 minutes. Two-hour [3H]TdR pulse labeling was carried out to measure DNA repair synthesis immediately after the second exposure to 4NQO. The expected level of unscheduled [3H]TdR incorporation following the second exposure should be equivalent to the sum of the repair synthesis still proceeding after the first exposure, plus the Ievel of repair synthesis observed immediately after a single exposure for 60 minutes. It was found that if the second exposure was

MOLECULAR BIOLOGY OF THE CARCINOGEN,

4NQO

149

within 30 minutes of the first, the level of DNA repair synthesis was far below the expected value, and the second exposure seemed to cause only a very small increase in repair synthesis. However, after a “refractive period,” the level of repair gradually increased to the expected level. This “refractive period was about 4 hours, and DNA repair synthesis due to the first exposure to 4NQ0 was quite high in this period. A second exposure to 4NQ0 during this period resulted in decreased cell survival and more chromosome aberrations. For instance, a single exposure to 4NQ0 reduced the cell survival to 71%,and the expected value after two exposures was SO%,whereas in fact it was only 5%. Analysis of DNA repair replication with excision repair at a molecular level elucidated the patch size of nucleotide replacement ( Regan and Setlow, 1973, 1974). From the patch size in cultured human fibroblasts, repair was classified into two categories, ionizing-type repair and UVtype repair. Ionizing-type repair was achieved by insertion of a few nucleotides and involved DNA polymerase and DNA ligase. This ionizing or “short p a t c h type of repair is completed within 30-60 minutes after the original stimulus. UV-type repair was achieved by extensive excision of bases, and 100 nucleotides were inserted over a period of 20 hours. Chemical agents that modify DNA could also be classified into two categories on the basis of the type of repair they induced. 4NQ0 induced both types of repair replication in normal human cells (Regan and Setlow, 1974). Observation of unscheduled DNA synthesis resulting from excision repair in mammalian cells is useful as a prescreening method for chemical carcinogens ( Regan and Setlow, 1973; Stich, 1975; A. Mitchell, personal communication).

2. Postreplication Repair Mouse leukemia cells, L5178Y, show low activity of excision (Fox and Fox, 1973) and repair replication (Painter and Cleaver, 1969). Moreover, they lack recombination repair (Lehman, 1972). Makino and Okada (1974) investigated the effects of exposure of these cells to 2 x M 4NQ0 for 20 minutes. They found that this decreased incorporation of [3H]TdR into the acid-insoluble fraction in DNA replication by 40%. Alkaline sucrose density gradient centrifugation of DNA labeled for 20 minutes from those cells revealed the presence of small segments of DNA. These segments of less than 5 to 6 X lo7 daltons were products of newly initiated replication during the labeling period. Elongation of these newly synthesized short segments was slower than in untreated cells. After a chase period of 2 hours, the 20-minute labeling pattern of DNA resembled that of the bulk of the DNA. This postreplication

150

MINAKO NAGAO AND TAKASHI SUGIMURA

repair observed after 4NQ0 treatment was similar to that found after UV irradiation by Lehman ( 1972). In postreplication repair, normal replication proceeds with gaps opposite “damage” induced by some agent in the parental DNA strands. Then gap-filling replication occurs without a recombinational process. The size of the repaired gaps was estimated to be 800 nucleotides (Lehman, 1972).

3. Repait-Deficient Strain of Human Cells Stich and San (1971) reported that after treatment with 4NQ0 cultured XP fibroblastic cells showed less DNA repair synthesis than cultured normal skin fibroblasts. This finding is very similar to results on UV-sensitive mutants of bacteria. The colony-forming ability of the XP-fibroblastic cells was also more sensitive to 4NQ0 and to UV light than that of normal human cells, as illustrated in Fig. 3 (Takebe ef d.,1972). When XP cells which had been transformed with SV40 virus, were incubated with [ 3H]4NQ0, the radioactivity was incorporated into cellular DNA. During further incubation without 4NQ0 of XP cells and normal cells after 4NQ0 treatment, the radioactivity in DNA was efficiently removed from the latter, but not from the XP cells (Ikenaga et al., 1975b). Similar results were obtained on 4NQ0 treatment of the wild strain and uorA- strain of E. coli (Ikenaga et d.,1975a) as described later. Lymphocytes from patients with XP also showed much less DNA repair synthesis after treatment with 4NQ0 than lymphocytes from healthy subjects (Jacobs et al., 197%). Regan and Setlow (1974) reported that, in repair replication induced

I

2

3 4

5 6 7

4NW CO”TWATl0N (xIO-? I h)

FIG.3. Fraction of colony formers plotted against concentration of 4-nitroquinoline 1-oxide (4NQO) (exposure for 1 hour) or UV dose. Normal cells: 0-0, 4NQO; 0--0 - ,UV. Xeroderma cells: 0---0,4NQO; 0- - - 0, UV.

MOLECULAR BIOLOGY OF THE

CARCINOGEN, ~NQO

151

by 4NQ0, fibroblastic cells from XP patients were defective in “longpatch repair, but showed similar ability to perform “short-patch” type repair to normal fibroblast cells. Fibroblasts from human cases of Fanconi’s anemia, which are thought to be defective in a specific exonuclease, showed the normal activity of DNA repair synthesis after treatment with 4NQ0 (Poon et al., 1974). X P cells also showed very low photoreactivating activity (Sutherland et al., 1975).

V.

Interaction of 4-Nitroquinoline 1 -Oxide and Its Derivatives with Nucleic Acids

4NQ0 is highly mutagenic, as described in the preceding section. Thus since it is generally accepted that carcinogenicity is closely related to mutagenicity, the interaction of 4NQO and its metabolites with DNA has attracted much attention. 4NQ0 and its metabolites have been found to cause three main alterations of DNA. These are the intercalation of quinoline in base pairs of DNA, the formation of covalently bound adducts between quinoline and bases, and strand scissions of DNA. Here attention is focused on adduct formation, which has been elucidated most recently. Strand scission of DNA induced by chemical reaction of 4HAQO is briefly reviewed in this article, and the production of strand scission in uiuo has been described in Section IV. Intercalation was reviewed comprehensively in a previous monograph on 4NQ0 by Nagata ( 1971).

A. In Vivo FORMATION OF QUINOLINE-BASE ADDUCTS When 4NQ0 or its derivatives were injected intraperitoneally into Donryu strain rats bearing Yoshida ascites hepatoma AH130 cells, quinoline bound covalently to DNA of the cells, as demonstrated fluorometrically. This was demonstrated with 4NQ0 and 6-chloro-4NQ0, both carcinogens, but not with 4AQ0 or 3-methyl-4NQ0 which are noncarcinogenic or only weakly carcinogenic. A fluorescent compound formed from 4NQ0 was found even in single-stranded DNA obtained by heating and rapid cooling of double-stranded DNA from cells treated with 4NQ0 (Matsushima et al., 1967; Tada et al., 1967). Ikegami et al. ( 1969/1970) injected [14C]4NQ0 intraperitoneally into rats bearing AH130 cells. Then they extracted the DNA from the AH130 cells and subjected it to cesium chloride equilibrium density gradient centrifugation under neutral and alkaline conditions. They found that the 14C radioactivity sedimented concomitantly with the peak of DNA. They removed the quinoline from the DNA by depurination at pH 1.6

152

MINAKO NAGAO AND TAICASHI SUGIMURA

and 37°C. They suggested that some metabolite of 4NQ0 interacted with the purine bases of DNA. Formation of quinoline-purine adducts were also elucidated in detail by Tada and Tada (1971) in studies i,n uivo using AH130 cells and [ H3]4NQ0.

B. ENZYMICACTIVATIONSTEPS REQUIRJSDFOR MODIFICATION OF DNA BY 4-NITnOQUINOLINE1-OXIDE 4NQ0 is first reduced to 4HAQO by soluble and microsomal oxidoreductases with oxidation of NADH or NADPH, as described in the review of Matsushima and Sugimura (1971). The enzymatic activation of 4HAQO was first discovered by Tada and Tada (1972). They found that the binding of ["]4HAQO to DNA and RNA in uitro was very greatly stimulated by the additions of the 105,000 g supernatant fraction of Yoshida ascites hepatoma cells AH130 and ATP and Mg.2+This in uitro reaction was not stimulated by adding 3'-phosphoadenosine 5'-phosphosulfate, SO,", CoA, and CH,COO-. Boiled supernatant showed no activity. The enzyme in the cytosof of AH130 cells was purified and identified as seryl-tRNA synthetase. The purified enzyme required L-serine, ATP, and Mg" and a sulfhydryl for binding of [3H]4HAQ0 to DNA (Tada and Tada, 1974). Prolyl-tRNA synthetase, obtained from ascites cells in the presence of L-proline and ATP, was also active in production of quinoline-nucleotide adducts. In the case of baker's yeast, only seryl-tRNA synthetase was effective whereas in the case of E . coli, both phenylalanyl- and seryl-tRNA synthetases were effective. Poly( A ) and poly( G ) were suitable acceptors (Tada et al., 1974). 4HAQO was activated by the reaction with serylAMP on the surface of seryl-tRNA synthetase, probably owing to formation of seryl4HAQ0, although the latter was not isolated. This seryl4HAQO was considered to react with purine bases, but not with pyrimidines. tRNA,, served as an acceptor of the seryl residue from seryl-AMP on the enzyme, but 4HAQO could replace tRNA,,, as acceptor of the aminoacyl residue. A synthetic seryl-AMP was also effective for the binding of L3H]4HAQ0 to nucleic acid without enzyme. In this type of pure chemical reaction, other aminoacyl-AMPS besides L-seryl-AMP were effective (Tada and Tada, 1975).

C. BIOLOGICAL A m m OF MOD IF^ DNA The transforming activity of Bacillus subtilis DNA was lost on incubation with 4HAQ0, even in the presence of 0.2 M dimethyl sulfoxide as a radical scavenger (see Section V,E ), if the incubation mixture con-

MOLECULAR BIOLOGY OF THE

CARCINOGEN, 4NQO

153

tained L-serine, seryl-tRNA synthetase, ATP, and Mg2+.Thus enzymically modified DNA could be repaired by host-cell reactivating enzymes of B . subtilis (hcr gene products), while damage of DNA produced chemically by 4HAQO in the absence of a radical scavenger could not (Tanooka et al., 1975). It is evident that the mechanism of loss of transforming activity caused by metabolically activated 4HAQO was different from the DNA strand scissions caused by the radical reaction of 4HAQO. The hcr strain of B . subtilis was 10-fold more sensitive to the lethal action of 4HAQO than the hcr+ strain. Transforming DNA from B . subtilis hcr- strain, treated in vivo with 4HAQO was more effectively repaired in recipients of the her+ strain than in those of the h c r strain (Ishii and Kondo, 1971; Tanooka and Takahashi, 1972). Tada et al. (1970) examined the template activity of modified DNA for DNA-dependent RNA polymerase highly purified from E . coli. The template activities of DNA isolated from 4HAQO-treated cells was decreased with increase in the amount of quinoline adducts on DNA strands. Incorporation of -p32P-nucleotidetriphosphate into RNA with modified DNA template were increased, indicating the initiation site or initiation frequency of RNA synthesis was increased with modified DNA. RNA chains were initiated almost exclusively by purine nucleoside triphosphates. Chain length of RNA synthesized with DNA from 4HAQO-treated cells was 320 nucleotides in contrast to 2200 nucleotides with DNA from nontreated cells. D. CHEMICAL STRUCTURES OF QUINOLINE-PURINE ADDUCTS The [ 3H]4HAQO-nucleicacid complex produced by a cell-free system in vitro, was hydrolyzed by treatment with acid or alkali. The adducts of the quinoline base from DNA of ascites cells treated with [14C]4NQ0 in vivo and the adducts of the quinoline base from DNA treated with 4HAQO in vitro behaved quite similarly on paper chromatography (Tada and Tads, 1972). Poly(A) and poly(G) were treated with [3H]4HAQ0 in the presence of activating enzyme system from baker’s yeast (Tada and Tada 1974). The products were hydrolyzed by treatment with trifluoroacetic acid at 165°C for 60 minutes, and the hydrolyzates were separated by paper chromatography. Three labeled compounds were isolated after treatment of poly(G) with [3H]4HAQ0. Two of these are guaninequinoline adducts, and the other is 4AQ0, which is thought to be liberated from an unstable adduct of guanine and quinoline by acid hydrolysis. In contrast, only a single product, quinoline-adenine adduct ( QA) was detected after treatment of poly( A ) with [3H]4HAQ0. When DNA

154

MINAKO NAGAO AND TAKASHI SUGIMURA

from E . coli cells had been treated with t3H]4NQ0, four radioactive compounds were separated by paper chromatography after hydrolysis. They corresponded to two kinds of guanine derivatives, 4AQ0, and one adenine derivative. These results are in agreement with those of in oitro experiments (Ikenaga et al., 1975a). Escherichia coli wild-strain H/r30 was capable of excising three purine-quinoline adducts from DNA, but the uvrA- strain Hs30R could not excise the quinoline adduct, as shown in Table V (Ikenaga et al., 1975, ) . Quite recently, the structure of the QA was elucidated by M. Araki et al. (personal communication). They obtained purified QA in a yield of 1.3 mg from 200 mg of poly( A ) treated with 4HAQO in the presence of activating enzyme system after hydrolysis with 1 N HCl at 100°C. The UV spectrum of QA was very similar to that of 3-substituted 4-aminoquinoline 1-oxide, and the nuclear magnetic resonance ( NMR ) spectrum showed that QA was the 3-substituted quinoline 1-oxide derivative and that the quinoline group was bound to the N atom of adenine. On the basis of these results, Araki et al. consider that the most plausible structure of QA is 3-( N-6-adenyl)-4-aminoquinoline1oxide ( Fig. 4 ) although the structure 3- (N-l-adenyl)-4-aminoquinoline 1-oxide is not completely ruled out. Mass spectroanalysis of the adduct supported the structure of QA thus proposed, 4-Acetoxyaminoquinoline 1-oxide has very high reactivity and yields the adenine quinoline adduct upon incubation with poly( A). 4-Acetoxyaminoquinoline 1-oxide, which is very unstable, was prepared from diacetvl-4HAQO by adding equimolar amounts of dithiothreitol in dimethyl sulfoxide solution. After hydrolysis of this poly ( A ) -quinoline

R m m s F : OF

TABLE V QUINOLINE-PURIXE ADDUCTSFROM DNA. 4NQ0 adduct (cpm/mg DNA)

Escherichia coli strain H/r30 Hs30R

a

Incubation time (min) 0 60 0 60

Data from Ikenaga et al. (19758).

Guanine adduct 1

2

Adenine adduct

4AQO

428 65 745 74 1

182 23 15.5 179

215 17 330 334

150 22 482 348

MOLECULAR BIOLOGY OF THE CARCINOGEN,

H

4NQO

155

1

0 0

b

FIG. 4. Plausible structures of quinoline-adenine adduct. ( a ) 3-( N-6-Adenyl ) -4aminoquinoline l-oxide; ( b ) 3- ( N-l-adenyl) -4-aminoquinoline l-oxide.

adduct only a single spot of quinoline-adenine product was obtained by paper chromatography. The adenine-quinoline adduct obtained had the same physicochemical properties, UV absorption, mass spectrum, and NMR spectrum as QA from 4HAQO-treated poly( A) with the enzyme system ( M. Araki, personal communication).

E. CHEMICAL INTERACrION OF 4NITROQUINOLINE 1-oXIDE AND ITS DERIVATIVES WITH DNA 4NQ0 and its derivatives also interact with double-stranded DNA in vitro. Conformational studies indicated that in this interaction the quinoline ring is oriented in parallel to the base-pair planes (Nagata et al., 1966) while the 4-nitro group is at an angle of approximately 50" to the plane of the quinoline ring (Paul et al., 1971). The interaction depends upon the ionic strength, indicating that the charge site of quinoline compounds may be involved (Paul, 1970). UV-flow dichroism studies using apurinic acid or apyrimidinic acid showed that the primary site of interaction involved purine residues (Nagata et al., 1968). Kawazoe et al. (1972) also studied this binding using 3H-labeled quinoline derivatives. A weakly carcinogenic pyridine derivative, 4NP0, did not interact with DNA in vitro (Okano and Uekama, 1967). Direct evidence of the in uitro interaction of 4HAQO with DNA was first obtained by Ono (1964). He showed that transforming DNA of B. subtilis was inactivated in vitro by incubation with 4HAQ0, but not by incubation with 4NQO. This was later confirmed by Tanooka et al. (1969). The T4 phage of E. coli (Ishizawa and Endo, 1967) and the P22 phage of S . typhimurium (Yamamoto et al., 1970a) were also inactivated by in vitro treatment with 4HAQ0, but not with 4NQO. Sugimura et al. (1968) reported that 4HAQO caused single-strand scissions of DNA on in vitro incubation, 4NQ0 or 4AQO was inactive.

156

MINAKO NAGAO AND TAKASHI SUGIMURA

Ishii and Kondo (1971) and Tanooka and Takahashi (1972) investigated the mechanism of inactivation of transforming DNA of B. subtilis by 4HAQO in uitro. This inactivation of transforming DNA required aerobic conditions (On0 et al., 1967) and 4HAQO readily produced free radicals in the presence of oxygen. The radical forms of 4HAQ0, R-N-OH or R-NH-0, were proved. The active component is considered to be HOO or HO., which is simultaneously produced (C. Nagata, personal communication ) . The inactivating effect of 4HAQO on transforming DNA was effectively prevented by 0.1 M methyl, ethyl, isopropyl, or butyl alcohol, dioxane, dimethyl sulfoxide, sodium thiosulfate, or mercaptoethylamine (Tanooka et al., 1969). The protection may be due to the scavenging or quenching of the active reactants produced from 4HAQO. The bacteriophages T4 and P22 were also inactivated in uitro by treatment with 4HAQ0, as mentioned above. The damaged DNA in these phages could not be repaired by host DNA-repairing enzymes (Ishizawa and Endo, 1967; N. Yamamoto et al., 1970). Also damage of transforming DNA produced in uitro by 4HAQO could not be repaired by host DNA-repairing enzymes (Ishii and Kondo, 1971; Tanooka and Takahashi, 1972 ).

VI. Interaction of 4-Nitroquinoline 1 -Oxide and Its Derivatives with Protein

4NQ0 itself binds covalently to sulfhydryl groups with release of nitrous acid (Endo, 1958; Nakahara and Fukuoka, 1959). When cultures of the mouse fibroblast line, L-P3 were incubated with M E3H]4NQ0 for 30 minutes, the 3H radioactivity was incorporated into the cellular protein fraction. The radioactivity in the protein fraction was more rapidly released from the cells than was that in the nucleic acid fraction. Radioactive metabolites of 4NQ0 bind to many soluble proteins (Andoh et al., 1971). The activating system, which is effective for modification of nucleic acid by 4NQ0, may also be effective for modikation of protein by this chemical (Tada and Tada, 1972). In the presence of a catalytic amount of 4HAQ0, reduced glutathione was oxidized by oxygen. Similarly, oxidation of glutathione with concomitant reduction of cytochrome c was noticed, and this was found to be due to repeated oxidoreduction of the catalytic amount of 4HAQO and its radical form (Hozumi, 1968; Hozumi et al., 1967; Matsushima et al., 1968; Matsushima and Sugimura, 1971).

MOLECULAR BIOLOGY OF THE

CARCINOGEN, 4NQo

157

VII. Recent Information on Carcinogenesis by 4-Nitroquinoline 1 -Oxide

The carcinogenicity of 4NQ0 was first established in 1957 by Nakahara, Fukuoka, and Sugimura. Reports on the carcinogenicities of this compound and its derivatives up to 1967 were summarized in “Chemistry and Biological Actions of 4-Nitroquinoline l-Oxide” ( Endo et al., 1971) . The present review covers only reports that were, not included in the above review. A. In Viuo CARCINOGENESIS Experimental induction of cancer in the glandular stomach is very difficult, although stomach cancer is a very common type of human carcinoma in some countries. In 1967, Sugimura and Fujimura succeeded in producing adenocarcinomas with high incidence in the glandular stomach of rats by administration of a low concentration of the strong mutagen MMNG in the drinking water. Induction of stomach cancer by 4NQ0, which is also mutagenic, was first reported by Baba et al. (1962). An adenocarcinoma of the glandular stomach was induced in only 1 of 33 rats by administration of 1 ml of a 0.1% ethanolic solution of 4NQ0 by stomach tube and then painting 20-methylcholanthrene on the skin of the back. Later, Mori (1967) reported the production of adenocarcinomas in the glandular stomach of mice by administration of 0.25 or 0.5 mg of 4NQ0 in 0.1 ml of 50%ethanol solution by stomach tube every 7-12 days. In this way adenocarcinomas of the glandular stomach developed in 2 of 42 mice, and squamous cell carcinomas of the forestomach in 11 of 42 mice. Mori and Ohta (1967) reported that administration of 0.25-0.5 mg of 4HAQO by stomach tube every 7-10 days produced adenocarcinomas in the glandular stomach in 13 of 63 mice. 4HAQO HC1 induced a slightly higher incidence of gastric lesions in mice (Mori et al., 1969~).Adenocarcinomas were also induced in the glandular stomach of rats by instillation of 1or 2 mg of 4HAQO-HCI ethanolic solution by gastric tube into female Buffalo rats (Mori et al., 1 9 6 9 ~ )In . this way, adenocarcinomas of the glandular stomach were induced in 7 of 15 rats. They assumed that ethanol solution first damaged the mucous barrier so that 4HAQO.HCl could penetrate into the mucosal layer of the glandular stomach and come into direct contact with epithelial cells. No tumors were found in the forestomach of these animals. Takahashi (1970) introduced 1 mg of 4NQ0 in 1 ml of 8%alkylbenzenesulfonate and 20% of ethanol into male Moriyama strain rats by intubation. Two cases of adenocarcinoma, 1 case of hemangiosarcoma, and 1 case of hemangioma were found in the glandu9

158

MINAKO NAGAO AND TAKASHI SUCIhlURA

lar stomach of the 15 rats treated in this way. Multiple papillomatous lesions, including 5 cases of squamous cell carcinoma, were found in the forestomach of all 15 rats. A second group of rats was administered the same dose of 4NQO in 20% ethanol without alkylhenzenesulfonate. No adenocarcinoma was found in these animals, although papillomas of the forestomach and 1 case of sarcoma of the liver were observed. Administration of alkylbenzenesulfonate certainly enhanced the induction of adenocarcinomas by 4NQO. Ito et aZ. (1969) administered 1 mg of 4NQO suspension by stomach tube to male Sprague-Dawley rats after removal of the submaxillary and parotid salivary glands. The 4NQ0 was suspended in a mixture of olive oil, Tween 80, and water. Three cases of squamous cell carcinoma of the forestomach, 1 case of adenocarcinoma, and 1 case of leiomyosarcoma of the glandular stomach were observed in the 10 effective rats. The adenocarcinoma was a poorly differentiated type. Ito et al. thought that saliva plays an important role in preventing direct contact of carcinogen with the stomach mucosa. MNNG usually induces adenocarcinomas in high frequency, but most of the tumors are well differentiated. Oral administration of 4NQ0 induces an undifferentiated type of adenocarcinoma with metastases, but in very low incidence. Thus on combined treatment with MNNG and 4NQ0, a high incidence of undifferentiated adenocarcinomas was expected. Sugiura (1973) administered 50 mg of MNNG per liter ad libitum in the drinking water to rats for 17 weeks and then 1 mg of 4NQ0 per milliliter in 20%ethanol and 8%of alkylbenzenesulfonate twice a week for 9 weeks. Among 14 treated animals, 8 developed adenocarcinomas. Four of these were undifferentiated adenocarciiiomas with metastases. Undifferentiated adenocarcinomas appeared very similar to human gastric cancer in their light and electron microscopic features and their metastatic behavior. 4NQ0 treatment apparently converted well-differentiated adenocarcinomas to undifferentiated types. Hayashi and Hasegawa (1971) reported the induction of pancreatic tumors in rats by 4HAQO.HCl. Male and female Sprague-Dawley rats were given a single intravenous injection of 6 mg, 9 mg, or 13 mg/kg of 4HAQO.HCl in 0.005 N HC1-saline, and 25 of the 35 treated animals developed pancreatic tumors. All but one of these were exocrine, and only one was an islet cell tumor. Ito (1973) induced kidney tumors in rats by implantation of cotton impregnated with 4NQO-beeswax. Histologic examination revealed that the tumors were mostly transitional cell tumors. The relations of the structures and carcinogenicities of 4NQ0 derivatives have been investigated by many workers. Nakahara et al. (1958)

MOLECULAR BIOLOGY OF THE CARCINOGEN,

4NQO

159

found that 4-nitroquinoline (4NQ) was not carcinogenic and that an oxygen atom on the ring nitrogen was essential for the strong carcinogenicity of 4NQO derivatives. However, Mori et al. (1969b) reported the positive carcinogenicities of 2- and of 4-nitroquinoline (2NQ and 4NQ) (Mori et al., 1969d). Repeated subcutaneous injections of a total of 17 mg of BNQ as an aqueous solution with 10%lecithin into female ICR strain mice resulted in formation of lung tumors in 44% of the animals, but did not induce any malignant change at the site of injection. In contrast, repeated subcutaneous injection of a total of 17 mg of 4NQ induced sarcomas at the site of injection in 57% of the ICR strain of mice treated. Moreover, 10 of 21 mice developed lung cancer and 4 of 21 developed ovarian cysts. Mori et al. claimed that 2NQ and 4NQ were definitely carcinogenic. Recently, quinoline was proved to be carcinogenic. Male SpragueDawley rats were continuously administered a diet containing 0.05%, 0.1%, or 0.25% quinoline. The 0.05% quinoline diet induced hepatomas in 27% of the rats and hemangiosarcomas in 55%,within 40 weeks. The 0.1% and 0.25%diets induced hepatomas and hemangiosarcomas in the liver. 2-Chloroquinoline did not induce any tumors when given under similar experimental conditions ( N. Ito, personal communication) . Sat0 et al. (1970b) showed that subcutaneous injection of 11.6 pmoles of o-acetyl-HAQ induced sarcomas at the site of injection in 17 of 20 DDD mice. They also observed high incidences of subcutaneous sarcomas (13%)and lung adenomas (50%) in DD mice given a single subcutaneous injection of 53 pmoles of o-acetyl-HAQ within 24 hours after birth. These results also indicate that the N-oxide group was not essential. The carcinogenicity of o,o’-diacetyl-4HAQO was reported by Kawazoe and Araki (1967) and by Sat0 et al. (1970b). 3-Methyl4NPO was found to be carcinogenic (Araki et al., 1971). Repeated subcutaneous injection of a total of 42 mg of the former compound induced fibrosarcomas in 8 of 20 mice and a polymorphous cell sarcoma in 1 mouse. Hoshino et al. (1969) reported the carcinogenicities of 4,7-dinitroquinoline l-oxide and 3-methyl-4NQ0 on mouse skin using the summatior, tcchnique. Nomura and Okamoto (1972) injected 4NQO solution directly into the abdominal cavity of mouse fetuses with a microsyringe. Injection of 4NQ0 on day 14 or 15 of gestation resulted in a high rate of abortions and stillbirths. Its injection on day 16 or 17 resulted in live offspring, which all developed pulmonary tumors in the twenty-first week after birth. A single subcutaneous injection of 4NQ0 into pregnant mice on day 7 of pregnancy did not induce any tumor.

160

MINAKO NAGAO AND TAKASHI SUGIMURA

B. IMMUNITY It is thought that the immunosuppressive state of the host favors the development of tumors, as stated by Prehn and Main (1957). Many chemical carcinogens, such as polycyclic hydrocarbons ( Prehn, 1963; Rubin, 1971), aminoazo dyes ( Baldwin and Glaves, 1968), and urethane (Parmiani et al., 1969), depress the immunological response. The immunosuppressive activity of 4NQ0 has been well documented (Phillips, 1972; Nakashima and Ono, 1972; Outzen and Prehn, 1973). Humoral immunosuppression was observed after subcutaneous or intraperitoneal injection of 4NQ0, by using plaque-forming spleen cells (Jerne et d., 1963) or by measuring the response to bacterial a-amylase (Onoue et al., 1963). Suppression of the immune response by 4NQ0 or 4HAQO was dose dependent. Noncarcinogenic or weakly carcinogenic analogs, such as 4-chloroquinoline l-oxide, 4NP0, and 4AQ0, did not cause immunosuppression. Phillips (1972) reported that rapidly dividing bone marrow cells were affected most by 4NQO. Outzen and Prehn (1973) studied the effect of 4NQ0 on the cellmediated immune response. 4NQ0 was administered subcutaneously 30 days before transplantation of the skin of male C57BL/6 mice to recipient females. The survival time of the skin grafts as distinctly prolonged by treatment with 4NQO. Subcutaneous implantation of a Millipore filter impregnated with 4NQ0 suspension in paraffin caused immunosuppression and subcutaneous tumor development in C57BL/ 6 and C3H mice. c . In VitfO CARCINOGENESIS BY, AND EFFECTOF, 4-NITROQUINOLINE 1-Oxum ON in Vitro VIRALCARCINOGENESIS Malignant transformation in uitro by chemical carcinogens was first observed on treatment of a Syrian hamster embryonic cell culture with polycyclic hydrocarbon by Benvald and Sachs (1963, 1965). This phenomenon was confirmed by Dipaolo et ul. (1969), who also found a correlation between morphological transformation and acquisition of a malignant character. Malignant transformation of cultured cells with 4NQ0 was first observed independently by Sato and Kuroki (1966) and Kuroki et al. (1967) and by Kamahora and Kakunaga (1966, 1967) using hamster embryonic cells which showed very low spontaneous malignant transformation. It was later confirmed by Kinoshita et al. (1968). Rat embryonic cells and rat liver parenchymal cells were also transformed by 4NQ0

MOLECULAR BIOLOGY OF THE CARCINOGEN,

4NQO

161

(Namba et al., 1969; Namba and Sato, 1970; Katsuta and Takaoka, 1972). NEP cells and the WNE culture strain, both originating from strain A mice, were also transformed by 4NQO. NEP cells were derived from pulmonary tissues of 5 embryos and WNE cells were derived from whole tissue of one embryo (Toyoshima et al., 1971). 3-Methy1-4NQ0, which is very weakly carcinogenic and 4AQ0, which is almost noncarcinogenic, failed to transform cultured cells ( Kuroki M M 4HAQO or 10-5.5to and Sato, 1968). Concentrations of 4NQ0 were sufficient to induce malignant transformations of hamster embryonic cells (Kuroki and Sato, 1968) and the A31-714 subclone of BALB/3T3 cells ( Kakunaga, 1974), respectively. The process of malignant transformation by chemical carcinogen was carefully analyzed by Kakunaga (1974). A31-714 cells did not produce transformed foci when kept under nonreplicating conditions after 4NQ0 treatment. The maximal transformation frequency was obtained when the cells had undergone more than 4 cell generations after 4NQ0 treatment. When the cells were kept in a nongrowing state after 4NQ0 treatment, they lost the capacity to exhibit transformation. However, cell that underwent one cell replication within 24 hours after 4NQOtreatment retained the ability to transform even when subsequently kept in the nongrowing state for as long as 6 days. Similar results were obtained by Kuroki and Sat0 (1971) using hamster embryonic cells. Kakunaga ( 1973) succeeded in quantitative measurement of malignant transformation with 4NQ0 and its derivatives using the clone cell line A31-714 BALB/3T3. The process in which cell multiplication was required for malignant transformation was defined as the “progressive process” (Kakunaga and Kamahora, 1969; Kuroki and Sato, 1968). Pretreatment of 3T3 cells with 4NQ0 significantly enhanced the efficiency of transformation by SV40 treatment, although 4NQ0 alone induced no transformation under the experimental conditions. It should be mentioned that viral transformation was enhanced when cells treated with 4NQ0 were infected with SV40 within the period of unscheduled DNA synthesis induced by 4NQ0 (Kakunaga and Miyashita, 1972). Stich et al. (1972) also reported a similar enhancement of transformation of Chinese hamster cells by adenovirus type 12, or SA7 after pretreatment with 4NQ0 as shown in Table VI. This enhancement decreased greatly 12 hours after 4NQ0 pretreatment. DNA repair synthesis initiated by 4NQ0 was not stimulated or suppressed by viral infection, but the virus enhanced induction of chromosome aberrations by 4NQ0 (Table VI), Chinese hamster cells transformed by SV40 virus after pretreatment with 4NQ0 contained SV40 T antigen (Diamond et al., 1974). The integration of SV40 DNA into cellular DNA increased in cells pretreated

162

MINAKO NAGAO AND TAKASHI SUGIMURA

TABLE VI H \MSTER

ItESPONSI.:S O F CCLTE'KED S Y R I . I N

(A111Z

OR

SA7),

COMHIN.%TIONS O F

Virus" 4NQ0 Virus"

+ 49QO

C E L L S TO ADICNOVIRKJS

4-NITROQUINOLINE l - O X l D E

AD12

AND

(4NQ0), .\ND

4NQOa

DNA repair

Chromosome aberrations

(grains/nucleus)

(%I

0 61

37 2(1

-57

.)J

Transformation frequencyb

--

13 0 300

Data taken from Stich et al. (197.5). colonies per 2 X 1 0 6 surviving cells. Human adenovirus type 12(AI)12) was used for studies on DNA repair and chromosomes: simian adenovirus (SA7) was employed for experiments on transformation.

* Number of

with 4NQ0 (Hirai et al., 1974). It was also found that 4NQ0 pretreatment enhanced nuclear penetration of SV40. Thus the primary effect of 4NQ0 on virus transformation may be on early events, and increased integration might be a secondary effect. Contrary to this, it has been reported that 4NQ0 inhibits transforming activity of oncogenic virus in mouse embryonic cell cultures (Fujimoto, 1973).

D. DECARCINOGENESIS BY 4-NITROQUINOLINE 1-OXIDE Treatment of the cultured mammary adenocarcinoma cells of mice, line FM3A/ B with 4NQ0 yielded the nontransplantable clone M6 (Koyama and Ishii, 1969). It is known that nontransplantable cell lines sometimes arise from cultures of transplantable tumor cell lines. However, it is likely that 4NQ0 or 4HAQO treatment results in a significantly enhanced yield of this kind of nontransplantable ( decarcinogenized ) cell line. The term of decarcinogenesis was proposed by Sugimura { 1970).

E. OTHERBIOLOCICE F F E COF ~ ~4-NITROQUIh'OLINE 1-OXIDE

A good correlation between the cytocidal actions on N F sarcoma cells and the carcinogenicities of 4NQ0 and 42 of its derivatives were reported by Tokuzen et al. ( 1970). S . Yamamoto et al. (1970) observed that intracerebral injection of 4NQ0 into mice induced obesity: 0.04 mg of 4 N Q 0 and 0.01 mg of

MOLECULAR BIOLOGY OF THE CARCINOGEN,

4NQO

163

4HAQO induced 40% obesity in mice in 4 weeks whereas 8AQ0, 3methyl-8NQO and 4NP0 which are noncarcinogenic, or very weakly carcinogenic did not cause obesity. The effects of intracerebral injections of other carcinogens, such as P-propiolactone, N-nitrosomethylurea, 3,4benzopyrene, and N-hydroxy-N-2-fluorenylacetamide,on obesity have not been observed. VIII. 4-Nitroquinoline 1 -Oxide and Microbial Screening Method for Carcinogen

Rapid screening tests for carcinogens using microbial systems are now widely carried out. 4NQ0 was proved to be positive in the following rapid screening tests on mutagenicity: the reverse mutation test using the WP2 trp- mutant of Escherichia coli (Clarke, 1971; McCalla et al., 1974; Yahagi et al., 1974), Hs30R arg- mutant (Kondo, 1974a), Salmonella typhimurium TA1538, TA100, and TA98 his- mutant ( Ames et al., 1973; McCann et al., 1975), the forward mutation test using Neurospora crassa (Matter et al., 1972). In rapid screening tests for carcinogens, based on repair systems (Slater et al., 1972), 4NQ0 has proved to be a very useful positive control substance. Tests in which it has been used include repair tests on E . coli (Nagao and Sugimura, 1972), B . subtilis (Kada et al., 1974) S. typhimurium (Ames et al., 1973), and Saccharomyces cereoisiae (Nagao and Sugimura, 1972). IX. Conclusions

4NQ0 is a mutagenic and carcinogenic substance that is fairly soIuble in water. In both microbes and mammalian cells it is activated metabolically first by reduction to 4HAQO and then by further steps of activation. Most other potent carcinogens, including aromatic hydrocarbons, dialkyl nitrosamine, acetylaminofluorene, and azodyes, are metabolically activated by mammalian enzymes but not by microbial enzymes. This is one advantage of use of 4NQ0 as a test carcinogen. Another advantage is that the mechanisms of its molecular, biological, and genetic actions are better known than those of most other carcinogens. Alkylation of nucleic acid by methylated carcinogens, such as dimethylnitrosourea have also been fairly well investigated, but methylation of nucleic acid is a normal regulatory mechanism in microbial and mammalian cells. However, formation of a quinoline-base adduct is a quite unique reaction, which is distinguishable from normally occurring modifications of nucleic acid.

164

MINAKO NAGAO AND TAKASHI SUGIMURA

The relation between mutation and carcinogenesis is a topic of great current interest, so that it seems appropriate to review available information on 4 N Q 0 at this time. The biological significance and effects of 4 N Q 0 still require further study. Future studies should include investigations on the thresholds of carcinogens, since these are now a most urgent problem not only from scientific but also from social viewpoints. The genetic effects of carcinogens like 4 N Q 0 have been well documented, so these compounds provide convenient tools for studies along this line.

ACKNOWLEDGMENTS Many investigators, whom we wish to thank, have contributed to the information presented in this chapter. Our laboratories are supported partly by grants from the Ministry of Education, Science and Culture, the Ministry of Health, and the Princess Takamatsu Cancer Research Fund. The authors wish to express their sincere thanks to Dr. War0 Nakahara, President of National Cancer Center for his encouragement during the preparation of this manuscript.

REFERENCES Ames, B. N., Lee, F. D., and Durston, W. E. (1973). Proc. Nut. Acad. Sd. US. 70, 782-786. Andoh, T., and Ide, T. (1972). Cancer Res. 32, 1230-1235. Andoh, T., Kato, K., Takaoka, T., and Katsuta, H. ( 1971). Int. 1. Cancer 7,455-467. Araki, M . , Koga, C., and Kawazoe, Y. ( 1971). Gann 62,325327. Baba, T., Misu, Y., and Takayama, S. (1962). Gann 53,381-387. Baldwin, R. W., and Glaves, D. (1968). Rep. Brit. Cancer Campaign 46, 236. Benvdd, Y., and Sachs, L. (1963). Nuture (London) 200, 1182-1184. Berwald, Y., and Sachs, L. (1965). J. Nut. Cancer Inst. 35, 641-661. Bootsma, D., De Weerd-Kastelein, E. A., Kleijer, W. J., and Keijzer, W. (1975). I n “Molecular Mechanisms for the Repair of DNA” (P. C. Hanawalt and R. B. Setlow, eds. ). Vol. 27, Part B, pp. 725-728. Plenum, New York. Bridges, B. A. (1969). Annu. Reo. Nucl. Sci. 19, 139-178. Bucking-Throm, E., Duntze, W., Hartwell, L. H., and Manney, T. R. (1973). Exp. Cea Res. 76, 99-110. Clarke, C. H. (1971). Mutat. Res. 11, 247-248. Cleaver, J. E. ( 1968). Nature (London) 218, 652-656. Cleaver, J. E. ( 1969). Proc. Nut. Acad. Sci. U . S . 63,428-435. Cleaver, J. E. (1971). I n “Nucleic Acid-Protein Interactions” (D. W. Ribbons, J. F. Woessner, and J. Schultz, eds. ), pp. 87-112. North-Holland Publ., Amsterdam. Cleaver, J. E., and Thomas, G. H. (1969). Biochem. Biophys. Res. Commun. 36, 203-208. Cook, S . , and McGrath, J. R. (1967). Proc. Nut. Acad. Sci. U.S. 58, 1359-1365. Diamond, L., Knorr, R., and Shimizu, Y. (1974). Cancer Res. 34, 2559-2604. Dipaolo, J. A., Donovan, P. J., and Nelson, R. ( 1969). J . Nut. Cancer Inst. 42, 867-876. Endo, H. (1958). Gann 49,151-156.

MOLECULAR BIOLOGY OF THE CARCINOGEN,

4NQo

165

Endo, H., Ishizawa, M., and Kamiya, T. (1963). Natunoissenschaften 50, 525-526. Endo, H., Ono, T., and Sugimura, T. (1971). Recent Results Cancer Res. 34, 1-98. Epstein, S. S., and St. Pierre, J. A. (1969). Tozicol. Appl. P h a m c o l . 15, 451-460. Evans, R. G., and Norman, A. (1968). Radiat. Res. 36,287-298. Fahring, R. (1970). Mutat. Res. 10, 509-514. Fahring, R. ( 1973). Agents Actions 3,99-110. Felkner, I. C., and Kadlubar, F. (1968). 1. Bacteriol. 96, 1448-1449. Fox, M., and Fox, B. W. (1973). Int. 1. Radiot. B i d . 23,359-376. Freidberg, E. C. (1972). Mutat. Res. 15, 113-123. Freidberg, E. C., and Clayton, D. A. (1972). Nature (London) 237, 99-100. Freidberg, E. C., and King, 1.J. (1971).1. Bacterial. 106, 500507. Fujimoto, J. ( 1973). J . Nut. Cancer Inst. 50,79-85. Gudas, L. J., and Pardee, A. B. (1975). PTOC.N d . Acad. Sci. U.S.72,2330-2334. Hahn, G. M., Yang, S. J., and Parker, V. ( 1968). Nature (London) 220,1142-1144. Harm, W. (1963). Virology 19,66-71. Hartman, P. E., Levine, K., Hartman, Z., and Berger, H. (1971). Science 172, 1058-1060. Hayashi, Y., and Hasegawa, T. (1971). Gann 62,329330. Hirai, K., Defendi, V., and Diamond, L. (1974). Cancer Res. 34, 34974500. Horikawa, M., Nikaido, O., Tanaka, T., Nagata, H., and Sugahara, T. (1970). Exp. Cell Res. 59, 147-152. Hoshino, H., Kawazoe, Y., and Fukuoka, F. ( 1969). Gann 60,523-527. Hozumi, M. (1968). Biochem. P h a m c o l . 17, 769-777. Hozumi, M., Inuzuka, S., and Sugimura, T. (1967). Cancer Res. 27, 1378-1383. Hunnable, E. G., and Cox, B. C. (1971). Mutat. Res. 13,297409. Ide, T., and Andoh, T. (1972). Cancer Res. 32, 1236-1242. Ikegami, S., Nemoto, N., Sato, S., and Sugimura, T. ( 1969/1970). Chem.-Biol. Interact. 1, 321430. Ikenaga, M., Ichikawa-Ryo, H., and Kondo, S. (1975a). I. Mol. B i d . 92, 341356. Ikenaga, M., Ishii, Y., Tada, M., Kakunaga, T., Takebe, H., and Kondo, S. ( 197513). In “Molecular Mechanisms for the Repair of DNA” (P. C. Hanawdt and R. B. Setlow, eds. ), Vol. 27, Part B, pp. 763-771. Plenum, New York. Isaka, H. ( 1970). Gann 61,193-196. Isaka, H. (1975). Gann Monogr. 17,31-38. Ishii, Y., and Kondo, S. ( 1971).Mutat. Reg. 13, 193-199. Ishizawa, M., and Endo, H. (1967). Biochem. P h a m c o l . 16,637-646. Ishizawa, M., and Endo, H. (1970). Mutat. Res. 9, 134-137. Ishizawa, M., and Endo, H. ( 1971). Mutat. Res. 12, 1-8. Ishizawa, M., and Endo, H. (1972). Gann 63,511-515. Ising, U., and Levan, A. (1957). Acta Pathol. Micr~biol.Scand. 40, 3-24. Ito, N. (1973). Act0 PathOZ. lap. 23, 87-109. Ito, M., Yamada, S., Suzuki, H., and Nagayo, T. (1969). Gann 60, 223-225. Jacobs, A. J., O’Breien, R. L., Parker, J. W., and Paolilli, P. (1972a). Znt. J. Cancer 10, 118-127. Jacobs, A. J., O’Breien, R. L., Parker, J. W., and Paolilli, P. (1972b). Mutat. Res. 16, 420-424. Jerne, N. K., Nordin, A. A., and Henry, C. (1963). In “Cellbound Antibodies” (A. Bernard and H. Koprowski, eds.), pp. 109-125. Wistar Inst. Press, Philadelphia, Pennsylvania.

166

MINAKO NAGAO AND TAKASHI SUGIMURA

Kada, T., Tutikawa, K., and Sadaie, Y. (1972). Mutat. Res. 16, 165-174. Kada, T., Moriya, M., and Shirasu, Y. (1974). Mutat. Res. 26,243-248. Kakunaga, T. (1973). Int. J. Cancer 12,463-473. Kakunaga, T. (1974). Int. J. Cancer 14,736-742. Kakunaga, T., and Kamahora, J. (1969). Symp. Cell Chem. 20, .135-148. Kakunaga, T., and Miyashita, K. (1972). Symp. Cell Biol. 23,95-102. Kamahora, J., and Kakunaga, T. (1966). Proc. Jap. Acad. 42, 1079-1081. Kamahora, J., and Kakunaga, T. ( 1967). Biken J. 10,219-242. Katsuta, H., and Takaoka, T. (1972). J. Nut. Cancer Inst. 49, 1563-1576. Kawazoe, Y., and Araki, M. ( 1967). Gann 58, 485-487. Kawazoe, Y., Huang, G. F., Araki, M., and Koga, C. ( 1972). Gann 63,161-166. Kemp, C. L. (1972). Arch. Microbiol. 86, 255-264. Kemp, C. L., nd Malloy, K. M. (1973). Mutat. Res. 20, 191-200. Kinoshita, R., Goetz, I. E., and Mori, J. (1968). Proc. Amer. Ass. Cancer Res. 9, 37. Kobalizoba, C. B. (1973). Genetika 9, 110-115. Kondo, S. (1973). Genetics 74, Suppl., 109-122. Kondo, S. ( 1974a). Mutat. Res. 26, 235-241. Kondo, S. (1974bf. Genetics 78, 149-161. Kondo, S. (1975). Aduan. Biophys. 7,91-162. Kondo, S., Lchikawa, H., Iwo, K., and Kato, T. (1970). Genetics 66, 187-217. Koske, R. E., and Stich, H. F. (1973). Mutat. Res. 19,265-298. Koyania, K., and Ishii, K. (1969). Gann 60, 367372. Kraemer, K. H., Coon, H. G., Petinga, R. A., Barrett, S. F., Rahe, A. E., and Robbins, J. H. (1975). Proc. Nut. Acad. Sci. US.72, 59-63. Kurita, Y., Yoshida, T.H., and Moriwaki, K. (1965). Jap. J. Genet. 40,365-376. Kuroki, T., and Sato, H. (1968). J . Nut. Cancer Inst. 41,53-71. Kuroki, T., and Sato, H. ( 1971). In “Oncology” ( R. L. Clark et al., eds. ), Vol. I, pp. 91-103. Yearbook Publ., Chicago, Illinois. Kuroki, T., Goto, M., and Sato, H. (1967). Tohoku J. Exp. Med. 91, 109-118. Latajet, R., Elias, C., and Buu-Hoi, N. P. (1949). C . R. SOC.Biol. 143, 776778. Lehman, A. R. (1972). J. MoZ. BioZ. 66, 319437. Lehmann, A. R., Kirk-Bell, S., Arlett, C. F., Paterson, M. C., Lohman, P. H. M., De Weerd-Kastelein, E. A., and Bootsma, D. (1975). Proc. Nut. Acad. Sci. U.S. 72, 219-223 Levan, A., and Hauschka, T. S. (1953). J. Nut. Cancer Inst. 14, 1-43. Lwoff, A. (1953). Bacterial. Reo. 17,269337. McCalla, D. R., Voutsinos, D., and Olive, P. L. (1974). Mutat. Res. 31, 31-37. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N. (1975). Proc. Nut. Acad. Sci. US. 72, 979-983. Makino, F., and Okada, S. ( 1974). Mutat. Res. 23, 387-394. Mashima, S., and Ikeda, Y. (1958).Appl. Microbiol. 6, 45-49. Matsushima, T., and Suginiura, T. ( 1971). Recent Results Cancer Res. 34,53-60. Matsushinia, T., Kobuna, I., and Sugimura, T. ( 1967). Nature (London) 216,508. Matsushima, T., Kobuna, I., Fukuoka, F., and Sugimura, T. (1968). Gann 59, 247-250. Matter, B. E., Ong, T. M., and de Serres, F. J. (1972). Gann 63, 265-267. hlifuchi, I., Morita, T., Yanagihara, Y., Hosoi, M., and Nishida, M. (1963). l a p . I. Microbiol. 7 , 69-79. Mori, K. (1967). Gann 58, 389-393. Mori, K., and Ohta, A. (1967). Gann 58, 551554.

MOLECULAR BIOLOGY OF THE CARCINOGEN, 4 N Q o

167

Mori, K., Ohta, A., Murakami, T., Tamura, M., and Kondo, M. (1969a). Gann 60, 151-154. Mori, K., Kondo, M., Tamura, M., Ichimura, H., and Ohta, A. (196913). Gunn 60, 609-610. Mori, K., Ohta, A., Murakami, M., Tamura, M., and Kondo, M., and Ichimura, H. ( 1 9 6 9 ~ )Gann . 60, 627-630. Mori, K., Kondo, M., Tamura, M., Ichimura, H., and Ohta, A. (1969d). Gann 60, 663-664. Morita, T., and Mifuchi, I. (1970). Bwchem. Biophys. Res. Commun. 38, 191-196. Morita, T., and Mifuchi, I. ( 1974). Gann 65,27-32. Mortimer, R. K., and Manney, T. R. ( 1971). In “Chemical Mutagens” (A. Hollaender, ed.), Vol. I, pp. 289-310. Plenum, New York. Moseley, B. E. B., Mattingly, A., and Copland, H. J. R. (1972). J . Gen. Microbiol. 72, 329-338. Moustacchi, E. (1971). MoZ. Gen. Genet. 114,50-58. Moustacchi, E., and Marcovich, H. (1963). C . R. Acud. S c i . 256,5646-5648. Nagai, S. (1969). Mutat. Res. 7,333-337. Nagao, M., and Sugimura, T. ( 1972). Cancer Res. 32,2369-2374. Nagata, C. (1971). Recent Results Cancer Res. 34, 17-31. Nagata, C., Kodama, M., Tagashira, Y., and Imamura, A. (1966). Biopolyrn. 4, 409-427. Nakahara, W., and Fukuoka, F. ( 1959). Gann 50,l-15. Nakahara, W., Fukuoka, F., and Sugimura, T. (1957). Genn 48, 129-137. Nakahara, W., Fukuoka, F., and Sakai, S. (1958). Gann 49,3341. Nakai, S., and Matsumoto, S. (1967). Mutat. Res. 4, 129-136. Nakai, S., and Mortimer, R. K. (1969). Mol. Gen. Genet. 103,329438. Nakashima, S., and Ono, S. (1972). Gann 63, 111-117. Namba, M., and Sato, J. (1970). Gann 61,583587. Namba, M., Masuji, H., and Sato, J. (1969). Jap. J. E r p . Med. 39, 253-265. Nomura, T., and Okamoto, E. (1972). Med. J. Osaka Uniw. 22, 245-249. Okabayashi, T. (1953).Hakko Kogaku Zusshi 31,373-375. Okabayashi, T. ( 1955). Hakko Kogaku Zasshi 33,513-516. Okabayashi, T., and Yoshimoto, A. (1962). Chem. Phamt. Bull. 10, 1221-1232. Okabayashi, T., Yoshimoto, A., and Ide, M. (1964). Chem. Pharm. Bull. 12, 257-261. Okano, T., and Uekama, K. (1967). Chem. Pharm. Bull. 15, 1251-1253. Ono, T. (1964). Tanpakushitsu, Kakusan, Koso 9, 1122-1128. Ono, T., Iwamura, Y., and Ohashi, M. (1967). Proc. Int. Cancer Congr., Qth, 1966 p. 200. Onoue, K., Okada, Y., Nakashima, S., Shimada, K., and Yamamura, Y. (1963). J. Biochem. (Tokyo) 53, 472478. Outzen, H. C., and Prehn, R. T. (1973). Cancer Res. 33,408-410. Painter, R. B., and Cleaver, J. E. (1969). Rudiat. Res. 37, 451466. Parmiani, G., Colnaghi, M. I., and Porta, G. D. (1969). Proc. SOC. Exp. @oZ. Med. 130, 828-830. Paul, J. S. (1970). Mol. Pharmucol. 6, 315-322. Paul, J. S., Montgomery, P. OB., and Louis, J. B. (1971). Cancer Res. 31, 413-419. Phillips, J. M. ( 1972). Int. J. Cancer 9, 3947. Poon, P. K., O’Brien, R. L., and Parker, J. W. (1974). Nature (London) 250, 223-225. Prakash, L., and Sherman, F. (1973). J. Mol. Biol. 79, 65-82. Prakash, L., and Sherman, F. (1974). Genetics 77, 245-254.

168

MINAKO NAGAO A N D TAKASHI SUGIMURA

Prakash, L., Stewart, J. W., and Sherman, F. (1974). J. Mol. Biol. 85, 51-65. Prehn, R. T. ( 1963). Can. Cancer Conf. 5, 387-395. Prehn, R. T., and Main, J. M. (1957). J. Nut. Cancer Inst. 18, 769-778. Radman, M. (1975). In “Molecular Mechanisms for the Repair of DNA” (P. C. Hanawalt and R. B. Setlow, eds.). Vol. 27, Part A, pp. 355-367. Plenum, New York. Rasmussen, R. E., and Painter, R. B. ( 1966). J. Cell Biol. 29, 11-19. Regan, J. D., and Setlow, R. B. (1973). In “Chemical Mutagens” (A. Hollaender, ed.), Vol. 111, pp. 151-170. Plenum, New York. Regan, J. D., and Setlow, R. B. ( 1974). Cancer Res. 34,3318-3325. Regan, J. D., Trosko, T. E., and Carrier, W. L. (1968). Biophys. J. 8, 319-325. Robbins, J. H., Kreemer, K. H., Lutzner, M. A., Festoff, B. W., and Coon, H. G. ( 1974). Ann. Intern. Med. 80, 221-248. Roberts, J. T., Crathron, A. R., and Brent, T. P. (1968). Nature (London) 218, 970-972. Roman, H., and Jacob, F. (1957). C . R. Acad. Sci. 245, 1032-1034. Rubin, B. A. (1971). Progr. Exp. Tumor Res. 14, 138-195. Rupp, W. D., Zipser, I., von Essen, C., Reno, D., Prosnitz, L., and Howard-Flanders, P. (1970). In “Time and Dose Relationships in Radiation Biology as Applied to Radiotherapy,” pp. 1-13. Brookhaven Nat. Lab. Publ. BNL 50203, C. 57. Rupp, W. D., Wilde, C. E., Reno, D. L., and Howard-Flanders, P. (1971). 1. MoZ. Biol. 61, 25-44. Sato, H., and Kuroki, T. (1966). Proc. l a p . Acud. 42, 1211-1216. Sato, K., Saito, T., and Enornoto, M. (1970b). Jup. J. Exp. Med. 40, 475-478. Schmid, W. ( 1966). E r p . Cell Res. 42, 201-204. Schwaier, R., Nashed, N., and Zimmermann, F. K. (1968). MoZ. Gen. Genet. 102, 290-300. Schwarzacher, H. G., and Schnedl, W. ( 1965). Cytogenetics 4, 1-18. Sedgwick, S. C . (1975). Psoc. Nut. Acud. Sci. U.S.72, 2753-2757. Sekiguchi, M., Yasuda, S., Okabo, S., Nakayama, H., Shimada, K., and Takagi, Y. ( 1970). J . Mol. Biol., 47, 231-212. Skavronskaya, A. G., Andreeva, I. V., and Kiryushkina, A. A. (1973). Mutut. Res. 18, 259-266. Skavronskaya, A. G., Andreeva, I. V., and Kiryushkina, A. A. (1974). Mutat. Res. 23, 275-277. Slater, E. F., Anderson, M. D., and Rosenkranz, H. S. (1971). Cancer Res. 31, 970-973. Slonimski, P. P., and Ephrussi, B. (1949). Ann. Inst. Pasteur, Purls 77, 47-63. Slonimski, P. P., and Perrodin, C., and Croft, J. H. (1968). Biochem. Biophys. Res. Commun. 30, 232-239. Stich, H. F., and San, R. H. C. (1970). Mutut. Res. 10,389-404. Stich, H. F., and San, R. H. C. (1971). Mutut. Res. 13,279-282. Stich, H. F., San, R. H. C., and Kawazoe, Y. (1971). Nature (London) 229,416419. Stich, H. F., Hammerberg, O., and Casto, B. (1972). Can. J. Genet. Cytol. 14, 91 1-917. Stich, H. F., Stich, W., and San, R. H. C. (1973). Proc. Soc. E r p . B i d . Med. 142, 1141-1144. Stich, H. F., Keiser, D., Laishes, B. A., San, R. H. C. and Warren, P. (1975). Cunn Monogr. 17, 3-15. Sugimura, T. (1970). In “Chemical Tumor Problems” (W. Nakahara, ed.), pp. 269-284. Jap. SOC.Promort. Sci., Tokyo.

MOLECULAR BIOLOGY OF THE CARCINOGEN, 4NQO

169

Sugimura, T., and Fujimura, S. ( 1967). Nature (London) 216,943-944. Sugimura, T., Otake, H., and Matsushinia, T. (1968). Nature (London) 218, 392493. Sugimura, T., Okabe, K., and Kodania, M. (1969). J. Bacterial. 97, 964-965. Sugiura, H. (1973). Nagoya Med. J. 18,157-168. Sutherland, B. (1974). Nature (London) 248, 109-112. Sutherland, B. M., Runge, P., and Sutherland, J. C . (1974). Biochemistry 13, 47104715. Sutherland, B. M., Rice, M., and Wagner, E. K. ( 1975). Proc. Nut. Acad. Sci. U.S. 72, 103-107. Sutou, S. (1973). Mutat. Res. 18, 171-178. Sweet, D. M., and Moseley, B. E. B. (1974). Mutat. Res. 23,311418. Swinton, D. C., and Hanawalt, P. C. (1973). Biochim. Biophys. A d a 294, 385495. Tada, Ma., and Tada, Mi. (1974). Gann 65,281-284. Tada, Ma., Tada, Mi., and Takahashi, T. (1967). Biochem. Biophys. Res. Commun. 29, 469477. Tada, Ma., Tada, Mi., and Takahashi, T. (1970). Collect. Pap., 23rd Annu. Symp. Fundam. Cancer Res. pp. 216-227. Tada, Ma., Morikawa, M., and Tada, Mi. ( 1974). Proc. Jap. Cancer Ass. p. 74. Tada, Mi., and Tada, Ma. ( 1971). Chem-Biol. Interact. 3, 225-229. Tada, Mi, and Tada, Ma. (1972). Biochem. Biophys. Res. Commun. 46, 1025-1032. Tada, Mi., and Tada, Ma. ( 1975). Nature (London) 255,510-512. Takahashi, M. (1970). Gann 61, 27-33. Takebe, H., Furuyama, J., Miki, Y., and Kondo, S. (1972). Mutat. Res. 15, 98-100. Tanoolfa, H., and Takahashi, A. (1972). Chem.-Bid. Interact. 5, 351-360. Tanooka, H., Kawazoe, Y., and Araki, M. ( 1969). Gann 60,537543. Tanooka, H., Tada, M., and Tada, M. (1975). Chem.-Bid. Interact. 10, 11-18. Tokuzen, R., Ark, M., Saneyoshi, M., and Fukuoka, F. (1970). Gann 61, 601-603. Toyoshima, K., Tsuji, H., and Tsubura, Y. ( 1971). J. Nara Med. Ass. 22, 162-174. Trosko, J. E., and Chu, E. H. Y. (1975). Aduan. Cancer Res. 21,391425. Trosko, J. E., and Mansour, V. H. (1968). Radiat. Res. 36, 333443. Warren, P. M., and Stich, H. F. ( 1975). Mutat. Res. 28, 285-293. Witkin, E. M. (1966). Science 152, 1345-1353. Witkin, E. M. (1969a). Mutat. Res. 8, 9-14. Witkin, E. M. (196913).Annu. Rev. Genet. 3, 525-552. Witkin, E. M. (1974). Proc. Not. Acud. Sci. U.S. 71, 1930-1934. Wolff, S., and Cleaver, J. E. (1973). Mutat. Res. 20,71-76. Yahagi, T., Nagao, M., Hara, K., Matsushima, T., Sugimura, T., and Bryan, G. T. ( 1974). Cancer Res. 34, 2266-2273. Yamagata, K., Oda, M., and Ando, T. ( 1956). Hukko Kogaku Zasshi 34, 378-381. Yamamoto, K., and Ishii, Y. (1974). Mutat. Res. 22, 81-83. Yamamoto, N. ( 1969). Virology 38,447456. Yamamoto, N., Fukuda, S., and Takebe, H. (1970). Cancer Res. 30, 2532-2537. Yamamoto, S., Mizutani, T., Kaneuchi, C., and Shirasu, Y. (1970). Proc. SOC. Exp. BWZ. Med. 133, 303-306. Yee, B., Tsuyumu, S., and Adams, B. G. (1972). Biochem. Biophys. Res. Commun. 49, 1336-1342. Yoshida, T. H., Kurita, Y., and Moriwaki, K. (1965). Gann 56, 523528. Zimmermann, F. K. ( 1971). Mutat. Res. 11, 327-337. Zimmermann, F. K., and Schwaier, R. (1967). MoZ. Gen. Genet. 100, 63-76.

This Page Intentionally Left Blank

EPSTEIN-BARR VIRUS A N D N O N H U M A N PRIMATES: NATURAL A N D EXPERIMENTAL INFECTION* A. Frank, W. A. Andirnan, and G. Millerz Departments of Pediatrics end Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut

.

. .

. . .

I. Introduction . . . . . . . . 11. EBV Reactive Antibodies in Nonhuman Primates . . . . . . A. Antibody to Viral Capsid Antigen . . . . . . . . B. Antibody to Other EBV-Related Antigens . . . . . . . C. Significance of Serologic Findings . . . . . . . . 111. Lymphoblastoid Cell Lines (LCL) from Nonhuman Primates . . . A. Spontaneous LCL from Old World Monkeys and Apes . . . . B. In Vitro Transformation of Nonhuman Primate Lymphocytes by EBV . C. Cytologic and Cytogenetic Characteristics of Simian LCL . . . D. Expression of EBV in Simian LCL . . . . . . . . IV. Experimental Infection of Nonhuman Primates with EBV . . . . A. Historical Background . . . . . . . . . . . B. Characteristics of Various Inocula Containing EBV . . . . . C. Host Range of Experimental Infection . . . . . . . D. Clinical and Pathologic Responses . . . . . . . . E. Serologic Responses . . . . . . . . . . . F. Demonstration of EBV in the Pathologic Lesions . . . . . G. Significance of Experimental Infection and Tumorigenesis in Nonhuman Primates by EBV . . . . . . . . . . . . V. Summary and Conclusions . . . . . . . . . . . References. . . . . . . . . . . . . .

.

171 172 172 175 176 177 178 179 181 182 186 186 188 190 192 194 194

195

197

199

1. Introduction

The purpose of this review is to analyze interrelationships between Epstein-Barr herpes virus (EBV) and primates other than man. Aspects of the discovery of the virus, its association with Burkitt lymphoma, infectious mononucleosis, and nasopharyngeal carcinoma, and its biologic properties studied in uitro have been covered in other reviews, including one by M. A. Epstein earlier in this series (Epstein, 1970; Klein, 1973; Miller, 1974). Studies of EBV in nonhuman primates have been useful in many respects. They have provided clues about the evolution of this highly Supported by Grants from the American Cancer Society VC107, Damon Runyon Memorial Funds DRG-1147, and from the National Institutes of Health CA-12055, CA-16038, AI-11611, HD-00177. 'An Investigator of the Howard Hughes Medical Institute. 171

172

A. FRANK, W. A. ANDIMAN, AND G. MILLER

adapted human parasite. Infection of nonhuman primate cells in uitro has yielded a regular source of cell-free virus for laboratory studies and has also clearly demonstrated the importance of host-specified control mechanisms in the regulation of expression of viral information in transformed cells. Experimental infection of primates was successful once adequate amounts of transforming virus were available and once the virus’ host range was better defined by means of in uitro transformation. Subsequent study allowed direct demonstration of the capacity of EBV to cause lymphoreticuloproliferative disease. The identification of SUSceptible primate hosts now offers promise of detailed analysis of the pathogenesis of EBV-induced disease and of the comparison of the pathogenic properties of different EBV-related viruses. II.

EBV

Reactive Antibodies in Nonhuman Primates

Primates were examined for serologic evidence of EB virus infection shortly after the discovery of the virus in cell lines established from Burkitt lymphomas and the development of techniques to measure antibodies to it (Gerber and Birch, 1967). These studies have been pursued for several reasons: Identification of seronegative species or individuals is important in the selection of animals or tissues to be used for experimental infection with human EB virus. Identification of seropositive species or individuals could suggest the presence of analogous, perhaps related, agents in nonhuman primates that might serve as epidemiological or experimental models for EB infection in man. The possibility also exists that nonhuman primates are natural reservoirs for EB viruses and they could play a role in transmission in certain geographic locales. A. ANTIBODYTO

VIRAL CAPSID

ANTIGEN

Tables IA, IB, IC summarize published data (and some previously unpublished data from our laboratory) about the prevalence of antibody to EB viral capsid antigen (VCA) in the sera of Old and New World nonhuman primates. The VCA was present in human lines, either the EB, or P,J-HR-lK strains of Burkitt lymphoma cells; the antigen was detected by the indirect immunofluorescence technique ( Henle and Henle, 1966). A fluorescein-conjugated antihuman IgG has been used in most instances. Dunkel et al. (1972) compared results obtained when primate sera were assayed for EBV antibody with antihuman and antimonkey fluorescein-conjugated antisera prepared in a goat. With the latter reagent, 2- to 4-fold higher titers and a slightly increased number of seropositive animals were detected.

173

EBV AND NONHUMAN PRIMATES

TABLE IA ANTIBODY (Ab) REACTIVITY TO EPSTEIN-BARR VIRALCAPSIDANTIQEN I N SERAOF OLD WORLDNONHUMAN PRIMATES No. with Ab/No. tested Species@ ~~~

~~

Prosimians Lemur macaco Galago crassicuaudatus Nycticebus coucang Old World Monkeys (Cercopithecoidea) Cercopithecus pygerythrus Miopitheeus talapoin Macaea cylopis Macaca fascieularis Maeaca mulatta Macaca radiata Maeaca (mixed) Papio cynocephalus Great apes (Pongidae) Pongo pongo P a n troglodytes Gorilla gorilla Hylobates lar a

(%I

Common name

Referencesb

~

Lemur Galago Slow loris

a a1 b C

African green Talapoin Taiwan Cynomolgus Rhesus Bonnet Cynomolgus Baboon

4/10 1/8 88/282 48/65 24/52 5/6 3/75 9/13

Orangutan Chimpanzee Gorilla White-handed gibbon

4/7 (57) 95/104 (91) 16/16 (100) 41/110 (37)

(40) (13) (31) (74) (46) (83) (4) (69)

Classification according to Buettner-Janusch (1963).

* Key to references: (a) Gerber and Lorene, 1974; (b) Dunkel et al., 1972; (c) G. Miller, unpublished, 1975; (d) Chu et al., 1971; (e) Landon and Malan, 1971; (f) Naito et al., 1971; ( 9 ) Kalter et al., 1972; (h) Kalter et al., 1973; (i) G. Miller and G. vanwagenen, unpublished, 1974; (j) Werner et al., 1972a; (k) Goldman et al., 1968; (1) Levy et al., 1971; (m) Stevens et al., 1970.

The data in Tables IA and IB indicate striking patterns in the distribution of anti-EBV reactivity in the sera of the order Primates. In the most primitive species, the prosimians (galagos, lemurs, and lorises of the Old World), and in New World monkeys (Cebidea), no antibodies are detected. By contrast, prevalence of antibody in Old World monkeys ( Cercopithecoidea) and great apes (Pongidae ) is very high. Antibodies to VCA are detected in 31-74%of sera from adults of individual macaque species and in more than 90% of sera from members of the Pongidae subfamily (orangutans, gorillas, and chimpanzees ) . The absence of antibodies to VCA among New World monkeys is not due to any peculiarities of the assay for antibodies, since these species, when experimentally infected (see below) with EBV, can be shown to develop antibody (Shope et al., 1973; Falk et al., 1974).

174

A. FRANK, W. A. ANDIMAN, AND G . MILLER

Ansfi:NcE;

TABLE IB ANTIBODYRE.WTIVITY TO EPSTEIN-BARR VIR.IL CAPSIDANTIGEN SERA O F NEW WORLD N O N H U M AP N R I M A T E S (CEBIDEA)

OF

IN

Species Hapalidae Saguinus mystax Saguinus oedipus Saguinus myncollis

1

S . oedtpus S. myncollis S . fusicollis Cebidae Alouatta palliata Ateles geofroyii Lagothriz lagotricha Saimiri seiurea

Common name

No. with Ab/No. tested

Referencesa

AZoustached marmoset (tamarin) Cotton top marmoset (tamarin) White-lipped marmoset (tamarin )

0/49

a

0 /30

b -f

0/11

R

Mixed lot

0!108

a

Mantled howler monkey Spider monkey Woolly monkey Squirrel monkey

0/5 0/42 0/8 11/19

Key to references: (a) Gerber and Lorenz, 1974; (b) Kalter et al., 1972; ( c ) Shope el al., 1973; (d) Miller el al., 1974b; (e) Falk et al., 1974; (f) Kalter el al., 1973; (9) Werner et a!., 1972a; (h) W. A. Andiman and G. hliller, unpublished, 1975; (i) Dunkel

et al., 1972; (j) Shope and Miller, 1973; (k) G. Xiller and L. V. XIelendes, unpublished, 1974. TABLE IC EFFECTOF AGE O N THE DISTRIBUTION OF ANTIBODYTO EPSTIXN-BIRR VIRALCAPSIDANTIGENAMONG MAC.~QUE MONKEYS

Species Macaca mulatta (rhesus)

3facaca fascicularzs (cynomolgus) .%facacacyclopis (Taiwan)

Age

No. with antibody/ No. tested

Percent positive

Newborn Juvenile Adult Newborn

22/24 6/41 24/52 17/17

92 15 46 100

Juvenile Adult Yewborn Juvenile Adult

0/17 48/6.5 .5/6 2/4 881282

References"

0 74 83 50 31

a Key to references: (a) Landon and Malan, 1971; (b) G . hliller, G. vanwagenen, and D. M. Horstmann, unpublished, 1974; (c) Cohen et al., 1974; (d) Kalter et al., 1972; (el Kalter el al., 1973; (f) Dunkel et al., 1972; (9) Chu et al., 1971.

EBV AND NONHUMAN PFUMATES

175

The pattern of distribution of antibody to VCA among macaque species demonstrates an effect of age on antibody prevalence (Table IC). Antibodies are detected in newborn rhesus, cynomolgus, and Taiwan monkeys, suggesting transplacental acquisition of antibody. Two studies conducted in breeding colonies of rhesus monkeys found that newborns lose antibody over the first year of life and reacquire antibody upon exposure to other monkeys (Landon and Malan, 1971; G. Miller, G. van Wagenen, and D. M. Horstmann, unpublished, 1974). Juvenile members of the rhesus and cynomolgus species have a considerably lower prevalence of antibodies than adult populations, a finding which indicates that infection is acquired toward maturity.

B. ANTIBODY TO OTHEREBV-RELATED ANTIGENS Some primate sera have also been tested for antibodies to EBV-related cell-associated antigens by complement fixation ( C F ) (Gerber and Birch, 1967; Gerber and Rosenblum, 1968; Gerber and Lucas, 1972; Gerber and Lorenz, 1974; Shope et al., 1973). In general, the results confirm those obtained with immunofluorescence techniques for anti-VCA antibodies, namely, seropositivity among Old World monkeys and seronegativity among New World monkeys. Gerber and Lorenz, however, detected reactivity by CF between certain marmoset sera and antigens prepared from EBV-carrying human cell lines. Shope et al. (1973) failed to detect CF antibodies in twelve marmoset sera until the animals were given EBV. Discordant results have aIso been reported for the detection of reactivity to “early antigens” in nonhuman primate sera. Dunkel et al. (1972) failed to detect antibodies to “early antigens” in sera from Old World monkeys, the majority of which were reactive with capsid antigens. Gerber and Lucas ( 1972), however, found antibody to “early antigens” in 80% of chimpanzee, 50% of rhesus, and 40% of cynomolgus sera. Of possible significance, “early antigen” was obtained in the former study by superinfection of Raji cells with the P,J-HR-l EBV strain whereas in the latter study, early antigens were obtained by bromodeoxyuridine ( BUdR) induction of Raji and NC37 cells. Some effort has been made to study EBV reactive antibodies in chimpanzee sera by immunodiffusion (Goldman et al., 1968; Stevens et al., 1970). In particular, these studies have focused on the problem of antigenic relatedness between EBV in human cell lines and a morphologically identical virus identified in continuous lymphoid cell lines from chimpanzee blood. Chimpanzee and human sera show a “line of identity” when allowed to react with antigen prepared from chimpanzee cell lines. However, not all components of the soluble precipitating antigen complex found in human cell lines is detected by chimpanzee sera.

176

A.

FRANK,

W. A. ANDIMAN, AND G . MILLER

In preliminary unpublished work from our laboratory, we determined the capacity of nonhuman primate sera, with antibodies determined by other methods, to neutralize EBV. Two of 4 sera from adult rhesus monkeys and 2 of 2 sera from newborn monkeys neutralized EBV when tested at a 1:8 dilution. The neutralization test involved inhibition of formation of lymphoid cell lines (Miller et al., 1972b). Further measurements of neutralizing antibody in nonhuman primate sera are needed in order to compare viral envelope antigens and thus help to resolve the question of relatedness of EBV’s found in nonhuman primates and man.

C. SIGNIFICANCE OF SEROLOGIC FINDINGS There are several possible explanations for anti-EBV activity in the sera of Old World monkeys and apes. Infection of captive monkeys by contact with human EBV excretors is very difficult to exclude, especially in studies dealing with laboratory animal populations, but seems unlikely to be the principal reason for the presence of EBV antibodies. The most compelling evidence for natural transmission of EBV dirgctly among nonhuman primates is that chimpanzees immobilized in their jungle habitat, “freshly caught” baboons, and rhesus bled shortly after capture by methods effective in preventing measles infection all have been seropositive (Levy et al., 1971; Kalter et al., 1972, 1973; Gerber and Rosenblum, 1968).Two rhesus infants bled serially by us lost placentally transmitted antibody and did not seroconvert even after extensive human contact in a monkey nursery visited daily by children until placed in a large room with other monkeys. This result again suggests that contact with primates other than man is responsible for the presence of EBV-reactive antibodies. It is possible that Old World primates are infected with agents which share immunologic reactivity with human EBV but which are not identical to it. Continuous lymphoblastoid cell lines have been spontaneously derived from peripheral blood leukocytes of chimpanzees (Landon et al., 1968b; Schable et al., 1974) (see Section 11). A fraction of the cells in such lines harbor a herpes virus and an antigen that crossreacts with EB capsid antigen. It seems likely that this virus might account for some of the anti-EB activity in chimp sera. Another herpes virus has been detected in leukocytes and oropharyngeal secretions of rhesus monkeys (Frank et al., 1973). However, in the limited number of monkey and human sera studied there was no correlation between seropositivity to EBV and to the rhesus leukocyte-associated herpes virus. We are not aware of systematic studies which have attempted to correlate EBV

EBV AND NONHUMAN PRIMATES

177

reactivity in Old World primate sera with antibodies to other herpes viruses indigenous to these species such as Herpes simiae and simian cytomegaloviruses. Artificial antisera prepared against H. simiae, H. tamarinus, H. hominis, and SA8 were nonreactive with EBV by immunofluorescence ( Landon and Malan, 1971). According to recent studies, there is no homology between EBV DNA and that of simian cytomegalovirus (Huang and Pagano, 1974). In conclusion, seroepidemiologic studies of EBV infections of nonhuman primates demonstrate that infection with EBV or related viruses is common in Old World monkeys in their natural habitat. The exact modes of transmission, the disease producing potential, and the structural relatedness of these viruses to human EBV is unknown. The absence of naturally occurring EBV reactive antibodies in prosimians and New World monkeys suggests that this group of viral agents became associated with primates at a time in evolution after the separation of the Old and New World monkey lines (about 50 million years ago). Ill. Lymphoblastoid Cell lines (LCL) from Nonhuman Primates

One of the properties of EBV is to permit continuous proliferation of infected lymphocytes in uitro (Henle et al., 1967; Pope et al., 1969; Miller et al., 1971). This property is demonstrated in two general ways. First, when leukocytic cells from infected individuals are placed in vitro they may form lymphoblastoid cell lines. Such lines do not become established from blood leukocytes of EBV seronegative subjects. (Even when EBV genome-free cell lines have been established, such as T-cell lines from leukemic patients the donors have been EBV seropositive (Minowada et al., 1972). The success of establishing LCL from seropositive subjects is dependent on a number of factors such as the time interval since the original infection, the number of leukocytes cultivated and the source of leukocytes. Lymph nodes appear to be a better source of EBV-infected cells than peripheral blood. The second general method of demonstrating the “immortalizing property of EBV is to add suspensions of the virus in uitro to lymphocytes of nonimmune individuals. After an interval of time which varies from a few days to several weeks, depending on the amount of biologically active EBV added, LCL appear in culture. Studies of LCL from the blood leukocytes of nonhuman primates can be appraised from the two perspectives of spontaneous LCL formation and that induced by in uitro addition of EBV. Spontaneous LCL formation has been described only in Old World monkeys and apes. Transformation induced by addition of EBV has been carried out with

178

A. FRANK, W. A. .4NDIMAN, AND G . MILLER

cells of New World primates which are uniformly EBV seronegative and with cells of seronegative gibbon apes.

A. SPONTANEOUS LCL FROM OLDWORLDMONKEYS AND APES (Table IIA) A leukocyte suspension culture arising ‘‘spontaneously’’from the buffy coat of a baboon was derived by Landon et al. ( 1968a). The line was composed of “lymphoblasts” after an initial 2 4 weeks of growth in “macrophage-like monolayers.” No virus was seen by electron microscopy, and the line was apparently not further studied. The peripheral blood of rhesus monkey with myelogenous leukemia associated with procarbazine treatment from birth yielded a lymphoblastoid cell line (OGara et al., 1971). The line showed herpes virus particles on electron microscopy but was subsequently lost. A similar line, obtained from another procarbazine-treated rhesus monkey, demonstrated neither herpes particles, EBV immunofluorescent antigens nor soluble C F antigens (Dunkel and Myers, 1972). Attempts to grow lymphocyte lines from normal untreated rhesus monkeys seropositive for EBV have been unsuccessful even when feeder layers and mitogens have been used (Landon et al., 1968b; Dunkel and Myers, 1972; G. Miller, unpublished). Four lymphoblast lines derived by Landon et al. from normal uninoculated EBV-seropositive chimpanzees bear a striking resemblance to the human LCL (Landon et al., 1968b). All four lines started out as macrophagelike monolayers but later became free-floating and lymphoid in morphology. Electron microscopy revealed herpes particles in 1-2% of the cells of all four lines. VCA was detected by immunofluorescence in 13%of the cells of one line with antibody-positive human and chimp sera (Goldman et aZ., 1968). A precipitogen, possibly related to one found in human LCL, was also detected in the LCL of chimp origin (Stevens et al., 1970). One of us (GM) observed the spontaneous development of lymphoblastoid cell lines in 2 of 5 cultures from the buffy coat of a stump-tailed macaque. One of the cell lines contained VCA which reacted with EBpositive human sera but was not studied further. Agents other than EBV, including other lymphotropic primate herpes viruses such as H. saimiri and H. ateles have proved capable of causing the formation of continuous LCL (Rabson et al., 1971). Recently, permanent lymphoblastoid transformation of mouse cells by Abelson murine leukemia virus has been demonstrated ( Rosenberg et al., 1975). A variety of different viruses may ultimately be shown to induce lymphoblast lines. Therefore, one must point out that EBV may not have been impor-

179

EBV AND NONHUMAN PRIMATES

tant in the initiation of all the cell lines listed in Table IIA. In fact, only the stump-tail macaque and chimpanzee cell lines might be considered presumptive EBV carriers, since EBV-related antigens were found in them. B. I n Vitro TRANSFORMATION OF NONHUMAN PRIMATELYMPHOCYTES BY EBV Table IIB lists seven species of nonhuman primates whose cells have been established as continuous cell lines following in vitro exposure to EBV. It is notable that six of the seven species in which this type of experiment has been successful are New World primates. It has also been possible to transform lymphocytes from gibbon apes. It seems important to note that attempts to establish lymphoblastoid cell lines from rhesus monkey blood leukocytes after exposure to EBV have been uniformly unsuccessful ( G. Miller, unpublished). This finding may indicate that, although rhesus monkeys have antibodies that react with EBV capsid antigen, their peripheral blood cells may not contain receptors for EBV of human origin. Several different strains of EBV have been used to transform monkey lymphocytes. Most studies in our laboratory have been conducted with the 883L EBV strain and its derivative produced in marmoset cells designated B95-8 (Miller and Lipman, 1973a). This virus originated in the blood leukocytes of an elderly lady with posttransfusion mononucleosis. TABLE IIA SPONTANEOUS LYMPHOBLASTOID CELLLINES FROM PERIPHERAL BLOOD OF OLD WORLDPRIMATES AND GREATAPES Expression of herpeslike virus5 Species

Common name

Macaca mulatta

Rhesus

Macaca speciosa Papio cynocephalus Pan troglodytes

Stump-tail macaque Baboon Chimpanzee

Viral particles

Capsid antigen

Other Referantigens encesb

Present Absent ND Absent Present

ND Absent Present ND Present

ND Absent ND ND Present

a b C

d e, f

a ND, not done or not stated. b K e y to references: (a) O’Gara et al., 1971; (b) Dunkel and Myers, 1972; (cb) G. Miller, unpublished; (d) Landon et al., 1968a (abst-r.); (e) Landon et al., 1968; ( f ) Schable et a/., 1974.

LYMPHOllL~\STOID C C L L

TABLE I I B LINESESTARLISHEI) FIZOM PICRIPHENIL BLOODOF NONHUMAN PIZIM.\TES hFTICI< EXPOSUI~E TO EPSTEI N-BARIZ Vrrius (EBV) in Vilro

?

2b

Expression of herpeslike virusa

2

Species

Common name

Viral particles

Saguinus oedipus Saguinus myncollis Saguinus sciurea A ot us t r zvirgat us Cebus albifrons Lagothriz lagotricha Hylobates lar

Cotton-top marmoset (tamarin) White-lipped marmoset (tamarin) Squirrel monkey Owl monkey Capuchin monkey Woolly monkey Gibbon

Present Present Present Present Present Present NL,

EBV 1)NA Present NI) Present

ND NI) NI) Present

EBV

EBV

capsid antigen

nuclear antigens

Present Present Present Present Present Present Present

Present Present Present NI) N1) Present

ND

"8

Biologieally active virus"

Refercneesc

Present Present Present N 1) N 1) Present N1)

a, b, e d d, e d d f R

3

NI), not done or not stated. In the transformation assay. Key to references: (a) Miller and Lipman, 1973a; (b) Miller et al., 1974a; (c) Pritchett el al., 1973; (d) Falk el al., 1974; (e) Miller et al., 1972a; (f) W. A. Andiman and G. Miller, unpublished, 1975; (g) Werner, 1972a. a

? 4

5

-%

z

0

zP

!i

EBV AND NONHUMAN PRIMATES

181

We also transformed marmoset cells with throat washings obtained from mononucleosis patients and more recently with virus obtained from Burkitt lymphoma cell lines (Miller et aZ., 1973). The Kaplan mononucleosis strain and the Maku and P3J Burkitt lymphoma strains have also been used (Werner et al., 1972a; Falk et al., 1974). In order to establish primate LCL, the technique of cocultivation of an X-irradiated EBV-containing lymphoblastoid cell line with primary monkey lymphocytes has often been necessary. Most human lymphoblastoid cell lines release minute amounts of extracellular transforming virus; use of lethally X-irradiated lymphoblasts as the source of virus apparently increases the amount of virus to which the primary leukocytes are exposed. Many experiments on the immortalization of nonhuman primate lymphocytes have employed “feeder layers” of diploid human cell strains derived from embryo or placenta and also a mitogen, phytohemagglutinin. The variables of mitogenic stimulation and the “feeder layer” have not yet been systematically studied; however, it would be of great interest to know whether blast cell formation induced by these substances enhances the susceptibility to transformation or merely permits the cells to survive long enough in vitro to be infected and transformed.

C. CYTOLOGIC AND CYTOCENETIC CHARACTERISTICS OF SIMIANLCL Simian LCL, particularly those from marmosets and squirrel monkeys, demonstrate certain cytologic differences from comparable human peripheral blood cells transformed in vitro by EBV. In particular, the simian lines tend to grow as partial monolayers with a fraction of floating and a fraction of attached cells (Miller et al., 1972a). The tendency of simian LCL to attach to glass or plastic is not the result of the lines consisting of a mixture of cell types because single cell clones derived from simian LCL exhibit the same behavior. The marmoset cell lines have a greater tendency than human lines to form niultinucleate giant cells. Multinucleate cells have intranuclear inclusion bodies in nearly all nuclei. A very low frequency of inclusion-bearing cells are found in single cells of the lines. The multinucleate cells also appear to be preferential sites of production or accumulation of viral capsid antigen, for these cells more frequently contain the viral capsid antigen than do the single cells (Miller et at., 1972a; Falk et at., 1974). In general, simian LCL, like their human counterparts, have exhibited diploid or near-diploid karyotypes. Dr. Herman Lisco ( unpublished) has studied, in detail, the karyotype of squirrel monkey and cotton-top marmoset cells transformed in uitro by EBV. These ceIls demonstrated a rather high level (2530%) of chromosome aberrations consisting of

182

A.

FRAKK,

W. A. ANDIMAN, AND G . MILLER

chromatid breaks, dicentric chromosomes, chromosome pulverization, etc., and nearly one-fourth of the cells in these cultures were hypodiploid. No marker chromosomes could be identified. Some effort has been devoted to identification of lymphocyte markers on the cell surface of simian LCL. Falk et al. (1974) demonstrated surface immunofluorescent staining with an antiimmunoglobulin in 6 simian LCL. Such staining was not detected in one marmoset and two squirrel monkey lines. Jondal and Klein (1973) demonstrated that the B95-8 cotton-top marmoset cells contained receptors for erythrocytes coupled with antibody and complement (EAC rosettes), and there were trace amounts of immunoglobulin on this line. None of the simian LCL have formed rosettes with sheep red cells ( E rosettes). Taken together, these results imply that simian LCL are derived from B cells, as are human LCL containing EBV. However, since simian LCL adhere to glass and plastic ( a property not held by human lymphoblasts) and because EB virus has mainly induced reticulum cell lymphoproliferation when inoculated in marmosets, the exact origin of the cells in the peripheral blood of New World primates which are transformed by EBV should at this point in time be considered unresolved. It is at least worthy of speculation that one of the target cells of EBV in marmoset blood is a “reticulum cell,” which is a histiocytic rather than a lymphocytic precursor. D. EXPRESSION OF EBV IN SIMIAN LCL (Table IIB) All available evidence indicates that the herpeslike virus found in simian LCL established after in uitro exposure to EBV is indeed EBV and not an endogenous monkey virus: No reactivity is detected between the sera of leukocyte donor animals and the LCL themselves. This indicates the absence of preexisting antibody to virus-associated antigens in the lines. The whole spectrum of EBV-associated antigens is found in simian lines, and, as far as has been determined, these antigens crossreact completely with comparable antigens in human lines. For example, antibody titers to VCA in human sera are the same whether measured on the P,J-HR-l human Burkitt lymphoma line or the B95-8 marmoset line. Furthermore, a “line of identity” has been found in immunodiffusion tests with the soluble precipitinogen found in the same two cell lines ( Miller et a]., 1 9 7 4 ~ ) Finally, . the envelope antigens of virus released from simian lymphoblastoid cell lines are identified by human sera. Antibodies that neutralize the marmoset-derived virus are absent before infectious mononucleosis and appear thereafter ( Miller, 1974). EBV DNA has been detected in several nonhuman lymphoblastoid

EBV AND NONHUMAN PRIMATES

183

cell lines formed in uitro after exposure to transforming virus. Werner et al. (1972a) detected EBV DNA with a probe of complementary RNA prepared from the virus released by the human P3J-HR-1 line. Approximately 1 0 3 0 genome equivalents of EBV DNA per transformed gibbon cell was found. Pritchett et al. (1975) have detected EBV DNA in the marmoset line B95-8. They have compared the relatedness of the DNA obtained from the virus released by the B95-8 cells and that released by the HR-1 cells. Their results suggest that all the sequences present in the B95-8 DNA are represented in the HR-1 virus, but that the B95 virus lacks homologous sequences amounting to approximately 15%of the HR-1 genome. They suggest that the DNA of the marmosetproduced virus contains a deletion with respect to the HR-1 DNA. More analysis is required to determine whether these differences in the DNA reflect transforming or nontransforming virus biotypes, or virus produced in simian as opposed to human cells or viruses from mononucleosis versus Burkitt lymphoma.

1. Activation of Viral Capsid and Early Antigens in LCL of Different Primate Species Both human and nonhuman primate LCL which are “producers” of EBV consist of a mixture of cells; most of them are not productive of virus, but a small fraction, varying from less than 0.1%to 15% are virus producers. As evidenced by cloning, all cells contain the viral genome and all express the EB nuclear antigen EBNA (Reedman and Klein, 1973) . Spontaneous activation of the genome and production of nucleocapsids occurs only periodically. The events leading to spontaneous activation of VCA are not understood, but cellular DNA synthesis and cell growth are required. Studies with simian LCL demonstrate the role of the host cells in determining the rate of spontaneous activation of the genome. When EB virus is used to transform cells from man and different primate species, there are marked differences in the rate of spontaneous activation of synthesis of VCA in the transformed cells. In lines derived from adult human leukocytes, less than 1%of the cells make VCA; if the cells were originally obtained from human umbilical cord blood, VCA is not found (Miller and Lipman, 1973b). By contrast 5-15% of transformed marmoset cells exhibit VCA. Transformed squirrel monkey cells appear to be intermediate between human cells and cotton-top marmoset cells. Woolly monkey cells are very much like human transformants as are gibbon cells. Only 0.1-1% of the cells in lines from these species spontaneously produce capsid antigen (Werner et al., 1972a; W. A. Andiman and G. Miller, unpublished, 1975). All clones of a marmoset

184

A. FRANK, W. A. ANDIMAN, AND G. MILLER

line exhibit as high a level of VCA as their parent; all daughter clones of a human line containing the same EBV strain have a low level of VCA. These findings indicate that the regulation of activation of VCA is a genetic property inherent in the cell which harbors the virus (Miller and Lipman, 1973b). In general, all LCL, of human and simian origin, tend to show a decrease in the fraction of cells with capsid antigen as the line is maintained in the laboratory, probably as a result of selection against producer cells. In some human lymphoblastoid lines the appearance of “early antigens” may be stimulated by treatment with halogenated pyrimidines or by addition of concentrated virus of the superinfecting biotype (Henle et al., 1970c; Gerber, 1972; Gerber and Lucas, 1972; Hamper et d., 1972). In general, studies thus far indicate that simian lymphoblastoid cell lines are relatively unresponsive to the induction of early antigens by either method (Falk et al., 1974; W. A. Andiman and G. Miller, unpublished, 1975). Among human cell lines there is great variation in the inducibility of early antigens; the control of this set of viral genes is also closely modulated by the host cell (Klein and Dombos, 1973).

2. Release of Extracellular Biologically Active EBV by Simian LCL The high yield of transforming virus obtained from EBV-converted marmoset cells is due to several factors which have been identified. The fraction of cells in marmoset LCL which are activated to produce virus is as high or higher than the P,J-HR-1 cell line, which is the most highly activated human cell line. Transformed marmoset cells release more mature enveloped extracellular virus per activated cell than most htiman producer cell lines. Thus the process of viral envelopment and release takes place more efficiently in marmoset cells. The biological type of virus produced by marmoset cells is transforming in its capacity. Table I11 shows the relationship between the number of cells activated to make VCA and the yield of extracellular virus per activated cell in human, squirrel monkey, and marmoset LCL and their clones transformed by virus from the same source. The yield of extracellular virus per activated cell is best for marmoset cells and is approximately 1 transforming unit per activated cell. A point to be emphasized is that the release of transforming virus from marmoset LCL does not seem due to a permanent change in the virus but rather to differences in controls imposed by the host cell which harbors the viral genome. The same strain of virus, after a sojourn in marmoset cells, when placed again in human cells causes nonproductive transformants. Several different transforming strains of EBV have demonstrated similar behavior, i.e., nonproductive transformation of human

185

EBV AND NONHUMAN PRIMATES

TABLE I11 COMPARISON OF THE ACTIVATION OF EPSTEIN-BARR VIRALCAPSIDANTIGEN (VCA) A N D THE YIELDOF EXTRACELLULAR VIRUS IN HUMANA N D NONHUMAN P R I M A T E LYMPHOBLASTOID CELL LINES TRANSFORMED BY THE SAME EBV STRAIN5

Type of material Human adult lines Clones of one human line Human neonatal lines Squirrel monkey Clones of one squirrel monkey line Cotton-top marmoset lines Clones of one cotton-top marmoset line

Number of lines or clones testedb 5 6

Fraction of cells VCAc

Estimated yield of transReleased forming infectious virus virus/cell (TD6o/ml)" with VCAc

1 6

0.012 0.018 Nil 0.088 0.040

Nil 4,000 5,000

0.002 0.004 Nil 0.045 0.096

5 5

0.06 0.144

350,000 99,800

5.833 0.786

4

20 84

Data from Miller and Lipman (1973a,b). Lines are derived from different individuals of the same species; clones are derived in each case from one line. Median values for the lines or clones. b

cells and productive transformation of marmoset celb (Miller and Lipman, 1973b). The actuaI reasons, either at a cellular or biochemical level, for the differences in the rates of activation and release of extracellular virus by transformants originating from different species is a problem unexplored in its basic aspects. Whether these differences are due to fundamental differences in the type of peripheral blood cell which is transformed or due to qualitative differences in the control mechanisms or a mixture of both, is not known. Until two EBV-transformed cell strains releasing comparable number of viral particles were available, it was not feasible to compare the biological properties of different EB strains. However, it is now possible to state with certainty that there are at least two different biological EBV variants. Transformed simian cells (line B95-8) release virus with the capacity to immortalize lymphocytes and to stimulate DNA synthesis in lymphocyte cultures (Miller et al., 1974a). By contrast, the prototype P, J-HR-1 virus obtained from Burkitt lymphoma cells has neither the capacity to stimulate DNA synthesis nor cause the establishment of

186

A. FRANK,

W. A.

ANDIMAN,

AND G . MILLER

LCL. Furthermore, the transforming biotype induces only EB nuclear antigen (EBNA) in the EBV genome-free BJA-B line, whereas the abortive infecting biotype induces both EBNA and EA (Klein et d.,1974). The P,J-HR-1 line, however, did release transforming virus early in its laboratory history, suggesting that both types of viral particles may be produced by the same fine ( Henle et aE., 1967; Gerber et aE., 1969b). IV. Experimental Infection of Nonhuman Primates with EBV

A. HISTORICAL BACKGROUND Experimental inoculation of monkeys with substances derived from mononucleosis patients can be divided into two major phases: those experiments done prior to a general recognition of the association of EB virus with infectious mononucleosis ( I M ) , Burkitt’s lymphoma (BL), and nasogharyngeal carcinoma ( NPC ) and those experiments performed subsequent to the recognition of these associations. The earliest experiments attempted to find a specific causative agent for a disease ( I M ) that was clinically felt to be a nosologic entity. The difficulty of this type of experimentation in the absence of any markers for an etiologic agent are illustrated in Table IVA, which lists several attempts (experiments Nos. 1-5) to infect the most commonly used laboratory primate, namely, Macaca mulatta, with a host of biological and pathologic products derived from patients with IM and later from BL. As can be seen from the table, more than 50 rhesus monkeys have been inoculated and, in general, have exhibited either no responses or ones that must be considered nonspecific. Two animals developed splenomegaly, and one lymphadenitis and fever. Only one of the animals developed antibody rise to EB VCA. The difficulties in this type of experimentation in the absence of a definite marker should be apparent: It is now known that many individuals of the Old World monkeys have naturally acquired antibodies that cross-react with EBV, and therefore many of these animals might be considered immune. Several of the studies employed newborn animals with maternal antibodies, and these too may have been immune. The inocula used may, with the exception of study No. 7, have contained minimal amounts of virus or none at all, despite the fact that the patient-derived materials were chosen with logic and were used fresh. Some studies employed samples obtained from the nose and throat of acute mononucleosis patients, but it is now known that fewer than 10 transforming units of EBV per milliliter of throat washing are detected in the usual infectious mononucleosis case (Lipman et aE., 1975) and there is no virus in the nose. Blood and

TABLE IVA CHRONOLOGY OF ATTEMPTSTO INFECT Macaca mulalta WITH MATERIALS THOUGHT TO CONTAIN THE ETIOLOGIC AGENT OF INFECTIOUS MONONUCLEOSIS (IM) AND WITH EPSTEIN-BARR VIRUSIEBV) ~

Expt. No.

Inoculum (strain)

No. of animals

Age

EBV antibody status when inoculated

No. develop- Clinical and ing anti- pathological response VC An

1

Lymph node emulsion

1

Adult

Unknown

ND

2

Multiple samples from acute I M cases Sera from acute I M cases Blood from acute I M cases Buffy coat from I M patients

4

Adult

Unknown

Unknown Adult

Unknown Unknown

3 4 5

6

7

Mixture of Burkitt’s lymphoma cell lines (Raji PaJ-HR-1) Cell-free EBV (B958)

16 2 12

4 8

Baby 10 Weeks

2

Yearling Newborn

2

a

VCA, viral capsid antigen; ND, not done or not stated.

~

_

_

_

_

~

References Wising (1939)

ND

Lymphadenitis Fever None

Bang (1942)

ND ND

None Splenomegaly

Evans et al. (1953) Joncas et al. (1966)

Seronegative

0

None

Gerber et al. (1969a)

Low level maternal antibodies Seronegative Maternal antibody

1

None

Cohen et al. (1974)

None

G. Miller, G. vanwagenen, and D. M. Horstmann (unpublished)

188

A.

FRANK, W. A.

ANDIMAN,

AND G. MILLER

various components of the blood have been the most frequently used inocula. These too probably have very low infectivity for a variety of reasons. EBV in the peripheral blood leukocytes may be in a nonproductive state during the acute phase of mononucleosis, and the cells might need to be placed in uitm before infectious virus is produced (Rickinson et al., 1974). If present in the serum the virus may be mixed with antibody. Even though most blood donors are EBV antibody-positive and therefore presumed EBV carriers, the frequency of posttransfusion heterophile-positive mononucleosis is very low ( Henle et uZ., 1970b). Even when seronegative macaques were used and when the inoculum was known to contain biologically active EBV, the rhesus monkey proved to be refractory to infection (experiment No. 7). Thus, Table IVA illustrates that some primate species may not be susceptible to in uiuo infection with human strains of EBV even though members of these same species have EBV-reactive antibodies. The evidence that ultimately linked the herpeslike virus, EBV, with IM, BL, and NPC was initially derived from comprehensive epidemiologic and serologic studies (Henle et al., 1968, 1969, 1970a; Evans et al., 1968). Once these associations became evident and serologic tools were available to assess the response of inoculated primates a more definitive design of experimental infection in nonhuman primates could be made. The inocula were more carefully selected beforehand for the presence of EBV antigens and of biologically active virus. EBV-specific serologic tests were used to detect inapparent infection in the absence of gross pathologic changes. The ability of the virus to cause continuous proliferation of lymphocytes was used to characterize the course of experimental infection by the derivation of LCL from peripheral blood, lymph nodes, and other sites of inoculated animals. OF VARIOUS INOCULA CONTAINING EBV B. CHAMCTEIUSTICS (Table IVB) The successful infection of nonhuman primates was dependent on a number of characteristics of the virus inoculum as well as of the experimental host. Too few experiments have been done to be able to evaluate definitively the importance of various factors associated with the inocula, such as the strain of virus employed or whether the inoculum was given in the form of EBV-converted cell lines or cell-free virus. In some instances, crude cell extracts of human LCL have been used. The P,J-HR-l EB virus strain, presently incapable of cell transformation in uitro, has not induced antibodies or disease in cotton-top marmosets in three different laboratories (Falk et al., 1974; Wolf et al., 1975; Miller et al., 1974a). However, in early experiments, when the P,J-HR-l strain

TABLE IVB

PRIMATE SPECIES THATHAVEBEENEXPERIMENTALLY INFECTED AFTER INOCULATION OF EPSTEIN-BARR VIRUS(EBV) As DEMONSTRATED BY DEVELOPMENT OF ANTIBODIES,RECOVERY OF VIRUS, A N D OCCURRENCE OF PATHOLOGIC LESIONS No. develNum- oping ber antiNo. of animals 04 VCA (site, time) ani- antifrom which Route“ mals bodies virus recoveredd iv, “it” 3 3 0

Species Gibbon

Inoculum (strain) Cell-free EBV (P,J-HR-l)

iv, sc

4

iv, sc

2

Owl monkeys

Autochthonous EBVconverted cell lines Alloseneic EBV-converted cell lines (Maku, Kaplan) Cell extract (EB,)

iP

3

1

Autochthonous EBV converted cells (883L) Cell-free EBV (B95-8)

iv, ip, sc

4

4

0

iv, ip, sc

12

8

5 (lymph nodes blood, s leen, 3-9 weeis)

Cell-free EBV (HR-1)

iv, ip, sc

1

0

Allogeneic EBVconverted cells (Kaplan) Cell-free EBV (HR-1) Cell-free EBV (B95-8)

ip, irn

2

iv ND

Cell-free EBV (Kaplan)

im

Cotton-top marmoset

1

No. (type) of pathologic lesions 2 (tonsillitis)

References Werner et al. (1972b)

1 (blood, 4 weeks)

None

Werner et al. (1972a)

1 (lymph node,

Epstein et al. (1973a,b)

0

1 (“reticuloproliferative” hyperplasia of lymph nodes) 1 (reticulum cell lymphoma) 4 (reticulum cell lymphoma) 5 (lymphoid hy perplasia) None

2

NDc

None

4 5

0 ND

ND ND

3

1

None Falk et al. (1974) Falk et al. (1974) 1 (lymphoma mixed cell) 1 (lymphosarcoma) Wolf et al. (1975)

2 3b

14 weeks)

1 (also viral DNA in tumor) 0

Shope et al. (1973) Miller et al. (1974b) Miller et al. (1974b) Falk et al. (1974)

Wolf et al. (1975) None Cell-free EBV (HR-1) im 3 0 Routes: iv, intravenous; it, intratonsillar; sc, subcutaneous; ip, intraperitoneal; im, intramuscular. One additional animal with anti-viral capsid antigens (VCA) 1: 10 before inoculation developed a rise in antibody. c ND, not done or not stated. d In the form of LCL with EBV antigens.

m

z

z

2

s

Ez

?I

v,

w

Cn

(D

190

A. FRANK, W. A. ANDIMAN, AND G . MILLER

was capable of cell transformation, the virus also induced antibodies upon inoculation into gibbons (Werner et aE., 1972a,b). It would appear from this evidence that only transforming virus is capable of infecting primates. The earliest examples of lymphoproliferative disease in nonhuman primates were induced by a strain of EBV, B95-8, which was derived from marmoset peripheral blood leukocytes transformed in uitro by a mononucleosis-derived strain (Shope et al., 1973). This strain has the advantage of releasing a high titer of virus with transforming ability into the medium of the culture. Experimental infection has also been achieved with virus obtained from a clone of B95-8 cells and from a line obtained from a tumor induced by B95-8 virus. On the basis of antigenic tests as well as study of viral DNA, the B95-8 strain of EBV is very closely related to the prototype human strain (see Section 11). It is important to note that experimental lymphoproliferative disease has also been induced in New World monkeys with the human virus strains EB, and Kaplan, which have never been passaged through cells of another host (Epstein et d.,1973a; Wolf et al., 1975).

C. HOSTRANGEOF EXPERIMENTAL INFECTION (Tables IVB, IVC ) On the basis of published studies, nonhuman primates fall into three groups with respect to susceptibility to infection by EBV. Clear-cut evidence has been obtained for successful infection of three species: the gibbon ape, the owl monkey, and the cotton-top marmoset (Table IVB ). The evidence which indicates that experimental infection has taken place in these species is the development of persisting EBV-specific antibody, the recovery of EB virus from inoculated animals, and the development of some form of Iymphoproliferative disease. In the second category (Table IVC) are three primate species that appear to be of low susceptibility to experimental infection in v i m even though their cells can be transformed in uitro. In this category are the squirrel monkey, the white-lipped marmoset, and the lagothrix or woolly monkey (Table IVC). Antibodies to EBV can be induced in the squirrel monkey by hyperimmunization, but they do not regularly form after inoculation of cell-free virus (Shope and Miller, 1973). Only infrequently have white-lipped marmosets developed antibody responses of low level. Similarly, lagothrix monkeys, when given cell-free virus or autochthonous EBV-converted cells, developed an extremely low antibody response, which is probably the result of an immune response to the inoculated antigen rather than to any viral replication (W. A. Andiman and G. Miller, unpublished, 1975). In only one instance has EBV virus been recovered from inoculated animals of these three species. A cell line

TABLE IVC PRIMATE SPECIESWHOSECELLSARE SUSCEPTIBLE TO EPSTEIN-BARR VIRUS(EBV) in Vitro APPEAR RESISTANT TO EXPERIMENTAL INFECTION in Vim

Species Squirrel monkey Squirrel monkey White-lip marmoset White-lip marmosetb White-lip marmosetb White-lip marmosetb Woolly monkey

Inoculum Autochthonous EBVconverted cells (883L) Cell-free EBV (B95-8) Autochthonous EBVconverted cells (Kaplan) Allogeneic EBVconverted cells (Kaplan) Xenogeneica EBVconverted cells (Kaplan) Cell-free EBV-HR-1 Cell-free virus Autochthonous EBVconverted cells

Two squirrel monkey lines; 1 human line. Newborn or neonatal. One week after intrathecal inoculation. d ND, not done or not stated. c In the form of LCL with EBV antigens.

No.

No. deNo. from veloping which virus antibodies recoverede

BUT

THAT

References

3

0

6

0

NDd

I

I

ND

G. Miller and L. V. Melendez (unpublished) Falk et al. (1974)

4

1

ND

Falk et al. (1974)

Shope and Miller (1973)

3

0

ND

Falk et al. (1974)

2 4 2

0 3 2

ND 0

Falk et al. (1974) W. A. Andiman and G. Miller (unpublished, 1975)

1 C

i

F

3

*1 tr

2

3 E

2, 'd

??

z

%

R

192

A. FRANK, W.

A. ANDIMAN,

AND G. MILLER

was obtained from a lagothrix monkey 1 week after intrathecal inoculation with autochthonous EBV converted cells. The recovery of this cell line probably represents recovery of the inoculated cells. In the third category are animals that are resistant to infection with EBV, particularly Old World monkeys. (Table IVA). Factors governing susceptibility to infection by this virus are obviously complex; however, they must include the availability of cells which can be infected by EBV and might include the extent to which the infected cells propagate more virus. For example, EBV-transformed cells of the lagothrix, a species which is resistant to infection with EBV, release very little extracellular virus. By comparison the cells of the most susceptible species, the cotton-top marmoset releases large quantities of extracellular virus. Thus far, no systematic inquiry has been made, using seronegative animals and the same virus inoculum, to explore in detail the host range in nonhuman primates.

D. CLINICALAND PATHOLOGIC RESPONSES A spectrum of pathologic responses, including inapparent infection, hyperplasia of mesenteric lymph nodes, and malignant lymphoma of the mesenteric and mediastinal lymph nodes, has been observed among individuals of the most studied susceptible species, the cotton-top marmoset (Miller et uZ., 1974b). At least two cell types are involved in both the hyperplastic and malignant changes of involved lymph nodes. The earliest changes appear to be enlargement of centers of the germinal follicles in the cortical region of the lymph node. There is a marked increase in the number of reticulum cells. In some instances, the entire node including the capsule and extranodal sinuses may be replaced by a sheet of reticulum cells (Fig. 1). On the other hand, some parts of involved lymph nodes demonstrate predominantly lymphoblastic proliferation in which the lymphoblasts are heavily infiltrated by histiocytic cells giving a starry sky appearance which is reminiscent of Burkitt lymphoma. Epstein et al. (1973a) have emphasized that, in the lymphoproliferative disease of an owl monkey after EBV inoculation, there was marked heterogeneity of cell types found in affected nodes. Plasma cells, reticulum cells, lymphocytes, and eosinophiles were all present in excess. In mme marmosets extensive hyperplasia of lymph nodes, which is apparent between week 4 and week 8 after inoculation, ultimately regresses and the animal recovers completely. In at least two instances we have studied, normal lymph node architecture was evident at autopsy a year or more after inoculation, whereas considerable lymphoid hyperplasia was prevalent at the time of a biopsy taken in the second month after inoculation.

EBV AND NONHUMAN PRIMATES

193

FIG.1. Histologic section from a mesenteric lymphoma that occurred after inoculation of a cotton-top marmoset with Epstein-Barr virus. The cytology is that of a reticulum cell sarcoma. ~200.

194

A. FRANK, W. A. ANDIMAN, AND C. MILLER

The mode of spread of EBV in the infected animal is primarily local even though the virus can be recovered from blood leukocytes 3-5 weeks after inoculation (Shope, 1975; Miller, 1975). Both Wolf et al. (1975) and Falk et al. (1974) observed that the major lymph node pathology occurred in nodes that drained the site of inoculation. Similarly, in marmosets given EBV intraperitoneally, the major pathology is found in the mesenteric lymph nodes. Gibbons inoculated with EBV directly into the tonsil developed tonsillitis ( Werner et al., 1972b). The factors that contribute to the varied pathologic responses are unknown. In cotton-top marmosets lymphoma has occurred more often in animals that were also given a regimen of azathioprine and prednisone for immunosuppression, but it has occurred also in nonimmunosuppressed animals. It would be expected that age might play a significant role in the pathologic responses, but this has not been studied in detail. Animals used for these studies are nearly all caught in the wild and have indigenous parasites and other diseases. Until laboratory bred stocks of New World monkeys become regularly available it will be difficult to dissect the contribution of many components of the host response. It is to be emphasized, however, that the host is crucial in determining the outcome, since the response of animals given exactly the same inoculum has ranged from inapparent infection to lymphoma. E. SEROLOGIC RESPONSES Most, but not all, members oi susceptible species listed in Table IVB have demonstrated EBV antibody responses following inoculation. Usually, antibodies to VCA have been measured although evidence has also been obtained for the development of antibodies to EBV-associated complement-fixing antigens and early antigens. Gibbons inoculated with autochthonous cultured lymphoblasts developed anti-VCA antibodies 1-5 weeks after inoculation; maximum titers were reached at approximately 6 weeks after inoculation (Werner et al., 1972b). In cotton-top marmosets, there was a delay of 4-6 weeks before the appearance of antibody; and titers never reached very high levels except in those animals that developed lymphoma. Lymphoma was accompanied by a rise in antibody titer and the appearance of antibodies to the EBV-specified early antigen. Two animals with lymphoma died before antibodies developed (Miller et al., 1974b).

F. DEMONSTRATION OF EBV

IN THE

PATHOLOCIC LESIONS

Virions and capsid antigens are not found directly in the lymphomatous or hyperplastic lymph nodes. However, in two tumors composed

EBV AND NONHUMAN PRIMATES

195

of a uniform population of reticulum cells, the EB nuclear antigen was detected in most of the cells in an imprint (Miller and Coope, 1974). Wolf et al. (1975) have recently demonstrated EBV complementary DNA by the technique of DNA reassociation kinetics in a lymphosarcoma induced in marmosets by the KapIan EBV strain. When lymph nodes from inoculated animals have been placed in cell culture, continuous LCL have formed which are productive of VCA and which release mature virus (Shope et al., 1973; Epstein et at., 197313). Work in progress in our laboratory indicates that the virus released from the cell lines is biologically and antigenically similar to theJ3BV in the inoculum. A cell line obtained from an inoculated owl monkey has been shown to contain EB viral DNA on the basis of annealing with EBV cRNA (Epstein et al., 1975). The cell-virus relationship in the marmoset lymph nodes is analogous to that which occurs in EBV infections of man. Cell lines have been obtained from cotton-top marmoset lymph nodes exhibiting hyperplasia as well as those with lymphoma. Furthermore, EB virus containing cell lines have been recovered from histologically normal spleens and from peripheral blood of infected animals, which developed no other disease. Recovery of EBV-containing cell lines from the pathologic lymph nodes is not, by itself, conclusive proof that the lesions were induced by EBV since these lines could be derived from relatively few cells which might be present in normal and pathologic lymphoid cells. Recently, Wolf et al. (1975) and Shope (1975) have performed in vivo virus neutralization experiments on a small scale and have found that when EBV is premixed with antibody the lymphoproliferative lesions do not occur. This evidence too suggests that indeed EBV is responsible for the lymphoproliferative disease.

G. SIGNIFICANCE OF EXPERIMENTAL INFECXION AND TUMORIGENESIS IN NONHUMAN PRIMATES BY EBV The development of lymphoproliferative disease and the demonstration of the viraI genome in the affected lymph nodes provides direct evidence of the disease-producing potential of EBV. A number of parallels between experimental infection of nonhuman primates and natural infection of man have been noted. There is a spectrum of responses. The cell-virus relationship and certain aspects of the pathology of involved lymph nodes are similar. Taken together, this evidence shows that the ability to induce lymphoproliferative disease in primates is a biological property of EBV. The preliminary results obtained so far indicate that this lymphoproliferative response is not restricted to only one species of nonhuman primate, nor is it limited to only one strain of the virus. There

196

A. FRANK, W. A. ANDIMAN, AND G. MILLER

A SUMMARY

OF

Primate class Prosimians New World monkeys Old World monkeys Great apes a

TABLE V RESPONSES O F NONHUMAN PRIMATES TO EPSTEIN-BARR VIRUS(EBV) OR ITSCLOSERELATIVES~ Natural Lymoccurphocyte rence of EBV transEBVreactiv- forma- Experi- Lymphoprolifreactive Sponta- ity in tion in mental antineous simian vitro by infecerative bodies LCL LCL EBV tion disease

-

+ +

-

+ +

NA NA

+ +

-

+ +

ND

+ +

NA

+

NA +b

LCL, lymphoblastoid cell lines; NA, not applicable; ND, not done.

* Tonsillitis only.

is clear evidence that both virus and host differences will ultimately be important in defining the pathologic response. One question which was germinal to experiments in nonhuman primates was: “Are peripheral blood leukocytes transformed in oitpo by EBV oncogenic when replaced in the autochthonous host?” The answer to this question must still be considered unresolved. Three of four cottontop marmosets failed to develop tumors in response to inoculation of autochthonous EBV converted cells, and so did 4 gibbons (Shope et al., 1973; Werner et al., 1972a). One cotton-top marmoset developed a lymphoma after a long latent period of 7.5 months. Since marmoset cells transformed by EBV release virus, it cannot be stated with certainty whether the cells converted in uitro were the source of the tumor or whether the tumor was induced by virus released from the inoculated cells. Nonetheless there now seems to be little doubt of the tumorigenic potential of the virus per se. While New World primates offer exciting laboratory models for the study of tumorigenesis their use must be viewed conservatively in terms of translating the observations to human disease. It is clear from work with Herpes saimiri and Herpes ateles that the oncogenicity of these viruses is predominately controlled by the host response; the same virus may be oncogenic in one species but not in another (Laufs et al., 1975). It is well to bear in mind this problem of crossed-species oncogenicity considering the experiments with EBV. However, EBV infection of marmosets appears to be different from that obtained with Herpes saimiri.

EBV AM) NONHUMAN PRIMATES

197

In the latter infection oncogenicity is the rule even with very small inocula; in the former the response is much more variable and the percentage of “takes” in the form of lymphoma much less. This finding indeed suggests that the marmoset may be an appropriate experimental model on which to base further experiments designed to elucidate the pathogenesis of EBV infections in man, since variability of host response is so characteristic of human infections with this agent. The nature of the pathogenesis of lymphoproliferative disease which follows EBV infections in man or in nonhuman primates is still poorly understood. A variety of indirect effects of the virus could be postulated, such as activation of endogenous (presumably RNA) tumor viruses or depression of normal “immunologic surveillance” mechanisms. There is as yet no evidence for these mechanisms. Some of the events are surely attributable to direct transformation by the virus of lymphoid and related elements into cells with increased growth potentid. However, other mechanisms may be operative as well. Some lymphoid hyperplasia, particularly the transient form, may reflect primarily a response of sensitized lymphocytes to relatively few transformed cells. V. Summary and Conclusions (Table V )

Antibodies that cross-react with EBV capsid antigen and also with soluble EBV-associated complement-fixing antigens are found in sera from Old World monkeys and apes but not in sera from prosimians or New World monkeys. Old World monkeys and apes trapped in the wild show signs of infection with EBV or an antigenic relative which is, therefore, presumably enzootic among them. Lymphoblastoid cell lines that contain EBV-like viruses and cross-reacting antigens can readily be obtained from bIood leukocytes of chimpanzees and on occasion have been derived from baboons and macaques. This is further evidence for natural acquisition of EBV-related viruses in Old World monkeys and apes. By contrast, spontaneous lymphoblastoid transformation of blood leukocytes has not been observed in New World monkeys or prosimians. Is the EB virus that infects Old World monkeys and apes identical to human EBV? A direct answer to this question will come from a comparison of the structure of EBVs derived from lymphoid cell lines of diverse origins. However, some indirect evidence suggests that EBV reactivity in Old World monkeys is not due to the human virus. Leukocytes of seronegative macaque monkeys cannot be infected with high concentrations of EBV of demonstrable capacity to infect human and New World monkey cells. Furthermore, antibody-negative macaque in-

198

A. FRANK, W. A. ANDIMAN, AND

G. MILLER

dividuals cannot be induced to form antibodies after inoculation of amounts of EBV that are capable of infecting marmosets. One possible explanation is that Old World monkeys, in evolution, have acquired their own EBVs and are not susceptible to the type that now infects man, On the other hand, receptors for human EBV and susceptibility to infection has been retained by apes (gibbons). Thus, it may turn out that EBVs recovered from apes will closely resemble the human variants. Another important unanswered question is whether the EBV-like viruses naturally found in baboons, macaques, and apes cause disease in their natural host. There is no direct information on this point, although it is germane to mention that at least one EBV-like agent was identified in leukocytes from a rhesus monkey with leukemia following procarbazine treatment. Cells of New World monkeys and apes are “immortalized,” i.e., grow indefinitely after they are experimentally infected by EBV in uitro. EBVtransformed cells from various New World monkey species differ in the degree to which they yield extracellular virus; cotton-top marmoset cells are the most permissive and woolly monkey cells the least permissive studied so far. Although the hypothesis has not been rigorously tested, it is worth speculating that lymphoid cells of the different species of primitive tamarins and marmosets will prove to be more permissive than those of the more advanced cebid monkeys. It would be of interest to learn whether the extent of upermissiveness” of New World monkey cells to human viruses could provide clues about the evolution of primates. Not all species whose cells can be transformed in uitro can be experimentally infected. However, cotton-top marmosets, owl monkeys, and gibbons can be infected with EBV. Lymphoreticuloproliferative disease has occurred in marmosets and owl monkeys. The disease has varied from malignant lymphoma of the reticulum cell or mixed reticulum cell and lymphobiastic type, to extensive but reversible lymphoid hyperplasia to inapparent infection. Gibbons have manifested tonsillitis after inoculation. Different animals given exactly the same inoculum have shown this spectrum of pathologic responses. Thus host differences, unexplored in their basic nature, would seem crucial in determining the outcome of experimental infection. EBV has been demonstrated in the experimental lesions in the form of the viral genome and the EB nuclear antigen. Virus-producing cell lines have been derived from tumors and hyperplastic lymph nodes. Thus the essential characteristics of the EB-host relationship observed in man has been reproduced in a laboratory animal.

EBV AND NONHUMAN PRIMATES

199

ACKNOWLEDGMENT We are indebted to Mrs. V. Thompson for her invaluable help with the manuscript.

REFERENCES Bang, J. (1942). Acta Med. Scand. 111,291402. Buettner-Janusch, J. ( 1963 ). “Evolutionary and Genetic Biology of Primates,” Vol. 1. Academic Press, New York. Chu, C.-T., Yang, C.-S., and Kawamura, A., Jr. (1971). Appl. Microbiol. 21, 539-540. Cohen, M. H., Bernstein, A. D., and Levine, P. H. (1974). Oncology 29,353463. Deinhardt, F., Falk, L. A., and Wolfe, L. G. (1974). Cancer Res. 34,1241-1244. Dunkel, V. C. (1973). Ann. Clin. Lab. Sci. 3,424428. Dunkel, V. C., and Myers, S. L. (1972). 1. Nut. Cancer Inst. 48, 777-782. Dunkel, V. C., Pry, T. W., Henle, G., and Henle, W. (1972). J. Nut. Cancer Inst. 49, 435. Epstein, M. A. (1970). Aduan. Cancer Res. 13, 383411. Epstein, M. A., Hunt, R. D., and Rabin, H. (1973a). Int. J. Cancer 12,309418. Epstein, M. A., Rabin, H., Ball, G., Rickinson, A. B., Jarvis, J., and Melendez, L. V. (197313).lnt. J. Cancer 12, 319432. Epstein, M. A., zurHausen, H., Ball, G., and Rabin, H. (1975). Int. J. Cancer 15, 17-22. Evans, A. S., Evans, B. K., and Sturtz, V. (1953). Proc. SOC. Exp. Biol. Med. 82, 437-440. Evans, A. S., Niedennan, J. C., and McCollum, R. W. (1968). N. Engl. J. Med. 279, 1121-1127. Falk, L. A. (1974). Lab. Anim. Sci. 24, 183-192. Falk, L. A., Wolfe, L., Deinhardt, F., Paciga, J., Dombos, L., Klein, G., Henle, W., and Henle, G. ( 1974). Int. J . Cancer 13,353-376. Frank, A. L., Bissell, J. A., Rowe, D. S., Dunnick, N. R., Mayner, R. E., Hopps, H. E., Parkman, P. D., and Meyer, H. M. (1973). J. Znfec. Dis. 128,618-629. Gerber, P. (1972). Proc. Nut. Acad. Sci. US.69, 83-85. Gerber, P., and Birch, S. M. (1967). Proc. Nut. A c d . Sci. U.S. 58, 478-484. Gerber, P., and Lorenz, D. (1974). Proc. SOC. Exp. Biol. Med. 145, 654-657. Gerber, P., and Lucas, S. (1972). Proc. SOC. Exp. Biol. Med. 141, 431-435. Gerber, P., and Rosenblum, E. N. (1968). Proc. SOC. Exp. Biol. Med. 128,541546. Gerber, P., Branch, J. W., and Rosenblum, E. N. (1969a). Proc. SOC. Exp. Biol. Med. 130, 14-19. Gerber, P., Whang-Peng, J., and Monroe, J. H. (1969b). Proc. Nut. Acad. Sci. US. 63, 740-747. Goldman, M., Landon, J. C., and Reisher, J. I. (1968). Cancer Res. 28, 2489-2495. Goodheart, C. R. (1973). Cancer Res. 33, 1443-1445. Hampar, B., Derge, J. G., Martos, L. M., and Walker, J. L. ( 1972). Proc. Not. Acad. Sci. U.S. 69, 78-82. Henle, G., and Henle, W. (1966). 1. Bacteriol. 91, 1248-1256. Henle, G., and Henle, W. (1967). Cancer Res. 27, 2442-2446. Henle, G., Henle, W., and Diehl, V. (1968). Proc. Nut. Acad. Sci. US.59, 94-101. Henle, G., Henle, W., Clifford, P., Diehl, V., Kafuko, G. W., Kirya, B. G., Klein, G., Morrow, R. H., Munube, G. M. R., Pike, P., Tukei, P. M., and Ziegler, J. L. (1969). 1. Nut. Cancer Znst. 43, 1147-1157.

200

A. FRANK, W. A. ANDIMAN, AND C. MILLER

Henle, W., Diehl, V., Kohn, G., zurHausen, H., and Henle, G. (1967). Science 157, 1064-1065. Henle, W., Henle, G., Ho, H.-C., Burtin, P., Cachin, Y., Clifford, P., deschryver, A., de T h P , G., Diehl, V., and Klein, G. (197Oa). J. Nut. Cuncer Inst. 44, 225-23 1. Henle, W., Henle, G., Scriba, M., Joyner, C. R., Harrison, F. S., von Essen, R., Paloheimo, T., and Klemola, E. (1970b). N. Engl. J. Med. 282, 1068-1074. Henle, W., Henle, G., Zajac, B. A., Pearson, G., Waubke, R., and Scriba, M. ( 1 9 7 0 ~ ) . Science 169, 188-190. Huang, E., and Pagano, J. S. ( 1974). J. Vird. 13,642-645. Jarvis, J. E. (1974). Brit. J. Cancer30,164-167. Joncas, J.. Lussier, G., and Pavilanis, V. (1966). Can. Med. Ass. 1. 95, 151-154. Jondal, M., and Klein, C. (1973). J. Exp. Med. 138, 1365-1378. Kalter, S . S., Heberling, R. L., and Ratner, J. J. (1972). Nature (London) 238, 353-35-1. Kalter, S. S., Heberling, R. L., and Ratner, J. J. (1973). Bibl. Huernatol. (Basel) 39, 871-875. Klein, G. (1973). In “The Herpesviruses” (A. S. Kaplan, ed.), pp. 521-555. Academic Press, New York. Klein, G., and Dombos, L. ( 1973). Int. 1. Cancer 11, 327437. Klein, G., Sugden, B., Leibold, W., and Menezes, J. ( 1974). Interoirology 3,232-244. Landon, J. C., and Malan, L. B. (1971). J. Nut. Cuncer Inst. 46, 881-884. Landon, J. C., Ellis, L. B., and Fabrizio, D. P. A. (1968a). Proc. Amer. Ass. Cuncer Res. 9, 39. Landon, J. C., E h , L. B., Zeve, V. H., and Fabrizio, D. P. A. (196813). 1. Nut. Cancer Inst. 40, 181-186. Laufs, R., Steinke, H., Steinke, G., and Petzold, D. (1975). J. Nut. Cancer Inst. 53, 195-199. Levy, J. A., Levy, S. B., Hirshaut, Y., Kafuko, G., and Prince, A. (1971). Nature ( London) 233, 559-560. Lipman, hl., Andrews, L., Niederman, J., and Miller, G. (1975). J . Infec. Dis. 132, 520573. Miller, G. (1974). J . Infec. Dis. 130, 187-205. Miller, G. ( 1975). In “Oncogenesis and Herpesviruses,” Vol. I1 (in press). Miller, G., and Coope, D. (1974). Trans. Ass. Amer. Physicians 87, 205-218. Miller, G., and Lipman, M. (1973a). Proc. Nut. Acud. Sci. US. 70, 190-194. Miller, G., and Lipman, M. (1973b). I . E r p . Med. 138, 1398-1412. Miller, G., Lisco, H., Kohn, H. I., and Stitt, D. (1971). Proc. SOC. Exp. Biol. Med. 137, 1459-1465. Miller, G., Shope, T., Lisco, H., Stitt, D., and Lipman, M. (1972a). Proc. Nut. Acad. Sci. US. 69, 383487. Miller, G., Niederman, J. C., and Stitt, D. A. (1972b). J . Infec. Dis. 125, 403-406. Miller, G., Niederman, J. C., and Andrews, L. ( 1973). N . Engl. J. Med. 288, 229-232. hliller, G., Robinson, J., Heston, L., and Lipman, M. (1974a). Proc. Nut. Acad. Sci. U.S. 71,4006.1010. Miller, G., Shope, T., and Coope, D. (1974b). I n “Mechanisms of Virus Disease” (W. S. Robinson and C. F. Fox, eds.), Vol. I, pp. 429-454. Benjamin, New

York.

EBV AND NONHUMAN PRIMATES

201

Miller, G., Robinson, J., and Heston, L. ( 1 9 7 4 ~ )Cold . Spring Harbor Symp. Quunt. Biol. 39, 773-781. Minowada, J., Ohnuma, T., and Moore, G. E. (1972). J. Nut. Cancer Inst. 49, 891-895. Naito, M., Ono, K., Doi, T., Kato, S., and Tanabe, S. (1971). B i k m J . 14, 161-166. OGara, R. W., Adamson, R. H., Kelly, M. G., and Dalgard, D. W. ( 1971). J. Nut. Cancer IW.46, 1121-1130. Pope, J. H., Horne, M. K., and Scott, W. (1969). Int. J. Cancer 4, 255-260. Pritchett, R. F., Hayward, S. D., and Kieff, E. D. (1975). J . ViroZ. 15, 556569. Rabson, A. S., O’Connor, G. T., Lorenz, D., Kirschstein, R. L., Legallais, F. E., and Tralka, S. T. (1971). J. Nut. Cancer Illst. 46,1099-1109. Reedman, B. M., and Klein, G. (1973). Int. J . Cancer 11,499520. Rickinson, A. B., Jarvis, J. E., Crawford, D. H., and Epstein, M. A. (1974). Int. J . Cancer 14, 704-715. Robinson, J., and Miller, G. (1975). J. Virol. 15, 1065-1072. Rosenberg, N., Baltimore, D., and Scher, C. D. (1975). Proc. Nut. Acad. Sci. U.S. 72, 1932-1936. Schable, C. A., Murphy, B. L., Berquist, K. R., Gravelle, C. R., and Maynard, J. E. (1974). Infec. Immitnity 10, 1443-1444. Shope, T. ( 1975). In “Oncogenesis and Herpesviruses,” Vol. 11 (in press). Shope, T. C., and Miller, G. (1973). J. Exp. Med. 137, 140-147. Shope, T., Dechairo, D., and Miller, G. (1973). Proc. Nat. Acad. Sci. U S . 70, 2487-2491. Stevens, D. A., Pry, T. W., Blackham, E. A., and Manaker, R. A. (1970). Proc. SOC. E x p . Biol. Med. 133, 879-683. Werner, J., Henle, G., Pinto, C. A., Haff, R. F., and Henle, W. (1972a). Int. J. Cancer 10, 557-567. Werner, J., Pinto, C. A., Haff, R. F., Henle, W., and Henle, G. (1972b). J. Infec. Dis. 126, 678-681. Wising, P. J. (1939). Acta Med. Scand. 98,329-339. Wolf, H., Werner, J., and zurHausen, H. (1975). Cold Spn’ng Harbor Symp. Quant. BioZ. 39, 791-796.

This Page Intentionally Left Blank

TUMOR PROGRESSION AND HOMEOSTASIS

Richmond

T.

Prehn

The Institute for Cancer Research, The Fox Chore Cancer Center, Philadelphia, Pennsylvania

. . .

.

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

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

. . . . .

I. Introduction . . . . . 11. Initiation: The First Step in Tumor Progression . . A. The Clonal Nature of Initiation . . . B. The Latent Tumor Cell or Clone . . C. Induction versus Selection; Genetic versus Epigenetic Change . 111. The Subsequent Steps in Tumor Progression . . . A. The Clonal Nature of Subsequent Steps . . . B. Latency during Progression . . . . . . C. Induction versus Selection in Progression; Genetic versus Epigenetic Change in Progression . , IV. Immunity as a Homeostatic Mechanism . . . A. Introduction . . . . . . B. Induced versus “Spontaneous” Tumors. . . C. Immunological Selection and Surveillance . D. Virally Induced Tumors and Lymphoreticular Neoplasms . . E. Is Subliminal Irnmunogenicity Adequate for Surveillance? . . F. Immunostimulation of Tumor Growth . . . G. Metastasis . . . . . . . H. Conclusions concerning Immunity. . V. Concluding Remarks . . . . . . References . . . . . . .

.

.

.

.

.

.

. . . .

. .

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

. . . .

.

203 204 204 207 211 213 213 213

215 218 218 219 220 222 225 228

231 231 233 233

1. Introduction

The idea that cancer evolves as a series of sequential heritable cellular changes is quite old. It probably had its genesis in the common cIinical observation that human neoplasia often appears to undergo change during its clinical course, such that what was originally a relatively benign tumor of low grade is transformed over a period of weeks or years into a highly malignant and now rapidly lethal disease. It must be emphasized that what I am discussing is not a change in the size or extent of the tumor, but a change in the biological properties of its cells. Transplantation studies have made it clear that the change resides primarily in the tumor cells themselves, usually not in an altered host. This process ‘This investigation was supported by Public Health Service Research Grant Nos. CA-08856, CA-06927, CA-05255, CA-13456, RR-05539 from the National Institutes of Health, and by an appropriation from the Commonwealth of Pennsylvania. 203

204

RICHMOND T. PREHN

of change with time in the biological attributes of the tumor cells was extensively described in the mouse mammary tumor system (Foulds, 1954), and the term “neoplastic progression” was applied. It is widely believed, although still debated, that all tumors may pass through such a process of progression, perhaps in some cases much condensed, and that possibly no malignancy ever results from a single comprehensive change. The fact that tumors undergo progression over a time course that may extend over many years and numerous steps, implies that at each step of the process there are homeostatic mechanisms operating with considerable but imperfect success to limit or reverse the process. Unfortunately, these postulated mechanisms are mostly unknown while some that have been investigated extensively may prove to be of relatively minor importance. In this paper I will largely limit my discussion to those areas of tumor progression and homeostasis actually impinged upon by my own work or that of one or more of my students or postdoctoral associates, in particular, Dr. E. Andrews, now at Cornell; Dr. G. Bartlett, now at Hershey; Dr. M. Basombrio, now at Buenos Aires; Dr. M. LappC, now at Hastings on Hudson; Dr. G. Slemmer, now at Vancouver; and Dr. H. Outzen, still associated with me in Philadelphia. 11. Initiation: The First Step in Tumor Progression

A. THECLONAL NATUREOF INITIATION According to Willis ( 1960), cancer arises as the result of cellular changes over a large area or field. This conclusion was the result of clinical observation, and the accuracy of the observations is beyond question. There can be no doubt that clinical cancer often appears to arise over a wide area rather than focally, and if focal, may sometimes be at least highly multifocal. A striking example of this is the cancer that sometimes arises in an old burn scar. Sometimes the entire broad area of the scar is occupied, seemingly all at once, by the cancer tissue. However, despite this common clinical observation, there are many experimental data showing that, regardless of appearances, the essential nature of the process is clonal. Cancer is usually the result of the clonal amplification of a heritable alteration, which occurred in a single cell. The evidence for this statement is quite extensive and persuasive. Some of the best data concerning the clonal nature of neoplasia arises from a study of glucose-6-phosphate dehydrogenase ( G-6PD ) polymorphisms and their occurrence in neoplasms. This enzyme occurs in several isomeric forms determined by a structural gene on the X chromosome.

TUMOR PROGRESSION AND HOMEOSTASIS

205

It follows, since one X is suppressed in somatic cells, that in heterozygous females, approximately half of the somatic cells will express one form of the enzyme, the other half will express the other. Consequently, if such a female develops a tumor, all the tumor cells, if of monoclonal origin, should exhibit one and the same isozyme. If the tumor had originated from multiple normal ancestor cells, both isozymes would be expected to be present. A variety of tumors have been examined, and the answer has usually been clear; they are, with few exceptions, monoclonal ( Fialkow, 1972). The basically monoclonal nature of a malignant neoplasm is also supported by the fact that all the neoplastic cells can sometimes be shown to carry other markers indicating a common neoplastic precursor cell, for example, an atypical chromosome that is unlikely to have arisen in multiple ancestors. In chemically induced tumors, each tumor can be shown to be antigenically unique and non-cross-reactive, but all the cells of each individual tumor do cross react. Again, this seems to be strong evidence for the clonal origin of each tumor (Prehn, 1970). Neoplastic transformation in tissue culture can frequently be visualized directly as a very focal and almost certainly clonal process. Also, when “initiating carcinogen is painted on the skin of a susceptible mouse followed by a “promotor,” numerous neoplasms can be produced in each animal, each arising as a small punctate lesion with no apparent involvement of intervening areas of skin (Berenblum, 1954). A similar picture is seen in mouse breast oncogenesis. In response to chemical oncogen or virus, numerous small focal hyperplastic lesions are produced throughout the mammary gland with no apparent alteration of intervening tissue (Pullinger, 1952; Bern et aE., 1958). A similar pAttem is seen in chemically induced hepatoma formation (Farber et al., 1975; Kitagawa and Pitot, 1975). Although the evidence is strong that neoplasia is basically a clonal process, it must be recognized that if multiple clones of transformed cells were originally present in a field of tissue, the resulting gross tumor might still be derived largely from only one of them. The growth rates of the clones could hardly be identical, and therefore the most rapidly proliferating would soon predominate in the lesion. It is probable that tumors induced in high frequency by strong carcinogens may often begin as a multiclonal disease which rapidly, by selection, becomes essentially monoclonal (Prehn, 1970). Direct evidence of this was obtained by an analysis of mouse sarcomas induced by the subcutaneous implantation of paraffin pellets containing the carcinogen 3-methylcholanthrene ( Prehn, 1970). Samples from widely separated areas of primary tumors were propagated as separate

206

RICHMOND T. PREHN

sublines by transplantation in syngeneic mice. These separate sublines were tested as to whether or not they were of common origin by examining their antigenic specificities. It is known that chemically induced multiple primary tumors are very seldom cross-reactive ( Basombrio, 1970; Globerson and Feldman, 1964). The question was whether or not the tumor sublines would behave in the manner of independently derived tumors, and each be antigenically specific. It was found that thesublines isolated from the primary tumors did indeed occasionally show individuality in antigenic specificity in the manner of independently derived tumors. It can be inferred that, because of the vagaries of the sampling procedure, the percentage of primary tumors of multiclonal origin must actually have been quite high. In contrast, in no instance could non-cross-reactive subclones be isolated from subsequent tumor-passage generations ( Prehn, 1970 ) . If initiation, or the first heritable change in the cells, is essentially of the focal, clonal nature I have described, why did Willis and other observers arrive at the opposite conclusion? Several possible explanations can be offered, any one or more of which may have obtained in any given instance. The first explanation has already been discussed, namely that some tumors, under conditions of strong carcinogen application and/or high host susceptibility may indeed begin as multifocal lesions or among many cells in a field. This may be the case in some neoplasms of the breast in which multiple primary tumors in one susceptible individual are not rare. The second explanation of the apparent arisal of a tumor by change of many cells over a large area lies in the effect of neoplastic cells on normal cells. It was pointed out by Ewing (1940) that cancer cells could impose a neoplastic appearance on surrounding normal cells (collateral hypertrophy). Experimental verification of this conclusion was provided by Argyris and Argyris ( 1962), who inoculated a transplantable mouse tumor into the dermis and showed that the normal epithelium above the transplant, although not in direct contact with the tumor, became hyperplastic. Appropriate controls established that the effect was not due to mechanical stress, but to some diffusible influence. The third possible explanation for the seeming arisal of some tumors over a large area has recently been experimentally documented by Slemmer (1974). He demonstrated that, under some circumstances in mouse breast oncogenesis, already heritably abnormal cells could apparently migrate through the ductal tree while still maintaining a grossly normal appearance, and their subsequent growth would give the illusion of tumor arisal simultaneously over a wide area of the tree. One caution must be expressed concerning the conclusion that there

TUMOR PROGRESSION AND HOMEOSTASIS

207

is no continuous recruitment of normal cells into tumor cells in the manner proposed by Willis ( 1960). Transplantation studies in genetically controlled mice make it clear that, in a growing tumor, cancer cells are derived from cancer cells, not from the cells of the normal host, but this rule is violated in the case of some tumors of viral etiology in which the cells are productive, i.e., are actively shedding the oncogenic virus into the cellular environment ( Siegler, 1970).

B. THELATENT TUMOR CELLOR CLONE DeCossk has described a function of the immune reaction which holds target cells reversibly in a nondividing state (DeCossk and Gelfant, 1968). Other instances are common in which only a part of the neoplastic phenotype is unexpressed, the proliferative aspect, but the cell or clone remains recognizable morphologically as neoplastic. Perhaps the most dramatic of such examples is provided by mammary tumor development in the mouse. The literature on the mammary tumor of the mouse is very extensive, and its review here would simply detract from the points to be made. An entrk to this literature can be found in a recent publication by Slemmer (1974). The experimental technique that permitted the nature of the latency phenomenon in this system to be explored was provided by the transplantation methods developed by DeOme et al. (1959) and his colleagues. A brief description of this technique and its application is required to understand the conclusions that have been reached regarding latency. The mouse mammary gland develops by the growth of a ductal tree beginning at the nipple and extending into a well defined and circumscribed subcutaneous fat pad, the mammary fat pad. Prior to puberty, the gland consists of a rudimentary ductal tree that extends only a few millimeters from the nipple. At puberty, a rapid proliferation of the ducts begins by growth at terminal end buds, and the ducts rapidly extend into and through the fat pad, until the pad is well filled by the branching ductal tree. Attempts to transplant and obtain growth of ducts in other areas of the body fail, although some growth may be obtained in other fatty tissues, such as the “brown fat.” Transplantation of the entire gland-containing fat pad was achieved by Prehn (1953) and subsequently in the rat by Dao et al. (1964). Transplantation with subsequent growth is readily obtained if, prior to puberty, a fragment of duct is placed in a distal, and thus gland-free portion of the mammary fat pad of a syngeneic female. Transplantstion with growth can also be achieved when a ductal fragment is placed in the fat pad of an

208

RICHMOND T. PREHN

adult syngeneic female recipient if that fat pad had previously been “cleared of endogenous mammary gland. Clearing is accomplished by the excision of the nipple area and the adjacent mammary rudiment prior to puberty. A fragment of duct placed in an “uncleared” fat pad, i.e., a fat pad already filled with endogenous gland, fails to grow. When oncogenesis is produced in the mouse mammary glands by feeding chemical oncogens, by action of the mammary tumor virus, and/or by hormonal treatment, the initial lesion appears to be a small benign tumor, the so-called hyperplastic nodule. Numerous focal lesions of this type can often be visualized scattered throughout the ductal tree. They grow to a size of one to several millimeters, and then cease to enlarge. This state of dormancy or latency usually persists for long periods, perhaps throughout the entire life of the mouse. Whether or not they may sometimes regress is not known. It is only rarely that one of these lesions undergoes progression and acquires the capacity for renewed growth. The transplantation procedures of DeOme have permitted the nature of this dormant or latent period to be explored. If a hyperplastic nodule is transplanted to an uncleared fat pad, the transplant will persist indefinitely and usually not grow, just as it would have done had it been left in situ. However, when transplanted to a cleared fat pad, the hyperplastic nodule grows and eventually fills the fat pad with an abnormal hyperplastic grand (benign tumor). Such transplanted tumors usually grow slowly in the cleared fat pad and, like normal‘gland, will not grow beyond the confines of the pad. Such transplantation studies, showing that the nodule will grow only in cleared fat pads, make it apparent that the heritably abnormal cells of the hyperplastic nodule are, like normal gland cells, inhibited from growth by the surrounding normal ducts. They can thus remain latent for extended periods, until further progression endows them with the capacity to overcome the inhibition. The nature of the inhibitory principle, apparently elaborated by the normal ducts, is completely unknown. It does appear, however, that a major distinction between the normal and the hyperplastic nodule tissue is that the former both elaborates and responds to the inhibitory principle; the hyperplastic nodule usually responds to, but does not elaborate the principle. The recent work of Slemmer (1974)has added a new dimension to the study of the growth potential of the hyperplastic nodule. His analysis has suggested that the mammary gland is probably composed of three distinct lineages of epithelial cells: the ductal, the alveolar, and the myoepithelial. The initial neoplastic change may occur in any one of these three. The most interesting finding was that, in the early hyper-

TUMOR PROGRESSION AND HOMEOSTASIS

209

plastic benign lesion, the neoplastic cells were often associated with one or both of the other normal epithelial cell types. This association was obligatory, the neoplastic component being unable to grow in a cleared fat pad, except in association with the normal population. Since Young et al. (1971) have shown that normal mammary ducts grow poorly when transplanted to old, endocrine-deficient animals, it is possible, although still unproved, that the growth of an early breast neoplasm may sometimes be endocrine-dependent because of the endocrinedependency of the associated and necessary normal cell component. Latency in the breast tumor system is apparently not influenced by the immune mechanism-immunity in this system will be discussed at some length in Section IV. The problems connected with initiation, latency, and progression have been extensively examined in oncogenesis of mouse skin, particularly in the case of chemical etiological agents. The work of Rusch and Kline ( 1946), Rous and Kidd ( 1941), and subsequently of Berenblum and Haran ( 1955) established the so-called “two-stage hypothesis.” If a chemical oncogen, for example an oncogenic hydrocarbon, is applied to the skin of a mouse in a suboncogenic dosage, the skin will appear overtly normal after the initial inflammation, and consequent generalized hyperplasia, has subsided. However, despite the lack of overt changes, a profound and long lasting change has, in actuality, occurred. If, at some subsequent time, which may be as long as a year or more, a second similarly small dosage of the oncogenic chemical is applied, neoplasia may rapidly ensue. In other words, the effects of the chemical oncogen are cumulative even when the interval between applications is very long (Boutwell, 1964). Berenblum postulated that the invisible change represented by “initiation” was due to the presence in the oncogen-treated skin of “latent tumor cells” ( Berenblum and Haran, 1955). These could subsequently be made to proliferate and develop into gross neoplasms by the second application of the chemical, in this case, acting as a “promoter.” The reason for believing that the first application of oncogen produced latent, rather than some type of defective or incomplete tumor cells, was that promotion, i.e., the second step, did not apparently require a chemical oncogen. In the classical system, initiation was produced with a subthreshold dose of a hydrocarbon oncogen; promotion was accomplished by the repeated application of the highly irritating substance, croton oil. Croton oil alone, in any dosage, was only marginally oncogenic, and if the experimental procedure was reversed, i.e., if the croton oil preceded the hydrocarbon, few if any tumors were produced.

210

RICHMOND T. PREHN

The interpretation that initiation produced complete latent tumor cells was reinforced by the work of Lapp6 (1968), who showed that a very effective promoter was simple skin grafting. Apparently all that was required for promotion was the production of compensatory hyperplasia in the skin that was previously initiated. As in the case of Berenblum’s work, if the skin grafting preceded the oncogen by an interval sufficient that the hyperplasia had subsided to the normal resting state, no tumors were produced. A variety of other forms of irritation, such as simple wire brushing, will also serve as a promoter (Deelman, 1927). However, promotion is apparently not cumulative unless the applications of promoter are made at short intervals (Boutwell, 1964). It certainly does seem that initiation and promotion may be qualitatively distinct processes. It perhaps should be noted at this juncture that there is a difference between promotion with a complete oncogen, an agent that can serve as both initiator and promoter, and promotion with a pure promoter-an agent, such as skin grafting or croton oil, which has little or no capacity to initiate. In the latter case, the great majority of the neoplasms (SO-SO%) are benign papillomas which undergo spontaneous regression after a few days or weeks. If a potent oncogen is used for both steps, the total number of lesions may actually be less, but a higher percentage of them will be carcinomas that can grow progressively and kill the host (Shinozuka and Ritchie, 1967). Although many chemical oncogens may both “initiate” and then “promote” the growth of previously transformed cells, there is a class of agents, typified by urethane, the members of which have the power to “initiate” or transform, but little or no capacity to promote. Studies of urethane oncogenesis in the skin of the mouse have led to the conclusion that urethane is nearly, but not completely, a pure initiator (Salaman and Roe, 1953). Studies in my laboratory (R. T. Prehn, unpublished) have shown that a similar situation exists in the mouse lung. Lung adenomas can be induced by urethane, but the growth of already transformed cells or clones is not promoted. In contrast, a hydrocarbon oncogen, given by mouth, both initiates and promotes the adenomas (R. T. Prehn, unpublished). Lappe (1969) was able to show that at least some benign papillomas initiated by a hydrocarbon oncogen and promoted by skin grafting, were serially transplantable. The techniques are not yet available to permit analysis of the detailed type accomplished with the hyperplastic breast nodules of the mouse, but the possible role of immunity as a homeostatic mechanism has been explored. The available data suggest that, as in the breast, immunological surveillance does not operate in

TUMOR PROGRESSION AND HOMEOSTASIS

211

this system except under certain conditions which will be discussed in Section IV.

C. INDUCXION VERSUS SELECIION; GENETICVERSUS EPICENETIC CHANGE When a chemical oncogen is painted on the skin of a mouse, punctate focal lesions arise, surrounded by apparently normal intervening skin. Likewise, when an oncogenic virus is added to a culture of normal embryo target cells, scattered foci of transformation appear. The percentage of target cells so transformed is limited regardless of the multiples of viral units applied. One reasonable explanation of these data may be that, for one reason or another, only a minority of the cells exposed to the oncogenic agent have the capacity to give rise to a transformed clone or tumor (Stoker and MacPherson, 1961). In the case of chemical oncogens, I advanced the theory several years ago that the chemical might be not really a transforming agent, but merely a selector of transformed cells already present in the population prior to application of the chemical (Prehn, 1964). The theory was based upon the observation that normal cells are regularly much more susceptible to the toxic effects of chemical oncogens than are neoplastic cells (Alfred et al., 1964). Thus, application of an oncogen would provide neoplastic cells with a great competitive advantage and perhaps liberate them from the inhibition of surrounding normal cells, an inhibition discussed in a previous section. However, subsequent work by Huberman and Sachs (1966) and by DiPaolo et al. ( 1969, 1971) was, in some cases, able to separate oncogen toxicity from tumorigenicity and Heidelberger (1973) was able to show that under ideal conditions 100% of target tissue culture cells could be transformed. These experiments suggest that the chemical oncogen, like the oncogenic virus, when it produces transformation, does something more than merely select previously existing variants. Although the theory that a chemical oncogen selects only previously existing variants now seems untenable, the word “only” needs emphasis. That the chemical (and perhaps in some cases the virus also) exerts a selection pressure in favor of the transformed cells, cannot be doubted. Not only are the chemicals and viruses often lethal to normal cells, they may also depress the immunological defenses of the host and so favor the proliferation of transformed, but antigenic, clones ( Malmgren et d.,1952). The fact that under most circumstances only a small portion of a target population is susceptible to induced transformation still needs explanation. While other explanations are certainly possible, I venture

212

RICHMOND T. PREHN

to suggest that perhaps the cells preferentially capable of responding to some oncogenic agents may be those that have already undergone the first of the sequential steps in tumor progression. These variants may already be “primed” and have fewer steps remaining before they manifest transformation or tumorigenicity. Thus, for example in the experiments of Heidelberger ( 1973), so-called “initiation” by a chemical agent may not really be the true first step. If the oncogen preferentially works on already variant cells, the widespread correlation between an inbred mouse strain’s spontaneous incidence of a particular tumor type, and its susceptibility to carcinogeninduced tumors of the same type would be explained. For example, strain A mice are very susceptible to spontaneous lung adenomas; the incidence is vastly increased by a chemical oncogen. In such cases the oncogen seems to be merely accelerating a process that would have occurred without it, and it is easy to think of the oncogen as acting upon cells already “determined to develop into tumor. Also, it seems evident that the true initial step may, in the case of some congenital tumors, be present in the germ line (Knudson, 1974). Thus, in summary, it appears that the initiation of tumor by chemical oncogen or virus involves the induction of heritably stable variants, but that the “inducer” may often act preferentially on cells already predisposed to undergo the further changes leading to actual tumor formation. Actual examples of this have recently been described (Naha and Ashworth, 1974; DiPaolo et al., 1969,1971 ). Whether or not the variant clone that eventually gives rise to tumor, originates by genetic or epigenetic change, i.e., by mutation or abnormal differentiation, cannot yet be determined. The two mechanisms are not mutually exclusive. The arguments in favor of abnormal differentiation have been well summarized by Pierce (1974). They seem to depend upon whether or not those tumors that sometimes resolve by differentiation to a normal state (crown gall in plants, teratocarcinoma in mice, and neuroblastoma in children) are representative of tumors in general. In this connection it should be recalled that mouse skin papillomas usually regress. Lapp6 has occasionally been able to observe a thin monolayer of apparently normal epithelium around the residual keratin pearls that mark the site of such a regression (M. A. Lappk, unpublished). Farber et al. ( 1975) has also tentatively described the apparent resolution, by a return to a normal morphology, of the oncogen-induced hyperplastic foci of incipient hepatomata in rats. In my laboratory, Outzen et al. ( 1975b) has observed, in a chemically induced frog “sarcoma,” sequential changes from an undifferentiated malignant lesion to a benign neuroma.

TUMOR PROGRESSION AND HOMEOSTASIS

213

111. The Subsequent Steps in Tumor Progression

A. THECLONAL NATURE OF SUBSEQUENT STEPS

In the previous section, it was pointed out that whether or not the initial step in the oncogenic process was clonal was often not clear. The situation with regard to the subsequent steps in tumor progression is much less ambiguous. The mammary tumor system of the mouse is probably the system in which the events of progression can most easily be observed and analyzed. Progression in mouse tumors was extensively analyzed by Foulds (1956a-d) and the basic features were described in a classical series of papers. Most notable was the fact, pointed out by Foulds and others, that a mouse breast neoplasm often exhibited a variety of morphologies in various parts of the lesion. These variant areas were often homogeneous spheres of tissue which could hardly be considered as anything but proliferative clones. Foulds thus came to the conclusion that tumor progression was due to the selection of a large number of essentially independently assorting characters. That the variants observed in different parts of a tumor were indeed heritably stable subclones was shown, in a different tumor system, by Henderson and Rous (1962). These investigators fragmented tumors of mixed morphologies into small pieces. The pieces were transplanted individually and each was shown to possess a marked tendency to propagate the morphology of the particular parental fragment. The work in the mouse mammary tumor system makes the same points even more elegantly ( Slemmer, 1974). As was already described, hyperplastic nodde tissue transpIanted to “cleared fat pads propagates to fill the pad forming a so-called “hyperplastic outgrowth line.” Several different morphologies are seen. The striking observation is that further variants can be observed grossly to arise as punctate lesions in the hyperplastic outgrowth line. These variants can be isolated and similarly propagated and breed “true.” The frequency of arisal of such variants, which in some cases are malignant, is a characteristic of the particular hyperplastic outgrowth line, but their appearance is essentially a random process, both with regard to time and location.

B. LATENCY DURING PROGRESSION In Section I1 it was pointed out that the variant clone that initiates the neoplastic process may remain latent, as in the “latent tumor cells” of initiated skin, or as in the hyperplastic nodules of the mouse breast which remain in a stationary growth phase for very extended periods

214

RICHMOND T. PREHN

of time. It is also clear from the work of Slemmer (1974) that variant subclones, the product of tumor progression, may also enter similarly extended stationary periods. When a new variant arises in the breast, its growth is determined by its response to the “spacing factors” of the normal breast. To recapitulate, the normal ductal tree apparently elaborates some principle that inhibits normal ductal growth. This principle, which serves as a ductspacing factor during the growth of the normal ductal tree, also usually inhibits the growth of new variant clones of breast epithelial cells. The new variants seem to respond to the factor, but not to elaborate it themselves. They are therefore inhibited whenever they come too close to normal ducts, and their size is thus limited to a few millimeters. When this constraint is removed by transplantation to a cleared fat pad, growth ensues. New variant subclones which can be isolated from the hyperplastic outgrowth line are often malignant, i.e., they would not be constrained by the presence of normal ducts, were such present, and they will grow outside the fat pad. However, in other sublines, varying degrees of the capacity to override the inhibition of the spacing factor are encountered. In the untransplanted hyperplastic nodule, no further variants will be noted until one occurs that can override, at least to some extent, the inhibition of the spacing factor. Other variants that might arise will not grow and are probably irrelevant. It should be noted that variant formation in the growth-inhibited in situ hyperplastic nodules is probably low compared with that in an outgrowth line in a cleared fat pad, simply because the amount of tissue at risk is comparatively small. On the basis of what has been learned about progression in the mouse breast tumor, an explanation can be offered for the common observation of prolonged latency in human breast cancer after surgery. It is sometimes observed that after mastectomy, a patient may occasionally remain well for periods as long as 15 years, only to eventually develop a recurrence of tumor in the operative scar. It is usually thought that some peculiarity of the local environment changes at that time to permit the growth of the surgically seeded tumor cells. I think it perhaps more likely that the explanation lies in tumor progression. I have pointed out that the early hyperplastic lesions in the mouse cannot grow outside the environment of the fat pad, and for this reason we term them benign. I have also pointed out that malignancy arises as the result of further variation in these early lesions. Some of the hyperplastic outgrowth lines that cannot grow or grow poorly outside the fat pad would be diagnosed, with justification, as malignant by the pathologist-he has learned by experience that such lesions can

TUMOR PROGRESSION AND HOMEOSTASIS

215

be expected to metastasize. I suggest, however, that the true state of affairs may be that these lesions are in themselves often benign at the time of examination, but can be expected to give rise to variants that are indeed malignant and which are the elements that actually grow in a metastasis. The pathologist is thus making a forecast, rather than necessarily diagnosing the actual present state. The cells the surgeon accidentally seeds in the scar or the cells that seed naturally in distant organs to remain latent for many years may not be, and perhaps never were, endowed with the capacity to grow to any appreciable extent outside the fat pad. They therefore remain latent until further random progression occurs among them. This concept of the long latency of some metastatic human tumors, while I think quite reasonable in view of the lesions of the mouse, is entirely speculative. It is unfortunately a somewhat pessimistic theory inasmuch as it suggests that prolonged latency and subsequent metastatic recurrence may be innate functions of random changes in the tumor cells themselves, not in the host.

C. INDUCXION VERSUS SELECTION IN PROGRESSION; GENETICVERSUS EPIGENETIC CHANGE IN PROGRESSION I have pointed out earlier that the sporadic, random, clonal nature of the later steps of tumor progression seems very clear, at least in some systems, and is probably more firmly established than is true in the case of the initial event of the oncogenic process. In the initial event, the question of whether an oncogenic agent merely selected from among previously existing variants seems to have been answered in the negative. It will be recalled from the earlier sections, that the oncogen not only selects, but the evidence is good that it also can induce change or transform. This same question concerning induction versus selection has been asked in reference to the later steps of tumor progression. Some of the earliest and most definitive work was reported by Klein and Klein (1956). This work showed that the change in a mouse tumor from the solid to the ascitic form was due to a relatively stable, heritable, alteration in the tumor cells. If a number of tumor sublines were serially transplanted and exposed at intervals to the selective pressure of the intraperitoneal environment, conversion to the ascitic form was observed to occur at a very variable and unpredictable pace among the different sublines; in other words, it appeared to be a random process. The conclusion was thus made that the change must have depended upon the sporadic arisal of stable variants in the tumor cell population, and that these arose independently of the selective pressure of the peritoneal location. A similar conclusion was reached in another system by Law

216

RICHMOND T. PREHN

(1954), who applied a modified fluctuation test to another form of tumor progression. It seems clear from the preceding that tumor progression is due, at least in some cases, to the random appearance in the neoplastic cellular population of hereditarily stable variants with a selective advantage. This conclusion is reinforced by the observation of Lappk that a mouse skin papilloma possesses throughout its life a constant probability of progressing to malignancy. In the absence of further oncogen treatment, the number of malignancies is directly determined by the “papilloma days” that the mouse is at risk (Lapp6 and Prehn, 1969). The variants probably arise spontaneously and may be selected for by a carcinogen. There seems to be no critical evidence to show that an oncogen or other environmental change actually induces later steps in progression; an oncogen’s action in progression, in contrast to initiation, could be the pure selection of spontaneous variants. It should be noted that few systems have been critically examined by a form of fluctuation test (Luria and Delbruck, 1943), which is often difficult to perform in the context of tumor progression. However, there is some evidence to suggest that a potent oncogen can do more to influence progression than merely to select. The evidence suggesting induction, as well as selection, by an oncogen, of the successive steps in tumor progression is derived from both skin and breast. In the mouse skin, application of a carcinogen in low dosage followed by a nononcogenic promoter leads to the production of numerous benign papillomas. Larger dosages of the carcinogen result in more of them being malignant (Shubik, 1961). These facts could be nicely explained if one postulated that the carcinogen actually induces the successive steps in tumor progression (they can also occur spontaneously but more slowly) and that each successive step occurs with greater frequency or probability than the preceding one. There thus tends to be an induced cascade effect in transformed clones. Coupled with this; the carcinogen tends to be toxic to more normal cells (Alfred et al., 1964), and therefore some potential tumor clones might be eliminated by a high oncogen dose at a very early stage of progression, Thus, the net effect of these two processes, induction and selective toxicity, would be to favor, at high doses of oncogen, fewer lesions but those of higher malignancy. A similar situation may occur in the breast. If the oncogen is applied directly to the gland (high dose?) only malignant carcinomas, rather than hyperplastic nodules, are produced (Sinha and Dao, 1975). The situation is thus quite analogous to that in mouse skin and would be difEicult to explain if the subsequent steps in progression were due to mere selection of spontaneously occurring variants.

TUMOR PROGRESSION AND HOMEOSTASIS

217

I have already discussed the question of whether or not “initial” transformation by an oncogen is really the first step or whether the oncogen preferentially acts upon cells already abnormal. If the latter, then induction of the later steps of tumor progression seems established. Support for this idea comes from the observation by Basombrio and Prehn (1972a) that mildly tumorigenic 3T3 cells can be further transformed by a chemical oncogen. There is one example of neoplastic progression in which the mechanism of selection of previously existing variants is almost certainly not adequate to account for the observations. I refer to what is called the Barrett-Deringer phenomenon ( Barrett and Deringer, 1950). The phenomenon involves a change in the ability of a transplanted tumor to grow despite a moderate histocompatibility barrier. In the original experiment, a tumor that arose in an inbred mouse strain was transplanted to an F, hybrid formed by outcrossing that strain. The other parental strain forming the F, was quite resistant to the growth of the allografted tumor. The tumor, of course, grew well in the F, in accord with the “laws of transplantation.” After passage in the F,, the tumor was transplanted into resistant backcross mice, i.e., mice produced by crossing the F, with the tumor-resistant parental strain. Owing to Mendelian segregation, some backcross mice would be expected to grow the tumor, and others not. In fact, the extent of the growth of a tumor in resistant backcross mice has been used as a measure of the degree of histocompatibility difference between the tumor and the resistant allogeneic inbred strain (Prehn and Main, 1958). The Barrett-Deringer phenomenon consists of the observation that the percentage of resistant backcross mice growing the tumor was increased when the tumor was previously passaged through the F,, rather than transplanted directly from the syngeneic host. The alteration in the tumor was stable; once the change had occurred, passage of the tumor in the strain of origin did not restore the original transplantation characteristics. The observation has been repeated with a number of different tumors with essentially the same result, although with some tumors the change may be in the opposite direction, i.e., less growth in the back-cross animals (for review, see Klein and Klein, 1957). The Barrett-Deringer phenomenon seems to be an example of tumor change or progression that may not be explicable on the basis of the selection of preexisting, sporadically occurring, cellular variants. It has been shown that a very brief sojourn in the F, is all that is necessary for the full manifestation of the effect, and it is also independent of the number of tumor cells exposed to the F, environment ( a very small inoculum of tumor undergoes the change equally as well as a large

218

RICHMOND T. PREHN

inoculum, suggesting that the effect is not due to the existence in the inoculum of a small number of preexisting variant cells). The change is very constant and reproducible with no detectable element of sporadicity. It thus appears that the change is in the nature of a rapid adaptation of the tumor cell population, not a selection of randomly appearing cellular variants (Klein and Klein, 1957). It is still possible, however, that the adaptive change is induced in only a small portion of the tumor cells rather than in most of the population. The nature of the adaptation-inducing mechanism in the Barrett-Deringer phenomenon is not known. However, it has been shown by Klein that if the tumor cells are within the protective confines of a diffusion chamber ( Algire et al., 1954) while in the F, host, the adaptation still occurs (Klein and Klein, 1957 ). The change is thus not dependent upon cell-to-cell interaction within the F,. A further observation by Sanford (1965) was that the tumor change in the F, did not take place if those animals had been immunologically crippled by X-irradiation. In her work, the change observed, as a result of F, passage, was a decreased rate of tumor growth in the strain of origin; the effect on growth in backcross mice was not tested. As far as it has been possible to analyze progression in the autochthonous host, the observations favor the theory of the selection of random variants. However, the Barrett-Deringer phenomenon, produced by exposure of the tumor to a grossly foreign environment, seems to demonstrate that host-induced stable adaptations may occur in a tumor cell population. It may be well to remember that most of the changes associated with organ differentiation in normal ontogeny are presumably due to some form of induction rather than the selection of randomly occurring variants.

IV. immunity as a Homeostatic Mechanism

A. INTRODUCTION The role of immunity as a defense against the growth of cancer cells has been the subject of intense investigation for nearly 20 years. During that period the prevalent opinion has varied from “it has no role” to “it is the major defense” and now perhaps back again to “it is a homeostatic mechanism of questionable importance.” Certain it is that 20 years of endeavor have left the role of the immune mechanism in cancer more controversial than ever. The possibility that immunity might play a major role in cancer stems

TUMOR PROGRESSION AND HOMEOSTASIS

219

from two observations. The first was that certain tumors in animals were caused by infectious agents, viruses; immunity certainly must play a role in controlling such agents. Second, there was the observation that most, perhaps all, tumor cells were at least potentially antigenic in the animal of origin (Main and Prehn, 1957). It is only this second observation that leads to hypotheses concerning the role of immunity as a homeostatic mechanism in relation to the cancer cells per se. It is only this role that has been of immediate concern to my laboratory and that will be examined in what follows. This is not intended in any way to minimize the possible importance of the immune mechanism in controlling cancer by controlling the spread of oncogenic viruses. There is little merit in reviewing the entire field of tumor immunology; this has been done by a number of authors (Haughton and Amos, 1968; Hellstrom and Hellstrom, 1969; Klein, 1973; Baldwin, 1973; Herberman, 1974). Rather, I shall confine myself to an examination of a few controversial issues, to which my laboratory has contributed, and discuss the implications of the findings in relation to the broad field of tumor homeostasis.

B. INDUCED VERSUS “SPONTANEOUS” TUMORS The first point to be made is that there is a profound immunological difference between so-called “spontaneous” tumors and those induced in the laboratory by chemical, viral, or physical means. It appears that, virtually without exception, those tumors that appear sporadically with low incidence and without known cause have little or no capacity to effectively immunize animals syngeneic to the animal of origin. Mice “immunized with such tumors usually grow a challenge inoculum of that same tumor as well or nearly as well as do the nonimmune controls. Immunization with these tumors produces little evidence of immunity effective against the growth of the challenge tumor cells; whatever immunogenicity they may exhibit is relatively weak (Main and Prehn, 1957; Baldwin, 1966; Hammond et al., 1967; Peters, 1975; RCvksz, 1960). The situation with regard to induced laboratory tumors is usually quite different. If tumors are induced, for example, with a standard oncogenic dose of 3-methylcholanthrene such that most of the animals will become tumorous within 3-6 months, most of the tumors can be shown to be immunogenic in mice syngeneic with the animal of origin (Main and Prehn, 1957). Such mice, immunized with tumor cells, are often highly resistant to the growth of a challenge inoculum. The immunity, in the case of chemical induction, is highly specific, being limited to the particular immunizing tumor and usually not cross-reactive with

220

RICHMOND T. PREHN

other tumors even of the same etiology, histology, organ, or animal of origin. The immunogenicity, as measured by the degree of resistance to the growth of the challenge inoculum, is very variable from tumor to tumor, ranging from nondetectable in some cases to nearly absolute immunity in others (Main and Prehn, 1957; Prehn, 1960; Old et d., 1962; Bartlett, 1972). I have recently been able to show that in the case of 3-methylcholanthrene (MCA) the average immunogenicity of the induced tumors is directly related to the concentration of the chemical (Prehn, 1975a). As the concentration was reduced, the tumor incidence declined, the average latency before tumor appearance lengthened, and the average immunogenicity decreased even when the latency was held constant. The more the concentration was reduced, the more these induced tumors modeled, as far as immunogenicity is concerned, the so-called spontaneous. The relationship of immunogenicity to dose of oncogen and to latent period suggest that the characters immunogenic and tumorigenic arise as independent variants. This idea is further supported by the fact, previously mentioned, that lines of 3T3 cells that are more tumorigenic in uiuo than are normal cells, can, nonetheless, be transformed by a chemical carcinogen. Each clone, separately transformed, produces a tumor of different antigenic specificity ( Basombrio and Prehn, 1972a). Thus, it is quite clear that the initial change which makes 3T3 cells more tumorigenic than are completely normal cells occurred prior to the antigenic transformation produced by the chemical.

C. IMMUNOLOGICAL SELECTIONAND SURVEILLANCE Since it is known that most oncogens, and in particular MCA, are iinmunodepressants ( Malmgren et al., 1952; Prehn, 1963; Stjernsward, 1967), one could entertain the hypothesis that the above facts support the concept of immunological surveillance ( Burnet, 1970). Thus, with a high concentration of chemical, immunodepression would permit the growth of highly immunogenic transformed clones. At lower concentrations, or at longer times after the MCA administration, when immunological recovery had occurred, surveillance would be more effective, fewer transformed clones would grow, and those that did grow would tend to be thosc of lesser immunogenicity. According to this formulation, the lack of immunogenicity of spontaneous tumors, as compared with those induced by higher concentrations of chemical, is exactly in accord with the expectations of the surveillance hypothesis. The idea that the inverse relationship that exists between latency

TUMOR PROGRESSION AND HOMEOSTASIS

221

(i.e., the period of time between initial exposure to MCA and the appearance of gross tumor) and immunogenicity (Prehn, 1962, 1969a; Old et al., 1962) might be due to imfhunoselection was challenged by Bartlett (1972), who tested whether or not the relationship would still be found if immunological selection were eliminated from the system. The method was to expose mouse cells to MCA within the confines of intraperitoneal diffusion chambers. The exposed cells were subsequently transplanted to the subcutaneous tissues of mice. Thus, at least the early stages of the oncogenic process took place within the immunologically isolated confines of diffusion chambers, a site presumably free from the possibility of immunological surveillance and selection (Prehn and Main, 1956). Tumors arose from the transplants after varying periods. It was found that under these conditions there was no detectable correlation between the latency and the immunogenicities of the tumors. Therefore, the conclusion seemed justified that the usually observed inverse relationship between the latency and the immunogenicity was indeed a function of immunological selection. Similar results were obtained by Parmiani et al. (1973). Concomitant with the above study, I examined the immunogenicities of tumors that arose among cells in tissue culture [immunogenicity again defined as the degree of immunity to challenge tumor growth that could be elicited in mice by prior immunization with tumor (Prehn, 1971c)l. Inasmuch as the tissue culture environment was presumably completely free of any possibility of immunological surveillance and immunological selection, it was anticipated that tumors originating in such an environment would be uniformly highly immunogenic. Such was not the case. It was found that whenever transfonnation took place “spontaneously” in the cultures, the resulting tumors were not detectably immunogenic in uiuo.? Likewise, “spontaneous transformation” in diffusion chambers was also shown to result in nonimmunogenic tumors (Prehn, 1971c; Bartlett, 1972; Parmiani et al., 1971). In contrast, when the cultures In the initial series of cultures not exposed to MCA, the tumors that were presumably the result of “spontaneous” in uitro transformation were all found to be immunogenic. [A similar finding has been reported by Kieler et al. (1972).] The tumors were also found to be cross-reactive, suggesting that the immunogenicity was the result of contamination with an unknown virus, possibly unrelated to the transformational event. Subsequent to that series of cultures, the laboratory was moved to another city, the substrains of mice were changed, and a new technical staff performed the work. Immunogenicity of “spontaneously transformed” cultures was never encountered again. The probable explanation that immunogenicity in the initial series was due to contaminating virus is strengthened by the observation that immunogenicity can be deliberately imparted to a tumor by infection with a passenger virus (SvetMoldovsky and Camburg, 1965; Sjogren, 1964).

222

RICHMOND T. PREHN

were exposed to MCA, and when transformation presumably resulted from the action of the MCA, the tumors were usually highly immunogenic. Thus, in tissue culture and diffusion chamber oncogenesis, the immunogenicity was apparently a function of the presence or the absence of MCA, not a function of immunological surveillance. At first glance the results obtained with MCA and with “spontaneous” transformation in immunologically protected environments seem paradoxical. However, the conclusions to be drawn from the two types of experiment are not mutually exclusive. The Bartlett work suggested that immunological selection plays a role in determining the immunogenicities of the MCA-induced tumors, and that immunological surveillance is a factor in chemically induced oncogenesis. The tissue culture work suggested that the oncogen is directly involved and necessary in producing immunogenicity. Without the MCA, there is apparently little or no immunogenicity for the immunological selection to act upon. The lack of detectable immunogenicity in “spontaneous tumors,” even in the absence of an immunological surveillance mechanism, raises questions about the possible role of immunity in human cancer. Judging from the mouse model, as I have thus far described it, the possible role is presumably dependent upon the etiology of the tumor. If the tumors arise in high frequency as the result of a high flux of an oncogen-as may be the case in, for example, bronchogenic squamous cell tumors of the lung or ultraviolet-induced skin tumors-then one would expect that immunological surveillance might play a significant role. On the other hand, tumors that arise infrequently in response to very low levels of environmental oncogens or which may be truly spontaneous, and which therefore probably have little or no immunogenicity, would presumably be little affected by immunological defenses. I will discuss this argument in greater depth after a short digression concerning those tumors overtly induced by oncogenic viruses.

D. VIRALLY INDUCEDTUMOFSAND LYMPHORETICULAR NEOPLASMS The evidence in favor of the hypothesis that immunity serves as a defense against the growth of cells neoplastically transformed by oncogenic viruses is even greater than that concerned with tumors chemically induced. It is not always easy to decide whether the surveillance is operating against the spread of the virus or against the growth of transformed cells. However, much evidence suggests that it is, at least in some cases, the latter, and thus quite analogous to the situation in MCA induced tumors, This evidence has been extensively reviewed by Klein ( 1973) and Hellstrom and Hellstrom (1969).

TUMOR PROGRESSION AND HOMEOSTASIS

223

The most persuasive work concerns the small oncogenic DNA virus tumors, such as those induced by polyoma, SV40, and the oncogenic adenoviruses. Tumors induced by these agents carry relatively strong tumor-specific transplantation antigens ( Klein, 1973; Hellstrom and Hellstrom, 1969). Antilymphocyte serum treatment or newborn thymectomy profoundly increases the tumor incidence. Curiously, the incidence of polyoma or SV40-induced tumors in neonatally inoculated animals can be reduced by inoculation of a second virus dose or of irradiated tumor cells during the oncogenic latent period (Deichman, 1969). The implication is clear that immunity to the transformed clones can apparently influence the tumor incidence. The resemblance of these systems to oncogenesis with MCA seems striking. The tumors are highly immunogenic; however, in the case of the viruses the tumors are of course cross-reactive, and because of viral multiplication in the host, dose response studies of the type done with MCA are not yet available. In my opinion, great caution should be exercised in interpreting work done with lymphoreticular tumors, however induced, as being either for or against the immunological surveillance hypothesis. This is because of the fact that these are tumors of the immune organ itself. It has been known from many other organ systems that excessive physiological demand can itself result in neoplasia. For example, there is the classical work of Biskind and Biskind (1949) on oncogenesis in the ovary. The ovary, transplanted in the spleen of the gonadectomized rodent, eventually becomes tumorous because the feedback inhibition to gonadotropin production is interrupted by the destruction of ovarian hormones in the liver. It is possible that increased lymphoma production in immunodepressed animals or man is due to an analogous physiological stress, in this case antigenic stimulation of an organ that is crippled and unable to respond adequately. This idea has recently been placed on a more sophisticated level by Schwartz, who suggests that lymphomas in kidney transplant recipients or in graft-versus-host disease are the result of antigenic stimulation under conditions in which feedback inhibititon by regulatory suppressor T cells has been blocked (Schwartz, 1975). Such tumors may therefore not be a manifestation of a failure of the surveillance mechanism. These considerations complicate the interpretation of Marek's disease in chickens. This virally induced lymphoma can be largely prevented by immunization of the birds with a related but nonpathogenic turkey virus ( Nazerian, 1973). Although the incidence of disease is drastically reduced, the amount of Marek's virus detected in the birds is only moderately altered (Nazerian, 1973). This fact might suggest that the turkey virus inoculation increases immune surveillance against incipient tumor

22A

RICHMOND T. PREHN

cells per se, rather than against the virus. However, the lack of a dramatic effect on the Mareks virus is somewhat difficult to understand since the antigen on the tumor cell is either a part of, or determined by, the Marek‘s virus. Only in the former case would one expect a cross-reactive turkey virus to influence the growth of tumor cells, but in that event it should also markedly affect the virus. In explanation, it has been suggested that virus production is little affected by immunization because it occurs largely in a “sequestered site (Klein, 1973; Hellstrom and Hellstrom, 1969). If one were to speculate that the Marek‘s lymphoma is not a result of virally induced transformation, but is instead the result of an undampened but ineffectual immune reaction (perhaps due in part to a genetically determined lack of T-suppressor cells of the proper specificities) , the role of the turkey virus might be to activate T-suppressor cells-that could dampen the “run away” response to the Marek‘s virus. This is, of course, speculation, but it serves the purpose of showing that alternatives to the concept of a failure of immunological surveillance vis-8-vis tumor cells can be conceived even in Marek‘s disease, a malignant disease in which immunization is highly successful. If we disregard all evidence, for the reasons expounded above, derived from lymphoreticular neoplasms, the case for an effective immunological surveillance seems to depend almost entirely upon the fact that immunodepressive measures, such as neonatal thymectomy or antilymphocyte serum ( ALS ) , can sometimes increase the incidence of tumors in animals exposed to potent chemical oncogens or laboratory strains of oncogenic viruses (Klein, 1973; Hellstrom and Hellstrom, 1969; Prehn, 1974). In the case of some of the viruses, these data are much more persuasive than with the chemicals. In some of the chemically induced tumor systems, such as some hydrocarbon-induced tumors of the mouse, the available evidence suggests that the state of the immune mechanism influences neither the incidence nor the regression of the tumors to any appreciable degree (Outzen et d.,1975a; Andrews, 1971, 1974; Stutman, 1974). Even with the viruses, the situation is not clear. Virally induced mouse mammary tumors, for example, are much decreased following thymectomy or ALS, and this apparently is not a function of ovarian atrophy (Prehn, 1971a). On the other hand, there can be little doubt that regression in the Moloney sarcoma system is caused by a thymus-dependent immune reaction (Stutman, 1975). It is not yet known whether tumors induced by viruses, under conditions of natural infection, are highly immunogenic or are perhaps more analogous to those tumors produced by very low levels of chemical carcinogen. To summarize the discussion up to this point, there is good evidence

TUMOR PROGRESSION AND HOMEOSTASIS

2%

that the immune mechanism apparently plays a role in the regulation of the growth of certain tumors, particularIy those induced in the laboratory by high concentrations of certain, but not all (Baldwin, 1973), chemicals and by infection with laboratory strains of oncogenic viruses. (Contrary evidence from studies with the “nude” mouse will be discussed later. ) On the other hand, spontaneous tumors and tumors induced with low concentrations of chemical have little or no immunogenicity (for reasons having nothing to do with immunological selection) and thus would presumabIy be difficult targets for immunological surveillance. Naturally occurring viral tumors remain to be investigated in this regard.

E. Is SUBLIMINAL IMMUNOCENICITY ADEQUATE FOR SURVEILLANCE? The fact that spontaneous tumors have little or no detectable immunogenicity does not necessarily mean that immunological surveillance could not operate against them. It is well established that the effectiveness of an anticellular immunity is dependent upon the size of the target population. A level of immune response adequate to deal with a tumor cell population of a certain size may be overwhelmed by a larger. Thus, it could be postulated that an immunogenicity too low to be recorded in the usual immunization-challenge type of in uiuo test, might still be adequate to cope with a very small nidus of transformed cells. In other words, a subliminal immunogenicity might be adequate for the purposes of effective immunological surveillance. However, several lines of evidence suggest that this is probably seldom the case. The first evidence against the concept that a subliminal level of tumor immunogenicity might be adequate for the purposes of surveillance arises from a consideration of the effects of a very small antigenic stimulus upon the immune reactivity of an animal. It has been shown that a very small exposure to even a strong transplantation antigen, such as might be the result of the growth of a small nidus of immunogenic tumor cells, tends to condition the immunological mechanism in such a way that the immune response to that antigen is very small; it tends to remain small throughout the remainder of life regardless of the size of subsequent antigenic stimuli (Stillstrom, 1974). Perhaps this is a form of partial immunological tolerance, but the mechanisms of the phenomenon are not known. Whatever the mechanism, it seems likely that it is because of exposure to a minimal antigenic stimulus during early tumor development that the animal in which an autochthonous MCA induced primary tumor has been excised is much less capable of resisting subsequent challenge inoculations of that tumor than are syngeneic controls that had been initially immunized by having the tumor implanted ( Stjernsward, 1968; Basombrio and Prehn, 197213). One can conclude

226

RICHMOND T. PREHN

that much of the immunogenicity observed during the syngeneic transplantation of laboratory tumors is in reality a laboratory artifact imposed by the transplantation method of initial antigen presentation ( Andrews, 1974). If the result of a very small initial exposure to a strong antigen results in partial tolerance, it is hard to see how exposure to the very weak antigens of a nidus of developing spontaneous tumor cells could result in an effective surveillance response. That transplantation can induce immunological artifacts was demonstrated dramatically in studies concerning MCA-induced papillomas in mouse skin, work already alluded to in Section II,B. Lapp6 devised a method of inducing papillomas that consisted of exposure of the mouse skin to a low dose of MCA followed by the syngeneic grafting of the exposed skin (Lap@, 1968; Prehn and Lapp6, 1971). The grafting provided a physiological stimulus ( “promotion”) that rapidly elicited a crop of papillomas in the previously MCA-treated epithelium. Lapp6 was able to show clearly that the papilloma incidence and the rate of regression were influenced by the immunological capacity of the host animal. Immunologically crippled hosts developed more papillomas, and these persisted longer than was the case in normal or in immunologieally potentiated hosts. Apparently this was clear evidence of immunological surveillance. Subsequent work by Andrews (1974) showed, however, that the immunological effect on tumor incidence was probably dependent upon an artifact introduced by transplantation of the skin containing the incipient papillomas. In the absence of such transplantation, the incidence was completely uninfluenced by the immunological status of the animal. This conclusion is consistent with the observation that life long immunodepression with the aid of ALS did not influence hydrocarbon-induced papillomas ( Haran-Ghera and Lurie, 1971) . Furthermore, Andrews showed that regression of the papillomas still occurred even when the possibility of host immunity was almost certainly excluded ( Andrews, 1971). He produced immunologically crippled animals by adult thymectomy followed by X-radiation. The crippled mice were then given a graft of already MCA-initiated skin from an allogeneic donor. The grafting served as a promoter, and papillomas appeared in the usual fashion; they also regressed despite the fact that the allograft in which they resided was surviving healthily. It thus seems most unlikely that host immunity plays a necessary role in ordinary papilloma regression. Immunity can contribute when, and only when, the immune response is aroused by some procedure such as skin grafting in immunocompetent hosts. The in situ papilloma does not apparently arouse an immune reaction. In the case of the skin papilloma system, the lack of effective immuni-

TUMOR PROGRESSION AND HOMEOSTASIS

227

zation by the in situ, untransplanted neoplasm could be accounted for by the conditioning effect of a low initial exposure to the pertinent antigens, as already described, and/or to a second phenomenon best illustrated in the work of Slemmer in the mammary tumor system. I have already described the system in Section II,B,l. Slemmer (1972) was able to show that an antigenic MCA-induced hyperplastic nodule could be transplanted into an uncleared fat pad in a syngeneic mouse and there persist without growth or regression for many months and perhaps indefinitely. At any time during this period, the nodule tissue could be rapidly destroyed by simply implanting another fragment of the same nodule subcutaneously, i.e., outside any mammary fat pad. Clearly, in this system the immune mechanism was capable of destroying the incipient tumor whenever immunization took place. However, the lesion inside the fat pad did not immunize, although it was kept from growing by the inhibiting influence of the normal epithelium. It seems probable that within many and perhaps all epithelial organs, such as skin and breast, in which the parenchymal stem cells are separated from mesodermal elements by a basement membrane, even transplanted, antigenic cells immunize poorly, if at all. It has definitely been shown that both the mammary fat pad and the superficial skin epithelium are immunologically privileged sites in which immunization to cellular antigens is very slight (Blair and Moretti, 1967; Billingham and Medawar, 1950). Thus, in these sites one could not expect a nidus of weakly immunogenic tumor cells to be eliminated by an immune reaction. Even when the tumor cells are potentially highly immunogenic, i.e., MCA-induced, such elimination does not occur in the natural course of events. In the breast, immunization does not occur until the tumor mass is large and/or has escaped the fat pad. Under these conditions, surveillance could not be effective against weakly immunogenic nascent tumors. Further evidence suggesting that a subliminal immunogenicity in tumor cells may not be adequate for the purposes of surveillance derives from a consideration of two distinct but possibly related phenomena: “sneaking through and “immunostimulation.” The “sneaking through phenomenon was first described in relation to the growth of an allogeneic tumor, but has subsequently been confirmed by a number of laboratories with respect to completely syngeneic systems (Humphreys et al., 1962; Old et d.,1962; Potter et al., 1969; Marchant, 1969). In brief, the phenomenon consists of the observation of an anomalous behavior in the growth of transplanted tumor cells when these are implanted in varying dosages. With at least some immunogenic tumors it can be shown that a very small inoculum may grow better than a larger. Recent evidence suggests that the phenomenon is due to what can be called a specific, immunological, partial tolerance

228

RICHMOND T. PREHN

produced by the small inoculum ( Bonmassar et al., 1974). The phenomenon is thus closely related to the effect of an initial small antigen dosage already discussed; it can be demonstrated even in highly immunogenic systems. Again, a very low dosage of inoculated tumor cells probably mimics in situ tumor formation. It seems unlikely that a weakly immunogenic, in situ, untransplanted tumor can produce an effective immune resistance to its growth when this is unattainable by the inoculation of a small dose of highly immunogenic tumor cells.

F. IMMUNOSTIMULATION OF TUMORGROWTH Thus far the argument against the efficacy of immunity in surveillance against newly formed, weakly immunogenic, tumors has related to the ineffectiveness of a very weak antigenic stimulus, such as a newly developing tumor cell clone would provide. Immunostimulation is a phenomenon which suggests that a weak immune response may not only be ineffective in controlling tumor growth; it actually may make the tumor cells grow better. The original idea of immunostimulation was obtained by consideration of the rather extensive literature suggesting that a weak immunological reaction by the mother against the conceptus could result in larger placentas and larger and more numerous offspring (Prehn and Lapp6, 1971 ) . Although this literature remains controversial, it did suggest, by analogy, that the immune reaction might also be able to favor tumor growth. The initial tests of this hypothesis were done with the aid of Winn tests in immunologically crippled host mice (Prehn, 1972). It was found that small numbers of specifically immune spleen cells, when implanted together with tumor cells, produced better tumor growth than did similar numbers of normal or nonspecifically immune spleen cells. Subsequently, similar phenomena have been recorded in a variety of systems both in uitro and in uiuo (hledina and Heppner, 1973; Fidler, 1973; Jeejeebhoy, 1974; Kall and Hellstrom, 1975; Bray and Keast, 1975; Ilfeld et al., 1973; Bartholomaeus et al., 1974). Shearer has shown clearly that heterologous antibody can be directly stimulatory to target cells (Shearer, 1973). At present, the mechanisms are not known, and it appears that several different effector systems, i.e., T cells and/or antibody, may stimulate under different conditions or in different systems. It now appears that some of the work subsumed under the title of “enhancement” may be synonymous with immunostimulation rather than with “blocking,” a position not unlike that long championed by Kaliss (1965). If immunostimulation is a general phenomenon, and this is not yet established, it probably plays a crucial role in tumor biology. Any tumor, regardless of its immunogenic potential, should produce a weak immune

TUMOR PROGRESSION AND HOMEOSTASIS

229

response early in its development; the immune response must pass though a weak phase before it gets strong. The immune reaction would thus give tumors an initial impetus; the immunity might or might not, depending upon the circumstances, mature into an inhibitory type of reaction. That this actually occurs is supported by the report by Jeejeebhoy (1974). If nothing else, the existence of the phenomenon of immunostimulation suggests that the weak immune response, probably elicited by an in situ nidus of spontaneously transformed, weakly immunogenic, tumor cells, would not be likely to result in surveillance. On the contrary, immunostimulation might be more likely. Immunity might result in more tumors than would occur if there were no immune reaction at all. The “nude” mouse would seem to offer a definitive means of assessing the role of immunity in oncogenesis, and of determining the relative roles of surveillance and immunostimulation. The congenitally athymic “nude,” despite the fact that some of its lymphoid cells are theta-antigen positive, has no detectable ability to reject allografts or xenografts. This immunological deficit results in a short life-span in most conventional colonies; however, longevity can be extended and approach normal under pathogen-free or germ-free conditions ( Outzen et al., 1975a). Inasmuch as this mouse has no resistance to allografts, it would have no resistance to the growth of antigenic tumors unless the surveillance function and allograft immunity are completely different types of reactions. Although data have been accumulating slowly, there appears, thus far, to be no increase in tumors induced in nude mice by a hydrocarbon oncogen (Outzen et al., 1975a; Stutman, 1974). With the exception of lymphoreticular tumors, there has been, as yet, no increment of spontaneous tumors (Outzen et aZ., 1975a). This result is identical to that obtained by the life-long administration of antilymphocyte serum to normal mice (Nehlsen, 1971; Sanford et al., 1973). On the other hand, neither has there been a tumor deficit; the nude mice seem to be about as susceptible to tumor formation as are their heterozygous littermates. The data from the nude mouse seem to suggest that neither immunological surveillance nor immunostimulation plays a significant role in chemical oncogenesis. On the other hand, Moloney sarcoma virus-induced tumors do not regress in nudes as they usually do in conventional nonnude controls (Stutman, 1975). Although the published accounts of oncogenesis in nudes seem to denegrate the role of the immune response in hydrocarbon oncogenesis (and thus seem contrary to the implications of the Bartlett ( 1972) work previously discussed), there exist several as yet unpublished observations that suggest the possibility of a different conclusion. Reed has data to suggest that although the nude mouse is resistant to skin oncogenesis,

230

RICHMOND T. PREHN

produced by painting a hydrocarbon, it becomes susceptible when restored by thymus grafting ( N. Reed, personal communication, 1975). Although nonimmunological explanations have not been excluded, this result is consistent with the immunostimulation hypothesis, since it suggests that thymic function may facilitate oncogenesis. Also consistent with the immunostimulation interpretation is the general observation in many laboratories that transplanted allogeneic tumors do not grow as well as expected in nude mice (Skov et al., 1975; Maguire et al., 1975). Furthermore, tumors that metastasize in the strain of origin seldom do so in nudes ( Maguire et al., 1975). Nonimmunological explanations cannot yet be excluded; for example, macrophages have been reported to nonspecifically inhibit tumor growth in nudes (0.Stutman, personal communication, 1975). An as yet unpublished account, with a bearing on the role of the immune response in oncogenesis was not done in nudes, but in mice rendered immunoincompetent by thymectomy and whole-body radiation ( Prehn, 1975a). Partial restoration of immunocompetence by injection of normal syngeneic spleen cells resulted in accelerated oncogenesis with MCA as compared with controls that were either unrestored or completely restored. The result was thus exactly in accord with the immunostimulation hypothesis, but again nonimmunological explanations must be considered. If the increased tumor production in partially restored immunocrippled mice is indeed immunological, how is it that, apart from the observation of Reed, there has been no indication of a difference in induced-tumor susceptibility between nude mice and their heterozygous littermate controls? The only explanation I can presently advance is to point out that there was no significant difference between the tumor incidence in unrestored immunocrippled mice and the maximally restored group. Perhaps these groups were comparable to the nudes and the heterozygotes, respectively. If so, partial restoration experiments in nude mice will eventually give similar results. The immunostimulation hypothesis leads to a further prediction-one that in fact seems to have been realized by observations made before the theory was conceived. According to the theory, immunostimulation occurs only when the immune response is relatively “weak” (Prehn and LappC, 1971) . Consequently, the theory predicts that in tumor systems in which the immunogenicity of the tumors is low, measures that lower the immunological responsiveness of the animal still further will decrease the growth of transplanted tumors and the tumor incidence. This is in contrast to the usual laboratory tumor systems in which immunogenicity is high and immunodepression tends, if anything, to increase the tumor incidence, and certainly the “take” of transplanted tumors. In

TUMOR PROGRESSION AND HOMEOSTASIS

231

other words, if the immune reactivity of the system is already in the low or irnmunostimulatory range, further reduction will decrease tumor growth-if it is in the high or tumor-inhibitory range, a decrease will lead to decreased inhibition and perhaps to actual stimulation. This consequence of the immunostimulation theory, namely that immunocrippling procedures will result in decreased tumor growth in weakly immunogenic tumor systems, is actually seen among mammary tumors in mice. In this system much of the immunogenic potential of the tumors is due to viral antigens-but this potential cannot be appreciably realized in animals that are infected from birth by natural transmission of the virus from the mother. In such animals, although antiviral antibodies are formed, the virus persists throughout life. In these mice, transplantation studies reveal very little evidence of immunity capable of inhibiting tumor growth (Vaage, 1968; Morton et al., 1969; Prehn, 1969b). If the general immunological capacity of such mice is interfered with by any of a variety of means (X-rays, ALS, thymectomy, etc.), tumor growth and incidence are usually reduced ( Prehn and Lapp6,1971) . In contrast, these same tumors are markedly accelerated in their growth by similar measures when they are transplanted to syngeneic mice lacking the virus, mice in which they are markedly more immunogenic (Prehn and Lapp6,1971) .

G. METASTASIS

It has long been bruited that an immune response to tumor antigens might play a role in the prolonged latency of some neoplasms or in their slowness to metastasize. The fact that in the breast tumor system the immune mechanism can be aroused by tumor cells only when they leave the protective confines of the fat pad mighst support such a hypothesis. However, firm support of the hypothesis has not yet been achieved, although there are several reports that are highly suggestive. Perhaps outstanding among these is the report of Lewis et al. (1969), which we have recently been able to confirm (Bodurtha et d.,1975), that patient serum is uniformly inhibiting in culture to the autochthonous melanoma when, and only when, the tumor is regional in extent. The patient's serum loses this property in association with, and perhaps prior to, dissemination of the tumor. Whether or not this is a cause and effect relationship has not been determined, but the observation is highly suggestive.

H. CONCLUS~ONS CONCERNING IMMUNITY Throughout this discussion of tumor immunity I have made virtually no reference to the interesting work being pursued in so many labora-

232

RICHMOND T. PREHN

tories concerning the results of varied in uitro tests of tumor antigenicity and the immune status of patients or experimental animals. This is not because of lack of interest or familiarity-much work of this type is being pursued in my laboratory. However, I find the results to date confusing, and it is difEcult to draw firm conclusions from them. Most irritating is the fact that there has sometimes been a striking lack of correlation between the results of in uitro and in uiuo testing in mouse systems ( Baldwin, 1973). Lymphoid cells can be immunized against and can develop cytotoxicity against normal syngeneic targets ( Wekerle et al., 1973). Normal lymphoid cells tend to be as cytotoxic as are patient cells to target tumors (Takasugi et al., 1973; Berkelhammer et al., 1975; Jeejeebhoy, 1975). Also, I am distressed by the lack of tumor or even organ specificity so often encountered (Berkelhammer et d.,1975). I have no doubt that all the vaned phenomena described by the in vitro methods are real-blocking, unblocking, arming, etc. ( Herberman, 1974). However, I am uncertain of what role these phenomena play in the gesta1.t of the reaction in uiuo. Despite these problems, it is ultimately only by dissecting these processes in uitro that progress will be made. The present confusion will no doubt gradually resolve. Perhaps I can summarize this section on the immune reaction and cancer by stating my own tentative conclusions. 1. A cytotoxic or inhibiting immune reaction usually plays little or no role in determining the occurrence or nonoccurrence of tumors except under exceptional laboratory conditions, i.e., large doses of chemical oncogens, laboratory-selected viruses, etc. ( Naturally occurring viral tumors require much further investigation in this regard.) In other words, immunological surveillance is, at best, probably a very limited phenomenon, 2. In many systems immunity, especially in low titer, may directly stimulate rather than inhibit the growth of tumor cells. (When immunodepression increases the incidence of tumor, it is because of decreased surveillance or increased stimulation?) 3. Some human cancers that occur in high frequency as a result of potent environmental oncogens may, like the analogous laboratory tumors, be influenced by an immune reaction, i.e., ultraviolet-induced skin cancers and bronchogenic squamous cell tumors might fall in this category. However, the probability of immunostimulation rather than surveillance must be remembered. 4. Immunity may sometimes function late in the course of disease to limit blood-borne metastases. 5. The usual lack of effectiveness of immunity as a defense mechanism increases the chance that effective methods of improving the immune

TUMOR PROGRESSION AND HOMEOSTASIS

233

response (immunotherapy ) may be found. These may already be present in adjuvant immunotherapy with agents such as Bacillus Calmette-GuCrin ( BCG) (Berkelhammer et ul., 1975), but the possible hazards of immunostimulation must constantly be considered. V. Concluding Remarks

This survey of tumor initiation, progression, and homeostasis has omitted many topics and references that would belong in any comprehensive review. Indeed, a comprehensive review of such a broad subject would cover most of what is known of tumor biology. This survey has been limited to those topics of particular interest to me and thus reveals my biases. What I have presented is a personal view. However, even in the limited areas that I have chosen to discuss, this brief survey makes it apparent that knowledge has been accumulating at a rapid pace; it makes it even more apparent that our ignorance remains profound. Perhaps most critical is the lack of knowledge concerning homeostatic mechanisms other than the immune response. I have discussed one such in the regulation of growth in the mammary tumor system, but nothing is known concerning its mechanism of action. Perhaps one area for fruitful investigation is the role of nerves and regenerative capacity in oncogenesis (Prehn, 1971b). Recently, my colleagues and I have been able to show that oncogenesis in the frog by a hydrocarbon is markedly potentiated by nerve section-possibly via the critical effect of nerves on regenerative phenomena (Outzen et al., 1975b). Although the future of such studies is hard to predict, it is my feeling that, in the long run, important as immunological studies may be, they will be counterproductive if they so monopolize attention that other mechanisms of tumor homeostasis are thereby neglected.

REFERENCES Alfred, L. J,, Globerson, A., Benvald, Y., and Prehn, R. T. (1964). Brit. 1. Cancer 18, 159-164. Algire, G. H., Weaver, J. M., and Prehn, R. T. (1954). 1. Nut. Cancer Inst. 15, 493-508. Andrews, E. J. ( 1971 ). 1. Nut. Cancer Inst. 47,653-665. Andrews, E. J. ( 1974). 1. Nut. Cancer Inst. 52,729-732. Argyris, T. S., and Argyris, B. F. ( 1962). Cancer Res. 22,73-88. Baldwin, R. W. ( 1966). Int. 1. Cancer 1, 257-264. Baldwin, R. W. (1973). Advan. Cancer Res. 18,l-76. Barrett, M. K., and Deringer, M. K. (1950). 1. Nut. Cancer Inst. 11, 51-60. Bartholomaeus, W. N., Bray, A. E., Papadimitriou, J. M., and Keast, D. (1974). 1. Nut. Cancer Inst. 53, 1065-1072.

234

RICHMOND T. PREHN

Bartlett, G. L. ( 1972). 1. Nut. Cancer Ins#. 49,493504. Basombrio, M. A., and Prehn, R. T. (1972a). Int. J. Cancer 10,l-8. Basombrio, M. A., and Prehn, R. T. ( 1972b). Cancer Res. 32,2545-2550. Berenblum, I. (1954). Cancer Res. 14,471-477. Berenblum, I., and Haran, N. (1955). Brit. J. Cancer 9, 268-271. Berkelhammer, J. B., Mastrangelo, M. J., Laucius, J. F., Bodurtha, A. J., and Prehn, R. T. (1975). lnt. J. Cancer (in press). Bern, H. A., DeOme, K. B., Alfret, M., and Pitelka, D. R. (1958). Znt. Symp. Mammary Cancer, Proc., Znd, 1957 pp. 565-573. Billingham, R. E., and Medawar, P. B. (1950). Heredity 4, 141-164. Biskind, G. R., and Biskind, M . S. (1949). Amer. J. Clin. Pathol. 19, 501-521. Blair, P. B., and Moretti, R. L. ( 1967). Transplantation 5, 542544. Bodurtha, A. J., Chee, D. O., Laucius, J. F., Mastrangelo, M. J., and Prehn, R. T. (1975). Cancer Res. 35, 189-193. Bonmassar, A., Menconi, E., Goldin, A., and Cudkowicz, G. (1974). J. Nut. Cancer Insf. 53, 475-479. Boutwell, R. K. ( 1964). Progr. Exp. Tumor Res. 4,207-250. Bray, A. E., and Keast, D. (1975). Brit. J . Cancer 31, 170-175. Burnet, F. M. ( 1970). “Immunological Surveillance.” Pergamon, Oxford. Dao, T. L., Tanaka, Y.,and Gawlak, D. ( 1964). J. Nut. Cancer Inst. 32, 1259-1276. DeCossC, J. J., and Gelfant, S. (1968).Science 162,698-599. Deelman, H. T. ( 1927). Brit. Med. J. 1, 872. Deichman, G. I. (1969). Adoan. Cancer Res. 12, 101-136. DeOme, K. B., Faulkin, L. J,, Jr., Bern, H. A., Blair, P. B. (1959). Cancer Res. 19, 515-520. DiPaolo, J. A., Donovan, P. J., and Nelson, R. L. (1969). J . Nut. Cancer Inst. 42, 867-876. DiPaolo, J. A., Nelson, R. L., and Donovan, P. J. ( 1971). J. Not. Cancer Inst. 46, 171-181. Ewing, J. ( 1940). “Neoplastic Disease,” 4th ed. Saunders, Philadelphia, Pennsylvania. Farber, E., Hartman, S. P., and Solt, D. (1975). Proc. Amer. Ass. Cancer Res. 16, 3. Fialkow, P. J. (1972). Adoan. Cancer Res. 15,191-226. Fidler, J. ( 1973). J . Nut. Cancer Inst. 50, 1307-1312. Foulds, L. ( 1954). Cancer Res. 14, 327-339. Foulds, L. ( 1956a). J. Nut. Cancer I nst. 17,701-712. Foul&, L. (1956b). J . Nut. Cancer Inst. 17,713-754. Foul&, L. ( 1 9 5 6 ~ )J.. Nut. Cancer Inst. 17,755-782. Foulds, L. ( 1956d). 1. Nut. Cancer Inst. 17,783-802. Globerson, A., and Feldman, M. (1964). J. Not. Cancer Inst. 32, 1229-1243. Hammond, W. G., Fisher, J. C., and Rolley, R. T. ( 1967). Surgery 62, 124-133. Haran-Ghera, N., and Lurie, M . (1971). J. Not. Cancer Inst. 46, 103-112. Haughton, G., and Amos, D. B. (1968). Cancer Res. 28, 1839-1848. Heidelberger, C. ( 1973). Adcan. Cancer Res. 18, 317366. Hellstrom, K. E., and Hellstrom, I. (1969). Adoan. Cancer Res. 12, 167-223. Henderson, J. S., and Rous, P. ( 1962). J . Exp. Med. 115, 1211-1230. Herberman, R. B. ( 1974). Adoan. Cancer Res. 19,207-264. Huberman, E., and Sachs, L. (1966). Proc. Not. A d . Sci. US. 56, 1123. Humphreys, S. R., Glynn, J. P., Chirigos, M. A., and Goldin, A. ( 1962). J . Nut. Cancer Ins#. 28, 1053-1063.

TUMOR PROGRESSION AND HOMEOSTASIS

235

Ilfeld, D., Carnaud, C., Cohen, I. R., and Trainin, N. (1973). Int. J. Cancer 12, 213-222. jeejeebhoy, H. (1974). Int. J. Cancer 13,665-678. Jeejeebhoy, H . (1975). Int. J. Cancer 15, 867-878. Kaliss, N. (1965).Fed. Proc., Fed. Amer. SOC. Erp. Biol. 24,1024-1029. Kall, M. A., and Hellstrom, I. (1975). J. Immunol. 114, 1083-1088. Kieler, J., Radjikowski, C., Moore, J., and Ulrich, K. (1972). J. Nut. Cancer Inst. 48, 393405. Kitagawa, T., and Pitot, H. C. (1975). Cancer Res. 35, 1075-1084. Klein, G. ( 1973). Transplant. Proc. 5, 31-41. Klein, G., and Klein, E. (1956). Ann. N.Y. Acad. Sci. 63, 640-661. Klein, G., and Klein, E. ( 1957). Symp. SOC.Erp. Bid. 11,305-328. Knudson, A. G., jr. (1974). Amer. 1. Puthol. 77, 77-84. Lapp6, M. A. ( 1968). J. Nut. Cancer Inst. 40, 823-846. Lapp&,M. A. ( 1969). Nature (London) 223,82-84. LappB, M . A., and Prehn, R. T. (1969). Cancer Res. 29,2374-2378. Law, L. W. ( 1954). Cancer Res. 14, 695-709. Lewis, M. G., Ikonopisiv, R. L., Nairn, R. C., Phillips, T. M., Fairley, G. H., Bodenham, D. C., and Alexander, P. ( 1969). Brit. Med. J. 3,547552. Luria, S. E., and Delbruck, M. (1943). Genetics 28,491511. Maguire, H. C., Outzen, H. C., Custer, R. P., and Prehn, R. T. (1975). J. Nut. Cancer Inst. (in press). Main, J. M., and Prehn, R. T. ( 1957). J. N d . Cancer Inst. 19, 1053-1064. Malmgren, R. A., Bennison, B. E., and McKinley, T. W., Jr. (1952). Proc. Soc. Exp. Biol. Med. 79, 484-488. Marchant, J. ( 1969). Brit. J. Cancer 23,383-390. Medina, D., and Heppner, G. ( 1973). Ndure ( London) 242,329330. Morton, D. L., Goldman, L., and Wood, D. A. (1969). J. Nut. Cancer Inst. 42, 321-329. Naha, P. M., and Ashworth, M. (1974). Brit. 1. Cancer 30,448458. Nazerian, K. (1973). Aduan. Cancer Res. 17, 279-315. Nehlsen, S. L. ( 1971). Clin. Exp. Immunol. 9, 63-77. Old, L. j., Boyse, E. A., Clark, D. A., and Carswell, E. A. (1962). Ann. N.Y. Acad. Sci. 101, 80-113. Outzen, H. C., Custer, R. P., Eaton, G. J., and Prehn, R. T. (1975a). J . Reticuloendothel. SOC. 17, 1-9. Outzen, H. C., Custer, R. P., and Prehn, R. T. (197513). In “Immunologic Phylogeny” ( W . H. Hildemann and A. A. Benedict, eds.), pp. 383-386. Plenum, New York, 1975. Parmiani, G., Carbone, G., and Prehn, R. T. (1971). J. Nat. Cancer Inst. 46,261-268. Parmiani, G., Carbone, G., and Lembo, R. (1973). Cancer Res. 33, 750-754. Peters, L. J. (1975). Brit. J. Cancer 31,293300, Pierce, G. B. ( 1974). Amer. J. Pathol. 77, 103-118. Potter, C. W., Hoskins, J. M., and Oxford, J. S. (1969). Arch. Gesamte Virusforsch. 27, 73-86. Prehn, R. T. (1953). J . Nut. Cancer Inst. 13, 859-871. Prehn, R. T. (1960). Cancer Res. 20, 1614-1617. Prehn, R. T. (1962). 16th Annu. Symp. Fundam. Cancer Res., Uniu. Tex., M. D. Anderson Hosp. Tumor Inst. pp. 475-485. Prehn, R. T. (1963). J. Nut. Cancer Inst. 31,791-805.

236

RICHMOND T. PREHN

Prehn, R. T. (1964). J . Not. Cancer Inst. 32, 1-17. Prehn, R. T. (1969a). Ann. N.Y. Acad. Sci. 164,449457. Prehn, R. T. (1969b). J. Nut. Cancer Inst. 43, 1215-1220. Prehn, R. T. (1970). I . Nut. Cancer Inst. 45, 1039-1045. Prehn, R. T. (1971a). J. Reticubendothel. SOC. 10, 1-16. Prehn, R. T. (1971b). Progr. Exp. Tumor Res. 14, 1-24. . “Immune Surveillance” (R. T. Smith and M. Landy, eds.), Prehn, R. T. ( 1 9 7 1 ~ )In pp. 451462. Academic Press, New York. Prehn, R. T. (1972). Science 176, 170. Prehn, R. T. (1974). Clin. Immunobiol. 2, 191-203. Prehn, R. T. (1975a). J. Not. Cancer Inst. 55,189-190. Prehn, R. T. ( 1975b). In preparation. Prehn, R. T., and Lapp&,M. A. ( 1971). Transplant. Rev. 7 , 2 6 5 4 . Prehn, R. T., and Main, J. M. (1956). J. Not. Cancer Inst. 16, 1257-1261. Prehn, R. T., and Main, J. M. (1958). J. Nut. Cancer Inst. 20, 207-209. Pullinger, B. D. ( 1952). Brit. 1. Cancer 6, 78-79. R&v&sz,L. (1960). Cancer Res. 20, 443. Rous, P., and Kidd, J. G. (1941). J. Erp. Med. 73,365490. Rusch, H. P., and Kline, B. E. (1946). Arch. Pathol. 42,445-454. Sdaman, M. H., and Roe, F. J. C. (1953). Brit. J. Cancer 7,472-481. Sanford, B. H. (1965). Erp. Cell Res. 39,97-102. Sanford, B. H., Kahn, H. I., Daly, J. J., and Soo, S. F. (1973). J. Immunol. 110, 1437-1439. Schwartz, R. C. (1975). N . Engl. J. Med. 293,181-184. Shearer, W. T. (1973). Clin. Res. 21, 839. Shinozuka, H., and Ritchie, A. C. ( 1967).Int. 1. Cancer 2,77-84. Shubik, P. (1961). Acta Unio Int. Contra Concnrrn 17,2241. Siegler, R. ( 1970).Bib. Haemutol. (Basel) 36, 257-260. Sinha, D., and Dao, T. L. ( 1975). J. Nut. Cancer Inst. 54,1007-1010. Sjogren, H. 0. ( 1964). J. Nut. Cancer Inst. 32, 361-374. Skov, C. B., Holland, J. M., and Perkins, E. H. ( 1975). J. lmmunol. (in press). Slemmer, G. ( 1972). Nut. Cancer Inst., Monogr. 35, 57-71. Slemmer, G. (1974). J. Invest. D e m t o l . 63, 27-47. Stillstrom, J. (1974). Int. J . Cancer 13, 273-285. Stoker, M., and MacPherson, I. (1961).Virology 14,359-370. Stjemsward, J. (1967). J. Nut. Cancer Inst. 38,515526. Stjemswiird, J. ( 1968). J. Nut. Cancer Inst. 40, 13-22. Stutman, 0. ( 1974). Science 183,534-536. Stutman, 0. ( 1975). Nature (London) 253, 142-144. Svet-Moldovsky, G. Ya., and Camburg, V. I. (1965). Bull. Exp. Biol. Med. (USSR) 60, 1053-1055. Takasugi, M. R., Mickey, M. R., and Terasaki, P. I. (1973). Cancer Res. 33, 2898-2902. Vaage, J. (1968). Nature (London) 218, 101-102. Wekerle, H., Cohen, I. R., and Feldman, M. (1973). Nature (London), New Biol. 241, 25-26. Willis, R. A. (1960). I n “Pathology of Tumors,”3rd ed., p. 108. Butterworth, London. Young, L. J. T., Daniel, C. W., Medina, D., and DeOme, K. B. ( 1971).Exp. Gerontol. 6, 95-101.

GENETIC TRANSFORMATION OF ANIMAL CELLS WITH VIRAL DNA OF RNA TUMOR VIRUSES

Miroslav Hill and Jana Hillova Department of Cellular and Molecular Biology and Equipe d e Recherche No. 148 du C.N.R.S., Institute of Cancerology and Immunogenetics, Villeiuif, France

.

.

.

.

111. Virus-Specilk DNA in Virus-Infected and Uninfected Cells IV. Infectivity of the Viral DNA . . . . . . . A. Transfection Assay . . . . . . . . B. Efficiency of Transfection Assay . . . . . C. Genetic Content of Infectious Viral DNA . . . . D. Structure of Infectious RSV DNA . . . . . E. Minimum Size of Infectious RSV DNA . . . . V. Sizing the RNA Genome in Virus Particles . . . . VI. Search for Transforming Genetic Material . . . . A. Sarcoma Viruses . . . . . . . . . B. Leukemia and Leukosislike Viruses . . . . . C. “Spontaneous” Transformation in Vivo . . . . VII. Conclusions . . . . . . . . . . References . . . . . . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

I. Introduction

.

11. Endogenous Viruses

.

.

.

.

.

.

. .

.

.

.

.

.

.

.

.

.

.

. . . . . . .

.

. . . . . . .

.

. . . . . . .

237 239 243 246 247 257 262 268 270 271 274 275 279 282 287 289

I. Introduction

A metazoan organism is a highly organized multicellular system in which the cells have the capacity to recognize and follow signals of growth control that influence their mitotic activity and differentiation. Such normal cells may, however, give rise in uiuo to progeny tumor cells that multiply uncontrollably. In uitro, too, the mitotic activity of normal cells is regulated though the mechanisms of control may differ from those in uiuo. Cells that lose the ability to obey these signals are said to be transformed. All tumor cells, if able to grow in uitro, behave as transformed cells, whereas not all cells transformed in uitro can give rise to tumors isl uiuo. However, this latter failure is assumed to be due to the inability of the transformed cells to grow in the new host. It is believed that the processes involved in in uitro transformation, at least at the molecular level, are equivalent to the same processes involved in the initiation of a tumor. Hence, because studies of in uitro transformation allow a direct interaction between cell and transforming agent, malignant transformation is mostly studied in simplified models 237

238

MIROSLAV HILL AND JANA HILLOVA

using cells in culture, i.e., growing outside the organism. Under these conditions, only fibroblasts of some species, such as man and chick, are able to maintain a normal phenotype throughout their entire life-span in uitro and, at the same time, are unable to grow indefinitely as an established line in cell culture. Fibroblasts of other animal species, however, can be readily established as permanent lines in uitro. The establishment of a cell line sometimes goes together with the acquisition of a malignant phenotype; if so, the cell line is considered as “spontaneously’’ transformed. Normal cells can be transformed in uitro with chemical and physical carcinogens and, in addition, with tumor viruses. In such cases, as in the case of “spontaneous” transformation, the cells acquire new biological and biochemical properties allowing them to reach a high saturation density in monolayers, to form colonies in soft agar, to grow at a reduced serum concentration, to be agglutinated by lectins, etc. Transformed cells can be assayed for tumorigenicity in animals that will not immunologically reject the cell graft. The transition from a normal to a transformed phenotype may be either the consequence of a modged expression of the cellular genome, or of a modification in the genetic content of the cell. In other words, the genetic infohation for cancer may be either already present in the cell and repressed, or introduced into the cell genome by genetic transformation (i.e., the integration of new genes) or mutation. The former hypothesis is best known under the name of oncogene theory (Huebner and Todaro, 1969). These two alternatives delineate, in general terms, the central problem in the etiology of cancer. In this presentation we will discuss experiments showing that, upon infection with RNA tumor viruses, animal cells become genetically transformed in uitro. The transfection technique in particular has provided unambiguous evidence that there is a DNA intermediate in the life cycle of the RNA tumor viruses which is integrated into the cellular genome. To emphasize this common property of all oncogenic viruses, i.e., the ability to introduce viral sequences into the host cell DNA, the RNA tumor viruses may be regarded as DNA tumor viruses with an RNA intermediate. Transformation therefore can either be caused by the products of viral genes, integration simply serving to ensure their inheritance, or be a result of integration per se and the deletion of unique cell sequences. In the experiments described in this chapter we show that the ability of both the sarcoma and leukemia viruses to cause malignant transformation depends on the presence of new genetic material, introduced by the virus into the infected cell, which can be detected both by nucleic

DNA OF RNA TUMOR VIRUSES

239

acid hybridization and transfection techniques. Endogenous viruses, which by definition are harbored by all cells of the same animal species and released spontaneously or after treatment with physical and chemical inducers, cannot phenotypically transform cells. This is evidence against the possibility that integration alone could cause transformation. In the case of sarcoma viruses, there is conclusive evidence that the viral genetic material contains transforming genes that must be continually expressed in order to maintain the transformed phenotype of virus-infected cells. These transforming genes are not involved in virus replication. It is therefore probable that these genes were originally mutated cell genes whose covalent linkage to viral nucleic acid sequences was favored by strong selection and passage in the laboratory. The case of leukemia viruses differs in that no temperature-sensitive ( t s ) mutants have yet been found that pinpoint transforming genes. Leukemia viruses almost certainly lack transforming genes of the type found in sarcoma viruses, and it is quite possible that the genes responsible at least for certain leukemias are also involved in virus replication. Such a situation would help to explain how transforming genes evolved in vivo and would further strengthen an already strong case for the viral etiology of leukemia. This idea will be taken up in detail in Section VI. For the sake of brevity, we will not mention data that are not directly related to the discussed topics. For instance, we will deal with the sarcoma-leukemia viruses, i.e., with C-type viruses, and omit all the experimental evidence obtained in studies with the B-type RNA tumor viruses (recently reviewed by Bentvelzen, 1974; for morphology, see Bernhard, 1960; also the recent review by De Harven, 1974). The molecular structure of the RNA and DNA forms of the genome in C-type viruses has already been reviewed (Hill and Hillova, 1974; Hill et al., 1975). The reader is also referred to comprehensive reviews concerning the physiology and the molecular biology and genetics of RNA tumor viruses (Tooze, 1973; Temin, 197413; Gillespie et al., 1975) and concerning related topics, such as RNA-directed DNA polymerase (Temin and Baltimore, 1972; Green and Gerard, 1974), input and output of genetic material in cells producing C-type RNA tumor viruses (Hill, 1973), viruslike particles in human leukemia cells (Gallo and Gallagher, 1974), activation of mammalian leukemia viruses ( Hirsch and Black, 1974), structura1 components of RNA tumor viruses (Bolognesi, 1974), and viral and tumor cell antigens (Bauer, 1974). II. Endogenous Viruses

Until recently, RNA tumor viruses were copsidered simply to be infectious agents propagated by horizontal transmission from one host to

240

MIROSLAV HILL AND JANA HILLOVA

the other, or vertically from one generation to the next. This classical concept has been enlarged by the discovery of apparently ubiquitous endogenous viruses. In 1966 Dougherty and Di Stefan0 observed that leukosis-free chickens carry the group-specific (gs) antigen of avian tumor viruses. Later Weiss (1969) noted that synthesis of an infectious Bryan high-titer Rous sarcoma virus (BH-RSV) occurs only when the virus is growing in gs-positive chicken cells. H. Hanafusa et al. (1970) also recognized that there is some mechanism operating in chicken cells and specifically affecting the properties of BH-RSV. In an attempt to elucidate this mechanism, they have shown the presence, in normal chicken cells, of a genetic factor that shares some of the characteristics of avian leukosis viruses: it is responsible for the formation of the infectious form of BH-RSV and can be transferred, by the RSV or avian leukosis virus, from helper factor-negative to factor-positive cells. The factor was named chick helper factor (chf). It was later isolated in its infectious form from Rous-associated virus ( RAV)-infected chicken cells. This form, known as RAV-60, was allocated to a new subgroup E avian tumor viruses (T. Hanafusa et al., 1970). Recently H. Hanafusa et al. ( 1974) examined the oncogenicity, replication in susceptible hosts, and degree of sequence homology between viral RNA and chicken cell DNA and emphasized that RAV-60 is a genetic recombinant between the endogenous helper factor and an exogenous virus, rather than the endogenous virus itself. The occurrence of a chicken endogenous virus has been reported by Vogt and Friis (1971). These authors observed that some line 7 embryos release spontaneously infectious viral particles, named RAV-0, which share envelope properties with BH-RSV(chf) and RAV-60. The phenomenon was further analyzed in a series of genetic studies. Production of a complete RAV-0 in chicken cells (phenotype V+) seems to be controlled by dominant genes (Crittenden et al., 1974), though further studies are required before it is known whether these are only regulator genes or whether some of them are structural genes. So far the experimental data at least show that structural information for a complete virus is present in all chicken cells since, upon induction with physical and chemical carcinogens, chicken cells are able to produce endogenous viruses indistinguishable from RAV-0. This has been shown to occur in a variety of chicken cells, irrespective of whether these cells did, or did not, express gs antigen and the chf (Weiss et al., 1971). The hostrange of endogenous viruses of chick cells is restricted to subgroup E. Endogenous viruses recovered from pheasan.ts possess a different host range and have been designated to subgroup F (T. Hanafusa and Hanafusa, 1973) and C (Fujita et al., 1974) avian RNA tumor viruses.

DNA OF RNA TUMOR VIRUSES

241

Endogenous viruses were later identified in a variety of mammalian species. Most, if not all, strains of mice were shown to release C-type viruses either spontaneously or upon induction (see Tooze, 1973, for review). These viruses are found either to be able to grow in the cells of the same species, in which case they are called ecotropic, or unable to grow in the same species though able to grow in heterologous hosts, in which case they are called renotropic ( Levy, 1973). Ecotropic viruses, like exogenous murine leukemia viruses ( MuLV), are further divided into separate host-range classes. N-tropic viruses are able to multiply in NIH Swiss mouse cells, whereas B-tropic viruses grow preferentially in BALB/c cells. Xenotropic viruses are allocated to a distinct host-range class of X-tropic viruses. It was reported that, for example, BALB/c cells release all three host-range classes of endogenous viruses, i.e., nononcogenic N-tropic ( Aaronson et al., 1969; Todaro, 1972) and X-tropic (Benveniste et al., 1974c; Fischinger et al., 1974) viruses released from cell lines in vitro ( Aaronson and Dunn, 1974; Stephenson et al., 1974b), and oncogenic B-tropic viruses from aged and neoplastic tissues (Peters et al., 1972, 1973). These viruses apparently differ one from another in various portions of the viral genome according to nucleic acid hybridization studies (Callahan et al., 1974). Furthermore, xenotropic viruses released from different strains of mice differ in a type-specific virion p12 polypeptide (Stephenson et al., 1974a). Spontaneous or induced release of endogenous viruses was shown to occur in cell cultures from Chinese hamster (Lieber et al., 1973), rat (Teitz et al., 1971; Klement et al., 1973; Lieber et al., 1973), guinea pig (Nayak and Murray, 1973; Murray and Nayak, 1974), pig (Breese, 1970; Armstrong et al., 1971; Todaro et al., 1974), cat (Livingston and Todaro, 1973; Lieber et al., 1973), and baboon (Kalter et al., 1973; Benveniste et al., 1974b); only those viruses from the latter two animal species, however, multiplied successfully in vitro and were found to be xenotropic (Livingston and Todaro, 1973; Benveniste et al., 1974b). Nevertheless, the other virus isolates were shown to carry reverse transcriptase (Klement et al., 1973; Lieber et al., 1973; Todaro et al., 1974) and 60-70 S RNA (Klement et al., 1973; Nayak and Murray, 1973) characteristic of RNA tumor viruses, so that they could be considered as potentially infectious despite the apparent absence of permissive hosts. Some animal species appear to carry endogenous viral information that can only be expressed when the cells are transformed. For example, normal Syrian hamster cells have never been shown to be able to produce a C-type virus. However, release of viral particles has been reported to take place in hamster cells from spontaneous tumors (Stenback et

242

MIROSLAV HILL AND JANA HILLOVA

al., 1968), and from tumors induced by murine sarcoma viruses (Bassin et al., 1968; Klement et al., 1969; Kelloff et al., 1970; Sarma et al., 1970) or from hamster cells transformed in vitro by chemical carcinogens ( Freeman et al., 1971). These endogenous hamster viruses, if infectious, can be propagated in hamster cells (Kelloff et al., 1970; Freeman et al., 1971); noninfectious isolates were also reported and shown to be deficient in DNA polymerase activity (Peebles et al., 1972; Somers et al., 1973). A considerable body of evidence has been accumulated (see review by Tooze, 1973) concerning the inheritance of avian and murine endogenous viral information and the regulatory control of its expression. However, the biological role of this viral information both at the cellular level and at the level of the metazoan organism remains unknown. It seems likely that these viruses (except the ecotropic viruses endogenous in inbred strains of mice (cf. Gross, 1951; Peters et al., 1973; Stephenson et al., 1974c; Greenberger et d.,1975)) are unable to induce tumors; at least under routine conditions of bioassay. Thus in the absence of inducing agents (and/or carcinogens) they seem to behave as inoffensive companions of normal cells, and their existence raises important questions. The widespread occurrence of endogenous viruses was predicted by Huebner and Todaro (1969) in their oncogene hypothesis, which suggested that most, or all, vertebrate cells carry “endogenous virogenes” maintained either in an unexpressed form or expressed to varying degrees. Full expression of this genetic material gives rise to an endogenous virus. According to this hypothesis, a complete set of viral genes is an inherent portion of a normal cellular genome and, consequently, C-type particles are cellular products rather than independent biological entities. The hypothesis assumes that, among the structures produced by a normal cell, one known as C-type particle, is exceptional because it is endowed with the characteristic properties of an infectious agent. An alternative interpretation of the release of C-type particles from apparently normal cells necessarily presumes that the virus-producing cells have been infected with the virus or have inherited the viral genome from an infection taking place in the past. The latter concept was recently upheld by Gross ( 1974). Both these hypotheses have stimulated further predictions about the role of the virus. For instance, those who consider that the endogenous viruses are normal cellular constituents implicate a role for these viruses in embryogenesis, cytodifferentiation, and immunogenesis, whereas those who believe the viruses to be latent infectious agents regard the induced virus as representing a potential danger to its carrier. Before going fur-

DNA OF RNA TUMOR VIRUSES

213

ther in this discussion, however, we will first inquire how the viral information is stored in the infected cells. Ill. Virus-Specific DNA in Virus-Infected and Uninfected Cells

In view of the above discussion the term “uninfected cells” will be used to mean uninoculated cells that may still contain endogenous viral genomes. The idea that cells infected with RSV carried virus-specific DNA originated from Temin ( 1964b). He extracted ~ridine-~H-labeled RNA from virions and unlabeled DNA from both uninfected and RSV-infected cells. The RNA was hybridized with denatured DNA, and the fraction of RNA recovered in RNA-DNA hybrids was measured. Under these conditions the amount of RNA hybridizing with the DNA from RSVinfected cells was always higher than that found to hybridize with the DNA from uninfected cells. However, the extent of hybridization was rather low, not exceeding 0.3%of the input RNA. Temin’s experiments were later repeated by several authors, but because of the poor efficiencies obtained in these hybridizations no convincing proof for the existence of a virus-specific DNA was provided (see Duesberg, 1972, for review). Nevertheless, Bader (1966) was able to recognize that RSV RNA hybridizes more efficiently with the DNA from either RSV-infected or uninfected chicken cells than with the DNA from Escherichia ~ 0 1 i or hamster embryo. He also noticed a difference due to virus infection when he compared hybridization of viral 65 S RNA (but not 10 S RNA) and the DNA from RSV-induced hamster tumors with hybridization of the same 65 S RNA and the DNA from hamster embryos. His results suggested that virus-specific nucleotide sequences are present in the DNA of RSV-transformed hamster cells and, furthermore, in the DNA of both RSV-transformed and uninfected chicken cells. The explanation of these results came later: infectious virus-specific DNA was detected in RSV-transformed cells (Hill and Hillova, 1971c; also Section IV), and endogenous C-type viruses were recovered from normal chicken celIs (Section 11; see also Weiss, 1972, for further discussion and references). These new pieces of evidence encouraged several investigators to reexamine, by RNA-DNA molecular hybridization experiments, for the presence of virus-specificnucleotide sequences in the DNA of normal chicken cells and, furthermore, to estimate the number of virus genome equivalents per cell. Earlier estimates suggested that avian myeloblastosis virus (AMV) (Baluda and Nayak, 1970), RSV, or RAV (Rosenthal et al., 1971) infected chicken cells carry about twice as much virus-specific

244

MIROSLAV HILL AND JANA H I U O V A

DNA as uninfected cells. In further experiments, about 2-6 viral genome equivalents were found in the DNA of healthy gs positive as well as gs negative chickens, when measured both by the filter RNA-DNA hybridization technique of Gillespie and Spiegelman ( 1965) (see also Shoyab et aZ., 1974c, for the reliability of this technique) with the viral RNA in excess (Baluda, 1972; Baluda and Drohan, 1972) and, more accurately, by HNA-DNA reassociation kinetics (Neiman, 1973a; Shoyab et al., 1974a) with the cellular DNA in excess as described by Melli et at. (1971). Ring-necked pheasant, Japanese quail (Neiman, 1973a), or rat (Shoyab et al., 1974a) cells contained only about lo%, 4%, and none, respectively, of the avian RNA tumor virus genome equivalents. Upon viral infection, chicken cells could be shown to acquire additional virusspecific nucleotide sequences (Baluda, 1972; Baluda and Drohan, 1972; Neiman, 1972; Shoyab et al., 1974a), and new virus-specific nucleotide sequences occurred in mammalian cells (Hare1 et al., 1972; Shoyab et al., 1974a). After the discovery of reverse transcriptase by Temin and Mizutani (1970) and Baltimore (1970) the search for virus-specific DNA continued with the adoption of the technique described by Gelb et al. (1971b) of DNA-DNA reassociation. Viral DNA was synthesized in uitm by the viral enzyme using the endogenous RNA template of deterghnt-treated virions. Labeled double-stranded DNA product of the enzymic reaction was denatured and used as a probe to demonstrate virusspecific nucleotide sequences in unlabeled cellular DNA which would accelerate reassociation of the probe. Using this technique, Varmus et al. ( 1972) have shown RSV-specific sequences in uninfected chicken cells. However, they failed to demonstrate a difference in viral DNA content of uninfected and RSV-infected chicken cells. In contrast, Schincariol and Joklik (1973) used a single-stranded DNA probe [which unlike the double-stranded probe is representative of most, perhaps all, of the viral genome (Garapin et al., 1973)], and estimated from the reassociation rate of this probe with cellular DNA that uninfected chicken cells harbor only one RSV genome equivalent, although additional equivalents are found in these cells within 24 hours after RSV infection. These results agreed with those obtained in above-mentioned RNA-DNA hybridization studies and were confirmed by Varmus et al. ( 1 9 7 4 ~ )using three different techniques in a subsequent paper. In rat and mouse cells, the DNA-DNA reassociation technique detected no RSV-specific nucleotide sequences before RSV infection. Upon transformation with RSV, however, these cells were shown to acquire about two viral genome equivalents per diploid cell (Varmus et al., 1973a) and hold them covalently integrated in the chromosomal DNA (Varmus

DNA OF RNA TUMOR VIRUSES

245

et al., 197313). Integration of RSV-specific DNA was also shown to occur in RSV-transformed duck cells which normally contain no nucleotide sequences specific for RSV (Varmus et al., 1973b). At the same time, Markham and Baluda ( 1973) using KNA-DNA hybridization techniques, were able to detect an increase in the number of virus-specific nucleotide sequences in the high-molecular-weight DNA of chicken cells after AMV infection. Furthermore, free virus-specific DNA has been found early (but not late) in RSV (Varmus et al., 197313, 1974b) or AMV (Ali and Baluda, 1974) infections. In the case of mouse cells the hybridization experiments using acceleration of reannealing kinetics with a double-stranded MuLV DNA probe failed to demonstrate a difference in viral DNA content of normal and MuLV-infected cells (Gelb et al., 1971a). Furthermore, there was no detectable difference in the number of MuLV genome equivalents in the DNA from low and high leukemia-incidence strains of mice (Gelb et al., 1973). In contrast, single-stranded MuLV-specific DNA probes hybridized more extensively to virus-induced lymphoma DNA (Viola and White, 1973) and virus-infected mouse cell DNA (Scolnick et al., 1974a) than to normal mouse DNA. Early in infections with MuLV, mouse cells have been shown to carry nonintegrated virus-specific DNA (Lovinger et al., 1974; Gianni et al., 1975), RNA-DNA hybrids (Takano and Hatanaka, 1975), and RNA-DNA covalent hybrids (Leis et al., 1975) . I n situ hybridization technique localized MuLV-specific DNA in the chromocenters of interphase nuclei and also in the centromeric region of metaphase chromosomes (Loni and Green, 1974). To summarize, the search for virus-specific DNA in uninfected and virus-infected cells provided data consistent with the notion that uninfected cells carry endogenous virogens and, upon infection of these cells, new virus-specific DNA appears and becomes integrated into the cellular chromosome. However, the question whether this virus-specific DNA fully specifies the virus could not be resolved because (1) with DNA excess, for instance, only 60-80% of viral RNA [or endogenously synthesized single-stranded DNA (Varmus et al., 1974c)l hybridizes to the DNA of virus-infected cells (Neiman, 1972; Shoyab et al., 1974a), and ( 2) double-stranded DNA probes synthesized in detergent-treated virions do not completely represent nucleotide sequences of viral RNA (Garapin et al., 1973). Therefore, it is not clear whether various RNA-DNA and DNA forms so far isolated early from virus-infected cells, are fully representative of the viral genome. Furthermore, there exists no direct evidence that late in infection the viral RNA of virus progeny is transcribed from those virus-specific nucleotide sequences that reside in the host chromosomal DNA. That the integrated sequences

246

MIROSLAV HILL AND JANA HILLOVA

really represent full-length DNA copies of the viral genome has been shown by means of transfection experiments (Hillova et aZ., 1974b), as will be discussed below. IV. Infectivity of the Viral DNA

In the last decade several authors have observed that cocultivation (Simkovi&et al., 1962) or fusion (Svoboda et al., 1967; Vigier, 1967; Yamaguchi et al., 1967) with permissive cells rescued the virus from nonproducer RSV-transformed mammalian cells. It was clear, therefore, that a compkte RSV genome must persist in virus-transformed cells in a form permitting its propagation to progeny cells. The molecular nature of this persistent form of the viral genome was, at that time, a complete mystery. Studies of viral replication provided the first clues. No conclusive evidence could be obtained in favor of the replication of viral RNA via RNA intermediates, although an RNA complementary to the viral RNA has been encountered in virus-transformed cells (Biswal and Benyesh-Melnick, 1969). In an alternative concept, Temin (1964a) suggested that the intermediate for replication of RNA tumor viruses might be DNA. According to this author (Temin, 1971b) the strongest evidence for the existence of the DNA provirus was as follows: (1) a transient requirement for a DNA synthesis in early infection, ( 2 ) a continuous requirement for a DNA-dependent RNA synthesis during the release of the virus progeny, (3) a sensitivity to visible light of the virus growing in the presence of bromodeoxyuridine ( BUdR), and ( 4 ) the presence in virions of RNA-directed DNA polymerase. Formation of the provirus has been defined by Temin (1971b) as “formation, in a form resistant to inhibition of DNA synthesis, of a stable nonvirion structure containing viral information.” Available experimental data led us to believe first that there is a DNA intermediate in the viral replication and, second, that it is this DNA that gives rise to the virus upon fusion of RSV-transformed mammalian cells with permissive cells. At that time, it was known that purified DNA extracted from polyoma (DiMayorca et aZ., 1959) and SV40 (Gerber, 1962) virions readily infects permissive cells. Furthermore, a considerable body of evidence indicated that the DNA extracted from animal cells could be taken up by the recipient cells in uitm, and some observations even suggested that this DNA is biologically expressed, so that donor-specific changes are induced in the recipient cell phenotype (for reviews, see Bhargava and Shanmugam, 1971; Hill and Hillova, 1974). Supporting evidence for the latter findings has been obtained in biochemical studies showing the occurrence of single-strand stretches of foreign DNA covalently bound to nascent strands of the recipient DNA

DNA OF RNA TUMOR VIRUSES

247

(Hill and Hillova, 1971a). These data suggested that integrated viral DNA might, when introduced into recipient cells, give rise to virus-specific gene products and, when fully expressed, to the virus. This was shown to be the case in our very first experiment in which we found virus-producing foci of transformed cells in chicken cell cultures treated with the DNA from RSV-transformed mammalian cells (Hill and Hillova, 1 9 7 1 ~ )Infections . of permissive cells with a high-molecular-weight DNA extracted from SV40-transformed cells were later reported by Boyd and Butel ( 1972). The phenomenon is named tmnsfection, since animal cells are infected with naked viral nucleic acid instead of virus particles. Because of the unique replication cycle of the RNA tumor viruses in which there are two forms of the viral genome, viral RNA in virions is spoken of as RNA form of the viral genome, and viral DNA in virus-infected cells represents a DNA form of the viral genome (Hill and Hillova, 1974). Recent evidence has shown that the DNA form of some, perhaps all, RNA tumor viruses is infectious. Below we demonstrate that this is certainly the case with the avian tumor viruses, where the infectious DNA was most thoroughly studied, and also with mammalian tumor viruses, where so far analogous studies have been performed with infectious DNA of feline RD-114 or feline leukemia viruses, gibbon ape lymphosarcoma virus (Nicolson et d.,1975), and murine xenotropic virus (Scolnick and Bumgarner, 1975). As to the infectivity of the RNA form, the evidence is not clear, although a relatively unpurified preparation (e.g., still containing protein) of RNA extracted from AMV has been reported to be infectious (Veprek et ul., 1971).

A. TRANSFECTION ASSAY A transfection assay may be described by the simple scheme: DNA from virus-infected cell permissive recipient cell + virus. According to this scheme the virus is generated in the recipient cell from the donor DNA (see Section IV,C for further discussion). It is possible that before virus production starts the virus-specific nucleotide sequences of the donor DNA are first integrated into the host chromosome. Alternatively, these virus-specific nucleotide sequences are expressed before integration, which could occur if the cellular DNA-dependent RNA polymerase were able to transcribe unintegrated foreign DNA. Perhaps both pathways occur in transfections, for biochemical data show that donor DNA in recipient cells is preserved both in an integrated and nonintegrated state (Hill and Hillova, 1971a). We tentatively adopt the view that in most transfection experiments the viral DNA is fully expressed. Partial expressions may arise from

+

248

MIROSLAV HILL AND JANA HILLOVA

fragments or, less likely, from incomplete transcripts of the viral DNA In the case of full expression, a stable association between viral DNA and the host DNA is not prerequisite for a positive transfection assay because transfection would be manifested, even if the transfecting DNA was destroyed after the first transcriptional event, by the appearance of an infectious virus. This is not so in the case of partial expression in which no infectious virus would appear, and in which the transfected state would manifest itself only by the appearance of a clone of transformed cells. Therefore, transfections not producing the virus would necessarily imply that a stable association takes place between viral DNA and the host cell, such as integration of viral genes into the host DNA. Relevant experimental data are discussed in the Section IV,A,4. In this context it is worth adding that the DNA treatment of animal cells may have side effects inducing nonspecific changes (changes that also occur spontaneously in the same cells) in the phenotype 6f the recipient cells. Obviously, the cell lines spontaneously segregating transformed cells or releasing endogenous virus are not suitable for transfection assays, unless the specificity of transfection could be established from genetic markers, eg., temperature-sensitivity of the recovered vinis.

1. Infectious DNA of Avian RNA Tumor Viruses The first transfection assays clearly showed that no particular precautions are necessary during the extraction of the DNA able to infect chicken cells. The reasons are now obvious: the viral DNA is apparently small ( 6 X lo6 daltons only) and, furthermore, it is covalently linked to the cellular DNA. The infectious DNAs were prepared from nonproducer virus-transformed cells and from producer virus-infected or virus-transformed cells as listed in Table I. Whole cells (Hill and Hillova, 1971c, 1972b) or cell nuclei (Svoboda et al., 1973; Goubin and Hill, 1974) were lysed with sodium dodecyl sulfate and the lysate treated, with small modificaions, according to Mannur’s (1961) method or, in experiments by Montagnier and Vigier (1972) and Ogura d al. (1974a), further fractionated according to the Hirt’s (1967) procedure. The nucleoprotein was deproteinized with chloroform (Hill and Hillova, 1972b) or phenol (Svoboda et al., 1972) at room temperature, and treated with RNase. Purified DNA was slightly contaminated with RNA and proteins, and both contaminants could be further removed by banding the DNA samples at equilibrium in CsCl gradients (Hill and Hillova, 197213). The DNA was very heterogeneous in size, as shown by the sedimentation profile in a sucrose gradient (Hill et al., 1974). The average molecular weights of various infectious DNA samples varied from 5.3 X lo6 to 25.5 X lo6

DNA OF RNA TUMOR VIRUSES

249

(Hill et al., 1974). High-molecular-weight DNA (MW more than 60 x 106) seems to be less efficient in transfections and has to be sheared before use (Levy et al., 1974). 2. Recipient Cells

Transfection assays are performed using celIs that are permissive for viral replication and susceptible to the recovered virus. The reasons are obvious: a transfected cell should allow synthesis and release of virus particles coded by the DNA. This progeny can easily be detected only if horizontal spread and multiplication in susceptible cells occur. Table I shows that the cells from various avian species fulfill the abovementioned criteria and, accordingly, can be transfected with the DNA of avian RNA tumor viruses. Most transfection experiments were carried out using chicken embryo fibroblasts. The cells were obtained from chicken embryos and allowed to grow in monolayers. Secondary (Hill and Hillova, 1972b) or later (Cooper and Temin, 1974) passages of these cell cultures are convenient for transfection assay. Simple addition of the DNA into the culture medium never gave rise to the virus. Apparently the viral DNA is not inherently infectious when presented at the surface of healthy (nontreated) cells. In the past, various procedures have been elaborated in order to render animal cells more susceptible to the infection with viral nucleic acids. Among these procedures those using DEAE-dextran according to McCutchan and Pagano (1968) and, more recently, calcium phosphate as introduced by Graham and Van der Eb (1973) are the most efficient. To check for the capacity of cells of a given cell line to take up and store foreign DNA a biochemical assay is performed (Hill and Hillova, 1971a,b). Briefly, the cells in monolayer are pretreated with a DEAE-dextran solution, then overlayered with a solution of foreign th~midine-~H-labeled DNA, and 15-60 minutes later allowed to grow in the presence of thymidine-'". After about 24 hours the cells are lysed on the top of an alkaline sucrose gradient and centrifuged. Foreign DNA, if preserved in a nonintegrated state, is separated from the highmolecular-weight chromosomal DNA by sedimentation. Under these conditions, chicken cells (Hill and Hillova, 1971a,b) and also a variety of mouse and rat cells (J. Hillova and J. A. Levy, unpublished data) were found to take up and carry in a nonintegrated state foreign DNA for at least 24 hours. Such a fraction of DNA was missing in control cells receiving foreign DNA without the DEAE-dextran pretreatment. DEAE-dextran was shuwn to form a complex with nucleic acids in solution which is more resistant (aIthough not completely resistant) to the action of nucleases than naked RNA or DNA (Pagano et al.,

TABLE I PARENT A N D PROGENY AVIAN1tNA TUMOR VIRUSE:SI N TRANSFEC~WN ASSAYS

Parent virus Sarcoma viruses Crude P R

Crude P R PR-C (from XC PIt-C (from Wyke's lab) tdu4335Plt-C

Virus-infected donor cells (source of DNA)

. DNA recipients

Progeny virus Subgroup specificity

Transforming capacity

Rat X C cellsa

-

CEFb

c

+"

Rat X C cells CEF

+ + +

-

CEF, chj-d CEF

C

c

+

CEF

NTs

CEF

NT

+ +

-

CEF, gs-, Chj-

NT

+

CEF

u

+

CEF

A

+

CEF CEF

D D

+and +

+

CEF, gs-, chj-

D

+

+

Duck cellsd

NT

+

CEF CEF

Crude sIt

Rat embryo fibroblasts Chinese hamster ItSCH cellsA

SR-A

CEF

SR-D SR-D SIX-D

CEF Cloned RSVtransformed CEF CEF

SR-D

CEF

B77

Virus production in donor cells

+ + +

+and

Other characteristics

lteferenccs Hill and Hillova (1971c, 1972b), Svoboda et al. (1972). HloZhek and Svoboda (1972) Levy el al. (1974) Hillova et al. (1974a)

-1

IIillova et al. (1975) ts reverse

transcriptase

-1

Hillova et al. (1975) Cooper and Temin (1974) Svoboda et al. (1972). Hlo6dnek and Svoboda (1972) Vigier and Montagnier (1975) Hillova d al. (1974a) J. Hillova (unpublished data) Montagnier and Vigier (1972), Ogura el a!. (1974a), Cooper and Temin (1974), Vigier and Montagnier (1975) Cooper and Temin (1974)

CEF Syrian hamster 14B cells

SR-D tsSR-D (FU- 19) Leukosis viruses AMV

Chick myeloblasts Chick myeloblasts

AMV DNVi RAV-1 RAV-2

CEF

RAV-50

CEF

tdSR-D Reticuloendotheliosis viruses REV-T

CEF Chicken spleen cells CEF or pheasant cells

TDSNV

++ + + + + + + + +

+

Pheasant cells CEF

NT D

+

ts transforming

CEF, chf-

B

-

CEF

NT

Lacour et al. (1972)

CEF

NT

CEF

NT

-

Ne.phroblastoma zn vzvo Myeloblastosis in vivo Ne.phroblastoma an vzvo

CEF

NT

-

CEF

NT

-

CEF

D

-

CEF

Cytopathic effect

Cooper and Temin (1974)

CEF or duck cells or pheasant cells

Cytopathic effect

Cooper and Temin (1974)

function

Cooper and Temin (1974) Hill and Hillova (1972~) Ogura et al. (1974a,b)

Fourcade et al. (1974)

-

Vigier and Montaenier 71975) I. Ali and M. Hill (unpublished) Vigier and Montagnier (1975) Hillova et al. (1974a)

The XC cells produce Prague strain of Rous sarcoma virus (PR-C) upon fusion with chicken embryo fibroblasts (HloZBnek and Svoboda, 1972). * CEF, chicken embryo fibroblasts. c The symbol means that the virus is able to transform CEF in vitro. d In contrast, HloLBnek and Svoboda (1974) and Svoboda et al. (1974) were unable to transfect CEF, gs-, Japanese quail, and duck cells with the PR-C DNA from XC cells. c The virus was recovered in a transfection assay using the DNA from rat XC cells. I Infectious DNA segregated sarcoma and transformation-defective (Id) viruses. 0 NT, not tested. * The RSCH cells produce SchmidbRuppin strain of Rous sarcoma virus (SR-D) upon fusion with chicken embryo fibroblasts (HloZQnekand Svoboda, 1972). i An isolate from the crude AMV stock.

+

252

MIROSLAV HILL A N D JANA HILLOVA

1967; Maes et al., 1967; May et at., 1969). The solubility of DEAE-dextran-DNA complexes diminished with increasing molecular weight of the DNA, so that DNA of more than 35 S largely precipitated when added to DEAE-dextran solution (May et at., 1969). However, the formation of complexes with nucleic acids cannot itself explain the role of DEAE-dextran in transfections. Pagano (1970) supposes that DEAEdextran also affects the cell surface and makes it either more permeable to, or facilitates pinocytosis of, the DEAE-dextran-DNA complex. Labeled DEAE-dextran has been shown to become attached to the cell although it is not clear whether it enters the cells (Pagano and Hutchison, 1971). Surprisingly, the optimum concentration of DEAE-dextran (100 rglml) for the association of SV40 DNA with cells is much lower than that ( lo00 pglml) for infectivity (Howard et al., 1971). Furthermore, as mentioned above, a large proportion ( u p to 48%, Hill and Huppert, 1970) of foreign DNA entering recipient cells is preserved if the cells are pretreated with DEAE-dextran, and degraded if the pretreatment is omitted (Hill and Hillova, 1971a). The latter two pieces of evidence may mean that DEAE-dextran functions in some way even in the interior of cells. The procedure using calcium phosphate was originally introduced by Graham and Van der Eb (1973) to enhance infectivity of adenovirus DNA in KB cell monolayers. The DNA in a buffered saline-phosphate solution was precipitated upon addition of CaCl,, and the resulting suspension of DNA and calcium phosphate was administered to cell monolayers. The precipitate became adsorbed to cell membranes. Presumably the DNA entered cells through a calcium-requiring process which OCcurred at 37°C after exposure. It has been shown that the infectivity of the adenovirus DNA is about 100-fold greater with calcium phosphate than with DEAE-dextran (Graham and Van der Eb, 1973) whereas the infectivity of RSV DNA during transfection experiments is the same regardless of whether the calcium phosphate or DEAE-dextran is used (Table 11). This difference remains unexplained.

3. Virus Recovey In the first experiment a single exposure of DEAE-dextran-pretreated chicken cells to the DNA extracted from RSV-transformed rat cells gave rise to virus-producing foci of transformed cells (Hill and Hillova, 1 9 7 1 ~ )This . was rather fortunate since in following experiments several DNA exposures were needed in order to transfect every chicken cell culture receiving viral DNA and, furthermore, to establish that the applications of either the same DNA digested with DNase or the rat thymus DNA are inefficient (Hill and Hillova, 1972a,b). The same technique

253

DNA OF RNA TUMOR VIRUSES

TABLE I1 EFFICIENCY OF DEAE-DEXTRAN A N D CALCIUM PHOSPHATE PROCEDURES I N TRANSFECTIONS USING RSV DNA pg DNAa per cell culture

Transfection procedure DEAE-dextranb Calcium phosphated Carrier DNAe added, calcium phosphate

20

5

0.5

0.05

0.005

5/5"

5/5

5/5

5/5

5/5 1/ 5

0/5 0/5

5/5

2/5

1/ 5 0/5 1/5

0/5

DNA was extracted from a clone derived from a single chicken fibroblast, infected, and transformed with a single SR-D particle. The DNA sample was free of transformation-defective (td) virus DNA. * See Hillova et al. (1974a,b) for the technique of transfection assay. The number of transfected cultures per the number of DNA-treated cultures. See Graham and Van der Eb (1973) for the calcium phosphate procedure. Other conditions as in b. Rat thymus DNA was added to make 20 pg of DNA per cell culture throughout.

using repeated exposures furnished evidence that the virus is consistently produced even by those cultures that were treated with alkali-denatured instead of native DNA samples (Hillova et al., 1972). These experimental data allowed us to conclude that the infectious agent detected in the DNA samples from RSV-transformed cells is, in fact, the DNA. A standard transfection assay was introduced later (Hill and Hillova, 1972~;see also Hillova et al., 1974a,b, for further modifications). First a single addition of DNA is made to a DEAE-dextran-pretreated monolayer of chicken cells. After being exposed to the DNA the cells are passaged about 4 days later to prevent detachment of the overcrowded multilayer of cells from the plastic support. Foci of transformed cells appear from day 7 after the DNA treatment (Fig. 1).The use of sparse monolayers in transfection assays may render subsequent passages superfluous (Cooper and Temin, 1974), provided the cells are kept growing under the liquid medium to allow spreading and multiplication of the recovered virus. Under the conditions of the transfection assay, the viral DNA infects one or several cells and these cells start producing virus. If the virus is able to transform cells its presence is revealed by examination of the culture for foci of transformed cells and, furthermore, by the presence of a transforming virus in the culture medium. This examination of the cuIture medium is of particular importance now that nonproducer transformed foci have been encountered (Section IV,A,4). The virus

254

MIROSLAV HILL AND JANA HILLOVA

60

-

40-

: I-

20-

a W n

o

I

z 0

5

80PR.C DNA, denatured

60-

0

5

10

15

20

25

30

TIME (DAYS)

FIG. 1. Cumulative frequency of transfected chicken cell cultures plotted against the time at which the first foci of transformed cells were detected. Passages of cell cultures are indicated with roman numerals. Primary cultures (passage I ) were prepared at the 0 time. Bars under the passage numbers delineate time limits in which the passage of cells took place. DNA treatment of cells is indicated by an arrow. Schmidt-Ruppin strain of Rous sarcoma virus (SR-D) DNA was extracted from SR-D-transformed chicken cells while Prague strain RSV (PR-C) DNA originates from rat XC cells. The plots summarize data from several transfection assays, all performed under conditions as described (Hillova et al., 1974a,b). On the ordinate 100%accounts for 115 cell cultures transfected with SR-D DNA, and for 45 and 26 cell cultures transfected with native and alkali-denatured PR-C DNA, respectively. The dose of infectious DNA varied from 0.005 to 50 cg of DNA per culture.

may also be devoid of transforming capacity. In this case, its presence may be revealed either by an interference assay between the culture medium (assumed to contain the virus) and a transforming virus of the same antigenic subgroup as that shared by the DNA parent, or from an assay of reverse transcriptase activity according to Kelloff et al. (1972) in the high-speed pellet of the culture medium. These estimates are reliable only when confirmed by an electron microscopic examination of cells for the presence of budding and extracellular C-type particles. The transfection procedure discussed above provides at the moment only a qualitative answer about the infectivity of the assayed DNA samples. Obviously, it would be of interest to get a quantitative answer by directly counting the number of transfected cells in DNA-treated cell monolayers. At first sight, it might be possible to obtain such a

DNA OF RNA TUMOR VIRUSES

255

result in a standard focus (or infectious center) assay by covering DNAtreated cells with an agar overlay before the virus spreads or, less rigorously, by using cells resistant to infection with the virus under study in order to prevent spread of the virus released from transfected cells. However, the relevant experiments have not yet been performed, and various problems can be envisaged. For example, it is not clear whether all cells transfected with the DNA, and releasing the virus, are able to survive for enough time to give rise to a focus under the above conditions. Even if they can do this, we still do not know the length of the time lag between the uptake, by a cell, of the DNA and its expression in terms of virus release and cell transformation. This may be much longer in some cases than we allow for. We believe that the lag period in most (if not all) transfections exceeds that characteristic for infections initiated with virus particles. More intriguingly, lag periods in transfection assays are subjected, for unknown reasons, to rather large variations. The relevant experimental data are presented in Fig. 1. In transfected cultures the very first foci of transformed cells did not appear before day 6 whereas in virus-infected cultures (not shown) the foci readily occur in each culture 3-6 days after infection. No relatiomhip has been found between the DNA dose (i.e., the number of DNA copies of the viral genome) delivered per cell culture and the time of occurrence of the first foci. This is demonstrated in Fig. 2. Furthermore, Fig. 1 shows that at day 12 the foci developed in 69%and 65%of cell cultures expressing native and denatured, respectiveIy, DNA from PR-C-transformed rat cells, and, in contrast, in 92% of those expressing the DNA from SR-D-transformed chicken cells. Recovered PR-C and SR-D viruses have grown equally well when plated on chicken cells. The above difference may suggest, therefore, that transfections of chicken ceIIs with chicken cell DNA manifest themselves more rapidly than those with rat DNA. As shown in Fig. 1 the longest time periods between the DNA application and the occurrence of foci amounted to 26 and 18 days in cell cultures treated with mammalian and chicken DNA, respectively. Such lag periods are not reconcilable with the idea that in transfected cultures the virus was released before the first cell passage (i.e., at the latest 4 days after the DNA treatment), even if one postulates a low yield. On the contrary, it seems likely that certain cells have to go through several passages (and/or mitoses) before being able to produce the virus. Why these cells need such a long time remains a mystery.

4. Nonproducer Cell Transformation Transfection assays listed in Table I were all performed in permissive cells such as chicken, duck, or pheasant cells susceptible to the recovered

256

MIROSLAV HILL AND JANA HILLOVA

TIME (DAYS)

FIG. 2. Cumulative frequencies of chicken cell cultures transfected with various amounts of Schmidt-Ruppin strain of Rous sarconia virus (SR-D) DNA and plotted against the time at which the first foci of transformed cells were detected. On the ordinate, 100%accounts for 27, 36, and 35 cell cultures transfected with 20-50 pg, 5 fig, and 0.5 pg of infectious DNA per culture, respectively. See Fig. 1 for the DNA treatment and subsequent passages of cells, and for a plot totalizing SR-D DNA transfections irrespective of the DNA dose delivered per culture.

virus. In these assays, the appearance of foci of transformed cells following transfection was always accompanied by the release of an infectious sarcoma virus into the culture medium (see also Hill and Hillova, 1974, for review). A nonproducer transformation of permissive cells with RSV DNA rfiay also occur (see below), although, however, it seems to be a very rare phenomenon. In 1972 we tried, without success, to transform nonpermissive rat embryo cells with the RSV DNA (Hill and Hillova, 1974). We believed that such a negative result was equivocal, as the selective pressure may not have been high enough to allow overgrowing sf normal cells in the culture by the progeny of a single cell transformed with the DNA. Since then a nonproducer cell transformation has been reported by Karpas and hlilstein ( 1973), who demonstrated the occurrence of foci of transformed cells in mouse 3T3 cell monolayers treated with the DNA from murine sarcoma virus-transformed mouse and hamster cells. Transformed cells did not yield infectious sarcoma virus unless fused with Rauscher leukemia virus-producing 3T3 cells. In further experiments of this kind, Karpas and Kleinberg (1974) have obtained transformed cells that were, this time, devoid of a rescuable sarcoma virus but capable

DNA OF RNA TWMOR VIRUSES

257

of giving rise to tumors in bioassays. The occurrence of transformed cells was interpreted to show that sarcoma virus DNA was first integrated into the genome of mouse 3T3 cells, where, if complete, it could be rescued by the helper virus. Although suggestive, the observations of these authors need confirmation in further experiments by using a suitable mutant instead of a wild-type virus DNA in order to establish clearly that the transformation of DNA-treated mouse cells in vitro was specified by the genetic information of the parent sarcoma virus. More evidence can be drawn from experiments using chicken cells because these cells, unlike mouse cells, are unable to acquire spontaneously a transformed phenotype in uitro. Among numerous chicken cell cultures transfected with the RSV DNA, we have encountered one containing solitary foci of transformed cells (Fig. 3) which did not manifest the usual tendency to overgrow normal cells (I. Ali and J. Hillova, unpublished data). In fact, these transformed cells were unable to produce infectious virus. Again the sarcoma virus could be rescued in an infectious form when transformed cells were infected with a helper td virus. Thus, these cells carried a defective RSV genome probably because they derived from a single cell transformed, when treated with the DNA, with a fragment instead of full-length piece of RSV DNA.

B. EFFICIENCY OF TRANSFECTION ASSAY We have already mentioned that the transfection assay performed under conditions allowing the spread of the virus gives only a qualitative answer to the question of whether a given amount of the DNA applied into a cell culture is able, or not, to accomplish transfection. Under such conditions, it is not known how many transfection events take place per transfected culture. Nevertheless, rough evaluation of the infectivity of the DNA may be obtained from the ratio of transfected and DNA-treated cultures. We will show that in the case of certain DNAs such a procedure allows analysis of transfection kinetics.

1. Dose-Response Relationship The data from several different experiments are shown in Fig. 4. In this figure, the infectivity is expressed as a fraction of the number of transfected cultures, and plotted against the dose of infectious DNA. At the first sight, it is obvious that some DNAs are capable of achieving infections in all treated cultures. Two of these DNAs specified nontransforming viruses (Fig. 4C), the third one a sarcoma virus (Fig. 4B, plot 5 ) . Very similar dose-response relationship patterns have been obtained by Cooper and Temin (1974) in transfection assays using RSV

FIG. 3. A focus of nonproducer chicken cells transformed with Prague strain of Rous sarcoma virus (PR-C) D N A in chicken embryo fibroblast culture No. 718. The D N A was extracted from PR-C-transformed chicken cells. Transfection assay with this D N A was performed under usual conditions as described (Hillova d d., 1974a,b), and the cells were kept growing in uitro for 32 days. Photographed in living state with a bright field optics. ~ 8 6 .

DNA OF RN'A TUMOR VIRUSES

aa "

O

r

/

259

-

y g DNA

FIG.4. Dose-response relationship in transfection assays using infectious DNA of avian RNA tumor viruses. Each point refers to five (or more) chicken cell cultures (see Hillova et al., 1974a,b, for the conditions of transfection assay) receiving infectious DNA in various amounts from 0.0005 to 50 ag per culture as indicated on the abscissa. DNA-treated cell cultures were grown in uitro for 1 month; they were examined for the foci of transformed cells and, by electron microscopy, for C-type particles as described ( Hillova et d.,1974a). ( A ) Recovery of sarcoma and transformation-defective ( t d ) viruses. The DNA was extracted from uncloned SchmidtRuppin (SR-D) (plots la, l b ) and Prague (PR-C) (plots 2a, 2b) strain RSVtransformed chicken cells. The plots l a and 2a (closed symbols) refer to the recovery of sarcoma viruses whereas l b and 2b (open symbols) refer to the recovery of both sarcoma and td viruses. ( B ) Recovery of sarcoma viruses. The DNA was extracted from XC cells (for cells see Table I ) (plot 3), from uncloned PR-C( a strain from Wyke's laboratory, i.e., different from that in A ) transformed chicken cells (plot 4; I. Ali and M. Hill, unpublished data), and from cloned SR-D- (the same strain as in A ) transformed chicken cells (plot 5 ) . These DNAs, unlike those in A, gave rise only to sarcoma viruses. ( C ) Recovery of td and leukosis viruses. The DNA was extracted from tdSR-D- (plot 6) and RAV-2- (plot 7; I. Ali and M. Hill, unpublished data) infected chicken cells.

260

MJROSLAV HILL AND JANA HILLOVA

and reticuloendotheliosis virus ( REV) DNA, respectively. These authors processed their data by statistical analysis with a computer and reported that the occurrence of transfected cultures fits the theoretical curve for single-hit kinetics. Unfortunately the estimates of this kind are based on experimental data in a very narrow range from 0 to about 5 (see Poisson distribution ) transfection events per average culture. The data plotted in Figs. 4A and B raise a puzzling question. Why are some DNAs unable to transfect chicken cell cultures with a 100% efficiency even when applied in amounts 10- to 1000-fold higher than that efficient at the end-point dilution? The idea that higher concentrations than 25 pg of DNA per milliliter are toxic for chicken cells (Cooper and Temin, 1974) cannot account for the data in Fig. 4B, plots 3 and 4, particularly because experiments show that 20 r g of carrier DNA does not diminish the infectivity of viral DNA (Table 11). Finally, such an idea cannot explain the data in Fig. 4A. Cell cultures treated with various amounts (10-lo00 times exceeding that at the end-point dilution) of the DNA from uncloned RSV-transformed chicken cells, and scoped as negative with respect to the release of a sarcoma virus, were all found to produce a td virus (Fig. 4A, plots l b and 2b). This happened in spite of the fact that the amount of the DNA at the end-point dilution was about the same for both sarcoma and td virus recovery. To summarize the available data, some RSV DNAs may, on transfection, give rise to sarcoma virus in all DNA-treated cell cultures [e.g., the DNA from cloned SR-D-transformed chicken cells (Fig. 4B, plot 5, and Table 11) and, sometimes, that from PR-C-transformed rat XC cells (Hill and Hillova, 1974; Hillova et aE., 1974b)], whereas others may give rise to the virus only in a fraction of DNA-treated cultures [e.g., the DNA from uncloned PR-C- and SR-D-transformed chicken cells (Fig. 4,plots la, 2a, 4) and, sometimes, that from PR-C-transformed rat XC cells (Fig. 4B, plot 3)]. On the contrary, the DNAs of td and leukosis viruses, like tdSR-D (Hillova et d.,1974a; also Fig. 4C, plot 6), and RAV-2 (Fig. 4C, plot 7), give rise to the virus in every cell culture, and this happens under the same conditions as those used in transfections with the foregoing RSV DNAs. The td and leukosis virus DNAs are, therefore, more efficient than sarcoma virus DNAs, but only when applied at high concentrations; at the end-point dilution the efficiency of these two DNA species seems to be about the same. The following considerations may account for the apparent fluctuations in the efficiency of sarcoma virus DNAs. One may imagine that the overall efficiency of transfection assays may vary owing to factors such as the rate of cell multiplication, the degree of cell differentiation, the incidence of competent cells, etc. According to the Poisson distribution

DNA OF RNA TUMOR VIRUSES

261

only a small increase from 1 to 5 transfection units delivered on the average per cell culture should be sufficient to raise the fraction of transfected cultures from about 6% to 10%. Experimentally, however, an increase in the dose of infectious DNA does not always produce the predicted rise in efficiency, and this suggests that limiting factors are involved. In particular, variations in the overall amount of foreign DNA preserved nondegraded in recipient cells and the fraction of recipient cells competent to be transfected may be of crucial importance. We suspect that under optimum conditions of transfection assay these limiting quantities are just sufficient to allow transfection in each ( P < 0.01) DNA-treated cell culture (i.e., occurrence of 5 transfection events per average culture); thus under less favorable conditions the transfection will occur only in a fraction, never in a totality of DNA-treated cultures. Compatible with the view that a limited amount of infectious DNA can be preserved in competent cells is the idea that variations in the maximum frequency of transfection events reflects differences in the saturation level. Hence if one postulates a higher saturation level in chicken cells for td and leukosis virus DNA than sarcoma virus DNA, then this would explain why, when the DNA amount per culture is raised, the probability of recovering a td and leukosis virus increases, whereas the probability of recovering a sarcoma virus remains unaltered. This obviously raises the question why the saturation level of the two DNA species should differ. Does this suggest that there are two different compartments in the recipient cell for leukosis and sarcoma virus DNAs, or rather two different mechanisms operating in the expression of these DNA species? Taking into account biochemical studies that show that a recipient cell stores large amounts of DNA in a nonintegrated state whereas much smaller amounts (0.2% of recipient cell DNA content) are integrated into the host chromosomal DNA ( Hill and Hillova, 1971a), we tentatively suggest that sarcoma virus DNA must be integrated before it is expressed whereas td and leukosis virus transcription can be effectuated before integration.

2. Specific Infectivity The amount of infectious DNA at the end-point dilution was 0.05 pg, and this figure was highly reproducible in several transfection assays using the DNAs from virus-infected chicken cells and virus-transformed chicken or mammalian cells (cf. Fig. 4 ) . This amount of the DNA carries approximately lo5 DNA copies of the viral genome (i.e., 4 x lo4 and 1.5 X lo4 copies if it is assumed that there are about two copies (Schincariol and Joklik, 1973) and 2.5 x pg of DNA (Shapiro, 1970) per diploid chicken cell, and two copies (Hare1 et al., 1972) and about

262

MIROSLAV HILL A h 4 JANA HILLOVA

7x pg of DNA (Shapiro, 1970) per rat XC cell, respectively). In cell cultures transfected with 0.05 pg DNA one transfection event occurred, therefore, per 105 copies of the viral genome applied into the culture. One may hypothesize that the transfection event has been specified either by one or by two, three, etc., DNA fragments representing the totality or only parts of the viral genome, respectively. In the latter case the expression of each fragment, if independent, should occur with a probability of $'@, etc., in order that the transfection could be accomplished from two, three, etc., respectively, fragments and become manifest with an overall probability of as observed. is much higher than those A probability of (i.e., about 3 x governing infections with the DNA extracted from virions, e.g., that of 5 x [lo7 plaque-forming units (PFU)/pg] for SV40 (Pagan0 and Hutchison, 1971), 3 x ( 6 x lo5 PFU/pg) for polyoma virus (2.6 X lo3 PFUlpg) for herpes (Warden and Thorne, 196S), 4 x simplex virus (Sheldrick et at., 1973), and 1.5 x (40 PFU/pg) for adenovirus DNA (Graham and Van der Eb, 1973). For this reason, we consider the hypothesis of the double-hit or multi-hit mechanism in transfections of animal cells with the DNA of RNA tumor viruses as untenable, and conclude that these transfections occur as a single-hit phenomenon from one molecule of the viral DNA. At the end-point dilution, the fraction of 2040%of cell cultures transfected with 0.05 pg of DNA corresponds, according to Poisson distribution, to the delivery of 0.2-0.5 infectious units ( I U ) per average culture. This figure indicates that the specific infectivity of the DNA from avian RNA tumor virus-infected cells amounts to about 4-10 IU/pg as previously mentioned (Hill et al., 1974). It is important to point out that virtually the same value for the specific infectivity was obtained in other transfection assavs using RSV, REV (Cooper and Ternin, 1974), and feline RD-114 (Nicolson et aZ., 1975) DNAs.

m,

C. GENETIC CONTENTOF INFECTIOUS VIRALDNA The fact that DNA from virus-infected cells gives rise in recipient cells to the virus makes us ask whether the virus is entirely coded by the donor DNA which enters into recipients. The affirmative answer cannot be given without reserve, particularly because the chicken fibroblasts, readily used as recipients, all presumably carry endogenous viral genome (as discussed in Section 11). Thus, upon transfection, these cells may release particles carrying ( 1 ) an endogenous viral genome, ( 2 ) a genotypic mixture of endogenous genes and the genes borne on the infectious DNA, or ( 3 ) a genome entirely specified by the infectious DNA.

DNA OF RNA TUMOR VIRUSES

263

The experimental data shown below are all in support of the third eventuality. Whether or not it is possible to induce endogenous viruses with nonviral or viral DNAs in DNA-treated or DNA-transfected cells, respectively, is not known.

1. Transfer of Genetic Markers via Viral DNA from Parent into the Progeny Virus The available body of evidence concerning the recovery of parent genetic markers in viruses released from DNA-treated cell cultures is summarized in Table I. a. Subgroup Specificity. Avian sarcoma and leukosis viruses differ in the host-range, interference pattern, and neutralization by antisera. By virtue of these characteristics different laboratory strains of avian tumor viruses were classified into four subgroups, A through D (Vogt and Ishizaki, 1965, 1966; Ishizaki and Vogt, 1966; Duff and Vogt, 1969). Further subgroups E , F, and G were introduced later to classify endogenous viruses recovered from chicken and pheasant cells as mentioned in Section 11. It appears that the subgroup specificity of virions is determined by virus-coded envelope glycoproteins (Duesberg et al., 1970; Fleissner, 1971; see also Bolognesi, 1974, for review) essential for infectivity ( ScheeIe and Hanafusa, 1971) and providing a genetic marker which is transmitted from parent to progeny virus. In mixed infections, the subgroup specificity marker (also called host-range marker) may recombine with the transformation marker of the superinfecting virus (Vogt, 1971b; Kawai and Hanafusa, 1972b; Weiss et al., 1973). Table I gives a list of sarcoma and leukosis viruses of different subgroups so far successfully used in transfection assays as parent viruses. These viruses gave rise, upon infection of chicken or mammalian cells, to infectious DNAs. When examined in transfection assays these DNAs were found to produce viruses belonging, in all cases examined, to the same antigenic subgroups as those of the respective parent viruses (see the last coIumn of Table I for references). We conclude that the genetic information for the subgroup specificity of the parent virus is contained in its infectious DNA, and is transmitted following transfection to the virus progeny. b. Transforming Capacity. Early work showed that infectious DNA extracted from RSV-transformed cells may give rise, in recipi6nt cells, to a sarcoma virus (Hill and Hillova, 1 9 7 1 ~ ) .Later, various strains (Prague, Schmidt-Ruppin, B77, as listed in Table I ) of nondefective avian sarcoma viruses recovered in transfection assays were reported to have many of the properties of their parent viruses. It was shown for instance that the morphology of cells in vitro transformed by both

264

MIROSLAV HILL A h D JANA HILLOVA

viruses was the same, and that both parent and progeny virus had the capacity to generate pocks on the chorioallantoic membrane and sarcomas on the site of inoculation in vivo. This evidence strongly suggested that transforming genetic material is also carried on infectious DNA. The uncertainty about the viral origin of the transforming genes was resolved in experiments using a temperature-sensitive RSV mutant, as described later. The fact that nondefective avian sarcoma viruses spontaneously segregate transformation-defective ( t d ) derivatives ( Vogt, 1971a) raised the question whether the DNA from RSV-transformed cells could produce, besides sarcoma viruses, the td segregants. To look for the latter viruses, negative DNA-treated cultures ( i.e., lacking transformed cells) were systematically examined in ultrathin sections by means of electron microscopv for C-type particles. The results were consistently negative for the DNA extracted from nonproducer RSV-transformed mammalian cells (Hill and Hillova, 1974; Hillova et al., 1974a). In contrast, cell cultures treated with DNA samples extracted from producer RSV-transformed chicken cells were found to generate besides transforming viruses also nontransforming viruses. These nontransforming viruses resembled td segregants since, like those, they carried the same envelope antigens, banded at the same density, and possessed a somewhat smaller RNA than the transforming viruses recovered from the same sample of infectious DNA (Hillova et al., 1974a). These td viruses may originate either from a deficient transcription of infectious RSV DNA or, alternatively, from a complete transcription of a particular td virus DNA. The former possibility is obviously in contradiction with the data of the paragraph above, whereas the latter possibility is supported by the fact that crude stocks of nondefective RSV are usually contaminated with td segregants and, consequently, the chicken cells infected with these stocks may carry two species of infectious DNA, i.e., a RSV DNA and a td virus DNA. Further pieces of evidence for the latter view that td viruses are generated from a particular td virus DNA rather than from a RSV DNA have been obtained very recently. There are stocks of RSV, e.g., tsLA335 and its wild-tvpe parent PR-C, giving rise upon infection of chicken cells to an infectious DNA which was found to produce in transfections sarcoma viruses, but not td viruses (Hillova et al., 1975). Furthermore, stocks of SR-D segregating in transfection assays both sarcoma and td viruses gave rise only to sarcoma viruses when the infectious DNA was extracted from a cloned (instead of uncloned) population of RSV-transformed chicken cells derived from a single fibroblast infected and transformed in all probability by a single RSV particle. Exceptionally, td

DNA OF RNA TUMOR VIRUSES

265

viruses also occurred and we suppose that they arose from either double infection or contamination (when picking up the colony from soft agar) of the clones (two out of six) by a td virus or an uninfected cell, respectively (Hillova, 1975). We conclude that a partial expression of RSV DNA, with transforming genes omitted, is, at best, a rare event. Table I also includes transfection assays giving rise to leukosis viruses, such as RAV-1, RAV-2, and RAV-50. These assays furnished the important, but hardly surprising, evidence showing that no transforming virus arises after transfection with DNA from leukosis virus-infected cells. c. Capacity t o Znduce Leukemias. The relevant experiments are listed in Table I. In these experiments, the DNA was extracted from leukemic myeloblasts obtained from AMV-infected chickens and was added to chicken fibroblast cultures under the conditions of transfection assay. Recovered viruses were assayed for oncogenicity in uiuo. Two of them produced myeloblastosis (Lacour et al., 1972), and the third gave rise to nephroblastomas (Ogura et aZ., 1974a). Ogura et al. believe that the nephroblastomas are due to a nephroblastoma virus rather than to an AMV, and that their AMV stock contained, besides AMV, so-called myeloblastosis-associated viruses known to induce lymphoid leukosis, osteopetrosis, and nephroblastoma, but not myeloblastosis, in chickens (Smith and Moscovici, 1969). If so, the transfection assay allowed the issolation of an unipotent virus from a standard heterogeneous AMV (Ogura et al., 197413). Consistent with this interpretation, DNA from virus-induced nephroblastoma produced, in transfection assays, a virus capable of inducing nephroblastomas, but not leukemias, in bioassays ( Fourcade et al., 1974). Thus, the experimental data provided circumstantial evidence that the leukosis viruses recovered in transfection assays are oncogenic in vivo and give rise to tumors in all respects similar to those induced by parent viruses. Whether the genetic information for malignancy is specifically transferred in the infectious viral DNA or, for instance, rescued from donor cells was not investigated. Perhaps, further work using cloned (for the plaque assay, see Kawai and Hanafusa, 1972a; Graf, 1972) avian leukosis viruses and their conditional mutants, if available in the future, will be more rewarding. d. Conditional Lesions. Perhaps the best evidence for transfer of viral genes from a parent to a progeny virus via infectious DNA has been obtained in transfection assays using temperature-sensitive mutants. One of these mutants, named FU-19, was able to transform chicken fibroblasts at permissive temperature, but unable to maintain the transformed phenotype of virus-infected cells at the nonpermissive temperature ( Biquard and Vigier, 1970). The other mutant, ts-335 ( tsLA335PR-C

266

hiIROSLAV HILL AND JANA H I U O V A .ira,

DNA

RNA

hr. rt

1

a

b

tra.

hr, rt I

I

hr. rt

I

x

FIG.5. Schematic representation of the RSV genome. The DNA form of the genome is composed of a single DNA piece of about 6 x 10' daltons (Section IV,E) covalently bound to the chromosomal DNA (Section IV,D). Viral DNA is coding for transformation capacity tra, host-range specificity hr, and reverse transcriptase rt of the virus ( Section IV,C). Full-length RNA transcripts of the viral DNA (class u RNA subunits) are carried in RSV particles. Deletions occurring in the course of the replication cycle (probably during RNA + DNA transcription) of the virus produce transformation-defective ( td ) segregants which, unlike RSV, harbor smaller class b RNA subunits lacking the piece x, which contains the sarcoma transforming information. The R S A pieces a and b were first described by Duesberg and Vogt ( 1970), and the length of the x piece was estimated by Duesberg and. Vogt ( 1973b). It is still uncertain whether the transforming material tru is situated at the very end of the viral genome.

according to the system for numbering of mutants as proposed by Vogt et al., 1974), had a temperature-sensitive defect in viral functions required early in infection; the late viral functions were unaffected ( Wyke, 1973a; Linial and Mason, 1973). The virions of this mutant possessed a heat-labile RNA-directed DNA polymerase (Mason et al., 1974; Vercna et al., 1974). It is logical to assume that in FU-19 the lesion is localized in the transforming genetic material (portion x of class a RNA subunit in Fig. 5 ) , whereas in LA335 the lesion is outside of the transforming genetic material (i.e., in the portion b of class a RNA subunit in Fig. 5 ) . Therefore, these mutants offer a unique opportunity to follow the transfer, by means of infectious DNA, of genes from functionally different portions of RSV RNA. Table I shows that the DNAs extracted from chicken cells transformed with these mutants produced, in transfection assays, temperature-sensitive viruses unable to maintain cell transformation or to accomplish early functions, respectively, at the nonpennissive temperature. The latter virions carried a thermolabile reverse transcriptase similar to the enzyme of the parent LA335 (Hillova et al., 1975). These data furnished conclusive evidence that at least two viral genes coding for temperature-sensitive proteins are transmitted via infectious DNA to the progeny virus. Furthermore, the transfer of lesions supposedly residing on two functionally different portions of viral RNA

DNA OF RNA T U M O R VIRUSES

267

indicates that these portions are both represented in infectious viral DNA. This view is strongly supported by the following data. 2. Transfections in Heterologous Hosts It is important to ask whether or not the DNA efficient in transfections is a complete copy of the viral RNA, i.e., whether this DNA initiates by itself infections in recipient cells or depends on recombination with endogenous viral genome of recipient cells. To answer this question, transfections must be made in heterologous, yet permissive, hosts, and it must be shown that the host DNA contains no nucleotide sequences complementary to either the parent or the progeny virus. Svoboda et al. (1973) and Hloihek and Svoboda (1974) have attempted to transfect duck cells with RSV DNA, but without success. Probably there were technical reasons underlying the negative results (see also Table I ) , since more recently Cooper and Temin (1974) reported successful transfections initiated with RSV DNA in duck as well as pheasant cells and, moreover, with reticuloendotheliosis virus, strain T (REV-T), and Trager duck spleen necrosis virus (TDSNV) DNAs in chicken cells. It has been shown that RSV apparently lacks nucleotide sequences complementary to the DNA of duck (Varmus et al., 1973b) or pheasant ( Neiman, 1973a) cells and, similarly, that reticuloendotheliosis viruses like REV-T and TDSNV lack representation in chicken cells (Kang and Temin, 1974). However, there is no evidence concerning the progeny viruses synthesized in these cells after the DNA treatment. For instance, sarcoma viruses recovered in transfections of pheasant cells may be suspected to harbor nucleotide sequences specific for an endogenous virus reported in pheasant cells by T. Hanafusa and Hanafusa (1973) and Fujita et al. (1974). It is worth adding, however, that the occurrence of new nucleotide sequences in the progeny virus does not necessarily mean that the infectious DNA is defective and is complemented with cellular or endogenous viral genes. Indeed in infections initiated with virus particles host cellspecific nucleotide sequences are believed to occur in the virus progeny (Shoyab et al., 1975), for this would explain why avian (Altaner and Temin, 1970) or murine ( Aaronson, 1971) tumor viruses recovered after passage through cells of foreign species in vitro differ in host-range and/ or serological characteristics from the parent virus. In conclusioh, no evidence has been encountered suggesting defectiveness in the infectious viral DNA. On the contrary, experimental data are all consistent with the view that this DNA represents the full-length genome of RNA tumor viruses.

268

MJROSLAV HILL AND JANA HILLOVA

D. STRUCTURE OF INFECTIOUS RSV DNA The technique of transfection assay has offered an opportunity for the investigation of the location and structure of viral DNA in RSV-transformed cells. For this purpose, established rat XC cells transformed with a nondefective PR-C have been chosen as representative of nonproducer cells that carry the viral genome by vertical transmission through cell generations in vitro. The DNA of XC cells (XC-DNA) has been extracted either from whole cells or from purified nuclei. In transfection assays, the former DNA which contained both nuclear and cytoplasmic DNA species exhibited about the same infectivity as the latter which lacked the cytoplasmic components (Goubin and Hill, 1974). Similar results were obtained by Svoboda et al. ( 1973). Clearly the bulk, if not all, of RSV DNA seems to be associated with nuclear structures. However, the possibility of a minor fraction of viral DNA residing in the cytoplasm of XC cells, although unlikely, cannot yet be eliminated. The aim of more recent experiments has been to elucidate the secondary structure of the infectious DNA. Preliminary trials showed that the XC-DNA extracted under native conditions and producing the virus in transfection assays, surprisingly, continued to give rise to the virus even when denatured with alkali (Hillova et al., 1972). This original observation was later confirmed (Cooper and Temin, 1974; Nicolson et al., 1975), though negative results with denatured DNA were also reported (Levy et al., 1974; Svoboda et al., 1974). In our laboratory, the infectious agent was found to band as single-stranded DNA when the denatured XC-DNA was centrifuged in CsCl density gradients at neutral pH (Hill and Hillova, 1974). It seems likely, therefore, that single strands of XC-DNA are infectious to the same extent as doublestranded helices of the same DNA. However, the fractionation in CsCl gradients (densities of single- and double-stranded DNA differ by 16 mg/cm3) did not completely rule out an alternative possibility that an alkali-resistant double-stranded RSV DNA structure contaminated the band of single strands. Conclusive evidence showing infectivity of singlestranded DNA was obtained later. We have also tested to see whether the infectious agent, when extracted under native conditions from XC cells, was in double- or, less likely, single-stranded conformation before being treated with alkali. A sample of crude XC-DNA was adsorbed on hydroxyapatite and serially eluted with phosphate-formamide buffer solutions of stepwise increasing molarity. Under these conditions the infectious agent behaved as doublestranded DNA (Hill et al., 1974). It is thus unlikely that viral DNA in RSV-transformed cells occurs in a single-stranded form.

DNA OF RNA TWMOR VIRUSES

269

A further important question concerned the state of viral DNA in XC cells; does it persist as free double-stranded circular or linear molecules or, alternatively, as integrated genetic material? Two types of experiments were performed to examine this question. In the first one the XC-DNA was banded at equilibrium in alkaline CsCl gradients in order to separate single-stranded DNA (density 1.756 gm/cm3) from possible double-stranded alkali-resistant circles [expected density 1.784 gm/cm3 as in the case of SV40 DNA (Weil and Vinograd, 1963)1. The material from pooled gradient fractions (six pools through the whole gradient) was assayed for infectivity. It was found that the infectious agent bands, under alkaline pH, as single-stranded DNA, and apparently does not band as alkali resistant circular DNA (Hill and Hillova, 1974; Hill et al., 1974). Furthermore, the treatment of DNA samples with S1 single-strand specific nuclease destroyed the infectivity of alkali denatured RSV DNA but did not affect that of native XC-DNA ( G . Goubin, personal communication) . These results have conclusively shown that XC-DNA can be converted into single-strands without losing its capacity to initiate infections in chicken cells. The second experimental approach consisted in separation of lowmolecular-weight cellular DNA species from chromosomal DNA by sedimentation in an alkaline glycerol gradient as described by Sambrook et al. (1968). If integrated by alkali-resistant covalent bonds the viral DNA would sediment with chromosomal DNA. On the other hand, nonintegrated DNA, if present, would sediment slower than the chromosomal DNA and, consequently, would be found in gradient fractions between the peak of chromosomal DNA and the gradient surface. Accordingly, the XC cells were deposited on the top of a glycerol gradient, lysed under alkaline conditions, and centrifuged. The infectious agent was recovered from gradient fractions corresponding to the peak of chromosomal DNA (110 S). On the other hand, no infectious material was detected sedimenting outside of this peak, i.e., in the upper and lower portions of the gradient ( Hillova et al., 1974b). These results provided evidence that, in XC cells, the RSV DNA is covalently bound to the chromosomal DNA. It is worth pointing out that so far the integrated state of infectious viral DNA in the cellular chromosome has been established only in the above-described case of nonproducer rat RSV-transformed XC cells. Nevertheless, it is tempting to hypothesize that the integration of viral DNA also occurs in producer virus-transformed or virus-infected cells. In agreement are the following findings. When the DNA from RSV-transformed chicken cells was fractionated by means of Hirt’s procedure ( Hirt, 1967), the infectious agent sedimented together with chromosomal DNA in the Hirt’s pellet and was apparently absent from the Hirt’s

270

MIROSLAV HILL AND JANA HILLOVA

supernatant ( 14ontagnier and Vigier, 1972). Authors working with molecular hybridization techniques could detect virus-specific nucleotide sequences covalently integrated in the high-molecular-weight DNA of productively infected cells, but failed to reveal nonintegrated DNA (Varmus et al., 197313; Markham and Baluda, 1973) except in early infections (Varmus et al., 1973b, 1974b; Ali and Baluda, 1974; Lovinger et al., 1974; see also Section 111). However, these two types of experiments could not entirely eliminate the possibilities of noncovalent association of full-length copies and covalent integration of partial copies, respectively, of the viral genome in virus-producing cells. More evidence has been reported by Guntaka et al. (1975), who found that both the occurrence of virus-specific nucleotide sequences in the host chromosomal DNA and the production of the virus in the host cells are inhibited by a factor of 6-8 when the cells are infected with the virus in the presence of ethidium bromide. These authors believe that the dye intercalates into DNA duplexes, and by preventing (or reversing) the formation of supercoiled DNA [which has already been demonstrated in early infections with avian sarcoma viruses (Varmus et al., 1974a) and murine leukemia viruses (Gianni et al., 1975)] inhibits integration and subsequent expression of the viral genome. No direct tests of this idea have yet been performed and it is premature, therefore, to generalize that the integration of viral DNA into the cellular chromosome, even' if occurring in most (perhaps all) viral infections, is the only pathway of RNA tumor virus replication.

E. MINIMUM SIZE OF INFECTIOUS RSV DNA It has been shown earlier that a single-hit mechanism may reasonably explain the efficiency observed in transfection assays with the DNA at the end-point dilution (Section IV,B,B) as well as the kinetics of transfection (Cooper and Temin, 1974). Hence a single piece of infectious DNA may transfect a normal cell. If infectious, such a piece necessarily carries a complete set of viral genes and, in addition, nucleotide sequences of cell origin. These latter sequences cannot be selectively removed, although breakage at random may considerably reduce their length, so that the minimum size of DNA pieces required for infectivity would roughly correspond to the size of the DNA form of the viral genome. So far the relevant experiments have been performed in two laboratories. Cooper and Temin (1974) used infectious DNAs extracted from RSV-transformed or TDSNV-infected chicken cells. The DNA was sheared by passage through syringe needles of different diameters, sized by electrophoretic migration in agarose gels, and assayed for infectivity

DNA OF RNA TZTMOR VIRUSES

271

in chicken cell cultures. The authors have observed that the infectivity of RSV DNA dramatically diminishes when the average molecular weight of the DNA drops from 9 lo6 to 5 X lo6. Surprisingly, the infectivity of TDSNV DNA was found to be more sensitive to the shearing than that of RSV DNA, From these data Cooper and Temin (1974) estimated that the minimum molecular weight of RSV DNA roughly corresponds to 6 lo6 and that of TDSNV DNA to 10 to 20 X106. Obviously, their estimates are based on the assumption that the minor fraction of DNA molecu!es with a molecular weight much higher than average could not contribute to the infectivity of the sheared samples of infectious DNAs. In a more direct approach, G. Goubin and M. Hill (unpublished data) assayed for infectivity the fractions of RSV DNA in a very narrow range molecular weight of 3-8 million. Briefly, the DNA was extracted from XC cells and purified on hydroxyapatite to remove denatured single-stranded structures. During this procedure the average molecular weight of the DNA was reduced by shearing so that crude XC-DNA of 25.5 lo6 was eluted as double-stranded DNA of 5.3 X lo6 (see Hill et a,?., 1974, for the respective sedimentation profiles). This DNA was then fractionated by sedimentation through a linear sucrose gradient. Gradient fractions were pooled into three samples to separate the material sedimenting (1) between the bottom of the tube and the leading moiety of the DNA peak, ( 2 ) at the leading moiety of the DNA peak, and ( 3 ) between the trailing moiety of the DNA peak and the gradient surface, respectively. The samples were further examined with respect to the infectivity and the molecular weight. The results were conclusive: the DNA molecules of 8 as well as 6 million daltons were infectious whereas those of 3 million daltons were devoid of infectivity. It can be concluded that the 6 x lo6 daltons is the minimum size of infectious DNA pieces which could specify, in transfected cells, the synthesis of the RSV. Consequently, the molecular weight of the fulllength DNA copy of the RSV genome is 6 x106, or less, indicating the maximum genetic complexity of viral RNA of about 3 million daltons.

x

x

x

V. Sizing the RNA Genome in Virus Particles

The possible structure and organization of the genome in RNA tumor viruses has been thoroughly discussed by Vogt (1973) and reviewed on many occasions (Temin, 1974b; Green and Gerard, 1974; Hill and Hillova, 1974). However, despite considerable effort, the experimental data cannot be fitted into a coherent and widely accepted picture. We will discuss the salient features concerning the size of the viral genetic material.

272

hmosuv

HILL AND JANA HILLOVA

In 1965 Robinson et al. characterized the sedimentation behavior and the base composition of fast-sedimenting 60-70 S viral RNA, and they concluded that this RNA is single-stranded with a molecular weight of about 10'. The reliability of the estimate was based on the assumption that the hydrodynamic behavior of the 60-70 S RNA in gradients does not significantly differ from that of reference single-stranded tobacco mosaic virus RNA. This assumption could not be directly tested. Later, it was found that 60-70 S RNA could be considered as an aggregate rather than a single polyribonucleotide since, under denaturing conditions, it dissociates irreversibly into RNA molecules migrating as singlestranded RN.4 of molecular weight 3 x loGin polyacrylamide gels and sedimenting with a coefficient of about 35 S in sucrose gradients (Duesberg, 1968; Montagnier et al., 1969; Erikson, 1969). The melting of fast-sedimenting RNA into 35 S RNA is accompanied by the release of 4 S RNA (Erikson and Erikson, 1971) and 5 S RNA (Faras et al., 1973) molecules, which constitute about 3 4 % and l%,respectively, of the 70 S RNA complex (Faras et aZ., 1973). Early electron microscopic examinations of the 60-70 S viral RNA have revealed a heterogeneous population of extended molecules with an average contour length of 8.3 pm (Granboulan et ul., 1966) and the upper length limit of 14 p n ~(Kakefuda and Bader, 1969). However, it now appears that the experimental conditions used allowed the extension of contaminating double-stranded DNA molecules, not the viral RNA ( Weber et al., 1975). More evidence has been obtained in recent studies in which a variety of techniques were used to cause spreading and extension of single-stranded RNA. The RNA has been coated and extended for instance with the bacteriophage T4 gene-32 protein. In this complex the molecular weight-to-length ratio calculated for the RNA amounts to 870,000 daltonsImicrometer (Delius et al., 1973). The native 60-70 S and denatured 35 S RSV RNA molecules when complexed with this protein showed a considerable heterogeneity in length. The native RNA exhibited a network structure while the denatured RNA resembled a linear polyribonucleotide. The average contour lengths of 6.8 pm and 3.2 pm indicated molecular weights of about 6 million for native and 3 million for denatured RNA of the Rous sarcoma virus (Mange1 et al., 1974). Chi and Bassel (1975) have also observed dissociation on heating of the highly folded structure of 60-70 S AMV and RSV RNAs into smaller molecules, of which the largest ones had molecular weights of 2.9 X106 and 3.5 x lo6,respectively. In further studies the size of RNA subunits was examined. In one such study (Kung et ul., 1974) the secondary structure of 60-70 S viral RNA was disrupted by glyoxal, and the RNA subunits were described as well extended filaments with a contour length in the case of RSV

DNA OF RNA TUMOR VIRUSES

273

corresponding to 3.28 x lo6 daltons; an unusual value of 5 x lo6 daltons has been obtained in the case of RD-114. In experiments by Jacobson and Bromley ( 1975), the RSV RNA was denatured by dimethyl sulfoxide (DMSO) and sedimented through a sucrose gradient. RNA molecules of 33 S were spread and shown to have a contour length of, on the average, 2.86 pm and a molecular weight of 3.12 X10" as estimated from comparison with a standard MS2 RNA. Apparently in all these studies the estimates of the size of denatured RSV RNA gave reasonably close values. Lower values (2.2 x lo6 and 2.6 x 106 daltons maximum) reported by Weber et al. (1974, 1975) for AMV and RSV RNAs, respectively, were perhaps due to the stringent conditions used to denature the 6&70 S RNA complex. Using another approach, Bellamy et al. (1974) determined, from measurements of sedimentation behavior and hydrodynamic parameters, that, according to the Svedberg equation, the molecular weight of RSV and AMV particles was 296 x lo6 and 256 X lo6, respectively. Each particle was assumed to be composed of 1.9% (Quigley et al., 1971) or 2.17% (Bonar and Beard, 1959) RNA, respectively. Thus, when subtracting the low-molecular-weight RNA species, the molecular weight of the 6&70 S RNA would lie, according to these authors, in the range of 3.8 to 4.8 million for both RSV and AMV. The foregoing paragraphs show that there is controversy concerning the size of the 60-70 S RNA and, on the other hand, agreement concerning the size of the denatured 35 S RNA. The latter may weigh, according to the electrophoretic migration (Duesberg and Vogt, 1973b) and contour length (see above) analyses, about 3 x10" daltons. It is important to remember that the infectious RSV may be specified with a DNA of about 6 x lo6 daltons (Section IV,E), which corresponds in size to a viral RNA of 35 S. According to this evidence one molecule of the 35 S RNA may be considered as representative of an entire viral genome. The relevant question is thus how many molecules of that size make up a 60-70 S RNA complex; in other words what is the degree of ploidy in the viral particle. So far three to four, two, or only one 35 S RNA molecule may account for molecular weights of 10, 6, or less than 4.8 million, respectively, which were all quoted above as possible sizes of the 60-70 S RNA. Two 35 S RNA subunits per virion is the minimum amount still compatible with the supposed subunit structure of the viral 60-70 S RNA, as seen in electron micrographs (Mange1 el al., 1974; Kung et al., 1974; Chi and Bassel, 1975) and as postulated from the occurrence of heterozygotes ( Weiss et al., 1973). Alternatively, the virions may carry less than two full-length 35 S RNA molecules, in which case they would be partially polyploid (or haploid). The occurrence of heterozygotes would be explained by the insertion of two cores

274

hiIROSLA\’ HILL AND JANA HILLOVA

[as occasionally seen in murine (Nermut et al., 1972) or avian (D. Dantche\r, personal communication ) leukemia viruses] of different viruses in the same virus particle. Direct evidence that viral 60-70 S RNA shares more than one full-length genome is lacking. Instead, experiments have been performed to measure the complexity of the viral RNA. One of the possible approaches consists in the separation of an RNase digest of a labeled viral RNA by a two-dimensional “fingerprinting” procedure and the base composition analysis and radioactivity measurement of the oligonucleotides. Unique oligonucleotides are assumed to occur each only once; thus they are present in equimolar amounts through the unique nucleotide sequence. The molar yield of these oligonucleotides in the digest refers to an apparent chain length (Fiers et al., 1965) called sequence complexity of the RNA. The analyses of the RSV RNA performed last year in different laboratories gave concordant results : the estimated complexity of the viral genome was 3.5 10” daltons in PR-B (Beemon et al., 1974), 3.4 loF daltons in SR (Billeter et aZ., 1974), and 3.3 x lo6 daltons in PR-C (Quade et a!., 1974). Another approach using RNA-DNA hybridization kinetics to estimate the complexity of RNA molecules according to Bishop (1969) and Birnstiel et al. (1972) yielded contradictory results. The reassociation rate of leukemic cell DNA immobilized on filters and hybridized to AMV 35 S RNA in excess suggested a complexity of viral RNA of 3.3 million daltons when compared with ribosomal 18 S and 28 S RNA standards (Baluda et aZ., 1974). This value agrees with the results above. By contrast, a complementary in uitro synthesized viral DNA has been reported to anneal to an excess of RSV RNA with a reaction rate suggesting a complexity of 9.3 X lo6 daltons (Taylor et al., 1974), when standardized with a poliovirus RNA. Similar sequence complexities have been derived from RNA-DNA reassociation kinetics in reactions using murine leukemia virus (Fan and Paskind, 1974) or visna virus (Haase et al., 1974) RNAs. The latter estimates, which contradict Baluda et al. (1974), clearly favor a nonrepetitive viral genome of approximately lo7 daltons and thus cannot be reconciled with the size of infectious RSV DNA of 6 x lo6 daltons or the complexity in fingerprint patterns of RSV RNA of about 3 x lo6 daltons. The reliability of the conflicting data furnished in these RNA-DNA hvbridization experiments needs further examination.

x

x

VI. Search for Transforming Genetic Material

RNA tumor viruses are characterized by their capacity to replicate and to induce tumors. Both functions may be mutually independent.

DNA OF RNA TUMOR VIRUSES

275

For instance, leukemia viruses usually do not transform fibroblasts although they readily infect and replicate in them. The reasons why certain viruses transform only some types of somatic cells are unknown. We hope that an insight into this mystery may be gained by examining the physiology of the virus and its genetic material. In general, virus-induced tumors are probably, though not necessarily, caused by proteins coded for by the virus. When a virus infects a cell, the viral genome becomes integrated into the cellular chromosome, thus bringing about a genetic transformation of the cell bearing the virus. As a consequence, the cell may adopt a transformed phenotype. This occurs only in certain cells, seemingly as a cellular reaction on the products of viral genes which are specific for induction of malignancy. There is, however, an alternative possibility that none of the viral genes is specifically involved, and the occurrence of a transformed phenotype in virus-infected cells results from impaired cellular gene ( s ) function at the site of integration of the viral DNA. At present, several lines of evidence clearly show that the ability of sarcoma viruses to transform cells is due to the presence of a transforming genetic material in the viral genome. In leukosis viruses, however, the presence of specific transforming genes has not yet been directly proved, and consequently neither of the above mechanisms can be a priori eliminated.

A. SARCOMA VIRUSES Among RNA tumor viruses avian sarcoma viruses are most suitable for in uitro studies of the virus-induced cell transformation. These viruses transform chicken fibroblasts in culture and give rise to pocks on chorioallantoic membrane of embryonated eggs and to sarcomas at the site of injection in uivo. That the viral genetic material is responsible for the transformation of virus-infected cells has been proved in studies using t s mutants. Unlike the wild-type virus, these mutants are unable to maintain the transformed phenotype of cells at the nonpermissive temperature: the foci of transformed cells, which developed at the permissive temperature, disappear upon the shift to the nonpermissive temperature, and reappear when shifted back to the permissive temperature (Biquard and Vigier, 1970; Martin, 1970). Thus the transformation seems to be under continuous control of viral genes. Its occurrence requires protein synthesis, but not the synthesis of DNA or RNA (Kawai and Hanafusa, 1971; Biquard and Vigier, 1972). On the other hand, its disappearance apparently does not require newly synthesized macromolecules ( Biquard and Vigier,

276

MIROSLAV HILL AND JANA HILLOVA

1972). According to complementation assays, the ts mutants carrying the defect in transforming information have been divided into two (Kawai et al., 1972) and, more recently, four (Wyke, 1973b) groups, suggesting that more than one and perhaps four distinct gene functions are prerequisite in order to accomplish and maintain the transformed state in virus-infected cells. Nonconditional replication defective ( r d ) mutants have been isolated from avian sarcoma virus stocks either untreated (Kawai and Yamamoto, 1970; Kawai and Hanafusa, 1973) or treated with UV light (Kawai and Yamamoto, 1970; Toyoshima et al., 1970), y-rays (Gold&, 1970), or chemical mutagens (Weiss, 1972). These mutants are able to transform fibroblasts in uitro. However, their ability to replicate is impaired (see Duesberg et al., 1975, for deletion in a naturally occurring rd mutant) so that the transformed cells either release noninfectious particles which, like BH-RSV, can be complemented with a helper virus, or fail to synthesize a rescuable virus at all (Toyoshima et al., 1970; GoldC, 1970). The most immediate impression is that a lesion in viral genes involved in the replication cycle of the virus does not represent an obstacle for the establishment of the transformed state in virus-infected cells. This is probably true except for genes essential for early steps in viral infection. For example, the BH-RSVa, which apparently lacks besides the surface glycoproteins ( Scheele and Hanafusa, 1971) an active RNA-directed DNA polymerase (H. Hanafusa and Hanafusa, 1971; H. Hanafusa et al., 1972), can be rescued in an infectious form when complemented for the glycoprotein and the enzyme by a superinfecting helper virus (H. Hanafusa and Hanafusa, 1968). Thus the presence of the gene products, but not the genes themselves, in virions is in this case sufficient to effectuate early functions required for cell transformation. Similarly t s mutants carrying a temperature-sensitive lesion in early functions are unable to infect and transform cells at the nonpermissive temperature, although at that temperature they are able to maintain a transformed state if established at the permissive temperature. Two of these mutants, tsLA335 and tsLA337, have been shown to carry a temperature-sensitive DNA polymerase (Mason et al., 1974; Verma et al., 1974). The early viral functions are presumably required to bring about the RNA + DNA transcription and perhaps also the integration of an intermediate RNA-DNA (or DNA) structure into the cellular chromosome. Hence these early functions are not required if viral DNA rather than the rd mutant is introduced into the cells. Occasionally this may happen in transfection assays since the DNA molecules used in these assays are sheared at random and, consequently, certain broken molecules may contain transforming information and lack

277

DNA OF RNA TUMOR VIRUSES

some genetic material essential for synthesis of C-type particles. One such DNA fragment was probably responsible for a nonproducer transformation of chicken cells that we have described in Section IV,A,4. It may be said, therefore, that the cells receiving RSV DNA can acquire and maintain the transformed phenotype even when unable to synthesize an infectious sarcoma virus. This strongly suggests that the viral transforming information, when in a DNA form, acts itself as a carcinogen (i.e., without being dependent on viral genes involved in replication of the virus ) . Consistent with this view is the evidence showing that the loss of transforming function does not impair the capacity of the virus to replicate. When a nondefective RSV is grown in a chicken cell culture infected with a single particle, then the progeny virus harvested from this culture contains, besides RSV, a td derivative unable to transform fibroblasts (Vogt, 1971a). The defectiveness of this derivative is best explained by a genetic deletion affecting transforming genes. In support of this idea, uncloned RSV, i.e., a population composed of transforming and td particles, has been shown to contain a 60-70 S RNA which, upon denaturation, melts into two size classes of RNA subunits (Duesberg and Vogt, 1970). The larger class a subunits apparently belong to the nondefective RSV because they can be isolated without concomitant class b subunits when the virus is harvested from a clonal population derived from a single fibroblast transformed with a single RSV particle (Duesberg and Vogt, 1973a). The smaller class b subunits are characteristic of td viruses (Duesberg and Vogt, 1970, 1973a). Further analyses by oligonucleotide fingerprinting, cross RNA-DNA hybridization ( Lai et al., 1973), and competitive hybridization (Neiman et d.,1974b) techniques suggested that class a subunits contain all the genetic material of class b subunits plus a certain material x [corresponding in size to about 124: of the class a RNA subunit (Duesberg and Vogt, 1973b)l required for sarcomatogenous transformation, i.e., a = b x (Lai et al., 1973) as schematically represented in Fig. 5. It was possible to hypothesize that the deletions giving rise to td viruses may develop either from incomplete RNA+DNA or DNA + RNA transcriptions. However, recent data obtained in transfection experiments (see Section IV,C,l,b) argue against the latter possibility and suggest that RSV DNA always generates RSV RNA, though RSV RNA, upon viral infection, can give rise to either RSV or td virus DNA. The foregoing experimental results lead us to assume that the transforming genetic material carried in RSV is nonessential for viral replication. This material seems to be sufficient on its own to effectuate cell

+

278

MIKOSLAV HILL AND JANA HILLOVA

transformation and, when in a DNA form, to do so without the aid of early viral functions. Thus it is hard to avoid the idea (as proposed by Vogt, 1972) that the sarcoma transforming genes are of nonviral origin, and have arisen from cells. This is based on the assumption that there are recombinations between the viral and host cell genome, an idea that could also account for the observed phenotypic changes ( Altaner and Temin, 19.70; Aaronson, 1971), and the occurrence of hostspecific nucleotide sequences (Shoyab et al., 1975) in RNA tumor viruses passaged through foreign host cells. Murine sarcoma viruses, the Harvey (1964) and Kirsten (Kirsten and Mayer, 1967) strains, have arisen from rat passages of Harvey and Kirsten MuLV, respectively, whereas the Moloney strain ( Moloney, 1966) was recovered from mouse passage of the Moloney MuLV. Recent molecular hybridization experiments have shown that both Harvey and Kirsten sarcoma viruses carry rat-specific nucleotide sequences ( Scolnick et al., 1973; Scolnick and Parks, 1974), while Moloney sarcoma virus derives its nucleotide sequences exclusively from the mouse genome (Scolnick et d.,1973; Okabe et aE., 1973). More recently, Roy-Burman and Klement ( 1975) compared nucleotide sequences in Kirsten sarcoma and leukemia viruses and suggested that the sarcoma virus lacks about 30%of the leukemia virus-specific nucleotide sequences which are replaced with rat-specific sequences. It would be of interest to know whether such a phenomenon could arise in vitro. In relevant experiments, sarcoma-specific nucleotide sequences of the Kirsten sarcoma virus have been found to share homology with a particular 30 S RNA species found in normal rat NRK cells and Fisher rat embryo cells (Tsuchida et al., 1974), but are barely detectable in RT2lc rat cells (Scolnick et al., 1974b). According to the latter authors both NRK and RT2lc cells (both derived from Osborne-Mendel rats) contain in their DNA a full complement of rat Kirsten sarcoma virus-specific sequences, but express different levels of these sequences in their RNA. When NRK cells were infected with a Moloney MuLV, the rat genetic information specific for Kirsten sarcoma virus occurred in the virus progeny and could be detected in the high-molecular-weight viral RNA (Scolnick et al., 1974b). However, so far no focus-forming virus was encountered even when NRK cells infected with a Kirsten MuLV were propagated for a long time in tit70 ( Roy-Burman and Klement, 1975). In this context it is worth adding that C-type viruses are known to carry a small amount of cellular RNA species which are incorporated accidentally into budding virus particles. However, a nonrandom incorporation seems to take place as well, for example, in the case of tRNAs (Wang et al., 1973). Recently Fidanih et al. (1975) have observed

DNA OF RNA TUMOR VIRUSES

279

a surprisingly large amount of cellular (mostly ribosomal) RNA in a simian sarcoma-associated virus growing in human rhabdomyosarcoma cells. Friend MuLV encapsulates from mouse globin-producing erythroleukemia cells globin messenger RNA, and carries this RNA in a free, 9 S form, and also in association with the 60 S viral HNA complex (Ikawa et al., 1974). Finally, it may be mentioned that in the endogenous reaction in uitro the viral enzyme was reported (Garapin et al., 1973) to transcribe, besides viral RNA, the low-molecular-weight RNA species like 4 S and 5 S RNA associated with the 60-70 S RNA complex and the 7 S RNA occurring free in the virions. The possibility exists, therefore, that a leukemia virus is able to turn into a sarcoma virus because of its capacity to pick up and recombine with cellular messengers. If it is the case we should ask, however, whether the sarcoma-specific messenger RNA originates from normal ( or mutated ) cellular genes or endogenous virogenes. These possibilities remain unexplored. Instead, circumstantial evidence is available showing that a MuLV may induce sarcomas in several strains of mice in viuo, and a sarcoma virus can be isolated from these sarcomas (Ball et al., 1973). Hopefully, further in uitro experiments will soon be forthcoming. B. LEUKEMIAAND LEUKOSISLIKE VmusEs Unlike sarcoma viruses, leukemia viruses are generally unable to transform fibroblasts in uitro. However, they may cause a productive infection in sensitive cells in vitro without directly killing them. How they induce a malignant growth in uiuo is far from understood. Only recently are we beginning to believe that the viral genetic material causes, in a direct way, malignant transformation of the target cell. The first relevant observation was made by Hackett and Sylvester ( 1972). These authors noticed that murine leukemia vinises were able to transform certain lines of BALB/3T3 cells. This was confirmed by Fenyo and Grundner (1973), who showed that Moloney leukemia virus released from lymphoma cells in uitro infected and transformed 3T3 cells. There are now hints that only a special class of leukemia virus particles can transform in uitro. Abelson and Rabstein (1970) isoIated an agent ( Abelson virus), associated with Moloney leukemia virus, which causes a rapidly progressive lymphoblastic leukemia of bone marrowderived lymphocytes in mice. Cell-free extracts of Abelson tumors were found to contain two species of virus particIes that differed in their capacity to transform 3T3 cells (Scher and Siegler, 1975). The important observation was made that clones of virus-transformed cells did not produce detectable C-type particles unless infected with a helper murine

280

MHOSLAV HILL AND JANA HLLLOVA

leukemia virus. They behaved, therefore, as though the agent responsible for their transformation (and for Abelson lymphomas) was defective for virus replication. Other experiments were performed in order to examine directly the capacity of leukemia viruses to transform target cells in uitro. Sklar ct al. (1974) infected mouse splenocytes in a suspension culture in uitro with the Abelson isolate and transplanted these cells into recipient mice of different karyotype. The resulting tumors contained cells of donor karyotype (with or without concomitant cells of host karyotype) indicating that the transformation of cells was initiated in uitro. In confirmation, Rosenberg et al. (1975) were able to recognize transformation by Abelson virus in murine lymphoid cells growing in long-term cultures in uitro. Van den Berg et al. (1975) observed that incubation of mouse bone marrow cells with Rauscher leukemia virus induced formation of erythroid colonies in semisolid cultures. Haas et al. (1975; Haas and Hilgers, 1975) propagated a natural radiation leukemia virus from C57BL/6 mice in mouse thymocytes and mouse embryo fibroblasts in uitro, and found that the virus which was able to replicate in thymocytes was unable to do so in fibroblasts and vice uersa. The virus particles growing in thymocytes were leukemogenic in duo, whereas those multiplying in fibroblasts were devoid of the capacity to induce leukemias. This may provide further evidence for the presence of two (or several) viral entities in extracts of leukemic tissues (Haas et d.,1975; Haas and Hilgers, 1975). Alternatively, however, this could be a result of the loss of transforming function when a leukemia virus is passaged in nontarget cells. Another indication that leukemia viruses bear transforming genes comes from studies that examine the genetic relatedness between these viruses and their hosts by means of nucleic acid hybridization techniques. The leukemia viruses are found to be either partially or not at all related to the genome of their natural hosts. For example, the nucleotide sequences of AMV (Shoyab et al., 1974b), avian leukosis viruses (H. Hanafusa et d.,1974; Neiman et al., 1974a,b, 1975), and td segregants of avian sarcoma viruses (Neiman et al. 1974b) are to a varying extent, but never completely, represented in normal chicken cells; the same is the case for murine leukemia viruses (Viola and White, 1973; Scolnick et aE., 1974a) in normal mouse cells. Ecotropic murine viruses, e.g., those of AKR mice, although fully represented in cells derived from the parent mouse strain are only partially represented in the cells of other strains of mice (Chattopadhyay et al., 1974; Lowy et al., 1974) and therefore cannot be regarded as true endogenous viruses. Other viruses, like woolly monkey and gibbon ape viruses (Scolnick et al.,

DNA OF RNA TUMOR VIRUSES

281

1974a; Benveniste et al., 1974a) and those of feline sarcoma-leukemia virus (Quintrell et al., 1974) are examples of oncogenic viruses carrying very few if any nucleotide sequences in common with the genome of the animal species from which they were isolated. These experimental data show that leukemia viruses, like sarcoma viruses, introduce upon infection new genetic material into the host cell. In confirmation, Sweet et al. (1974) have detected new sequences in splenic DNA of leukemic mice presumably introduced by the Rauscher MuLV which caused the disease. We believe that it is these distinctive nucleotide sequences absent from the normal cell that are responsible for malignant transformation. It is possible that in the case of leukemia viruses the transforming genes code for “continuous inducers” ( or derepressors in the case of negative control) of the mitotic activity, which can be recognized only in certain differentiated target cells, perhaps because they interfere with, or are activated by, the substances specifically synthesized in these cells. Similarly (although there is no precedent for this) they could act as triggers of an irreversible sequence of events leading to transformation. The establishment of the transformed state, therefore, would depend as much upon ability of the cell to acquire a leukemic phenotype under the action of viral genes as upon the presence and the expression of these integrated viral genes. Latent periods of many months between time of injection of the virus and the appearance of the tumor suggest either that among virus-infected cells only a few of them are competent, or that target cells are infected with the virus when undifferentiated and must differentiate before the oncogenic action of the virus can take effect. Strong inferential evidence that the leukemia viruses are carrying transforming genes is derived from the fact that true (see above) endogenous, and apparently nononcogenic, viruses differ in nucleotide sequences from their exogenous counterparts ( Benveniste and Todaro, 1973; Quintrell et al., 1974) and, furthermore, unlike exogenous viruses they are fully represented in all the normal cells of the same animal species. The evidence from hybridization experiments, for instance, shows that avian RAV-0 is fully represented in chicken cells (Neiman, 1973a; H. Hanafusa et al., 1974), cat CCC or RD-114 viruses in cat cells (Neiman, 197313; Benveniste and Todaro, 1974a; Gillespie et al., 1975), endogenous guinea pig virus in guinea pig cells (Nayak, 1974), baboon M7 virus in baboon and other Old World monkey (but not human and New World monkey) tissues (Benveniste et al., 1974a,b; Benveniste and Todaro, 1974a,b; Sherr et al., 1974a), and xenotropic murine virus in normal mouse cells (Benveniste and Todaro, 1974a ). The possibility, however, that the endogenous virus could still be oncogenic if inoculated into foreign animal

282

MIROSLAV HILL AND JANA HILLOVA

species has not been examined, although a yn‘ori under normal conditions (e.g., without carcinogens) it must be considered as unlikely. If they could induce tumors, we would regard the malignancy to be due to inopportune integration of viral genes and destruction of the continuity of the proximal cellular nucleotide sequences. Evans et d.( 1974) showed that in AMV-infected chicken cells the virus may integrate within unique cell sequences. If endogenous viruses could do the same, the elimination of a structural gene rather than the presence of a new viral gene would be responsible for transformation. Another question concerns the origin of the transforming genes in the leukemia viruses. That the leukemia viruses have arisen by mutation from endogenous virogenes would be suggested if it were found that the genes involved in leukemogenesis are also functioning in viral replication. If the transforming genes were not so involved then this would implicate the existence of transforming material of nonviral origin, which is carried and perpetuated in C-type particles in the same way as bacterial genes in transducing phages. Both alternative possibilities are discussed in Section V1,C. As concerns the oncogenic woolly monkey, gibbon ape, and cat viruses, no endogenous counterparts have so far been found to enable explanation of their origin.

TRANSFORMATION in Vioo C. “SPONTANEOUS” There are essentially two ways in which one can imagine involvement of C-type viruses in “spontaneous” tumors: (1) unrecognized infection by an exogenous virus, and ( 2 ) mutation in either endogenous virogenes or cellular genes which subsequently recombine with endogenous virogenes. A t the present time, it seems likely that both occur, although the evidence favors the first explanation for at least certain types of human leukemias. Shortly after the discovery of the RNA-directed DNA polymerase in RNA tumor viruses (Temin and Mizutani, 1970; Baltimore, 1970), numerous studies were undertaken with the aim of finding a similar enzyme in cells (see Green and Gerard, 1974, for review). Gallo et al. (1970) reported a reverse transcriptase activity apparently residing in lymphoblasts of acute leukemia patients, but not in those of normal donors. This important observation was extended, and evidence emerged that leukemic cells, particularly in patients with acute myelogenous leukemia, contain an enzyme indistinguishable from that of C-type viruses. Briefly, the leukemic cells were shown to contain a particular intracytoplasmic fraction composed of particles that band at the density of 1.16

DNA OF RNA TUMOR VIRUSES

283

gm/cms which is typical of RNA tumor viruses, and carry a reverse transcriptase activity ( Gallo et al., 1973). Moreover, they contain a 60-70 S RNA which was detected in a “simultaneous assay” (according to Schlom and Spiegelman, 1971) using the RNA as template for the endogenous synthesis of DNA (Baxt et d.,1972). The enzyme purified from viruslike particles of human leukemic cells resembles viral enzyme: it efficiently transcribes viral 70 S RNA templates and utilizes synthetic primer-templates in a manner characteristic of viral RNA-directed DNA polymerases (Samgadharan et d.,1972; Gallagher et al., 1974) and is distinct from RNA-primed DNA polymerase activities found in normal human lymphocytes (Reitz et al., 1974). When isolated from human leukemic cells the reverse transcriptase occurs in a form with molecular weight of 130,000-140,000 (Mondal et al., 1975), which could be dissociated to give a characteristic form of the enzyme (when extracted from primate C-type virions (Gallagher et al., 1974)) with a molecular weight of 70,000. In this respect, the enzyme in human leukemic cells resembles that contained in gibbon ape virus-infected lymphosarcoma cells ( Mondal et al., 1975). Furthermore, unlike other known cellular DNA polymerases, the human leukemic cell enzyme is strongly inhibited by antisera to reverse transcriptase of woolly monkey or gibbon ape oncogenic viruses, but not by antisera to the reverse transcriptase of avian, mouse, and cat sarcoma-leukemia viruses and of cat endogenous RD-114 virus (Todaro and Gallo, 1973; Gallagher et al., 1974). Interestingly, the above antisera efficient against the human and simian enzymes do not cross-react with the enzyme of the endogenous baboon virus (Sherr et al., 1974b). More recently, Sherr and Todaro (1975) have detected in human leukemic cells, by radioimmunoassays, antigens related to the major protein (p30) of the woolly monkey and gibbon ape viruses. Very little antigen, if any, was found related to the p30 protein of the baboon virus. This evidence strongly suggests that the viruslike agent in human leukemia cells is antigenically more related to primate sarcoma-leukemia viruses than to the endogenous primate virus. Current interest is directed at the elucidation of the origin of nucleotide sequences in the viruslike particles of human leukemias. Baxt et al. (1972) first observed that DNA endogenously synthesized in these particles hybridizes, in part, to the RNA of the Rauscher MuLV but not to that of mouse mammary tumor virus or AMV. These results were confirmed and extended by others, who showed that as much as a half of the DNA nucleotide sequences synthesized in particles of acute myeloblastic leukemia hybridized to the RNA from either woolIy monkey or mouse sarcoma viruses, a lesser amount to the RNA of murine leu-

284

MIROSLAV HILL AND JANA HILLOVA

kemia viruses, and a hardly detectable amount or none to the RNA of other sarcoma and leukemia viruses (Gallo et al., 1973; Miller et al., 1974). In reciprocal hybridization experiments, the RNA from human leukemic cells was shown specifically to hybridize, although to a very low extent, with an endogenously synthesized DNA of mouse .( Hehlmann et al., 1972) and woolly monkey (Gallo et al., 1973) tumor viruses. Also hybridization was found in control experiments in which a large amount of the RNA from normal instead of leukemic leukocytes was used (Gallo and Gallagher, 1974). It seems likely that human leukemic cells do not contain a full-length genome of any of the known simian or murine sarcoma-leukemia viruses since high-molecular-weight RNA of these viruses does not hybridize to more than a small extent (10% maximum) with the nuclear DNA of either leukemic or normal leukocytes ( Gallo and Gallagher, 1974 ) . The putative virus in human leukemic cells may differ in most, though not all, nucleotide sequences from all the other RNA tumor viruses. In this case, experiments that are directed at showing a different abundance of genetic material of a known virus between leukemic and normal cells are irrelevant. It is more important to identify new sequences present in leukemic cells that are absent in normal cells. Baxt and Spiegelman (1972) have elaborated a recycling procedure to investigate these new sequences. A DNA probe synthesized endogenously in viruslike particles of leukemic cells was first hybridized with normal DNA in order to separate the leukemic sequences from normal sequences, and the remaining material, unable to hybridize, was recycled and annealed with leukemic DNA. The results, which are striking although not confirmed, demonstrate that up to 4O!Z of the endogenous DNA probe is specific for leukemic cells. Later, by using the same procedure, Baxt et al. (1973) were able to detect, in two instances, the presence of a leukemia-specific DNA in leukocytes of the leukemic, in distinction to the nonleukemic, members of identical twins, This finding strongly suggests that additional leukemia-specific information is inserted into the DNA of leukemic individuals subsequent to fertilization. If confirmed, such evidence will clearly rule out any hypothesis that invokes vertical transmission, through the germ line, of the causal agent of human leukemias. The same idea, i.e., that tumor cells carry a transforming genetic information, was at the origin of experiments by Karpas and Tuckerman (1974). These authors introduced, by transfection, a DNA from human rhabdomyosarcoma into normal human embryo fibroblasts, and observed that, unlike control fibroblasts, those treated with the DNA acquired the capacity to grow at high population density and to survive crisis and continue growing in oitm for over 14 months. Unfortunately, these

DNA OF RNA TUMOR VIRUSES

285

observations, although promising, are too preliminary to be greeted without skepticism. More evidence about the transfer of malignancy from one cell to another arises from the karyological studies of leukemic patients who received bone marrow grafts from a donor of the opposite sex. The results were alarming: they showed that the leukemia could recur in cells with the donor karyotype (Thomas et al., 1972). The concept of the viral origin of human leukemias suffered from the failure to find complete C-type particles in leukemic cells, though electron microscopic studies did provide evidence for the presence of viruslike particles in the cell cytoplasm (see Cawley and Karpas, 1974, for demonstration and further discussion). It was therefore suggested that human cells are unable to build-up full virions. An important observation was made by Mak et al. (1974a), who showed that the reverse transcriptase activity of the intracellular particulate fraction increased when leukemic cells were cultured for several days in uitro. Surprisingly, viruslike particles previously found in cells could be now also recovered from the culture medium. Extracellular particles resembled C-type viruses in several respects including density, morphology, presence of an RNA-dependent DNA polymerase (Mak et al., 1974b), and the presence of a high-molecular-weight RNA in a complex (presumably) form of 70 S (Kotler et al., 1973; Mak et al., 1975). Most important, this RNA (like that of the intracellular particles previously reported by Gallo et al., 1973) was found to share nucleotide sequences homologous in greatest extent to the RNA of primate sarcoma viruses, and in a much lesser extent to that of murine sarcoma-leukemia viruses. No homology was detected with the RNA of an avian sarcoma virus (Mak et al., 1975). These findings strongly suggested that extracellular particles harvested from the culture medium have arisen from leukemic cells. It was not clear, however, whether they are actively produced by leukemic cells or only released when these cells are maintained under in uitro conditions. Gallagher and Gallo (1975), who examined this problem, were able to maintain the exponential growth and myeloid differentiation of leukemic cells in uitro by means of a factor occurring in the medium of a particular culture strain of whole human embryo cells. Fresh leukemic cells from one patient with an acute myelogenous leukemia subjected to electron microscopic examination were found free of detectable virus. However, when these cells were kept growing in uitro they started producing virus, as revealed in ultrathin sections which showed budding viruses on the cell surface and free C-type particles in the culture medium. The particles banded at the density of 1.16 gm/cms (characteristic

286

MIROSLAV HILL AND JANA HILLOVA

of RNA tumor viruses) and possessed a reverse transcriptase antigenically related to that of simian sarcoma-leukemia viruses. In this respect the enzyme of the particles released from cultured cells resembled the enzyme previously detected in viruslike particles of fresh human leukemic cells. Although the virus was repeatedly obtained from cells of this one patient, cells from 17 other patients were cultured and analyzed similarly, and though some contained components related to the exogenous simian virus family they failed to yield detectable virus (Gillespie and Gallo, 1975). It seems likely, therefore, that leukemic cells do not necessarily start building up virions when subjected to in uitro growth conditions even in those cases in which intracellular viruslike particles have been demonstrated. The origin of both the virus released from leukemic cells and of the intracellular viruslike particles in freshly harvested leukemic cells is obviously a problem of considerable importance. So far the above-quoted hybridization experiments and immunological studies suggested that the “human” virus was more closely related to exogenous oncogenic primate viruses than to the representative of endogenous (and probably nononcogenic) primate virus. Thus, we are considering the possibility that human leukemia is caused by an exogenous virus, though the means by which it is transmitted and the way it enters and causes the malignancy are to say the least a mystery. At the beginning of this section we have emphasized that a cell may obtain transforming genetic material either as a consequence of the infection by an exogenous virus or by mutation of a normal cellular gene. Let us consider some implications of these two possibilities. It has been shown that with all probability exogenous RNA tumor viruses are vectors of transforming genes and, consequently, may spread cancer among members of the same or different animal species by infection. However, only a few C-type viruses have so evolved. These may originate from spontaneous animal tumors that release a virus, derived either from a mutated endogenous virus or an endogenous virus that has recombined with a mutated cellular gene (see Section VI,A, on sarcoma viruses), which infects healthy animals. There is presumably some selective advantage for those particles that carry and conserve the transforming genes. The virus may also acquire, by recombination with the host, new genetic material coding for properties advantageous in further transmissions. These new properties may, for instance, help overcome immunity barriers. In the case of leukemia viruses, such as those of cat and primate, a host of origin, and the recombination events that lead to their evolution, need to be identsed in order to explain their complete lack of relatedness to the respective endogenous viruses.

DNA OF RNA TUMOR VIRUSES

287

A cell may also turn malignant without the aid of an exogenous virus by means of somatic mutation induced by physical or chemical carcinogens followed by inefficient (cf. Hart and Setlow, 1974) and faulty (cf. Radman, 1975) repair. The rate of somatic mutations will increase if the genes coding for enzymes concerned with DNA maintenance and fidelity of replication (Springgate and Loeb, 1973) are affected. Among such random genetic changes, at least some will occur that affect the normal control of cell division. A special case of somatic mutation is provided when mutation in an endogenous virus promotes malignant growth and, occasionally, the release from the transformed cell of an oncogenic virus. The best example is the occurrence of leukemia viruses in mice that carry “spontaneous” tumors (e.g., Peters et aZ., 1973) or tumors induced by exposure to X-rays (Gross, 1958; Lieberman and Kaplan, 1959) and chemical carcinogens (Irino et al., 1963; Toth, 1963; Haran-Ghera, 1967; Ball and McCarter, 1971). Temin (1971a, 1974a) has postulated that transforming genes can arise from successive DNA + RNA + DNA information transfers. At present, no evidence supports the idea that reverse transcriptase introduces errors in transcripts or that amplification of nucleotide sequences alters the cellular genetic content in uiuo. Springgate et al. (1973) and Sirover and Loeb (1974) have shown that purified AMV and MuLV reverse transcriptases make errors in in uitro transcription of synthetic polyribonucleotides. This evidence, however, more likely indicates that in uitro reaction with reverse transcriptase is not a good model for the in uiuo replication. Temin’s idea, therefore, could only provide another explanation of how mutations may be generated in endogenous virogenes. No such system for generating errors could give rise to extensive new sequences coding for transforming proteins without extremely strong selection, and no one has yet suggested why such a formation of transforming sequences should be an advantage for either virus or host. The possibility also exists of recombination events occurring between endogenous virogenes and transforming genes of oncogenic DNA viruses. So far, however, this possibility is not supported by any experimental data. Rather Kufe et al. (1973) reported that RNA-containing viruslike particles in Burkitt’s tumors are apparently devoid of nucleotide sequences specific for the Epstein-Barr virus. VII. Conclusions

C-type RNA tumor viruses have been isolated from naturally occurring animal tumors and also from normal tissues. Infectious forms of these viruses carry an RNA-directed DNA polymerase. Early in infection the

288

MIROSLAV HILL AND JANA HILLOVA

enzyme transcribes viral RNA into the DNA, which becomes covalently bound to the host cell chromosome. Virus-specific DNA can be detected by means of molecular hybridization techniques. Conclusive evidence about the synthesis and integration of full-length DNA copies of the viral genome has been obtained in transfection assays which show that chromosomal DNA of RSV-transformed cells is able to infect chicken cells. The molecular weight of infectious double-stranded RSV DNA was estimated to be about 6 x lo6 daltons, which agrees with most estimates of the genetic complexity of the viral RNA. It appears, therefore, that RSV particles which seem to carry more than one molecule of 35 S RNA, are polyploid. Several points have not yet been elucidated. It is not clear whether the RNA-DNA and double-stranded DNA forms isolated in early infections are all precursors of the integrated viral DNA. A possibility exists that virus-infected cells contain besides full-length viral DNA also partial transcripts that may escape detection in transfection assays. One particular example is the generation of td viruses from a defective RNA +-DNA transcription of a nondefective RSV. Finally, the integration mechanism inserting viral DNA into the cellular chromosome remains unknown, and there is an uncertainty as to whether the integration step is a prerequisite for the synthesis of the progeny virus. Cells infected with an exogenous virus acquire new genetic material; hence these cells are considered to be genetic transformants. The case of endogenous viruses remains obscure though there is a possibility that endogenous viruses entered animal cells during phylogenesis. Alternatively, endogenous viruses may be the products of normal cellular genes. Such a point of view has to be reconciled with the fact that certain ecotropic viruses endogenously carried in healthy mice are able to induce tumors. Otherwise normal cells seem to be devoid of the transforming genes and must be subjected to carcinogens or infected with exogenous viruses in order to acquire a transformed phenotype. It is assumed that the acquisition of a malignant phenotype is due to a genetic change (mutation) provoked by carcinogens in a normal cellular genome, or to the integration of exogenous viral transforming genes into the cellular genome. The possibility is also emerging that a mutation giving rise to malignancy may affect endogenous virogenes or cellular genes that can be picked up by the endogenous virus. This may explain why avian leukosis viruses are genetically related to the endogenous virus and, furthermore, why transforming genes of sarcoma viruses are not involved in the building-up of virus particles. It is not clear, however, whether endogenous viruses represent a natural reservoir for accidental evolution of exogenous viruses or, alternatively, whether endogenous and exogenous viruses diverged early in evolution from a common ancestor. Some

DNA OF RNA TUMOR VIRUSES

289

oncogenic C-type viruses, isolated for instance from cat, primate, and human tumors, lack genetic relatedness to their natural hosts, and no endogenous counterparts of these viruses have so far been identified. Thus we are facing a possibility that certain animal tumors (including human leukemias) are, in fact, due to products coded for by transforming genes of exogenous RNA tumor viruses.

ACKNOWLEDGMENTS The authors express their gratitude to Drs. V. Klement, P. Sheldrick, and N. Stedman for their helpful suggestions and criticism, and to N. Stedrnan for his continuous assistance in the preparation of the manuscript. Research carried out in the authors’ laboratory was supported in part by a Contract ATP 73.4.430.18 from I.N.S.E.R.M. and a Contract A 655 1800 from C.N.R.S.

REFERENCES Aaronson, S. A. ( 1971). Ndure (London) 230, 445-447. Aaronson, S. A,, and Dunn, C. Y. (1974). J . Virol. 13, 181-185. Aaronson, S. A., Hartley, J. W., and Todaro, G. J. (1969). Proc. Not. Acad. Sci. U.S. 64, 87-94. Abelson, H. T., and Rabstein, L. S . (1970). Cancer Res. 30, 2213-2222. Ali, M., and Baluda, M. A. (1974). J. Virol. 13, 1005-1013. Altaner, C., and Temin, H. M. ( 1970). Virology 40, 118-134. Armstrong, J. A., Porterfield, J. S., and De Madrid, A. T. (1971). J . Gen. Virol. 10, 195-198. Bader, J. P. (1966). Zn “Subviral Carcinogenesis” (Y. Ito, ed.), pp. 144-155. Aichi Cancer Center, Nagoya. Ball, J. K., and McCarter, J. A. (1971). J . Not. Cancer Inst. 46, 751-762. Ball, J. K., Harvey, D., and McCarter, J. A. (1973). Nature (London) 241,272-275. Baltimore, D. ( 1970). Nature (London) 226, 1209-1211. Baluda, M. A. (1972). Proc. Nat. Acad. Sci. US.69, 576-580. Baluda, M. A., and Drohan, W. N. (1972). J. Virol. 10, 10021009. Baluda, M. A., and Nayak, D. P. (1970). Proc. Not. Acad. Sci. U.S.66, 329-336. Baluda, M. A,, Shoyab, M., Markham, P. D., Evans, R. M., and Drohan, W. N. (1974). Cold Spring Harbor Symp. Quant. B i d . 39,869-874. Bassin, R. H., Simons, P. J., Chesterman, F. C., and Harvey, J. J. (1968). Znt. J . Cancer 3, 265-272. Bauer, H. (1974). Aduan. Cancer Res. 20,275-341. Baxt, W. G., and Spiegelman, S. (1972). Proc. Nut. Acad. S c i . US.69,3737-3741. Baxt, W. G., Hehlmann, R., and Spiegelrnan, S . (1972). Nature (London), New Biol. 240, 72-75. Baxt, W. G., Yates, J. W., Wallace, H. J., Jr., Holland, J. F., and Spiegelman, S. (1973). Proc. Nat. Acad. Sci. U.S.70,2629-2632. Beemon, K., Duesberg, P., and Vogt, P. (1974). Proc. Nut. Acad. Sci. U.S. 71, 4254-4258. Bellamy, A. R., Gillies, S. C., and Harvey, J. D. (1974). J. Virol. 14, 1388-1393.

290

MIROSLAV HILL AND JANA HILLOVA

Bentvelzen, P. ( 1974). Biochim. Biophys. Acta 355,236-259. Benveniste, R. E., and Todaro, G. J. ( 1973). Proc. Nut. Acad. Sci. U.S. 70, 3316-3320. Benveniste, R. E., and Todaro, G. J. (1974a). Nature (London) 252, 170-173. Benveniste, R. E., and Todaro, G. J. (1974b). Proc. Nut. Acad. Sci. U.S. 71, 4513-4518. Benveniste, R. E., Heinemann, R., Wilson, G. L., Callahan, R., and Todaro, G. J. ( 1974a). J . Virol. 14, 56-67. Benveniste, R. E., Lieber, hl. hl., Livingston, D. M., Sherr, C. J., and Todaro, G . J. ( 1974b). Nature (London) 248,17-20. Benveniste, R. E., Lieber, M. M., and Todaro, G. J. (1974~).Proc. Nut. Acad. Sci. US. 71, 602-606. Bernhard, W. (1960). Cancer Res. 20, 712-727. Bhargava, P. hl., and Shanmugam, G. (1971). Progr. N u c ~ .Acid Res. Mol. Biol. 11, 103-192. Billeter, M. A,, Parsons, J. T., and Coffin, J. M. (1974). Proc. Not. Acad. Sci. US. 71, 35603564. Biquard, J.-M., and Vigier, P. ( 1970). C. R. Acad. Sci. 271,2430-2433. Biquard, J.-M., and Vigier, P. ( 1972). Virology 47, 444-455. Bimstiel, hl. L., Sells, B. H., and Purdom, I. F. (1972). J. Mol. Biol. 63, 21-39. Bishop, J. 0. (1969). Biochem. J. 113,805-811. Biswal, N., and Benyesh-Melnick, M. (1969). Proc. Nut. Acad. Sci. US. 64, 1372-1379. Bolognesi, D. P. (1974). Aduan. Virus Res. 19,315-359. Bonar, R. A., and Beard, J. W. (1959). J . Nut. Cancer Inst. 23, 183-197. Boyd, V. A. L., and Butel, J. S. (1972). J. Virol. 10,399-409. Breese, S . S., Jr. (1970). Arch. Gesamte Virusforsch. 30, 401404. Callahan, R., Benveniste, R. E., Lieber, M. M., and Todaro, G. J. (1974). J. Virol. 14, 1394-1403. Cawley, J. C., and Karpas, A. ( 1974). Eur. J. Cancer 10,559-562. Chattopadhyay, S . K., Lowy, D. R., Teich, N. M., Levine, A. S., and Rowe, W. P. (1974). Proc. Nut. Acad. Sci. U.S.71,167-171. Chi, Y. Y.,and Bassel, A. R. (1975). ViroEogy, 64,217-227. Cooper, G . M., and Temin, H. M . (1974). 1. Virol. 14, 1132-1141. Crittenden, L. B., Smith, E. J., Weiss, R. A., and Sarma, P. S. (1974). Virology 57, 128-138. de Harven, E. (1974). Aduan. Virus Res. 19,221-261. Delius, H., Westphal, H., and Axelrod, N. ( 1973). J. Mol. Biol. 74, 677-687. DiMayorca, G. A., Eddy, B. E., Stewart, S. E., Hunter, W. S., Friend, C., and Bendich, A. (1959). Proc. Nat. Acad. Sci. US.45, 1805-1808. Dougherty, R. M., and Di Stefano, H. S. (1966). Virology 29,586-595. Duesberg, P. H. (1968). Proc. Nat. Acad. Sci. US.60, 1511-1518. Duesberg, P. H. (1972). Adoan. Biosci. 8, 145-156. Duesberg, P. H., and Vogt, P. K. (1970). Proc. Nat. Acad. Sci. U.S. 67, 1673-1680. Duesberg, P. H., and Vogt, P. K. (1973a). Virology 54, 207-219. Duesberg, P. H., and Vogt, P. K. (1973b). J. Virol. 12, 594599. Duesberg, P. H., Martin, G. S., and Vogt, P. K. (1970). Virology 41, 631-646. Duesberg, P. H., Kawai, S., Wang, L.-H., Vogt, P. K., Murphy, H. M.,and Hanafusa, H. (1975). Proc. Nat. Acad. Sci. US.72, 1569-1573. Duff, R. G., and Vogt, P. K. (1969). Virology 39, 18-30. Erikson, E., and Erikson, R. L. (1971).J. Virol. 8,254-256.

DNA OF RNA TUMOR VIRUSES

291

Erikson, R. L. ( 1969). Virology 37, 124-131. Evans, R. M., Baluda, M. A., and Shoyab, M. ( 1974). Proc. Nut. Acad. Sci. US. 71, 3152-3156. Fan, H., and Paskind, M. (1974). J. Virol. 14,421429. Faras, A. J., Garapin, A. C., Levinson, W. E., Bishop, J. M., and Goodman, H. M. ( 1973).J. Virol. 12, 33-42. Fenyo, E. M., and Grundner, G. (1973). Int. J. Cancer 12, 452-462. Fidaniin, H. M., Drohhn, W. N., and Baluda, M. A. (1975). 1.Virol. 15, 449457. Fiers, W., Lepoutre, L., and Vandendriessche, L. (1965). J. Mol. Biol. 13,432450. Fischinger, P. J., Blevins, C. S., and Nomura, S. (1974). J. Virol. 14, 177-179. Fleissner, E. ( 1971). J . Virol. 8, 778-785. Fourcade, A., Huynh, T., and Lacour, F. (1974). J. Virol. 14,407-411. Freeman, A. E., Kelloff, G. J., Gilden, R. V., Lane, W. T., Swain, A. P., and Huebner, R. J. ( 1971). Proc. Nut.Acud. Sci. U.S. 68,2386-2390. Fujita, D. J., Chen, Y. C., Friis, R. R., and Vogt, P. K. ( 1974). ViroZogy 60,558-571. Gallagher, R. E., and Gallo, R. C. (1975). Science 187,350353. Gallagher, R. E., Todaro, G. J., Smith, R. G., Livingston, D. M., and Gallo, R. C. (1974). Proc. Nut. Acad. Sci. US.71, 1309-1313. Gallo, R. C., and Gallagher, R. E. (1974). Ser. Huematol. 7,224-273. Gallo, R. C., Yang, S. S., and Ting, R. C . (1970). Nature (London) 228,927-929. Gallo, R. C., Miller, N. R., Saxinger, W. C., and Gillespie, D. (1973). Proc. Nut. Acud. Sci. US.70, 32193224. Garapin, A. C., Varmus, H. E., Faras, A. J., Levinson, W. E., and Bishop, J. M. ( 1973). Virdogy 52, 264-274. Gelb, L. D., Aaronson, S. A., and Martin, M. A. (1971a). Science 172, 1353-1355. Gelb, L. D., Kohne, D. E., and Martin, M. A. (1971b). J. Mol. B i d . 57, 129-145. Gelb, L. D., Milstien, J. B., Martin, M. A., and Aaronson, S. A. (1973). Nature (London), New Biol. 224, 76-79. Gerber, P. (1962). Virology 16, 96-98. Gianni, A. M., Smotkin, D., and Weinberg, R. A. (1975). Proc. Nut. Acad. Sci. US. 72, 447451. Gillespie, D., and Gallo, R. C. ( 1975). Science 188, 802411. Gillespie, D., and Spiegelman, S. (1965). J. Mol. Biol. 12, 829-842. Gillespie, D., Saxinger, W. C., and Gallo, R. C. (1975). Progr. Nucl. Acid Res. Mol. Biol. 15, 1-108. Gold&,A. ( 1970). Virology 40, 1022-1029. Goubin, G., and Hill, M. (1974). C . R. Acad. Sci. 278, 685-688. Graf, T. ( 1972). Virolugy 50, 567578. Graham, F. L., and Van der Eb, A. J. ( 1973). Virology 52,456-467. Granboulan, N., Huppert, J., and Lacour, F. (1966). 1. Mol. Biol. 16, 571-575. Green, M., and Gerard, G. F. ( 1974). Progr. Nud. Acid Res. M d . Biol. 14, 187434. Greenberger, J. S., Stephenson, J. R., Moloney, W. C., and Stuart, S. A. (1975). Cancer Res. 35, 245-252. Gross, L. ( 1951). Proc. SOC. Exp. Biol. Med. 76,2742. Gross, L. (1958). Acta Haemutol. 19, 353-361. Gross, L. (1974). Proc. Nut. Acad. Sci. US.71,2013-2017. Guntaka, R. V., Mahy, B. W. J., Bishop, J. M., and Varmus, H. E . (1975). Nature (London) 253, 507-511. Haas, M., and Hilgers, J. (1975). Proc. N a t . Acud. S c i . US. 72, 35463550. Haas, M., Hilgers, J., and DeclAve, A. (1975). 9th Meet. Eur. Tumour Virus Group Abstracts, p. 135.

292

MIROSLAV HILL AND JANA HILLOVA

Haase, A. T., Garapin, A. C., Faras, A. J., Taylor, J. M., and Bishop, J. M. (1974). Virology 57, 259-270. Hackett, A. J., and Sylvester, S. S. (1972). Nature (London), New Biol. 239, 164-1 66. Hanafusa, H., and Hanafusa, T. ( 1968). Virology 34, 630-636. Hanafusa, H., and Hanafusa, T. (1971). Virology 43,313-316. Hanafusa, H., Miyamoto, T., and Hanafusa, T. (1970). Proc. Nut. Acad. Sci. U.S. 66, 314-321. Hanafusa, H., Baltimore, D., Smoler, D., Watson, K. F., Yaniv, A., and Spiegelman, S. (1972). Science 177,1188-1191. Hanafusa, H., Hayward, W. S., Chen, J. H., and Hanafusa, T. (1974). Cold Spring Harbor Symp. @ant. Biol. 39, 1139-1144. Hanafusa, T., and Hanafusa, H. (1973). Virology 51, 247-251. Hanafusa, T., Hanafusa, H., and Miyamoto, T. (1970). Proc. Nat. Acad. Sci. U.S. 67, 1797-1803. Haran-Ghera, N. (1967). Proc. Soc. Erp. B i d . Med. 124, 697-699. Harel, L., Harel, J., and Frezouls, G. (1972). Biochem. Biophys. Res. Commun. 48, 796-801. Hart, R. W., and Setlow, R. B. (1974). Proc. Nat. Acad. Sci. U.S.71,2169-2173. Harvey, J. J. (1964). Nature (London) 204, 1104-1105. Hehlmann, R., Kufe, D., and Spiegelman, S. (1972). Proc. Nut. Acad. Sci. U.S. 69, 435-439. Hill, M. ( 1973). Biomedicine 18, 453-458. Hill, M., and Hillova, J. (1971a). Nature (London), New Biol. 231, 261-265. Hill, M., and Hillova, J. (1971b). I n “Informative Molecules in Biological Systems” (L. Ledoux, ed.), pp. 113-120. North-Holland Publ., Amsterdam. Hill, M.,and Hillova, J. ( 1 9 7 1 ~ )C. . R. A c d . Sci. 272, 3094-3097. Hill, M., and Hillova, J. (1972a). Aduan. Biosci. 8, 159-165. Hill, M., and Hillova, J. (1972b). Nature (London), New Biol. 237, 35-39. . 49,309413. Hill, M., and Hillova, J. ( 1 9 7 2 ~ )Virology Hill, M., and Hillova, J. (1974). Biochim. Biophys. Acta 355, 748. Hill, M., and Huppert, J. ( 1970). Biochim. Biophys. Ado 213, 26-35. Hill, M., *Hillova,J., Dantchev, D., Mariage, R., and Goubin, G. (1974). Cold Spring Harbor Symp. Quant. Biol. 39, 1015-1025. Hill, M., Hillova, J., Goubin, G., Manage, R., and Dantchev, D. (1975). Bull. Cancer 62, 183-194. Hillova, J. (1975). To be published. Hillova, J., Goubin, G., and Hill, M. (1972). C . R. Acad. Sci. 274, 1970-1973. Hillova, J., Dantchev, D., Manage, R., Plichon, M.-P., and Hill, M. (1974a). Virology 62, 197-208. Hillova, J., Goubin, G., Coulaud, D., and Hill, M. (1974b). J . Gen. Virol. 23, 237-245. Hillova, J., Mariage, R., and Hill, M. (1975). Virology 67, 292-296. Hirsch, M. S., and Black, P. H. (1974). Aduan. Virus Res. 19, 265-313. Hirt, B. (1967). J. Mol. Biol. 26, 365-369. HloiPnek, I., and Svoboda, J. (1972). J. Gen. Virol. 17, 55-59. HloiPnek, I., and Svoboda, J. (1974). Folk Biol. (Prague) 20,369-377. Howard, B. V., Estes, M. K., and Pagano, J. S. (1971). Biochim. Biophys. A d a 228, 105-116. Huebner, R. J., and Todaro, G. J. (1969). Proc. Nut. Acad. Sci. U.S.64, 1087-1094. Ikawa, Y., Ross, J., and Leder, P. (1974). Proc. Nut. Acod. Sci. U.S.71, 1154-1158.

DNA OF RNA TUMOR VIRUSES

293

Irino, S., Ota, Z., Sezaki, T., Suzaki, M., and Hiraki, K. (1963). Gann 54, 225 and 237. Ishizaki, R., and Vogt, P. K. (1966). Virology 30, 375-387. Jacobson, A. B., and Bromley, P. A. ( 1975). J . Virol. 15, 161-166. Kakefuda, T., and Bader, J. P. ( 1969). J . Virol. 4, 460-474. Kalter, S. S., Helmke, R. J., Panigel, M., Heberling, R. L., Felsburg, P. J., and Axelrod, L. R. (1973). Science 179, 1332-1333. Kang, C.-Y., and Temin, H. M. (1974).J . Virol. 14, 1179-1188. Karpas, A., and Kleinberg, D. (1974). Eur. J . Cancer 10, 551-553. Karpas, A,, and Milstein, C. ( 1973). Eur. J. Cancer 9,295-299. Karpas, A., and Tuckerman, E. (1974). Lancet 1, 1138-1141. Kawai, S., and Hanafusa, H. ( 1971). Virology 46,470-479. Kawai, S., and Hanafusa, H. (1972a). Virology 48, 126-135. Kawai, S., and Hanafusa, H. ( 1972b). Virology 49, 37-44. Kawai, S., and Hanafusa, H. (1973). Proc. Nut. Acad. Sci. US. 70, 3493-3497. Kawai, S., and Yamamoto, T. (1970). Jap. J . E z p . Med. 40,243-256. Kawai, S., Metroka, C. E., and Hanafusa, H. (1972). Virology 49, 302-304. Kelloff, G. J., Huebner, R. J., Lee, Y. K., Toni, R., and Gilden, R. (1970). Proc. Nut. A d . Sci. U.S.65, 310-317. Kelloff, G. J., Hatanaka, M., and Gilden, R. V. (1972). Virology 48, 266-269. Kirsten, W. H., and Mayer, L. A. (1967). J . Nut. Cancer Inst. 39, 311-335. Klement, V., Hartley, J. W., Rowe, W. P., and Huebner, R. J. (1969). J . Nat. Cancer Inst. 43, 925-934. Klement, V., Nicolson, M. O., Nelson-Rees, W., Gilden, R. V., Oroszlan, S., Rongey, R. W., and Gardner, M. B. (1973). Int. J. Cancer 12, 654-666. Kotler, M., Weinberg, E., Haspel, O., Olshevsky, U., and Becker, Y. (1973). Nature (London), New Biol. 244, 197-200. Kufe, D. W., Peters, W. P., and Spiegelman, S. (1973). Proc. Nut. A d . Sci. U.S. 70, 3810-3814. Kung, H. J., Bailey, J. M., Davidson, N., Vogt, P. K., Nicolson, M. O., and McAIIister, R. M. (1974). Cold Spring Harbor Symp. Quant. Biol. 39, 827-834. Lacour, F., Fourcade, A., Merlin, E., and Huynh, T. (1972). C . R . Acad. Sci. 274, 2253-2255. Lai, M. M. C., Duesberg, P. H., Horst, J., and Vogt, P. K. (1973). Proc. Nut. Acad. Sci. U.S.70, 2266-2270. Leis, J., Schincariol, A., Ishizaki, R., and Hurwitz, J. (1975). J . Virol. 15, 484-489. Levy, J. A. (1973). Science 182, 1151-1153. Levy, J. A., Kazan, P. M., and Varmus, H. E. (1974). Virobgy 61, 297302. Lieber, M. M., Benveniste, R. E., Livingston, D. M., and Todaro, G. J. (1973). Science 182, 56-59. Lieberman, M., and Kaplan, H. S. (1959). Science 130,387-388. Linial, M., and Mason, W. S. ( 1973). Virology 53, 258-273. Livingston, D. M., and Todaro, G. J. (1973). Virology 53, 142-151. Loni, M. C., and Green, M. (1974). Proc. Nut. Acad. Sci. U.S.71, 3418-3422. Lovinger, G . G., Ling, H. P., Klein, R. A., Gilden, R. V., and Hatanaka, M. (1974). Virology 62, 280-283. Lowy, D. R., Chattopadhyay, S. K., Teich, N. M., Rowe, W. P., and Levine, A. S. (1974). Proc. Nat. Acad. Sci. US.71,35553559. McCutchan, J. H., and Pagano, J. S. (1968). J . Nut. Cancer Inst. 41, 351357. Maes, R., Sedwick, W., and Vaheri, A. (1967). Biochim. Biophys. Acta 134, 269-276.

294

MIROSLAV HILL AND JANA HILLOVA

Mak, T. W., Aye, M. T., Messner, H., Sheinin, R., Till, J. E., and McCulloch, E. A. (1974a). Brit. J. Cancer 29, 433-437. Mak, T. W., Manaster, J., Howatson, A. F., McCulloch, E. A., and Till, J. E. ( 1974b). Proc. Nut. Acad. S c i . US.71,4336-4340. Mak, T. W., Kurtz, S., Manaster, I., and Housman, D. (1975). Proc. Nut. Acad. Sci. U.S.72, 623-627. Mangel, W. F., Delius, H., and Duesberg, P. H. (1974). Proc. Nut. Acud. Sci. US. 71, 4541-4545. Markham, P. D., and Baluda, M. A. ( 1973). J. Virol. 12, 721-732. Marmur, J. ( 1961). J. Mol. Biol. 3,208-218. Martin, G. S. ( 1970). Nature (London) 227, 1021-1023. Mason, W. S., Friis, R. R., Linial, M., and Vogt, P. K. ( 1974). Virology 61, 559-574. May, E., May, P., and Cassingena, R. (1969). Biochim. Biophys. Actu 186, 136-144. Melli, M., Whitfield, C., Rao, K. V., Richardson, M., and Bishop, J. 0. ( 1971). Nature (London),New Biol. 231, 8-12. Miller, N. R., Saxinger, W. C., Reitz, M. S., Gallagher, R. E., Wu, A. M., Gallo, R. C., and Gillespie, D. (1974). Proc. Nut. Acud. Sci. US. 71, 31774181. Moloney, J. B. (1966). Nut. Cuncer Znst., Monogr. 22, 139-141. Mondal, H., Gallagher, R. E., and Gallo, €7. C. (1975). Proc. Nut. Acud. Sci. US. 72, 1194-1198. Montagnier, L., and Vigier, P. (1972). C . R. Acad. Sci. 274, 1977-1980. Montagnier, L., Gold&, A., and Vigier, P. ( 1969). J. Gen. Virol. 4, 449-452. Murray, P. R., and Nayak, D. P. (1974). J. Virol. 14, 679-688. Nayak, D. P. (1974). Proc. Nut. Acad. Sci. US.71, 1164-1168. Nayak, D. P., and Murray, P. R. (1973). J. Virol. 12, 177-187. Neiman, P. E. (1972). Science 178,750-753. Neiman, P. E. ( 1973a). Virology 53,196-204. Neiman, P. E. ( 1973b). Nature ( h d o n ) , New B i d . 244,62-64. Neiman, P. E., Wright, S. E., and Purchase, H. G. (1974a). Cold Spring Harbor Symp. Qlurnt. Biol. 39,875-883. Neiman, P. E., Wright, S. E., McMillin, C., and MacDonnell, D. (1974b). J. Virol. 13, 837-846. Neiman, P. E., Purchase, H. C., and Okazaki, W. (1975). Cell 4, 311-319. Nerrnut, M. V., Frank, H., and Schfifer, W. (1972). Virology 49, 345-358. Nicolson, M., Hariri, F., Krempin, M., and McAllister, R. (1975). Proc. Amer. Ass. Cancer Res. 16, 185. Ogura, H., Friis, R. R., and Bauer, H. (1974a). Z . Naturforsch. c 29, 437-441. Ogura, H., Gelderblom, H., and Bauer, H. (1974b). Intervirology 4, 69-76. Okabe, H., Gilden, R. V., and Hatanaka, M. (1973). Proc. Nut. Acud. Sci. U.S. 70, 392343927. Pagano, J. S. (1970). Progr. Med. Virol. 12, 1-48. Pagano, J. S., and Hutchison, C. A., 111. ( 1971). I n “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. 5, pp. 79-123. Academic Press, New York. Pagano, J. S., McCutchan, J. H., and Vaheri, A. (1967). J . Virol. 1, 891-897. Peebles, P. T., Haapala, D. K., and Gazdar, A. F. (1972). J. Visol. 9, 488-493. Peters, R. L., Hartley, J. W., Spahn, G. J., Rabstein, I.,. S., Whitmire, C. E., Turner, H. C., and Huebner, R. J. ( 1972). Int. J. Cancer 10,283-289. Peters, R. L., Spahn, G. J., Rabstein, L. S., Kelloff, G. J., and Huebner, R. J. (1973). Science 181, 665-667. Quade, K., Smith, R. E., and Nichols, J. L. ( 1974). ViroZogy 61, 287-291.

DNA OF RNA TUMOR VIRUSES

295

Quigley, J. P., Rifkin, D. B., and Reich, E. (1971). Virology 46, 106-116. Quintrell, N., Varmus, H. E., Bishop, J. M., Nicolson, M. O., and McAllister, R. M. ( 1974). Virology 58, 568575. Radman, M. ( 1975). In “Molecular Mechanisms for the Repair of DNA” (P. C. HanaWalt and R. B. Setlow, eds. ). Plenum, New York (in press). Reitz, M. S., Jr., Smith, R. G., Roseberry, E. A., and Gallo, R. C. (1974). Biochem. Biophys. Res. Commun. 57, 934-948. Robinson, W. S., Pitkanen, A., and Rubin, H. (1965). Proc. Nut. Acad. Sci. US. 54, 137-144. Rosenberg, N., Baltimore, D., and Scher, C. D. (1975). Proc. Nut. Acad. Sci. U.S. 72, 1932-1936. Rosenthal, P. N., Robinson, H. L., Robinson, W. S., Hanafusa, T., and Hanafusa, H. ( 1971). Proc. Nut. Acad. Sci. U.S. 68,2336-2340. Roy-Burman, P., and Klement, V. (1975). J. Gen. Virol. 28, 193, 198. Sambrook, J., Westphal, H., Srinivasan, P. R., and Dulbecco, R. (1968). Proc. Nut. Acad. Sci. US.60, 1288-1295. Sarma, P. S., Log, T., and Gilden, R. V . (1970). Proc. Soc. Erp. Biok Med. 133, 718-722. Sarngadharan, M. G., Sarin, P. S., Reitz, M. S., and Gallo, R. C. (1972). Nature (London),New Biol. 240, 67-72. Scheele, C. M., and Hanafusa, H. ( 1971). Virobgy 45,401-410. Scher, C. D., and Siegler, R. ( 1975). Nature (London) 253, 729-731. Schincariol, A. L., and Joklik, W. K. ( 1973). Virology 56, 532548. Scholm, J,, and Spiegelman, S. ( 1971). Science 174,840-843. Scolnick, E. M., and Bumgarner, S. J. (1975). J. Virol. 15, 1293-1296. Scolnick, E. M., and Parks, W. P. (1974). J . Virol. 13, 1211-1219. Scolnick, E. M., Rands, E., Williams, D., and Parks, W. P. (1973). J. Virol. 12, 458-463. Scolnick, E. M., Parks, W. P., Kawakami, T., Kohne, D., Okabe, H., Gilden, R., and Hatanaka, M. ( 1974a). J. Virol. 13,363369. Scolnick, E. M., Maryak, J. M., and Parks, W. P. (197413). J. Virol. 14, 1435-1444. Shapiro, H. S. (1970). In “Handbook of Biochemistry” (H. A. Sober, ed.), 2nd ed., pp. H-104 to H-116. Chem. Rubber Publ. Co., Cleveland, Ohio. Sheldrick, P., Laithier, M., Lando, D., and Ryhiner, M. L. (1973). Proc. Nut. Acad. Sci. US.70, 36213625, Sherr, C. J., and Todaro, G. J. (1975). Science 187, 855-857. Sherr, C. J., Benveniste, R. E., and Todaro, G. J. (1974a). Proc. Nut. Acad. Sci. US. 71, 37214725. Sherr, C. J., Lieber, M. M., Benveniste, R. E., and Todaro, G. J. (197413). Virology 58, 492-503. Shoyab, M., Baluda, M. A., and Evans, R. (1974a). J. Virol. 13, 331339. Shoyab, M., Evans, R. M., and Baluda, M. A. (1974b). J. Vird. 14, 47-49. Shoyab, M., Markham, P. D., and Baluda, M. A. ( 1 9 7 4 ~ ) J. . Virol. 14, 225-230. Shoyab, M., Markham, P. D., and Baluda, M. A. (1975). Proc. Nut. Acad. Sci. U.S.72, 1031-1035. SimkoviE, D., Vdentovh, N., and Thurzo, V. (1962). Neoplasms 9, 104-106. Sirover, M. A., and Loeb, L. A. (1974). Biochem. Biophys. Res. Commun. 61, 410-414. Sklar, M. D., White, B. J., and Rowe, W. P. (1974). Proc. Nat. Acad. Sci. US. 71, 40774081. Smith, R. E., and Moscovici, C. (1969). Cancer Res. 29, 1356-1366.

296

MIROSLAV HILL AND JANA HILLOVA

Soniers, K. D., May, J. T., Kit, S., McCormick, K. J., Hatch, G. G., Stenback, W. A., and Trentin, J. J. (1973). Interoirology 1, 11-18. Springgate, C. F., and Loeb, L. A. (1973). Proc. Nat. Acad. Sci. U S . 70,245-249. Springgate, C. F., Battula, N., and Loeb, L. A. ( 1973). Biochem. Biophys. Res. Commun. 52, 401406. Stenback, W. A., Van Hoosier, G. L., Jr., and Trentin, J. J. (1968). J. Viral. 2, 1115-1121. Stephenson, J. R., Aaronson, S. A., Amstein, P., Huebner, R. J., and Tronick, S. R. (1974a). Virology 61, 56-63. Stephenson, J. R., Crow, J. D., and Aaronson, S. A. (1974b). Virology 61, 411419. Stephenson, J. R., Greenberger, J. S., and Aaronson, S. A. ( 1 9 7 4 ~ ) J. . Virol. 13, 237-240. Svoboda, J., Machala, O., and Hloihek, I. (1967). Folia Biol. (Prague) 13, 155157. Svoboda, J., Hloihek, I., and Mach, 0.(1972). Folia Biol. (Prague) 18, 149-153. Svoboda, J., HloiAnek, I., Mach, O., Michlovi, A., Riman, J., and UrbLnkov6,-M. (1973). J. Gen. Virol. 21, 47-55. Svoboda, J., Hloznek, I., Mach, O., and Zadraiil, S. ( 1974). Cold Spring Harbor Symp. Quant. Biol. 39, 1077-1083. Sweet, R. W., Goodman, N. C., Cho, J.-R., Ruprecht, R. M., Redfield, R. R., and Spiegelman, S. (1974). Psoc. Nut. Acad. Sci. U.S. 71, 1705-1709. Takano, T., and Hatanaka, M. (1975). Proc. Nut. Acad. S c i . U.S. 72, 343347. Taylor, J. M., Varmus, H. E., Faras, A. J., Levinson, W. E., and Bishop, J. M. (1974). J. Mol. Biol. 84,217-221. Teitz, Y., Lennette, E. H., Oshiro, L. S., and Cremer, N. E. ( 1971). 1. Nat. Cancer Inst. 46, 11-23. Temin, H. M. ( 1964a). Nut. Cancer Inst., Monogr. 17,557-570. Temin, H. M. ( 1964b). Proc. Nut. Acad. Sci. US.52,323429. Temin, H. M. (1971a). J. Nut. Cancer Inst. 46,111-VII. Temin, H. M. (1971b). Annu. Reo. Microbiol. 25, 609-648. Temin, H. M. ( 1974a). Cancer Res. 34,2835-2841. Temin, H. M. (1974b). Aduan. Cancer Res. 19,47-104. Temin, H. M., and Baltimore, D. (1972). Aduan. V i m Res. 17, 129-186. Temin, H. M., and Mizutani, S. (1970). Nature (London) 226, 1211- 1213. Thomas, E. D., Bryant, J. I., Buckner, C. D., Clift, R. A., Fefer, A,, Neiman, P., Ramberg, R. E., and Storb, R. (1972). Transplant. Proc. 4, 567570. Todaro, G. J. (1972). Nature (London),New Biol. 240, 157-160. Todaro, G. J., and Gallo, R. C. (1973). Nature (London) 244, 206-209. Todaro, G . J., Benveniste, R. E., Lieber, M. M., and Sherr, C. J. (1974). Virology 58, 65-74. Tooze, J. ( 1973). “The Molecular Biology of Tumor Viruses.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Toth, B. (1963). Proc. SOC.Exp. Biol. Med. 112, 873-875. Toyoshima, K., Friis, R. R., and Vogt, P. K. (1970). Virology 42, 163-170. Tsuchida, N., Gilden, R. V., and Hatanaka, M. (1974). Proc. Nut. Acad. Sci. U.S. 71, 45034507. Van den Berg, K. J., Nooter, K., and Bentvelzen, P. (1975). 9th Meet. Eur. Tumor Virus Group Abstracts, p. 68. Varmus, H. E., Weiss, R. A., Friis, R. R., Levinson, W., and Bishop, J. M. (1972). Proc. Nut. Acad. Sci. US.69,20-24.

DNA OF RNA TUMOR VIRUSES

297

Varmus, H. E., Bishop, J. M., and Vogt, P. K. (1973a). J. Mol. Biol. 74, 613-626. Varmus, H. E., Vogt, P. K., and Bishop, J. M. (1973b). Proc. Nut. Acud. Sci. U.S. 70, 30674071. Varmus, H. E., Guntaka, R. V., Deng, C. T., and Bishop, J. M. (1974a). Cold Spring Harbor Symp. Quunt. Biol. 39, 987-996. Varmus, H. E., Gantaka, R. V., Fan, W. J. W., Heasley, S., and Bishop, J. M. ( 197413). Proc. Nut. Acud. Sci. U.S. 71, 3874-3878. Varmus, H. E., Heasley, S., and Bishop, J. M. ( 1 9 7 4 ~ ) J. . Virol. 14, 895-903. Veprek, L., Beard, D., Langlois, A. J., Ishizaki, R., and Beard, J. W. (19i ). 1. - Nut. Cancer Inst. 46, 713-729. Verma, I. M., Mason, W. S., Drost, S. D., and Baltimore, D. (1974). Nature Lond o n ) 251, 27-31. Vigier, P. ( 1967). C . R. Acud. Sci. 264, 422-425. Vigier, P., and Montagnier, L. (1975). Int. 1. Cancer 15,67-77. Viola, M. V., and White, L. R. ( 1973). Nature (London) 246, 485-487. Vogt, P. K. ( 1971a). Virology 46, 939-946. Vogt, P. K. ( 1971b). Virology 46, 947-952. Vogt, P. K. ( 1972). J. Nut. cancer Inst. 48,3-9. Vogt, P. K. (1973). In “Possible Episomes in Eukaryotes” (L. G. Silvestri, ed.), pp. 35-41. North-Holland Publ., Amsterdam. Vogt, P. K., and Friis, R. R. ( 1971). Virology 43,223-234. Vogt, P. K., and Ishizaki, R. (1965). Virology 26, 664-672. Vogt, P. K., and Ishizaki, R. (1966). Virology 30, 368-374. Vogt, P. K., Weiss, R. A., and Hanafusa, H. (1974). J. Virol. 13, 551-554. Wang, S., Kothari, R. M., Taylor, M., and Hung, P. (1973). Nature (London), New Biol. 242, 133-135. Warden, D., and Thorne, H. V. ( 1968). J. Gen. Virol. 3,371377. Weber, G . H., Heine, U., Cottler-Fox, M., and Beaudreau, G. S. (1974). Proc. Nut. A d . Sci. U.S. 71, 1887-1890. Weber, G . H., Heine, U., Cottler-Fox, M., Garon, C. F., and Beaudreau, G. S. ( 1975). Virology 64,205-216. Weil, R., and Vinograd, J. (1963). PTOC. Nut. Acud. Sci. U.S. 50, 730-738. Weiss, R. A. ( 1969). J. Gen. Virol. 5, 511528. Weiss, R. A. (1972). In “RNA Viruses and Host Genome in Oncogenesis” (P. Emmelot and P. Bentvelzen, eds.), pp. 117-135. North-Holland Publ., Amsterdam. Weiss, R. A., Friis, R. R., Katz, E., and Vogt; P. K. ( 1971). Virdogy 46, 926938. Weiss, R. A,, Mason, W. S., and Vogt, P. K. (1973). Virology 52, 535-552. Wyke, J. A. (1973a). virology 52, 587-590. Wyke, J. A. ( 1973b). Virology 54,28-36. Yamaguchi, N., Takeuchi, M., and Yamamoto, T. (1967). Jup. J. Ezp. Med. 37, 83-86.

This Page Intentionally Left Blank

SUBJECT INDEX A

Adenoviruses, 91-130 assembly of, 100-101 genetics of, 91-130 complementation, 105-109, 115 characterization, 105-115 maps for, 113-115 recombination tests, 109-113 mutants of functional studies, 118-123 isolation, 101-105 multiple, 105 mutagenic procedures, 103-104 phenotypes, 116-123 selection, 101-105 temperature-sensitive, 116-117 transformation by, 121-123 types, 101-103 protein synthesis in, 99-100 replication of, 95-101 of DNA, 98-99, 119-120 transcription of, 97-98 of mutants, 120 virion of, 92-95 Adherentes junctions, structure and function of, 39-42 Adhesion, at call junctions, 75-76 Adrenal gland tumors, cell junctions in, 44 Albinism, genetics of, 16-17 Amber mutations, by 4-nitroquinohne 1-oxide, 135 Apes, Epstein-Barr virus in, 173 ATP, role in gap junction permeability, 64-66 Avian tumor viruses as endogenous virus, 240 infectious DNA of, 248-249

B Bacteria, carcinogen-induced DNA damage in, repair of, 143-145 299

Bacteriophages carcinogen-induced DNA damage in, repair of, 146 induction by 4-nitroquinoline 1-oxide, 138 Barrett-Derringer phenomenon, in tumor progression, 217-218 Base-pair change mutations, by 4-nitroquinoline 1-oxide, 133-136 Bladder, see Urinary bladder Blood cells, cell junctions between, 57-59 Bone tumors, cell junctions in, 44 Breast tumors cell junctions in, 44 genetics of, 3-7

C Calcium, intracellular, control of gap junction permeability and, 64-66 CAMP,in gap junction permeability, 64-66 Cancer. (See Q ~ Tumors) O human, genetic aspects of, 1-21 intercellular junctions in, 23-89 carcinogenesis, by 4-nitroquinoline l-oxide, 131-169 Carcinogens, Cnitroquinoline 1-oxide in screening tests for, 163 Carotid body tumors, cell junctions in, 45 cell division, postconfluence inkbition of, 74 Cell junctions, in cancer, 23-89 Chromosomes aberrations of leukemia and, 9-10 from 4-nitroquinoline 1-oxide, 140143 endoreduplication of, induction by 4-nitroquinoline 1-oxide, 141-143 Cigarette smoking, lung cancer and, 18 Clonal nature, of tumor initiation, 204207

300

SUBJECT INDEX

Colorectal cancer, genetics of, 10-12 Contact inhibition movement concept, of tumor cells, 73-74 Craniopharyngioma, cell junctions in, 45 C-type viruses, as endogenous viruses, 21 1 D

Deletion mutations, by 4-nitroquinoline 1-oxide, 136137 Desmosonie, ultrastructure of, 39-42 Differential adhesion theory, of tumorcell locomation, 78 DNA of adenoviruses, replication of, 98-99 4-nitroquinoline 1-oxide-damaged, repair of, 143-151 of RNA tumor viruses, 237-297 genetic content, 262-267 infectivity, 2 4 6 2 7 1 structure, 268-270

E Ear tumors, cell junctions in, 45 Electron microscopy, of membranes, 26-30 Embryos, cell junctions in development of, 70-71 Endogenous viruses, RNA tumor viruses as, 240-243 Endoreduplication, of chromosomes, induction by 4-nitroquinoline 1-oxide, 141-143 Epididymal tumors, cell junctions in, 45 Epstein-Barr virus, 171-201 inocuIa containing, 188-190 lymphocyte transformation by, 179-181 in nonhuman primates, 171-201 experimental infection of, 1 8 6 1 9 7 in pathogenic lesions, 194-195 reactive antibodies, 172-177 significance of, 1 7 6 1 7 7 serology of, 194 tumorigenesis by, 195-197 Eye tumors, cell junctions in, 45 F

Fallopian tube tumors, cell junctions in, 45

Frameshift mutations, by 4-nitroquinoline 1-oxide, 135, 136 Freeze-etching, nomenclature of, 28 Freeze-fracturing, of membranes, 28-30

G Gap junctions in cell communication, 62-64 genetic aspects of, 69-70 isolation of, 38-39 metabolic coupling at, 66-67 permeability control in, 64-66 structure and function of, 35-39 in tumors, 52-53 Gardner syndrome, in colorectal cancer, 11 Gastric cancer, genetics of, 12-13 Genetics of adenoviruses, 91-130 of gap-junction occurrence, 69-70 of human cancer, 1-21 Gibbon, Epstein-Barr infection of, 189 Growth control of, intercellular junctions in, 61-70 postconfluence inhibition of, 74 H

Homeostasis immunity in, 218-233 tumor progression and, 203-236 Human cancer, genetic aspects of, 1-21 Hypopharyngeal tumors, cell junctions in, 45 I

Immunity as a homeostatic mechanism, 218-233 to tumors, 231-233 Immunological surveillance, 220-222, 232 subliminal, 225-226 Immunostimulation of tumors, 228-231 Infectious mononucleosis, Epstein-Barr virus and, 187 Initiation, as first step in tumor progression, 204-212

301

SUBJECX INDEX

Initiation mutations, by 4-nitroquinoline 1-oxide, 135 Intercellular junctions adherentes type, 39-42 adhesion at, 75-76 in cancer, 23-89 in cell-to-cell communication, 61-70 classification of, 3 2 4 3 complexes of, 42-43 in embryonic development, 70-71 function of, 24-25 gap (nexus) type, 35-39 in growth control, 61-70 miscellaneous types, 43 in nonmalignant growth disorders, 60-61 occludentes type, 33-35 plasma membrane and, 30-32 in tissue cultures, 60-61 in tumor metastases, 74-75 in tumors, 43-61 biological behavior, 71-75 Intestinal tumors, cell junctions in, 45

K Kidney tumors, cell junctions in, 45 L

Laryngeal tumors, cell junctions in, 45 Leukemia( s ) chromosomal aberrations and, 9-10 from DNA of RNA tumor viruses, 265 genetics of, 7-10 Leukemia viruses, transforming genetic material of, 279-282 Leukosis viruses, transfection assay of, 251 Leukosislike viruses, transforming-genetic material of, 279-282 Liver tumors, cell junctions in, 45-46 Locomotion, of tumor cells, cell junctions and, 71-74 Locomotory paralysis theory, of tumorcell lacornotion, 72-73 Lung cancer cell junctions in, 46 genetics of, 17-19 smoking and, 18

Lymphoblastoid cell lines cytological properties, 181-182 from nonhuman primates, Epstein-Barr virus effects on, 177-179 Lymphoreticular neoplasms, virally induced tumors and, 222-225 M

Macula adherens in tumors, 5 3 5 6 ultrastructure of, 39-42 Mammary tumors (murine), progression from clones, 207-211 Marek's disease, immunity to, 223-225 Marmoset, Epstein-Barr infection of, 189, 191 McNutt-Weinstein model of gap junction, 37 Membranes, ultrastructure of, 26-32 Metastases cell junctions in, 59-60, 74-75 immune response and, 231 Mitotic gene conversions, by 4-nitroquinoline 1-oxide, 137 Monkeys, Epstein-Barr virus in, 173-175, 189 Mouth tumors, cell junctions in, 47 Mutagen, 4-nitroquinoline 1-oxide as, 133-139 Mutual adhesion theory, of tumor-cell locomotion, 72

N Nasopharyngeal tumors, cell junctions in, 47 Negative staining, of membranes, 27-28 Nervous system tumors, cell junctions in, 47 Nexus junctions, structure and function of, 3 5 3 9 4-Nitroquinoline 1-oxide, 131-169 in carcinogen screening tests, 163 carcinogenesis by, 157-163 immunity and, 160 in uitro, 160-162 in uiuo, 157-159 chromosome aberrations induced by, 140-143

302

SUBJECT LNDEX

decarcinogenesis by, 162 derivatives of, as mutagens, 139 DNA damage by, repair of, 143-151 DNA modification by, 151-153 biological activity, 152-153 interaction with nucleic acids, 151-156 chemical, 155-156 interactions with protein, 156 niokeular biology of, 131-169 mutagenic activity of, on organisms, 133-139 phage induction by, 138 Nucleic acid hybridization technique, in reverse transcriptase studies, 244246 Nucleic acids, 4-nitroquinoline 1-oxide interaction with, 151-156

0 Occludentes junctions structure and function of, 33-35 in tumors, 5 M 7 Ochre mutations, by 4-nitroquinoline 1-oxide, 135 Ovarian tumors, cell junctions in, 47 Owl monkeys, Epstein-Barr infection of, 189

P Pancreatic tumors, cell junctions in, 48 Parathyroid tumors, cell junctions in, 48 Pineal gland tumors, cell junctions in, 48 Plant cells, carcinogen-induced DNA damage in, repair of, 146 Plasma membrane cell junctions and, 3 0 4 2 electrochemical potential across, 65-66 Prosimians, Epstein-Barr virus in, 173 Prostate gland tumors, cell junctions in, 48 Protein, 4-nitrquinoline 1-oxide interaction with, 156

Q Quinoline-base adducts, formation by 4-nitroquinoline 1-oxide, 151-152

R

factor, loss caused by 4-nitroquinoline 1-oxide, 137-138 Reticuloendotheliosis viruses, transfection assay of, 251 Retinoblastoma, genetics of, 13-15 Reverse transcriptase, of RNA tumor viruses, 244 RNA tumor viruses as endogenous viruses, 240 RNA genome size in, 271-274 viral DNA of, genetic transformation by, 237-297 of individual viruses, 274-289 “spontaneous,” 282-287 Rous-associated virus, as endogenous virus, 240 Rous sarcoma virus as endogenous virus, 240 infectious DNA of minimum size, 270-271 structure, 268-270

p

S Salivary glands tumors, cell junctions in, 48 Sarcoma viruses transfection assay of, 250-251 transforming genetic material of, 275279 Skin tumors, cell junctions in, 48 Smoking. lung cancer and, 18 Soft tissue tumors, cell junctions in, 48 Squirrel monkey, Epstein-Barr infection of, 191 Stomach cancer cell junctions in, 49 genetics of, 12-13 Surgery, effect on tumor dissemination, 77 T

Temperature-sensitive mutants, of adenoviruses, 116-117 Testes, tumors of, cell junctions in, 4 9 5 0 Thin-section electron microscopy, of membranes, 26-27

303

SUBJECT INDEX

Thymus tumors, cell junctions in, 50 Thyroid tumors, cell junctions in, 50 Tissue culture, intercellular junctions in, 60-61 Transepithelial permeability, malignant transformation and, 77 Transfection assay of viral DNA, 247-257 dose-response relationship, 257-261 efficiency, 257-262 specific infectivity, 261-262 Transformation malignant, transepithelial permeability and, 77 by viral DNA of RNA tumor viruses, 237-297 nonproducer cells, 255-257 Tumor ( s ) . ( See also Cancer ) cell junctions in, 43-61 cells, coupling between, 67-69 dissemination by surgery, 77 immunostimulation of, 228-231 induced vs. “spontaneous,” 2 19-220 invasion by, cell junctions in, 74-75 metastases of, cell junctions in, 59 Tumor progression, 203-236 genetic vs. epigenetic change in, 215218 homeostasis and, 203-236 induced vs. “spontaneous” tumors, 219-220 induction vs. selection in, 211-212 initiation in, 204-212 latency during, 213-215

metastasis and, 231 steps in, 213-218 Tumorigenesis, by Epstein-Barr virus, 195-197 U

Ureter tumors, cell junctions in, 50 Urinary bladder tumors, cell junctions in, 50 Uterine tumors, cell junctions in, 50-51 V

Vaginal tumors, cell junctions in, 51 Virion, of adenoviruses, 92-95 Virus, tumors induced by, lymphoreticular neoplasms and, 222-225 W

Woolly monkey, Epstein-Barr infection of, 191 X

Xeroderma pigmentosum cells from, 4-nitroquinoline 1-oxide effects on, 140-)-14,1 genetics of, 15-16 Y

Yeast, carcinogen-induced DNA damage in, repair of, 145-146

CONTENTS OF PREVIOUS VOLUMES Carcinogenesis and Tumor Pathogenesis I . Berenblum Electronic Configuration and Carcino- Ionizing Radiations and Cancer genesis Austin M . Bmes C. A. Codson Survival and Preservation of Tumors in Epidermal Carcinogenesis the Frozen State E. V. Cowdy James Craigie The Milk Agent in the Origin of Mam- Energy and Nitrogen Metabolism in mary Tumors in Mice Cancer L. Dmochowski Leonard D. Fenninger and G. Burroughs Mider Hormonal Aspects of Experimental Tumorigenesis Some Aspects of the Clinical Use of T. U. Gardner Nitrogen Mustards Caloin T. Klopp and Ieanne C . BateProperties of the Agent of Rous No. 1 man Sarcoma R. J. C. Harris Genetic Studies in Experimental Cancer L.w. Low Applications of Radioisotopes to Studies of Carcinogenesis and Tumor Me- The Role of Viruses in the Production of tabolism Cancer Charles Heidelberger C. Oberling and M . Guerin The Carcinogenic Aminoazo Dyes Experimental Cancer Chemotherapy James A. Miller and Elizabeth C. C. Chester Stock Miller AUTHOR INDEX-SUB JECT INDEX The Chemistry of Cytotoxic Alkylating Agents Volume 3 A4. C.1. Ross Nutrition in Relation to Cancer Etiology of Lung Cancer Albert Tannenbaum and Herbert Richard Doll Siloerstone The Experimental Developnient and Plasma Proteins in Cancer Metabolism of Thyroid Gland Richard J. Winder Tumors Harold P. Morris AUTHOR IXDEX-SUB JECT ISDEX Electronic Structure and Carcinogenic Activity and Aromatic Molecules: Volume 2 New Developments A. Pullman and B., Pullman The Reactions of Carcinogens with MacSome Aspects of Carcinogenesis romolecules P. Rondoni Peter Alexander Pulmonary Tumors in Experimental AniChemical Constitution and Carcinogenic mals Activity Michael B. Shimkin G. M. Badger Volume 1

304

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues

Sidney Weinhouse AUTHOR INDEX-SUBJECT INDEX

Volume 4

Advances in Chemotherapy of Cancer in Man Sidney Farber, Rudolf Toch, Edward Manning Sears, and Donald Pinkel The Use of Myleran and Similar Agents in Chronic Leukemias D. A. G. Gdton The Employment of Methods of Inhibition Analysis in the Normal and Tunlor-Bearing Mammalian Organism Abraham Goldin Some Recent Work on Tumor Immunity P. A. Gorer Inductive Tissue Interaction in Development Clifford Grobstein Lipids in Cancer Frances L. Haven and W . R. Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A. Lacassagne, N . P. BuuHoi, R. Daudel, and F. Zaidela The Hormonal Genesis of Mammary Cancer 0. Muhlbock AUTHOR INDEX-SUB JECT INDEX

Volume 5

Tumor-Host Relations R. W . Begg Primary Carcinoma of the Liver Charles B e m n Protein Synthesis with Special Reference to Growth Processes both Norma1 and Abnormal P. N . Campbell

305

The Newer Concept of Cancer Toxin Waro Nakahara and Fumiko Fukuoka Chemically Induced Tumors of Fowls P. R. Peacock Anemia in Cancer Vincent E . Price and Robert E. Greenfield Specific Tumor Antigens L. A. Zilber Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizabeth K. Weisburger and John H. Weisburger AUTHOR INDEX-SUB JECX INDEX

Volume 6

Blood Enzymes in Cancer and Other Diseases Oscar Bodansky The Plant Tumor Problem Annin C. Braun and H e n y N. Wood Cancer Chemotherapy by Perfusion Oscar Creech, Jr. and Edward T. Krementz Viral Etiology of Mouse Leukemia Ludwick Gross Radiation Chimeras P. C. Koller, A. J . S. Daoies, and Sheila M. A. Doak Etiology and Pathogenesis of Mouse Leukemia J . F. A. P. Miller Antagonists of Purine and Pyrimidine Metabolites and of Folic Acid G. M . Timmis Behavior of Liver Enzymes in Hepatocarcinogenesis George Weber AUTHOR INDEX-SUB JECr INDEX

Volume 7

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

306

CONTENTS OF PREVIOUS VOLUMES

Mechanisms of Resistance to Anticancer Agents R. W. Brockman Cross Resistance and Collateral Sensitivity Studies in Cancer Chemotherapy Dorris 1. Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W. M. Court Brown and lshbel M. Tough E thionine 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. Negroni AUTHOH INDEX-SUB J ECT INDEX

Volume

8

The Structure of Tumor Viruses and Its Bearing on Their Relation to Viruses in General A. F. Howatson Suclear Proteins of Neoplastic Celis Harris Busch and William J. Steele Nucleolar Chromosomes: Structures, Interactions, and Perspectives M. J. Kopac and Gladys M. Mateyko 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 Hofman AUTHOR ISDEX-SUB JECT INDEX

Volume

9

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

The Relation of the Immune Reaction to Cancer Louis V. Caso Amino Acid Transport in Tumor Cells R. M. johnstone and P. G. Scholefield 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

Volume 10

Carcinogens, Enzyme Induction, and Gene Action H. V. Gelboin In Vitro Studies on Protein Synthesis by Malignant Cells A. Clark Griffin The Enzymatic Pattern of Neoplastic Tissue W. Eugene Knox Carcinogenic Nitroso Compounds P. N. Magee and J. M. Barnes The Sulfhydryl Croup and Carcinogenesis J. S. Hasrington The Treatment of Plasma Cell Myeloma Daniel E. Bergsagel, K. M. Grifith, A. Haut, and W. J. Stuckley, Is. AUTHOR INDEX-SUB JECT INDEX

Volume 1 1

The Carcinogenic Action and Metabolism of Urethan and N-Hydroxyurethan Sidney S . Miruish Runting Syndromes, Autoimmunity, and Neoplasia D. Ke& Viral-Induced Enzymes and the Problem of Viral Oncogenesis Saul Kit

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 William Regelson Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Joseph C . Arcos and Mary F . Argus AUTHOR INDEX-SUB JECT INDEX CUMULATIVE INDEX

Volume 12

Antigens Induced by the Mouse Leukemia Viruses G . Pastemak Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses G . I . Deichman Replication of Oncogenic Viruses in Virus-Induced Tumor Cells-Their Persistence and Interaction with Other Viruses H . Hanafusa Cellular Immunity against Tumor Antigens Karl Erik Hellstrom and lngegerd Hellstrom Perspectives in the Epidemiology of Leukemia Irving L. Kessler and Abraham M. Lilienfeld AUTHOR INDEX-SUB JECT INDEX

Volume 13

The Role of Immunoblasts in Host Resistance and Immunotherapy of Primary Sarcomata P . Alexander and J. G . Hall Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Oswald Jarrett

307

The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Haim Ginsburg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gajanan V . Sherbet The Characteristics of Animal Cells Transformed in Vitro Ian Macpherson Role of Cell Association in Virus Infection and Virus Rescue I. Svobodu and I . HktZcinek Cancer of the Urinary Tract D. B. Clayson and E . H . Cooper Aspects of the EB Virus M . A. Epstein AUTHOR INDEX-SUB JECT INDEX

Volume 14

Active Immunotherapy Georges Mathk The Investigation of Oncogenic Viral Genomes in Transformed Cells by Nucleic Acid Hybridization Ernest Winocour Viral Genome and Oncogenic Transformation: Nuclear and Plasma Membrane Events Georges Meyer Passive Immunotherapy of Leukemia and Other Cancer Roland Motta Humoral Regulators in the Development and Progression of Leukemia Donald Metcalf Complement and Tumor Immunology Kusuya Nishioka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G . 1. Abeler Low Dose Radiation Cancers in Man Alice Stewart AUTHOR INDEX-SUB JECT INDEX

308

CONTENTS OF PREVIOUS VOLUMES

Volume 15

1,3-Bis( 2-chloroethyl)-l-nitrosourea (BCNU ) and Other Nitrosoureas in Cancer Treatment: A Review Stephen K . Carter, Frank M. Schabd, Jr., Lawrence E. Broder, and Thomas P. Johnston

Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J. S. Butel, S. S. Tevethia, and 1. L. Melnick AUTHOR INDEX-SUB JECT INDEX Nasopharyngeal Carcinoma ( N P C ) I . H . C. Ho Transcriptional Regulation in Eukaryotic Volume 17 Cells A. J. MacGillivray, J. Paul, and G. Polysaccharides in Cancer : Glycoproteins and Glycolipids Threlfall Viiai N . Nigam and Antonio Cantero Atypical Transfer RNA's and Their OriSome Aspects of the Epidemiology and gin in Neoplastic Cells Etiology of Esophageal Cancer with Ernest Borek and Sylvia I . Kerr Particular Emphasis on the Transkei, Use of Genetic Markers to Study Cellular South Africa Origin and Development of Tumors Gerald P. Warwick and John S. H&in Human Females ington Philip J . Fidkow Genetic Control of Murine Viral LeuElectron Spin Resonance Studies of Carkemogenesis cinogenesis Frank Lilly and Theodore Pincus Harold M . Swartz Some Biochemical Aspects of the Rela- Marek's Disease: A Neoplastic Disease of Chickens Caused by a Herpestionship between the Tumor and the virus Host K. Nazerian V . S. Shapot Mutation and Human Cancer Nuclear Proteins and the Cell Cycle Alfred G. Knudson, Jr. G a y Stein and Renato Baserga Mammary Neoplasia in Mice AUTHOR INDEX-SUB JECT INDEX S. Nandi and Charles M . McGrath AUTHOR INDEX-SUB JECT INDEX

Volume 16

Polysaccharides in Cancer Viiai N . Nigam and Antonio Cantero Antitumor Effects of Interferon Ion Gresser Transformation by Polyoma Virus and Simian Virus 40 Joe Sambrook Molecular Repair, Wound Healing, and Carcinogenesis : Tumor Production a Possible Overhealing? Sir Alexander Haddow The Expression of Normal Histocompatibility Antigens in Tumor Cells Alena Lengerood

Volume 18

Immunological Aspects of Chemical Carcinogenesis R. W . Baldwin Isozymes and Cancer Fanny Schapira Physiological and Biochemical Reviews of Sex Differences and Carcinogenesis with Particular Reference to the Liver Yee Chu Toh Immunodeficiency and Cancer John H. Kersey, Beatrice D. Spector, and Robeft A. Good

CONTENTS OF PREVIOUS VOLUMES

Recent Observations Related to the Chemotherapy and Immunology of Gestational Choriocarcinoma K. D. Bagshawe Glycolipids of Tumor Cell Membrane . Sen-itiroh Hakomori Chemical Oncogenesis in Culture Charles Heidelberger AUTHOR INDEX-SUB JECT INDEX

Volume 19

Comparative Aspects of Mammary Tumors 3. M . Hamilton The Cellular and Molecular Biology of RNA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses, and Their Relatives Howard M . Temin Cancer, Differentiation, and Embryonic Antigens: Some Central Problems 1. H. Coggin, Jr. and N . G. Anderson Simian Herpesviruses and Neoplasia Fredrich W . Deinhardt, Lawrence A. Falk, and Lauren G. Wolfe Cell-Mediated Immunity to Tumor Cells Ronald B. Herberrnun Herpesviruses and Cancer Fred Rapp Cyclic AMP and the Transformation of Fibroblasts Ira Pastan and George 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 Principles of Immunological Tolerance and Immunocyte Receptor Blockade G. 3. V. Nossal

309

The Role of Macrophages in Defense against Neoplastic Disease Michael H. Levy and E. Frederick Wheelock Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis P. Sims and P. L. Grover Virion and Tumor Cell Antigens of C-Type RNA Tumor Viruses Heinz Bauer Addendum to “Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing?” Sir Alexander Haddow SUBJECT INDEX

Volume 21

Lung Tumors in Mice: Application to Carcinogenesis Bioassay Michael B. Shimkin and Gary D. Stoner Cell Death in Normal and Malignant Tissues E. H. Cooper, A. J. Bedford, and T. E. Kenny The Histocompatibility-Linked Immune Response Genes Baruj Benucerraf and David H. Katz Horizontally and Vertically Transmitted Oncomaviruses of Cats M . Essex Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms Keen A. Raferty, 3r. Selection of Biochemically Variant, in Some Cases Mutant, Mammalian Cells in Culture G. B. Clements The Role of DNA Repair and Somatic Mutation in Carcinogenesis lames E. Trosko and Ernest H. Y. Chu SUBJECT INDEX

310

CONTENTS OF PREVIOUS VOLUMES

Volume 22

Renal Carcinogenesis 1. M. Hamilton Toxicity of AntineoplasticAgents in Man: Chromosomal Aberrations, Antifertility Effects, Congenital Malformations, and Carcinogenic Potential Susan M . Sieber and Richard H . Adumson

Interrelationships among RNA Tumor Viruses and Host Cells Raymond V. GUden Proteolytic Enzymes, Cell Surface Changes, and Viral Transformation Richard Roblin, lih-Nan Chou, and Paul H . Black Imrnunodepression and Malignancy Osias Stutmn SUBJECT INDEX

A 8 C D E F G H

6 7 B 9 O 1 2 3

1 4

J 5