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ADVANCES IN CANCER RESEARCH VOLUME 15

Contributors to This Volume

L.

Renato Baserga

J.

Ernest Borek

J. Paul

J. S. Butel

V. S. Shapot

Philip J. Fialkow

Gary Stein

J. H. C. Ho

Harold M. Swartz

Sylvia J. Kerr

S. S. Tevethia

A. J. MacGillivray

G. Threlfall

Melnick

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet 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 75

(29

ACADEMIC PRESS

New York and London

1972

COPYRIGHT 8 1972, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRl"EN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS INC. 111 Fifth Avenue,

New qork, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD.

24/28 Oval Road, London NW17DD

Lmmy

OF

CONORESS CATALOO CARDNUMBER:52-13360

PRINTED IN THE UNlTED STATES OF AMERICA

CONTENTS CONTRIBUTORS TO VOLUME15 . CONTENTSOF PREVIOUS VOLUMES

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ix xi

Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of The Viral Genome

J . S. BUTEL.S. S . TEVETHIA. AND J . L . MELNICK I. Introduction . . . . . . . . . . . . 11. Oncogenic Potential of SV40 in Vivo . . . . .

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57 57 80 88 89

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93 101 124 146 150

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163 168

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I11. Transformation of Mammalian Cells in Vitro by SV40 IV . Phenotypic (Antigenic) Changes in Tumor and Transformed Cells V . Genotypic Changes in Tumor and Transformed Cells . . . VI . Conclusions and Summary . . . . . . . . . . References . . . . . . . . . . . . . .

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Nasopharyngeal Carcinoma (NPC)

J . H . C . Ho I . Introduction I1. Histogenesis I11. Etiology . IV . Conclusion References

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Transcriptional Regulation in Eukaryotic Cells

A . J . MACGILLIVRAY. J . PAUL. AND G . THRELFALL

I. Introduction . . . . . . . . . . . . I1. Eukaryotic Chromosomes . . . . . . . . I11. Control of Transcription in Eukaryotic Cells . . . . IV . Theories of Transcriptional Regulation in Eukaryotic Cells References . . . . . . . . . . . .

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Atypical Transfer RNA’s and Their Origin in Neoplastic Cells

ERNESTBOREKAND SYLVIAJ . KERR

I . Introduction . . . . . . . I1. The tRNA Methylases of Tumor Tissues V

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vi

CONTENTS

I11. Transfer RNA of Tumor Tissues . . . . . . . IV . Regulation of tRNA Methylase Activity . . . . . V . Elevated Excretion of Modified Purines and Pyrimidines in Tumor-Bearing Animals and Humans . . . . . VI . The tRNA Methylases in Reverted Oncogenic Systems . . . . . . . . VII . An Attempt a t Interpretation References . . . . . . . . . . . .

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172 178

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180 184 185 187

Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females

PHILIP J . FIALKOW I . Introduction . . . . . . . . . . . . . I1. Interpretation of G-6-PD Phenotypes in Tumors . . . . I11. Hematopoietic Neoplasms . . . . . . . . . IV . Carcinomas . . . . . . . . . . . . . V . Benign Tumors VI . Study of Genetic Markers in Established Tieaue Culture Lines VII . Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . .

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191 195 199 209 213 219 221 223

Electron Spin Resonance Studies of Carcinogenesis

HAROLD M . SWARTZ I . Introduction . . . . I1. The ESR Technique . . 111. Experimental Results . IV . Summary and Conclusions References . . . .

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227 228 233 248 248

Some Biochemical Aspects of the Relationship between the Tumor and the Host

V . S. SHAPOT I . Introduction . . . . . . . . . . . . . . I1. Respiration of Tumors in Vivo . . . . . . . . . I11. Glucose Levels in Ascites Cancer Cells and Their Medium in Vivo IV . Glycolysis and Destructive Tumor Growth . . . . . . V . Relative Glucose Deficiency of the Tumors Growing in the Body VI . Tumor as a Glucose Trap in the Body . . . . . . . VII . The Problem of Cancer Cachexia . . . . . . . . VIII . Conclusion . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

253 254 257 262

264 268 279 281 282

vii

CONTENTS

Nuclear Proteins and the Cell Cycle

GARYSTEINAND RENATO BASERGA

I . Introduction . . . . . . I1. The Biochemistry of the Cell Cycle I11. The Control of Cell Proliferation . References . . . . . . AUTHOR INDEX. SUBJECTINDEX

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CONTRIBUTORS TO VOLUME 15 Numben in parentheses refer to the pages on which the authors’ contributions begin.

RDNATO BASERGA, Department of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania (287)

ERNEST BOREK, Departments of Microbiology and Surgery, University of Colorado Medical Center, Denver, Colorado (163)

J. S. BUTEL,Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas (1) PHILIPJ. FIALKOW, Departments of Medicine and Genetics, University of Washington, Seattle, Washington (191)

J. H. C . Ho, Medical and Health Department Institute of Radiology, Queen Elizabeth Hospital, Kowloon, Hong Kong (57)

SYLVIA J. KERR,Departments of Microbiology and Surgery, University of Colorado Medical Center, Denver, Colorado (163) A. J. MACGILLIVRAY, Beatson Institute for Cancer Research, Glasgow, Scotland (93) J. L. MELNICK,Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas (1) J. PAUL, Beatson Institute for Cancer Research, Glasgow, Scotland (93) V. S. SHAPOT, Institute of Experimental Clinical Oncology, Academy of Medical Sciences, MOSCOW, USSR (253)

GARYSTEIN,Department of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania (287)

HAROLD M. SWARTZ, Departments of Radiology and Biochemistry, The Medical College of Wisconsin, Milwaukee, Wisconsin (227) S. S. TEVETHIA, Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas (1)

G. THRELFALL, Beatson Institute for Cancer Research, Glasgow, Scotland (93) ix

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CONTENTS OF PREVIOUS VOLUMES Volume 1

Carcinogenesis and Tumor Pathogenesis I. Berenblum Ionizing Radiations and Cancer Austin M . Brues Survival and Preservation of Tumors in the Frozen State James Craigie Energy and Nitrogen Metabolism in Cancer Leonard D. Fenninger and G. Burroughs Mider Some Aspects of the Clinical Use of Nitrogen Mustards Calvin T. Klopp and Jeanne C. Bateman Genetic Studies in Experimental Cancer

Electronic Configuration and Carchogenesis C. A . Coulson Epidermal Carcinogenesis E. V . Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice L. Dmochowski Hormonal Aspects of Experimental Tumorigenesis T . U. Gardner Properties of the Agent of b u s No. 1 Sarcoma R. J . C . Harris L. W . Law Applications of Radioisotopes to Studies of Carcinogenesis and Tumor The Role of Viruses in the Production Metabolism of 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-SUBJECT INDEX The Chemistry of Cytotoxic Alkylating Agents M. C. J . Ross Volume 3 Nutrition in Relation to Cancer Etiology of Lung Cancer Albert Tannenbaum and Herbert Richard Doll SilVeTStO?W The Experimental Development and Plasma Proteins in Cancer Metabolism of Thyroid Gland Richard J . Winzler Tumors AUTHOR INDEX-SUBJECT INDEX Harold P. Morris Electronic Structure and Carcinogenic Activity and Aromatic Molecules: Volume 2 New Developments A. Pullman and B . Pullman The Reactions of Carcinogens with Macromolecules Some Aspects of Carcinogenesis Peter Alexander P. Rondoni Chemical Constitution and Carcinogenic Pulmonary Tumors in Experimental Activity Animals Michael B. Shimkin G. M . Badger

xi

xii

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues Sidney Weinhowe AUTHOR INDEX-SUBJECT

Volume 4

INDEX

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 . Weis burger

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. Galton AUTHOR INDEX-SUBJECT INDEX The Employment of Methods of Inhibition Analysis in the Normal and Tumor-Bearing Mammalian OrganVolume 6 ism Abraham Goldin Blood Enzymes in Cancer and Other Diseases Some Recent Work on Tumor Immunity P. A. Gorer Oscar Bodansky Inductive Tissue Interaction in Develop- The Plant Tumor Problem ment Armin C. Braun and Henry N. Wood Clifford Grobstein Cancer Chemotherapy by Perfusion Lipids in Cancer Oscar Creech, Jr., and Edward T . Frances L. Haven and W . R. Bloor Krementz The Relation between Carcinogenic Viral Etiology of Mouse Leukemia Activity and the Physical and Ludwik Gross Chemical Properties of Angular Radiation Chimeras Benzacridines P. C. Koller, A. J . S. Dauies, and A. Lacassagne, N . P. Buu-Hoi, R . Sheila M . A . Doak Daudel, and F . Zajdela Etiology and Pathogenesis of Mouse The Hormonal Genesis of Mammary Leukemia Cancer J . F . A. P. Miller 0. Miihlbock Antagonists of Purine and Pyrimidine AUTHOR INDEX-SUBJECT INDEX Metabolites and of Folic Acid G. M . Timmis Behavior of Liver Enzymes in HepatoVolume 5 carcinogenesis George Weber Tumor-Host Relations AUTHOR INDEX-SUBJECT INDEX R. W . Begg Primary Carcinoma of the Liver Charles Berman Volume 7 Protein Synthesis with Special Reference to Growth Processes both Normal Avian Virus Growths and Their Etiologic Agents and Abnormal J . W . Beard P . N . Campbell

CONTENTS OF PREVIOUS VOLUMES

...

Xlll

Mechanisms of Resistance to Anticancer The Relation of the Immune Reaction to Cancer Agents Louis Caso R. W . Brockman Cross Resistance and Collateral Sensi- Amino Acid Transport in Tumor Cells R. M. Johnstone and P. G. Scholefield tivity Studies in Cancer Chemotherapy Studies on the Development, BiochemDorris J . Hutchison istry, and Biology of Experimental Hepatomas Cytogenic Studies in Chronic Myeloid Harold P. Morris Leukemia W. M . Court Brown and Ishbel M . Biochemistry of Normal and Leukemic Tough Leucocytes, Thrombocytes, and Bone Marrow Cells Ethionine Carcinogenesk I . F. Seitz Emmanuel Farber Atmospheric Factors in Pathogenesis of AUTHOR INDEX-SUBJECT INDEX Lung Cancer Paul Kotin and Hans L. Falk Progress with Some Tumor Viruses of Volume 10 Chickens and Mammals: The Prob- Carcinogens, Enzyme Induction, and lem of Passenger Viruses Gene Action G. Negroni H . V. Gelboin AUTHOR INDEX-SUBJECT INDEX I n Vitro Studies on Protein Synthesis by Malignant Cells A. Clark Griffin Volume 8 The Enzymatic Pattern of Neoplastic The Structure of Tumor Viruses and Its Tissue Bearing on Their Relation to Viruses W . Eugene Knox in General Carcinogenic Nitroso Compounds A. F. Howatson P. N . Magee and J . M . Barnes Nuclear Proteins of Neoplastic Cells The Sulfhydryl Group and CarcinoHarris Busch and William J . Steele genesis Nucleolar Chromosomes : Structures, J . S. Harington Interactions, and Perspectives The Treatment of Plasma Cell Myeloma M . J. Kopac and Gladys M . Mateyko Daniel E. Bergsagel, K . M . Grifith, Carcinogenesis Related to Foods ConA. Haut, and W . J. Stuckey, Jr. taminated by Processing and Fungal AUTHOR INDEX-SUBJECT INDEX Metabolites H . F. Kraybill and M. B. Shimkin Experimental Tobacco Carcinogenesis Volume 1 1 Ernest L. Wynder and Dietrich Hoffmann The Carcinogenic Action and Metabolism of Urethan and N-Hydroxyurethan AUTHOR INDEX4UBJECT INDEX Sidney S. Mirvish Runting Syndromes, Autoimmunity, and Volume 9 Neoplasia D. Keast Urinary Enzymes and Their Diagnostic Viral-Induced Enzymes and the Problem Value in Human Cancer of Viral Oncogenesis Richard Stambaugh and Sidney WeinSaul Kit house

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xiv

CONTENTS O F 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-SUBJECT

INDEX

CUMULATIVE INDEX

Volume 12

Antigens Induced by the Mouse Leukemia Viruses G. Pasternak 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 . Hannfusa Cellular Immunity against Tumor An tigens Karl Erik Hellstrom and Ingegerd Hellstrom Perspectives in the Epidemiology of Leukemia Irving I . Kessler and Abraham M . Lilienfeld AUTHOR INDEX-SUBJECT

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

The Function of the Dclayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Haim Ginsburg Epigenetic Processcs and Their Relevance to the Study of Neoplasia Gnjanan V . Sherbet The Characteristics of Animal Cells Transformed in Vitro Inn Macpherson Role of Cell Association in Virus Infection and Virus Rescue J . Svoboda and I . Hloz'cinek Cancer of the Urinary Tract D. B . Clayson and E . H . Cooper Aspects of the EB Virus M . A. Epstein AUTHOR INDEX-SUBJECT

INDEX

Volume 14

Active Immunotherapy Georges Math6 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 M e y e r Passive Immunotherapy of Leukemia and Other Cancer Roland M o tta Humoral Regulators in the Development and Progression of Leukemia Donald Metcalj Complement and Tumor Immunology Kusuya Nishwka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G . I. Abeler Low Dose Radiation Cancers in Man Alice Stewart AUTHOR INDEX-SUBJECT

INDEX

ONCOGENICITY AND CELL TRANSFORMATION BY PAPOVAVIRUS SV40: THE ROLE OF THE VIRAL GENOME1 J. S. Butel, S. S. Tevethia,' and J. L. Melnick Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas

I. Introduction . . . . . . 11. Oncogenic Potential of SV40 in Vivo

. . . . . . . . . . . . . . . A. Role of the Host Animal . . . . . . . . B. Role of Defective Viral Genomes . . . . . . . C. Factors Affecting Viral Oncogenicity . . . . . . . 111. Transformation of Mammalian Cells in Vitro by SV40 . . . . A. Transformation of Permissive and Nonpermissive Cells by SV40 B. Transfocmation by Defective SV40 . . . . . . . C. Double Transformation of Cclls . . . . . . . . D. Reversion of Transformed Cells . . . . . . . . IV. Phenotypic (Antigenic) Changes in Tumor and Transformed Cells . . . . . . . . . . . . . A. Tumor Antigen B. Tumor-Specific Transplantation Antigen (TSTA) . . . . C. Surface Antigen . . . . . . . . . . . . D. Relationship of S-Antigen to TSTA . . . . . . . V. Genotypic Changes in Tumor and Transformed Cells . . . . A. Rescue of Infectious Virus from Transformed Cells . . . B. State of the Viral Genome in Transformed Cells . . . . C. Transcription of the Viral Genome in Transformed Cells . . . D. Basis for Lack of Virus Production by Transformed Cells . . . . .

E. Consideration of Whether the Viral Genome Is Essential for Maintenance of Transformation . . . . . VI. Conclusions and Summary References . . . . . . . .

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I. Introduction

Simian virus 40 (SV40) has undergone intensive study by many investigators in the decade following its discovery (Sweet and Hilleman, 1960). It is one of the most, if not the most, well-understood model tumor virus containing deoxyribonucleic acid (DNA). 'Supported in part by research grant CA 04600 from the National Cancer Institute and research contract PH 43-68-678 within the Special Virus-Cancer Program of the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. * Recipient of Research Career Development Award 5-K3-CA 38,614 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. 1

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A number of attributes have encouraged the selection of SV40 as the viral agent for so many studies: it can be readily propagated and accurately assayed in tissue culture; it can transform cells in witro as well as induce tumors in wivo; i t has a limited content of genetic information; and its nucleic acid can be isolated in an infectious form. It is a member of the papovavirus group (Melnick, 1962) and the family Papotraviridae (International Committee on Nomenclature of Viruses, 1971). Several recent reviews have covered different aspects of SV40 tumorigenesis (Rapp and Melnick, 1966; Black, 1968; Deichman, 1969; Rapp, 1967, 1969), so this chapter will not attempt to recapitulate all the recorded facts about the virus. Rather, we have posed questions pertinent to the phenomenon of oncogenesis and surveyed the literature for possible answers. The subjects considered, among others, are: ( a ) the role of complete and defective viral genomes in oncogenesis and transformation; ( b ) the antigenic changes which accompany transformation and their relationship to each other; (c) the virogenic and genotypic changes in transformed cells; ( d ) the failure to recover virus from tumor cells; ( e ) the requirement for a persisting viral genome to ensure the maintenance of the transformed state, and (f) the factors which affect malignancy of transformed cells. Obviously, all the answers to the above questions are not yet known, but some intriguing clues have been obtained. We have attempted to evaluate critically the available knowledge of SV40 oncogenesis and to assess the implications for carcinogenesis by DNA-tumor viruses as a whole. I I . Oncogenic Potential of SV40 in Vivo

HOSTANIMAL The oncogenic potential of SV40 has been demonstrated only in hamsters (Eddy et al., 1962; Girardi et al., 1962; Ashkenazi and Melnick, 1963; Black and Rowe, 1964). Efforts to induce tumors by SV40 in mice, guinea pigs, rats, rabbits, and monkeys have been unsuccessful (Eddy, 1964). The latent period of tumor induction by SV40 in hamsters ranges from 3 months to more than a year, depending upon the concentration of virus employed and the age of the animal a t the time of inoculation. The incidence of tumors in animals inoculated as newborns is usually over go%, but it is variable in older animals. Girardi et al. (1963) reported that 23% of hamsters inoculated a t 1 month of age ultimately developed tumors with an average latent period of 360 days. In other studies, A. ROLE OF

THE

PAPOVAVIRUS

SV40

3

tumors failed to appear in hamsters inoculated at 3 weeks of age, although some hamsters inoculated a t 4 months of age developed tumors when held for long periods (Allison et al., 1967). Newborn hamsters are susceptible to the oncogenic effects of SV40 whether inoculated by the subcutaneous, intracerebral, intraperitoneal, or intrathoracic route. However, no tumors were observed when newborn hamsters were inoculated by the oral and intranasal routes (Eddy, 1964). Of the preceding methods of inoculation, the subcutaneous route is the one of choice since neoplasms are visible and can be scored soon after appearance. There seems to be some correlation between the concentration of the virus inoculum and the latent period for tumor development when animals are injected by the subcutaneous route (Eddy et al., 1962). DNA isolated from SV40-infected monkey cells is also oncogenic in these animals (Boiron et al., 1965). Histologically, SV40-induced tumors in subcutaneous tissues, lungs, and kidneys have been designated as undifferentiated sarcomas, although some fibrosarcomas have been reported (Eddy, 1964). Intracerebral inoculation of the virus into newborn hamsters resulted in the development of ependymomas (Kirschstein and Gerber, 1962). Induction of ependymomas by SV40 in mastomys has also been reported (Rabson et al., 1962) although no evidence was presented that the observed neoplasms were related to SV40. X-Irradiation of adult animals prior to virus inoculation enhances their susceptibility to tumor induction (Allison et al., 1967). Also, adult animals inoculated in the cheek pouch with SV40 were found to develop tumors after a latent period of only 97 days, as compared with a latent period of 490 days when animals of the same age were inoculated by the subcutaneous route. All tumors which developed were SV40 tumors based on the presence of SV40 transplantation antigen (Allison et al., 1967). The study indicated that potentially malignant cells can remain dormant in the animal for a long period of timc and that depression of the immune response of the host will result in the growth of such “dormant” cells. Tumor induction in mice by SV40 has not been demonstrated, even by the use of immunosuppressed animals (Tevethia, 1971). The virus can infect mouse cells rather easily, as shown by ( a ) transformation experiments in vitro using 3T3 mouse cells (Black and Rowe, 1963b; Todaro and Green, 1964) ; ( b ) the elevated enzyme levels and increased DNA synthesis following infection of mouse kidney cells (Kit et al., 1966b), and (c) the ability of the virus to induce SV40 specific transplantation immunity in mice (Kit e t al., 1969; Wesslh, 1970). It appears

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J. S. BUTEL, S. S. TEVETHIA, AND J. L. MELNICK

that a mouse cell may possess many of the characteristics of transformed cells after infection with SV40 and yet not be able to grow as a tumor cell.

B. ROLEOF DEFECTIVE VIRALGENOMES Viral defectiveness may be divided into two broad categories-conditional and nonconditional. Conditionally defective viruses are unable to complete the cycle of replication under certain conditions, such as in a nonpermissive host cell, in the absence of a helper virus, or at an elevated temperature [ temperature-sensitive (ts) mutants]. I n contrast, nonconditionally defective viruses are not able under any known conditions to complete a replicative cycle. In this section, the significance in oncogenesis is considered of both conditionally defective helper-dependent viruses as well as other viral preparations which may, or may not, be nonconditionally defective. The defective viruses may be conveniently subdivided into those which are produced by some exogenous treatment of the virus stock, such as irradiation, and those which occur naturally (Table I). TABLE I TYPES OF DEFECTIVE SV40 GENOMES SHOWN TO BE ONCOGENIC A. Produced by exogenous treatment 1. Irradiated virus 2. Hydroxylamine-inactivated virus B. Naturally occurring 1. T-antigen-inducing defective particles 2. PARA(defective SV40)-adenovirus hybrid population

Defendi and Jensen (1967) demonstrated that, after inactivation by ultraviolet or by gamma radiation, SV40 not only retained its oncogenicity for newborn hamsters, but actually exhibited an enhanced tumorproducing capability when compared to that of the untreated virus. Most of the virus in the irradiated samples had been inactivated as determined by infectivity assays. Similar results were reported by Altstein et al. (1967a) using hydroxylamine-inactivated virus. The authors postulated that their results could be explained if the oncogenic potential of SV40 is actually due to the presence of defective particles in virus stocks. After inactivation of the virus preparation, the concentration of these hypothetical defective particles would increase, and these, in turn, would be responsible for the observed enhancement in oncogenicity. However, defective virions may not be the only explanation for oncogenicity because of some other unexplained observations: ( a ) complete virus genomes can be recovered from some transformed cells (Section V,A), ( b ) nononcogenic variants of defective SV40

PAPOVAVIRUS

SV40

5

(PARA) have been described (see below) and (c) inactivated virus preparations that exhibit increased transforming capacity in vivo do not do so in vitro (Section 111,B). One other possible explanation offered for enhanced levels of oncogenicity is that tumors produced by defective viruses may lack tumor-specific transplantation antigens (TSTA) , the absence of which allows the tumors to grow without being influenced by the hosts’ immune mechanisms. However, no experimental evidence has been provided to support this claim. Naturally occurring SV40 particles which are defective for the synthesis of late viral proteins and infectious virus, but which are able to induce the synthesis of SV40 tumor (T) antigen, have been described (Sauer e t al., 1967; Altstein e t al., 196713; Uchida e t al., 1968). The concentration of defective particles in preparations of SV40 has been shown to increase after serial passage using undiluted inocula (Uchida et al., 1966) . The naturally occurring defective particles were oncogenic in newborn hamsters (Uchida and Watanabe, 1969). The tumors induced by the defective viruses contained SV40 T-antigen. A defective SV40 genome (PARA) carried by an unusual strain of human adenovirus type 7 has been described (Huebner et al., 1964; Rowe and Baum, 1964; Rapp e t al., 1 9 6 4 ~ )For . a current review of the properties of PARA-adenovirus populations, see Rapp (1971). The PARA genome is defective in that i t cannot code for SV40 coat protein; it is encased in adenovirus capsids supplied by helper adenovirions present in the mixed population (BoBye e t al., 1966; Butel and Rapp, 1966a; Rowe and Baum, 1965). PARA-adenovirus 7 produces SV40 type tumors in newborn hamsters (Huebner e t al., 1964; Rapp et al., 1966a), induces the synthesis of SV40 T-antigen in vitro (Rowe and Baum, 1964; Rapp e t at., 1 9 6 4 ~ )and ~ induces SV40 TSTA in weanling hamsters (Rapp et al., 1966b, 1967a). The discovery and subsequent characterization of PARA provided convincing evidence that late functions associated with the synthesis of capsid proteins and maturation of progeny virions are not required for the induction of SV40 T-antigen, TSTA, or oncogenicity. The tumors induced by the original PARA-adenovirus 7 population had latent periods similar to those induced by parental SV40, but the oncogenic potential vaned following “transcapsidation” (Rapp e t al., 196523) to a series of different human adenoviruses (Rapp et uZ., 1968). PARA particles which had been transcapsidated to adenovirus types 14, 16, and 21 were nononcogenic, whereas transcapsidant PARA-adenovirus population types 1, 2, 3, 5, and 6 were oncogenic in newborn hamsters. However, both the nononcogenic and oncogenic PARA-adenovirus populations were capable of inducing SV40-specific transplantation immunity in weanling animals. When PARA was transcapsidated from

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J . S. BUTEL, 6 . S. TEVETHIA, AND J . L. MELNICK

the nononcogenic adenovirus 21 population to adenovirus 6, it remained nononcogenic, whereas PARA from the oncogenic adenovirus 6 population retained its oncogenic potential upon transcapsidation to adenovirus type 21 (Rapp et al., 1970). Thcse results suggested that variants of PARA which differed in oncogenicity were present in the original population of PARA-adenovirus 7. Such variants were then isolated from the parental PARA-adenovirus 7 population by two successive plaque purifications in monkey kidney cells; 20 of the 112 cloned lines of virus tested were shown to be nononcogenic (Rapp et al., 1969). The nononcogenic variants of PARA were capable of transforming hamster cells in vitro (Butel et al., 1971c; Rapp and Duff, 1971) ; however, the transformed cell lines which were established varied in transplantability (Butel et al., 1 9 7 1 ~ ) . When PARA was transcapsidated from adenovirus type 7 to the highly oncogenic Huie strain of adenovirus type 12, it produced SV40 tumors in newborn hamsters with a short latent period of only 5-7 weeks (Butel et al., 1971a). All the early-appearing tumors induced by PARAadenovirus 12 contained SV40 T-antigen. Parental SV40 and PARAadenovirus 7 did not induce any tumors within the same time period. Artificial mixtures of adenovirus 12 and SV40 or adenovirus 12 and PARA-adenovirus 7 did not induce any tumors containing SV40 Tantigen within 5-7 weeks. Early SV40 tumors induced by PARAadenovirus 12 contained SV40 TSTA, ruling out the absence of antigen as the cause of the early appearance of the SV40 tumors. One possible explanation for the results with PARA-adenovirus 12 was that recombination had occurred between a portion of the adenovirus 7 region of the PARA genome and a portion of the adenovirus 12 genome. Such recombinant particles might contain the adenovirus 12 marker (s) responsible for the rapid appearance of tumors, and, in the process of tumor development, the linked SV40 information would be expressed. There is no direct proof yet available, however, that the postulated recombinational event actually occurred. Defective viruses can be very useful tools for determining viral functions necessary for oncogenicity. They can be replicated with the aid of a helper virus which, if it is nononcogenic, does not interfere with subsequent tests in vivo. With a conditionally defective virus which may have lost part of the parental genome, the system is not complicated by any possibility of leakiness or reversion on the part of the mutant. I n addition, strict conditions do not have to be imposed to ensure defectiveness as is the case, for example, with temperature-sensitive mutants. With the ts mutants, unfortunately, the body temperature of the intact animal required for oncogenicity studies is frequently that of the permissive temperature.

PAPOVAVIRUS

SV40

7

C. FACTORS AFFECTING VIRALONCOGENICITY As described above, if SV40 is inoculated into hamsters within 24 hours of birth, tumors will develop in a majority of the animals with latent periods ranging from 3 to 6 months. Without exception, all SV40induced tumors are antigenic and contain TSTA a t the cell surface (see Section IV,B). Polyoma virus can induce the development of tumors in certain strains of mice if inoculated into newborns. However, C57B1, strain A, and C3H/Lw mice are resistant t o polyoma virus oncogenesis (Law, 1969). If the resistant strains were made immunologically deficient by thymectomy a t birth, they became susceptible to polyoma virus tumor induction. Similar results were obtained when the resistant strains were treated with antilymphocytic serum. The immunologic capacity of the thymectomized mice and the resistance to polyoma virus oncogenesis could be restored by intravenous injection of syngeneic spleen or lymph node cells from intact animals, The role of immunocompetence in resistance to SV40 oncogenesis in hamsters was demonstrated by Allison et al. (1967))when they showed that tumor development in hamsters was dependent upon the age of the animal at the time of virus inoculation. SV40 produced tumors in adult animals only after X-irradiation of the host. Tumors also developed when virus was inoculated into the cheek pouch of animals (an immunologically privileged site). These studies suggested the following sequence of events upon inoculation of the virus into an adult animal: ( 1 ) cells are transformed, ( 2 ) TSTA a t the transformed cell surface sensitizes the host, and (3) potential tumor cells are eliminated. This sequence of events was first postulated by Habel (1962) and is supported . thymocyte by data in the report by Tevethia et al. ( 1 9 6 8 ~ )Antihamster serum, a potent suppressor of cellular immunity, prevented the sensitization of animals to SV40 TSTA when administered to hamsters during the period of virus immunization. The failure to become sensitized to TSTA was demonstrated by the fact that treated animals were unable to reject a challenge of SV40-transformed cells. In addition, spontaneous regression of primary tumors induced by SV40 in hamsters has been observed (Tevethia et al., 1968a; Deichman, 1969). The appearance of primary tumors in hamsters inoculated as newborns requires discussion in view of the fact that SV40 tumors have long latent periods and contain TSTA. The development of immunologic tolerance to SV40 TSTA can be ruled out, since SV40 oncogenesis can be blocked by immunization of the animals during the latent period with either virus or virus-transformed cells (Goldner et al., 1964; Girardi, 1965; Tevethia et al., 1968b). Such immunization, however, is ineffective

8

J . S. BUTEL, S. 8 . TEVETHIA, AND J . L. MELNICK

in thymectomized animals (Girardi and Roosa, 1967). Hamsters undergoing viral oncogenesis do not become sensitized to SV40 TSTA before the appearance of palpable tumors (Deichman and Kluchareva, 1964). However, animals bearing tumors become sensitized and can reject a challenge of SV40-transformed cells at a site distant from the primary tumor (Lausch and Rapp, 1971), which may explain the absence of widespread metastases in tumor-bearing animals. There are several possible mechanisms to explain the growth of primary SV40 tumors: 1. A transformed focus in a newborn animal may be established before development of immunocompetence. If the transformed focus reaches a certain critical size, it may sensitize the host without being rejected. However, this mechanism is unlikely since SV40 tumors have long latent periods (100-200 days) and the animals become immunocompetent long before the appearance of palpable tumors (Friedman and Goldner, 1970a). Also, infection of animals with SV40 does not lead to generalized immunosuppression (Friedman and Goldner, 1970b). 2. The amount of TSTA present in the developing focus of transformed cells might not be enough to sensitize the host. By the time the concentration of TSTA becomes sufficiently high to sensitize the host, the tumor might already have grown to a size such that it cannot be rejected by the immune lymphocytes. I n support of this, Deichman and Kluchareva (1964) demonstrated that hamsters inoculated with SV40 as newborns were as susceptible to challenge with SV40-transformed cells as control animals. The fact that SV40 oncogenesis can be prevented by immunization of hamsters during the latent period with either SV40 or transf,ormed cells indicates a block a t the efferent level of the immunological arc. Insufficient TSTA to sensitize the animals undergoing viral oncogenesis can be ruled out since only a single inoculation of virus during the latent period is enough to prevent tumor appearance. Deichman (1969) advanced a hypothesis to explain the difference between newborn and adult animals with respect to the development of transformed foci. She proposed that the development of TSTA in virusinfected cells and the transformation of the cells are separate functions. She postulated that TSTA is synthesized in cells of both newborn and adult animals infected with SV40. In newborn animals, however, TSTA would disappear from most of the cells perhaps because of the fast rate of cell division; the number of cells which ultimately transform would not be large enough to sensitize the host. In contrast, in adult animals TSTA would persist long enough to sensitize the host. I n support of this hypothesis, Kluchareva et al. (1967) showed that hamster cells infected

PAPOVAVIRUS

SV40

9

with SV40 in vitro developed SV40 TSTA which could immunize a host. 3. Blocking antibody (K. E. Hellstrom and Hellstrom, 1970) may be responsible for the growth of primary SV40 tumors. Such antibody molecules bind to TSTA and protect the antigenic sites from the action of immune lymphocytes. The possibility that blocking antibodies are responsible for the growth of primary tumors before they become palpable is unlikely, for the following reasons: ( a ) Blocking antibodies have been demonstrated only in tumor-bearing animals and cannot be detected before the development of the tumor or after the tumor is surgically removed. ( b ) Animals undergoing SV40 oncogenesis behave as relatively nonsensitized animals before the appearance of palpable tumors. Blocking antibodies may play an important role, however, in guarding the tumor cells from the action of immune lymphocytes once the animal has been sensitized. 4. The failure to demonstrate immunity to SV40 TSTA in hamsters undergoing SV40 oncogenesis may be explained by the immobilization of the immunoblasts in the lymph nodes draining the tumor site (Alexander et al., 1969). The release of the immunoblasts was achieved either by surgical removal of the tumor or by immunization of the host with tumor cells. These results may explain why SV40 oncogenesis can be prevented by immunization of the host during the latent period. Although it is well established that SV40 can induce oncogenesis in v i m , the role of the viral genome in the acquisition of malignant potential by cells transformed in vitro is not known. Transformed hamster cells can usually be readily transplanted in adult animals. I n contrast, mouse cells transformed by SV40 can be transplanted in syngeneic animals only with great difficulty, frequently requiring depression of the immune response of the host (Takemoto et al., 1968a). Prolonged cultivation in vitro of the transformed cells is also often necessary before a successful transplant in vivo can be achieved (Kit et al., 1969; Wesslkn, 1970). Enders and Diamandopoulos (1969) observed that hamster heart cells transformed by SV40 became highly transplantable after passage in vivo. Sixteen clones were isolated from the original population of transformed cells and a wide variation was found in the transplantability of the clonal lines. Some clones were highly transplantable whereas others failed to produce tumors unless more than a million cells were inoculated per animal. When the lines of apparent low transplantability were passaged in vivo, the oncogenic potential of the cells increased markedly. These studies indicated that cells of high oncogenicity may be selected in viva and that cellular mutations may be responsible for the increase in oncogenic potential. The authors concluded that the viral genome did

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J . S. BUTEL, S. S . TEVETHIA, AND J . L. MELNICK

not appear to be responsible for the increase in transplantability of the transformed cells. Variation in the transplantability of hamster lung and kidney cells transformed by various PARA-adenovirus hybrid populations has been observed by Butel et al. ( 1 9 7 1 ~ ) Virus . clones which appeared to be nononcogenic in newborn hamsters were able to transform hamster cells in vitro, but the transplantability in weanling hamsters of early passages of the transformed cells varied greatly. Some of the cell lines were readily transplantable whereas other lines transformed by the same clone of virus were either not transplantable or could be transplanted only with difficulty. The mechanism responsible for the observed differences in transplantability of transformed cells is not known. However, immunological factors which may operate a t either the level of the host or that of the tumor cell may be important. All cells transformed by SV40 contain TSTA which mediates the development of specific resistance against transplantation of the transformed cells. Most lines of SV40-transformed mouse cells are not transplantable in the syngeneic host, but can be transplanted in immunosuppressed animals (Takemoto et al., 1968a; Tevethia, 1971) indicating the role of immunological surveillance on the part of the host. This hypothesis is supported by the observation that an animal bearing a progressively growing SV40-transformed cell transplant is immune to a second transplant of the same cells a t a distant site. This concomitant immunity is viral-specific (Lausch and Rapp, 1971). However, SV40-transformed cells can become immunoresistant and are then capable of growing in immune animals. Deichman and Kluchareva (1966) reported the loss of SV40 TSTA from cells taken from metastatic tumors. SV40-immunized animals were unable to reject tumor cells derived from lung metastases. However, these cells were not tested for their capacity to immunize hamsters against a challenge of an immunosensitive cell line to definitely prove they lacked SV40 TSTA. There is the possibility that the cells from the lung metastases were simply immunoresistant in view of the fact that Tevethia et al. (1971) have isolated immunoresistant variants from a population of SV40-transformed cells. These variants are not rejected by SV4O-immunized animals but can immunize hamsters against a challenge of immunosensitive tumor cells, showing that the immunoresistant cells do contain TSTA. Major factors that may contribute to immunorcsistance include (a) a faster rate of cell growth in v i m , ( b ) lower concentrations of TSTA, ( c ) masking of TSTA by mucopolysaccharides, and ( d ) interference by blocking antibodies. Beyond these reports, which suggest that immunological factors may

PAPOVAVIRUS

SV40

11

play a role, very little is kn0w.n about the event(s) which determine the malignancy of a transformed cell. One factor which may affect the transplantability of transformed cells may be surface changes other than SV40 TSTA. These changes may be reflected in either increased or decreased transplantability of tumor or transformed cells. Deichman and Kluchareva (1966) reported that SV4O-transformed cells, when grown in vitro in the presence of sera from SV40 tumor-bearing animals, became immunoresistant. This change was not observed when the cells were grown in the presence of sera from hamsters which had rejected SV40 tumors or from animals bearing polyoma tumors. These findings strongly suggest the presence of antigens which can act as TSTA but are capable of “modulation.” Conclusive evidence favoring this hypothesis is still lacking. Ill. Transformation of Mammalian Cells in Vifro by SV40

Viral transformation has been defined as a n induced inheritable change in the properties of a cell, accompanied by the loss of regulatory controls of cell growth. The criteria for transformation of cells generally include the following (Enders, 1965) : (a) loss of contact inhibition, ( b ) altered morphology, (c) increased growth rate, ( d ) increased capacity to persist in serial subcultures, ( e ) chromosomal abnormalities, ( f ) increased resistance to reinfection by the transforming virus, (9) emergence of new antigens, and ( h ) capacity to form neoplasms. Black (1968) recently summarized the details of various SV40-host cell transformation systems. Consequently, only certain features of transformation by SV40 will be emphasized a t this time to provide a background for subsequent sections of the chapter.

A. TRANSFORMATION OF PERMISSIVE AND NONPERMISSIVE CELLSBY SV40 As discussed above, SV40 is maximally oncogenic in vivo in newborn hamsters. The narrow host range of the virus has been widely extended by in vitro transformation experiments. In addition to transformation of hamster cells in culture (Black and Rowe, 1963a; Rabson and Kirschstein, 1962; Ashkenazi and Melnick, 1963; Shein e t al., 1963), it has also been reported that SV40 can transform cells of human (Koprowski et al., 1962; Ashkenazi and Melnick, 1963; Shein and Enders, 1962), mouse (Black and Rowe, 1963b; Todaro and Green, 1964; Kit et al., 1966b), rabbit (Black and Rowe, 1963b), rat (Diderholm et al., 1966), bovine (Diderholm et al., 1965), guinea pig (Diderholm et al., 1966), and monkey (Fernandes and Moorhead, 1965; Koprowski et al., 1967; Wallace, 1967; Margalith et al., 1969; Shiroki and Shimojo, 1970) origin.

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J. S. BUTEL, S . 6 . TEVETHIA, A N D J. L . MELNICK

I n the hamster species, which is the most thoroughly studied transformation system for SV40, cells from a variety of organs and tissues have been shown to be susceptible to virus-induced transformation. Types of cells which have reportedly been transformed by SV40 include the following: ( a ) kidney (Black and Rowe, 1963a; Rabson and Kirschstein, 1962; Shein et al., 1963), ( b ) lung (Diamandopoulos and Enders, 1965; Nachtigal and Butel, 1970), (c) liver (Diamandopoulos and Enders, 1965), ( d ) heart (Enders and Diamandopoulos, 1969), ( e ) skin and subcutaneous tissue (Diamandopoulos et al., 1969), ( f ) lens epithelium (Albert e t al., 1969), (9) astrocytes (Shein, 1968), ( h ) prostate (Paulson e t al., 1968), and (i) pineal (Wells et al., 1966). The latter two types of transformed cells retained a certain level of enzymatic function (tartrate-inhibited acid phosphatase and hydroxyindole-0methyltransferase, respectively). Obviously, SV40 exhibits little, if any, particular tissue tropism in the in vitro studies described above since cells derived from nine different types of differentiated tissue were successfully transformed. Cells from patients with diseases associated with chromosomal abnormalities are highly susceptible to transformation by SV40. Human diseases which have yielded transformation-susceptible cells include Fanconi’s anemia (Todaro e t al., 1966), Down’s syndrome (Todaro and Martin, 1967; Potter et al., 1970) , and Klinefelter’s syndrome (Mukerjee et al., 1970). Using high concentrations of virus ( 1 0 9 . O TCID50/ml.), Todaro and co-workers observed that about 0.02-0.03% of normal human fibroblasts would transform. I n contrast, the cells from patients with Down’s syndrome were about three times as susceptible to transformation while those derived from people suffering with Fanconi’s anemia were at least ten times more susceptible. However, the mere presence of extra chromosomes per se was shown not to be sufficient to increase the susceptibility of cells to transformation (Payne and Schmickel, 1971). A triploid human fibroblast strain was no more susceptible to SV40 than normal human cells, based on the frequency of induction of T-antigen. It was speculated that the heightened susceptibility to transformation of the trisomic cells might be due to cellular replication errors which facilitated integration of the viral genome since the cells have chrornosome abnormalities. A recent report, however, suggests an alternative explanation for the observed differences in susceptibility of human cells to transformation by SV40 (Aaronson, 1970a). All the differences between various cell strains were eliminated when isolated SV40 DNA was employed. These results suggest that most human cells are resistant to transformation by SV40 because of a block a t an early step in infection, such as adsorption, penetration, or uncoating.

PAPOVAVIRUS

SV40

13

The preceding catalog of species of cells which have been transformed by SV40 contains cells which fall into three categories: (a) permissive, ( b ) semipermissive, and (c) nonpermissive. Permissive cells are those which are highly susceptible to SV40 and in which the virus undergoes a typical replicative cycle. Monkey kidney cells are permissive for SV40 (Hsiung and Gaylord, 1961; Mayor et al., 1962; Carp and Gilden, 1966; Kit et al., 1966a; Sauer et al., 1967). Human cells represent the semipermissive system. Small amounts of infectious virus are produced by the infected cells. Yet, even when free viral nucleic acid is used to infect the human cells, the yields of virus do not attain the levels produced by monkey cells (Swetly et al., 1969; Kit et al., 1970). All the other species of cells fall into the third category, nonpermissive. These cells are not susceptible to SV40 and do not produce detectable amounts of progeny virus. It has been shown with both hamster and mouse cells (Swetly et al., 1969) that even after exposure to SV40 DNA no infectious progeny virus can be detected in the cultures. The above distinction between permissive and nonpermissive types of cells is made in anticipation of the discussion below, in Section V,D. One cannot say that a transformed cell is resistant to superinfection by the transforming virus if the normal cells derived from that species are naturally resistant to the virus. It is much easier to transform nonpermissive cells by SV40 than those derived from permissive species. The mouse 3T3 cell line is sensitive to transformation by SV40 and constitutes the most widely employed quantitative transformation system for the virus (Black, 1966a ; Todaro and Green, 1966a). I n the 3T3 system, one transforming unit of virus corresponds to about lo3 infectious units and about lo5 physical particles. When high concentrations of virus are used, a t least 10% of the 3T3 population will transform. I n contrast, transformation of semipermissive human diploid cells is less efficient. An equivalent input of virus will transform only about 0.03% of the human cells. In general, laboratory manipulation of some type is used in order to achieve transformation of permissive cells. Tricks on the part of the researcher are necessary because the inoculum virus tends to replicate and destroy all the cells in the culture. Examples of such manipulation employed in successful studies include heavy ultraviolet irradiation of the inoculum virus (Shiroki and Shimojo, 1970), high multiplicities of infection (Margalith et al., 1969), and very low multiplicities of infection (Koprowski et al., 1967; Wallace, 1967; Fernandes and Moorhead, 1965). Transformation of permissive cells is a very rare event, far too rare to quantitate. Therefore, it has not been possible to differentiate between

14

J. S.

BUTEL,

S. S. TEVETHIA, AND J.

L.

MELNICK

the two alternative possibilities: ( a ) that a unique mutant cell in the population has been transformed or ( b ) that the virus has mediated a rare molecular event which aborted the normal replicative cycle and resulted, instead, in transformation of the permissive cell. BY DEFECTIVE SV40 B. TRANSFORMATION

Defective particles of SV40 can induce oncogenesis in vivo (see Section I1,B). Similarly, defective SV40 can mediate transformation in vitro as demonstrated by the following examples of defective virions: ( 1 ) PARA (defective SV40) -adenovirus hybrid population (Black and White, 1967; Black and Todaro, 1965; Diamond, 1967; Duff and Rapp, 1970a; Nachtigal and Butel, 1970; Butel et al., 1971c; Duff et al., 1970), ( 2 ) hydroxylamine-inactivated virus (Altstein et al., 1967a), and ( 3 ) Tantigen-forming defective virions (T-particles) of SV40 (Uchida and Watanabe, 1969). All stocks of SV40 naturally contain defective particles which arc capable of inducing the synthesis of T-antigen, but not of V-antigen (Sauer et al., 1967; Altstein et al., 1967b; Uchida et al., 1968). It remains to be determined whether the defective particles are genetically stable and perpetuated during passage or whether they are produced randomly during each replicative cycle of the virus. In either event, defective particles are prevalent. Since transformation is a relatively rare event (requiring a minimum of lo3 to > lo* PFU of virus per transformation event in the most susceptible cell systems (Black, 1966a; Todaro and Green, 1966a; Aaronson and Todaro, 19681, defective particles could conceivably be the agents which mediate the transformation event (s) . Irradiation experiments also indicate that the entire viral genome is not required for transformation. Aaronson (1970b) used human cells as the assay system with SV40 so that three viral functions (T-antigen, V-antigen, and transformation) could be determined under identical conditions. Infectivity was about 4 times more sensitive and the synthesis of V-antigen about 3 times more sensitive to inactivation by ultraviolet (UV) light than the property of transformation. The relative UV-sensitivity of the synthesis of T-antigen was more difficult to assess because the survival curve changed with time (time after infection before cells were stained for T-antigen). It was concluded, however, that UVsensitivity of T-antigen was intermediate between those of V-antigen and transformation. Previous reports had also indicated that T-antigen synthesis and thymidine kinase-inducing activity appeared to be more UV-resistant than plaque-forming ability by SV40 (Butel and Rapp, 1966b; Carp and Gilden, 1965; Carp et al., 1966). However, Yamamoto and Shimojo (1971) have reported that, owing to apparent multiplicity

PAPOVAVIRUS

SV40

15

reactivation, it is not possible to accurately estimate the size of the Tantigen gene on the basis of UV-irradiation data. Aaronson’s (1970b) above estimate of the proportion of SV40 genome involved in transformation compares well with that reported for polyoma virus by Latarjet e t al. (1967). Other authors had previously reported larger target sizes for the property of transforming ability by polyoma virus (Benjamin, 1965; Basilico and di Mayorca, 1965). Since only a portion of the genome is needed to mediate transformation, defective particles present in virus populations may be responsible for the oncogenicity of the virus. The concept that defective particles may be the transforming entities is considered further in Section V,D. C. DOUBLE TRANSFORMATION OF CELLS Transformation of a given cell by one member of the papovavirus group does not preclude its further transformation by a second member of the same virus group. Todaro and Green (1965) presented evidence based on colonial morphology that SV40 could further transform 3T3 mouse cells initially transformed by polyoma virus. Several isolated clones of the doubly transformed 3T3 cells were shown to contain specific T-antigens of both viruses (Todaro et al., 1965). Takemoto and Habel (1966) showed that SV40-induced hamster tumor cells became doubly transformed after superinfection with polyoma virus. This latter study demonstrated the presence of the specific transplantation (TSTA) and tumor (T) antigens induced by both viruses in the doubly transformed cells. Cells can be doubly transformed by SV40 and a human adenovirus, as well. Cells transformed either in vivo or in vitro by the PARA (defective SV40) -adenovirus hybrid populations sometimes contain both SV40 and adenovirus T-antigens and generally induce antibody against both SV40 and adenovirus T-antigens in tumor-bearing hamsters (Rapp et al., 1966a, 1969; Butel et al., 1971c; Richardson and Butel, 1971; Duff and Rapp, 1970a; Black and White, 1967). The morphology of the tumors and transformed cells will sometimes be characteristic of either adenovirus or SV40-transformed cells or, on occasion, histologically intermediate (Rapp et al., 196713; Igel and Black, 1967; Black et al., 1969). The most convincing evidence, however, that both SV40 and adenovirus information can be present in the cultures of PARA-transformed cells is that both adenovirus 7 and SV40 specific RNA’s were detected in such cultures after cloning (Levin e t al., 1969a). Therefore, the same cell can contain both SV40 and adenovirus genetic information. It should be recalled, though, that the PARA system is unique because the defective SV40 and the defective adenovirus 7

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J. S. BUTEL, S. S. TEVETHIA, AND J. L. MELNICK

DNA’s appear to be linked (Baum et al., 1966; Rowe and Pugh, 1966; Levin et al., 1969a; Kelly and Rose, 1971). Therefore, integration of the SV40 portion of the PARA genome would carry into the chromosome the adenovirus 7 region also. However, cells transformed by a transcapsidant PARA-adenovirus 12 population sometimes contained both adenovirus 7- and adenovirus 12-specific RNA in addition to the SV40specific RNA (Levin et al., 1969a). This indicates that both PARA and adenovirus 12 genomes can be integrated into the same host cell. It has recently been reported (Rhim et al., 1971) that rat embryo cells infected with Rauscher leukemia virus show enhanced susceptibility to transformation by SV40. The underlying mechanisms responsible for the phenomenon remain to be determined. One important facet of multiple transformation by viruses has not yet been studied. That area concerns the number of different SV40 genomes which can be present in the same transformed cell. Such information would indicate how many different integration sites there are for SV40 in a single cell. Such a study could be approached using genetically defined temperature-sensitive mutants of SV40. The antigenic, genotypic, and virogenic properties of cells transformed in vitro by SV40 are analyzed in the following sections of this chapter.

D. REVERSION OF TRANSFORMED CELLS The transformation of a normal cell into a cancer cell has generally been held to be an irreversible change. Recently, however, Braun (1970) drew together examples from experimental systems ranging from plants to frogs, newts, mice, hamsters, and humans, and showed that the reversal of the neoplastic state can and does occur. For the purposes of this chapter, only those tumors induced by viruses will be considered with respect to the property of reversion. Nowcver, because of a dearth of information concerning the SV40 system, other viruses will also be considered in this section. Treatment of transformed cells with FUdR a t different cell densities resulted in the selection of certain cell variants (Pollack et al., 1968). These variant derivatives exhibited a flattened morphology, attained lower saturation densities, and, with the two variants tested, reduced tumor-producing capacity in vivo. This study included 3T3 mouse cells transformed by either SV40 or polyoma and a hamster tumor cell line originating from a polyoma virus-induced tumor. The variant clones still contained the virus-specific T-antigens and, with a t least one derivative cell line, still yielded infectious SV40 following fusion with monkey kidney cells. It would be informative to determine the transforming capabilities of the rescued virus. Such experiments would indicate

PAPOVAVIRUS

SV40

17

whether the observed reversion was due to a mutation of one or more essential genes of the transforming virus or whether reversion was due, instead, to an alteration in the expression of the essential virus genes in the transformed cells. Hamster embryo fibroblasts transformed by polyoma virus have reportedly undergone a reversion of properties characteristic of transformation (Rabinowitz and Sachs, 1968, 1969a,b, 1970a,b; Inbar et al., 1969). These variants showed a decreased cloning efficiency in soft agar, a decreased saturation density, and a loss of the ability to multiply a t 41°C. The variants also acquired the ability to form colonies on glutaraldehyde-fixed normal cells, manifested contact inhibition toward one another, and inhibited the parental transformed cells. The variant cells had an altered cell surface membrane in that they were no longer agglutinated by the carbohydrate-binding protein concanavalin A. The variants produced in vitro were less tumorigenic than the parental transformed cells, while those produced in vivo were more tumorigenic. All the variant cells continued to synthesize polyoma T-antigen. Eight different variant cell lines were shown to have suffered a partial or complete loss of detectable levels of polyoma-specific transplantation antigen. Further, it was observed that the reversion was initially unstable but that it could be stabilized by inhibiting cell multiplication to prevent a crowded condition. Sachs (1965) has proposed that the presence of the transplantation antigen on the cell surface results in a change in the control mechanism for cell replication. The revertant cells characterized by Sachs and his colleagues support his hypothesis because the variant cells behaved like normal cells in that they exhibited both contact inhibition and an absence of virus-specific transplantation antigen (Rabinowitz and Sachs, 1970a). However, there are other known examples which do not support this hypothesis. Hare (1967) reported that the lp-D strain of polyoma virus does not induce virus-specific transplantation immunity in weanling hamsters, yet is oncogenic. Furthermore, cells derived from lp-D-induced tumors do not contain polyoma transplantation antigen. The sp-D strain of polyoma virus, also lacking the marker for TSTA, was able to efficiently transform cells in vitro (Hare, 1967). An example with SV40 which fails to support the foregoing hypothesis is a fibroblastic line of H-50 cells (Tevethia, 1971). The cells are morphological revertants which are fibroblastic in appearance, attain similar saturation densities to that of the parental line, and have a reduced chromosome complement. Nevertheless, these revertant cells are highly transplantable and contain high levels of SV40 transplantation antigen. Eagle e t al. (1970) studied a variety of cell lines with respect to maximum

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J . S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

population density, inhibition by normal cells, serum requirement, and growth in soft agar and attempted to correlate such properties with xenogeneic transplantability. They concluded that none of the properties studied appeared to be a valid in vitro criterion for predicting tumorigenicity. However, it would be interesting to do such a study within a single species, e.g., SV4O-transformed hamster cells, the in vitro properties of which could then be correlated with transplantability in vivo in weanling hamsters. I n contrast to the cells studied by Rabinowitz and Sachs which still contained polyoma T-antigen, the revertant cells studied by Marin and Macpherson (1969) appeared to have lost the viral genes present in the parental cells. The revertant cells were less tumorigenic and both polyoma-specific T- and transplantation antigens were lost or reduced. The cells could be retransformed by polyoma virus. This latter fact ruled out the possibility that reversion was a consequence of the loss of cell genes required for the expression of the transformed phenotype. Some relevant experiments concerning reversion of transformed cells have been performed with the RNA-containing tumor virus Rous sarcoma virus (RSV). Macpherson (1966) reported the isolation of revertants from BHKPl cells transformed by the Schmidt-Ruppin strain of RSV. The revertant cells showed altered morphology, were not tumorigenic, did not contain detectable levels of COFAL antigen, and had decreased plating efficiencies in soft agar. The revertant cells appeared to be resistant to retransformation by RSV. Nevertheless, it appeared that, in this particular system, reversion was due to a loss of viral genes. A temperature-sensitive ( t s ) mutant (TI) isolated from the SchmidtRuppin strain of RSV (SR-RSV-A) has provided a good model for study (G. S. Martin, 1970). Cells transformed a t the permissive temperature by the mutant virus and then shifted to the elevated, nonpermissive temperature rapidly ( 2 4 hours) reverted back to a nontransformed phenotype. The t s mutant was able to replicate well a t the elevated temperature. It would appear from these experiments that the expression of some viral gene is required for the maintenance of the transformed state and the gene product is temperature-sensitive in the case of mutant TI. Furthermore, that gene product is evidently not required for viral replication. Toyoshima and Vogt (1969) also reported the detection of two ts mutants of RSV (strain B77) which could not transform cells a t the elevated temperature. As with Martin’s T1 mutant, cells transformed a t the permissive temperature and then shifted to the nonpermissive temperature reverted back to a normal phenotype. These mutants, however, could not replicate at the elevated temperature so they did not

PAPOVAVIRUS

SV40

19

allow a separation of viral functions required for replication and those essential for transformation. From these few scattered reports one can conclude that reversion of virus-transformed cells probably can and does occur. Some of the reports appear to have detected complete reversion, possibly due to a loss of the transforming viral genes. Some of the other observed cases of reversion would more accurately be denoted partial reversion in that some phenotypic properties were altered but virus-specific antigens were still present. In these latter examples, transplantability of the cells was sometimes lessened and sometimes increased. The available data are too few and too preliminary a t this time to prompt any generalizations about either the properties of cells which have reverted or the mechanism (s) of the observed reversion. IV. Phenotypic (Antigenic) Changes in Tumor and Transformed Cells

Transformation of mammalian cells by SV40 is accompanied by the synthesis of virus-specific proteins which are antigenic in the syngeneic host. These antigenic proteins are not a part of the mature virion but are specifically induced by the transforming virus. These virus-induced nonvirion antigens can be classified into two categories based upon their location within the transformed cell : (a) tumor (T) antigen-located in the nucleus of transformed cells. ( b ) surface antigen-located a t the surface of transformed cells. The surface antigens can be further subdivided: (i) tumor-specific transplantation antigen (TSTA)-detected in vivo by the transplantation rejection test, and (ii) S-antigen-detected by serological tests in vitro, using antibody from hamsters immunized with SV40 or SV40-transformed cells. In addition, the transformed cells may acquire some properties which are cryptic in normal cells. Of all thc changes that a cell undergoes during transformation by an oncogenic virus, acquisition of specific antigens a t the cell surface is one of the most important. In this section, we will attempt to analyze the nature of these antigens arid their relationship to each other and consider whether the changes are extrinsic (coded for by the virus) or intrinsic (due to derepression of the host cell genome). A. TUMOR ANTIGEN The T-antigen in SV40 tumor cells was first demonstrated by means of the complement fixation test using sera from hamsters bearing tumors induced by SV40 (Huebncr et al., 1963; Koch and Sabin, 1963). The nuclear localization of T-antigen was later established using the indirect immunofluorescence technique (Rapp et al., 1964a; Pope and Rowe,

20

J. S. BUTEL, S. S. TEVETHIA, AXD J . L. MELNICK

1964). A typical pattern of nuclear fluorescence by SV40 T-antigen is shown in Fig. 1. The T-antigen present in transformed cells was directly related to the transforming virus when it was shown to be synthesized in acutely infected monkey kidney cells (Rap]) e t al., 1964b; Sabin and Koch, 1964; Hoggan e t al., 1965). Kinctic studies showed that T-antigen appears 12-24 hours after infection with SV40, prior to the appearance of viral capsid antigen and progeny virions. The synthesis of T-antigen is not affected by inhibitors of DNA synthesis but is inhibited by actinomycin D, suggesting that i t is due to an early function of the viral genome (Rapp e t al., 1965a). T-antigens extracted from productively infected or transformed cells

FIG.1. Inimunofluorescence detection of SV40 T-antigen localized in nucleus of rell. X 400.

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SV40

21

originating from different species are antigenically similar. Conversely, T-antibodies obtained from various sources all give a typical intranuclear reaction when tested against cells either transformed or infected by SV40 (Rapp e t al., 1964a,b; Tevethia, 1970). These observations suggest that the synthesis of T-antigen is coded for by the viral genome. This hypothcsis is supported by the fact that isolated SV40 DNA is also able to induce the synthesis of T-antigen (Black and Rowe, 1965). Furthermore, three mutants of defective SV40 (PARA) have been described which induce the synthesis of an antigenically identical T-antigen in the cytoplasm of infected cells rather than in the nucleus (Butel e t al., 1969). The cytoplasmic T reaction is shown in Fig. 2. Subsequent studies by Duff et al. (1970) and Richardson and Butel (1971) showed that the

FIG.2. Immunofluorescence detection of SV40 T-antigen localized in cytoplasm cell. X 400.

22

J. S. BUTEL, S. 8 . TEVETHIA, AND J . L. MELNICK

cytoplasmic SV40 T-antigen was present in cells transformed by the variant viruses, as well as in cells from tumors induced by these variant viruses in newborn hamsters. The cytoplasmic localization of T-antigen in transformed or tumor cells was stable upon multiple passages in vitro. No apparent differences were detected when the properties of cytoplasmic SV40 T-positive cells were compared t o those of nuclear T-positive cells (Richardson and Butel, 1971). A summary of the results of that comparative study is given in Table 11. Although all the available evidence supports the concept that information for the synthesis of SV40 T-antigen resides in the viral genome, the host cell nevertheless does appear to exert some influence upon the expression of the T-antigen. Nachtigal et al. (1970) demonstrated that lung cells derived from hybrid animals originating from a cross of the Syrian and Rumanian species of hamsters did develop Tantigen when transformed by SV40. In contrast, after exposure to SV40, lung cells from Syrian hamsters frequently acquire the property of unlimited growth in vitro but do not develop detectable levels of T-antigen (Diamandopoulos and Enders, 1965; van der Noordaa and Enders, 1966; Nachtigal and Butel, 1970). SV40 T-antigen is heat labile and resistant to DNase and RNase, but is susceptible to trypsin (Gilden et al., 1965). The molecular weight of T-antigen has been estimated to be approximately 200,00(1-250,000 by Sephadex G-200 gel chromatography (Gilden e t al., 1965) and 70,000TABLE I1 COMPARISON OF PROPERTIES OF HAMSTER TUMORCELLLINESCONTAINING SV40 TUMORANTIQEN IN EITHER THE NUCLEUS OR T m CYTOPLASM OF CELLS

Propertya Contain nuclear SV40 T-antigen by IF Contain cytoplasmic SV40 T-antigen by IF Contain SV40 T-antigen by CF Contain SV40 S-antigen by IF Stable nuclear localization of T-antigen Stable cytoplasmic localization of T-antigen Indefinite life-span in vitro Readily transplantable in vivo Elicit SV40 T-antibody in vivo Induce SV40 transplantation immunity (immunogenic) Rejected by SV40 or PARA-immune animals (immunosensitive ) 0

T

=

Nuclear T-positive cell line

Cytoplasmic T-positive cell line

+ 0 + ++ 0 + + + + +

+ + + 0 + + + + + +

0

tumor; S = surface; IF = immunofluorescence; CF = complement fixation.

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SV40

23

80,000 by use of an immunoabsorbent (Del Villano and Defendi, 1970). Its physical properties indicate that T-antigen is not identical to any of the enzymes involved in DNA biosynthesis (Kit et al., 1967). The biological function of T-antigen remains obscure. Recently, a presumably new SV4O-specific antigen, designated the U-antigen, has been described (Lewis et al., 1969). This antigen has been localized in the perinuclear region of cells infected with a strain of adenovirus type 2 carrying a defective SV40 genome (AdB'ND,). Sera from monkeys immunized with cells infected with Ad2+ND, react with an intranuclear antigen present in both SV40-transformed and SV40infected cells (Lewis and Rowe, 1971). Antibody to U-antigen is also detected in sera from SV40 tumor-bearing hamsters. I n contrast to Tantigen, U-antigen is heat-stable. The existence of U-antigen and the difficulty in obtaining specific antisera (Lewis and Rowe, 1971) reveal that future studies aimed at characterizing T-antigen will need to be carefully designed and evaluated.

B. TUMOR-SPECIFIC TRANSPLANTATION ANTIGEN(TSTA) The first evidence for the presence of TSTA in cells transformed by DNA viruses was provided when Sjogren et al. (1961) and Habel (1961) demonstrated that mice or hamsters inoculated with polyoma virus were capable of rejecting a challenge of polyoma tumor cells. TSTA was later demonstrated in SV40-induced tumor cells using the transplantation rejection technique (Khera et al., 1963; Habel and Eddy, 1963; Koch and Sabin, 1963; Defendi, 1963). The transplantation rejection test involves immunizing hamsters with one to three doses of live SV40 and later challenging immune and nonimmune hamsters with varying numbers of transplantable SV40 tumor cells. The results are generally expressed as the number of cells required to produce tumors in 50% of the animals (TPD,,). A 10-fold difference in the TPD,, of the immune animals when compared to that of the control, nonimmunized group is taken as evidence of the presence of TSTA in the tumor cells. TSTA is not a capsid antigen since antiviral antibodies have no effect on the growth of SV40 tumor cells (Khera et al., 1963). Also, SV40 tumor cells carrying TSTA were shown to be free from both infectious virus and virion antigens (Melnick e t al., 1964). Live virus is essential in inducing immunity against the tumor cells. Habel (1962) postulated that virus, upon inoculation into adult hamsters, transforms certain cells in the animals which then develop TSTA identical to those present on tumor cells subsequently used for challenge. The virus thus codes for the synthesis of new antigens unrelated to capsid antigen which sensitize the host against challenge of SV40 tumor cells.

24

J . S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

The TSTA present in cells transformed by SV40 in vitro have been shown to be identical to those present on virus-induced tumor cells (Khera et al., 1963). The evidence that SV40 TSTA is present at the surface of transformed cells is 2-fold: ( a ) the rejection of tumor cells by immune animals required contact between sensitized lymphoid cells and TSTA (Tevethia et al., 1970b; Coggin et al., 1967), and ( b ) cell membranes isolated from SV40 tumor cells induced a state of immunological tolerance to SV40 TSTA in newborn hamsters (Tevethia and Rapp, 1966). The tolerance was demonstrated by the failure of adult hamsters, which had received cell membranes as newborns, to be immunized by SV40 against a challenge of SV40 tumor cells. The membranes from SV40 tumor cells were later shown to immunize adult hamsters against SV40 tumor cells (Coggin et al., 1969). The TSTA in SV4O-transformed cells can also be demonstrated by immunogenicity tests. Transformed cells are tested either for their ability to immunize adult hamsters against a challenge of transplantable SV40 tumor cells or for their ability to block SV40 oncogenesis in hamsters inoculated as newborns with the virus (Girardi, 1965; Lausch et al., 1968). The oncogenicity of SV40 can also be prevented by inoculating hamsters in the latent period before tumor appearance with nontransplantable syngeneic or xenogeneic SV40-transformed cells. Using this method, Girardi (1965) demonstrated the cross-reactivity between the TSTA of SV40-transformed human cells and that of SV40-transformcd hamster cells. F. Jensen and Defendi (1968) used the same technique to demonstrate the absence of SV40 TSTA in simian cells transformed by ultraviolet light-irradiated defective SV40 (PARA), However, it should be pointed out that these latter cells were not tested for their ability to immunize hamsters against challenge with an immunosensitive SV40 tumor cell line. This is an important point, since Lausch et al. (1968) showed that hamster cells transformed by defective SV40 (PARA) were not effective at preventing SV40 tumorigenesis, yet were rejected by SV40-immunized hamsters. Recently, Girardi and Defendi (1970) showed that SV40 TSTA was synthesized during the cytolytic cycle of the virus in monkey cells. The synthesis of TSTA could be blocked by inhibitors of protein synthesis and by actinomycin D, but not by inhibitors of DNA synthesis.

c. SURFACE ANTIaEN Since in vivo tests for the measurement of immunosensitivity and immunogenicity of SV40-transformed cells are time-consuming, many efforts have been made to develop in vitro tests for the detection of

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SV40

25

SV40 TSTA. The attempts to devise in vitro tests fall into two general categories: ( a ) cytotoxic tests which use specific antibodies or immune lymphocytes, and ( b ) serological tests based on the detection of antigenantibody interaction. Immunofluorescence and mixed agglutination tests belong to this latter category. A tumor virus may bring about changes in transformed cells other than the development of TSTA, and some of these changes may represent new antigenicities. Therefore, it cannot be said that different in vitro tests all measure TSTA. For this reason, the antigen or antigens detected by in vitro tests will be designated as surface or S-antigen(s) . I n this section, we will analyze the S-antigen(s) with respect to their detection, specificity, and nature. Tevethia et al. (1965) first demonstrated S-antigen in SV40-transformed cells by the indirect immunofluorescence test using sera from SV40-vaccinated hamsters that had rejected a transplant of virus-free SV40 tumor cells. Live cells were used in the immunofluorescence test, and the reaction was localized at the cell surface in the form of a ring around the cell (Fig. 3 ) . The reaction was specific for SV4O-transformed cells since antibody against S-antigen did not react with either normal hamster cells or cells transformed with unrelated viruses. The S-antigen was present whether the hamster cells had been transformed by SV40 in vivo or in vitro. Antibody against S-antigen was present in hamsters which had rejected a challenge of transformed cells, but was absent from tumor-bearing animals. Tevethia et al. (1968a) later demonstrated that hamsters synthesized S-antibody when immunized with SV40 alone, thereby ruling out the participation of isoantigens in the membrane reaction. Even though the hamsters that were used in the preceding

FIG.3. Immunofluorescence detection of SV40 S-antigen localized a t surface of cell. X 400.

26

J . S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

studies were not inbred, this probably was not a serious detriment because it has not been possible to demonstrate isoantibody in hamsters (Billingham and Silvers, 1964). Kluchareva et al. (1967) confirmed the presence of S-antigen in SV40-transformed cells, and further established the specificity of the reaction by using sera which were prepared in hamsters inoculated with live SV40 during the latent period of viral oncogenesis. This latter approach avoided any possible isoantigen incompatibilities in the test system since the antibody was from animals which had never been exposed to foreign cells. A similar membrane reaction was later demonstrated in polyoma virus-transformed cells by the immunofluorescence test (Malmgren et al., 1968; Irlin, 1967). The S-antigen of polyoms virus-transformed cells was specific for that virus. Hayry and Defendi (1968) and Metzgar and Oleinick (1968) independently demonstrated the presence of S-antigen in SV40-transformed cells by the mixed hemadsorption technique. The S-antigen detected with this method was virus-specific and the antibody against the antigen did not react with cells transformed by unrelated viruses. The authors also showed that the S-antigen in SV40-transformed human cells would cross-react with the S-antigen of SV40-transformed hamster cells, further establishing the specificity of the antigen. Tevethia et al. (1970a) confirmed the cross-reaction between the S-antigens of SV40-transformed human and hamster cells by means of the immunofluorescence test using sera prepared according to the method of Hayry and Defendi (1968). Tevethia e t al. (1970a) used the colony inhibition test originally described by I. Hellstrom and Sjogren (1965) to demonstrate specific S-antigen in SV40-transformed cells. The sera prepared in hamsters either against SV40-transformed human or marmoset cells or against purified SV40 was active only against SV40-transformed cells. The observed inhibition of colony formation was dependent upon the presence of active guinea pig complement. There was a positive correlation between results obtained using the colony inhibition and the immunofluorescence tests, suggesting that the two tests may be detecting the same or similar antigen. These results were confirmed by Smith et al. (1970) , who demonstrated S-antigen by the in vitro cytotoxic test using sera from mice hyperimmunized with syngeneic nontransplantable SV40transformed mouse cells. The sera were cytotoxic only against SV40transformed cells. Recently, Wright and Law (1971) used the cytotoxic test and humoral antibody prepared in mice by the repeated inoculation of syngeneic SV40-transformed cells to demonstrate the presence of tumorspecific antigens a t the cell surface. SV40-transformed cells labeled with chromium-51 were selectively killed with the antisera in the presence of rabbit complement. The fact that only the cells which possessed

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SV40

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SV40 TSTA were killed by the cytotoxic antibody suggests that the cytotoxic action of humoral antibody may be directed against TSTA. Since TSTA and S-antigen(s) are both present a t the cell surface, there has been a temptation to assume that the in vitro tests are actually measuring TSTA. Tevethia et al. (1968b) were the first to point out the lack of relationship between TSTA and S-antigen in certain SV40exposed hamster cell lines which became oncogenic. The cell lines in question were derived by Diamandopoulos et al. (1968) by exposing cloned hamster embryo fibroblasts to SV40. Four of the seven virusexposed cell lines became oncogenic and developed SV40 S-antigen. However, only two of the cell lines also contained intranuclear T-antigen. Neither T- nor S-antigen was detected in lines established from cultures which had not been exposed to SV40. When the various lines were tested for the presence of SV40 TSTA, only S'T' cell lines were rejected by SV40-immunized hamsters ; S'T- cells were not rejected by the immune animals (Tevethia et al., 1968b). This finding, plus the observation that S'T- cells were unable to block SV40 oncogenesis in hamsters when inoculated during the latent period (Tevethia et al., 1968b) , suggested that the S'T- cells lacked detectable amounts of SV40 TSTA. Wright and Law (1971) using hyperimmune mouse sera in the cytotoxicity test also failed to demonstrate SV40 TSTA in S'T- cell lines. The S'T- cells have subsequently been shown to lack detectable amounts of both SV40 messenger RNA (Levin et al., 196913) and SV40 DNA (Levine et al., 1970). The absence of detectable quantities of SV40 genetic information in the S'T- cells suggests that the S-antigen in these cells may not be coded for by a persistent viral genome. One possibility, proposed by Tevethia et al. (1968133, is that the Santigen in the S'T- cells may be the result of derepression of the host cell genome by the virus. Duff and Rapp (1970b) recently demonstrated that sera from pregnant hamsters reacted specifically with SV40-transformed hamster cells, indicating the presence of some derepressed embryonic antigens on SV40-transformed cells. It was somewhat puzzling that the sera from pregnant hamsters did not react with cells transformed either by adenoviruses or by chemical carcinogens. Even if each tumor virus depresses only a specific region of the host cell chromosome, pregnant hamsters should be exposed to all these antigens during embryogenesis, The definite proof that the S reaction is due, altogether or in part, to an embryonic antigen is still lacking, since no attempts have been made to determine whether anti-S sera will react with embryonic cells. Baranska e t al. (1970) demonstrated cross-reactions between SV40transformed mouse cells and mouse eggs using antimouse egg sera prepared in guinea pigs. The sera also reacted with a tumor line induced by a chemical carcinogen, but not with normal cells. The nature of the antigen detected remains obscure.

28

J. S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

It is also possible that, upon infection of hamster cells, SV40 either directly or indirectly activates an unrelated virus previously latent in the hamster cells (Huebner and Todaro, 1969), which, in turn, is responsible for the membrane reaction. Another possibility is that cells infected with SV40 may undergo abortive transformation, but in the process conditions are altered such that S-antigen synthesis is initiated. A recent report (Vasconcelos-Costa, 1970) indicated that cells transformed by adenovirus 12 may lose T-antigen but retain S-antigen. It would be of great interest to ascertain whether those cells also lose adenovirus genetic information and TSTA. Recent studies by Hayry and Defendi (1970) using the mixed hemadsorption test have suggested that S-antigen is a normal cell antigen which is specifically unmasked by SV40 during transformation. This conclusion was based on the observation that, after brief treatment with trypsin, spontaneously oncogenic or polyoma virus-transformed cells reacted with SV40 S-antibody. This antigen on the non-SV40-transformed cells could also be exposed by treatment of the cells with chemotrypsin, but not with ficin, papain, or neuraminidase. Similar reactivity was detected using the immunofluorescence test. On the basis of adsorption studies, it was concluded that normal cell antigens cross-reacted with the SV40 antigen. However, complete absorption of anti-S activity from the sera using either SV40-transformed cells or trypsin-treated nontransformed cells was not achieved. Burger (1969) has described presumably “tumor-specific” sites a t the surface of SV40-transformed cells. The sites were demonstrated by an agglutination test, using agglutinin isolated from wheat germ lipase. The agglutinin binding sites which were present on transformed cells were cryptic in normal cells, but could be unmasked by treatment with trypsin. The agglutination reaction could be inhibited with N-acetylglucosamine. Agglutination of transformed cells by Concanavalin A has been reported by Inbar and Sachs (1969). Normal cells were agglutinated only after mild trypsinization, again suggesting the cryptic nature of the agglutination sites on normal cells. The authors claimed that the sites were specifically exposed during viral transformation. SV40-infected 3T3 cells wcre also agglutinated by Concanavalin A, but only if they had undergone a t least one cell generation (Ben-Bassat et al., 1970). Of interest is thc report (Benjamin and Burger, 1970) that the change in tlic cell membrane which renders a cell agglutinable by Concanavalin A was not detected after infection with mutants of polyoma virus previously denionstrated to be defective in their ability to transform cells. The agglutination sites were acquired by cells after lytic infection with the wild-type polyoma virus.

SV40

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29

D. RELATIONSHIP OF S-ANTIGENTO TSTA I n view of the many surface changes which have been described with SV40-transformed cells, their relationship to each other and to TSTA is considered below and summarized in Table 111. TABLE 111: RELATIONSHIP BETWEEN SURFACE CHANGES OBSERVED AFTER TRANSFORMATION BY SV40 ~~

Surface change

Properties Specific for transforming virus Appear after viral transformation Present on normal cells in cryptic form Forssman antigen Antigenic in syngeneic hosts

Normal Agglutination cell sites TSTA S-antigen antigen 0

+ + ? 0

+ +

+ +

0

0

+0

+

0

Embryonic antigen

0

0

+ +

+

0 ?

? ?

?

The agglutination sites described by Burger (1969) and Inbar and Sachs (1969) may not be TSTA, since all tumor cells bind wheat germ agglutinin and Concanavalin A regardless of whether they are transfoimed by SV40 or by other tumor viruses. I n contrast, TSTA is specific for the transforming virus. Also, normal cells possess the agglutination sites in a cryptic form, and TSTA has not been demonstrated in normal cells. The possibility that the agglutination sites are similar to the Santigen detected in vitro is also unlikely because cells transformed by polyoma and other viruses do not react with SV40 anti-S sera but do bind the agglutinins. I n addition, SV40-transformed cells which have been pretreated with anti-S serum and then washed to remove the excess serum can still be agglutinated by Concanavalin A a t a rate similar to that of untreated cells or of cells pretreated with normal hamster serum, indicating that there are agglutination sites on the tumor cells which are not the S-antigen (Tevethia, 1971). The agglutination sites may also differ from the sites containing the normal cell antigen described by Hayry and Defendi (1970). The normal cell antigen is exposed a t the surface of SV40-transformed cells but is cryptic in polyoma and spontaneously transformed cells, whereas agglutinin sites are exposed in all the transformed cells. It is not yet

30

J. S. BUTEL, S.

S. TEVETHIA, AND J .

L. MELNICK

known whether the agglutination sites are antigenic in syngeneic hosts. The relationship between the normal cell antigen described by Hiiyry and Defendi (1970) and SV40 TSTA is not clear a t this time, but the available cvidence suggests that the two are different. Experiments carried out in the authors’ laboratory, as well as by others, indicate that spontaneously oncogenic cells or normal cells that are treated with typsin do not immunize hamsters against a challenge of SV40 tumor cells. Furthermore, if the normal cell antigen were to act as TSTA, then animals should be tolerant to it unless the antigen is masked during both embryonic and adult life (which is unlikely), and SV40 would not be able to immunize hamsters against a challenge of SV40 tumor cells. The transplant rejection test used to detect TSTA is dependent upon the fact that SV40 can immunize adult animals. It is not known whether sera prepared in hamsters against trypsinized normal, polyoma-transformed, or spontaneously oncogenic cells will react with nontrypsinized SV40-transformed cells. Such tests would help clarify a possible identity between normal cell antigens and SV40 S-antigen. The L‘normal” cell antigen is not a Forssman antigen such as that described by Robertson and Black (1969), because extensive treatment of SV40 S-antisera with sheep red blood cells failed to adsorb antibody against the normal cell antigen (Hayry and Defendi, 1970). Duff and Rapp (1970b) discovered that sera from pregnant hamsters reacted with the membrane of SV40-transformed cells in the immunofluorescence test, indicating that embryonic antigens are present a t the surface of the transformed cells. Since the pregnant animals developed antibody against these antigens, the synthesis of the antigens in the developing embryo must be shut off before the development of the lymphoid system. Although there is no direct evidence that embryonic antigens may act as SV40 TSTA, Coggin e t al. (1970) reported that hamsters immunized with 10-day-old hamster or mouse embryo cells temporarily stopped the replication of SV40-transformed cells. These experiments were carried out by placing Millipore chambers containing the transformed cells in the peritoneal cavities of immune animals. In a previous study Coggin and Ambrose (1969) demonstrated that antibody diffuses into the chambers and produces a cytostatic effect on the tumor cells. The effect is only temporary and, after 5 days, the multiplication of the tumor cells in immunized animals is equal to that in the controls. The evidence that the immunofluorescence and the cytotoxic tests using humoral antibody may be measuring SV40 TSTA is circumstantial. Tevethia et al. (1970a) showed that antibody prepared in hamsters against SV40-transformed cells or against purified SV40 reacted with the S-antigen of SV40-transformed cells by the immunofluorescence test and

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SV40

31

also reacted in the complement-dependent colony inhibition test. TSTA is apparently detected in the colony inhibition test in which the inhibition of tumor cell growth in vitro is measured. This opinion is based on the reasoning that the colony inhibition test in vitro and the reaction of the immune host in vivo against the target tumor cells are similar in that both result in the death (inhibition) of the neoplastic cells (I. Hellstrom and Sjogren, 1966). Further, the common antigens shared by SV40transformed human and hamster cells demonstrated by Tevethia et al. (1970a) using the in vitro immunofluorescence and colony inhibition tests are compatible with the common antigens previously demonstrated by the use of in vivo techniques (Girardi, 1965; F. Jensen and Defendi, 1968). Common antigens between polyoma virus-transformed mouse and hamster cells have also been demonstrated by in vivo and in vitro tests (I. Hellstrom and Sjogren, 1966). Further evidence that some in vitro tests may be detecting TSTA was provided by Girardi (1967) when he demonstrated that sera from SV4O-immunized animals gave a positive reaction by immunofluorescence and also enhanced the growth of SV40 tumor cells in immune animals. This immunological enhancement is thought to proceed through immunoglobulin molecules which cover TSTA sites and thus protect the tumor cells from the action of immune lymphocytes (K. E. Hellstrom and Hellstrom, 1970). More direct evidence that the cytotoxic test with immune serum may be detecting SV40 TSTA was provided by Smith et a2. (1970). They isolated a solubilized SV40 TSTA which could immunize animals and which could also absorb the cytotoxic activity of immune serum. Recently, Klietmann and Seemayer (1971), using the mixed hemadsorption test, showed marked differences in the concentration of specific surface antigen(s) on hamster cells transformed by normal SV40 and those transformed by UV-irradiated SV40. UV-irradiated virus also had a reduced capacity to induce SV40 transplantation immunity in vivo. The cells which had lower concentrations of surface antigen(s) as measured by the mixed hemadsorption test showed equal rates of growth in intraperitoneally implanted diffusion chambers in either immune or normal hamsters. These cells, however, were not checked for the presence of SV40 TSTA by determining their capacity to immunize animals against a challenge of immunosensitive tumor cells. Transformation of normal cells by oncogenic viruses may produce cellular changes other than the development of TSTA. These changes could result in an alteration of surface structure, which in turn would create new antigenic sites either specified by the viral genome or by the derepressed host cell genome. Since TSTA is present a t the surface of cells transformed by oncogenic DNA-containing viruses, it appears to be a t least one of the antigens detected by the various in vitro tests.

32

J . S. BUTEL, S. S. TEVETHIA, AND J .

L.

MELNICK

Isolation and characterization of the various cell membrane antigens in pure form will help elucidate the relationships between the various surface changes that have been observed after transformation by SV40. V. Genotypic Changes in Tumor and Transformed Cells

A. RESCUEOF INFECTIOUS VIRUSFROM TRANSFORMED CELLS Cells transformed either in vivo or in vitro by SV40 are generally virus-free, although occasional tumors and cell lines have been reported to spontaneously release small amounts of infectious virus (Sabin and Koch, 1963a,b; Gerber and Kirschstein, 1962; Black and Rowe, 1963a; Ashkenazi and Melnick, 1963). SV40 tumors which had been allowed to grow in an animal for long periods of time more frequently contained detectable amounts of infectious virus than did tumors taken soon after transplantation (Sabin and Koch, 1963b). The minute amounts of virus found in extracts of such tumors amounted at most to 109.3TCID,,/gm. of tumor tissue. This small accumulation of virus, supposedly from those rare cells which, out of the millions in the tumor, spontaneously released a small amount of virus, probably had not had time to reach detectable levels in tumors excised less than 4 weeks after transplantation. Procedures known to induce lysogenic bacteria to produce infectious bacteriophages, such as exposure to mitomycin C, proflavin, hydrogen peroxide, and X-irradiation, were only minimally effective when applied to SV40 tumor cells (Sabin and Koch, 196313; Gerber, 1964; Melnick et al., 1964; Burns and Black, 1968, 1969; Rothschild and Black, 1970). Combinations of inducing agents were not more effective a t induction of infcctious SV40 than were the individual agents alone. I n addition, there was no dose-response relationship between the concentration of inducer employed and the yield of virus obtained (Rothschild and Black, 1970). Yields of SV40 following induction by mitomycin C were in the range of 10-1000 PFU/lO' cells. Thus, only the very rare tumor cell had been induced to release infectious virus in these experiments. Gerber (1966) discovered that recovery of infectious SV40 was more efficient if the tumor cells were placed in direct contact with susceptible indicator cells, such as primary African green monkey kidney cells. The sensitivity of the indicator cell system was increased by the use of inactivated Sendai virus to form heterokaryons (Harris and Watkins, 1965) of tumor and indicator cells (Gerber, 1966; Watkins and Dulbecco, 1967; Koprowski et al., 1967). The fusion technique increased the yield of virus about 1000-fold over simple cocultivation, but the vast majority of the heterokaryons were nonproductive (Watkins and Dulbecco, 1967; Koprowski et a,!., 1967). Therefore, while this is the most sensitive means

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SV40

33

of virus rescue currently available, it also succeeds in inducing only a rare tumor cell to produce virus. The frequency of successful virus recoveries and the amount of virus rescued in the fusion experiments ( a ) varied widely between clonal lines of transformed cells derived from the same parental cell line (Watkins and Dulbecco, 1967)) ( b ) varied a t different passage levels of the same clone (Watkins and Dulbecco, 1967), (c) did not correlate with the number of viral DNA equivalents estimated to be present in each transformed cell (Westphal and Dulbecco, 1968), and ( d ) was not dependent upon the multiplicity of infection at the time transformation occurred (Kit and Brown, 1969). Burns and Black (1968) observed considerable heterogeneity with respect to inducibility by mitomycin among a series of clonal lines derived from an SV40-transformed hamster kidney cell line. The fluctuation in rescue obtained at different passage levels of a given cell line suggests a possible change in the status of the viral genome in the cell (see Section V,B). However, some SV40-transformed cell lines have consistently resisted repeated attempts to rescue infectious virus (Black, 1966b; Koprowski et al., 1967; Melnick et al., 1964; Westphal and Dulbecco, 1968; Dubbs and Kit, 1968; Kit and Brown, 1969). It has been suggested that these lines might have been derived from cells transformed by defective viruses and that the entire genome is not present. An alternative explanation for the nonyielding lines is that there has been a mutation which precludes viral replication, e.g., the absence or inability to function of an enzyme required to release the viral genome from integration. I n line with this reasoning, it would be interesting to know whether SV40 can be rescued, and how readily, from transformed xeroderma pigmentosum cells which appear to be deficient in the enzyme(s) involved in host cell reactivation (Aaronson and Lytle, 1970) since these might be the enzymes involved in excision of the integrated viral genome. Single cell clones have been isolated from many SV40-transformed cell lines. The vast majority of such clonally derived lines were found to yield small amounts of infectious SV40 (Gerber, 1966; Black, 1966b; Dubbs et al., 1967). These studies revealed that nearly every SV40transformed cell contained the complete viral genome. The fact that many of the single cell clones were established in the presence of SV40 antiserum ruled out any essential role of extracellular virus in the maintenance of transformation. The cloning experiments do not rule out the possibility, however, that some exceptional cell lines might contain only defective viral genetic complements. I n support of this hypothesis, Knowles et al. (1968) reported that virus could be rescued when two different virus-free transformed cell

34

J. S . BUTEL, S . S. TEVETHIA, AND J. L. MELNICK

lines were fused in the presence of monkey kidney cells. It was postulated that this rescue occurred because the transforming genomes were defective a t different loci. However, the rescued progeny were not characterized to reveal whether this rescue was a consequence of complementation or of recombination of the defective genomes. If complementation, one would expect the yield of rescued progeny to be a mixture of defective viruses unable to replicate independently. If due to recombination of defective genomes, one would expect the rescued virus to consist of complete infectious virions. T o adequately study this phenomenon, it would be necessary to utilize cells transformed by genetically marked parental viruses differing in recognizable properties. This would enable one to prove that recombination of transforming genomes had occurred if the two genetic markers appeared linked in the rescued virus. “Partial” induction of SV40-transformed cells has been achieved by heat shock (45°C. for 30 minutes), which induced the synthesis of SV40 viral antigen, but not infectious virus, in transformed BSCl cells (Margalith e t al., 1970b). Oddly enough, the synthesis of the viral antigen after heat treatment was not inhibited by actinomycin D, although SV40 capsid antigen synthesis is inhibited by the antibiotic in productively infected cells (Margalith et al., 1970a). The mechanism of this heat induction phenomenon is not clear, but a plausible explanation is that only a portion of the transforming viral genome has been derepressed and possibly only one of the viral coat protein polypeptides synthesized. It is not yet known whether the heat induction procedure results in the detachment of an integrated viral genome (see Section V,B) prior to capsid antigen synthesis. In general, virus which has been recovered from tumor cells has resembled the parental transforming virus (Tournier et al., 1967; Dubbs et al., 1967; Takemoto et al., 1968b). However, distinctive genetic markers were not available for these studies. In one interesting exception (Todaro and Takemoto, 1969), rescued virus was more efficient at transformation than the parental type. The authors postulated that the enhanced efficiency of transformation of the rescued virus could be due either to a selection of more efficient transforming viruses from the original stock or to some host-induced modification of the virus. There is no direct evidence available that the infectious virus recovered in any of the rescue experiments is the virus responsible for the transformation event, the genome of which has been excised from integration and replicated. Extrachromosomal copies of the virus may exist, and these may be detected in the virus recovery attempts. Rescue experiments to date have not succeeded in eliciting infectious virus from more than a minor proportion of a transformed cell population. One type of

PAPOVAVIRUS

SV40

35

evidence which would be supportive would be the isolation of specialized transducing particles containing a portion of the viral genome linked to a region of the host cell DNA. Such particles would be analogous to Xdg (Campbell, 1957) and would arise as a result of excision a t the LLwrong’’ sites on the chromosome. It has been shown that SV40, under the proper growth conditions, will encapsidate host cell DNA to form pseudovirions (Trilling and Axelrod, 1970; Levine and Teresky, 1970), so it is reasonable to assume that linked viral and cell DNA could also be encapsidated if it were the correct size. In addition, it has been demonstrated that polyoma pseudovirions are adsorbed to, and uncoated by, host cells (Osterman et al., 1970) and that the monkey cell DNA in SV40 pseudovirions is able to enter the nuclei after infection of mouse embryo cells without loss of physical integrity (Grady e t al., 1970). Therefore, transduction should be possible. The SV40 pseudovirions associated with virus grown in monkey cells appear to be analogous to generalized transducing bacteriophages in that they appear to contain only host DNA and do not show a preference for the region of host DNA incorporated (Grady et al., 1970). Experiments designed to detect transduction by either rescued virus or pseudovirions cannot be carried out until a wide variety of genetic markers have been identified for the host cell. One interesting virus which should be considered with respect to the possibility that host cell DNA might be carried in virus stocks and express certain functions in infected cells is the MAC-adenovirus hybrid population (Butel et al., 1966). MAC (the monkey cell-adapting component) is defective and can replicate in simian cells only in the presence of a helper adenovirus because MAC is dependent upon the adenovirus for the provision of a capsid. In turn, MAC complements the human adenovirus so that it can replicate in simian cells. MAC can be transcapsidated to heterologous types of adenoviruses (Butel and Rapp, 1967). Although similar to PARA in many respects, MAC does not carry the genetic information for any known SV40 markers, including T-antigen (Butel and Rapp, 1967) and transplantation antigen (Rapp et al., 1967a). In addition, no SV40 DNA could be detected in cells infected with MAC-ftdcnovirus 7 (Butel, 1967). So, all available evidence indicates that MAC did not originate from SV40. Furthermore, sera which reacted with three different groups of simian adenovirus tumor antigens failed to react with GMK cells infected with MAC (Butel, 1967). Therefore, the origin of MAC remains obscure. However, it is conceivable that the defective genetic material of MAC is actually of host cell origin which, in some way, became associated with adenovirus type 7 under conditions of mutual dependence such that its persistence was assured.

36

B.

J. S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

STATE O F THE VIRAL

G ~ N O MI NE TRANSFORMED CELLS

Since all, or nearly all, transformed cells can produce infectious virus, but do so only rarely, what is the state of the viral genome in such cells? Assays of nucleic acid isolated from SV40-transformed cells have failed to reveal the presence of any infectious viral DNA (Sabin and Koch, 1963b; Kit et al., 1968). Reich et al. (1966) first reported that SV40-transformed hamster cells contain DNA with homology t o SV40 mRNA prepared in vitro. This complementary RNA bound to some extent to normal hamster cell DNA, but the extent of binding was greater in the transformed cells. Westphal and Dulbecco (1968) confirmed these observations using RNA prepared from highly purified component I of virus DNA as template and were able to estimate the number of viral DNA equivalents per transformed cell. They found a wide spectrum ranging from 7 equivalents in one SV40-transformed mouse cell line (SV3T3-56) to as many as 58 in a hamster tumor cell line (H-50). The estimated number of equivalents was stable for a given cell line even when grown under variable physiological conditions. A more recent report (Levine e t al., 1970), using a further refined hybridization system, lowered the estimate of SV40 viral DNA equivalents/cell in the H-50 cell line to 9. Other SV40-transformed cell lines contained 2-5 DNA equivalentsjcell. Although the estimates vary, these reports, plus an additional one (Tai and O’Brien, 1969), all indicated that there were apparently multiple copies of the SV40 genome per transformed cell. I n contrast, analysis of renaturation kinetics of DNA ( C o t values) resulted in an estimation of only 1-3 SV40 DNA equivalents per transformed cell (Gelb et ul., 1971). Such estimates, of course, do not indicate whether or not some of the “equivalents” might be defective (incomplete) viral genomes, especially since virus has never been rescued from the H-50 tumor cell line (Melnick e t al., 1964) in spite of trials in many laboratories. What, then, is the state of these multiple copies of viral nucleic acid within the transformed cells? Using the DNA-RNA hybridization technique and an SV40-transformed line of 3T3 mouse cells, Dulbecco and his colleagues (Westphal and Dulbecco, 1968; Sambrook et nl., 1968) established the following points about the physical state of the viral DNA: ( a ) It is in the nucleus; no hybridizable DNA was detected in cytoplasmic extracts. ( b ) It is associated with the cellular chruinosomes. Chromosomes were isolated from metaphase cells, and the DNA isolated from the chromosomes was shown to hybridize to the same extent as DNA from interphase cells. (c) I t is not in the form of circular molecules. After centrifugation to equilibrium in cesium chloride

PAPOVAVIRUS

SV40

37

in the presence of ethidium bromide, the hybridizable material was found in the cellular DNA band in the gradient. ( d ) It is not present in the form of free molecules the size of a single SV40 genome. The Hirt (1967) salt precipitation method, which separates large and small molecules of DNA, showed that the hybridizable regions were found in the precipitate of cellular DNA. ( e ) The viral and cellular DNA’s are not separated by alkaline denaturation and centrifugation in a sucrose gradient. The main conclusion to be drawn from these studies is that the SV40 DNA is apparently covalently bound to the chromosomal DNA of the transformed cell. The site or sites of insertion of the multiple copies of the genome are still unknown. The fluctuation in the rescue of infectious SV40 a t different passage levels of the same cell line suggests that the state of the viral genome might possibly vary in the transformed cells. If the rare cells which release infectious virus do so because the viral DNA is detached from the host chromosome, the quantity of viral nucleic acid present in a free state would be too small to be detected by the biochemical tests employed in the studies just described. I n addition to the above evidence which points strongly to the fact that the transforming viral DNA is predominantly, if not always, integrated into the host cell genome, a totally different type of evidence supports the same concept. Weiss et al. (1968) prepared somatic hybrids between mouse cells and SV40-transformed human cells. On passage of cells in culture, the chromosomes of human origin were gradually lost from the hybrid cells, and this gradual loss was correlated with a reduction of SV40 tumor antigen in the hybrid cells. The T-antigen was not compIetely lost until all or nearly all the human chromosomes had been lost. Hybrid cells which had lost T-antigen were capable of synthesizing the antigen after reinfection by SV40, thereby showing that the loss of T-antigen had not been due to the loss of a cellular gene required for the expression of the viral genome (Weiss, 1970). Karyological analyses of the hybrid cells failed to implicate a single specific integration site in the human chromosomes. Rather, the results suggested that the multiple viral genomes were scattered among various human chromosomes. Wherever the integration sites may be, there must be different ones for each virus, since both hamster and mouse cells can be doubly transformed by SV40 and polyoma virus (see Section II1,C). The use of interferon has also suggested the integration of the viral genome into that of the host cell. The induction of T-antigen by SV40 in 3T3 cells is sensitive to inhibition by interferon. However, serial passage of SV40-transformed 3T3 cells in the presence of interferon had

J . S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

38

no effect on the synthesis of T-antigen (Oxman et al., 1967). Previous investigations had established that fixation of the transformed state in the infected cell required only one cell generation (Todaro and Green, 1966b) and that treatment of the cells a t the time of infection with interferon would prevent transformation (Todaro and Baron, 1965). The use of synchronized cell cultures revealed that if the cells were not synthesizing DNA, transformation remained interferon-sensitive, but if the cells were rapidly synthesizing cellular DNA (S phase), transformation readily became interferon-resistant (Todaro and Green, 1967). Since there is evidence that interferon blocks translation of viral mRNA (Marcus and Salb, 1966; Joklik and Merigan, 1966), these observations can be explained by the assumption that the viral DNA becomes integrated into the cellular chromosomes during the S phase of cell growth. Once integrated, the viral mRNA is masked by attached regions of cell rnRNA so that interferon no longer recognizes i t as being viral in origin. C. TRANSCRIPTION OF THE VIRALGENOME IN

TRANSFORMED CELLS

The viral DNA shown by the above experiments to be present in SV40-transformed cells was obviously being transcribed to some degree. This was evident in that virus-specific antigens were associated with such transformed cells (Section IV). It was important to determine the degree to which transcription did occur. Benjamin (1966) first reported that a small fraction of pulse-labeled RNA from SV40-transformed cells was able to hybridize with SV40 DNA. Aloni et al. (1968) modified the hybridization technique and compared the virus-specific RNA in transformed mouse cells with the mcssenger RNA (mRNA) synthesized in BSC-1 cells productively infected with SV40. They found that mRNA formed during lytic infection prior to viral DNA synthcsis (“early” RNA) was different from the mRNA present after the onset of viral DNA replication (“late” RNA). Approximately one-third of the SV40 genome was represented in the early RNA whereas a t least 75% of the genome was represented in the late RNA. Competition experiments between virus-specific RNA from transformed cells and late RNA from infected cells suggested that only about onethird of the genome was transcribed in the transformed cells. Unfortunately, competition experiments between early RNA and transformed cell RNA were not technically feasible a t that time. However, they did show that the base composition of the virus-specific RNA in transformed cells was not identical to the basc composition of either the early or the late mRNA in productively infected cells. Oda and Dulbecco (1968) and Sauer and Kidwai (1968) both confirmed the existence of early and late mRNA after infection with SV40.

PAPOVAVIRUS

SV40

39

Although approximately the same proportion of viral genome was represented in early RNA and virus-specific transformed mouse cell RNA, the two types of RNA were not identical. A late gene seemed to be transcribed in the transformed cells. Similar results were obtained with transformed mouse cells, but approximately 80% of the SV40 genome appeared to be transcribed in a transformed green monkey kidney cell line. I n all cases, it appeared that the lack of expression of certain viral genes in transformed cells was at the level of transcription. A more recent study of the regulation of SV40 gene activity in transformed cells utilized randomly labeled lytic mRNA (M. A. Martin and Axelrod, 1969). A series of SV40-transformed mouse cell lines were analyzed. The extent of transcription in the individual lines varied, ranging from 30 to 100% of that seen during lytic infection. In the latter cell line, there must be some block beyond the point of transcription which prevents the synthesis of infectious virus. This study also emphasized that the extent of transcription of the SV40 genome is variable from one transformed cell line to the next, even within a single species of host cell. Of interest is a recent report (Sauer, 1971) which demonstrated that in an SV40-transformed green monkey kidney cell line, production of late viral mRNA sequences was not prevented when DNA synthesis was inhibited. This was in contrast to a productive infection by SV40 in which the inhibitors of DNA synthesis prevented the appearance of late mRNA. The mechanism(s) responsible for the apparent differences in transcriptional control is not known a t this time. High molecular weight heterogeneous RNA that contains virusspecific RNA occurs in the nucleus of transformed mouse cells (Lindberg and Darnell, 1970). Polysomal mRNA of lower molecular weight also contained virus-specific RNA. The authors suggested that the large nuclear molecules may be precursors of the cytoplasmic mRNA. Of particular interest is the fact that the largest nuclear molecules containing SV40 sequences were longer than one SV40 genome; therefore, either cellular RNA must be covalently linked to virus-specific RNA, or several SV40 DNA molecules must be integrated in tandem. The latter possibility was not supported by the chromosome data described above (Weiss et al., 1968; Weiss, 1970), but different transformed cell lines were employed in the two studies. If cellular mRNA regions are shown to be adjacent to the virus-specific sequences, this would be very formidable evidence in support of the concept of integration of the viral genome into that of the host cell. It would also strengthen the postulated rnechanism described above of the interferon resistance ( a ) of T-antigen synthesis in transformed cells and ( b ) of transformation after S phase growth of SV40-infected 3T3 cells. Evidence for such polycistronic

40

J. S. BUTEL, 6 . S. TEVETHIA, AND J . L. MELNICK

“viral-cell” RNA molecules has, in fact, been obtained with RNA from adenovirus 2-transformed rat embryo cells (Green et al., 1970).

D. BASISFOR LACKOF VIRUS PRODUCTION BY TRANSFORMED CELLS The preceding sections have established that the majority of SV40transformed cells, if not all, contain one or more copies of the viral genome, probably in an integrated state. Most cell lines can be induced to release small amounts of infectious virus, but only a small fraction of the individual cells in a given cell line appear to synthesize virus in such induction experiments. By far, the general rule is that the transformed cells do not produce infectious virus. What, then, is the mechanism by which transformed cells prevent the completion of the virus replicative cycle? This important question is central to the understanding of the mechanism of viral carcinogenesis and can be viewed from three different perspectives. ( a ) A virus-specific “repressor” is synthesized and the presence of this material in some way blocks the replicative cycle. ( b ) The transformed cells may lack some essential factor required by the virus for replication. ( c ) Defective viral genomes which are inherently unable to replicate because of a lack of certain genetic information may be the transforming agents (see Section II1,B) , It will be recalled that in Section II1,A a distinction was made between cells which were permissive and cells which were nonpermissive for SV40. Normal cells derived from nonpermissive species are not susceptible even to the naked viral nucleic acid (Swetly et al., 1969). Certain species of cells seem to lack essential factors required for SV40 replication and are, therefore, nonpermissive. Alternatively, a cellular “repressor” could be present, making the system nonpermissive. I n either case, it is meaningless at this point to say that transformed, nonpermissive cells are resistant to superinfection when the normal cells from that species cannot be productively infected. There are no data available to indicate whether the replication of a superinfecting SV40 genome in transformed cells is stopped p i o r to the block imposed by the “natural resistance” of the nonperrnissive cells. In contrast, permissive cells generally support viral replication. However, when such permissive cells are transformed (see Section II1,A) and cell lines are established, the lines tend to be virus free. These cell lines would, therefore, appear to be the most logical ones to study in an attempt to delineate which events determine whether a cell becomes transformed or productively infected following exposure to SV40. Based on the information currently available about transformed permissive

PAPOVAVIRUS

SV40

41

cells, it is difficult to distinguish between the alternate hypotheses proposed above, One can assume that since the parental cells were susceptible to SV40, proving that all essential replication factors were present, there must be some repressorlike substance present. However, since transformation of permissive cells is a rare event, such transformation may occur in those rare deficient cell mutants which lack the essential replication factors, and thus they are analogous to nonpermissive cells. Many of the SV40-transformed monkey kidney cell lines will not yield virus under any conditions which have been employed (Margalith et al., 1969; Rapp and Trulock, 1970; Fernandes and Moorhead, 1965; Shiroki and Shimojo, 1970; Koprowski et al., 1967; Butel et al., 1971b). These cell lines may well have been transformed by defective SV40 particles. However, some SV40-transformed monkey kidney cell lines, as well aa many SV40-transformed human cell lines, will yield infectious virus (Koprowski et al., 1967; Kit et al., 1970; Ashkenazi and Melnick, 1963). Therefore, one cannot conclude that permissive cells can be transformed only by defective particles. Cassingena and colleagues (1968, 1969) described the presence of a specific “repressor,” protein in nature, in cells of different species transformed by SV40, as well as in cells productively or abortively infected by SV40. This repressor was extracted from transformed cells by disrupting the cells by sonic oscillation, followed by removal of cell debris by centrifugation. The repressor in the supernatant fluid was found to inhibit plaque formation by SV40 in monkey kidney cells. The inhibitory activity could be detected only if polylysine or polyornithine was employed to increase the permeability of the host cell membrane to the extract. The repressor activity was specific for SV40; it had no effect on plaque formation by vesicular stomatitis virus, poliovirus, vaccinia, or herpes virus. The correct dilution of repressor was critical. The concentrated extracts did not show inhibitory activity. Maximum plaque reduction achieved in these tests a t the most potent dilution of extract was in the range of 50%. Conversely, other investigators, using a variety of approaches, have failed to obtain results compatible with the existence of a cytoplasmic virus-specific repressor. F. C. Jensen and Koprowski (1969) were able to rescue infectious SV40 when virus-free transformed hamster or mouse cells were fused with virus-free transformed monkey or human cells. If a repressor substance were present in the transformed cells, such completion of viral replication should not have occurred. It was found (Swetly et al., 1969) that transformed monkey and human cells were susceptible to infection by SV40 DNA although they were resistant to complete SV40 and that when uptake of virus was enhanced, a productive

42

J. S. BUTEL, S. S. TEVETHIA, AND J. L. MELNICK

cycle of infection proceeded (Barbanti-Brodano et al., 1970). Using a line of stable monkey kidney cells (BSC-1) transformed by SV40, Rapp and Trulock (1970) found it was resistant to superinfection by complete SV40 but supported the replication of a defective SV40 genome (PARA) enclosed in an adenovirus capsid. Experiments with transformed permissive cells in the authors’ laboratory (Butel et al., 1971b) have confirmed and extended those described above. Four monkey cell lines transformed by complete or defective SV40 were analyzed with respect to susceptibility to superinfection. A spectrum of susceptibility was observed. Two cell lines transformed by defective SV40 (defective T-antigen-inducing particles of SV40 and irradiated PARA-adenovirus 7, respectively) were sensitive to superinfection by complete SV40. The remaining two cell lines, transformed by complete SV40, were resistant to challenge by SV40. However, one did support the replication of SV40 DNA, confirming the observations of Barbanti-Brodano et al. (1970) that some transformed cell lines are resistant to SV40 apparently because of surface changes. However, the fourth cell line was markedly resistant to SV40 DNA, suggesting that alterations a t the cell surface are not the only explanation for resistance to superinfection in SV40-transformed cells. As Rapp and Trulock (1970) had observed with one cell line, all the transformed cell lines in this study were susceptible to superinfection by PARA. Experiments in the authors’ laboratory (Butel and Guentzel, 1971) have also confirmed the observation that extracts of SV40-transformed cells reduce plaque formation by SV40 in the presence of polyornithine. However, the same extracts, when added to SV4O-infected cultures every 6 hours in the presence of polyornithine had no effect on the kinetics of the viral replicative cycle. There was no change in the duration of the latent period, the rate of increase of infectious virus, the time a t which peak virus titers were obtained, or the final yield of virus from the cultures. Of course, i t is possible that an improper concentration of extract or improper multiplicity of infection (-1 PFUJcell) was employed in the growth studies which resulted in a negation of the repressor activity. The rescue of virus following fusion of transformed and susceptible cells is not the consequence of a phenomenon akin to “zygotic induction” in lysogenic bacteria. Wever et al. (1970) showed that when virus replication was activated by fusion of transformed cells with permissive monkey kidney cells, the initial site of synthesis was in the nucleus of the transformed cell. Only secondarily were the permissive cell nuclei in the heterokaryons infected with virus. The speculated series of events

PAPOVAVIRUS

SV40

43

which may lead to the rescue of infectious virus from an SV40-transformed cell is presented diagrammatically in Fig. 4. The technical problems involved in obtaining transformation of a permissive cell line by SV40 should be considered. If live virus is used initially, transformed cells which may arise but are not resistant to superinfection will eventually be destroyed by the virus in the culture. If inactivated (e.g., heavily UV-irradiated) virus is used to infect the cells, transformation would very likely be mediated by defective genomes. Consequently, technical problems are such that the transformed permissive cells currently available may not be representative of all the different types of transformation theoretically possible. Experimental conditions would tend to select those cells resistant to superinfection or those transformed by defective viruses. NORMAL SUSCEPTIBLE S V 4 0 - TRANSFORMED CELL UYYYYY HOST CELL CHROMOSOME

-m VIRAL DNA x

ESSENTIAL FACTOR OR ANTIREPRESSOR VIRUS PARTICLE

@@ ( 1 ) Cell fusion mediated by inactivated Sendai Virus

( 7 ) Release of rescued infectious virus

Secondary cycle of sv40 reDlication in normal cell nucleus

( 2 ) Transfer of ( a ) essential factors , or ( b ) antirepressor from normol cell to transformed cell

t

(3)Release of viral oenome J trom integration-

G (J& i-ji

( 5 ) Transfer of ( a ) virus or ( b ) viral DNA from Ironsformed cell nucleus to normal \ cell nucleus

/(4)

Replication of S V 4 0 in tronsformed cell nucleus

F I ~4.. Speculated series of events which lead to rescue of infectious virus from SV4O-transformed cells.

44

J . 8. BUTEL, 8. 8. TEVETHIA, AND J . L. MELNICK

One can visualize that an entire spectrum of transformed permissive cells should be obtainable, the variation noted by Butel et al. (1971b) being only part of that spectrum. If particles defective a t different points in the genome were to mediate transformation, the resulting transformed cell lines should exhibit variations in properties, such as differences in susceptibility to superinfection by virus or viral nucleic acid or differences in the extent of surface changes. The realization of these many variables makes it difficult to generalize since very few transformed permissive cell lines have been studied in detail. At this time, the diverse available evidence can best be synthesized in the following way: The nonpermissive cells lack some essential factor required for virus replication. These factors or, alternatively, an antirepressor for the nonpermissive cell-eoded repressor, may be supplied by permissive cells following fusion and heterokaryon formation, so that virus rescue can occur. Transformed permissive cells may possess a block which prevents the replication of the resident genome. This may well be some type of repressor substance, but it does not behave in tests like a lambda bacteriophage repressor molecule. Defective viral genomes may well be responsible for some observed cases of transformation. Obviously, this area, which is critical to our understanding of transformed cells, warrants further attention. A series of temperature-sensitive mutants defective in different viral functions would be one logical approach to the problem.

E. CONSIDEXUTIONOF WHETHER THE VIRALGENOME Is ESSENTIAL FOR MAINTENANCE OF TRANSFORMATION The strongest evidence in support of an affirmative answer to the above question is G. S. Martin’s t s mutant of RSV described in Section II1,D. His results (1970) strongly suggest that a viral gene not involved in the replication of the virus is required for the maintenance of the transformed state. This separation of replication and transformation by a tumor virus is supported by other evidence. Also working with RSV, both Gold6 (1970), using SR-RSV-D, and Toyoshima et al. (1970), using B77, irradiated the parental viruses and succeeded in isolating virus variants unable to transform cells but able to replicate well. I n addition, they also detected defective variants of virus able to transform cells but unable to replicate. While neither group was able to positively rule out the possibility that the nontransforming virus was actually a contaminating avian leukosis virus preexistent in the parental RSV stock, each felt that was an unlikely explanation and favored the interpretation that the virus-producing and cell-transforming capabilities of RSV had been separated.

PAPOVAVIBUS SV40

45

I n Section III,D, examples of complete reversion of polyoma and RSV-transformed cells were described which appeared to be due to a loss of viral genes. Those reports would, therefore, suggest that the presence of a t least part of the viral genome is required for the maintenance of the transformed state. The vast majority of SV40-transformed cells synthesize virus-specific antigens (Section IV) and virus-specific mRNA(Section V,C) and frequently yield virus under proper conditions (Section V,A) , leaving little doubt that viral information is, indeed, present. There are a few perplexing exceptions, however, which preclude any simple conclusions that the persistence of viral genetic material is obligatory to maintain transformation. Diamandopoulos and Enders (1965) reported that lung and liver cells from Syrian hamsters underwent transformation following exposure to SV40, but no T-antigen or infectious virus could be detected. These results were subsequently confirmed by others (van der Noordaa and Enders, 1966; Sahnazarov et al., 1970; Nachtigal and Butel, 1970). Hamster embryo fibroblasts were used in analogous experiments and four of seven virus-exposed cultures developed SV40 S-antigen while only two synthesized SV40 T-antigen (Diamandopoulos et al., 1968). The S+Tcells were found to lack detectable levels of SV40 transplantation antigen (Tevethia et al., 1968b), complementary RNA (Levin et al., 1969b), and viral DNA (Levine et al., 1970). The specificity of the S-antigen reaction was discussed in Section IV,C. These studies are pertinent in that we are faced with cells which transformed following exposure to SV40, but which lack demonstrable amounts of viral DNA or complementary RNA. Until such time as it is definitely ruled out, the possibility exists that exposure to SV40 activated a latent hamster tumor virus (Huebner and Todaro, 1969). Another possible mechanism is that the virus mediated a somatic mutation of the cell genome. However, Braun (1970) considered this possibility in depth and, after showing the diversity of tumors able to revert to normal phenotype, concluded that mutation was an unlikely explanation for neoplasia. Two experimental approaches are readily apparent which could be pursued to answer this question. One would be the use of temperaturesensitive mutants of SV40 to delineate one or more specific viral genes required for the maintenance of transformation. Cells transformed by such mutants should revert to normal under restrictive conditions where the temperature-sensitive gene product would be inactive. A second approach would be to study one of the cell types, such as Syrian hamster lung cells which exhibit the unique response to SV40.

46

J. S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

The key question is whether the observed cases of transformation were actually mediated by SV40 in spite of the absence of T-antigen in the transformed cells. After transformation by PARA (defective S V N ) , hamster lung cells are capable of synthesizing SV40 T-antigen (Nachtigal and Butel, 1970). These results demonstrate that there is no intrinsic host cell block which prevents the expression of this virus-specific marker. It is possible that several different types of virus-host cell interactions can occur after exposure to SV40. Once the spectrum of virushost interactions is established, the next step would be to determine the relative significance of each type of interaction. The type of interaction most common in the SV40-hamster system might be least common in the human system, and vice versa. It is, of course, critical for both the diagnosis and treatment of human tumors to know whether the persistence of the viral genome is required for maintenance of transformation. VI. Conclusions and Summary

This chapter has attempted to evaluate the available knowledge pertaining to SV40 oncogenesis. A summary of the different tests which can be employed to detect virus-induced changes in SV40-transformed cells is given in Fig. 5. Inoculation of such cells into susceptible hosts usually results in the production of tumors, the ultimate criterion necessary to establish that malignant transformation has indeed occurred. No in vitro test is known that will accurately predict whether a cell with all the properties of a transformed one will actually be transplantable

in vivo. Fusion or cocultivation of the transformed cell with normal susceptible cells may sometimes succeed in the rescue of infectious virus. The optimum conditions required to elicit production of virus from a majority of the transformed cells have not yet been established. Transformed cells are usually immune to superinfection by the transforming virus. The mechanism underlying this resistance to SV40 requires further delineation. Nucleic acid hybridization experiments indicate that multiple copies of the SV40 genome are present, probably integrated into the host cell chromosome. The integration sites are not known. Virus-specific mRNA is present in the transformed cells. The extent of transcription of the viral genome seems to vary from one transformed cell line to the next. No relationships have been established yet between the extent of transcription in a given transformed cell line and the number and/or magnitude of virus-specific changes of that cell line. Another in vivo test, the transplant rejection test, serves to detect

PAPOVAVIRUS

A. Intracellular changes 1. T-antigen a. Immunofluoreacence b. Complement fixation 2. Viral DNA

a. Hybridization

3. Viral-specific mRNA a. Hybridization

4. Infectious viral genome a. Rescue by fusion 5. Immunity to SV40

a. Superinfection

SV40

47

B. Cell surface changes 1. Santigen a. Immunofluoreacence b. Mixed hemadsorption c. Colony inhibition d. Cytotoxicity 2. TSTA a. Immunogenicity b. ImmunoseIlsitivity c. Neutralization by immune 1YmPhOCY~ d. Colony inhibition? 3. Normal cell antigens a. Immunofluorescence b. Mixed hemadsorption 4. Agglutination sites a. Concanavalin A b. Wheat-germ agglutinin 5. Embryonic antigens a. Immunofluoreacence 6. Immunity to SV40 a. Superinfection

FIG.6. Methods of detection of virus-induced changes in SV-40-transformed cells.

the presence of the tumor-specific transplantation antigen in the transformed cell. When this antigen is present, the cell is incapable of growing in animals immunized by the transforming virus. A series of in vitro tests detect a variety of surface changes on the transformed cells. These tests include immunofluorescence, agglutination, cytotoxicity, colony inhibition, and mixed hemadsorption. An important goal is to develop an in vitro test which can detect TSTA, and the colony inhibition test seems to be closest of those available. The relationships between the surface changes detected on SV40-transformed cells by the various tests are discussed in Section IV,D and summarized in Table 111. Results obtained using the tests outlined above enable us to visualize a transformed cell, as shown in Fig. 6. The transforming genome is integrated into the cellular chromosome in the nucleus. SV40-specific mRNA is found in both the nucleus and cytoplasm. T-antigen is synthesized and localized in the nucleus. A virus-specific repressor may or may not be present. If such repressor molecules do exist, they could conceivably be in either the nucleus or the cytoplasm or in both places. A multitude of changes occur a t the cell surface, including the appearance of TSTA, S-antigen, agglutination sites, embryonic antigens, and

48

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J. S. BUTEL, S. S. TEVETHIA, AND J. L. MELNICK

HOST CELL CHROMOSOME

0

Hm VIRAL DNA

0

it

8

\ .

VIRAL mRNA 1 - A NTI G EN REPRESSOR SWO VIRUS PARTICLE

UNCOVERED NORMAL CELL ANTIGENS

sANTIGEN CI

MALIGNANT

AGGLUTINATION SITES

Fia. 8. Diagrammatic comparison of normal and SV40-transformed cells.

normal cell antigens. The cell is immune to superinfection by SV40. Finally, it may be malignant. By comparison, all these changes are absent or masked in a normal cell, which in addition, is susceptible to infection by SV40. In spite of many efforts, the virus-mediated change which is the key to the neoplastic conversion of a normal cell into a cancer cell remains unknown at this writing. It may be one or a combination of the changes detected in the tests described in the preceding sections of this chapter, or it may be an event which has escaped detection so far. The last few years have seen a large increase in the storehouse of. knowledge pertaining to properties of papovavirus-transformed cells. Preliminary results obtained with mutant viruses and variant transformed cells, coupled with the advent of new tests to detect virusinduced changes, suggest that key events related to virus-induced carcinogenesis are on the verge of becoming unraveled.

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Rhim, J. S., Greenawalt, C., Takemoto, K. K., and Huebner, R. J. (1971). Nature New Biol. 230, 81-83. Richardson, L., and Butel, J. S. (1971). Int. J. Cancer 7, 75-85. Robertson, H. T., and Black, P. H. (1969). Proc. SOC.Exp. Biol. Med. 130, 363-370. Rothschild, H., and Black, P. H. (1970). Virology 42, 251-256. Rowe, W. P., and Baum, 8. G. (1964). Proc. Nat. Acad. Sci. U . S. 52, 1340-1347. Rowe, W. P., and Baum, S. G. (1965). J. Exp. Med. 122,955-968. Rowe, W. P., and Pugh, W. E. (1966). Proc. Nat. Acad. Sci. U . S. 55, 1126-1132. Sabin, A. B., and Koch, M. A. (1963a). Proc. Nat. Acad. Sci. U. S. 49, 304-311. Sabin, A. B., and Koch, M. A. (196313). Proc. Nat. Acad. Sci. U. S. 50, 407417. Sabin, A. B., and Koch, M. A. (1964). Proc. Nat. Acad. Sci. U. S. 52, 1131-1138. Sachs, L. (1966). Nature (London) 107, 12721274. Sahnazarov, N., GraEe, L. H., Nachtigal, M., and Ionescu-Homoriceanu, S. (1970). R e v . Roum. Injramicrobwl. 7, 79-85. Sambrook, J., Westphal, H., Srinivasan, P. R., and Dulbecco, R. (1968). Proc. Nat. Acad. Sci. U.S. 80, 1288-1295. Sauer, G. (1971). Nature New Biol. 231, 135-138. Sauer, G., and Kidwai, J. R. (1968). Proc. Nat. Acad. Sci. U. S. 61, 125f3-1263. Sauer, G., Koprowski, H., and Defendi, V. (1967). Proc. N a t . Acad. Sci. U. S. 58, 594806. Shein, H. M. (1968). Science 159, 1476-1477. Shein, H. M., and Enders, J. F. (1962). Proc. Nat. Acad. Sci. U. S. 48, 1164-1172. Shein, H. M., Enders, J. F., Levinthal, J. D., and Burket, A. E. (1963). Proc. Nal Acad. Sci. U.S. 49, 28-34. Shiroki, K., and Shimojo, H. (1970). Personal communication. Sjogren, H. O., Hellstrom, J., and Klein, G. (1961). Exp. Cell Res. 23, 204-208. Smith, R. W., Morganroth, J., and Maor, P. T. (1970). Nature (London) 227, 141-146. Sweet, B. H., and Hilleman, M. R. (1960). Proc. SOC.E z p . B i d . Med. 105, 420-427. Swetly, P., Barbanti-Brodano, G., Knowles, B., and Koprowski, H. (1989). J. Virol. 4, 348-356. Tai, H. T., and O’Brien, R. L. (1969). Virology 38, 698-701. Takemoto, K. K., and Habel, K. (1966). Virology 30, 20-28. Takemoto, K. K., Ting, R. C., Ozer, H. L., and Fabisch, P. (1968a). J. Nat. Cancer I m t . 41, 1401-1409. Takemoto, K. K., Todaro, G. J., and Habel, K. (1968b). Virology 31, 1-8. Tevethia, S. 5. (1970). J. Immunol. 104, 7278. Tevethia, S. S. (1971). Unpublished observations. Tevethia, S. S., and Rapp, F. (19as). Proc. SOC.Exp. B i d . Med. 123, 612-615. Tevethia, S. S., Katz, M., and Rapp, F. (1965). Proc. SOC.Ezp. B i d . Med. 119, 896-901. Tevethia, S. S., Couvillion, L. A,, and Rapp, F. (1968a). J. Immunol. 100, 3!%362. Tevethia, 6. S., Diamandopoulos, G. T., Rapp, F., and Enders, J. F. (198813). J. Immunol. 101, 11924198. Tevethia, S. S., Dreesman, G. R., Lausch, R. N., and Rapp, F. (196%). J. Immunol. 101, 1105-1110. Tevethia, S . S., Crouch, N. A., Melnick, J. L., and Rapp, F. (1970a). Int. J . Cancer 5, 176-184. Tevethia, S. S., Lausch, R. N., and Rapp, F. (1970b). In “Immunity and Tolerance in Oncogenesis,” pp. 183-199. 4th Quadrennial Int. Conf. Cancer, Perugia, Italy.

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NASOPHARYNGEAL CARCINOMA (NPC) J. H. C. Ho Medical and Health Department Institute of Radiology, Queen Elizabeth Hospital, Kowloon, Hong Kong

I. Introduction . . . . 11. Histogenesis . . . . 111. Etiology . . . . . A. Early Cases . . . B. Genetic Factors . . C. Environmental Factors IV. Conclusion . . . . References

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78 88 89

I. Introduction

Nasopharyngeal carcinoma (NPC) has recently attracted worldwide interest because a high titer of antibodies to Epstein-Barr virus (EBV), which is suspected to have oncogenic properties, has been found with unusual frequency among patients with this neoplasm compared with controls from the general population and also with patients with head and neck tumors other than NPC (de Schryver et al., 1969; W. Henle et al., 1970). It is of interest also that immune responses by the host against tumor-associated antigens have been shown in a number of neoplasms including NPC (Klein, 1970). This neoplasm is rare among most groups of people, but Chinese, particularly those originating from the southern province of Kwangtung, have a very high risk of developing the disease. Lilly (1966) has shown that in inbred strains of mice the incidence of lymphomas and leukemias is linked to a genetically determined susceptibility to tumor induction by oncogenic viruses. The discovery of an association between a virus infection and NPC which has a predilection for an ethnic group has, therefore, stimulated much interest in the nature of the association, which may shed some light on the tantalizing question whether viruses, which are now known to give rise to a variety of tumors in certain mammals and birds, also cause cancer in man. It is the purpose of this chapter to review the etiology of NPC. It. Histogenesis

Many types of nasopharyngeal cancers have been found, but carcinoma is by far the predominant type in people of all races. I n China 57

58

J. H. C. H O

and Southeast Asia, where the incidence is exceptionally high, a t leaat 99% of the malignant neoplasms have been reported to be carcinomas (Liang, 1964; Yeh, 1967; Shanmugaratnam and Muir, 1967). In Australia, Scott and Atkinson (1967) detected essentially no difference in the histopathological distribution of nasopharyngeal cancers among Caucasian and Chinese patients. In their series of 213 histologically proven cases of malignant neoplasms, 202, or approximately 95% of them, are carcinomas. I n America, where the incidence of nasopharyngeal cancers is low, the ratio of carcinoma to sarcoma is between 8 : l and 9 : l (Fletcher and Million, 1965; Vaeth, 1960). I n England, also a lowincidence area, the ratio of carcinoma (epithelioma including lymphoepithelioma) to sarcoma is 3.5:l in a series of 208 cases with known histology reported by Lederman (1961). Also in Kenya, Africa, carcinoma is the predominant neoplasm (Clifford, 1965). Table I shows the histological types of neoplasms arising primarily in the nasopharynx of 1606 cases diagnosed a t Queen Mary Hospital, Hong Kong, during the 5-year period 1959-1963. More than one case of reticulum cell sarcoma was diagnosed initially, but later development showed all of them, except one, to be undifferentiated squamous carcinoma. It is anticipated that, if electron microscopy is used routinely, there will be a reduction in the frequency of nasopharyngeal lymphoreticular neoplasms being diagnosed, a t least in Chinese patients. Svoboda et al. (1967) have shown by electron microscopy that the undifferentiated carcinomas, including the so-called lymphoepithelioma, often show evidences indicative of their squamous origin. Although similar evidences-desmosomes and cytoplasmic filamentous inclusionsare occasionally encountered in malignant lymphoma and chordoma (Friedman, 1967), the mere fact that most nasopharyngeal carcinomas

TABLE I HISTOLOQICAL TYPESOF NASOPHARYNQEAL CANCERS DIAQNOSED AT QUEENMARYHOSPITAL (1959-1963) No. of

% Carcinomas (squamous, undifferentiated and anaplastic) Malignant melanoma Reticulum cell sarcoma Not histologidly confirmed

From Ho (1972), by permission from the editor.

1571 2 1 32

98 2

-

-

1606

100

NABOPHARYNGEAL CARCINOMA (NPC)

59

contain more than one histological type of malignant cells in the same tumor and that typical squamous features are not uncommonly found in sections among predominantly undifferentiated carcinoma cells has led to the belief, which is now generally held, that the undifferentiated carcinoma including lymphoepithelioma are histogenetically variants of squamous carcinoma, the predominant neoplasm in the nasopharynx. It is in patients with this neoplasm, and not others, arising in the nasopharynx that an association with EB virus infection has been discovered, and also it is this cancer that has a predilection for people of Chinese descent. Ali (1967) in his study based on the histological examination of nasopharyngeal mucous membranes obtained at 100 medicolegal autopsies from apparently healthy individuals between ages of 10 and 80, found that under normal conditions only 60% of the total nasopharyngeal epitheIia1 surface was lined by stratified squamous epithelium and that the proportion of squamous epithelium in the nasopharynx appeared to be constant after the first decade of life. Liang et al. (1962) proposed, “Squamous metaplasia would be a prerequisite for the formation of different types of nasopharyngeal carcinoma.” This hypothesis is based on their finding that among 54 cases of malignancy (including precancerous change, carcinoma in situ, and early invasive carcinoma) arising from the nasopharyngeal mucosa, 27, or 50%, arose from the squamous epithelium of the mucosa, and that at the frequent site of carcinomatous origin, i.e., the superior two-thirds of the nasopharynx, the mucosa was normally lined by cylindrical cell epithelium. Further, in their histological examination of 300 complete nasopharyngeal mucosa specimens of cadavers above the age of 15 years, Liang et al. found that 129 specimens showed multiple small foci of squamous metaplasia. They, therefore, held squamous metaplasia to be of great significance in the histogenesis of NPC. On the other hand, they stated in the same paper that, with the exception of cylindrical cell carcinoma which arose from the surface cylindrical cell epithelium of the nasopharyngeal mucosa (including the lining of the crypts), all types of nasopharyngeal carcinoma (including squamous cell carcinoma) could arise either from the cylindrical cell epithelium or from the squamous cell epithelium. This finding was confirmed by Ch’en (1964a) from a study of 90 biopsies of the nasopharyngeal mucosa which showed malignant change. In another study Ch’en (1964b) found that anaplasia could start from a single cell or from a group of cells in the undifferentiated basal layer of the mucosal epithelium, or in the middle layer already beginning to differentiate, or in the well-differentiated superficial layer. Shanmugaratnam and Muir (1967) also found that all forms of nasopharyngeal carcinoma, both

60

J. H. C. H O

classical squamous cell carcinomas and undifferentiated carcinomas, might arise from squamous, transitional, or respiratory epithelium. Experience in Hong Kong is in agreement (Ho, 1972). It would appear, therefore, that carcinoma could start de novo without the cells first undergoing squamous metaplasia. Multicentricity of foci of carcinomatous change is not uncommonly seen in Hong Kong cases. Occasionally a new carcinoma may even appear on the opposite side of the nasopharynx from one to a few years after a course of radiotherapy which appeared to have caused the original tumor to disappear. These are unlikely to be recurrences of tumors too small to be noticed during the initial examination, because the smaller the tumor the greater is the chance of its being eradicated or controlled, and in the radiation treatment of NPC the whole nasopharynx is as a rule given the same high dose. Such new carcinomas raise a very important question: If radiotherapy had no prophylactic effect on the process of carcinomatous transformation in these cases, did it play any part in causing or accelerating the transformation, since radiation is known to be carcinogenic? The short interval between irradiation and the development of an apparently new carcinoma does not exclude such a possibility since it is not unlikely that other parts of the nasopharyngeal mucosa might already be primed by the time the first carcinoma was treated or already in the late stage of carcinomatous transformationprecancerous stage, in which case the interval can be very short. A similar example is to be found in the radiation treatment of carcinoma of the buccal mucosa. A new carcinoma may develop in the originally normal looking buccal mucosa on the opposite side, which already had received a good dose of radiation when the first tumor was treated. That a radical course of radiation treatment has little or no prophylactic effect on cells at a certain phase of carcinomatous transformation, a t least in some cases, is beyond doubt. It is, however, of importance to ascertain whether it has any promoting effect on the malignant change. Animal experiments designed to study the effect of radiation on cells at different stages of malignant transformation might help in producing an answer. 111. Etiology

A. EARLY CAB= It is not known whether NPC is largely a product of our environment within the last one and a half centuries or has afflicted man since antiquity, as has been claimed by Clifford (19701, who thinks that the oldest pathological specimens of NPC at present known were derived from

NASOPHARYNGEAL CARCINOMA (NPC)

61

inhabitants of Northeast Africa and the Middle East from the period 3500-3000 B.c., on the basis largely of the works of Wells (1963, 19641, Krogman (1940), Smith and Dawson (1924), and Derry (1909). The most important evidence is supposed to be found in the works of the first three, but close scrutiny of these works reveals considerable doubt that they are indicative of NPC, and not of other diseases. Wells (1964) stated that only 3 or 4 cases of indubitable carcinoma have been recognized among the tens of thousands of ancient Egyptian mummies and skeletons that have been examined, and that hardly a score of such cancers have been identified from all the cemeteries of the pre-Renaissance world. He thought that several of this very small number appeared to be of nasopharyngeal origin. Through the courtesy of Dr. J. C. Trevor, Director of the Duckworth Laboratory, and the kind cooperation of Professor J. Mitchell and Mr. J. A. Fairfax Fozzard of the University of Cambridge, the author had the opportunity to examine radiographs and photographs of probably the most important specimen, a skull (No. 236) kept a t the Duckworth Laboratory. This specimen shows only destruction of the posterior part of the left maxillary alveolus forming the floor of the left maxillary sinus, the adjacent part of the hard palate and pterygoid laminae, with antemortem loss of the 2nd and 3rd molars. These findings are shown in Figs. 1 and 2. The destruction is indicative rather of carcinoma or myeloma of the maxillary alveolus or of the floor of the maxillary sinus than of NPC, as the destroyed parts are situated in front of the nasopharynx, whereas the bones immediately overlying the nasopharyngeal cavity, which are the ones most commonly involved, were spared. Furthermore, the multiple circular holes in the tables of the cranial vault unaccompanied by any evidence of sclerotic bony reaction around them are far more typical of myelomatosis than NPC metastases. A “strongly probable one” according to Wells (1964) was from Tepe Hissar, Iran, ca. 3000 B.C. (Krogman, 1940). The specimen referred to is 33-23-36, the skull of a male of Mediterranean type from Hissar 11. It shows extensive destruction of the left facial bones-maxilla, palatine, and zygoma. There is an extension of the destruction to the floor of the left orbit. The left maxillary sinus is completely obliterated; the floor of the left nasal fossa is penetrated, and the penetration extends to the right of the midline. All upper teeth on the left side and the right central incisor are missing. The mandible is, however, intact, a fact which, according to Krogman, testifies against injury being the cause. He stated: “It may be conjectured whether the condition is primarily due to sinus infection, brought about by dental disease.” There was no description of destruction of bone in the immediate vicinity of the naso-

62

J. H. C. H O

Fro. 1. Photograph of skull specimen No. 236. Dotted line indicates the nasopharyngeal boundaries on the right side (R). Destruction of bone is limited to the floor of the left maxillary sinus and adjacent hard palate and pterygoid laminae.

NASOPHARYNGEAL CARCINOMA ( N P C )

63

FIG.2. Superoinferior X-ray projection of skull specimen No. 236. Dotted line indicates the nasopharyngeal boundaries on the right side (R).Site of lesion is snowed.

pharynx. There are, therefore, no grounds to interpret from these findings that the primary condition could be nasopharyngeal carcinoma. Wells (1964) thought that one of the Romano-Egyptian cases described by Smith and Dawson (1924) was another example of carcinoma of nasopharyngeal origin. The case referred to was reported to be a male

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J. H. C. HO

pre-Christian Nubian (ca. fourth to sixth century, A.D.) with extensive destruction of the base of skull from the cribiform plate to the basiocciput almost reaching the foramen magnum. The site and appearance of the destruction are consistent with a diagnosis of either carcinoma of the sphenoid sinus or nasopharyngeal carcinoma. It is, however, difficult and sometimes impossible to differentiate between the two even in examining patients. Judging from the evidence presently available, we are still far from sure whether nasopharyngeal carcinoma had afflicted human inhabitants of Northeast Africa and the Middle East some 5000 years ago. Since NPC is a prevalent disease in China, and in the province of Kwangtung has even earned the name of “Kwangtung tumor,” it would be pertinent to search old Chinese medical writings for reference to the disease. In a book of 50 volumes called, “Aetiology and Symptomatology of Various Diseases” written by CHOU Uen Fung or CHOU Yuan Fang ( # $ $), a royal physician of the Sui Dynasty (A.D. 589-617), there is a description of various types of superficial tumorous swellings. Only in “shu-lu,” or rat tumor, was there a description of the disease appearing in the neck with its root in the lungs. Jung and Yu (1963) found it impossible to determine whether all, some, or none of the cases of “lo li” (glandular enlargement of the neck) were carcinomatous metastases rather than tuberculosis or other diseases. In The Encyclopaedia of Chinese Medical Terms edited by Wu (1921) a disease, called “shih ying” or (‘shih jung” disease, meaning, respectively, loss of nutrition is described as belonging to one of the four fatal diseases and has the following clinical picture: Masses appear in front of and behind the ear and in the neck. They are stony hard, immobile, neither hot nor cold to the touch and painless at first. As they grow in sire pain gradually appears and fungation sets in. The discharge is sero-sanyineous but not pussy. Ultimately, a crater lined with necrotic tissue is formed. By this time pain becomes intense. Brisk bleeding from the ulcer soon occurs and the patient may die of haemorrhage after 1 or more bleeding episodes or of gradual loss of nutrition.

There is no doubt from this description that the masses are metastatic, not tuberculous, lymph nodes, and that they are most likely caused by a primary carcinoma in the nasopharynx. No mention is made in this encyclopedia as to when this disease was first reported in Chinese medical writings. Now that the name of the disease is known, the task of searching for such information is made easier. It is hoped that workers in mainland China and Taiwan, where one can get access to old Chinese medical writings, will do some work in this direction.

65

NASOPHARYNGEAL CARCINOMA ( N P C )

B. GENETICFACTORS 1. Sex Incidence In most countries the male to female incidence ratio is over 2:1, but in Sweden it is below 2:l. The crude and age-standardized annual incidence rates of nasopharyngeal carcinoma (with all other neoplasms of the nasopharynx excluded) for different parts of the world where such figures are available are given in Table I1 for comparison. TABLE I1 SEX INCIDENCE RATIOOF NABOPHARYNOEAL CARCINOMA Crude rates per 100,000 Population HongKong (Chinese) Singapore (Chinese) Sweden

Age-standardized rates per 100,OOO

Male

Female

Ratio M:F

Male

Female

Ratio M:F

20.68

9.42

2.19:l

24.66

10.36

2.38:l

1968

-

-

-

22.2

8.9

2.49:l

1959-65

0.76

0.49

0.30

1.86:l

Period 1965-9

1.55:l

0.56

2. Racial Susceptibility Nasopharyngeal carcinoma is rare in most parts of the world except in China and many parts of Southeast Asia inhabited largely by people of mongoloid stock who have had close relationship with Chinese for many centuries dating back to the early Ming dynasty in the 14th century. Although Japanese and Koreans are also mongoloid people and have had an even longer period of association with Chinese, incidence among the Japanese is rare (Miyaji, 1967), and probably this applies also to the Koreans (Clifford, 1970). Although the frequency of the disease is highest among people of Chinese descent both in and outside China, in China itself there is a drop in frequency from south to north according to the relative frequency expressed as percentages of all malignant tumors diagnosed by biopsies reported by major hospitals and medical schools in certain cities and provinces. This is illustrated in Table I11 and Fig. 3. The difference in frequency in different parts of China could be due to geographic reasons or to ethnic differences. It is, therefore, important to determine whether Chinese originating in different parts of China and living in the same locality have any significant differences in susceptibility. Hong Kong is not the ideal place for such a study because of the

J. H. C. H O

66

TABLE 111: RELATIVE FREQUENCY OF NABOPIIARYNGEAL CARCINOMA I N CHINESE MAINLAND AND TAIWAN ESTIMATED AS PERCENT OF ALL MALIQNANT TUMORS DIAGNOSED BY BIOPSY Percent of all cancers by biopsy

Male

Female

56.9 of 3010 3 1 . l o f 672

17.4 of 4O26 10.0of 748

16.2 of 1010 23.2 of 5436 7 . 3 of 8332 6 . 7 o f 879

4 . 7 of 1068 6 . 2 of 8636

5 . 1 of 2738 7 . 9 of 1562 4 . 0 of 5137

-

Place South Canton (Kwangtung province) Kwangei province Central Fukien province Taiwan Shanghai (Kiangeu province) Sian (Shensi province) North Tsinan (Shangtung province) Tientein (Hopei province) Peking

-

-

From Ho (1972), by permission from the editor.

constant influx of refugees. However, this influx became insignificant after 1962. The incidence rates by place of origin for Chinese males and females domiciled in Hong Kong in 1969 are given in Tables IV and V. Excluded in the analysis are patients who came from outside Hong

IOOOE

I 10°E

FIG.3. Map of China.

I2OOE

1309

67

NASOPHARYNQEAL CARCINOMA (NPC)

INCIDENCE RATES OF &INESE

Place of origin

Hong Kong Canton, M a w , and adjacent places Sze Yap area Chiu Chau area Elsewhere in Kwangtung and Kwangsi Elsewhere in China outside Kwangtung or Kwangsi

TABLE IV NASOPHARYNQEAL CARCINOMA FOR HONQKONQ &LEB BY PLACE OF ORIQIN, 1969 Population 1966 byNo. of c e n s u ~ ~ CBBB~C

Crude rate per100,OOO

Agestandardized rateper100,000

120,620s 875,530

1od 182

8.29 20.78

15.05 25.69

355,650 214,410 115,440

68 26 28

19.11 12.12 24.25

25.27 17.66 28.54

137,140

11

8.02

10.99

__

,, Barnett (1966). b Include an unknown number of non-full-blood Chinese. c Include only cases seen at Medical and Health Department Institute of Radiology. All full-blooded Chin-.

Kong for medical consultation or treatment. The rates for people of Chiu Chau origin of both sexes are significantly lower than those for people who claim origin from other parts of Kwangtung with Hong Kong excluded ( P < 0.01). The people from Chiu Chau are ethnically more TABLE V INCIDENCE RATESOF NASOPHARYNOEAL CARCINOMA FOR HONQKONQ CHINESE FEMALES BY PLACE OF ORIGIN, 1969

Place of origin

Hong Kong Canton, M a w and adjacent p l a w Sze Yap area Chiu Chau area Elsewhere in Kwangtung and Kwangsi Elsewhere in China outside Kwangtung or Kwangsi

Population 1966 bycensus0

No. of cases0

Crude rate per 100,OOO

Agestandardized rate per 1 0 0 , ~

124,63@ 875,110

6d 78

4.81 8.91

9.56

347,210 184,230 103,240

23 3 9

6.62 1.62 8.71

7.40 2.17 10.29

143,300

4

2.79

3.92

6.54

Barnett (1966). Include an unknown number of non-full-blood and Chinese. 0 Include only cases seen at Medical and Health Department Institute of Radiology. d All full-blooded Chinese. 0

b

68

J. H. C. H O

closely related to the people from Fukien than those from other parts of Kwangtung. The rates for people originating from parts of China outside Kwangtung and Kwangsi are significantly lower than those for people from Kwangtung and Kwangsi with Chiu Chau and Hong Kong excluded ( P < 0.01 for males and P = 0.025 for females). Hong Kong was a part of Kwangtung until it was ceded to Great Britain in 1841. These findings are in general agreement with those obtained in an earlier study on the incidence of nasopharyngeal cancer in the Chinese population of Hong Kong in 1961 (Ho, 1967a) and also with those reported by Mekie and Lawley (1954) on the Chinese population in Singapore. They found that the Teochews (people from Chiu Chau) and the Hokkienese (people from Fukien province) had lower frequencies of nasopharyngeal cancer than the Cantonese, Kheks, and people from Hainan Island. The Kheks are people scattered in different parts of China, but the majority of them are from Kwangtung. Muir and Shanmugaratnam (1967) have shown that in multi-racial Singapore the age-adjusted minimum incidence rates for the 12-year period 1950-1961 were the highest for Chinese and lowest for Indians and Pakistanis, those for Malays being in between. This finding is confirmed by Shanmugaratnam (1970) in a later analysis of the incidence rates based on histologically diagnosed cases only for Chinese, Malays, and Indians in Singapore for the 5-year period 1960-1964. The crude rates per 100,000 per annum for the 3 groups are, respectively, 13.3, 3.2, and 0.4, and the corresponding age-standardized rates 20.2, 5.8, and 0.2 for males. For Chinese and Malay females the crude rates are 6.4 and 0.9, and the age-standardized rates 9.0 and 2.0, respectively, there being no cases among Indians. Singapore, unlike Hong Kong, has relatively few immigrants. Chinese living there have been found to have risks according to their places of origin in China similar to those in Hong Kong. Furthermore, Indians who are normally of low risk and living in Singapore among Chinese and Malays do not share their high risk of developing the disease. It would appear, therefore, that it is the ethnic factor, rather than the geographic locality of domicile, which determines the risk. 3. Eject of Distant Migration on People of High Risk

Chinese in Hong Kong or Singapore constitute the majority of the local population and have, therefore, retained largely the customs and ways of life of their ancestors. Chinese in Australia, Hawaii, and California are in the minority and because of the vast distance between these places and the Orient they are likely to retain less. This applies especially to those born in their countries of adoption.

NASOPHARY NGEAL CARCINOMA ( N P C )

69

I n Australia, Scott and Atkinson (1967) found little difference in the risk of suffering from the disease whether a person of Chinese descent is born in Australia or outside. On the other hand, Worth and Valentine (1967) reported incidence rates for the population over the age of 14 years of Chinese descent to be 35.1 per 100,000 for males born in China and Hong Kong and 10.2 for those born in Australia. For females they are, respectively, 29.1 and 11.1. The corresponding crude rates for those of all ages are, respectively, 31.6 and 7.1 for males and 19.2 and 7.8 for females. Their report was based on an analysis of a series of 15 Chinese cases during a 10-year period 1953-1963. However, they pointed out that of the 11 China-born male cases, it was possible only to ascertain that five had been in Australia more than 5 years when diagnosed, one had been in Australia only 1 month, and the duration of residence was unknown for the other five. If these six were not immigrants but patients who went to Australia from Southeast Asia for treatment or other purposes and excluded from the list then the rate for Chinese males over 14 would be only 16.0 which is only 57% higher than the corresponding rate for non-Australian-born Chinese males. In such a small series a difference of this magnitude is without significance. In a place like Sydney, where all the case material was obtained, it is not at all unusual for Chinese from Southeast Asia traveling there during the 1953-1963 period to seek treatment because of the inadequate radiotherapy facilities in their place of domicile at the time. I n Hawaii, Quisenberry and Reimann-Jasinski (1967) analyzed a series of 14 cases of Chinese descent reported in the Hawaii Tumor Registry during 1960-1962; they found rates for 9 subjects born in the United States including Hawaii to be 6 times higher than for subjects born elsewhere. An annual rate of nasopharyngeal cancer of 54.2 per 100,000 for all ages and both sexes combined for Chinese other than those born in the United States including Hawaii is about 3.5 times higher than the corresponding rate of 15.2 per 100,000 and over 3 times the age-standardized rate of 17.2 for the mean total population of Hong Kong during the 5-year period 1965-1969 with patients from elsewhere excluded. This makes one wonder whether this group might have included Chinese patients who went to Hawaii for medical purposes. An alternative explanation is that Chinese by migrating to Hawaii had become more susceptible to the disease whereas the local-born Hawaiians of Chinese descent were no more susceptible than the Chinese in Hong Kong-a most unlikely explanation. I n the State of California, Zippin et al. (1962) investigated the place of birth of 31 Chinese males reported to the California Tumor Registry during a 16-year period 1942-1957; they found the ratio of observed-to-

70

J. H. C. H O

expected ( O / E ) number of cases by age group to be more than 8 times higher in Chinese under the age of 55 born outside United States than in those born inside. No difference was found between the two groups over 55. They stated that a difference in coverage might exist between Chinese born in the United States and those born outside, but they did not mention whether cases in the latter group might have included patients who went to California from the Far East specifically for treatment. These patients are likely to be in the younger age group because the older patients are less fit as well as less willing to make the trip. If such patients were unknowingly included, the O/E ratio would be unnaturally high. In addition, two assumptions were made in the calculation of the O / E ratio. One is that the age-sex-specific rates of New York State applied equally well to each of the age-sex-race-nativity-specific groups in California. This is not valid because the incidence rate for Chinese males reaches a peak at the age period of 50-54, after which the rate declines sharply (Fig. 5) whereas for Caucasian males the peak is reached one to two decades later. The second assumption is that the. age-distribution of the United States-born and foreign-born Chinese populations in California according to the 1950 census constitute the mean age-distributions for the two groups during 1943-1957. This is not necessarily valid. Finally, as the authors themselves have rightly emphasized, caution must be exercised in the interpretation of results based on numbers so small as those in some categories of their series. Buell (1965) , on the other hand, studied the California mortality records of deaths from cancer of the nasopharynx in 67 men and 13 women of Chinese descent during the 14 years from 1949 through 1962. The number of cases analyzed is again small and spread over a long period. He found that the risk of nasopharyngeal cancer in the local-born Chinese is considerably higher than in the white population, but lower t,han that in the immigrant Chinese. The factor of increase is about 20fold for both men and women of Chinese descent born in the United States, and 30- to 40-fold for the men and women born in China. Buell further stated that the Chinese immigrants to California have carried with them as much, if not more, risk of cancer of the nasopharynx as the immigrants to Singapore. While a lower incidence among the United States-born Chinese would support an environmental hypothesis, both Zippin et al. (1962) and Buell were of the opinion that it did not rule out a genetic etiology. It could be the result of a genetic-environmental interaction or a selection against a genotype as a cause of a reduction in the filial generation. Buell feels that there is no doubt that the immigrant generation had a lower fertility than their fathers, for, even as late as 1950, the ratio of

NASOPHARYNGEAL CARCINOMA (NPC)

71

single adult men to single women was 2:l and several decades earlier the ratio was several times higher. He finds evidence that marriage was often postponed, as revealed by the disparity in the parental age on some birth certificates of Chinese, a paternal age of 40 or 50 and a maternal age of 20 or 30 being not infrequent. Also according to Buell, about 28% of the nasopharyngeal cancer cases were reported to have died unmarried, and some married men were separated from their wives for long intervals, partly due to the Extension Acts of 1882 and 1924 (Lee, 1960). I n conclusion, it could be said that we are still left in doubt whether distant migration has altered the risk of Chinese born in their country of adoption, but the risk of those born in their place of origin appears to be unaffected. Further studies are called for.

4. Risk in People of Part-Chinese Ancestry Ho (196713)reported a crude average annual incidence rate estimated to between 20.0 and 26.7 per 100,000 during 195S1963 among Hong Kong “Macaonese,” a term which had been used by the Macao Government until a few years ago to describe “local” Portuguese as distinct from “continental” Portuguese. Macao has been colonized by Portugal for over 4 centuries, and the “Macaonese” are largely products of intermarriage between the two races, but all of them are Catholics by religion. Many of them have migrated to Hong Kong. In fact, the great majority of the people of Portuguese nationality in Hong Kong are “Macaonese.” According to a communication dated August 15, 1966 from the Commissioner of Registration of the Hong Kong Government, the number of persons resident in Hong Kong of Portuguese nationality who had registered with his department for Hong Kong Identity Cards comprised of 833 males and 735 females over the age of 6, and of 117 males and 121 females between the ages of 6 and 17 years. Those below the age of 6 were not registered, and it is not possible to ascertain the number of those who have adopted British nationality, and have not registered as Portuguese. Those registered as Portuguese included, on the other hand, a small proportion of “continental” Portuguese. With the help of some of the members of their community, it has been estimated that the total Portuguese population in Hong Kong was between 3000 and 4000 a t the time of the study. Even taking the lower estimate of 20.0 per 100,000, the crude incidence rate is unusually high, being higher than the highest of the crude rates for the various ethnic groups of Chinese in Hong Kong, e.g., 13.2 for the people originating from the Sze Yap area (Ho, 1967a). There was probably an underestimation of the “Macaonese” population accounting for its unusually high incidence,

72

J. H. C. H O

but there could be no doubt that it was much higher than that for the other non-Chinese ethnic groups in Hong Kong, because among the 53,230 persons classified as non-Chinese in the 1961 census there were five cases of nasopharyngeal carcinoma diagnosed during the 5-year period 1959-1963-four of them “Macaonese,” and one a Malay. There was not a single case among the rest, which include Europeans in the British civil service and armed forces and the foreign business community, Gurkhas in the Gurkha Infantry Brigade, Indians, Pakistanis, and other minority racial groups. I n fact, the author in over 20 years’ practice as a radiotherapist in Hong Kong has seen only one case of NPC among European Caucasians and none among the Gurkhas, Indians, and Pakistanis, although their total number far exceeds that of Macaonese in the population. In Thailand, Garnjana-Goochorn and Chantarakul (1967) engaged in a prospective survey of 1000 consecutive cancer patients in the Tumour Clinic of the Siriraj Hospital in Dhonburi with the intention of finding the relative frequencies of nasopharyngeal cancer among the three racial groups-Chinese, Chinese of part-Thai ancestry, and Thais. Out of the 1000 cases, there were 170 Chinese, 195 Chinese of mixed descent, 628 Thais, and 7 of other nationalities. Nasopharyngeal cancer was found in 27 Chinese (15.9%),20 Chinese of mixed descent (10.3%), and 29 Thais (4.6%). From this study they estimated the ratio of relative frequencies in the 3 groups to be respectively 3.4:2.2:1.0, and believed that this estimation was as near the correct proportion as they could get in Thailand. They further reported that nasopharyngeal cancer constituted 3.5% of all malignant neoplasms diagnosed a t Siriraj Hospital during a 6-year (1957-1962) period. Thais are mongoloid people, mostly Buddhists, but Portuguese are Caucasian Catholics. Yet in both, the products of intermarriage with Chinese appear to inherit at least a part of the high risk of their Chinese ancestors. 5. Familial Aggregations

Reports on familial aggregations of cases of nasopharyngeal carcinoma in literature are scarce. Pang (1959) records finding two pairs of related cases, mother-son and sister-brother combinations, in a series of 34 consecutive cases of nasopharyngeal cancer, including 27 Chinese, in Hawaii. Buell (1965) found one pair, mother-son combination, in his study of 80 certificates of death due to nasopharyngeal cancer in California. Ho (1967b) found 15 instances of familial aggregations in two separate series of 1180 cases of nasopharyngeal carcinoma. I n eight instances two brothers in the family had the disease, in one instance

73

NASOPHARYNGEAL CARCINOMA (NPC)

three brothers, in two instances a pair of brother and sister, in one instance the son and his father, one nephew and aunt, one sister and her brother, and finally in one instance a female patient had a family aggregation of cases extending over three successive generations. A pedigree study, retrospective and prospective, of this family is shown in Fig. 4. The family was originally from Canton, but generation I1 had settled in Hong Kong. Nos. 3 and 4 of this generation, the two younger brothers of the paternal grandfather of the propositus, died of a disease with a clinical history highly suggestive of NPC, e.g., nasal bleeding, regurgitation of food through the nose, and loss of voice. These are the only symptoms recalled by I11 (No. 1) and related by members of generation IV. When a Chinese of Canton origin dies of a disease with these symptoms, it is most likely NPC with involvement of the last four cranial nerves. The father of the propositus was a proven case of NPC; he was treated at Queen Mary Hospital by the late Professor K. K. Digby, who advised him to go to Shanghai for radiotherapy as there was no such facility in Hong Kong at that time. He died at the age of 38, leaving a wife with seven children and a concubine with three children. There has been no contact between the wife and the concubine, who live separately, since his death. However, it is known from hearsay that all 3 children by the concubine are now over 40 and none suffer from NPC, whereas six of the seven children by the wife had developed the disease verified by biopsy. The wife is still alive a t 78 and well. 0-0

1

n

Ip

1. 2. 1 2 8 dx50

D D D dead

4. 5. 6O 7. eo go ion 4x28 dx42 dx35 dx34 %W" W W " "AW" D D A3R AW D AW a l i v e & w e l l A3R a l i v e without recurrence

3.

t age at death 7

no d a t a

''

dx age at diagnosis

hearsay

0

NPC

I'not v e r i f i e d

FIQ. 4. Pedigree study of a family with aggregation of NPC cases extending over three successive generations. From Ho (1972), by permission from the editor.

74

J . H. C. H O

Pedigree studies of Chinese patients in Hong Kong are handicapped by the fact that many of them have lost contact with some or even all of their kinfolk as a result of wars, rebellions, and revolutions which have ravaged China over the last several decades. Some patients do not know the causes of death of relatives because of inadequate diagnostic service. Others were adopted when very young and know nothing about their relatives. Under the assumption that these handicaps probably apply equally to family studies of NPC patients and of patients suffering from other cancers (OC) in a large series, a study has been carried out a t the Medical and Health Department Institute of Radiology, Hong Kong, in 1969 to determine whether close blood-linked relatives of NPC patients have a higher risk of developing NPC than those of patients suffering from other cancers. The results of this study are given in Tables VI and VII. The source of data in the study consists mainly of medical records; these are, as a rule, inadequate for genetic studies, as clinicians are usually more concerned with medical diagnosis and treatment than with obtaining a detailed family history of diseases. T o minimize this inadequacy the medical staff has been requested to inquire for family histories of NPC in both groups of patients. Positive questions were asked in order to counterbalance the memory bias of patients who tend to remember relatives suffering from a disease similar to their own better than those suffering from other diseases. In doubtful cases, the patients were recalled for further questioning by the author. A greater risk of getting NPC is found in relatives of NPC patients than in relatives of patients suffering from other cancers. It is interesting to note that 3 TABLE VI FREQUENCY OF FAMILY HISTORYOF NPC IN PATIENTS WITH NPC AND IN THOSE WITH OTHER CANCERS (OC) DIAGNOSED AT THE MEDICALAND HEALTHDEPARTMENT INSTITUTE OF RADIOLOGY, H O N Q HONG,1969 Cancer

NPC

oc

Families with history

Families with no history

12” 2

385 687

-

14 X’ = 14.77785 ( P < 0.001) t

-

Total

397‘ 689”

-

1072 1086 = 3.864234 (P= 0.00011)

From Ho (1972),by permission from the editor. Of the 12 families, 3 are “boat” people. b Seventy-two were excluded because a family history wm unobtainable, unreliable, or not obtained. In this group, 515 c a m were excluded, 230 for the above reasons and 285 because the cancers are sex-determined, e.g., gynecological, penile, etc.

75

NAsOPHARY NGEAL CARCINOMA (NPC)

TABLE VII FAMILIAL AQOREQATIONB OF NP CARCINOMA Families 1

2

Vertid

PATIENTS SEEN IN 1969

Horizontal

+

Daughter (II/2M 4F) and father Son (V/2M 3F) and mother.

Remarks

1 Female paternal 1st cousin

+

3 4

IN

2 Siters (I and II/6M

Son (IV/4M

+ 5F) and

+ 3F)

-

“Boat” people

mother 5

6

+

Son (V/4M

+ 3F) and

father

+

7 8

9 10

11

2 Paternal 1st cousins: Male (III/3M 2F) and M OM)

+

Daughter (IV/lM 5F) and fathero Son (only child) and father

-

Mother (II/lM daughter

+ 3F) and

12 Total = 7

Male (V/3M 2F) and brother (11) and brother (11

Male (only child) and his half-brother by same father; latter’s halfbrother by same mother was well

“Boat” people

-

+

Brother (?/4M 1F) and 1 brother Total = 7

“Boat” people

From Ho (1972), by permission from the editor. 0 D iagnosis was based on typical history only. (II/2M 4F) means propositus is the second child (11) of a family of two sons and four daughters.

+

of the 12 NPC families with aggregations of cases are “boat” people, a very small group of people who have a tendency to marry within their group and have been living in boats, junks, and sampans for centuries until recently when some of them have settled ashore and intermarried with land dwellers. The marine population of Hong Kong, according to the 1966 By-Census (Barnett, 1966), constitutes only 2.76% of the total population and consists largely of “boat” people. Table VII shows the directions of aggregation in the 12 families. If we accept the two unverified cases with only hearsay typical clinical histories (mother of propositus No. 2, and father of propositus No. 8) as positive cases, then the incidence of aggregations in this series is as

76

J. H. C. H O

great in the vertical as in the horizontal direction. From sheer numbers at risk, one would expect the aggregation in the horizontal direction to be greater than in the vertical. It is also more likely for a patient to know of or remember diseases suffered by relatives belonging to the same generation than by those in earlier generations. Diagnostic facilities were also poorer in the early days. Duration at risk is on the side of the older generations but not to a great extent, as the risk of getting NPC declines after the fifth decade in Chinese except in recent years, when a second but lower peak has become increasingly apparent in the seventh decade. It would appear in the present and past studies that if there were vertical transmissions in NPC they are not sex-linked. The random nature of the aggregations is indicative of a multifactorial etiology, and if genes are involved they are likely to be polygenic. 6. Nasopharyngeal Carcinoma in Twins No report of nasopharyngeal carcinoma occurring in twins has been found in the literature. Of some 6000 cases of nasopharyngeal carcinoma diagnosed at the Queen Mary and Queen Elizabeth Hospitals, where the Medical and Health Department Institute of Radiology is based, during a 20-year period 1951-1970 there was only one verified instance of NPC occurring in a pair of twins, probably dizygotic, of Sze Yap origin. One member, who died of NPC, was a patient of the Institute. The other, who migrated to Canada over 10 years ago, received radiotherapy in Canada for NPC 8 years after the death of his twin brother a t the age of 41, 1.5 years after the clinical onset of his disease. There are an elder and a younger brother and three younger sisters. None of them or their parents are known to have suffered from the same disease. The eldest brother is alive and well. Nothing more is known about the others. A twin birth occurs in about every 100 deliveries among Chinese in Hong Kong. At Tsan Yuk Maternity Hospital there were 673 twin deliveries during the 10-year period 1959-1968. Of these the nature of the twinning is recorded in the case of only 631. Three hundred and fifty (55.6%) of them are biovular and 281 (44.4%) are uniovular (Chun and Lee, 1970).

7 . ABO Blood Group Distribution If a genetic etiology is suspected, it is a call for more data, not for immediate speculations. So far only the ABO group distribution in NPC cases and normal controls have been investigated (Ho, 196713; Clifford, 1970). Clifford, comparing the ABO blood group distributions in 233 Kenyan patients with nasopharyngeal carcinoma and in controls, found a significance level of 3% in the comparison A/O and considers this to

77

NASOPHARYNGEAL CARCINOMA (NPC)

TABLE VIII ABO BLOODGROUPDISTRIBUTION IN PATIENTS WITH NASOPHAF~YNGEAL CARCINOMA AND CONTROL^ Population (all Chinese)

0

A

B

AB

NPC patients: males NPC patients: females Total Percentage Controls (Grimmo and Lee, 1961) Controls (Tong el al., 1963) Total Percentage

291 99 390 39% 258 5,747 6,005 41.6%

197 69 266 26.6% 181 3,650 3,831 26.5%

205 69 274 27.4% 180 3,515 3,695 25.6%

57 13 70 7% 51 856 907 6.3%

Total 750 250

1,OOO 100% 670 13,768 14,438

100%

From Ho (1972), by permission from the editor. For A/O comparison: t = 0.82093 ( P = 0.41); for B/O comparison: t = 1.62801

(P = 0.104).

be highly suggestive that, in Kenya, group-A persons are “protected” or a t less risk of nasopharyngeal carcinoma and that persons with other blood groups are consequently relatively a t greater risk. He feels that there seems to be an inherited susceptibility to, or protection against, nasopharyngeal carcinoma in some Kenyan Africans. I n Hong Kong a study of the ABO blood group distribution in loo0 consecutive Chinese patients with nasopharyngeal carcinoma failed to reveal any significant difference in the distribution in this group when compared with normal controls in the A/O or BJO comparison. The results are given in Table VIII. Shanmugaratnam (1970) also failed to find any significant difference between NPC cases and controls in Singapore. The distributions of ABO blood groups in Chinese in different parts of China, Singapore, Sumatra, New York City, and Hong Kong are given in Table IX for comparison. There is no great difference in the distributions between those reported by Grimmo and Lee (1961) and Tong et al. (1963) for Chinese in Hong Kong and those reported by Dormanns (1929) and Allen and Scott (1947) for Chinese in Canton and in Singapore, respectively. A very high incidence of the disease is found in all three places. I n the Yangtse River region, Hunan, Hupeh, and Kiangsu, where the relative frequency is lower (Hu and Yang, 1959), there is a definite excess of A group over B, but then in Peking, where the relative frequency is lowest, it is just the reverse. It would appear, therefore, that if certain genes are associated with a greater susceptibility to nasopharyngeal carcinoma in Chinese they are unlikely to be associated with the ones which determine the possession of blood

78

J . H. C. H O

TABLE IX ABO BLOODGROUPDISTRIBUTIONB AMONG CHINEBE OUTSIDEOF HONGKONQ" AND AMONG CHINESEIN HONGKONG Percentage Place China Yangtge River Hunan Hupeh Kiangsu Kwangtung Canton Canton Peking Peking Hong Kong Singapore Sumatra (East coast) New York City

Authors

Size

0

A

B

AB

29.5 1096 42.9 Yang (1925)" 93 43.01 31.18 Yang (1928)" 197 42.13 32.49 Yang (1928)" Yang (1928)" 228 52.63 21.05 196 40.82 32.65 Yang (1928)" 992 45.87 22.78 Dormanns (1929) 101 45.54 29.70 Alley and Boyd (1943)" 1000 30.70 25.10 Liu and Wang (1920)" 427 65.34 4.68 Hung and Steffan (1928)" 670 38.51 27.02 Grimmo and Lee (1961) Tong el al. (1963) 13788 41.74 26.51 Allen and Scott (1947) 624 43.11 24.04 592 40.20 25.00 Bias and Verhoef (1924)O

19.7 7.9 19.35 6.45 17.26 8.12 15.79 10.53 18.37 8.16 25.20 6.15 18.81 5.94 34.20 10.00 19.67 10.30 26.86 7.61 25.53 6.22 27.72 5.13 27.33 7.26

Levine and Wong (1943)"

25.33

150 30.00 34.00

10.67

From Ho (1972), by permission from the editor. Cited in Mourant and Domaniewska-Sobczak, 1958.

0

group A, B, or 0 substance. A study of the HLA pattern in Southern Chinese and non-Chinese living in Singapore will soon be conducted. 8. Physical Anthropology

There have been regrettably few attempts to survey the physical characteristics of patients with nasopharyngeal carcinoma for comparison with unaffected subjects matched for ethnic origin, sex, and age. Walsh (1967) concluded from his study that to date no single physical characteristic has been found that is common to the population of those countries with a high incidence of the disease and absent from others. He emphasized, however, that this does not mean that the possibility has been exhausted of finding a unique multifactorial set of characteristics in those populations with high risk, and that many more surveys must be undertaken and submitted to computer analysis.

C. ENVIRONMENTAL FACTORS 1. External Factors a. Inhalants. Dobson (1924) postulated that the high incidence of the disease in Chinese was related to the poorly ventilated houses in

NASOPHARYNGEAL CARCINOMA (NPC)

79

which they lived and inhaled much of the domestic smoke from burning grass, wood, tobacco, candles, incense (joss sticks), kerosene, and peanut oil lamps. Then Clifford and Beecher (1964) proposed that the inhalation of smoke from burning wood from exotic trees (eucalyptus and wattle) and indigenous acacias in ill-ventilated huts for several hours a day over a period of years may have some bearing on the distribution and incidence of the disease in Kenya, especially when significant quantities of carcinogenic substances, such as benzopyrene and benzanthracene, have been found in the soot taken from the roof of the huts of 46 patients with nasopharyngeal cancer on analysis by Hoffman and Wynder at the Sloane-Kettering Institute, New York. On the other hand, Booth et al. (1968) found similar living conditions among the one million and more people living in the Highlands of Australian New Guinea, but nasopharyngeal carcinoma is a rarity among them. It might be argued that the smoke in the two cases contains different substances, but then the crude annual incidence rates of nasopharyngeal carcinoma for most Kenyan tribes are below 0.5 per 1OO,OOO, and even among the Nandi tribe, which has the highest rate, the rate was only 0.94 (Clifford, 1967), which is only slightly higher than for the Swedes, who live in well-ventilated houses. Furthermore, Ho (1967a) found the incidence rates for the marine population in general and the “boat” people in particular, who live and spend most of their lives in sampans and junks and cook their food in open air, to be significantly higher than that for the land dwellers in Hong Kong. It would appear, therefore, unlikely that domestic smoke plays a significant role in the genesis of nasopharyngeal carcinoma, and certainly it cannot account for the high incidence in Hong Kong Chinese. Opium has also been suspected since it was first introduced into China in the early part of the nineteenth century through Canton, the capital of Kwangtung province. Digby (1951) observed that many Chinese who developed nasopharyngeal cancer were not opium smokers, and Polunin (1967) quotes a personal communication to him by Leong (1964) who estimates that he has seen about 5000 Chinese opiumsmoking men in Singapore, following them up for a period of a year each. Their average age was 48 years, and they had smoked opium for about 20 years, but he did not find a single case of nasopharyngeal cancer among them. Shanmugaratnam and Higginson (1967) found 87% of 63 Chinese male patients with nasopharyngeal carcinoma never smoked opium as against 84% of matched controls. In a retrospective survey of the records of 685 patients with nasopharyngeal carcinoma diagnosed during the 2-year period 1962 and 1963 a t the Medical and Health Department Institute of Radiology, Hong Kong, only four patients admitted to having smoked opium. There may have been some underrecording, but it is unlikely to be more than slight. I n a later study

80

J. H. C. H O

in 1969 there were 6 cases of opium addiction and three of heroin among 471 cases of nasopharyngeal carcinoma. None of the rest indicated that they had smoked opium at any time. In contrast there were four cases of opium addiction and 2 of heroin among 173 cases of bronchial carcinoma, and 11 cases of opium addiction among 47 cases of laryngeal carcinoma. There is, therefore, no evidence to suggest that opium plays a part in the causation of the high incidence of the disease in Chinese. Oriental incense or joss sticks are burnt not only in Chinese Buddhist and Taoist temples but also on a small scale in many Chinese homes in Hong Kong, China, Southeast Asia, and the United States. Those used in Hong Kong are made from sawdust from sandal-wood imported from Hainan Island and other parts of Kwangtung, Indonesia, India, and Australia in factories in Macao and Hong Kong. During the IO-year period 19551964, and average of 71.4 tons of joss sticks were exported or reexported from Hong Kong to the United States per year according to the Hong Kong Department of Commerce and Industry. Sturton e t al. (1966) found a difference (significant a t the 5% level) between incense burning and nonincense burning for male cases under 50 years of age and for male cases as a whole, but not for those over the age of 50, when a group of 29 male NPC cases was compared with a group of 38 male cases of other cancers. There was, however, no significant difference in the female patients of the two groups. Perhaps, the latter finding is more important than the former, because it is the females, not the males, who attend to the burning of joss sticks in Chinese homes. Furthermore, the incense smoke hypothesis is not in keeping with the finding by Ho (1967a,b) that NPC is rare among Buddhist monks, nuns, and Taoist temple attendants, who spend much of their time in incense-laden atmosphere. In fact, among over 3500 cases of nasopharyngeal carcinoma diagnosed a t the Medical and Health Department Institute of Radiology during the ll-year period 195&1966, there were only one Buddhist nun and one Taoist temple keeper. According to Sturton et a,?. (1966) there were 2000 temples and shrines in the city of Hangchow in central China and about 10,000 monks and a smaller number of Taoist monks or priests in a population of about 800,000 in that city before the institution of the present Chinese Government. Sturton (1965), in his work as a doctor and radiologist from 1921 to 1952 in Hangchow, did not observe any higher incidence of nasopharyngeal cancer than one would find in a large general hospital in England. The apparently high incidence of nasopharyngeal carcinoma in Macaonese who are traditionally Catholics and do not burn joss sticks in their homes or go to Buddhist or Taoist temples for worship, the relatively high incidence in Malays in Southeast Asia, who are traditionally Muslims and do not burn joss sticks, and the

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high incidence in the “boat” people of Hong Kong, who are less exposed to the smoke from burning joss sticks by virtue of their living in open boats than the land dwellers, are other contradicting evidences. It is, liowever, not possible to draw any conclusion at this stage that, because nasopharyngeal carcinoma appears to be rare in Buddhist monks, nuns, and Taoist temple attendants, joss sticks may have a protective action against the risk of getting the disease. These people belong to a small occupational group and the actual incidence of nasopharyngeal carcinoma in them has yet to be determined. Of the other inhalants, mention must be made of antimosquito coils which have been in common use in Chinese homes in China for many years and in Hong Kong until recent years, when urban areas have been rid of mosquitoes. The coils are still being used in some rural homes. Shanmugaratnam and Higginson (1967) did not find any significant difference between a group of 100 patients with nasopharyngeal carcinoma and a group of matched controls of the same number in their use of antimosquito coils in Singapore. Neither did they find any significant difference between the two groups in their use of cigarettes, pipe tobacco, snuff, and Chinese medicinal balms, oils, drops, and powders for inhalation or intranasal applications. The use of tobacco among Chinese is not as prevalent as among people of western nations, and the introduction of its use as a social habit in China occurred only within this century. Consequently, it could not have been responsible for the high frequency of the disease in China which most probably have existed for a longer period. b. Ingestants. We must not look for carcinogenic inhalants in the environment only of people a t high risk. It has been shown by Druckrey et al. (1964a) that compounds of the nitrosamine group can act systematically and are organ specific. They have further shown that a single dose of such a carcinogen can initiate a train of events which will culminate in cancer development after a long latent period without a further dose (Druckrey et al., 1964b). Bonser (1967) suggests that nitrosamine is not itself carcinogenic, but has to be converted enzymatically to an active carcinogenic metabolite. The enzymes capable of effecting such a conversion are different for different nitrosamines, so that the location of specific enzymes determines the site of the cancer. Nitrosamines are formed when nitrates are used as food additives or preservatives. Fish preserved in salt containing nitrate has been for many years a common and favorite item of food among most Chinese, rich or poor, in or outside China in Southeast Asia or the United States. This is also true for Macaonese in Hong Kong and Macao and to a less extent the Malays in Malaysia. Shanmugaratnam and Higginson (1967) noted

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no significant difference in food habits between a group of 100 male patients with nasopharyngeal cancer and a group of matched normal controls. The frequency of use of rice, and a variety of other foodstuffs, including salted and dried food, was approximately the same in the two groups. They pointed out, however, that their negative findings are not conclusive. Further epidemiological studies aa well as investigation into their carcinogenic properties are called for. It is more important to determine whether Chinese who never eat salted fish or food have the same risk of getting the disease as those who do than to compare the frequencies of eating such food between nasopharyngeal cases and matched controls. c. Nasopharyngeal Carcinoma Occurring in Both Marital Partners. During the 14-year period 1956-1969 only two verified instances of the disease occurring in both the husband and the wife of a family are found among 5070 cases diagnosed at the Institute of Radiology, Hong Kong. The husband in the first instance developed symptoms of the disease a t the age 39, just over 6 years after marriage, and the wife a t the age of 36 about 10% years after marriage. Both of them are Catholic, have no family history of nasopharyngeal cancer, and lived together in Hong Kong after their marriage. The husband in the second instance developed symptoms a t the age of 52, and the wife a t the age of 40. Neither of them had a family history of the disease. The exact time of their marriage is not known, but the number of years between marriage and symptomatic onset cannot have been less than 13 years in the case of the wife and 18 years in the case of the husband, judging from the age of their only surviving daughter. The length of stay in Hong Kong before onset was 14 years in the case of the wife and 20 years in the case of the husband. Both have since died, and their daughter is untraced. This incidence must be considered as minimal, because after the death of a spouse the surviving one-might return to China or migrate elsewhere or would not return to the Institute which had failed to cure his or her spouse of the disease to seek treatment. There could not be many such cases because there are few places available for treatment, and the Institute is the only place in Hong Kong where the poor can get free treatment as well as social welfare benefits. Among the 5070 cases, about 200 are patients who came from China or Southeast Asia specifically for treatment, after which they returned to their homes. Of the 121 who came from Southeast Asia during the period 1964-1969, many are, however, still in contact with the Institute. Nasopharyngeal carcinoma is a prevalent disease. Even allowing for some probable underrecording, one cannot but be impressed by the infrequency of such occurrences. It would appear, therefore, that those environmental factors,

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which affect husband and wife alike and are found in most homes, are of little etiological importance, at least during adult life. d. Occupation and Socioeconomic Level. In Singapore, Shanmugaratnam and Higginson (1967) found a wide range of occupations to be represented in both nasopharyngeal cancer and control groups in a retrospective survey, and Polunin (1967) found a lack of a clear-cut association between ways of living and nasopharyngeal cancer. I n Hong Kong, the carcinoma does not appear to occur more frequently in any particular occupation, as in the case of Singapore. In the highly competitive Hong Kong society, where much free enterprise exists, there is a definite possibility that someone who came originally from a poor family could become rich later in life, and vice versa. It is more important to study the early part of the life of a subject in a survey, than the later part. There are in Hong Kong, however, sufficient cases of nasopharyngeal carcinoma who are known to belong to the upper, middle, and lower socioeconomic levels throughout their preceding lives to justify the impression that a higher or lower risk is not associated with any socioeconomic level. Andrews and Michaels (1968a,b) reported three cases of nasopharyngeal carcinoma in Canadian bush pilots, a very small occupational group. One is of French extraction, one is Finnish, and one English, and they had been on their jobs for 10, 28, and 25 years, respectively. Such flying in unpressurieed aircraft subjects the pilots to very frequent and rapid air pressure changes which are likely to produce “otitic barotrauma,” which is thought to be a precursor of nasopharyngeal carcinoma. All three were cigarette smokers. It was thought that cigarette smoke and other potentially carcinogenic substances may be specifically directed to the nasopharyngeal area as a result of pressure changes. It was also pointed out that there was a greater speed of pressure changes experienced by these pilots than that experienced by divers. There was, however, no subsequent report of another case in these pilots or in other groups of airmen working under similar conditions of barometric change, e.g., crop-duster, air taxi, and helicopter pilots. There must be many such airmen all over the world, although the climatic conditions under which they work may not be similar to those for the Canadian bush pilots. For example, the Canadian air in winter is very cold and dry (Lancet Annotations, 1968). Although coincidence cannot be ruled out, the finding of three cases in so small an occupational group certainly deserves further investigation. 2. Internal Factors a. Malnutrition and Vitamin Deficiency. Contrary to the findings by Clifford (1965) in Kenyans, a good nutritional state without clinical

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evidence of vitamin A or B group deficiency is the rule rather than the exception in Hong Kong patients except in the terminal stage of the disease. Many of them also gave no history of malnutrition or chronic ill health during childhood. b. Hormonal Factors. Kenyans with nasopharyngeal carcinoma had significantly lower plasma levels of dehydroepiandrosterone sulfate (DS) as compared with the control group, but plasma levels of androsterone sulfate (AS) in both groups were similar (Wang et al., 1969; Clifford, 1970). In Singapore, similar low levels of DS and AS were noted in Chinese patients with nasopharyngeal carcinoma, but studies on the plasma levels of DS, AS, cortisol, and transcortin in apparently normal male subjects from racial groups with high (Chinese), intermediate (Malays), and low (Indians) incidence of the disease revealed no differences (Wang et al., 1969). Plasma estrogens were, however, not measured in the studies carried out in Kenya or Singapore. c. Chronic Infection of the Upper Respiratory Passage and Vasomotor Rhinitis. Although nasal sinus infection is a common complication of established nasopharyngeal carcinoma, only a minority of the patients in Hong Kong gave a past history of chronic upper respiratory infection or vasomotor rhinitis. Vasomotor rhinitis may be common in Chinese, but it is certainly very rare as an antecedent disease in nasopharyngeal carcinoma as seen in Hong Kong. d. Virus. If a virus is involved in the causation of nasopharyngeal carcinoma it has to be one which is ubiquitous to explain the regular high incidence of the disease in Chinese living in widely scattered parts of the world. Furthermore, to explain why Indians, who live in similar environments in Singapore as Chinese and Malays, have a much lower risk of the disease than the other two racial groups, the virus has to be one to which Chinese and, to a lesser extent, Malays are more susceptible than Indians, or, alternatively, one which functions together with other cocarcinogenic or carcinogenic factors, which are peculiar to or selectively affect Chinese and Malays. So far, only an immunological relationship between Epstein-Barr virus (EBV) , a member of the herpes group, and nasopharyngeal carcinoma has been found. Old et al. (1966) found in the sera of nasopharyngeal carcinoma (NPC) patients precipitating antibodies similar to those present in Burkitt’s lymphoma (BL) sera. De Schryver et a2. (1969) found in the sera from African and Chinese postnasal (same as nasopharyngeal) carcinoma an unusual frequency of high titer of membrane reactive antibodies (Anti-MA) when EBV-carrying permanent lymphoblastoid cell lines derived from Burkitt’s lymphoma and infectious mononucleosis (IM) were used as

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target cells. Much lower frequencies were found in control sera from African healthy individuals and patients with neoplastic diseases other than NPC and nonneoplastic diseases, and from Indian donors with buccal, oro- and hypopharyngeal carcinomas. W. Henle et al. (1970) found that 100% of the sera from East African and Chinese patients with NPC had antibodies against E B viral capsid antigens (VCA) and that 84% of them had high titer ( 2 1:160) with a geometric mean titer (GMT) of 1:340, whereas only 10% of the controls from the general population, unmatched for sex and age, and 14% of the patients with head and neck tumors other than N P C showed high titer, with a G M T of 1 : l O and 1:41, respectively. They further found that when the NPC patients from Hong Kong were grouped according to the stage of the disease, presumed to correlate with the tumor burden, the incidence of high titers increased successively from 45% in stage I to ultimately 100% in stage V. The GMT also rose correspondingly from 1:103 in stage I to 1:788 in the stage V. G. Henle and Henle (1972) detected antibodies to early EBV-induced antigens (EA) in infectious mononucleosis, Burkitt’s lymphoma, and nasopharyngeal carcinoma. Whereas in infectious mononucleosis the antibodies to EA disappear usually within 6 months, in Burkitt’s lymphoma and nasopharyngeal carcinoma they usually persist and are frequently found at high titers. EpsteinBarr virus is ubiquitous on a worldwide basis, but no tumor has been induced to date by EBV-carrying materials in laboratory primates. There are, however, some observed effects of EBV infection in vitro which are consistent with-but by no means proof of-an oncogenic potential for the virus. There is a possible analogy with the herpes virus producing malignant proliferation of lymphoid cells in Marek’s disease of fowls, but the virus has not yet been recovered from disrupted cells, although it can spread from lymphoma cells to kidney cells when they are ruptured. I n nasopharyngeal carcinoma, herpes-type viral particles, similar to the EBV particles observed in cultures from B L and IM, have been observed in some of the degenerating lymphoblastoid cells derived from cultures of biopsy specimens from Hong Kong (de-The et al., 1969, 1970). A new human virus, unassignable to any known morphological group, has been observed in cultures of a nasopharyngeal carcinoma from Kenya (Epstein, 1972). This virus was found only in suspension cultures of lymphoblastoid cells released from the original monolayer after 105 days in vitro. Since nasopharyngeal carcinoma is of epithelial, not of lymphoreticular origin, EBV or a similar virus may play no role in the etiology of the disease. If this is so, an explanation has to be found for its close immunological association with EBV, which

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is not found in carcinomas arising elsewhere in the head and neck including other parts of the Waldeyer’s ring and tumors other than carcinoma arising in the nasopharynx. If, on the other hand, nasopharyngeal carcinoma provides only a favorable medium for the multiplication of this virus, we should find among the NPC patients a proportion of HTV-negative persons, reflecting the HTV-negative portion of the general population. Furthermore, if the virus were merely a passenger, then why other tumors, such as leukemias, lymphomas, Hodgkin’s disease, reticulum cell sarcoma, and multiple myelomas should not offer similarly favorable habitats. Unfortunately, the increase in anti-EBV titers with the advancement of the disease does not differentiate between a passenger role and a causal relationship. T o determine whether EBV or a similar herpes-type virus plays a role in the genesis of NPC, it is essential to study first the natural history of the virus in man, its prevalence in populations of high and low risks for NPC in different as well as the same parts of the world, its mode of spread, etc. Such a study has already commenced in Southeast Asia, Japan, and France. Then a prospective seroepidemiological study may be called for in an attempt to establish the type of association, causal or noncausal, direct or indirect, which exists between the suspected virus and NPC. As separate exercises, it is of great importance to obtain long-term cultures of the epithelioid cells derived from NPC for investigating the possible presence of herpes-type viral particles or their indicators in such cells, and also look for them in peripheral lymphocytes. 3. Pattern of Age Distribution

Figure 5 gives the age distributions of nasopharyngeal carcinoma in Chinese men in Hong Kong and Singapore and the male population of Sweden for comparison. There is much similarity in the Hong Kong and Singapore patterns, both of which show a rapid, almost uninterrupted and fairly regular increase in incidence after 20-24 years of age, two decades earlier than in Sweden and also earlier than most other epithelial cancers. The incidence then declines after 50-54 in both places, again two decades earlier than that in Sweden. This would suggest that the disease in Hong Kong and Singapore Chinese was not due to continued exposure to an external carcinogen throughout life, as is postulated for most of the common epithelial cancers, or, alternatively, that the susceptibility to the carcinogen is influenced by an internal factor, possibly hormonal in nature, which is responsible for the rapid increase in incidence soon after adolescence and the decrease after 50-54 years of age. Doll (1970) in a personal communication to the author commented that the Hong Kong nasopharyngeal carcinoma pattern is very similar to that

:I

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80 r

20

/:-

FIG.5. Male age-specific incidence rates of nasopharynge41 carcinoma for Sweden and for Chinese in Hong Kong and Singapore. Only transitional cell, squamous, and undifferentiated carcinomas are included for Sweden. K e y : -per 100,OOO (Hong Kong), 2019 cases (1965-9) ; --- per 2 million (Sweden), 202 cases (195% 65); . . . . per 100,ooO (Singapore), 839 cases (1950-81). From H o (19721, by permission from the editor.

of cancer of the uterine body and suggested that the possibility of hormonal factors contributing to both should be borne in mind. Figure 6 shows the age incidence curves for nasopharyngeal carcinoma in the two sexes in Hong Kong, where 98.7% of the population are of Chinese descent (Barnett, 1966), and Fig. 7 shows the curves in Sweden. In both places the curves for the two sexes differ only in height but not greatly in shape; the Swedish curves, however, differ significantly both in height and shape from the corresponding ones for Hong Kong. I n Sweden the curves reveal a pattern similar to that for bronchial carcinoma in cigarette smokers and most of the common epithelial cancers.

”i 20

10

FIQ.6. Age-specific incidence rates of nasopharyngeal carcinoma by sex in Hong Kong, 1960-1964 and 1% ! 51-969. Patients from elsewhere are excluded from the calculation. K e y : 19604 , male, 1492 cases; 1965-9 ---, male, 2019 cases; I-.... , female, 672 cases; 1966-9 -. .-, female, 887 cases.

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40

t

10

F I ~ 7. . Age-specific incidence rates of nasopharyngeal carcinoma (transitional cell, squamous, and undifferentiated) in Sweden by sex, 1959-1965. K e y : - male, 202 caaes; female, 129 caaes. -.a-

IV. Conclusion

The predominant cancer arising in the nasopharynx of people of different races is a carcinoma of the squamous type showing, in the majority of cases, poor differentiation or absence of differentiation. It is this tumor which shows a predilection for people of Chinese descent, especially those from the southern provinces, and an immunological relationship with EBV ar a similar virus. Chinese migrants in different parts of the world appear to have as much risk of the disease as Chinese inside China including Hong Kong, but whether the risk for people of Chinese descent born and raised in their countries of adoption is altered is not clear. Results of previous studies are far from conclusive. People of part-Chinese ancestry tend to share partly the high risk of their Chinese ancestors. Close blood-linked relatives of nasopharyngeal carcinoma patients have been found to have a higher risk of the disease than those of patients with other cancers, and the aggregation of nasopharyngeal carcinoma appears to be a t least as frequent in the vertical direction as in the horizontal. These are all highly suggestive, but not necessarily, the result of gene action. They may be the result of certain social customs, dietary habits, family recipes for treating minor ailments, etc., passed down to subsequent generations and shared by members of the same generation. We should, therefore, look for environmental factors likely to affect all ethnic groups of high risk. That such factors are also common in people of low risk does not necessarily exclude the possibility of a causative role played by these factors, because they may act together with other factors, especially genetic, which are present only in people of high risk, but their rarity or absence would suggest that they

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may be of etiological importance. Of the environmental factors, fish and probably eggs preserved in salt, which may be a source of carcinogenic or cocarcinogenic nitrosamines acting specifically on the nasopharynx of people with a genetically determined susceptibility, are traditionally common and favorite items in the diet of Chinese, especially those from Kwangtung, whether in China, Hong Kong, Southeast Asia, Australia, or the United States. The Chinese salted fish is always steamed or fried. In Japan, a low incidence area, a different kind of salted fish is often eaten, but it is always baked over a hot iron grill instead. The Chinese salted fish and eggs and the herpes-type virus which has an immunological relationship with nasopharyngeal carcinoma well deserve a thorough investigation. Chinese incense or joss sticks by virtue of the fact that they are commonly used by people of high risk should also be investigated as a possible etiological factor, although evidence so far obtained is all negative. There are still many unknown factors and missing links. All we can say a t present is that the etiology of nasopharyngeal carcinoma is most likely to be multifactorial, and that if a genetic factor were involved it is certain to be polygenic, not sex-linked or related to the ABO blood-group, at least in Chinese. There is an old saying: “A pinch of salt is worth a pound of precept.” What is needed now are more data, not speculation. ACKNOWLEDGMENTS The author is grateful to Dr. the Hon. Gerald Choa, Director of the Medical and Health Services of Hong Kong, for his permission to publish this paper; to Dr. J. K. Craig for helpful information; to Dr. C. C. Lin for histological diagnosis; to the medical staff of the Medical and Health Department Institute of Radiology for their care in obtaining family histories of the cases; to Mr. C. M. Lam for statistical assistance; to Mr. R. Abessor for preparing Figs. 3-7; to Mrs. P. Liu for careful secretarial assistance; to Messrs. K. Fung and H. K . Tam for collection of data, and Messrs. K. W. Leung and A. Lam for photographic assistance. The author is also indebted to the Swedish Cancer Registry and Professor Nils Ringertz, Scientific Surveyor of the Registry, for generous cooperation in supplying Swedish data; to Professor J. Mitchell, F. R. S., and Mr. J. A. Fairfax Fossard of Cambridge University for supplying photographs and radiographs of skull No. 238 kept at Duckworth Laboratory, Cambridge, and to Mr. J. C. Trevor, Director of the Laboratory, for the facilities afforded; to Professor R. Doll of Oxford University for helpful advice; to Professor K. Shanmugaratnam for supplying Singapore data; and finally to Dr. L. Atkinson for helpful information regarding conditions in Australian New Guinea.

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Wells, C. (1963). J . Laryngol. 77, 201-265. Wells, C. (1864). Brit. Med. 3. 1, 1611-1612. Worth, R. M., and Valentine, R. (1967). In “Cancer of the Nasopharynx” (C. 9. Muir and K. Shanmugaratnam, eds.), UICC Monogr. Ser. No. 1, pp. 73-76. Munksgaard, Copenhagen. Wu, C. H. (1921). “The Encyclopaedia of Chinese Medical Terms,” Vol. 1, p. 756 (in Chinese). Commercial Press, Shanehai. Yeh, 9. (1967). In “Cancer of the Nasopharynx” (C. S. Muir and K. Shanmugaratnam, eds.), UICC Monogr. Ser. No. 1, pp. 147-152. Munksgaard, Copenhagen. Zippin, C., Tekawa, 1. S., Bragg, K. U., Watson, D. A., and Linden, G. (19a2). 3. Nat. Cancer h a t . 2Q, 485-990.

TRANSCRIPTIONAL REGULATION I N EUKARYOTIC CELLS A. J. MacGillivray, J. Paul, and G. Threlfall Beatron Inrlituto for Cancer Research, Glargow, Scotland

I. Introduction . . . . . . . . . . . . . . A. Molecular Biology and Cancer . . . . . . . . B. Changes in Transcription during Differentiation . . . . C. General Aspects of Transcription and Its Control in Prokaryotes . 11. Eukaryotic Chromosomes . . . . . . . . . . . A. General Structure of Chromosomes . . . . . . . B. DNA in Eukaryotes . . . . . . . . . , . C. Chromosomal Proteins . . . . . . . . . . D. Chromosomal RNA . . . . . . . . . . . E. Miscellaneous Components of Chromatin . . . . . . F. Role of Chromosomal Constituents in the Structure of Chromatin 111. Control of Transcription in Eukaryotic Cells . . . . . . A. Template Properties of Chromatin . . . . . . . . B. Chromosomal Proteins in the Control of Transcription . . . C. Eukaryotic RNA Polymerases . . . . . . . . . IV. Theories of Transcriptional Regulation in Eukaryotic Cells . . . A. Free DNA in the Genome . . . . . . . , , B. Derepression by Polyanions . . . . . . . . . C. Other Mechanisms . . . . . . . . . . . References . . . . . . . . . . . . . .

.

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.

. . .

. . ,

. .

. . . .

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93 93 95

97

101 101 104 106 118 119 120 124 124

129 141 146 146 147 149 150

1. Introduction

A. MOLECULAR BIOLOGY AND CANCER The rapid development of basic ideas and new techniques in molecular biology have only recently made it possible to pose very precise questions about the nature of the abnormalities which characterize cancer cells. Some of the most meaningful questions emerge from studies of developmental biology, particularly from attempts to understand the nature of the differentiated state. It is now well established that, although the differentiated cells in an animal may all have the same genetic information within them (Gurdon and Laskey, 1970), the differentiated state may nevertheless be highly stable and survive clonal cultivation in vitro (Coon, 1966; Cahn and Cahn, 1966; Konigsberg, 1963) or lengthy serial transplantations (Hadorn, 1965). Like normally differentiated cells, cancer cells very often preserve characteristic features of the differen93

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tiated cells from which they were derived, again even in conditions of prolonged culture in vitro (Yasamura et al., 1966). On the other hand, it is a characteristic of cancer cells that they diverge from normality. This is, of course, frequently seen as a loss of differentiated features, but it has recently become clear that the acquisition of functions which are not normally exhibited by cells of the type from which the cancer cells developed is not uncommon. For example, fetal antigens may appear in tumors from adult tissue (Gold and Freedman, 1965), and hormones may be produced by nonhormone-secreting cells which have become neoplastic (Lipsett, 1965). Accordingly, the old idea has reemerged that cancer might be a disorder of cell differentiation. Not only is this a concept which provides a useful framework for the scientific investigation of the disease, but there is the suggestion that some tumors can undergo spontaneous cure through differentiation (Nelson, 1962 ; Smithers, 1962). Current ideas about the mechanisms involved in differentiation have been very much influenced by studies on the regulation of protein synthesis both in prokaryotes and eukaryotes. It is now clearly recognized that in mammalian cells the same basic rules as in bacteria probably apply in that genetic information for protein synthesis is included in DNA; this is transcribed to messenger RNA, and this in turn is translated into protein by a machinery involving ribosomes, activating enzymes, transfer RNA and other factors. I n eukaryotic cells there may be additional mechanisms not identified in prokaryotes. The chromosomes are much more complex and are segregated from the cytoplasm in the cell nucleus. Many of the new species of RNA made in the nucleus never leave it but are rapidly broken down, and their function is not known. Whereas ribosomes become attached to messenger RNA while it is still being transcribed from DNA in bacteria, in eukaryotes the RNA has to leave the cell nucleus and enter the cytoplasm before it is translated into protein. It is possible therefore to identify many levels a t which controls of protein synthesis could act in the course of cell differentiation. Changes could occur in DNA itself: regulatory steps could occur a t the level of transcription; the breakdown of RNA in the nucleus and its transport to the cytoplasm could provide a controlling point; and finally there could be control a t the level of translation of messenger RNA to protein. I n fact, there is good evidence that all these mechanisms can operate in the establishment of the differentiated state. I n this review we wish to consider regulation a t one level only, that of transcription, because there is much evidence to suggest that some very important controls operate there which are particularly relevant to normal differentiation and possibly to the distorted differentiation which is seen in cancer.

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B. CHANGES IN TRANSCRIPTION DURING DIFFERENTIATION The evidence that transcriptional controls play an important role in differentiation comes mainly from studies using the technique of RNAJ DNA hybridization. McCarthy and Hoyer (1964) hybridized rapidly labeled RNA from a variety of mouse tissues to DNA in competition with samples of unlabeled RNA from the same tissues. These studies indicated differences in RNA populations from one tissue to another. Later Whiteley et al. (1966) performed similar experiments on the rapidly labeled RNA in developing sea urchin embryos. They obtained evidence for changes in the RNA populations of the cells a t different stages of development. Similar experiments were carried out by Glisin et a2. (1966). Denis (1966) carried out investigations of the same kind on rapidly labeled RNA from unfertilized eggs and developing embryos of Xenopus laevis, and showed the same behavior. Daniel and Flickinger (1971) obtained similar findings for developing frogs. These studies and many others seem to indicate rather strongly that different species of RNA are made in different cells, and, since there is good reason to believe that the cells have the same DNA contents, it is a reasonable conclusion that transcriptional controls play an important part in differentiation. There are also many studiev which indicate that alterations of the physiological state of organs results in changes in transcription. Again most of these studies have used RNA/DNA hybridization. Church and McCarthy (1967a) found that changes in RNA occur during embryonic liver development. They also found (Church and McCarthy, 1967b,c) that changes in RNA synthesis could be detected in the liver following partial hepatectomy. In their studies they obtained evidence that some of the RNA’s were restricted to the nucleus in normal liver but were permitted to appear in the cytoplasm in regenerating liver. Wyatt and Tata (1968) studied RNA synthesis during induced metamorphosis in Xenopus and showed changing patterns of RNA synthesis (which they interpreted as reflecting preferential synthesis of ribosomal RNA) in response to thyroxine. Drews and Brawerman (1967) also found that new species of RNA were synthesized in the liver after treatment with cortisol. Turkington (1970) has reported changes in RNA during differentiation of mammary cells. There is thus a large body of evidence from DNA/RNA hybridization experiments to suggest that transcriptional controls operate in eukaryotes. The main limitation to interpretation of these experiments resides in the fact that it is now known that only RNA transcribed from repetitious DNA was studied (see Section 113). Evidence for transcriptional control comes from another direction

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in experiments carried out by Borun et al. (1967). These authors studied the synthesis of a messenger RNA which has been provisionally identified as histone messenger. This makes its appearance in the cytoplasm shortly before histone synthesis commences in the cell cycle, and disappears shortly before histone synthesis ceases. Evidence for intermittent transcription of RNA also comes from studies by D. Martin et al. (1969) on the induction of tyrosine aminotransferase during the cell cycle. These lead to the conclusion that a repressor molecule is synthesized only during certain parts of the cell cycle. Both of these studies, and other similar studies, can be explained in other ways, but the most likely interpretation is that transcriptional control results in the synthesis of different RNA molecules a t different times during the cell cycle. Observations of this kind have led several groups to study the patterns of RNA synthesis in normal and tumor cells with a view to determining whether differences in RNA can be identified. Some of the earlier studies were directed to identifying the presence of viral messenger RNA in cells transformed by tumor viruses, and these led, for example, to the demonstration of polyoma messenger RNA in cells transformed with polyoma virus (T. L. Benjamin, 1966). Kidson and Kirby (1964) were the first to look a t the patterns of RNA synthesis in a transplanted spontaneous mouse hepatoma, as compared with normal mouse liver. They used the technique of countercurrent distribution and obtained highly reproducible patterns from normal liver but progressive alterations in the messenger RNA profile of hepatomas during progression of the tumor. They concluded that many regions of the hepatoma genome which were undergoing transcription were different from those transcribed in normal liver. Jackson and Sels (1968) expressed doubts about the validity of studies of this kind because of their failure to obtain reproducible results. S. Jacob and Busch (1967) compared the template activity of chromatin from hepatoma cells with that from normal liver and reported that it was greater. Subsequently several RNAJDNA hybridization studies have been done to compare the rapidly labeled RNA from cancer and normal cells. Drews et al. (1968) reported a deficiency of some RNA classes in hepatoma nuclei as compared with nuclei from normal rat liver. Chiarugi (1969) compared nuclear RNA from rat liver, Yoshida ascites hepatoma cells, and Morris 5132 hepatoma cells and found them all to differ. Church et al. (1969) compared RNA synthesized in mouse liver with that synthesized in the Taper hepatoma. Their studies suggest a more diverse selection of molecules in the hepatoma cells than in normal liver. Similar results were reported by Mendecki et al. (1969) for rat liver hepatomas. Neiman and Henry (1969) compared the RNA from

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normal lymphocytes with that from chronic lymphocytic leukemic lymphocytes and again showed differences between them. Turkington and Self (1970) in a similar way compared RNA from normal mammary tissue with breast cancer cells. They too concluded that new species of RNA were synthesized in cancer cells. All the above experiments were performed by RNA/DNA hybridization under conditions which would permit hybridization only of RNA complementary to repetitious DNA, and they are therefore subject to the same limitations of interpretation as the experiments mentioned earlier. On the other hand, Roche et al. (1969) used polyacrylamide gel separations to compare RNA from nuclei of normal human kidney and several renal neoplasms and reported several additional RNA species in the tumor nuclear RNA. A few other general observations have been made about differences between tumor and normal cells which could be relevant to transcription and may be mentioned here. For example Sluyser (1968) claims that benzopyrene reacts in a fairly specific way with histones, while J. A. Smith et al. (1970) claim that breast cancers can be distinguished from each other and from normal tissue by their acidic nucleoprotein content. Those with the highest content of acidic nucleoproteins included some of the fastest growing tumors. While many of these experimental observations are capable of alternative explanations, taken together they add up to a considerable body of evidence to the effect that changed transcriptional patterns are characteristic both of normal differentiation and of neoplastic change. The case for studying transcription in relation to cancer is, therefore, clear. Before entering into consideration of special features of mammalian cells, some general aspects of transcription, mainly in prokaryotes, will be outlined. C. GENERAL ASPECTSOF TRANSCRIPTION A N D ITS CONTROL IN PROKARYOTES The central role of DNA-dependent RNA polymerase in the synthesis of RNA in bacterial cells was recently reviewed extensively by Richardson (1969). This section will therefore summarize the means whereby the enzyme itself may exert control over RNA transcription, and discuss briefly how it may interact with other controlling elements in the regulation of gene expression. DNA-dependent RNA polymerase catalyzes the synthesis of polyribonucleotide chains from nucleotide precursors by the formation of 3‘,5’-phosphodiester bonds (Hurwitz et al., 1960; A. Stevens, 1960; Weiss, 1960). Apart from its substrate, the 5’-triphosphates of guanosine,

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adenosine, cytidine, and uridine, the enzyme requires a DNA (or synthetic polydeoxyribonucleotide) template. Other requirements include a divalent metal ion (usually MnZ+or MgZ+),and the reaction rate is optimal a t pH 7.8 in a medium of moderate ionic strength. The Escherichiu coli enzyme comprises four main subunits, each being a single polypeptide chain, with a combined molecular weight of about 5 x lo5 daltons in the monomeric form. The two largest subunits have molecular weights of 165,OOO and 155,000 (p’ and p, respectively) and the smallest, a, is about 40,000. Together with u (Burgess et ul., 1969), molecular weight about 90,000 daltons, they form the functional unit of the enzyme (“complete” enzyme), which can be represented as follows: p’p az u. Sigma factor is known to be involved in the selection of correct initiation sites a t which the enzyme binds to the DNA template, whereas p’ is thought to be the template binding site of the enzyme. p is almost certainly involved in initiation of RNA chains, since it binds very strongly with rifampicin (an inhibitor of initiation by bacterial polymerase), and it contains binding sites for u and a. No specific role has yet been allocated to the a subunit, though its presence is required for activity, at least in the “reconstituted” enzyme (Heil and Zillig, 1970). The transcription process involves four well-defined steps (see Chambon, 1968), viz., the binding of the enzyme t o the DNA template, the initiation of polymerization (which involves the binding of the first nucleotide with the enzyme-template complex to give an initiation complex), elongation of the RNA chain on the template and finally, termination, which is accompanied by liberation of the nascent RNA molecule and the enzyme, which can then presumably reinitiate RNA synthesis. A great deal of information has recently accumulated on the role of u factor in the initiation process. It appears that the “core” enzyme (i,e,, complete enzyme minus U ) becomes bound to specific initiation sites on the DNA under the influence of u, which has been shown to associate transiently with the core (Travers and Burgess, 1969). The core enzyme itself can also bind to DNA and catalyze RNA synthesis, though much less efficiently than complete enzyme and with little specificity. For example, if the core enzyme is used to transcribe in vitro from a T 4 DNA template, it transcribes a t a low rate, and the RNA is synthesized randomly from both strands of the DNA. In contrast, when complete enzyme is used with the same template, it transcribes only the “pre-early” phage genes and with high efficiency (Milanesi et al., 1969; Bautz et al., 1969). Similar results regarding the function of u were obtained by Goff and Minkley (1969) in a study of its effect on transcription of the T7 phage genome by E . coli polymerase.

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The importance of u in strand selection is evident from these experiments, since a characteristic feature of in vivo transcription is the predominantly asymmetric nature of the RNA product. Studies of the temperature dependence of the initiation reaction (Hinkle and Chamberlin, 1970; Zillig et al., 1970) suggest that u may be involved in local denaturation of the DNA double strand in the vicinity of initiation sites thus allowing polymerase molecules access to single-stranded regions. It is doubtful whether in each cell type there is a multiplicity of u factors each with specificity for a particular site on the genome, though the appearance of new u factors has been reported in bacteriophageinfected cells. I n particular, the synthesis of RNA from a group of phage genes (“delayed early”) in T4 infected E . coli cells can be strongly correlated with the disappearance of the host u factor and the appearance of a new specificity factor (Travers, 1969, 1970a). This is accompanied by sequential modification of the a and p’ subunits of the host RNA polymerase (Walter et al., 1968; Bautz e t al., 1969; Travers, 1970b), presumably to allow interaction with the new sigma factor. A change in polymerase subunit structure has also been observed in sporulating Bacillus subtilis cells. Early in the sporulation process the template specificity of the enzyme is markedly altered so that it will no longer transcribe phage DNA (Losick and Sonenshein, 1969). The polymerase of vegetative cells requires a u factor for activity on phage DNA, but this factor is ineffective with enzyme from sporulating cells, indicating a change in the core enzyme. It was found that one of the @ subunits of the core enzyme from sporulating cells was considerably smaller than either of the p subunits of the vegetative enzyme (Losick et al., 1970). It is predictable that a new u factor is required for the modified polymerase to be able to transcribe genes associated with the sporulation process, although this has not yet been reported. Finally, it has been shown recently that when T7 bacteriophage infects E . coli cells, among the first phage genes to be transcribed is one which specifies a completely new RNA polymerase, required for transcription of the “late” genes (Chamberlin et al., 1970). A phagespecified RNA polymerase has also been recently identified in T3infected E . coli cells (Dunn et al., 1971). Both of these enzymes seem to be much simpler than E . coli polymerase, comprising a single polypeptide chain of molecular weight about 110,OOO daltons. It is apparent then that there are a t least two ways of directly changing the specificity of transcription in phage-infected cells by modification of the core enzyme in one or more of its subunits so that it will interact only with a new specificity factor, and by the appearance of a completely new polymerase molecule. These are examples of positive control mediated by

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the enzyme itself and factors which influence its ability to bind and initiate transcription a t specific sites in the genome. The studies by F. Jacob and Monod (1961) of the synthesis of E. coli lactose-metabolizing enzymes provide a clear example of negative control of gene transcription. Jacob and Monod postulated that a repressor molecule, the product of a “regulatory” gene for the lactose operon, can inhibit the synthesis of messenger RNA on the structural genes by binding strongly to an operator region of the lac operon. The structural genes are transcribed (“switched-on”) only when the repressor molecule is prevented from interacting with the operator region for example by combining instead with an inducer molecule such as lactose. A protein molecule having the properties expected of such a repressor has been isolated from E. coli cells (Gilbert and Mueller-Hill, 1966). It binds strongly to a specific region of the DNA, and even more strongly with specific inducer molecules (Gilbert and Mueller-Hill, 1967). This kind of mechanism is known to operate in the control of other E . coli operons and also in the mechanism whereby, in E. coli cells infected by phage A, the protein specified by the C, gene of the phage switches off the expression of other phage genes, enabling the phage genome to remain dormant within the host cell (Ptashne, 1967a,b). Recently a further control over gene activation in the E. coli lac operon was detected. During catabolite repression of induction of the lactose enzymes by glucose, a precipitous drop in cyclic AMP content of the cells was observed (Makman and Sutherland, 1965). The repressive effect of glucose was reversed by addition of cyclic AMP (Perlman and Pastan, 1968; Ullman and Monod, 1968). A protein factor was purified from the cells which has a strong affinity for cyclic AMP. It is thought that a combination of cyclic AMP with this protein (known as the CAP protein) exerts a positive control over the production of lac mRNA, though its mode of action remains unknown (Zubay et al., 1970). It could presumably interact with RNA polymerase on the promoter site for the lac operon, in some way enhancing the synthesis of the lac messengers (Perlman e t al., 1970). There is also a role for inhibitory factors in the control of RNA biosynthesis, and an instance of this is the inhibition of RNA synthesis in stringent strains of E . coli starved of an amino acid. An unusual nucleotide, guanosine tetraphosphate (ppGpp) accumulates rapidly in the cells (Cashel, 1970), and there is some evidence that it may specifically inhibit the production of ribosomal RNA. Less is known at present regarding the termination of RNA synthesis and release of nascent RNA molecules, i.e., the process which ensures that RNA molecules of the correct size are produced and released from

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the transcription complex. It can be shown that RNA made on a X phage DNA template by E . coli polymerase has a broad range of sizes, from 20 to 40s. However, when a protein factor isolated from E . coli cells is added, there are two main peaks, 7 S and 12 S, which correspond to early gene products found in &infected E . coli (Roberts, 1969). The protein has been designated p factor and presumably acts in conjunction with core enzyme and u to ensure synthesis of molecules of the correct length specified by the genes being transcribed. Furthermore, in the presence of p factor, RNA is released from the RNA-enzyme-DNA complex (transcription complex) at the end of the reaction. Richardson (1970) has shown that in the presence of 0 . 2 M KC1 (and absence of p) termination of RNA chains also occurs with the release of RNA and enzyme from the transcription complex. The polymerase molecules can reattach to the DNA template and reinitiate the synthesis of more RNA molecules. However, the RNA molecules formed under these conditions are on average 1.3 times larger than those made under lower salt (0.05M KCl) conditions. It would appear that the mechanism of chain termination by high salt conditions is probably not the same as that mediated by p factor, though it is possible that reinitiation in vitro in the presence of 0 . 2 M KC1 may have some relevance to reinitiation carried on under the physiological conditions in E . coli and other cells. A novel aspect of transcription has recently emerged with the demonstration of RNA-dependent DNA polymerase activity in many RNA tumor viruses (Baltimore, 1970; Temin and Mizutani, 1970). This could provide a mechanism by which viral genetic information encoded in an RNA strand can be transcribed into DNA, with subsequent insertion into the genome of the host cell resulting in transformation. Evidence has now accumulated to support this idea, including the identification of a hybrid RNA:DNA molecule in newly infected cells, which is presumably an intermediate in the formation of double-stranded DNA (Spiegelman et al., 1970a). II. Eukaryotic Chromosomes

A. GENERAL STRUCTURE OF CHROMOSOMES Chromosome structure has been reviewed recently by Hearst and Botchan (1970) and by Ris and Kubai (1970). The chromosomes of eukaryotic cells consist of DNA, an assortment of proteins and RNA. Only one major group of proteins, the histones, has been well characterized. Contracted metaphase chromosomes can be isolated fairly easily. During interphase the chromosomes are much more extended and hydrated. They cannot usually be studied micro-

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scopically, but they can be isolated as “chromatin,” the nucleoprotein complex obtained after the removal of cytoplasmic and soluble nuclear components. This may be prepared in a number of ways, e.g., by repeated extraction of the tissue with saline buffers followed by density gradient centrifugation (Bonner et al., 1968a) or by extraction of purified nuclei (Paul and Gilmour, 1968; Shaw and Huang, 1970). The general composition of chromatins prepared by such procedures is given in Table I. TABLE I CHEMICAL COMPOSITION OF CHROMATINS Source

DNA

RNA

Histone

Pig cerebellum Pig pituitary Rat liver Rat kidney Chicken liver Chicken erythrocyte Pea bud Calf thymus

1 1 1 1 1 1 1 1

0.13 0.108

1.6 1.56 1.15 0.95 1.17 1.08 1.10 0.89

0.04 0.06

0.03 0.02

0.05 0.05

Nonhistone protein 0.5 0.45 0.95 0.70 0.88

0.54 0.41 0.21

Reference

Shaw and Huang (1970) Shaw and Huang (1970) Elgin and Bonner (1970) Elgin and Bonner (1970) Elgin and Bonner (1970) Elgin and Bonner (1970) Elgin and Bonner (1970) Paul and Gilmour (1968)

Two types of chromosomes which can be readily studied microscopically during interphase are the giant chromosomes of insects and the lampbrush chromosomes of maturing oocytes. Most studies on the marphological aspects of interphase chromosome structure have been done with these, but chromatin has been commonly used for biochemical or physical studies. The details of chromosomal structure have not yet been worked out, but it has been recognized that the basic unit is a fiber 100-200 A. in diameter. Ris (1961) originally suggested that this was composed of a double fiber which itself contained two DNA molecules, but the suggestion made by DuPraw (1965) that there existed a basic 30 A. nucleoprotein fiber which formed a supercoiled strand of slightly more than 200 A. is now more generally accepted. Structures of this kind have been described by Solari (1968) in sea urchin sperm and by Abuelo and Moore (1969) in human lymphocytes. A similar structure has also been proposed by Wilkins et al. (1959) and by Pardon et al. (1967) based on X-ray diffraction studies of chromatin which indicated that the primary nucleoprotein fiber had a diameter of 35 A. and was coiled into a helical structure, More direct observation of structures with the dimensions predicted from these studies has been obtained in electron microscopical studies which have revealed equispaced electron dense and less dense bands lying adjacent and parallel to the envelope of nucleated erythrocytes (Davies, 1968; Davies and Small, 1968).

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When either isolated chromatin or intact interphase nuclei are studied by electron microscopy, no evidence of a higher order of organization can be discerned. However, since chromosomes segregate exactly like DNA during mitosis (Taylor et al., 1957), there must be a very highly ordered arrangement which has so far defied recognition. One particular fundamental question which remains to be resolved satisfactorily concerns the strandedness of individual chromatids. These behave genetically as though they are single and clearly regulatory mechanisms,for transcription would be easier to understand if they were. Evidence to support this idea has been advanced by Callan and MacGregor (1958) and Gall (1963), on the basis of studies with lampbrush chromosomes in amphibians in which it is possible to examine the single chromatid and to observe the way in which i t responds to enzymes such as proteases and DNA. On the other hand, some genetic findings are difficult to explain on the theory that chromatids are single-stranded and, although ingenious mechanisms have been advanced to explain how single-stranded chromosomes could behave in a way compatible with the occasional unusual patterns of segregation obtained in ascospores of Neurospora, this phenomenon would be more readily explained on the basis of doublestrandedness of the chromosome. Evidence for multistrandedness or a t least double-strandedness is by no means negligible (Wolff, 1969). One of the most important pieces of evidence is the appearance of isolabeled chromosomes a t the second mitosis after labeling (LaCour and Pelc, 1958; Peacock, 1963) and of subsequent mitoses (Deaven, 1968). Halfchromosomes have been observed in living cells (Bajer, 1965) and in disrupted chromosomes (e.g., Trosko and Wolff, 1965). Moreover, the evidence for single-strandedness based on the kinetics of digestion by DNase (Gall, 1963) has been challenged (Hecidle and Bodycote, 1968). These remarks refer to the interphase chromosomes of most eukaryotes. There is, of course, little doubt that the polytenic chromosomes of diptera are multistranded to a high degree. The subject is reviewed in a fairly recent paper (Heddle, 1969). What has been discussed above refers to the general structure of chromosomes. In interphase cells it can be recognized that some chromatin is more compact than the rest; this is referred to as heterochromatin; in general it is much less active in RNA synthesis than euchromatin, which is less densely packed. No precise information is available about the molecular differences between these two ; in particular very little is known about the ultrastructure of areas being transcribed in cells from vertebrates. The only hard information we have about this comes from the dramatic pictures by Miller and Beatty (1969) showing transcription of genes in Triturus oocytes. The examples they have

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studied involve sites which are being very actively transcribed indeed, and in which the chromosome is covered with a procession of polymerase molecules, to most of which is attached a strand of nascent messenger RNA. Since these increase in length from the initiation site to the termination site, the appearance has been likened to a “Christmas tree.” Obviously, there are enormous gaps in our knowledge of the general structure of eukaryotic chromosomes which will have to be filled before we have a good understanding of the control of transcription in these cells. The situation is somewhat better when we turn to look a t some of the details of the molecules which make up chromosomes. B. DNA IN EUKARYOTES In those prokaryotic genomes which have been studied, evidence has been obtained that certain sequences are reduplicated. However, the extent of reduplication is rather small and does not usually amount to more than two or three copies of a given gene. In eukaryotic cells there is much more DNA. For example, the human genome is about 1000 times bigger than that of Escherichia coli and there is evidence for a very high degree of reduplication of some sequences in it. This evidence comes not from genetic studies, but from physicochemical studies of the reannealing of denatured DNA. When DNA is heated it separates into two complementary strands. If this denatured material is then maintained at a temperature a little below the melting temperature, the strands reassociate, each strand of DNA with its complementary partner. Wetmur and Davidson (1968) showed that the rate-limiting reaction in the reannealing process behaves as a second-order reaction, It is characteristic of second-order reactions that the half-life of the reaction is inversely proportional to the concentration of reactant. Hence the more dilute the DNA solution, the longer the reaction will take. In the reannealing reaction the reacting component can be defined as being the minimum length of nucleic acid in which no long sequence of nucleotides is repeated. I n a relatively simple DNA molecule (such as a bacterial genome) a unique sequence of nucleotides might amount to 42 million daltons whereas in a complex DNA the unique nucleotide run might be 1000 times larger. If one took a given concentration by weight of the two, then the first would anneal very much faster than the second, since it would have a higher concentration of “unique” sequences. The rate of annealing by itself can therefore be used as an index of complexity. If one makes the simple assumption that the genome of an organism consists of a unique set of nucleotides without any reduplication, then the complexity of a DNA should be proportional to genome size; this is what is found for viral and bacterial DNA’s. By this criterion one

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would expect a mammalian DNA to be about 1000 times more complex than the DNA of Escherichia coli, and consequently one would expect that in the same conditions a given concentration of mammalian DNA would anneal about 1000 times slower. When experiments of this kind were first done with mammalian DNA, it was appreciated that there was an anomaly (McCarthy, 1967) in that a large part of the mammalian DNA annealed about as fast as bacterial DNA. The situation was analyzed in great detail by Britten and Kohne (1968), who introduced the use of the “Cot curve,” i.e., a plot in which reannealed DNA is measured against the product of the initial concentration of DNA (C,) X time ( t ) , usually placed on a logarithmic scale. Application of this analysis to eukaryotic DNA’s revealed discontinuities in reannealing. It became apparent that, besides DNA which was not repeated in the genome (unique or nonrepetitious DNA), there was a large amount of DNA which was highly repetitious in that it annealed very much more rapidly. The amount of this repetitious DNA varies from one organism to another. The degree of repetitiousness also varies but is often of the order of 10,OOO to 100,000; not infrequently, there are several groups of repetitious sequences. Britten and Kohne further showed, by studying the thermal stability of renatured DNA that the individual components of a given set of repetitious genes were not identical. Although they were similar enough to anneal with each other, there was sufficient mispairing of bases to lower the melting temperature of the hybrid. Accordingly, Britten and Kohne considered that these repetitious sequences occurred in “families” of related sequences. The distribution of repetitious DNA throughout the genome is of considerable interest. Britten’s group have tried, by shearing, to determine how long the unique and repetitious sequences are and have come to the conclusion that, except for a very highly repetitious component, the two are very intimately intermixed (Britten and Smith, 1970). A similar conclusion can be drawn from the results of Thomas et al. (1970), who found that when eukaryotic DNA was sheared and reannealed, either after degradation with exonuclease or after chain separation, a high proportion of the DNA occurred in circular structures. This provided evidence for linear repetition of nucleotide sequences a t relatively short distances. The function of the repetitious sequences is not, in general, known but there is some special information about some of them. When DNA is equilibrated in a cesium chloride density gradient, in addition to the main peak there occur in some species a small, heavy satellite, now known to contain the genes for ribosomal RNA, and a small, light satellite. The small, light satellite has been found to contain a partic-

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ularly highly repetitious class of DNA. This DNA has a base composition which suggests that it could never give rise to a messenger RNA which would be translated into protein (Southern, 1970). It is associated with the constitutive heterochromatin of the cell (Yunis and Yasmineh, 1970; Yasmineh and Yunis, 1970), and in in situ hybridization experiments it is found to be associated with the constitutive heterochromatin of the chromosome (Gall and Pardue, 1969). This DNA, then,probably has a structural role associated with the centromeric region of the chromosome. A degree of redundancy is also observed in the ribosomal genes, although it is not of nearly such a high order. There is now a considerable accumulation of evidence which indicates that the DNA sequences corresponding to the precursors of 2 8 s and 1 8 s RNA are separated by spacer sequences the whole complex being repeated several hundred times. There is also some evidence that sequences coding for messenger RNA may be highly repetitious. Williamson et al. (1970) found that the messenger RNA for globin hybridized with a fraction of DNA sufficient to account for about 1000 copies. Very recently A. Kedes and Birnstiel (1971) have obtained evidence that the sequences coding for histone messengers are similarly repetitious. These discoveries have, of course, to be taken into account in interpreting experimental findings, especially those based on RNA/DNA hybridization. C. CHROMOSOMAL PROTEINS 1. Histones

Much of the background to histone chemistry, characterization, and fractionation has been covered in recent reviews (Hnilica, 1967; Georgiev, 1969a; Stellwagen and Cole, 1969; Fambrough, 1969; E. L. Smith et al., 1970; Elgin et al., 1971; Johns, 1971). Hence for this discussion it is only necessary to summarize the situation and describe recent and relevant work. Many of the recent advances in histone chemistry have been due to the development of satisfactory methodology for their preparation and characterization. For example, the differential solvent extraction methods of Johns (1964) and ion-exchange chromatography procedures (Rasmussen et al., 1962; Kinkade and Cole, 1966a), together with reliable methods of electrophoresis (Johns, 1967a; Panyim and Chalkley, 1969a,b) have led to histones being characterized from many species and tissues (see Johns, 1971). It would appear that (a) histones occur only in eukaryotes and ( b ) there is little evidence for species or tissue specificity. They appear to consist of five main groups of proteins of rela-

TRANSCRIPTIONAL REGULATION IN EUKARYOTIC CELLS

107

tively low molecular weight (12,000-22,OOO) but of varying basicity. The main characterist'ics of these proteins are given in Table 11. Cell specificity of histones has been found in relatively few instances. Nucleated erythrocytes possess a specific serine- and lysine-rich histone (Neelin et al., 1964) [see Table 11, F2c(V)] which largely replaces the very lysine-rich F1 in these cells (Dick and Johns, 1969b), and may be connected with the lack of chromat'in template activity in mature erythrocytes. Certain other tissues have been shown to possess a specific very lysine-rich histone (Panyim and Chalkley, 1969b,c). In addition, in certain spermatozoa histones are replaced by arginine-rich protamines (Bloch, 1969). Although the proportions of histones and DNA are roughly equal (Table I), only a few studies have been carried out on the relative amounts of the various histone fractions in different tissues. In the chick embryo the proportions of the histone components vary during development until the adult pattern is reached (Lindsay, 1964; Agrell and Christensson, 1965; Kischer and Hnilica, 1967). In the embryo of the newt, however, no histones were observed until the early gastrula stage, when a constant amount of arginine-rich histones first appeared, followed by increasing amounts of slightly lysine-rich histones from late gastrula and finally by the lysine-rich fraction at the tail bud stage (Asao, 1969). A somewhat similar situation was found during embryogenesis in the sea urchin (Orengo and Hnilica, 1970). This sequence of events has been found to be associated in the newt with the appearance of the various histones in different regions of the embryo during development (Asao, 1970). Fambrough et a2. (1968) reported that the same pattern of histone fractions was found during development of the pea cotyledon except that the amount of histone F1 increased as maturation progressed. A few studies indicate that considerable variation may exist in the relative proportion of the histone fractions in various adult tissues (Hnilica et al., 1966; Johns, 1967a; MacGillivray, 1968). With the exception of the very lysine-rich histone F1, individual histone fractions probably consist of one molecular species (Fambrough and Bonner, 1969; Johns, 1971). The F1 histone fraction has been shown to be heterogeneous in electrophoresis (Panyim and Chalkley, 1969b) and ion-exchange chromatography systems (Kinkade and Cole, 1966a; Bustin and Cole, 1969a). Electrophoresis shows differences in the F1 components of mouse tissues, the complexity of the patterns being associated with the extent of phosphorylation of a parent histone molecule (Sherod et al., 1970). Chromatography on IRC-50, on the other hand, has revealed differences in the F1 complement from different species, but no convincing tissue differences in any one animal (Bustin and Cole,

? ?

TABLE I1 ~OMFQSXTION OF ~ ~ R O M APEOTEINS~ TIN ~

~

~~

~~~

Histon& Component

ASP (A)(P) (P) Ser (PI Glu @)(PI Pro (ap) GlY Ala Val (ap)

F1

F2b

F2a2

F2sl

2.5 5.6 5.6 3.7 9.2 7.2 24.3 5.4

5.0 6.4 10.4 8.7 4.9 5.9 10.8 7.5 Trace 1.5 5.1 4.9 4.0 1.6

6.6 3.9 3.4 9.8 4.1 10.8 12.9 6.3 Trace Trsce 3.9 12.4 2.2 0.9

5.2 6.3 2.2 6.9 1.5 14.9 7.7 8.2 Trace 1.0 5.7 8.2 3.8 2.1

t-Crs

0

Met ( w )

0 1.5 4.5 0.9 0.9

ne (ap)

Leu (ap) TYr Phe ( w )

F3

F2C(V)

4.2 6.8 3.6 11.5 4.6 5.4 13.3 4.4 1.0 1.1 5.3 9.1 2.2 3.1

1.7 3.2 11.9 4.3 4.7 5.3 16.3 4.2 0 0.4 3.2 4.7 1.2 0.6

A

9

Nonhistone proteins

B

pH5=

B+a’

HAP.

CSCl’

9.4 4.8 5.0 15.4 4.5 7.5 8.3 6.7 0.8 2.1 5.1 8.5 2.7 3.7

8.4 4.3 9.9 10.9 4.4 14.7 8.3 5.2 1.1 1.4 3.7 7.7 3.3 2.6

9.2 4.9 6.1 13.5 5.5 8.0 7.4 6.1 0.6 1.8 4.3 8.0 3.7 4.9

10.9 3.6 8.5 12.2 5.5 14.0 5.5 4.6

B r

5

”$

4

*d w

r 9

3

-

2.1 2.9 4.9 4.2 3.9

8

w

F! %i 9 F

r

26.8 0 Trace 1.8 3 6.2 28.6 4.6 46.0 21.5 2.1 Acetyl

LYfj

14.1 0 2.3 6.9 6 13.7 23.3 1.7 51.5 25.8 2.0

Pro Lys

10.2 0 3.1 9.4 7 16.4 22.7 1.4 43.3 27.6 1.6 Acetyl

Lys

0 Amino acid analysea are given as moles per 100 moles. *Johns (1971). Wang (1967a). 1 Elgin and Bomer (1970). MacGillivray e2 ol. (1971). W.Benjamin and GelIhorn (1968).

10.2 1.2 2.2 12.8 5 12.1 26.4 2.2 44.8

25.7 1.7 Acetyl Gly

9.0 1 .o 1.7 13.0 7 15.7 24.7 1.6 49.4 27.6 1.7 Ala

Ala

23.6

k! 3

7.7

4.8

-

6.7 -

6.2

3d

1.9 12.4

2.1 5.7

3.9

3.1

-

6.7

-

1.5 9.3

$

6.0 37.9 6.3 57.1 17.8 3.2

24.8 15.5 0.54 48.0 30.6 1.6

19.3 14.6 0.76 44.2 25.0 1.8 -

22.7 16.5 0.73 47.1 30.6 1.5

23.1 17.0 0.74 50.7 23.9 2.1

-

-

-

-

-

-

-

-

5.9

-

-

-

$ !a

f8! @

2

110

A. J . MACGILLIVRAY, J . PAUL, A N D G . THRELFALL

1968; Stellwagen and Cole, 1968; Kinkade, 1969). The F1 complement appears to be dependent on the physiological state of the animal (Stellwagen and Cole, 1968; Hohmann and Cole, 1969). Structural studies have been carried out on a purified component of rabbit thymus F1 (Bustin and Cole, 1970). These studies indicate an asymmetric distribution of amino acid residues such that the C-terminal portion of the molecule is highly basic (and possibly the site for binding to DNA) whereas the N-terminal portion has a more balanced composition. Other studies suggest that the C-terminus of F1 histone is invariant in structure, unlike the N-terminal region which may vary from one subfraction to another and possibly also depends on the species (Kinkade and Cole, 1966b; Bustin and Cole, 196913, 1970). Greenaway and Murray (1971) have suggested that these variations in structure may be genetically linked so that individual animals have different forms of F1 histones. Structural studies on the chick erythrocyte specific histone have shown microheterogeneity due to one genetically determined amino acid substitution near the N-terminus (Greenaway and Murray, 1971). It should be pointed out in this context that this specific erythrocyte histone varies considerably in analysis from one species to another (Edwards and Hnilica, 1968). The slightly lysine-rich histone fraction F2b has been obtained in purified form and its primary structure has been determined (Hnilica et al., 1970; Iwai et al., 1970). In this case the N-terminal region is the most basic part of the molecule and hence the most likely site for attachment to DNA. Many recent structural studies of histones have centered on the F2al histone. These have shown structural similarity of this fraction in various species and in both normal and tumor tissue (L. Desai et al., 1969; Leclerc et al., 1969; L. S. Desai and Foley, 1970; Sautiere et al., 1970a,c). The most significant studies, however, have resulted from the determination of the primary structure of the F2al histone of calf thymus, pig thymus, and pea seedling (Sautiere et al., 1970b; DeLange et al., 1969). The amino acid sequence of the calf and pig histones are identical, the majority of the basic residues being situated in the Nterminal region of the protein as in fraction F2b. However, it is of outstanding interest that the F2al histone of the more distant species, the pea, should contain the same sequence apart from two conservative substitutions (2% difference) (DeLange et al., 1969). The only similar comparison which has been made between plant and animal proteins concerns cytochrome c in which 30% of the residues differ between the two species (F. C. Stevens e t al., 1967; Yasunobu et al., 1963). Hence it would appear that during evolution the structure of this histone has been rigidly conserved, presumably because it has a highly specific

TRANSCRIPTIONAL REGULATION IN EUKARYOTIC CELLS

111

function to perform. Despite these manifestations of homogeneity, electrophoresis in systems of high resolution shows the F2al histone to be present in some tissues as a single component and in others as multiple components (Panyim and Chalkley, 1969b; Sung and Dixon, 1970). These tissue differences are probably associated with the varying degrees of acetylation and phosphorylation of lysyl residues in this histone (DeLange et al., 1969; Sung and Dixon, 1970). It is also interesting from the point of view of cell specificity that cow, but not pea, F2al histone should possess a methylated lysyl residue (DeLange et alJ969). Little structural work has been carried out on the F2a2 histone although it appears to have the same N-terminal peptide as F2al (Phillips, 1968; Sung and Dixon, 1970). It appears electrophoretically homogeneous (Johns, 1967a; Leclerc et al., 1969; Panyim and Chalkley, 1969a,b), but in high resolution systems it runs as a doublet due to microheterogeneity associated with phosphorylation (Sherod et al., 1970). Fraction F3 is the only histone to contain cysteine, the number of residues present depending on the species (Fambrough and Bonner, 196813). Since changes in the oxidoreduction state of this histone have been observed (see Section II,B,3), heterogeneity of this fraction could result from polymerization through disulfide bond formation. As before, microheterogeneity can be postulated since F3 histone possesses internal lysyl residues which are capable of being acetylated and methylated (Section II,B,3). Limited sequence studies have been performed on this histone (DeLange et al., 1970); these indicate little homology with F2al fraction. However, it is interesting to note that, unlike the other histones, Fraction 3 (and F2al) prepared from several species has constant electrophoretic mobility ; this may indicate some maintenance of structure during evolution (Panyim et al., 1970). I n summary, the characteristic properties (and possibly also the structure) of histones have been conserved during evolution in eukaryotes. This would tend to indicate that they have a specific function which cannot tolerate major changes in primary structure. Since they comprise a rather simple group of proteins, it is difficult to ascribe to them a complex role such as that of specific gene regulation; hence their actual function, although specific, may be of a much more general nature, e.g., as structural chromosomal protein. Modification of the structure of histones by phosphorylation, acetylation, etc., does introduce the possibility of a more varied role and this will be discussed later. 2. Nonhistone Proteins Several recent reviews describe the background and early work in this field (Hnilica, 1967; Georgiev, 1969a; Elgin et al., 1971) ; the study

112

A. J . MACGILLIVRAY, J. PAUL, AND G . THRELFALL

of nonhistone proteins is only now gaining momentum. They are defined as those proteins which remain associated with DNA after the histones have been removed. Owing to their ability to aggregate with themselves, histones, and nucleic acids, they have until recently been difficult to handle and characterize. Unlike histones, the amount of these proteins in chromatin varies with the tissue source and may be correlated with the level of RNA synthesis. For example, the nonhistone protein complement of maturing spermatozoa diminishes as the DNA template activity decreases (Marushige and Dixon, 1969). Conversely, the nonhistone proteins increase as the chromatin template activity increases in developing sea urchin embryos (Marushige and Ozaki, 1967). The results of Dingman and Sporn (1964) indicated that the amount of nonhistone chromatin proteins was less in mature than in immature chick cells. They also observed that treatment with certain carcinogens and hormones affected the level of these proteins in rat liver chromatin (Sporn and Dingman, 1966). Several methods are now used to characterize nonhistone proteins. These studies will be reviewed, and the implication of the results discussed. Wang (1966, 1967a) distinguished between chromosomal acidic proteins extracted from washed rat liver nuclei along with histones and DNA by 1 M NaCl and the nuclear residual proteins, which are insoluble under these conditions. The chromosomal acidic proteins are obtained in solution when the DNA and histones precipitate on reduction of the salt concentration to 0.14M. Of the four initial fractions obtained by ammonium sulfate and acid precipitation, three can be further separated into numerous components by chromatography on DEAE-cellulose and starch gel electrophoresis (Wang and Johns, 1968). The amino acid analysis of one fraction is shown in Table I. The presence of basic proteins in these fractions is indicated by protein unretained by DEAEcellulose and proteins electrophoretically cationic a t p H 8.3. The residual proteins can be solubilized by sodium deoxycholate and by procedures similar to those described for chromosomal acidic proteins. Pate1 et al. (1968) showed them to be highly heterogeneous as well. These two groups of proteins can be distinguished metabolically since the residual proteins show the highest specific activity in leucine-l'c labeling experiments (Kostraba and Wang, 1970). There is some evidence to suggest that the residual proteins are nucleolar in origin (Kostraba and Wang, 1970). Application of these techniques to the tissues of a number of species has shown tissue variations and differences between normal and tumor cells (Kostraba and Wang, 1970). Since the development of Wang's procedures, other workers have found a second fraction of nonhistone

TRANSCRIPTIONAL REQULATION IN EUKARYOTIC CELLS

113

proteins which can be extracted from chromatin by NaCl (0.35M) (Johns and Forrester, 1969; X. Wilhelm and Champagne, 1969; Spelsberg and Hnilica, 1971a). It would appear that these 0.35M NaCl soluble proteins are derived from the nuclear residual proteins (Johns and Forrester, 1969). Other authors have also claimed tissue differences in chromosomal acidic proteins prepared according to Wang’s procedure (19674 but separated by electrophoresis in an acrylamide-agarose system (Kruh et al., 1969,1970; Loeb and Creuzet, 1969, 1970; Dastugue et al., 1970). W. Benjamin and Gellhorn (1968) treated saline-washed rodent nuclei with dilute acid to remove histones. After the residue had been dispersed in CsCl at pH 11.6 the nonhistone proteins were separated from DNA by equilibrium density centrifugation and then applied to a polyacrylamide gel electrophoresis system run a t the same pH. Both mouse and rat proteins gave similar heterogeneous electrophoresis patterns, These preparations gave an acidic amino acid analysis (Table 11) and by 3*P labeling some of the electrophoresis bands appeared to be phosphoproteins. The work of Wang (1967a) and W. Benjamin and Gellhorn (1968) showed that some of the nonhistone proteins were phosphoproteins ; now a number of procedures have been developed for their isolation from chromatin. Langan (1967), Gershey and Kleinsmith (1969a), and Kleinsmith and Allfrey (1969) all used procedures based on that of Wang with a final purification step using calcium phosphate. Although these phosphoproteins appeared to be difficult to handle, solubilization in sodium dodecyl sulfate (SDS) followed by electrophoresis in an SDSacrylamide system has shown them to be highly heterogeneous and tissue specific (Platz et al., 1970). Shelton and Allfrey (1970) and C. T. Teng et al. (1970) have adopted a somewhat different procedure whereby phosphoproteins are extracted from dehistoned chromatin by phenol, a procedure used earlier by Steele and Busch (1963). Electrophoresis in an SDS-electrophoresis system has confirmed the tissue specificity of chromatin phosphoproteins. Using chromatins prepared according to Bonner et al. (1968a), Elgin and Bonner (1970) removed histones with dilute acid and then solubilized the residue in SDS a t pH 8. The DNA was removed by ultracentrifugation, and the SDS-nonhistone proteins were applied to an SDS-acrylamide gel electrophoresis system. The proteins from a number of tissues and species were found to be heterogeneous, the majority having molecular weights of the order of 5000-100,000, and to be remarkably similar from one organ to another. Very few tissue differences were found although pea bud nonhistone proteins appeared to possess only half of

114

A. J . MACGILLIVRAY, J . PAUL, AND G . THRELFALL

the vertebrate complement. The amino acid analyses of some of these components are given in Table 11. I n this laboratory we have attempted to develop a method of isolating the nonhistone chromatin proteins without exposing them to extreme conditions of pH, etc. (MacGillivray et al., 1971). Following upon the observations of Faulhaber and Bernardi (1967) and Hacha and Fredericq ( 1968), that salt-dissociated nucleoproteins could be chromatographed on hydroxylapatite (HAP) , we solubilized chromatins in 2 M NaCl-5 M urea-1 mM phosphate, pH 6.8 (Bekhor et al., 1969a) and applied the solution to HAP columns run in the same solution. The histones are unretained and the bulk of the nonhistone proteins are recovered from the column by increasing the phosphate concentration to 0.05 M . Table I1 gives the amino acid analysis of one such preparation. Since aggregation problems prevented the electrophoresis of desalted preparations in conventional systems, we then treated the nonhistone proteins with SDS as well as urea prior to electrophoresis in an SDS-urea-acrylamide system. Figure 1 shows that, in agreement with Elgin and Bonner (1970), the nonhistones are very heterogeneous and, compared with histones, are of a wide molecular weight range. However, the patterns of these proteins from different tissues and species are generally similar. Many of the proteins are of high molecular weight, but each chromatin extract has a discrete number of low molecular weight components which do not appear to be contaminating histones. A similar procedure for solubilizing cerebellum and pituitary chromatins in salturea was employed by Shaw and Huang (1970). After removal of the DNA by centrifugation or gel filtration, the proteins were separated on polyacrylamide gels at pH 2.7 in urea. Surprisingly, despite even freeze-drying such a mixture of proteins, an electrophoretic separation of histones and nonhistone proteins was obtained. Although a considerable amount of protein appeared to be unable to enter the gels, the patterns of the nonhistone proteins from both tissues were remarkably similar. Shirey and Huang (1969) attempted a separation of chromatin proteins using SDS alone. Sperm chromatin was solubilized in 1% SDS prior to removal of the DNA by ultracentrifugation. The ultracentrifuge supernatant contained most of the chromatin protein which was recovered by freeee-drying followed by removal of the SDS by urea dialysis and BaCl, precipitation procedures. Electrophoresis showed that all of the histone fractions had been recovered, together with one acidic protein fraction. However, the presence of nonmigratory material indicated that aggregation of histones and acidic protein had taken place to some extent during the fractionation procedure. Thus, in comparison with histones, the nonhistone proteins of chro-

TRANSCRIPTIONAL REGULATION I N EUKARYOTIC CELLS

115

FIQ. 1. Electrophoresis of nonhistone proteins from mouse tissues in a sodium dodecyl sulfate-acrylamide system. Curve A, Mouse kidney; B, mouse liver; C, mouse spleen. The complexity of the proteins may be compared with the pattern given by histones run in the same system (curve D). From MacGillivray et al. (1971), by permission fram the Federation of European Biochemical Societies.

matin appear to be more heterogeneous and of higher molecular weight. The degree of their complexity is difficult to estimate at present. The conditions employed by Wang (1967a), W. Benjamin and Gellhorn (1968), and Shaw and Huang (1970) may lead to aggregation, thus making the interpretation of electrophoresis data difficult to assess. In addition Sonnenbichler and Nobis (1970) have described the binding of histones to DNA in acid solutions so that by some criteria they appear as acidic proteins. This could complicate the results of the fractionation procedures which utilize acid extraction of histones as a first step (W. Benjamin and Gellhorn, 1968; Elgin and Bonner, 1970; Shelton and Allfrey, 1970). The use of detergent systems appears to avoid some of these problems, but this procedure separates proteins only on the basis of molecular weight differences (Shapiro et al., 1967; Weber and Osborn, 1969). Hence other forms of heterogeneity-e.g., differences in primary structure-will not be seen. If, however, we take the results of the SDS

116

A. J . MACGILLIVRAY, J . PAUL, A N D G . THRELFALL

procedures at face value, it appears that the nonhistone proteins show “limited heterogeneity” (Elgin and Bonner, 1970) in various species and tissues. This result is easy to rationalize since most chromatins are likely to possess similar enzymes (e.g., polymerases) , structural proteins, and perhaps contaminating membrane proteins. Demonstration of cell specificity may only come from differential extraction procedures similar to those described above for phosphoproteins. At this stage it should be pointed out that the distinction between histone and nonhistone proteins has been made entirely on the basis of chemical extraction procedures and from some aspects of their analysis. Thus dilute acid or strong salt will liberate histones, but not nonhistone proteins, from chromatin. Such a separation may be entirely arbitrary or reflect differences in binding to DNA since nonhistone proteins freed of DNA are soluble in acid (Wang and Johns, 1968; Shaw and Huang, 1970) and salt solutions (Wang, 1967a; Bekhor et al., 1969a; Shaw and Huang, 1970; MacGillivray et al., 1971). Likewise amino acid analysis shows histones to be basic, and nonhistone proteins to be acidic, proteins; hence, on the basis of isoelectric point data they also appear to be separate groups of proteins. If, however, their composition is assessed with respect to their content of polar and apolar residues, then the two groups of proteins appear similar except that some preparations of nonhistone proteins contain more apolar amino acids than the histones (Table 11).In fact, owing to the asymmetric distribution of basic amino acids, some portions of histones appear to be similar to enzymes and globular proteins (Bradbury and Crane-Robinson, 1971). 3. Synthesis and Turnover of Chromosomal Proteins

Early work suggested that the site of histone synthesis was within the nucleus since thymus nuclei were found to be capable of incorporating amino acids into histones by a puromycin-sensitive pathway (Allfrey et al., 1964; Reid and Cole, 1964). In support of this general impression, such nuclei were shown to contain ribosomes (Allfrey, 1963). However, more recent investigations with other cells have shown histonelike polypeptides on cytoplasmic ribosomes (Borun et al., 1967; Gallwitz and Mueller, 1969; Gurley et al., 1970b). In addition a 7-9 S RNA has been observed immediately before DNA synthesis and during histone synthesis in HeLa cells (Borun et al., 1967; Gallwitz and Mueller, 1969, 1970) and in sea urchin embryos at cleavage (L. W. Kedes and Gross, 1969). Nemer and Lindsay (1969) have also isolated slowly sedimenting polysomes which may be the sites of histone synthesis in sea urchin embryos. Hence, it seems very likely that histones are synthesized on cytoplasmic ribosomes in the orthodox way.

TRANSCRIPTIONAL RHXJLATION I N EUKARYOTIC CELLS

117

Although there appears to be little translational control of histone synthesis during the cell cycle (Pederson and Robbins, 1970), the major synthesis of histones occurs a t or near the time of DNA replication. There is some slight difference of opinion about the exact timing of these two events; e.g., Butler and Cohn (1963) and Orlova and Rodionov (1970) indicated that histone synthesis precedes that of DNA in regenerating liver whereas Takai et al. (1968) found concomitant synthesis of DNA and histones in the same cells. The synthesis of histones along with DNA in the S phase of the cell cycle has been confirmed in experiments using ascites (Yarbo, 1967), HeLa (Robbins and Borun, 1967), salivary gland (G. Stein and Baserga, 1970a), and cultured hamster ovary cells (Gurley and Hardin, 1968). Little is known concerning the site of synthesis of the chromosomal nonhistone proteins, and details of the timing of their synthesis during the cell cycle varys. G. Stein and Baserga (1970a) found maximum synthesis of these proteins prior to that of DNA, whereas in ascites cells their synthesis appeared to follow that of DNA and histones (Ingles, 1971). Halliburton and Mueller (1971) on the other hand, found the synthesis of nonhistone proteins in HeLa cells to come after DNA and histone synthesis. G. Stein and Baserga (1970b) have also reported that synthesis of nonhistone proteins takes place even during mitosis in HeLa cells. If either the histone or nonhistone proteins perform a regulatory function it might be expected that this would be revealed by a rather higher turnover than other proteins. However, Hancock (1969) found that labeled histones were not appreciably lost from chromatin during several cell cycles and Byvoet (1966) showed a similar turnover of both histones and DNA in rat tissues. This evidence would tend to indicate a somewhat general metabolic stability of the DNA-histone complex. However, in addition to the wave of histone synthesis near the time of DNA replication turnover of histones does occur to some extent during the rest of the cell cycle. Some workers indicate that all histone fractions turn over at the same rate (Dick and Johns, 1969a), whereas other workers have reported differential turnover of histone components (Chalkley and Maurer, 1965; Spalding et al.,1966). I n a study of histone synthesis in cultured Chinese hamster ovary cells, Gurley and Hardin (1969, 1970) found that during exponential growth only histone F1 turned over, the other histones being conserved for several generations. Moreover, part of the F1 histone complement appeared to be synthesized a t least 1 hour before being complexed with DNA; this indicates the probable existence of nonchromatin pools of histone in these cells (Gurley and Hardin, 1970; Gurley et al., 1970a). Interference with cell division

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A. J . MACGILLIVRAY, J. PAUL, AND G . THRELFALL

causes abnormalities in histone synthesis. Thus, according to Gurley et al. (1970a), after irradiation histones dissociate from chromatin but reassociate after some delay. I n preparation for the ensuing replication of nuclear constituents, histone F3 is synthesized some hours before reassociation in such a way that a large excess of this histone is ultimately complexed with DNA. Histone synthesis has been reported during inhibition of DNA synthesis (Spalding et al., 1966; Gurley and Hardin, 1968). On the other hand, in HeLa cells, the synthesis of lysine-rich histones has been found to be more dependent on DNA replication than that of the arginine rich fractions (Sadgopal and Bonner, 1969). Apart from experiments in which stimuli, such as hormones, induce the synthesis of specific acidic proteins in target tissues (Shelton and Allfrey, 1970; C. S. Teng and Hamilton, 19701, only a few studies have been carried out on the turnover of the nonhistone proteins. It appears that these proteins are synthesized during the entire cell cycle at a level higher than that of histones (Ingles, 1971; Halliburton and Mueller, 1971; G. Stein and Baserga, 1970b). Also, Holoubek and Crocker (1968) observed that the specific activity of DNA-associated acidic protdins was higher in ascites cells than that of histones, and Hancock (1969) found that there was a higher proportion of labeled nonhistone proteins in the chromatin of mitotic than in interphase cells. In HeLa cells Halliburton and Mueller (1971) found that nonhistone proteins turned over faster than histones, but in ascites cells Ingles (1971) observed similar turnover rates for both groups of proteins.

D. CHROMOSOMAL RNA When total chromosomal proteins are isolated, they contain a considerable amount of RNA. Huang and Bonner in 1965 first described a special class of RNA containing a high content of dihydrouridylic acid which they considered to be bound to histones covalently. They postulated that the histones were subunits of larger structures linked by this nucleic acid. W. Benjamin et al. (1966) described a similar RNA which was bound to histone, and was ribonuclease insensitive. Subsequently Bonner and Widholm (1967) found that chromosomal RNA hybridizes with a large amount of DNA, and they were able to demonstrate organ specificity between pea cotyledon and pea bud chromosomal RNA. The properties of calf thymus chromosomal RNA were described more recently by Shih and Bonner (1969). They found that it was bound not to histones but to the nonhistone proteins, contained 40 nucleotides, and had approximately 3-4 dihydrouridylic acid residues per chain. Dahmus and McConnell (1969) described similar material from rat ascites cells. This had an s value of 3.3 S and a high content of dihydroribothymidine;

TRANSCRIPTIONAL REGULATION I N EUKARYOTIC CELLS

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it hybridized with about 4% of nuclear DNA. They found that the turnover rate of this RNA was not high, and was similar to that of transfer RNA. A further unusual property of this RNA was reported by Bekhor et al. (1969b). They described observations which suggested that chromosomal RNA hybridized to double-stranded native DNA a t a low temperature (4°C.) in urea. The hybrid molecule formed was very stable and did not dissociate until the nucleic acid became denatured. The function of this RNA has been a matter for speculation. Bonner’s group (1968b) has repeatedly made the suggestion that it may provide a simple DNA recognition mechanism. Evidence for this will be discussed in a later section. On the other hand, Heyden and Zachau (1971) have evidence that a large part of the chromosomal RNA from calf thymus chromatin is simply tRNA.

E. MISCELLANEOUS COMPONENTS OF CHROMATIN The histone fraction of chromatin has already been described as consisting of relatively few components (Section II,C,l) whose functions appear to be structural (Section II,F) or to be involved in masking DNA (Section 111,B13).It is therefore quite likely that the few reports of the presence of enzymatic activity in histones, e.g., nucleases and proteases (Leslie, 1961; S.J. Martin et al., 1963; K. B. Tan e t al., 1969) are due to contamination by other proteins. The most probable source of these is the nonhistone fraction, the heterogeneity of which has already been referred to (Section II,C,2). Wang (1967a) showed that his chromosomal acidic protein fraction contained several enzymatic activities, e.g., dehydrogenase, transaminase, and ATPase. Since then the presence of enzymes more directly associated with nucleic acid metabolism has been described. An aggregate RNA polymerase enzyme was described in chromatin in the early 1960’s (Weiss, 1960; Huang and Bonner, 1962), DNA polymerase was subsequently isolated from several tissues by means of procedures based on Wang’s methodology (Pate1 et al., 1967; Wang, 1967b, 1968a; Howk and Wang, 1969). Such preparations have also been found to contain nucleotidy1 transferase activity (Wang, 1968b). DNase activity has been reported to be present in chromatin (Swingle e t al., 1969) and has been isolated from Wang preparations of nonhistone proteins (O’Connor, 1969). A number of other enzymes have been found in chromatin whose activities affect chromosomal proteins. Thus Langan (1967), Kleinsmith and Allfrey (1969), and King and Gordon (1969) demonstrated protein phosphokinase activity in chromatin nonhistone proteins, including phosphoproteins. A number of authors have described proteolytic enzymes

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in chromatin (Phillips and Johns, 1959; Furlan and Jericigo, 1967a,b; Panyim et al., 1968). Elgin et al. (1971) have suggested that these proteases may play a specific role in control mechanisms in assisting in the removal of histones from chromatin (cf. during the replacement of histones by protamine in sperm; Section II,B,3). I n this respect it is interesting to note that the susceptibility of histones to the proteolytic activity described by Bartley and Chalkley (1970) should depend on whether or not the histones are bound to DNA. Enzymes which methylate chromatin proteins have also been observed in nonhistone protein preparations (Comb et al., 1966; Burdon and Garven, 1971). Chromatin has also been found to contain enzymes which catalyze the methylation of its DNA (Burdon, 1971) and the acetylation of its histones (Racey and Byvoet, 1971). Nonhistone proteins have also been shown to contain receptor proteins for a number of compounds which stimulate target organs. Haussler and Norman (1969) demonstrated that a metabolite of vitamin D was localized among certain nonhistone proteins of intestinal mucosa. Likewise receptor proteins for some steroid hormones appear to be present in extracts of nonhistone proteins of intact nuclei, e.g., androgens (Fang et al., 1969; Mainwaring, 1969), estrogens (Jensen et al., 1968; King et al., 1969), and progesterone (O’Malley et al., 1970). As judged from the investigations using estrogen (Maurer and Chalkley, 1967; Rochefort et al., 1969; Shyamala and Gorski, 1969; Alberga et al., 1971), these hormone receptors are actually part of the nonhistone proteins of chromatin. It is interesting that these receptors should be extracted from chromatin (along with other nonhistone proteins) with 0.3 M salt, a procedure which does not alter the template properties of the chromatin (Spelsberg and Hnilica, 1971a). Since the nuclear hormone receptors may be cytoplasmic in origin (King et al., 1969), this might be thought to support the suggestion of Johns and Forrester (1969) that the proteins extractable from chromatin by this procedure are cytoplasmic contaminants. Finally Mondal et al. (1970) have reported that the nonhistone fraction of coconut chromatin contain factors which affect the indigenous RNA polymerase. These include initiation, rifampicin inhibitor and polymerase inhibitory activities.

F. ROLEOF CHROMOSOMAL CONSTITUENTS IN THE STRUCTURE OF CHROMATIN In the preceding sections of this chapter we reviewed aspects of the biological function of chromosomal proteins. Since strands of DNA double helix of considerable length are condensed into tightly packed

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metaphase chromosomes or into the looser structure of interphase chromatin, it is highly probable that some chromosomal proteins play a structural role. Attempts have been made to obtain information about the chemical structure of giant chromosomes and lampbrush chromosomes by eneymatic digestion and histochemical methods. With regard to the lampbrush chromosomes, in which all loci seem to be active in RNA synthesis, Izawa et al. (1963a,b) showed that the protein:DNA and RNA:DNA ratios were much higher than in other chromosomes. The nature of the proteins associated with nucleic acids in giant chromosomes has not been studied in any detail. More extensive work has been done with giant chromosomes. Both Swift (1964) and Gorovsky and Woodard (1967) produced evidehce that the histone:DNA ratio remained constant throughout the length of the giant chromosome, both in the condensed regions of heterochromatin and in the less condensed regions in the puffs. On the other hand, most of the nonhistone protein that could be detected occurred in Balbiani rings and puffs. Similarly, all the RNA could be detected in Balbiani rings and puffs. Pelling (1964) showed by autoradiography that RNA was in fact being synthesized or accumulated there, while Edstrom and Beermann (1962) found that dRNA was locusspecific on the basis of base compositions. Further studies on the characteristics of the RNA from puffs by Daneholt e t al. (1969a,b) showed that this RNA is of high molecular weight and is heterogeneous in size. It turns over rapidly with a complete turnover time of less than 45 minutes in puffs and of less than 30 minutes in Balbiani rings. In view of the speculation that acetylation of proteins might play an important part in gene activation, Clever and Ellgaard (1970) have studied acetylation of protein using autoradiography. They found no evidence for preferential incorporation of acetate in preexisting or in newly induced puffs. The information gained from these morphological studies is limited, and a much more detailed picture has now begun to emerge from biochemical and physical studies on chromatin. Much of the current work has been dealt with in recent reviews (Bradbury and Crane-Robinson, 1971; Fredericq, 1971). Using biochemical parameters, Murray et al. (1970) found little evidence for stretches of free DNA in sheared chromatin. However, some phosphate groups of DNA in chromatin are free to react, as shown by binding studies wit,h a number of reagents, e.g., Azure A, polylysine, toluidine blue, and specific anti-DNA sera. According to these studies, the fraction of the phosphate groups of DNA in chromatin which can react is of the order of 50% (Klein and Szirmai, 1963), 38-500/0 (Itzhaki, 1970; R. J. Clark and Felsenfeld, 1971), 28-60% (Miura and Ohba,

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1967; Salganik et al., 1969; Kurashina et al., 1970), and 1-5% (Stollar, 1970), respectively. Itehaki (1970) showed by polylysine titration that these phosphates occurred in lengthy stretches, a finding confirmed by R. J. Clark and Felsenfeld (1971). Frenster et al. (1963) described a technique for fractionating chromatin into “heterochromatin” and “euchromatin.” Both fractions contain similar amounts of histones, but euchromatin contains more nonhistone and phosphoproteins than heterochromatin (Frenster, 1965a ; Salganik et al., 1969). Spectroscopic and physical chemical techniques have been used to determine the structure of DNA-protein complexes, but most of these have involved nucleohistones ; i.e., little or nonhistone protein has been present. Some workers, with the exception of Permogorow et al. (1970), have indicated from circular dichroisim studies that the conformation of DNA is changed in nucleohistone (Fasman et al., 1970; Simpson and Sober, 1970) and chromatin (Henson and Walker, 1970a; Shih and Fasman, 1970), the effect being related to the presence of protein in the complex. Similar changes have been found in the optical rotatory dispersion spectra of the DNA of chromatin (Tuan and Bonner, 1969), and these have been ascribed to a slight alteration in the DNA double helix when it is part of the chromatin superstructure (Sponar et al., 1970). Henson and Walker (1970b) have shown that DNA is more asymmetrical than nucleohistone, histones F2b, F2a2, and F3 being involved in this effect. X-ray diffraction, infrared, and optical rotatory dispersion spectroscopy studies have shown that isolated or DNA-bound histone fractions possess both helical and random coil configurations, the amount of the former being estimated a t 20-50% in some fractions (Bradbury and Crane-Robinson, 1971). Nuclear magnetic resonance studies show that regions of histones F1, F2a, and F2b can undergo conformational changes and are responsible for histone-histone interactions. Such regions appear to be not the basic parts of the histone molecule, which probably bind to DNA, but the remainder of the polypeptide chain, whose composition is similar to that of enzymes and globular proteins (Bradbury and Crane-Robinson, 1971). This technique has further confirmed the impression that the basic region is the only part of histone F2b to bind to DNA, whereas all of the polypeptide chain of histone F1 is apparently capable of such an interaction (Boubuk et al., 1971). It is interesting to note that histone F1 possesses significant amounts of proline (Table 11) and has been reported to have an extended structure with little a helix content (Bradbury et al., 1967; Haydon and Peacocke, 1968). Studies using interphase and metaphase chromosomes (Littau et al., 1965; Mirsky et al., 1968; Sluyser and Snellen-Jurgens, 1970) indicate that this histone

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fraction may link DNA-containing fibrils together. Although Olins (1969) has provided evidence that the lysine-rich histones lie in the large groove of the DNA helix, the most tightly bound histones (arginine-rich) show an extended chain form which appears to lie parallel to the helix rather than to grooves themselves (Bradbury and Crane-Robinson, 1971). DNA still retains its native B-form in nucleohistone, but several studies already mentioned indicate that other aspects of its conformation are altered when it is part of the nucleohistone complex. The physical studies which indicate that histones cause DNA to assume a compact and symmetrical structure in nucleohistone have recently been reviewed by Fredericq (1971). Likewise Bradbury and Crane-Robinson (1971) have discussed the evidence that nucleohistone has a coiled or folded structure. Based on X-ray diffraction work in Wilkins’ laboratory (Pardon et al., 1967; Richards and Pardon, 1970) using stretched fibers, the structure of nucleohistone currently considered most likely is a “supercoil” in which the DNA double helix is folded upon itself to form a coil of diameter 100A and pitch 120 A. Bradbury and Crane-Robinson (1971) point out some discrepancies in the evidence and emphasize that the supercoil is a t present only a convenient working model. Other evidence also favors the supercoil structure and X-ray diffraction studies on reconstituted nucleohistones show that histones are required for this effect (Zubay and Wilkins, 1964; Palau e t al., 1967). Since heterologous nucleohistones prepared from DNA and histones from very different sources still show evidence of supercoiling, it seems unlikely that there are specific base sequence associations between DNA and histones (Garrett, 1968). Electron microscopic studies also support the concept of supercoil formation (see review by Bradbury and Crane-Robinson, 1971). Experiments using nucleohistone partially depleted of histones or reconstituted from DNA and histone fractions show that neither the F1 histone nor the erythrocyte-specific histone is involved in forming supercoils and that more than half of the histone complement has to be removed before the supercoil properties of chromatin are lost (Murray et al., 1970). Attempts have thus been made to determine which of the individual histone fractions is responsible for the supercoiled formation. Specific removal of the F2al and F2a2 histones has resulted in the loss of supercoil in nucleohistone, a result which has been reversed in some experiments by replacement of these histones (Bradbury and CraneRobinson, 1971). Moreover, nucleohistones reconstituted from DNA and histones show supercoiling properties using either a mixture of F2a1,2, and F3 histones or a mixture containing F2a2 and F3 histones (Richards and Pardon, 1970; Bradbury and Crane-Robinson, 1971). Bradbury and Crane-Robinson (1971) regard the formation of the supercoil-type of

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structure as resulting from histone-induced constraints “arranged both around the DNA molecule and displaced one from another in a regular manner along the molecule.” These constraints might be due to the basic regions of histones straining the DNA helix into a supercoil form or be associated with interactions between the portions of histone molecules which do not bind to DNA. Further degrees of condensation of nucleohistone fibers might then come about through a succession of histonehistone interactions or could be associated with cross-linking caused by F1 histones (Littau et al., 1965). The problem of how specific sequences of DNA are made available for transcription will be discussed in Section 111, but in the present context it may be remarked that ,Johns (1969) has suggested that supercoiling, by itself, may inhibit transcription by preventing progression of RNA polymerase dong DNA. Ill. Control of Transcription in Eukaryotic Cells

A. TEMPLATE PROPERTIES OF CHROMATIN 1. Heterochromatinization and Puffing

The first information to suggest that transcription might occur in localized regions of interphase chromosomes came from studies on “puffs” and “Balbiani Rings” in the giant chromosomes of Diptera. The field has been extensively reviewed elsewhere (Beermann, 1966; Clever, 1968; Berendes and Beermann, 1969), and it will suffice here only to mention the more important aspects. The terms “puffs” and “Balbiani rings” are used in a loosely interchangeable way, but more particularly the term “Balbiani ring” describes some of the larger swellings whereas the term “puff ,” although used rather generally, refers to smaller swellings. Early in the study of these structures it was observed that they were inconstant, but that the appearance of certain Balbiani rings and puffs was characteristic of certain stages of development in the larva. Beermann (1952) described differential puffing patterns in different organs. These observations led to the suspicion that puffs might be sites of gene activity, and this was much strengthened by the observation of Pelling (1964), who showed by autoradiography that puffs were sites of RNA synthesis. Edstrom and Beennann (1962) isolated RNA from individual puffs and obtained evidence for locus specificity. These studies in giant chromosomes have provided the best direct demonstrations of RNA synthesis of a presumably organ-specific nature occurring in localized sites in chromosomes. Some other observations of a similar nature have come from studies on heterochromatinization (see Brown, 1966). There is both

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genetic and autoradiographic evidence that transcription is much reduced, if it occurs at all, in heterochromatic (condensed) regions of chromosomes. Some of these regions, described as “constitutive” heterochromatin are always condensed whereas “facultative” heterochromatin may be condensed in some circumstances but uncondensed and active in RNA synthesis in others. Balbiani rings and puffs can be induced in giant chromosomes by treatment of the whole insect or isolated salivary glands with ecdysone. This work is reviewed by Clever (1966a, 1967, 1968). Juvenile hormone can also stimulate a Balbiani ring in Chironomus (Lezzi and Gilbert, 1969; Laufer and Holt, 1970). Izawa et al. (1963a) had shown that actinomycin D caused regression of lampbrush chromosome loops, and this suggested that similar experiments should be carried out with giant chromosomes. Clever (1966b, 1968) found in such experiments that actinomycin D interfered with certain puffs but not others; he suggested that the puffs appeared in sequence and that RNA synthesis by the primary puffs was essential for the occurrence of secondary puffs. Beermann (1963) had already reported that low doses of actinomycin D prevented puffing in Chironomus tentans whereas high doses prevented reduction in the size of the puffs. Clever (1964, 1966b) and also Ritossa and Pullitzer (1963) found that puromycin did not prevent puff formation; indeed, Clever found that certain puffs could be induced by cycloheximide, a n induction which could be prevented by actinomycin D. It is not easy to explain all these findings in a unified theory. Clever has inclined to the view that the appearance of secondary puffs in some way depends on RNA synthesis by primary puffs. Kroeger (1963) has interpreted the findings differently, since he has demonstrated that a rejuvenation pattern of puffing can be induced by changing the sodium/ potassium balance in the medium. He postulates that a primary effect of agents inducing puffing is an alteration of the ionic balance within the cells, to which the appearance of puffs is secondary. The arguments for this hypothesis are discussed in review papers by Kroeger and Lezzi (1966), Lezzi and Gilbert (1970), and Lezzi (1970). Berendes (1968) has shown that one of the earliest events in the formation of a puff is the accumulation of acidic proteins (previously synthesized) in the locus. This precedes RNA synthesis. 2. Activation of Nuclei

Probably associated with the activation of special sites in the genome is the general activation of chromatin which is observed when condensed facultative chromatin becomes less condensed. Very little RNA synthesis occurs in condensed heterochromatin; Hotta and Stern (1966) showed

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that in meiotic plant cells RNA synthesis was inversely related to the state of condensation of the chromosomes. Renewed interest in this problem has arisen from studies of nuclear transplantation and cell fusion. Gurdon (1968) has shown that when frog blastula nuclei are injected into oocytes they swell and the chromatin becomes more dispersed. This is associated with cessation of DNA synthesis and an increase in RNA synthesis. Zetterberg and Auer (1968) studied phytohemagglutinin-stimulated lymphocytes by cytochemical methods and deduced that a reduced number of arginine residues in histones was bound to DNA phosphate groups. Bolund et al. (1969) have investigated the behavior of chick erythrocyte nuclei introduced into the cytoplasm of HeLa cells (by fusion in the presence of inactivated Sendai virus). After fusion the volume of the nucleus and its dry mass increased until DNA synthesis began. During this time acridine orange binding increased and the temperature stability of DNA decreased. Rigler et al. (1969) developed methods for measuring heat denaturation of DNA in individual cell nuclei and showed that both activation of erythrocyte nuclei by fusion with HeLa cells and activation of lymphocytes with phytohemagglutinin resulted in striking changes in the melting profiles of nuclear DNP, the changes being evident as a reduction of melting temperature. Ringertz and Bolund (1969) showed that swelling of erythrocyte nuclei could be produced in intact red cells or red cell ghosts by washing and incubation in salt solutions or tissue culture media devoid of serum proteins. The changes which occurred in these nuclei were similar to those observed in nuclei of erythrocytes fused with HeLa cells. I n general these studies lead to the conclusion that quite major changes can occur in chromatin; they strongly suggest that the reduction in density of chromatin is a prerequisite for the resumption of RNA (and possibly DNA) synthesis. 3. Priming of R N A Synthesis b y Chromatin

Morphological studies on giant interphase chromosomes are made possible by their high degree of polyteny. Such studies cannot be performed on mammalian interphase chromosomes because they are not visible by ordinary microscopy. However, the possibility of exploring the occurrence of similar phenomena in mammalian cells emerged from studies of the RNA synthesized from isolated nuclei, or chromatin, with either endogenous or exogenous polymerases. Experiments to compare the template activity of chromatin with that of DNA were first carried out by Huang and Bonner (1962), Bonner and Huang (1963), and Frenster et al. (1963). They found that, while

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it was possible to obtain some RNA synthesis using a bacterial polymerase and labeled triphosphate as precursors, chromatin was clearly a much less effective primer than DNA. On the basis of this observation it was speculated that there might be a specific restriction to priming, but this was questioned by Sonnenberg and Zubay (1965), who demonstrated that by shearing chromatin to make it more soluble, the priming activity could be greatly increased. They therefore suggested that the inefficiency of chromatin as a primer was simply a measure of its insolubility in the buffers used in the enzyme assay. A more critical analysis was then carried out by Marushige and Bonner (1966), who used rat liver chromatin as a template for RNA synthesis and performed an absorption analysis of Michaelis-Menten type to study the interaction of template and RNA polymerase. Their evidence suggested that while RNA polymerase bound equally well to DNA and chromatin, transcription was less with the latter as primer. This conclusion and later studies by Shih and Bonner (1969) are based on the assumption that RNA polymerase binds only to DNA and not to any of the chromosomal proteins. However, there is some evidence that RNA polymerase is also bound by histones (Spelsberg and Hnilica, 1969a). Hence, interpretation of the observations is uncertain. The problem was clarified by a quite different approach. Paul and Gilmour (1966a) and Georgiev et al. (1966) performed hybridization experiments with the RNA produced in the reaction and found that chromatin-primed RNA was complementary to a smaller fraction of DNA than DNA-primed RNA. From this Paul and Gilmour (1966a) concluded that many of the DNA sequences in chromatin were not transcribed, and that this restriction of transcription was of a specific nature (i.e., the same sequences were transcribed from all molecules of chromatin present in the preparation). Subsequently Paul and Gilmour (1966b) showed that the RNA transcribed from chromatin was effectively competed out by natural RNA isolated from the tissue from which the chromatin was prepared whereas it was not effectively competed out by E . coli RNA. They went on to perform experiments with chromatin from different tissues and claimed to be able to demonstrate organ specificity. Subsequently, other groups have made similar observations. Ursprung et al. (1968) and K. D. Smith et al. (1969) prepared chromatins from mouse liver, kidney, and brain and showed that the RNA synthesized from these had a high level of tissue specificity, as determined by competition experiments. It may be noted that in the experiments which Paul and Gilmour had performed they used a Micrococcus lysodeikticus polymerase. Smith et al. found that the results obtained with a bacterial polymerase and mouse polymerase were identical. C. H.

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T a n and Miyagi (1970) have also shown differences in priming by chromatin from adult rat, liver, and kidney. They confirmed that RNA synthesized in vitro from chromatin was indistinguishable from natural RNA from the corresponding organism, using a double saturation technique to demonstrate organ-specific differences between the transcription products. Some somewhat similar experiments have been done with whole nuclei. For example, Sullivan (1968) isolated RNA made from bovine thymus nuclei (without adding exogenous polymerase). This RNA was hybridized and competed out by unlabeled RNA’s from thymus and other organs. Evidence of organ specificity was obtained. The main shortcoming of these experiments is that the conditions of RNA/DNA hybridization used could be expected to detect only hybridization between RNA and repetitious DNA. With the publication of Britten and Kohne’s work (1968) this became clear, and Melli and Bishop (1969) presented some direct evidence for it. Nevertheless, the RNA/DNA hybridization experiments do seem to provide evidence that some kind of restriction of transcription occurs in chromatin, that, a t least so far as repetitious sequences are concerned, this is organ specific, and that the template properties of isolated chromatin are related to the priming behavior of the intact nucleus in vivo. I n other words, the data provide evidence for a pattern of control of transcription in the whole cell which is perpetuated in isolated chromatin. These observations stimulated studies of the properties of chromatin itself. 4. Hormone Action and Chromatin

A useful review of this field is contained in O’Malley (1969). The observation that ecdysone and juvenile hormone can induce puffing in the giant chromosomes of insects has stimulated a great deal of work on the effects of hormones on the priming behavior of chromatin. Means and Hamilton (1966) reported that an early derepression of protein synthesis occurred in all uterine subcellular fractions within 30 minutes of estrogen action. They found that actinomycin D abolished this effect and reduced nuclear RNA-synthesis, but that cycloheximide did not do this and indeed resulted in an increased incorporation of uridine in nuclear RNA compared with control or estrogen-stimulated uteri. These observations are of course very similar to observations which have been made with giant chromosomes. At about the same time, K-H. Kim and Cohen (1966) reported that the administration of thyroxine to tadpoles resulted in the modification of chromatin in liver nuclei, so that it became a more efficient template for RNA synthesis, as measured by incorporation of nucleotides. C-S. Teng and Hamilton (1968) found that estrogens

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increased the amount of genetic material available for transcription in the uterus. They reported that the RNA and total protein content of chromatin increased while the relative concentration of histone decreased. They also found that the binding of the hormone to chromatin was increased. O’Malley and McGuire (1968) and O’Malley et al. (1968) showed that progesterone and estrogen treatment resulted in changes in the hybridizable RNA in chick oviduct cells. Hamilton (1968) reviewed the field at that time and drew the conclusion that estradiol bound to chromatin in the nucleus, that this resulted in the stimulation of the synthesis of chromosomal and ribosomal RNA and an acceleration in the rate of formation of ribosomal particles, and that this ultimately resulted in an accumulation of new polysomes. Hahn et a2. (1969) also found that estrogens induced the appearance of a new hepatic RNA species in immature fowls. At the same time, a new species of RNA was formed in the oviduct, but the species induced in the oviduct and the liver were not identical. There are, therefore, a number of reports which seem to indicate that one effect of estrogens and progesterone is the appearance of new species of RNA and that this is associated with changes in chromatin. The review by O’Malley (1969) summarizes much of this evidence. King et al. (1969) reported that the nuclear estradiol receptor was an acidic protein containing protein-bound phosphate, while C-S. Teng and Hamilton (1970) demonstrated that estrogen stimulation of the uterus of ovariectomized adult rats caused incorporation of tryptophan into a specific band of nuclear acidic proteins. A somewhat similar observation was made by Shelton and Allfrey (1970), who observed that the injection of cortisone into rats specifically enhanced the synthesis of a nonhistone protein of molecular weight 41,000. This was prevented both by puromycin and by low doses of actinomycin D ; the authors concluded that synthesis of certain chromosomal proteins was necessary for the induced synthesis of RNA following cortisone administration. There are, therefore, a number of reports which suggest that the effects of estrogen, and progesterone, and possibly cortisol, may be mediated through a nonhistone chromosomal protein which modifies the transcription pattern from the chromosomes. At least superficially, these phenomena seem to resemble in many respects the phenomena following stimulation of salivary glands with ecdysone.

PROTEINS IN THE CONTROL OF TRANSCRIPTION B. CHROMOSOMAL A number of recent authors have reviewed various aspects of this subject (Georgiev, 1969a; Stellwagen and Cole, 1969; Hearst and Bot-

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chan, 1970; Lezzi, 1970; Elgin et al., 1971). We shall examine the evidence for transcriptional control by chromosomal proteins in the following order: ( 1 ) effect of histones on DNA template activity; (9) result of partial deproteinization on transcription of chromatin; (3)reconstitution of chromatin from its components ; and ( 4 ) control through modification of histones. 1. Histones

The observation that bacterial RNA polymerase would transcribe mammalian DNA in vitro led a number of workers in the early 1960’s to examine the effect on DNA template activity of histones, long suspected to be gene regulators (Stedman and Stedman, 1950). In these experiments template activity was simply measured by estimating the rate of incorporation of labeled nucleotides into RNA in the presence of the template and a bacterial RNA polymerase. It was found that histones would inhibit the ability of DNA to act as a primer for RNA synthesis in vitro (Huang and Bonner, 1962; Butler and Chipperfield, 1967), but the efficiency of each histone fraction to do so varied from one laboratory to another (Allfrey et al., 1963; Barr and Butler, 1963; Hindley, 1963; Huang et al., 1964). Although Bonner and Huang (1966) reported that their nucleohistones were soluble in the RNA synthesis medium, others have found out that the insolubility of the DNA protein complex could render the primer unavailable to the polymerase enzyme (Sonnenberg and Zubay, 1965; P. R. Clark and Byvoet, 1970; Moskowitz et al., 1969, 1970). Johns and Hoare (1970) have suggested that the different DNA-histone ratios used in several of these experiments could result in precipitation of different amounts of DNA from the incubation mixture. Hence, different histones might appear to inhibit template activity. However, in the experiments of Butler and Chipperfield (1967) the reduction of template activity did not precisely parallel the amount of DNA precipitated, indicating that histones do have some real effect on DNA template. The inhibition of RNA polymerase itself by histone fractions has also been reported (Spelsberg et al., 1969). Moreover, finely dispersed deoxyribonucleoprotein is a more efficient primer for RNA synthesis than fibrous prepartions (Sonnenberg and Zubay, 1965; Roy and Zubay, 1966; Hoare and Johns, 1970). Thus it appears that technical problems can cause discrepancies in the results of the template assay used in these experiments. Apart from the studies of Skalka et al. (1966), little or no attempt was made to characterize RNA produced i n vitro or even to show that the product was in any way complementary to the DNA acting as primer. Characterization by DNA-RNA hybridhation of such RNA’s made from

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DNA-histone templates indicates that histones prevent most of the DNA sequences from being transcribed (see Section III,B,3). 2. Partial Deproteinization Experiments Another approach to this problem was to remove the protein components of chromatin, selectivity or otherwise, so that the release of this restriction might lead to the identification of the specificity-bearing molecules. Conveniently a number of procedures were developed to selectively remove histone fractions (e.g., Hindley, 1964; Johns, 1964, 1967b; Murray, 1966, 1969; Ohlenbusch et al., 1967; Fambrough and Bonner, 1968a; Murray et al., 1968; Johns and Diggle, 1969) and certain nonhistone proteins (Johns and Forrester, 1969) from chromatin. Such procedures should be carefully controlled (especially when rate of RNA synthesis is used to estimate template activity) so that alteration in the physical state of chromatin does not lead to apparent change in transcription. I n the first instance, salt and acid extraction procedures demonstrated that the removal of histone F1 caused no change, but the elimination of groups F2b and F2a,3 considerably increased the template activities of chromatin from a number of tissues (Hindley, 1964; Seligy and Neelin, 1970; Spelsberg and Hnilica, 1971a). In the chick erythrocyte much of the inactivity of the chromatin was abolished on removal of the serine-rich specific histone (Seligy and Neelin, 1970), thus implicating this protein in shutting off genes as red cell maturation progresses. Other workers, however, reported that dehistoning chromatin caused a gradual increase in template activity from the time fraction F1 was removed (Georgiev et al., 1966). Kurashina et al. (1970) demonstrated a similar effect using a nucleohistone purified from calf thymus and also judged that histone molecules were distributed randomly along the DNA chains. The amount of RNA synthesized in vitro by their partial nucleohistones was proportional to the number of free phosphate groups in the template, not to the amount of histone removed. DNA-RNA hybridization techniques were also used to further this approach. A considerable amount of nonhistone proteins can be removed from chromatin with 0.35M NaCl (Johns and Forrester, 1969). This step does not affect the specificity of the template restriction (Spelsberg and Hnilica, 1971a). Bonner et al. (1968b) showed that RNA synthesized on pea chromatin would saturate pea DNA to the extent of 2.5%. Removal of histone F1 increased this to 776, and the level continued to increase as more histones were progressively removed. Using both saturation and competitive hybridizations, Spelsberg and Hnilica (1971b) reported that removal of histone F1 caused no change in the

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type of DNA sequences transcribed from rat chromatins. Removal of the F2b or the F2a-3 groups of histones only partially derepressed the DNA, so that chromatin completely devoid of histones still possessed a considerably restricted template (Spelsberg and Hnilica, 1970, 1971b). The results of Paul and Gilmour (1968) and Seligy and Neelin (1970) using chromatins dehistoned by acid and salt also agree with the latter finding, but Marushige and Bonner (1966) claimed that the DNA in their rat liver chromatin when complexed only with nonhistone proteins possessed no such restriction. Seligy and Neelin’s and Marushige and Bonner’s studies involved rate measurements only. In contrast, Georgiev et al. (1966) reported that when F1 histone was removed by salt extraction, ascites cell chromatin was so derepressed as to resemble pure DNA. On the basis of a study of the growth of RNA chains synthesized in vitro, Koslov and Georgiev (1970) claimed that histone F1 molecules act as stop points and prevent the movement of RNA polymerase along DNA. Clearly there is some conflict in the conclusions reached by different workers, and this may be due in part to the use of different criteria. A serious shortcoming of some of these experiments, especially those depending on salt extractions in the 0.6-1.0M range is that in these conditions a state of mass equilibrium is established for a t least some DNAhistone complexes. They can, therefore, theoretically bind to regions to which they were not originally attached. A number of important issues arise through these studies. First, in general it appears that removal of the 0.35 M NaCl nonhistone proteins and histone F1 causes little or no alteration in the template specificity of chromatin. Removal of the arginine-rich histones, F2a3 in particular, causes some alteration of this property, but the significant residual restriction of the template appears to be associated with the nonhistone fraction remaining with the DNA. Second, as indicated earlier in this review, the conditions of hybridization used by, for example, Spelsberg and Hnilica (1970, 1971b) and Paul and Gilmour (1968) indicate the degree of restriction applied only to the repeated or redundant DNA sequences. Little is known concerning control over the unique type of DNA sequences. In this respect the results of Georgiev’s group are obviously a t variance with many of these points. There is considerable difficulty in comparing their results with those of others since in Georgiev’s hybridization experiments low RNA to DNA ratios are used. However, Georgiev (1969a) claims that his hybridization conditions give an estimate of the amount of RNA synthesized in vitro which has been transcribed from the redundant DNA sequences, Hence he suggests that these portions of the genome are masked by the F1 histones.

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3. Reassembly of Nuclear Proteins

The third strategy used to identify the components responsible for the specific restriction of chromatin template is the reconstruction of chromatins and nucleoproteins from previously isolated components. I n most of these studies DNA/RNA hybridization has been used to characterize RNA’s made in vitro on reconstituted templates. I n their original studies Paul and Gilmour (1968)dissolved components in 2 M sodium chloride and reconstituted nucleoproteins by a stepwise dialysis down to 0.2 M sodium chloride, after which the insoluble nucleoprotein was centrifuged and washed repeatedly with distilled water. Subsequently the technique was modified according to a recommendation by Bonner (1968). The main difference was that the components were taken up in 2 M sodium chloride, 5 M urea, 0.01 M Tris a t pH 8.3, preparatory to stepwise dialysis and washing as before. Using the former method, Paul and Gilmour (1968)showed that RNA transcribed from a nucleoprotein reconstructed from isolated DNA, histones, and a nonhistone fraction saturated the same amount of DNA as RNA transcribed from natural chromatin. Later, Gilmour and Paul (1969) showed that RNA transcribed from a nucleoprotein reconstituted from more highly purified component using the salt-urea method was indistinguishable from RNA transcribed from normal chromatin and that both were extensively similar to natural RNA from calf thymus, from which the chromatin and individual components were derived. I n other experiments Bekhor et al. (1969a) and Huang and Huang (1969) dissociated chromatin in salt-urea and reconstituted it by dialyzing out the salt and urea. They too obtained evidence that the reconstituted material behaved like the original chromatin. Paul and Gilmour (1968) exploited this reconstitution technique to investigate the functions of individual components. When they combined histones alone with DNA they found that most of the DNA in the reconstituted nucleohistone was not transcribed. On the other hand, when they dissociated chromatin into DNA and total proteins and then recombined these, they found that the normal transcriptional pattern was restored. Subsequently they separated the total protein fraction into an acid-soluble fraction (histones) and an acid-insoluble fraction (nonhistones and RNA). They found that if these fractions were added to DNA together, the original pattern of transcription could be restored. Material reconstructed in this way yielded RNA which, by competition experiments, was not distinguishable from RNA transcribed from native chromatin. If serum albumin was used instead of the nonhistone fraction, effective reconstruction was not obtained. In a further investigation

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Gilmour and Paul (1970) prepared fractions from several rabbit tissues and experimented with combinations of nonhistone proteins, DNA, and histones from different organs. These results suggested that the nonhistone fraction directed the organ specificity of template restriction. Paul and Gilmour (1968) also found that the residual material left after extracting histones from chromatin with acid had much greater template activity than chromatin (although it did show some restriction). When histones were added back to this, the reconstructed material behaved like natural chromatin, again suggesting that the nonhistone material remaining bound to DNA after acid extraction prevented the restriction of transcription which histones alone produced. Similar conclusions have been reached by Spelsberg and Hnilica (1970) using a slightly different approach. They found that, on extracting chromatin with 2 M sodium chloride a t pH 6, most of the histones were removed but most of the nonhistone proteins remained attached to DNA. When the residual nucleoproteins prepared in this way from two different organs were then combined with histones, the RNA synthesized from each template behaved like RNA transcribed from chromatin from the organ providing the DNA-nonhistone protein complex (which had been shown to exhibit organ specificity). I n both these experiments and the experiments of Paul and Gilmour (1968) there is evidence that dehistoned chromatin did not have the full template activity of pure DNA. This suggests that the nonhistone fraction possesses different components with properties of both inhibiting and promoting transcription of repeated sequences of DNA. Analysis shows that the nonhistone fractions prepared by both PaulJs and Hnilica’s groups are largely protein, with a small amount of RNA present. Since the proteins are retained by anion exchange resins at nearly neutral pH, they are probably acidic; recent preparations of our fractions showed an RNA content of approximately 1%. Some findings by Marushige and Bonner (1966) and Marushige et al. (1968) showed that the rate of RNA synthesis on dehistoned rat liver chromatin and on a nucleoprotein complex prepared from DNA and nonhistone proteins was not significantly different from that on DNA. These results are not necessarily in conflict with those of the groups of Paul and of Hnilica, since RNA/DNA hybridization was not used to test the nature of the RNA. I n the experiments of Marushige et al. (1968) the nonhistone proteins were prepared by an SDS-extraction procedure, and this could have led to denaturation of some of the components. Although there is general agreement that the nonhistone fraction of chromatin is of importance in establishing specificity of transcription, there are some differences concerning the nature of the molecules actually

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involved in DNA recognition. Bekhor et al. (1969a) and Huang and Huang (1969) reported findings which suggest that RNA plays an important part in this. Bonner has expressed the view that the requirement for 5 M urea in the reconstitution experiments may exist because some kind of hydrogen-bonding of RNA to DNA has to occur. [In this connection it should be noted that urea was not used in Paul and Gilmour’s (1968) original experiments.] As more direct evidence for the role of RNA, Bekhor et al. (1969a) treated chromatin with RNase; when this was used as a template, they showed that the RNase treatment had abolished specificity of transcription. The main problem in this kind of experiment is the complete removal of RNase; in their paper Bekhor et al. (1969a) merely stated that the chromatin was “relatively free of RNase.” Huang and Huang (1969) performed a similar kind of experiment; they attempted to degrade RNA by treating chromatin with zinc nitrate. Again they found that this destroyed the ability of the chromatin to reassemble accurately. Again the question which arises is whether treatment with zinc nitrate is sufficiently specific to enable the conclusion to be drawn that RNA is an essential component of the reassembly reaction. The results of other workers have tended to favor the protein components of the nonhistone fractions as being involved in controlling DNA template activity. Wang (1968~)demonstrated that a fraction of rat liver nonhistone proteins would reverse the inhibitory effect of histones on the rate of transcription. The addition of nonhistone proteins to chromatin increased the template activity, so that the RNA synthesized saturated DNA a t twice the level obtained with RNA from untreated chromatin (Wang, 1970). Kamiyama and Wang (1971) showed that a similar effect occurred when nonhistone proteins were added in vitro to heterochromatin. They reported that the new RNA so produced differed in base composition and coded for proteins which were different from that produced by the untreated chromatin. As this RNA also differed in analysis from that of controls, it appears that this form of activation of chromatin opens up new sites for transcription. Spelsberg and Hnilica (1969b) reported that acidic nuclear proteins, including phosphoproteins, would only prevent histones from inhibiting DNA template activity if the protein fractions were allowed to react before combing with the DNA. In a similar series of experiments C-S. Teng and Hamilton (1969) found that in order to restore histone inhibited template activity of endometrial chromatin, nuclear acidic or chromatin nonhistone proteins had to be added to the synthesis mixture before histones. Thus in some of these experiments the order of addition of components to the reaction mixture is important.

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Other findings which implicate nonhistone proteins in regulation of chromosomal function are concerned with evidence for differential synthesis and differential binding to DNA. c-S. Teng and Hamilton (1970) observed that estrogen stimulated the synthesis of nonhistone proteins in uteri, but not in livers. As judged by electrophoresis in SDS-acrylamide gels, this synthesis was largely confined to one species of protein. Similarly Shelton and Allfrey (1970) found that cortisol stimulated the synthesis of a protein of molecular weight 41,000 in rat liver chromatin. Berendes (1968) also reported that, during puff formation Drosophila larvae brought about by ecdysone or temperature shock, acidic proteins accumulate prior to the onset of RNA synthesis. Nonhistone proteins prepared from coconut chromatin have been found to contain activities which resemble the factors which control bacterial RNA polymerase (Mondal et al., 1970). The demonstration that repressor proteins can bind specifically to DNA of bacterial systems opens up the possibility that such a technique might be useful in identifying which of the chromatin nonhistone proteins control the mammalian template. Thus Kleinsmith et al. (1970) showcd that a small proportion of such proteins (prepared according to Wang’s procedure) would bind to DNA in a species specific manner. Likewise, C. T. Teng et al. (1970) found that chromatin phosphoproteins would similarly bind to DNA, tissue specificity being demonstrated by differences in the electrophoretic and 32P-labeling patterns of the proteins. Although it remains to be seen whether these proteins affect transcription in any way, the concept of small amounts of the nonhistone proteins binding specifically to DNA is an attractive one. In summary, the nonhistone fraction of chromatin appears to be biologically complex, since its components not only can restrict and promote transcription but also can determine organ specificity of the template. Evidence has been presented above that both the nonhistone proteins and chromosomal RNA may be responsible for this. It is by no means clear whether either of these is uniquely responsible for opposing the inhibitory effects of histones, and much further experimentation is needed. 4. Chemical Modification of Chromosomal Proteins

At present we must assume that the binding of chromatin proteins to DNA is associated with the function they perform, whether it be a structural or regulatory role. The observation that some of the chromatin proteins posscsscd modificd amino acids, e.g., methyllysine (Murray, 1964), and the finding that in some systems such groups were metabolically unstable (Kleinsmith et al., 1966a) have led to a series of investigations to determine whether such changes could affect

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the association of protein with DNA and so provide a mechanism for releasing areas of DNA unavailable for transcription. For example, it might be possible for derepression to take place by removal of histones from DNA as a result of the introduction of groups to the peptide chain which weakened the ionic links between DNA and protein. Modifications to be described here are acetylation, phosphorylation, methylation, and the reversible oxidation of thiol groups. Recently, ethylation and acylation of nuclear proteins have also been reported (Orenstein and Marsh, 1968; Friedman et al., 1969) ; Ramponi and Grisolia, 1970). a. Acetylation. Acetyl groups occur in histones as a result of two processes. The N-terminal acetyl groups of histone fractions F1 and F2a1,2 are incorporated into the protein a t synthesis (Liew et al., 1970; Marzluff and McCarty, 1970). These groups are apparently stable, unlike the metabolically unstable acetyls which are enzymatically added to <-amino groups of performed histones F2a1, F2a2, and F3 (Gershey et al., 1968; Vidali et al., 1968; B. G . T. Pogo et al., 1968; Marzluff and McCarty, 1970) and o-seryl groups of F3 (Nobara et al., 1968; B. G. T. Pogo et al., 1968). The degree of acetylation of a lysyl residue in histone F2al varies among species (Delange et al., 1969). The donor of these acetate groups is acetyl-CoA (Allfrey, 1970), and a number of enzyme systems have been described which Catalyze the reaction (Nohara et al., 1966; Bondy et al., 1970; Gallwitz, 1970a,b). Nonenzymatic acetylation of histones by acetyl-CoA has also been reported (Paik et al., 1970). To complete the turnover process, deacetylase enzymes are also known (Inoue and Fujimoto, 1969; Libby, 1970). For the investigation of the biological significance of these modifications, a number of systems have been studied. The early experiments of Allfrey et al. (1964) showed that chemically acetylated histone F3 was less effective than untreated histone in inhibiting DNA-dependent RNA synthesis in vitro. This suggested that acetylation of histones might be closely linked to the control of RNA synthesis. Following upon this, B. G. T. Pogo et al. (1966) showed that in lymphocytes the early induction of RNA synthesis brought about by phytohemagglutinin (PHA) was preceded by acetylation of histones, particularly the arginine-rich fractions. Although some workers were able to reproduce this effect (Mukherjee and Cohen, 1969; Cross and Ord, 1970), others have indicated that the increased histone acetylation is a nonspecific or artifactual effect of the PHA-lymphocyte system (On0 et al., 1968; Monjardino and MacGillivray, 1970). Takaku et al. (1969) have found that erythropoietin caused an increase in histone acetylation in mouse spleen. PHA apparently inhibits RNA synthesis in granulocytes and causes a concomitant deacetylation of histones (B. G. T. Pogo et al., 1967). puff formation in polytene chromosomes of Diptera is associated

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with an increased synthesis of RNA. This type of gene activation is not associated with histone acetylation according to some (Clever, 1967; Ellgaard, 1967; Clever and Ellgaard, 1970), although Allfrey e t al. (1968) have reported that this occurs. J. A. Wilhelm and McCarty (1970) found that different growth conditions would induce histone acetylation in HeLa cells but they could observe no alteration in the general pattern of histone acetylation, nor could they relate changes in histone acetylation with altered rates of RNA synthesis. In contrast, shortly after partial hepatectoiny histone acetylation occurs at about the same time as the synthesis of DNA-like RNA and the increase in RNA polymerase activity (B. G . T. Pogo et al., 1968). During the cell cycle acetylation of HeLa cell histones F2a, F2b, and F3 rises to a maximum coinciding with the end of DNA synthesis. On the other hand, acetylation of histone F1 rises to a maximum a t the same time as DNA synthesis. Thereafter the degree of histone acetylation diminishes during the rest of the cell cycle, with the result that histones F1 and F2b lose most of their labeled acetate groups whereas F2 and F3 retain about half of theirs (Shepherd et al., 1971). These findings correlate with the observations that histone F1 is synthesized before DNA in HeLa cells (Gurley and Hardin, 1970), that in interphase cells it does not contain internal acetate receptor sites but has an Nterminal acetate group, and that histone F2b is not normally associated with histone acetylation (see above). Hence it would appear that there is no general rule for the association of histone acetylation and the induction of RNA synthesis. This correspondence is not seen in some systems, and its presence is doubted in others. b. Phosphorylation. It has been known for some time that cell nuclei contain phosphoproteins. Recent investigations have shown that both histones and nonhistone proteins can be phosphorylated, e.g., W. Benjamin and Gellhorn (1968), Schiltz and Sekeris (1969), Turkington and Riddle (1969), and Gershey and Kleinsmith (1969b): A number of phosphokinases have been described which require histones or protamines as substrate (Langan and Smith, 1967; Jergil and Dixon, 1970), and a phosphotase with similar requirements has also been characterized (Meisler and Langan, 1969). In both histone and nonhistone proteins phosphorylation takes place largely a t serine residues, although phosphothreonine has also been found (Kleinsmith et al., 1966b; Ord and Stocken, 1966; Stevely and Stocken, 1966; W. Benjamin and Gellhorn, 1968; W. B. Benjamin and Goodman, 1969; Langan, 1969; Turkington ad Riddle, 1969). Such phosphorylation is energy dependent, and in some systems it is unstable since there is considerable turnover of phosphate groups (Kleinsmith et al., 1966a,b). The level of nuclear protein phosphoryla-

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tion has been found to be dependent on hormone action (Langan, 1968, 1969; Turkington and Riddle, 1969) and to decline during cell maturation (Gershey and Kleinsmith, 1969b). Different histone fractions are phosphorylated in different systems, e.g., histones F1 and F3 in rat liver (Ord and Stocken, 1966, 1969), F1 and F2a2 in mouse tissues (Sherod et al., 1970), and all histones in trout sperm and rat tissues (Gutierrea and Hnilica, 1967; Marushige e t al., 1969; Sung and Dixon, 1970). I n fact, studies in vitro have shown different sites to be available for phosphorylation in histone F1 from different tissues and species (Jergil et al., 1970). The phosphate content of histones, particularly F1, varies with the physiological state of the tissue or animal (Stevely and Stocken, 1968; Ord and Stocken, 1969; Fitzgerald e t al., 1970), so that there is an increased level in this fraction and in F3 during DNA synthesis (Ord and Stocken, 1968, 1969; Adams et al., 1970). In trout testes, protamines are phosphorylated in the cytoplasm immediately after synthesis (Ingles and Dixon, 1967; Marushige et al., 1969) and prior to their replacing histones on DNA. Since protamines of native sperm are not phosphorylated (Ingles and Dixon, 1967), this is taken to indicate that phosphorylation may be associated with the transport of newly synthesized nuclear proteins into the nucleus, However, phosphorylation of preexisting histones in chromatin does occur in several systems a t a time when RNA synthesis is increased. Thus Turkington and Riddle (1969) found that hormones, such as insulin and prolactin, would stimulate histone phosphorylation a t the same time as RNA synthesis was increased. Langan (1969) showed that RNA synthesis induced in rat liver by glucagon was associated with an increased phosphorylation of specific seryl residue in histone F1. In regenerating tissues phosphorylation of histones, particularly those other than F1, occurs before DNA synthesis and may be associated with new RNA synthesis (Ord and Stocken, 1969; Fitzgerald et al., 1970). RNA synthesis induced in lymphocytes by PHA increased the turnover rate of histone phosphate groups (Cross and Ord, 1970), thus confirming the increased phosphorylation of total nuclear phosphoproteins observed in the same cells (Kleinsmith e t al., 1966a). Little is known at present about the relationship of the observed phosphorylation of acidic phosphoproteins and gene activity. As has already been mentioned, Kleinsmith et al. ( 1966a) have shown increased phosphorylation of these proteins (together with histones) in PHAstimulated lymphocytes. I n studies with isolated phosphoproteins C. T. Teng et al. (1970) have demonstrated tissue-specific patterns of 3zPlabeling and have obtained evidence for species-specific binding of these proteins to DNA. This result could indicate that phosphorylation of

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chromatin proteins is involved in allowing the transcription of previously repressed genes. In this respect Steveley and Stocken (1968) found that phosphorylated F1 histone was less effective as an inhibitor of DNA-dependent RNA synthesis in vitro than nonphosphorylated F1. Also evidence from the trout testes system shows that phosphorylation of a serine residue a t the probable DNA-binding site of histone F2al could lead to detachment of the protein from DNA (Sung and Dixon, 1970). c. Methylation. Histones are methylated, after synthesis, at lysine and arginine residues (Allfrey et al., 1964; Paik and Kim, 1968; Burdon, 1971). A number of methylating enzymes have been described, e.g., by Comb et al., (1966), Paik and Kim (1968), and Burdon and Garven (1971) ; the donor of the methyl group is S-adenosylmethionine, which is derived in turn from methionine (Allfrey et al., 1964; Murray, 1964; S. Kim and Paik, 1965). Although the arginine-rich histones have been shown to be the best acceptors of methyl groups, other histones will suffice (Comb et al., 1966; Orenstein and Marsh, 1968; Paik and Kim, 1969; Burdon and Garven, 1971). Little is known about the biological role of this methylation except that in regenerating liver it occurs after the peak of DNA and histone synthesis (Tidwell et al., 1968). Since methylation would not necessarily cause a change in the overall charge of a peptide chain it is interesting that these changes should take place a t a time when chromatin is undergoing conformational changes leading to a compact structure and a curtailment of nucleic acid synthesis. d. Reversible Oxidation of Thiol Groups. I n considering the structure of histones, we have already mentioned that F3 is the only histone to contain cysteine and thiol groups. Since the number of these residues varies in different species (Fambrough and Bonner, 1968b), different degrees of polymerization of F3 can occur through the formation of disulfide bonds. The state of F3 thiols is apparently associated with structural changes in the nucleus during development (Ord and Stocken, 1968, 1970a,b) ; nucleic acid synthesis appears to require the F3 thiols to be reduced (Ord and Stocken, 1969). In differentiated tissues there appears to be a more distinctive correlation between the state of oxidation of these groups, the condensation of chromatin structure and repression of RNA synthesis. Thus, in liver, the thiol groups are largely reduced even during regeneration (Ord and Stocken, 1969), but in the inert bird erythrocyte chromatin F3 thiols may be oxidized (Vidali and Neelin, 1968). Interphase chromatin, on the other hand, contains mostly reduced F3 thiols whereas in metaphase chromosomes these histone groups are oxidized, since histone F3 is either polymerized or complexed with nonhistone protein (Sadgopal and Bonner, 1970a,b). Inactive dense

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heterochromatin contains more disulfide bonds than the active diffuse euchromatin (Ord and Stocken, 1966). Finally, oxidized histone is more effective in inhibiting DNA-dependent RNA synthesis in vitro than reduced histone (Hilton and Stocken, 1966). e. General Considerations of Histone Modification and Chromatin Template Function. The work reviewed in this section has indicated that only a very general correlation exists between histone modification and changes in chromatin template activity. One of the major problems of eukaryotic systems is that changes in transcription of single genetic loci in chromatin, as might occur, for example, during induction of a specific enzyme, are extremely difficult to locate and investigate by current techniques. Even if some of the systems mentioned above did yield more positive results, their biological complexity would be too great for them to be considered as “model” systems. Such a system would allow the investigation of the physical and biochemical changes in chromatin a t the site of unmasking of a sequence of DNA coding for a known protein. The question could then be posed: If histones repress DNA template function, what are the events which lead to their removal and so allow transcription to proceed? A biological system which has been used to determine the events leading to the complete removal of histones from DNA concerns the replacement of histones in trout testes by protamine during spermiogenesis (Marushige et al., 1969). During these events phosphorylation and acetylation of histones occurs in the basic regions of fractions F1, F2a1, and F2a2. By using space-filling models Sung and Dixon (1970) have shown that phosphorylation of the N-terminal serine together with acetylation of four lysine residues could neutralize the basic charge of the N-terminal part of testes histone F2al. As this region possibly lies along the major groove of DNA, any electrostatic interaction with DNA would be effectively abolished. Thus the modification of the probable DNA-binding site could lead to an “unzipping” of the histone from DNA or allow its degradation by protease. Such enzymes have been described in chromatin (see Section I1,E) and recently Bartley and Chalkley (1970) described how the susceptibility of histones to enzymatic degradation differs if they are bound to DNA. Although this testes system is a biologically extreme example, it presents obvious similarities to situations which occur during gene activation, during development, and a t cell division. C. EUKARYOTIC RNA POLYMERASES The synthesis of ribosomal type (GC rich) RNA is normally localized in the nucleolus, whereas RNA having a composition resembling DNA

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(AU rich) appears to be transcribed on extranucleolar chromatin. These observations led to the hypothesis that, although in prokaryotes there is probably only one form of RNA polymerase, there may be multiple forms of the enzyme in eukaryotes, each with specific synthetic functions. I n fact the experiments of Widnell and Tata (1964, 1966) with isolated rat liver nuclei suggested the presence of two forms of RNA polymerase, apparently requiring distinctly different incubation conditions for optimal activity. At low salt concentration and with Mgz+ as cofactor, the RNA synthesized was GC rich whereas a t higher ionic strength ( 0 . 3 M ammonium sulfate) in the presence of Mn2+,AU-rich RNA was made. It was predicted that the activity assayed a t low ionic strength was responsible for ribosomal RNA synthesis and that a t higher ionic strength a latent enzyme was activated which could synthesize DNAlike RNA (Widnell and Tata, 1966). It would be anticipated that the former activity might be localized in the nucleolus and the latter in the extranucleolar chromatin or nucleoplasm. Chambon et al. (1968) have shown that the aggregate enzyme from rat liver nuclei can synthesize either GC- or AU-rich RNA according to the conditions of ionic strength used for incubation. They found also that, under all incubation conditions tested, Mnz+was a better activator than Mgzt. Their interpretation of these results was that, a t increased ionic strength, dissociation of histones from the aggregate enzyme occurred, allowing greater amounts of the genome to be transcribed. Thus the shift from a GC- to AU-rich product might be accomplished by a single species of RNA polymerase, avoiding the necessity to invoke the unmasking of a latent RNA polymerase activity by high salt. They showed that treatment of the aggregate enzyme with polyethylene sulfonate, a polyanion which also releases histones, markedly increased the template efficiency and AU content of the RNA (Chambon et al., 1968). The results of A. 0. Pogo et al. (1967) confirmed by both biochemical and cytological means that the composition of RNA and sites of RNA synthesis in isolated regenerating rat liver nuclei can be influenced by specific divalent cations (Mn2+,Mg2+) and the salt concentration of the incubation medium. Nuclei incubated with Mg2+alone made RNA with a base composition characteristic of ribosomal RNA; electron microscopic autoradiography showed it was made exclusively in nucleoli. Following the addition of MnZ+ and 0.04M ammonium sulfate (a salt concentration which does not disrupt nuclear structure nor remove chromosomal protein), the base composition became more DNA-like and RNA synthesis was observed in extranucleolar chromatin as well as in the nucleolus. These results did not allow any clear conclusion to be drawn about

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the multiplicity of eukaryotic polymerase. However, Roeder and Rutter (1969) demonstrated the existence of two (or possibly three) distinct protein species with RNA polymerase activity derived from rat liver and sea urchin nuclei. Following solubilization of the total nuclear enzyme activity by sonication of isolated nuclei at high ionic strength, the activities were separable by DEAE-Sephadex column chromatography. Enzymes I and I1 displayed differing degrees of activity with Mn*+ and Mgz+ and also responded differently to increasing ionic strength. A third difference was in their template specificity with either native or denatured DNA. Subfractionation of whole nuclei into nucleolar and nucleoplasmic fractions showed that purified nucleoli contain predominantly enzyme I whereas the nucleoplasmic fraction is greatly enriched in I1, strongly suggesting that these polymerases are specifically localized within the nucleus (Roeder and Rutter, 1970a). It is likely that they play specific roles in regulation of gene transcription and changes in their relative concentrations have been observed during sea urchin development (Roeder and Rutter, 1970b), although as yet no such functions have been verified. RNA synthesis carried out in vitro by the separated, partially purified enzymes acting on calf thymus DNA as template gave products with similar base composition to each other and to the product of the Escherichia coli enzyme (Roeder and Rutter, 1969). However, it may be very difficult to simulate in vitro the physiological conditions required for specific action of the enzymes and this must await more sophisticated experiments. Enzyme multiplicity has been detected in nuclei of several other species ranging from amphibian oocytes (Roeder et al., 1970) to bovine thymus (Kedinger et al., 1970). The latter reported the separation, by methods analogous to those of Roeder and Rutter, of two polymerase activities from calf thymus nuclei which they designated A and B. They can almost certainly be equated with I and I1 of Roeder and Rutter whose nomenclature will be used here. It was demonstrated that enzyme I1 of calf thymus could be completely inhibited by amounts of an sntibiotic a-amanitin which had virtually no effect on enzyme I (or E . coli polymerase) (Kedinger et al., 1970). This was the first indication of a structural difference between the enzymes since a-amanitin appears to inhibit by binding strongly to the enzyme (Seifart and Sekeris, 1969; Seifart, 1969). Later results confirmed that inhibition was mediated by binding of the enzyme (S. T. Jacob et al., 1970a; Meihlac et al., 1970), and further evidence was presented by S. T. Jacob et al. (1970b) of the differential sensitivity of rat liver nucleolar and extranucleolar polymerase activities to a-amanitin, the former again being insensitive, the latter strongly inhibited. Recently, Roeder et a2. (1970) and Tocchini-Valentini and Crippa

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(1970) reported the existence in Xenopus tissues of two polymerase fractions, one of which was strongly inhibited by a-amanitin, the other being insensitive. The latter enzyme, I, could be isolated from purified oocyte nucleoli, and it was found to bind preferentially to ribosomal DNA whereas activity corresponding to enzyme I1 binds to only a small extent with either rDNA or rDNA-free bulk DNA (Tocchini-Valentini and Crippa, 1970). This seems to be an example of the eukaryotic enzyme binding specifically to the DNA which it normally transcribes in vivo. However, Gniazdowski et aZ. (1970) found that the calf thymus enzymes would bind to either native calf thymus DNA or T 4 DNA through their affinity for homologous template was much greater than with T 4 DNA. They also found that initiation of RNA synthesis was severely restricted on T 4 or DNA as compared with calf thymus DNA, although this could be markedly stimulated by mild DNase treatment of the DNA to produce “nicks,” or by the use of denatured DNA as template. Addition of E. coZi u factor does not stimulate transcription on phage DNA by eukaryotic polymerases (Gniazdowski et aZ., 1970; Furth et al., 1970; Blatti et al., 1970). This is not too surprising since there are likely to be structural and functional incompatibilities between bacterial u and eukaryotic enzyme. It is not yet known whether the eukaryotic enzyme as isolated contains specificity factors and this awaits clarification. In the light of this result, the findings of Crippa and TocchiniValentini (1970) that injection of E. coZi u factor into Xenopus oocytes greatly stimulates RNA synthesis is remarkable. It suggests that (I can recognize initiation sites on the Xenopus genome as well as being able to interact with the endogenous enzyme. Thus u may be overcoming a normal restriction on RNA synthesis in the oocyte by simply enabling more polymerase molecules to bind at initiation sites. When a-amanitin was injected with u into stage 4 oocytes, it was found that the stimulated RNA synthesis was partially sensitive to the inhibitor, indicating the presence of type I1 enzyme, though greater than 97% of RNA synthesis in the oocyte at this stage of development is diverted toward production of ribosomal RNA (Tocchini-Valentini and Crippa, 1970). It is interesting in this connection that E. coli polymerase appears preferentially to transcribe the same strand of purified Xenopus ribosomal genes that is transcribed in vivo by the homologous Xenopus enzyme (Reeder and Brown, 1969). Further the “spacer” regions of the ribosomal genes, which are not transcribed in vivo, are only transcribed to a small extent by the bacterial enzyme, the major products being 28 S and 18 S ribosomal RNA (Reeder and Brown, 1969). Several authors have found that the partially purified eukaryotic enzymes have a marked preference for denatured over native DNA as

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template, in contrast to the cruder enzyme (and bacterial enzymes) which is relatively inactive with denatured DNA (Roeder and Rutter, 1969; H. Stein and Hausen, 1970; S. T. Jacob et al., 1970b; Stirpe and Novello, 1970). This finding was partly explained by Stein and Hausen (1970), who showed that a fraction obtained from calf thymus during the purification of RNA polymerase greatly stimulated the enzyme activity with native DNA. Although the fraction can be partially purified and appears to be a protein (sedimentation constant 3 S) the kinetics of its stimulatory effect suggest that its action is probably different from that of bacterial u factors. Seifart (1970) has described a factor with similar properties from rat liver nuclei which stimulates the activity of rat liver polymerase I1 with native DNA templates. It is obvious that little is understood about the mechanisms involved in the specific initiation of RNA synthesis by eukaryotic polymerases. As with the bacterial enzyme, a detailed knowledge of the subunit structure of the enzymes will almost certainly help to clarify the situation, and this is now beginning to emerge. Multiple subunits have been detected by electrophoresis on SDS-polyacrylamide gels and their sizes appear to bear some resemblance to the bacterial subunits (Blatti et al., 1970; Chambon et al., 1970). The recent isolation of a mitochondria1 RNA polymerase from Neurospora crassa (Kuntzel and Schafer, 1971) will accelerate an understanding of this eukaryotic enzyme, since it is considerably smaller (molecular weight 64,000) than the known nuclear polymerases. As mentioned previously, RNA-dependent DNA polymerase (reverse transcriptase activity) was recently discovered in RNA tumor viruses, some of which induce leukemia in animals, and a similar enzyme was later demonstrated in lymphocytes of leukemic patients, although apparently not in normal human lymphocytes (Gallo et al., 1970). The possibility that the assay of this enzyme might provide a diagnostic tool for leukemia has been explored with disappointing results, as it appears to be present in a variety of RNA viruses including some which are not recognized as oncogenic (Stone et al., 1971; Parks et al., 1971). Even more recently Scolnick et al. (1971) have detected reverse transcriptase activity in mouse cells transformed by murine sarcoma virus and also in monkey cells infected with a nontransforming virus. Using poly(rA:dT) as template they found similar activity in control (nontransformed) mouse cell cultures and control (uninfected) monkey cell cultures, as well as in cultured human cells. Thus apparently normal cells may also contain the same activity as that found in RNA virions and RNA-virus transformed cells, although up to now this has been detected only by using a synthetic RNA-DNA hybrid template which

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greatly increases the sensitivity of assay for the viral enzyme (Spiegelman et al., 1970b). To be more sure of the identity of the reverse transcriptase activities from various sources, more detailed comparisons of the highly purified enzymes will be required. IV. Theories of Transcriptional Regulation in Eukoryotic Cells

Several hypotheses have been advanced to explain the organ-specific synthesis of RNA in eukaryotes. Few of these are exclusive of others, and it is quite likely that several mechanisms operate. For convenience of discussion, however, they are considered separately.

A. FREEDNA IN THE GENOME One of the early ideas about genetic regulation in eukaryotes was that there might be stretches of free DNA in the genome in which regulation occurred in the same way as in microorganisms. Evidence against the occurrence of large amounts of DNA in the genome is, however, readily available from electron microscopic studies on lampbrush chromosomes, giant chromosomes, and isolated chromatin. In early studies of isolated chromatin, Bonner and Huang (1963) did report that a small amount of DNA might be present. This conclusion was based on studies of the melting behavior of DNA and chromatin. Most of the DNA in chromatin melts at a much higher temperature than free DNA, because of reinforcement of the DNA double helix by bound protein. I n pea chromatin Bonner and Huang (1963) did observe a small amount of material which melted at a lower temperature, and this seemed to be correlated with a minor deficiency of histones. Moreover, Frenster (1965b) suggested that there might be evidence for some strand separation in DNA in chromatin. However, in chromatin from rat thymus and rat liver (Marushige and Bonner, 19661, no low melting component could be discerned. We ourselves have made many similar observations. The evidence from melting curves and electron microscopy now seems to argue against large stretches of entirely free DNA. However, R. J. Clark and Felsenfeld (1971) have raised this question again, on the basis of the observation that about half of the DNA in chromatin is accessible to nucleases, and about half of it can bind polylysine of rather high molecular weight. The first observations of this nature were made by Miura and Ohba (1967), who showed by dye binding just a little less than half of the phosphate groups of DNA in chromatin were free to react. Titration of chromatin with polylysine was first reported by Itahaki (1970) and Clark and Felsenfeld’s results essentially confirmed her report. Both of them concluded that rather large stretches of DNA were

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available for binding with polylysine. However, Itehaki interpreted her results in a rather different way from Clark and Felsenfeld. Now that the primary structure of histone F2al is known it is possible to construct space-filling models to study the way in which this histone might interact with DNA; this has been done by Richards and Pardon (1970). Their most likely configuration shows that with this particular histone only about 60% of the covered phosphate groups in DNA are likely to be neutralized by basic amino acid residues. It seems possible, therefore, that the histones may lie mainly in one (the large) groove of DNA (Simpson, 1970) and may cover most of it in this way, but satisfy only about half of the phosphate groups, the remainder being free to react, possibly with molecules occupying the other groove. Polylysine may well occupy the lesser groove, and Itehaki makes this interpretation of her findings. Some other evidence that there may be small exposed stretches of native DNA comes from fluorescent antibody studies. It is not possible with these to determine how large the naked portions might be, and they might, for instance, represent sites of DNA synthesis. It is significant that in the very many electron microscopic studies which have been done on chromatin, no extensive areas of naked DNA have been demonstrated. An estimate of 40% would seem to be much higher than could be compatible with other evidence but the possibility that up to 10% of DNA might not be protein bound cannot be excluded by present techniques. The idea of having stretches of free DNA is, of course, quite attractive because it makes it possible t o postulate that special positive and negative controlling factors operating through polymerases might be present. The analogy with sigma factors in bacteria is obvious.

B. DEREPRESSION BY POLYANIONS Frenster (1965a,b,c, 1969) made the suggestion that polyanions might oppose histone binding to DNA. He mentioned that different kinds of polyanions and, in particular, RNA might perform this function. Frenster, in his hypothesis, which was one of the first general hypotheses in relation to transcriptional control from chromatin, distinguished between ligands (like histones) with an affinity for double-stranded DNA and those (like RNA) which might have a special affinity for singlestranded DNA. He speculated that the first stage of regulation might be a loosening of histone binding by polyanions; this would permit local strand separation, which would then permit a “derepressor RNA” to base-pair with one DNA strand and allow transcription from the other, free, strand. I n the past five years two different classes of polyanions have been

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investigated as possible general derepressors. Bonner’s group in particular has speculated that the special class of RNA, chromosomal RNA, in chromatin may form a stereospecific complex with DNA and hence provide the recognition mechanism for sequences which are to be derepressed. In Frenster’s original model, the suggestion was made that the RNA might recognize single-stranded DNA by base pairing, and it was this attractive hypothesis that first drew attention to chromosomal RNA. However, Banner’s group (Dahmus and Bonner, 1970; Sivolap and Bonner, 1971) suggested that this RNA has the unusual property of being able to “hybridize” with double-stranded DNA (at low temperatures) and in an organ-specific manner. On the basis of these observations and of the general theories of F. Jacob and Monod (1961), Britten and Davidson (1969) have advanced an elaborate hypothesis to explain genetic regulation in eukaryotic cells. Their proposal has many features in common with the operon model of Jacob and Monod. Regulator genes are called by them “integrator genes,” and they propose that they contain a “sensor’’ component. When an inducer is bound to the sensor component, it leads to synthesis of RNA by the integrator (regulator) gene. This RNA is termed “activator” RNA; it is envisaged that it, in turn, interacts elsewhere in the genome with a “receptor gene” (analogous to the operator) to permit transcription from “producer genes” (which would be called structural genes in bacterial regulation). The two major ways in which this hypothesis differs from the Jacob and Monod hypothesis are that, first, the regulator is thought to be RNA and not protein, and second, an argument, is advanced that redundant sequences in DNA might be desirable for the sensor and integrator genes. The other hypothesis which has been proposed recently is that derepressing polyanions may be nonhistone proteins. The evidence for the role of nonhistone proteins has been reviewed in some detail. This hypothesis is based on the idea that nonhistone protein can interact stereospecifically with DNA. It is now known of course that a number of acidic proteins can do this and the best examples are the bacterial and viral repressors studied by Ptashne (1967a) and Gilbert and MuellerHill (1966). Evidence that eukaryotic nonhistone proteins can bind specifically to DNA has already been mentioned (C. T. Teng et al., 1970; Kleinsmith et aZ., 1970). Granted that stereospecific binding to DNA sequences can occur, i t is then possible to speculate on several modes of action of nonhistone proteins. On the one hand, there is quite good experimental evidence that some nonhistone proteins can neutralize the totally repressive effect of histones in DNA transcription. It has been suggested by Johns (1969) that the inhibition by histones of tran-

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scription is due to supercoiling of the DNA which provides a steric obstruction to movement of RNA polymerase along the DNA strand. It is not difficult to imagine how the binding of a nonhistone protein in the groove of DNA not occupied by histones might alter the physical environment in such a way that the molecule could unwind. There is, theoretically, no problem about RNA polymerase moving along a protein-coated molecule, as DNA polymerase must do this during replication of the chromosome. On the other hand, nonhistone prateins could act in an opposite way, as repressors, by binding rather more tightly to DNA, in the same way as bacterial repressors do; there is indeed evidence that some nonhistone protein may behave in this way. The main physicochemical difference between “derepression” and “repression” might lie merely in the binding energy, and this could conceivably lend itself to allosteric control. Models of regulation based on nonhistone proteins would therefore be very similar to models of regulation in bacteria in which proteins act as intermediates between regulator genes and structural genes. The main difference in this speculation is that some of the nonhistone proteins are seen to alter the configuration of nucleohistone in such a way that it becomes possible for an RNA polymerase to traverse it and transcribe RNA from the DNA. Based on either of these models, Georgiev (1969b) has proposed an ingenious system for regulation of transcription which provides an explanation for repetitious DNA. According to his model, each structural gene would be preceded by a large number of regulator sites and separated from the promotor site by them. The actual combination of regulators preceding the structural genes would vary from one locus to another. Repressor proteins produced elsewhere in the genome would interact with these regulator sites. For a gene to be transcribed, therefore, it would be mandatory that there should be no repressor protein for any regulator sequence preceding it. According to this model, RNA would be first formed as a giant molecule on the chromosome (and there is good evidence that this is so) and, before passing to the cytoplasm as messenger RNA, would be processed so as to remove the copies of the regulatory genes. C. OTHERMECHANISMS Consideration of this subject would not be complete without listing some of the other possibilities of regulation to which brief reference has been made. I n particular there is now good evidence that there may be multiple polymerases in animal cells and that some of those may

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be specific for the synthesis of different kinds of RNA. This field is still in its infancy and little more can be said. Again, mention must be made of gene amplification. It was observed some years ago that DNA synthesis occurred at some loci in the chromosomes of Rhynchosciara angelae a t a time when it was not occurring in other areas. This gave rise to the suggestion that there might be some independent replication of certain areas of the genome (Pavan, 1965). A similar phenomenon has been described in many other species, and a general discussion of this subject is given by Lima-de-Faria (1969). The occurrence of gene amplification has now been proved very beautifully in the amphibian oocyte, in which the ribosomal genes increase by a factor of several hundred relative to other DNA sequences. It is not yet known how general this mechanism may be. Finally, the reader must be reminded of the large body of evidence for modification of chromosomal proteins by methylation, acetylation, and phosphorylation, and the evidence that an altered ionic environment can change the binding of proteins to DNA. Where these fit in the general picture has yet to be determined. There is no shortage of ideas or facts in this field, but many of the issues which have been discussed are confused by conflicting results and the use of inadequate criteria in assessing them. Perhaps in such a complex field there is some inevitability about this, but there is a great likelihood that, with the rapid improvement of biochemical and physical methods which is occurring, there will be a great clarification of the field very soon. It may then be possible to investigate the relevance of transcriptional disturbances in cancer with precision.

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ATYPICAL TRANSFER RNA’S AND THEIR ORIGIN I N NEOPLASTIC CELLS Ernest Borek a n d Sylvia J. Kerr Departments of Microbiology and Surgery, University of Colorado Medical Center, Denver, Colorodo

I. Introduction . . . . . . . . . . . A. General . . . . . . . . . . . B. Structure and Synthesis of tRNA . . . . . C. The tRNA Methylases . . . . . . . 11. The tRNA Methylases of Tumor Tissues . . . . 111. Transfer RNA of Tumor Tieaues . . . . . . A. Hypermethylated tRNA’s in Tumor Tissues . B. Modified tRNA’s in Tumor Tissues . . . . IV. Regulation of tRNA Methylase Activity . . . . A. Natural Inhibitors of the tRNA Methylases . B. Hormonal Effects on tRNA Methylases C. Other Inhibitors of the tRNA Methylases . . . V. Elevated Excretion of Modified Purines and Pyrimidines in Tumor-Bearing Animals and H u m m . . . . VI. The tRNA Methylases in Reverted Oncogenic Systems VII. An Attempt a t Interpretation . . . . . . . References . . . . . . . . . . .

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I. Introduction

A. GENERAL Since the readers of this series may not be experts in molecular biology, a little background information may be in order. Every cell in a metazoan organism contains all the biological information available to that particular species. This information may be expressed by the synthesis of a huge variety of proteins with varied attributes in different amounts. However, after differentiation only the information for the synthesis of but ,a fraction of these proteins is expressed. For the selection of the specific proteins to be synthesized, both proksryotic and, in particular, eukaryotic organisms have become endowed with sophisticated mechanisms. One of these is the control of transcription. This is the term used for the conversion of the information in the DNA into information in the complementary messenger RNA’s. For the mechanism of this control, a powerfully illuminating hypothesis 163

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was proposed by Jacob and Monod (1961). The “operon hypothesis” states that in addition to the presence of structural genes in a given cell which contain the information for the synthesis of a particular protein, there are other genes which control the transcription of the structural gene. There is abundant evidence that the synthesis of some proteins in some prokaryotic cells is indeed controlled by this mechanism. It often happens when powerful hypotheses are promulgated that they obscure information which is accumulating in evidence for other hypotheses or other mechanisms. This certainly has happened in this instance. Everyone knows about the Jacob-Monod hypothesis but very few people are aware that there is evidence for regulation of protein synthesis a t another level-at that of translation. The term translation has been coined by Jacob and Monod to designate the process of protein synthesis by the interaction of ribosomes, transfer RNA, and messenger RNA. Probably the earliest evidence for posttranscriptional control came from studies of protein synthesis in the early stages of development of the sea urchin. Early workers had demonstrated that there is a very rapid increase in the rate of protein synthesis in the early cleavage stages of sea urchin embryos. Brachet et al. (1963) showed that this large stimulation of protein synthesis, as measured by amino acid incorporation, can also be achieved in enucleated sea urchin eggs which are artificially activated. An equally convincing demonstration that de novo synthesis of messenger RNA is not required in the fertilized sea urchin egg was performed by Gross et al. (1964). They permitted fertilization in the presence of sufficient actinomycin D to suppress any RNA synthesis, but, nevertheless, the embryos showed the usual burst of protein synthesis and continued to develop to the stage of normal blastulae. The conclusion from these two experiments is compelling: There must have been sufficient information in the cytoplasm prior to fertilization for the ensuing protein synthesis. Therefore, some control was exercised prior to fertilization for the nontranslation of the available information. Of course, an alternate mechanism could be offered in this instance. It is possible that the information-containing messenger RNA was in some kind of masked form, and for this reason could not be translated. However, unequivocal evidence for translational control came from the brilliant studies of Dintzis (1961). He demonstrated that the globin chains of hemoglobin are synthesized commencing with the free amino terminal. The study of the kinetics of the rate of incorporation of a particular amino acid into different positions in the protein chain proved that the early portion of the chain is synthesized rapidly, but the later portions of it are synthesized more slowly, There-

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fore, some rate-limiting mechanism must exist in the sequential synthesis of the total protein molecule. Translation is the most complex molecular mechanism known in the living cell. It requires messenger RNA, which is the simplest of the structures involved, even though it is laden with information. The ribosomes are complex structures composed of RNA and a t least a score of different proteins. Transfer RNA, as we shall discuss later, is the most complex biomacromolecule known. In addition to these major components, there are ancillary ones: enzymes which transfer amino acids to the transfer RNA. Moreover, there are a t least 8 soluble protein factors needed for the completion of the synthesis of a protein. Any or all of these numerous components could be a regulatory factor in protein synthesis. There are three different lines of biological evidence which point, a t least in those instances, to transfer RNA as a regulatory factor. The first of these was discovered by Wainwright and Wainwright (1967) a t Dalhousie University. Hemoglobin synthesis commences in the chick embryo after 2 7 3 0 hours of incubation of a fertilized egg. Wainwright was able to turn on hemoglobin synthesis in excised chick blastodiscs at an earlier time by the addition of a population of transfer RNA’s extracted from the later stage of the development of the chick when hemoglobin synthesis is already going on. Anderson and Gilbert ( 1969) have shown another instance of regulation of protein synthesis by tRNA. Both the a and p chains of hemoglobin can be synthesized in vitro, and the two can be separated and quantitated. Anderson showed that the relative amounts of each that are synthesized can be controlled selectively by the incorporation of certain fractions of a population of tRNA’s. Still another instance of the directing role of tRNA in protein synthesis comes from a demonstration by Hunter and Jackson (1970). They showed that in in vitro synthesis of hemoglobin by extracts of rabbit reticulocytes serious misplacements of amino acids occur when tRNA’s from Escherichia coli are used. Since the mRNA used was the natural one and the code is known to be universal some other attribute of the tRNA structure must be essential for the insertion of its cognate amino acid into the exact site in the polypeptide chain. B. STRUCTURE AND SYNTHESIS OF tRNA Transfer RNA is the pivotal molecule in protein synthesis. It is the link between the amino acids and the message-bearing nucleic acids. It, therefore, has more functions than any other macromolecule known. It must be recognized by the enzymes which place on i t the specific amino acids. It must recognize the code in the messenger RNA. It has

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an attachment function to the ribosomes. It is the agent of the initiation of protein synthesis as well as its termination. It can remedy some genetic errors. For these many functions, transfer RNA has been evolved to have an extraordinarily complex structure. I n addition to the four main bases-uracil, adenine, cytosine, guanine-which solely compose mRNA, tRNA contains some 40 different modified bases. These modifications include methyl groups, isopentenyl groups , pseudouridine, dihydrouridine, sulfur-containing bases, and others. It was shown in our laboratory that the most frequent of these modifications, the methylated bases, are formed not by the stepwise incorporation of preformed methylated nucleotides but by the enzyme-catalyzed addition of methyl groups to the preformed macromolecule. With the availability of this model, we can categorically say that all modifications of tRNA are achieved by enzymes with extraordinary specificity. For example, in every transfer RNA except that of mycoplasma, there is a methyl group on the uracil in the 23 position, from the 3’-terminal. This particular methylating enzyme can count. The number of methyl groups present in a transfer RNA varies from species to species. E . coli tRNA’s contain about 4, yeast tRNA’s about 6, and normal mammalian transfer RNA’s may contain as many as 8 or 10 methyl groups. The function of some of these methyl groups is now known: The methyl groups are involved in recognition by the amino acid charging enzyme (Shugart et al., 1968). The methyl groups in the transfer RNA may play a role in the codon recognition (Capra and Peterkofsky, 1968); the methylation serves as a kind of a shift key in a typewriter, determining to what codon the transfer RNA will respond. A third function which is well defined has been demonstrated with three different tRNA’s. Many tRNA’s have a base-methylated or otherwise modified next to the anticodon. If such a modification is missing from a transfer RNA it can still accept amino acids, but it cannot transfer it to a ribosome (Gefter and Russell, 1969). These are three of the known functions; but there are perhaps 8-10 methyl groups in mammalian transfer RNA. What is their function? Unfortunately, basic molecular biology has not advanced to the stage where it can give us an answer. The largest lacuna in our knowledge in this area is the interaction between the codon in the messenger RNA and the anticodon in the transfer RNA. The original Watson-Crick hypothesis would have had us believe that this is simply achieved by the hydrogen bonding of the three appropriate bases. However, the energy and precision is insufficient for alignment of 80 bases merely on the basis of complementarity of three bases. In the past few years, a whole series of protein factors have been discovered which are needed

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for in vitro protein synthesis. At the present time, we do not know what the recognition site is for any of these proteins, all of which may interact with transfer RNA.

C. THEtRNA METHYLASES In a companion review we present recent information about the biochemistry of the tRNA methylases (Kerr and Borek, 1972). I n this article we shall give only a very brief resume, adequate for the understanding of the aberrancy of these enzymes in tumor cells. The tRNA methylases are a complex family of enzymes which modify the structure of preformed tRNA, by the insertion of methyl groups into specific positions in the four main bases of tRNA. The enzymes are species specific, organ specific, base specific, and even site specific for particular bases. The discovery of the species specificity (Srinivasan and Borek, 1963) of the tRNA methylases was contrary to the prevailing concepts of the time. The machinery of protein synthesis was then visualized to be an almost universally identical mechanism in a variety of organisms. The species-specific insertion of methyl groups into tRNA, achieved a t great cost in energy, which renders their structure species specific, suggested to us that this must serve some species specific biological function, We suggested that the best systems in which to test whether some biological functions are thus served are systems undergoing changes in regulatory mechanisms (Borek, 1963). Alterations in the tRNA methylating enzymes have been found to date in the biological systems listed in Table I. It should be emphasized that in all cases the changes are not simply a change in the specific activity of the enzymes per unit amount of tissue or per cell, but rather some qualitative changes have appeared in these enzyme activities. Some of the base specific enzymes disappear completely, or new methylating capacities appear, or the enzymes with the same base target may seek out more sites or fewer sites for methylation. It should be emphasized that these changes in the enzymes, in turn, bring about qualitative alterations in the transfer RNA’s themselves. Where there may have been a uracil in a tRNA, there is now a methyluracil or thymine; an adenine may be altered to a methyladenine. In this manner even the primary structure of transfer RNA may be changed. A very interesting negative confirmation for the essential role of the methylated bases and of other modifications in protein synthesis has been provided by Stewart et ul. (1971). A glycine-specific tRNA which is functional in the synthesis of cell wall peptidoglycan, but cannot participate at all in in vitro protein synthesis, on analysis was found to have

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TABLE I MODULATIONS OF tRNA METHYLASES IN BIOLOQXCAL SYSTEMS System Phage induction Phage infection Insect metemorphosis Embryonic VB. neonatal tissue

Colonizing slime mold DVerentiating lens tissue Thyroxine-induced morphogeneais in the frog tadpole Ovariectomised uterus Mammary epithelial cell differentiation urchin embryogenesis

Reference Wainfan et (1.2. (1965) Wainfan et al. (1965) Baliga et al. (1965) R. L. Hancock et al. (1967); Simon et al. (1967); Kerr (1970) Pillinger and Borek (1969) Kerr and Dische (1970) Pillingsr et al. (1971)

Shama and Borek (1970); Wieaner et al. (1970) Turkington (1969) Sharma et al. (1971b)

but one modified base, a thiouracil. All the methylated bases and pseudouridine are absent from its structure. II. The tRNA Methylases of Tumor Tissues

The incentive to study the tRNA methylases in tumor cells came from observations of Magee and Farber (1962). These workers had shown that an alkylating carcinogen-dimethylnitrosamine-methylates transfer RNA more extensively than it methylates DNA. As must be well known to the reader, much attention has been focused on the alkylation of DNA ever since the discovery by Sir Alexander Haddow of the alkylation of DNA by alkylating carcinogens. Unfortunately, at the time, transfer RNA had not yet been discovered and its alkylation went unnoticed. The hypothesis that we formulated from the findings of Magee and Farber may be briefly stated as follows. A chemical alkylating agent which we have assumed to be causal in carcinogenesis by the alkylation of DNA also alkylates transfer RNA. May not this find a counterpart in methylation of tRNA or DNA by aberrant enzymes in tumor cells? (Borek, 1963). It was originally visualized that an oncogenic virus may introduce information for the synthesis of methylating enzymes foreign to the host. Such enrymes would be essentially rampant, foreign methylating agents. We first focused our attention on the enzymes that methylate tRNA. This decision was based both on theoretical and on heuristic considerations. I n the methylation of DNA, in mammals, it is most likely that only one enzyme and one base, cytosine, is involved. It seemed to us at the time that this system of modification has

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but a limited capacity for variety; moreover, the technology of the DNA methylases was in a primitive stage at the time. The DNA methylases, which are easily extracted in the soluble fraction from E . coli had eluded attempts a t detection from mammalian sources. It was shown only later by Sheid et a2. (1968) that in the mammalian cell, the DNA methylase is membrane bound and is insoluble. The technology of these enzymes unfortunately has still not gone beyond this observation. On the other hand, the tRNA methylases, owing to the number of enzymes and bases subject to modification, offer an almost infinite capacity for variation of structure in tRNA. Moreover, the tRNA methylases were known to be easily isolated and studied in vitro from mammalian cells. The enzymes in normal and tumor cells can be characterized by several parameters. When the crude enzyme complex is extracted from the tissues, one can determine in an in vitro system the rate of methylation of a heterologous substrate. (It should be emphasized that such an enzyme mixture, as a general rule, does not methylate homologous tRNA isolated from the same tissue. The transfer RNA’s are fully methylated in situ by the enzymes indigenous to that cell type.) One can also determine another parameter, the extent of methylation of a heterologous substrate. In such a study, the total number of methyl groups that can be introduced a t maximum concentration of enzymes at infinite time is determined. Still another characteristic of the enzymes which can be determined, is the relative amounts of the various bases methylated, for example, the ratio of the number of methyl groups introduced into adenine or guanine. Still another variable is the site specificity of the enzymes. There are two different kinds of site specificity: Different positions of the same base can be methylated. Thus, there is a guanine methylase which introduces methyl groups into position 7 and still another enzyme which introduces two methyl groups into position 2. The other type of site specificity is the ability to methylate the same base in different positions in the transfer RNA. Thus, there is clear evidence that in yeast cells there are three different enzymes which methylate uracil in different positions in the tRNA chain (Bjork and Svensson, 1969) and in liver there are two different enzymes which methylate guanine in different positions (Kuchino and Nishimura, 1970). So far, investigators have not used all these parameters in characterizing the enzymes of tumor cells versus normal cells. Indeed, most of the studies of tumor cell enzymes have been performed with the crude extracts of such tissues, measuring the totaI capacity of these extracts to incorporate methyl groups into a heterologous substrate. The neoplastic tissues whose crude enzyme extracts have been studied and were found to contain aberrantly high tRNA methylase capacities

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TABLE I1 ENHANCED tRNA METHYLASE CAPACITY IN NEOPLASTIC TISSUES Neoplasm Novikoff hepatoma Glioma Mammary carcinoma Human mammary carcinoma Human leukemia Mouse melanoma Human adenocarcinoma Myeloma Morris hepatoma 51230 Reuber hepatoma Mice spleen infected with murine leukemia RNA virus Rats transplanted with Dunning (R3323) leukemic cells Marek’s disease in fowl Mammary carcinoma of C3H mouse and Fischer rat Mouse hepatoma SV-40 induced tumor in hamster Adenovirus-12 i n d u d tumor in hamsters Nonneoplastic cell line of mouse embryo w neoplastic line and tumor tissue Ethionine-induced tumor in rats Tumors induced by dimethylnitrosamine in rat Polyoma transformed rat embryo cells SV40-trsnsformed m o w kidney cells Ehrlich asoitea Diethylnitrosamine-induced tumors of the primate Medulloblastoma Spongioblastoma ABtrocytoma Oligodendroglioma Glioblastoma

Reference

Tsutsui et al. (1966)

Nau el al. (1969) Kerr (1971) Sheid (1969) Silber el al. (1967) Baguley and Staehelin (1968) Mandel el al. (1969) Turkington and Riddle (1970)

R. L. Hancock (1967) Mittelman el al. (1967) McFarlane and Shaw (1968b) Gantt and Evans (1969)

R. J. Hancock (1968) Craddock (1971) Gallagher el al. (1971)

Kit el al. (1970) Kerr (1971) Gallo (1971)

Viale (1971) Vide (1971)

are listed in Table 11. I n all of these instances, the crude enzyme extracts could introduce anywhere from a 2- to a 10-fold increase in methyl groups compared to the closest normal counterpart of the neoplastic tissue, The smallest increment in the tumors in the list-a 2-fold one-

171

ATYPICAL TRANSFER RNA'B

TABLE I11 ISOLATION O F SPECIFICMETHYLATED BABESUBINGYEABTtRNA AB ACCEPTOR WITH ENZYME PREPARATIONB DERIVEDFROM SV40 TUMOR AND NORMAL TIBBUEB~ SV40 tumor (cpm)

Base NI-Me thyladenmine No-Methyladenosine 5-Methyluridine N'-Methylguanosine WJP-Dimethylguanine a

0

lo00 2500

9o00 880

Muscle (cpm)

Connective tissue (cpm)

50 0 0 2000 0

0 0 0 220 0

A

METHYL

Liver (cpm)

Thymus (wm)

100

125 0 0 5500 0

0 0 4500 0

From Mittelman et al. (1967).

was found with a mouse melanoma. There appears to be no correlation between the rate of growth of the tumor and the extent of hyperactivity of its tRNA methylases. Clearest evidence for both qualitative and quantitative changes in the tRNA methylase extracts of tumor cells can be seen from the work of Mittelman et aZ. (1967). I n Table 111, the bases methylated in yeast tRNA by extracts of normal tissues of the hamster and by extracts of an SV40 tumor are presented. Note the de novo appearance of three different labeled methylated bases after exposure of the tRNA to the enzymes from the tumor. Another important attribute of extracts of tumor tissue emerged from the studies of this group. tRNA usually does not serve as a receptive substrate for enzymes from the homologous source. This is to be expected since the tRNA may be presumed to have been fully methylated by the indigenous enzymes. However, tRNA isolated from normal hamTABLE IV PARTITION COLUMN CHROMATOGRAPHY OF NUCLEOSIDEB OF HAMSTER LIVERtRNA AFTER INCUBATION WITH !CUMOR ENZYYEB~ Fraction I Methylated adenmine I1 Adenosine and 5methyluridine I11 Uridine IV Methylaced guanosine V Gummine VI Cytidine a

From Mittelman et al. (1967).

counta pe) minute 30 280 11 2,449

68 84

172

ERNEST BOREK AND SYLVIA J. KEBB

ster liver when exposed to enzyme extracts of SV40 tumor could receive supernumerary methyl groups (Table IV). This is an additional confirmation of the qualitative changes in the specificities of the enzymes from tumor cells. 111. Transfer RNA of Tumor Tissues

A. HYPWMETHYLATED tRNA’s IN TUMOR TISSUES Conclusions drawn from in vitro studies of crude enzyme extracts are always suspect with respect to any relevance to the situation in vivo. A variety of factors can contribute to the result which may be but a vector of several artifactual components. Among these may be the release or the nonrelease of normal inhibitors or activators of the enzymes which exert their control in vivo. However, fortunately, there have been some analyses of the methylated base content of the tRNA’s themselves from tumor tissue. These analyses are tedious compared to the enzyme assays cited earlier, and therefore, their number is still all too low. The instances where tRNA’s have been analyzed from tumor tissue for their methylated base content, which was found to be abnormally high, are listed in Table V.* The TABLE V HYPERMETHYLATED tRNA’s IN TUMOR TISSUES Tumor

t

8180 Ascites m o m Adenocsrcinoma of the mow8 Ethionine-induced tumors Dimethylnitmsamin~inducedtumors Matoxin-induced tumors Burkitt lymphoma Six human brain tumors: Medulloblastoma Spongioblastoma Astrocytoma Glioblastoma Oligodendroglioma Neurinoma

Reference Bergquist and Matthew (1962) Craddock (1969) Bonney and Mittleman (1970)

Viale el al. (1967)

‘It is of interest to point out that the first two c a s e s t h a t of the analysis of tRNA’s from S180 ascites of the mouse and that adenocarcinoma of the mousewere published before we proposed the possibility that the nucleic acid methylating enzymes may be aberrant in tumor tissue. However, a t the time we were not aware of the existence of this pioneering contribution by Bergquist and Matthews (1962).

ATYPICAL TRANSFER RNA’S

173

most extensive analyses of the transfer RNA’s in tumor tissue have come from the laboratory of Viale in Genoa. Viale et al. (1967) studied six different human brain tumors. The tRNA’s were isolated from these tissues, and their methyl group content was compared with the methyl group content of tRNA’s isolated from normal human brain tissue obtained by frontal lobotomy. The extent of variation among the content of methyl groups in the tRNA’s from these different brain tumors is listed in Table VI. Two objections may be raised about the significance of the data reported thus far. The first of these is that the elevated methylase capacity and also the presence of excess methyl groups in the tRNA’s might merely be a function of the rate of growth of the tissue examined. The second objection that might be raised is that since the tRNA methylases are organ specific, the different extents of methylation may be but a characteristic of the cell lines from which the tumors originate. Both of these objections have been met by studies of cells in tissue culture. For example, Gantt and Evans (1969) studied a normal cell line of mouse embryos and its spontaneously transformed counterpart and found elevated tRNA methylase capacity in the neoplastic tissue. Pillinger and Wilkinson (1971) obtained essentially the same results from studies of a standard line of Syrian hamster kidney cells, BHK21. They compared the tRNA methylase capacity of normal cell lines with the same cell line which was spontaneously transformed during continuous culture, and which had definite loss of contact inhibition. The two cell lines grew at approximately the same rate but the transformed cell lines showed an elevation in tRNA methylase capacity. A third cell line was obtained from in vitro culture of cells from a cheek pouch tumor which developed after injection of lo‘ of the spontaneously transformed cells. This cell line had an even higher tRNA methylase capacity. Evidence that the elevated tRNA capacity is an attribute of the transformed state per se not of rate of growth or variety of cell lines, also comes from studies of the methylase capacity of cells transformed by DNA viruses. Gallagher et al. (1971) studied rat embryo cells and polyoma-transformed cells. The methylase capacities of the latter were highly elevated. Similar observations were made on mouse kidney cells transformed by SV40 by Kit e t al. (1970). Another line of evidence indicating that altered tRNA methylases are not an obligatory concomitance of changes in rate of growth comes from studies of regenerating liver by Rodeh et al. (1967).They found essentially no differences between normal adult and regenerating rat liver. Breier and Holley (1970)studied in vitro the tRNA methylases from normal cells in tissue culture and their polyoma transformed counter-

TABLE VI METEYLATED NUCLEOSIDEB IN tRNA’s FROM HUMAN BRAINAND BRAINTUMORSO Normal brain frontal lobe

Nucleosides ~

~

_

_

_

5-Methylcytidine Na-Methyladenosine Na-Dimethyladenosine 1-Methylguanmine NLMethylguauosine W-Dime thylgusnosine Iulbotymidine 1-Methylinmine 2’4-Methylcytidine 2’4-Methyladenosine 2’4-Methylguano13ine 2‘4-Methyluridine

_

SpongioMedulloblastoms blastoms

Grade

Grade

I

II-I11

I

II-I11

Grade

0.24

0.64

0.33

0.52 0.32 0.48

0.86 0.56 0.84

0.30 0.62 0.45 0.52 0.73 0.36 0.16 0.37 0.16 0.60 0.32

0.90

0.64

0.46

0.35

5.23

10.36

3.84

Glioblastoma Neurinoma

_

0.m 0.18 0.12 0.02 0.32 0.54 0.32 0.02 0.12 0.04

Eg ; i

0.74 0.52 0.08 0.32 0.26

0.72 0.54 0.32 0.88 0.72 1.04 0.36 0.04 0.20

0.06

0.08

0.36 0.14 0.08

0.30

0.26

0.25 0.03

0.38 0.12

0.26

0.84

0.05

0.04

0.40

0.38 0.32 0.57 0.72 0.66 0.51 0.15 0.18 0.12 0.44 0.16

2.08

3.86

5.40

4.74

7.17

4.54

0.45 0.36 0.46

From Vide (1971). b

Grade

Values are expressed as moles per 100 milliliters.

0.90

0.76 0.64

0.62 0.98 0.42 0.14 0.48 0.35

0.64

0.72 1.14

0.36 0.28

0.68

0.28 0.42

1.58 1.86 1.13 0.62 0.27

0.58 0.42

0.33 0.48 0.21 0.02 0.30 0.17

E 9

3

m .e

3 4

176

ATYPICAL TRANSFER RNA’S

part. They felt that their study “failed to provide evidence of a polyoma virus induced tRNA methylase.” Unfortunately, this conclusion was based on a narrow view of the specificities of the tRNA methylases we outlined above. We present one set of data from these authors in Table VII. When rat liver tRNA is used as a substrate for the enzyme from BHK cells and BHK polyoma-transformed cells, it is apparent (line 1 in Table VII) that the transformed cell line extracts produce twice as many N2-methylguanines as the enzyme from B H K cells. I n the dimethylstion of guanines in the 2 position (line 3) the ratio of the two ensyme activities is 72 to 29. Therefore, it is obvious that the enzymes from the transformed cells can seek out more sites for methylation in the rat liver tRNA. Even with the heterologous substrate, E. coli tRNA, there are large differences in enzyme activity from the two sources. For example, in the dimethylation of guanine (line 3) there is an increase from 9,000 to 12,000 by extracts of the transformed cells. I n the monomethylation of guanine in the same position, there is a decrease in the activity of the enzyme from the transformed cells from 20,000 to 16,000. The conclusion that these two sets of enzymes from the two sources are the same stems from a simplistic conception of these enzyme specificities. Two enzyme systems that methylate the same base in tRNA need not be the same. For example, it has been shown by Kuchino and Nishimura (1970) [for details, see our companion review (Kerr and Borek, 1972)] that there are two different enzymes which methylate guanine in different positions in the transfer RNA molecule. Even from the data presented by Breier and Holley (1970) one can conclude that the two enzyme systems have entirely different substrate specificities toward rat liver tRNA as well as toward E . coli tRNA. There is a TABLE VII METHYLATION UTILIZING THE BHI(Py AND BHK CELLLINESAS ENZYME SOURCE AT pH 8.80 BHKPy enzyme Product P-Methylguanine NI-Methylguanine N’,W-Dimethylguanine NLMethyladenine &Methylcytosine 0

From Breier and Holley (1970).

BHK enzyme

E . mli tRNA

Rat liver tRNA

E . coli tRNA

Rat liver tRNA

16,160S 115 12,000 88 560

156

20,000

74

62

96 9,000 82 620

42 29

* Valuee are expressed aa counts per minute.

72 0 0

0

0

176

ERNEST BOREK AND SYLVIA J. KERR

similar misinterpretation of data obtained by Kit et al. (1970). These authors have found a 4-fold enhancement of the capacity of tRNA methylases in extracts of an SV40-transformed line over extracts from noninfected mouse kidney or SV40 virus-infected kidney. However, the authors concluded that this is probably a derepression of host-cell enayme capacities related to growth. It is obvious from our discussion of the properties of the tRNA methylases above, that no such conclusion can be drawn unless it can be shown that the enzymes from the transformed cells have the same base-specific activities as the normal host’s, including the same site specificity of the enzymes. It may well turn out that the tRNA methylases in transformed cells or in solid tumors represent the total methylase capacity of the species which is normally expressed only in embryonic tissue. However, at this point there is no evidence to support such a conclusion. To be sure, the tRNA methylase capacity is known to be elevated in embryonic tissue, but no specific base analyses are available to draw any further conclusions. Should the tRNA methylases of tumors prove to be the same as the embryonic enaymes it will be interesting to compare the population of tRNA’s from the two sources, since the expression of genomic information is so high in both systems. A beginning along these lines has been made. Holland et al. (1967) and Yang (1971) have found that certain tRNA’s in tumor tissue which have no counterparts in the normal adult tissue are present in extracts of embryonic tissue. (For a fuller discussion see the next section.)

B. MODIFIED tRNA’s IN TUMOR TISSUES The identity or nonidentity of a specific tRNA from two sources can be analyzed by physicochemical methods. These depend upon the interaction of the tRNA with some solid chromatographic reagent and with a variety of solvents used to elute the RNA’s from such columns. Several different systems are used (Sueoka and Yamane, 1962; Gillam et at., 1967; Weiss and Kelmers, 1967), the details of which are of interest only to the technologist. The methods consist of charging the population of tRNA’s extracted from two sources-i.e., a tumor tissue and normal tissue-with the same amino acid and following the pattern of coelution. The origin of the tRNA is identified by the particular radioactive label of the amino acid used to charge it. If two samples of tRNA’s from the same tissue are labeled this way and are placed together on a chromatographic column, they elute identically. However, altered tRNA’s for the same amino acid are found in several tumor tissues. The tRNA alterations which have been reported in tumors are listed in Table VIII. It should be emphasiaed that this methodology

177

ATYPICAL TXANSFER RNA’S

TABLE VIII SPECIES IN NEOPLASTIC TISSUE^

~ E R E D TRANSFER RNA

Neoplasm

P388 Lymphocytio leukemia cells Adenovirue-7 transformed hamster cells, SV40 transformed hamster cells, and HeLa cells Mouse sarcoma-1 Ehrlich ascites cells Mouse Gcells and Row vim transformed hamster cells Mouse plasma cell tumors: Immunoglobulii-A producer (MPC 62) Immunoglobulin-G producer (MPC 47) Novikoff hepatoma Novikoff ascites cells Mouse plasma cell tumor: K - T y p immunoglobulin light chain producer G M Cells in culture and L-M cell induced tumor Leukemic human lymphoblasta Morris Hepatoma 5123 and 5123c Morris Hepatoma 5123d Mouse fibroaarcoma, mouse reticulum cell sarcoma, adenovirus-31 transformed hamster cells, Reuber hepatoma cells, and HTC hepatoma cells Malignant lymphomas and chronic lymphatic and myelogenous leukemias

Altered tRNA Species

Reference

His, Lys, Ser, Val Phe

Morton and Rogers (1965) Holland d al. (1967)

Phe Gly, Phe, Ser, Tyr TYr

Taylor d al. (1967) Taylor d ul. (1967) Taylor d al. (1968)

Ser

Yang and Novelli (1968)

Asn, His, Tyr Phe, Ser, Val Leu

Baliga d al. (1969) Goldman d al. (1969) Mushinski and Potter (1969)

Asp, His, Phe, Tyr Yang d al. (1969) Gln, Tyr Am, Gln, Phe His, Phe, Ser Asp, TYr

Gallo and Peatka (1970) Gonano d al. (1971) Volkers and Taylor (1971) Yang (1971)

Phe

Mittelman (1971)

does not discriminate between changes in the primary sequdnce of nucleotides in the transfer RNA and changes in modification, such as methylation. For such determinations it will be essential to isolate the tRNA’s in homogeneous form and to analyze both primary sequence and the distribution of the modifying groups. Such determinations have not as yet been performed. However, a tantalizing suggestion comes from the work of Goldman and Griffin (1970). They found that in vitro methylation of transfer RNA caused a shift in elution pattern of tRNA’s on reversed phase columns similar to that shown by tRNA from precancerous livers of rats which were fed a chemical carcinogen, 3‘-methyl-

178

ERNEST BOREK AND SYLVIA J. KERR

4-dimethylaminoazobenzene. These studies will obviously have to be extended to other systems. IV. Regulation of tRNA Methylase Activity

A. NATURAL I'NHIBITORB OF THE tRNA METHYLASES Since there are so many instances of alterations in the functional capacity of these enzymes, the question of the possible mechanisms which control them naturally arose. So far we have found two regulatory mechanisms: There are natural inhibitors of the tRNA methylases in normal adult tissue (Kerr, 1970; Chaney et al., 1970) and the enzymes can be under hormonal control (Sharma and Borek, 1970); Wiesner et al., 1970). The discovery of the natural inhibitors of the tRNA methylases and their properties are discussed in greater detail in our companion review (Kerr and Borek, 1972). Here we shall merely summarize that in a normal adult mammalian tissue there is a regulatory system of the tRNA methylases which has two components-a protein and a smaller molecule (Kerr, 1971). Neither of these components inhibits the tRNA methylases alone. It was found that the inhibiting factor is either absent from embryonic tissue or is inactive. The inhibitory factor was also found to be absent from three different tumors examined. These are Novikoff hepatoma, Ehrlich ascites, and a Morris hepatoma 5123C. The presence of this inhibitory system in normal adult tissue and its absence from tumor tissue suggests an alternate mechanism for the source of the aberrancy of the tRNA methylases in tumor tissue from that originally postulated. We postulated originally that an oncogenic virus may introduce new information for the synthesis of tRNA methylases foreign to the host. However, the apparent absence of the inhibitors from tumor tissues offers another mechanism. An oncogenic virus may repress the synthesis of the normal inhibitors of the tRNA methylases or eliminate them in some unknown fashion. Purification of these inhibitors is under way and a clue to their mechanism of action and of their inactivity in tumor tissue may be discerned from the structure of the purified inhibitor. R. M. Halpern e t aZ. (1971) have recently isolated a dialyzable inhibitor of the tRNA methylases from mammalian liver. The compound was identified as nicotinamide, which in a concentration of 5m.M was found to inhibit the complex of enzymes in a crude liver extract. This is an interesting observation demonstrating, as it does, possibly a new class of inhibitors of the tRNA methylases which, perhaps with appropriate modifications, might prove to be more effective inhibitors, and

ATYPICAL TRANSFER RNA’S

179

may perhaps serve in therapy. However, its acceptance as a normal regulatory agent in vivo must await answers to several obvious questions: The concentration of this vitamin in normal tissue; its apparent absence from tumor tissue ; its effectiveness against purified enzymes.

B. HORMONAL EFFECTS ON tRNA METHYLASES The rationale for exploring the effects of hormones on the tRNA methylases was rooted in the findings of the hyperactivity of enzymes in so many different tumors and the known palliative effects of ovariectomy in certain human malignancies. The biological system we studied was the uterus of normal and ovariectomized animals. We observed that in the ovariectomized uterus of the rat, rabbit, and pig the tRNA methylase capacities are reduced to as much as 50% of the extracts of the normal organs (Sharma et a2., 1971s). Estradiol administered in physiological doses restores the methylase capacity in the ovariectomized uterus equal to or above that of normal. It is interesting to note that neither ovariectomy nor the restoration of the hormone has any effect upon the enzyme activity in nontarget organs, such as the liver. Another example of hormonal control of the tRNA methylases has been reported by Turkington (1969), who observed elevated tRNA methylase capacity after the administration of insulin and prolactin to mammary glands in tissue culture. I n addition to the inhibition of the tRNA methylases, ovariectomy also produces an alteration in the population of tRNA’s in the uterus which can be detected by the physicochemical methods we discussed above. A novel serine specific tRNA appears in the tissues of the uterus of the ovariectomized pig (Sharma and Borek, 1970). Administration of estradiol eliminates this novel transfer RNA from the ovariectomized uterus. It will be interesting to study the molecular mechanisms of this hormonal activity since it represents a qualitative effect after the deprivation and the restoration of a hormone. There are not just more transfer RNA’s, but there is a unique seryl transfer RNA present in such tissue. Whether this novel seryl tRNA differs in modification or in primary sequence will not be answered until analysis of it is completed; however, its chromatographic behavior is what one would expect from a deficiency of methyl groups.

C. OTHERINHIBITORS OF THE tRNA METHYLASES Dr. Wainfan undertook in our laboratory an exploratory effort to search for inhibitors of the tRNA methylases which may be used in therapy. It had been reported by Hurwitz et al. (1964) that adenosine inhibits specifically the guanine tRNA methylases of E. coli. This ob-

180

ERNEST BOREK AND SYLVIA J . KERB

servation was confirmed and extended with other derivatives of adenine to mammalian enzyme extracts. It was found that not only adenosine but tubercidin-an analog of adenosine used in the chemotherapy of some cancers--also inhibits the tRNA methylases (Wainfan and Borek, 1967). More recently, Wainfan and Landsberg (1971) extended these studies to naturally occurring cytokinins, and they found that kinetin, isopentenyl adenosine, and zeatin riboside all inhibit the tRNA methylases. Whether these cytokinins have any such function in vivo is obscure a t present. It is interesting to note, however, that isopentenyl adenosine has been used successfully in short-term therapy of certain leukemias. Two other compounds used in cancer chemotherapy, 5-fluorouracil and chloramphenicol, have been shown to interfere with the methylation of tRNA in intact E . 00Zi cells (Lowrie and Bergquist, 1968; Gordon et al., 1964). It is also interesting to note that methotrexate depletes the methyl pool, as evidenced by the frequent fatty infiltration of the livers of patients on such therapy. Another inhibitor of the tRNA methylases was studied by Moore and Smith (1969). These investigators found that S-adenosylethionine inhibits tRNA methylases from both E . coli and mammalian sources in vitro. However, on the feeding of ethionine, an entirely different pattern emerged. Rats were kept on a diet of 0.2% ethionine for 1 month. At the end of this time they were sacrificed and the specific tRNA methylase activity of extracts of liver were compared to those of normal animals. In each of the three experiments performed there was a t least a 2-fold elevation in tRNA methylase activity in the rats which had been on the ethionine diet. These findings confirm those of R. J. Hancock (1968), who studied the effect of ethionine feeding on the tRNA methylase activity of the liver. But this is the earliest elevation of tRNA methylase activity reported. The changes in the enzyme capacity occur long before any cytological evidence of carcinogenesis by the ethionine in the diet is apparent. V. Elevated Excretion of Modified Purines and Pyrimidines in Tumor-Bearing Animals and Humans

That methylated purines are excreted in the urine of normal humans has been known since the pioneering work of Guttman and co-workers (Weissman et al., 1957). The number of these methylated purines which have been identified in normal urine is increasing. For the latest tally, see Table IX. Note that while the level of excretion of a modified nucleoside, pseudouridine-a nucleoside in which the ribose is attached not to its usual 3 position, but to the 5 position-may be as high as 50 mg./day, the four major nucleosides cytidine, uridine, guanosine, and

181

ATYPICAL TRANSFER RNA'S

PURINE8 AND PYRIMIDINE

No. 1 2 3 4 5 6 7

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

TABLE I X DERIVATIVEB EXCRETED I N NORMAL HUMANURINE" Compounds

mg./day

Adenine 1-Methyladenine No-Methyladenine Adenosine 1-Methyladenosine No-Me thy ladenosine N-[ (9-p-D-Ribofuranosyl-9Hpurin-6yl)carbomoyl] threonine (PCTR) Cytosine 3-Me thylcytosine Cytidme Deoxycytidine 2'4-Methylcytidine Guanine NtDimethylguanine 1-Methylguanine NrMe thylguanine 7-Methylguanine 8-H ydrox y-7-methylguanine Guanosine NrDimethylguanosine "-Me thylguanosine NtMethylguanosine Uracil 5-Acetamino-Bamino-3methyluracil 5-H ydrox ymethyluracil Uddine Pseudouridine Hypoxanthine 1-Methylhypoxanthine Inosine 1-Methylinosine Xanthine 1-Methylxanthine 7-Methylxanthine 1,3-Dimethylxanthie 1,7-Dimethylxanthine Uric acid 1,3-Dimethyluric acid N-(6purinyl)aspartic acid 5-Aminoimidazole-4-carboxamide 5Aminoimidarole-4-carboxamide riboside

1.4 0.3-0.4 Not quantitated Trace Not quantitated 2.1-5.2 0.3 Trace Not quantitated Trace Trace 0.2-0.5 0.3-1.3 Trace 0.6 0.3-0.6 2.7-7.8 1.6 Trace 1.1-2.2 0.4-0.6 0.2-0.3 7 6-10 Trace Trace 42-62 6.8 0.4-0.9 0.18 2.1-2.9 2.8-8.7

-

Trace

-

390-588 Trace 0.5-0.9 1.05 Not quantitated

182

ERNEST BOREK AND SYLVIA J. KERR

TABLE IX (Continued)

42 43

4A 45

0

1-Methylimidazole-4-acetic acid Imidazole-4-lacticacid Imidazole-4-acetic acid Allantoin

Trace 12.0 (mg. liter) 4 . 5 (mg. liter) 11.9

From Chheda (1970).

adenosine are excreted only in trace amounts. The reason for a minor tRNA component being a major excretion product is obvious: the other nucleosides can be recycled and reincorporated into nucleic acids. Pseudouridine cannot be recycled since it must be inserted into highly specific sites and there is no mechanism for the insertion of this or of any other modified base during the synthesis of the primary nucleotide chain. Adams et al. (1960) and Park e t al. (1962) both have observed elevated levels of methylated purines excreted by leukemic subjects. The origin of these modified purines and of pseudouridine was clarified after the discovery of the mechanism of methylation of transfer RNA. All the methylated purines which are excreted and pseudouridine are found mainly in the transfer RNA; rRNA contains only some of them in smaller amounts and variety. These products must, therefore, come from a turnover of transfer RNA. The rationale for their excretion is implicit in the mechanism of the synthesis of these compounds in transfer RNA. They must all be synthesized a t the macromolecular level. For example, the methylated purines, when fed to an organism, are not incorporated into the nucleic acid; they are excreted. This is obligatory, otherwise, the modified bases might be incorporated into tRNA randomly, and it is well known that their synthesis is highly specific for particular sites. Indeed, the kinases for their conversion to the triphosphates do not exist. After it became known that all these methyl groups in transfer RNA including, surprisingly, that of thymine, stem from the methyl group of methionine a new tool was offered for the study of the excretion of methylated purines in both normal and tumor-bearing animals. Mandel et al. (1966) observed increased excretion of “C methylated purines in rats bearing thymic lymphoma and mice bearing mammary carcinoma, McFarlane and Shaw (1968a) studied the excretion of methylated purines by hamsters bearing adenovirus 12-induced tumors and observed that while the weight of the animals increased only 1-5%, owing to the tumor growth, the excretion of the methylated purines increased by 200%. The elevation of the excreted methylated purines could stem either

RATIO OF

7-METEYMUANINE

TABLE X URINE

AND G E A T I N E I N TEE

OF

NORMAL AND TUMOR-BEARINO HUMANS~

*

Y -4

Kenyan patients Arab patients

Israeli normals 4.8 f 1.e

Kenyan normals

With cancer

With cirrhosis of the liver

With esophageal cancer

Withliver cancer

25

9.3 k 5 . 9

m

1

M

5.3 f 1.4

12.0 f 6.6

10.6 f 6 . 7

9.3 f 5 . 8

9.6 f 5 . 4

From Mirvish et al. (1971). * Values are expremed as micrograms of 7-methylguanine per milligram of creatinine. a

Without cancer 5 . 5 f 1.5

6 . 1 f 3.0

~

s

4

ul

184

ERNEST BOREK AND SYLVIA J. KEBg

from a more rapid turnover of transfer RNA in tumor tissue or from an increase in the extent of methylation of the tRNA's, or from both sources. More refined quantitation of the excretion products in normal and tumor-bearing animals could resolve these two possibilities. The adaptation of such quantitative studies to an approach to diagnosis is not implausible. Craddock (1970) reported some unpublished data of Mirvish et al. (1971) implying that elevated excretion of methylated purines does not accompany all solid tumors. Examination of the published data, however, does not support such a conclusion. Single urine specimens from a number of human subjects were examined; this is unfortunate because the breakdown of tRNA and the excretion of its products may be episodic. However, when the 7-methylguanine output is compared to the creatinine levels of the urine, an unequivocal elevation of the former in cancerous patients becomes apparent. A summary of the pertinent data is shown in Table X. Apparently no correlation was attempted between the size of the tumor and levels of excretion. Thus the smallest elevation was observed with the esophageal cancer. The size and any metastases of the tumors are, of course, essential parameters for any evaluation. VI. The tRNA Methyloses in Reverted Oncogenic Systems

A very interesting correlation between the methylation of nucleic acids and carcinogenesis has been reported recently by Magee and his collaborators (Fiume et al., 1970). As stated earlier, Magee did the pioneering work on the methylation of tRNA as well as of DNA by the alkylating carcinogen diniethylnitrosamine (DMNA). Magee's extensive research has indicated that this agent is not itself hepatotoxic, but that it is metabolized into a methylating intermediate compound which is the actual toxic agent. In an extension of these studies the effect of aminoacetonitrile on carcinogenesis by DMNA was investigated. This is a lathyrogenic agent which decreases the toxicity of several liver poisons, including DMNA. The administration of aminoacetonitrile inhibits the metabolism of DMNA, as measured by the conversion of "CH, to l4CO,, as well as the formation of liver lesions and tumorigenesis. The adjuvant also lowers significantly the methylation of both DNA and RNA in vivo by this alkylating carcinogen. While these observations are tantalizing, the real answer as to whether there is any causality between the reduction of methylation of nucleic acids and suppression of oncogenesis still eludes us. Another example of the correlation between the reversal of on-

ATYPICAL TBANSFER BNA’S

186

cogenicity and diminution of the tRNA methylase activity was provided by B. C. Halpern et al. (1970). They were able to reverse the oncogenicity of neoplastic cells prepared from the Walker 256 carcinoma by incubating these cells with DNA fragments extracted from the same tumor. The mechanism of such a reversal is utterly baffling and impossible to fit into the framework of our current concepts of regulatory mechanisms. (But if we accepted only phenomena which are consonant with current ideation, we would still be alchemists and medicine men.) Its general significance, of course, awaits confirmation and extension to other systems. Halpern and her co-workers incubated Walker 256 cells in tissue culture and after 48 hours added DNA extracted from the same cell line. During the next 50 hours, the growth rate of the controls and cells exposed to DNA were the same. However, the tRNA methylase activity underwent a profound decrease in the cells exposed to the DNA; in 72 hours the difference was more than one order of magnitude. The cells exposed to DNA were removed from that medium and returned to their usual milieu and were assayed for their tRNA methylase capacity. In 8 days their tRNA methylase activity was up to the level of the original control, the untreated Walker 256 cells. VII. An Attempt at Interpretation

Up to now we have dealt with more or less hard facts. May we now indulge very briefly in some soft speculation. Causality versus concomitance is a dilemma as old as science itself. What, if any, role these large changes in the enzymes that modify tRNA, and the changes in tRNA’s themselves in tumor cells, may have in oncogenesis, is impossible to state now with the information that is available. We are hindered in part by the large lacuna in our understanding of normal regulatory mechanisms which may exist a t the level of translation. Information is accumulating almost daily on the complexity of the process of protein synthesis. As stated earlier, in addition to the ribosomes with their numerous proteins as well as mRNA and tRNA, at least 8 different soluble proteins are known to be required for in vitro protein synthesis. The sites of attachment of these factors to the major components of the system are unknown. Therefore, it is impossible to conjecture what roles the modifications of tRNA may serve in such interactions. The discovery of the retroscriptase which can transcribe viral RNA into DNA and thus permit its integration into the host genome has unified tumor virology : All tumor-producing viruses are DNA viruses.

186

ERNEGT BOREK AND SYLVIA J . HERR

While this unity has brought us no closer to an understanding of the subsequent events which may oncogenize the host cell, it does offer a real advantage. We have a simple model system for study. It must be recalled that most of the body of knowledge we have about latent virus-host relationships was built on the seminal work of Lwoff with temperate phages. Indeed, Lwoff understood immediately the transferability of his findings to the cancer problem (Lwoff, 1951). Unfortunately, the great similarity in the two systems has become obscured by the latter-day neologisms of animal virologists. The process of the activation of the integrated virus which Lwoff called “induction” has become “rescue” of animal viruses; the provirus has become an “oncogene.” Unfortunately, our understanding of the molecular mechanism of induction has not progressed far enough during the past twenty years. Mutagens, carcinogens, radiation-both UV and X-and drugs which inhibit DNA synthesis are all inducing agents for the temperate phage system. We do know definitely that the integrated phage DNA need not be the primary target of the above agents. This is known from the phenomenon called “indirect induction” (Borek and Ryan, 1960). A fascinating analog of this phenomenon with mammalian viruses was performed by Watkins (1970), who was unaware of the phenomenon in bacteria. Watkins’ very important experiment may be summarized as follows: When transformed, nonpermissive cells (SV3T3) are fused with untransformed permissive cells (BSC), about 5% of the fusion products produce SV40 virus; if the transformed cells are treated with IUdR or 8-azaguanine before fusion, up to 90% of the fusion products produce SV40 virus. There is an analogy with respect to the tRNA methylases between lysogenized bacteria and virus-transformed mammalian cells as well. The tRNA methylases of lysogenized bacteria ( E . coli K,, A+) are different from those of the isogenic nonlysogenized organism ( E . coli K,, A-) (Wainfan and Visser, 1969). I n agreement with this, we know of no exception in the alteration of tRNA methylases in virus-transformed mammalian cells. I n lysogenic E . coli there are profound alterations in the tRNA methylases after induction. Whether this is also true during “rescue” of animal viruses from transformed cells has not been determined. It is obvious that many questions about the modifications of tRNA’s in tumor, and in normal cells as well, remain unanswered. So far only the methylasea have been studied, and there are several other modifying ensymes. The role of modified tRNA’s in translation must be studied in systems where subtle changes can be detected. Unfortunately, these am not yet available. In most in vitro methods currently used, simulated

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protein synthesizing systems are artificially forced to completion. Should some regulatory process hinge on structural conformations of tRNA’s then hypermethylation might vitiate such an orderly process. Methylation in biological systems appears to be an exquisitely balanced, functionally oscillating system. Alkylating agents can be carcinogens; they can also be carcinostatic agents. Diminution of methionine in the diet of rats will cause fatty infiltration of the liver; Stekol and Szaran (1962) showed a decade ago that excess methionine in the rat’s diet also causes fatty degeneration. We have confirmed this recently in the hamster; biweekly intraperitoneal injections of methionine produces limited growth and fatty degeneration of the liver (Sharma and Borek, 1971). As it has happened so often in the past, biological phenomena point out the need for new and subtle methodology for probing them, or we must await the fortuitous emergence of correlations between methylation and physiological function. Two such correlations have been published recently, oddly on functions of methylation not in tRNA, but in the much larger rRNA. Lai and Weisblum (1971) observed in Staphylococcus aureus that the development of resistance to erythromycin is accompanied by the appearance of a novel methylated base NO-dimethyladenine in the 2 3 s RNA. Since the seat of drug resistance to streptomycin is known to reside in the 23 S rRNA from genetic studies, the altered structural component discovered by Lai and Weisblum has a compelling interest. Another highly suggestive observation on rRNA methylation and function has been made by Torelli et al. (1970). I n the maturation of rRNA, methylation of 45s rRNA precedes its cleavage into the 2 8 s and 1 8 s molecule. Torelli and his co-workers studied the synthesis of rRNA in the blast cells of acute leukemia and observed that in such cells cleavage of the 45s particle is abnormally slow. They were able to correlate this with aberrant hypomethylation of the 45 S particle. The source of this hypomethylation will have to be studied. ACKNOWLEDGMENTS Our own work on the tRNA methylases has been supported during the past ten years, by grants from the American Cancer Society, The Damon Runyon Memorial Fund, the Milheim Foundation, the National Institutes of Health U. S. Public Health Service, and by contracts from the Atomic Energy Commission.

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Baguley, B. C., and Staehelin, M. (1968). Eur. J. Biochem. 6, 1.

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Baliga, B. S., Srinivasan, P. R., and Borek, E. (1965). Nature (London) 206, 555. Baliga, B. S., Borek, E., Weinstein, I. B., and Srinivasan, P. R. (1969). Proc. Nat. Acad. Sci. U.S. 02,899. Bergquist, P. L., and Matthews, R. E. F. (1962). Biochem. J. 85, 305. Bjork, G. R., and Svensson, I. (1969). Eur. J . Bwchem. 9,207. Bonney, A., and Mittelman, A. (1970). Personal communication. Borek, E. (1963). Cold Spring Harbor Symp. Quant. B i d . 28, 139. Borek, E., and Ryan, A. (1960). Biochim. Biophys. Acta 41,68. Brachet, J., Ficq, A., and Moyer, W. A. (1963). Ezp. Cell Res. 32, 168. Breier, B., and Holley, R. W. (1970). Biochim. Biophys. Acta 213, 365. Capra, J. D., m d Peterkofsky, A. (1968). J. Mol. BWl. 33,591. Chaney, S . Q., Halpern, B. C., Halpern, R. M., and Smith, R. A. (1970). Biochem. Biophye. Re8. Commun. 40, 1209. Chheda, G. B. (1970). “Handbook of Biochemistry,” 2nd ed. The Chemical and Rubber Co., Cleveland, Ohio. Craddock, V. M. (1971). Personal communication. Craddock, V. M. (1969). Biochim. Biophys. Acta 195, 351. Craddock, V. M.(1970). Nature (London) 226, 1264. Dintsis, H. M. (1961). Proc. Nat. Acad. Sci. U.S. 47, 247. Fiume, L., Campadelli-Fiume, G., Magee, P. N., and Holsman, J. (1970). Bwchem. J. 120, 60. Gallagher, R. E., Ting, R. C. Y., and Gallo, R. C. (1971). Proc. Soc. Ezp. Bwl. Med. 136, 819. Callo, R. C. (1971). Cancer Res. 31 (in press). Gallo, R. C., and Pestka, 8. (1970). J. Mol. Biol. 52, 195. Gantt, R.,and Evans, V. J. (1969). Cancer Res. 29, 536. Getter, M. L., and Russell, R. L. (1969). J . Mol. Biol. 39, 145. Gillam, S., Millward, S., Blew, D., von Tigerstrom, M., Wimmer, E., and Tener, G. M. (1987). Biochemistry 6, 3043. Goldman, M., and Griffin, A. c. (1970). Cancer Re8. 30, 1677. Goldman, M.,Johnston, W. M., and Griffin,A. C. (1969). Cancer Res. 29, 1051. Gonano, F.,Chiarugi, V. P., Pirro, G., and Marini, M. (1971). Biochemistry 10, 900.

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USE OF GENETIC MARKERS TO STUDY CELLULAR ORIGIN AND DEVELOPMENT OF TUMORS IN HUMAN FEMALES Philip J . Fialkow Departments of Medicine and Genetics. University of Woshington. Seattle. Washington

I . Introduction . . . . . . . . . . . . . . . A . Inactive-X Hypothesis . . . . . . . . . . . B . Glucose-6-Phosphate Dehydrogenase (G-6-PD) Isoenzymes . . . C . Inactivation at the G-6-PD Locus . . . . . . . . . I1. Interpretation of G-6-PD Phenotypes in Tumors . . . . . . A . Tumors with Double Enzyme Phenotypes (Both A and B Enzyme Types) . . . . . . . . . B . Tumors with Single Enzyme Phenotyprs (A or B Enzyme Types) . I11. Hematopoietic Neoplasms . . . . . . . . . . . A . Chronic Myelocytic Leukemia (CML)-Origin and Development . B . CMGUtilization of CML Cells to Study Gene Localization and Activation . . . . . . . . . . . . . C. Burkitt Lymphoma . . . . . . . . . . . . D . Other Hematopoietic Neoplasms . . . . . . . . . IV. Carcinomas . . . . . . . . . . . . . . . A . Metastatic Carcinoma of the Colon . . . . . . . . B. Carcinoma of the Cervix . . . . . . . . . . . C . Anaplastic Carcinoma of the Nasopharynx . . . . . . . D . Other Solid Malignant Tumors of the Head and Neck . . . . E . Summary . . . . . . . . . . . . . . . V . Benign Tumors . . . . . . . . . . . . . . A . Leiomyomas of the Uterus . . . . . . . . . . B . Ovarian Teratomas (Dermoid Cysts) . . . . . . . . C . Common Wart (Verruca Vulgaris) . . . . . . . . . D . Genetically Determined Tumors . . . . . . . . . E . Other Benign Tumors . . . . . . . . . . . . VI . Study of Genetic Markers in Established Tissue Culture Lines . . . A . Heteroploid Epithelial-Type Cell Lines . . . . . . . B . Burkitt Lymphoblastoid Cell Lines . . . . . . . . VII . Concluding Remarks . . . . . . . . . . . . . A . Implications of Clonal versus Multicellular Origin . . . . . B . Prospects for Future Studies . . . . . . . . . . References . . . . . . . . . . . . . . .

191 192 193 194 195 195 196 199 199 201 204 208 209 209 210 211 212 213 213 213 216 217 217 218 219 219 219 221 221 223 223

I . Introduction

Knowledge of whether neoplasms have a uni- or a multicellular origin and of how they subsequently develop may lead to a better understand191

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ing of their etiology and pathogenesis. Information relevant to these points can be obtained through study of tumors arising in organisms with two or more cell types distinguishable by their genetic markers. Tumors with a single cell origin arising in such organisms should consist entirely of one or the other cell type, whereas tumors with multiple cell origin may contain both types of cells. One approach is to study artificially produced organisms with fixed cellular mosaicism, such as “allophenic” mice “synthesized” by aggregating blastomeres from two genetically distinct embryos and transferring the resultant composites into the uteri of foster mothers (Mintz and Slemmer, 1969). Human tumors can be investigated in subjects with chimerism or mosaicism using either genetic and/or chromosomal markers. The primary purpose of this communication is to review studies of tumors derived from human females whose mosaicism is governed by X-chromosome inactivation. Development of the experimental technique used in these investigations depended upon the inactive-X hypothesis (reviewed in Lyon, 1968) and the demonstration that the glucose-6-phosphate dehydrogenase locus in many undergoes this type of inactivation (Beutler et al., 1962; Davidson e t al., 1963; DeMars and Nance, 1964).

A. INACTIVE-X HYPOTHESIS Mammalian females have two different cell types by virtue of the fixed genetic inactivation of one of the two X chromosomes which occurs early in embryogenesis in each somatic cell. The presumed sequence of events is schematically illustrated in Fig. 1. The female zygote has one maternally derived X (XM)and one of paternal origin (Xp). After Xchromosome inactivation occurs, only Xh‘ or Xp is genetically active in each somatic cell; the inactive X chromosome forms the nuclear chromatin, or Barr body. The initial choice of which X chromosome is to be inactive in a given cell is presumably random; however, once made, it is fixed for that cell and all its descendants. Thus, the adult female is a mosaic of two cell types-cells with an active XM and cells with an active Xp. Pure tumors with a single cell origin should consist of XMOT Xp cells, but neoplasms with a multiple cell origin may contain both XMand Xp cells. The two types of cells, XMand Xp, can be distinguished from one another in females heterozygous for alleles at an X-linked locus known to undergo inactivation, if the phenotype determined by each allele can be demonstrated a t the cellular level. In man, several X-linked loci fulfill these criteria, but at present only variant alleles at the locus for glucose-6-phosphate dehydrogenase (G-6-PD) are known to occur in high enough frequency to be generally useful.

GENETIC MARKERS FOR THE STUDY OF TUMORS Sperrn(XP)

193

Egg ( X u )

a

@

/ \

J \

@ ....... @ @ .; ... @ .:....,..:.. Embryo

A

P

P

A

M

A

/s,

M

P

M

P

M

Adult

FIG.1. Diagrammatic representation of the inactive-X hypothesis. See text for explanation. From Fialkow, P. J. Genetic Marker Studies in Neoplasia. In Genetic Concepts and Neoplasia ( A Collection of Papers Presented at the twenty-third Annual Symposium on Fundamental Cancer Research, 1969). The Williams and Wilkins Company, Baltimore, Maryland, 1970, p. 112.

B.

DEHYDROGENASE (G-6-PD) ISOENZYMES The common starch gel electrophoretic forms of G-6-PD from blood cell lysates are shown in Fig. 2. The B phenotype is seen in the vast majority of Caucasians, but in many black populations 3 M O % of males GLUCOSE-6-PHOSPHATE

B (1)

0 (2)

AB

(3)

A

(4)

A (5)

FIG.2. Starch gel electrophoresis phenotypes of glucose-6-phosphate dehydrogenase (GS-PD). The enzyme was derived from blood cells obtained from (1) and (2) G P / G P homozygotes, (3) GdA/GdB heterozygote, ( 4 ) Gd"/Gd" homozygote, and ( 5 ) GdA/GdA- heterozygote. The preparations contained different amounts of hemoglobin and different quantities of enzyme activity.

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have a variant enzyme (either type A or type A-). The A enzyme has faster electrophoretic mobility and only slightly less activity than the B type. The two isoenzymes, A and B, differ by a single amino acid substitution (Yoshida, 1967) ; their respective alleles are designated GdA and GdB. Since the G-6-PD locus is X-linked, males are hemizygous and have only one or the other enzyme type. The AB phenotype is observed only among females. The A- variant of G-6-PD has the same electrophoretic mobility as A, but its activity in red cells is only 8-20% or normal. The defect underlying the A- variant is a structural gene mutation resulting in a product which initially has normal enzymatic activity, but is more susceptible than the B or A types to degradation during cell aging (Yoshida e t al., 1967a). Thus, because they lack nuclei and do not have continued synthesis of nascent enzyme, red cells manifest the A- enzyme deficiency. Since nucleated cells have almost normal activity, they cannot be used to distinguish A from A- by measurement of enzyme activity. The electrophoretic variants are particularly well suited for studies of mosaicism since the minor component in a mixture of A and B can be detected even if it constitutes as little as 10% of the total activity (Nance, 1964). Other G-6-PD variants are prevalent in Mediterranean areas, but they cannot be conveniently distinguished from type B enzyme with electrophoretic techniques. Males with the severe G-6-PD deficiency of Mediterranean type have less than 10% of normal activity in their red cells and, in contrast to the A- type of G-6-PD deficiency, enzyme activity is also markedly decreased in nucleated cells. Approximately one-third of heterozygous females have enzyme activity levels in the normal range, and in 3% of heterozygotes activity levels are in the homozygote-deficient range (Stamatoyannopoulos et al., 1967; Stamatoyannopoulos, 1971). This overlapping somewhat limits the usefulness of the quantitative Mediterranean-type variants in tumor development studies.

C. INACTIVATION AT THE G-6-PD Locus I n heterozygotes for the B allele (GdB) and a variant allele such as A (GdA) or A- (GdA-), a given cell or clone of cells has only one of the two enzyme types seen in a mixture of their cells (Beutler e t al., 1962; Davidson et al., 1963; DeMars and Nance, 1964). For example, the electrophoretic pattern of an extract of cultured skin fibroblasts from a GdA/GdB heterozygote contains both types of enzymes, but clones derived from this culture by single cell platings exhibit only type A OT type B enzyme (Davidson e t al., 1963; DeMars and Nance, 1964). Applying this principle, it follows that neoplasms arising from many cells might contain both enzyme types, whereas those with a clonal

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origin should have only A or B type enzyme. This approach is best applied to the study of tumors in black females since nearly 45% of these women are either GdA/GdBor Gd-”/GdB heterozygotes. II. Interpretation of G-6-PD Phenotypes in Tumors

The potential use of the G-6-PD system in the study of neoplasms was suggested independently in 1964 by DeMars and by Gartler and Linder. In the first utilization of the system, Linder and Gartler (1965a) provided strong evidence for a single cell origin of uterine leimyomas. Before describing these and other studies, the difficulties which may arise in interpreting the G-6-PD phenotypes of tumors are briefly outlined.

A. TUMORS WITH DOUBLE ENZYME PHENOTYPES (BOTHA AND B ENZYME TYPES) Before concluding that a tumor contains both enzyme types (A and B) and thus has a multiple cell origin, the possibility must be considered that the actual tumor cells have a single enzyme phenotype, and that the second enzyme type is due to the presence of nonneoplastic cells “contaminating” the tumor specimen. Many solid invasive malignancies contain nontumor cells in sufficient quantities to make firm interpretation of double enzyme phenotypes extremely difficult. Consequently, careful histologic examination of the tumor samples is imperative. For this purpose, division of the tumor specimen into multiple small samples may prove satisfactory. For example, in a study of anaplastic carcinoma of the nasopharynx (see Section IV,C), the tumor biopsies were divided into several samples, each measuring 3-8 mm in diameter. Individual samples were then divided into three equal portions; the two outer pieces were combined and analyzed for G-6-PD, and the center piece was examined histologically. Another important factor in assessing the significance of the nontumor cell contribution to a double enzyme phenotype is the relative amount of A and B enzyme in normal tissue compared to that of the tumor. In the aforementioned study of carcinoma of the nasopharynx (Fialkow e t al., 1971b), all samples of one particular tumor biopsy (Case No. 918) had both A and B enzymes, and without any further study one might have concluded that this tumor had a multiple cell origin. However, normal tissue adjacent to the tumor had 80% A and 20% B enzyme. I n contrast, samples consisting predominantly of tumor cells had chiefly type B enzyme. These observations, while not entirely conclusive, suggest that the tumor actually originated in a single cell of type B.

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A more direct way to determine the source of the two enzymes of a double phenotype is to prepare a tissue culture. If the tumor cells themselves are of two types, both types should be found in tissue cultures derived from the tumors, and cloning experiments should show that some cells are A and others are B. However, before the double enzyme plienotypes in the tissue culture can be accepted as evidence for a multicellular origin of the tumor cells, the growth and morphologic characteristics of the cultures must provide evidence for their neoplastic nature. Unfortunately, tissue cultures derived from solid tumors often grow as normal fibroblastlike monolayers and presumably are derived from stromal cells. Theoretically, a tumor with a double enzyme phenotype could arise from a single cell if that cell had two active X chromosomes, one of maternal and the other of paternal origin. For example, the inactive X might be reactivated or the cell could be a fusion product of two cells. The possibility of reactivation can be tested by cytologic examination of tumors for nuclear chromatin bodies (Barr bodies) and/or latereplicating X chromosomes. Since the nuclear chromatin body is an expression of the inactive X chromosome, its presence in a tumor provides evidence that only one of the two X chromosomes is active. However, absence of a chromatin body in a cell does not necessarily imply absence of an inactive X (Klinger e t al., 1968). A reduced number of cells with chromatin bodies in a carcinoma of the colon with double enzyme phenotype has been cited as evidence for reactivation of the inactive X chromosome (Straub et al., 1969). In that study, there were no confirmatory cytogenetic data (e.g., no chromosome replication studies), nor were the results of in vitro cloning experiments reported. Furthermore, metastases from this neoplasm had both A and B single enzyme phenotypes indicating the presence of an inactive X chromosome (see Section IV,A) , In all other appropriately studied cases, the expected numbers of cells with Barr bodies have been observed in tumors with double enzyme phenotypes. Furthermore, if both X chromosomes were active in a single cell, an electrophoretic band with mobility intermediate between A and B type enzyme would probably be seen (Yoshida et al., 196713; Silagi et aZ., 1969). This type of band, representing a hybrid of A and B subunits, has not been reported in any human tumor. Thus, there are no firm data indicating activity of two X chromosomes in a malignant diploid cell in t h o . B. TUMORS WITH SINGLEENZYME PHENOTYPES (A OR B ENZYME TYPES) When the tumor extract has only a single electrophoretic component (A or B ) , other possibilities besides single cell origin of the tumor must be considered :

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1. X-chromosome loss. This cause has been excluded by appropriate cytologic techniques in all tumors examined with single enzyme phenotypes. 2. Nonspecific disturbance in gene expression. Whenever autosomal heterozygosity was expected from analyses of normal tissues, all tumors with a single G-6-PD type examined for markers a t autosomal loci have demonstrated heterozygous phenotypes. Thus, it seems unlikely that nonspecific regulatory disturbances associated with neoplasia underlie the hemizygous, single enzyme phenotypes observed for the X-linked G-6-PD locus. 3. Origin from adjacent cells of the same type. The probability that two adjacent cells will be of the same type depends on how much coherent clonal growth occurs during development of the tissue. If complete random cell migration occurs in a tissue composed of equal numbers of A and B cells, the chance that two contiguous cells will be alike is 0.5. This probability rises with increasing degrees of coherent clonal growth, since the daughter cells remain adjacent to one another. If this type of growth prevails during development of the tissue, there will be large clones or patches of adjacent cells which have the same enzyme type (A or B ) . The size of the patches can be estimated for a given tissue by statistically analyzing the results obtained from study of multiple small samples. Such estimates are based on the fact that the degree of heterogeneity in A: B composition between samples is inversely related to the number of clones or patches per sample. Estimates of the patch size for various mammalian tissues have been in the range of 15010,000 cells (Linder and Gartler, 1965b; Wegmann, 1970). If the patch size for the tissue in which the tumor arises is known, the probability that a single enzyme phenotype tumor will arise from two or more nearby cells with the same enzyme type can be calculated (Linder and Gartler, 1965a) (see Section V,A). 4. Repetitive sampling. If a considerable proportion of cells die during the growth of a tumor so that cell production only slightly exceeds cell death, even tumors with multicellular origin may eventually have only one enzyme type (Buhler, 1967). I t is unlikely that this explanation applies to many, if any, human tumors with single enzyme phenotypes. The time required for complete loss of one cell type presumably must be very long, and the predicted double enzyme phenotypes in small (presumably young) tumors and in the cores of old tumors have not been observed. 5. Selection. The possibility of selection is the most difficult problem to deal with in the interpretation of single enzyme phenotypes. The putative selective forces may operate in the initial transforming events or in the subsequent growth of the tumor; selection may be dependent

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upon G-6-PD phenotype or upon phenotypic differences determined by other X-linked loci. For example, conceivably some circumstances might favor the occurrence of the initial transforming event or the subsequent selective overgrowth of cells with one or another type of G-6-PD in GdA-/GdBheterozygotes (e.g., see Section 111,C). I n this case, a tumor with a B phenotype, for example, may have arisen from multiple B type cells. Similarly, a B phenotype may be the result of some selective advantage governed by a favorable allele at another locus on the same X chromosome as the G-6-PDB allele. An example of this type of selection in a nonneoplastic tissue was recently reported by Nyhan and co-workers (1970) in females heterozygous for the X-linked mutant allele which determines the Lesch-Nyhan syndrome. Affected males have profound deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in blood cells, but, rather than the expected intermediate levels, most heterozygotes have normal blood cell enzyme activity (Kelley et al., 1969; Nyhan et al., 1970). The probable explanation is that hematopoietic cells in which the active X chromosome bears the normal HGPRT allele have a selective advantage over cells with an active mutant allele. “Hemizygous” phenotypes may therefore be expected for all other loci linked to the one for HGPRT. For example, Nyhan and co-workers (1970) found only type B G-6-PD in the blood cells of two GdA/Gdn subjects heterozygous for H G P R T deficiency. As predicted, family studies indicated that the GdB allele was on the same X chromosome as the normal HGPRT allele. If it were not known that these women werc H G P R T heteroxygotes, and if they had leukemia, i t might be concluded on the basis of a hemizygous G-6-PD phenotype that the malignancy had a clonal origin. Fortunately, the allele governing severe HGPRT deficiency is rare, and the great majority of proven GdA/GdBheterozygotes have both A and B enzyme in white blood cells. However, alleles a t the HGPRT locus governing less drastic decreases in enzyme activity are more prevalent, and similar more “benign” alleles undoubtedly occur a t other X-linked loci. The resultant moderate or mild changes in enzyme activity would not cause selection to occur in normal tissues, but might well influence cell growth in malignant tissues. Selection as an explanation for single enzyme phenotypes is unlikely if it can be shown that in about half of the tumors from the same individual the enzyme type is A, and in the other half, B. If only a single tumor can be studied from each patient, but material is available from many patients with similar tumors, the finding of a 1:l proportion of A:B tumors makes selection based solely on G-6-PD type very unlikely.

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In this instance, however, selection for cells on the basis of phenotypic differences governed by other X-chromosomal loci cannot be excluded without ancillary evidence. 111. Hematopoietic Neoplasms

Diseases of the hematopoietic system are more easily studied than most solid tumors because the tissue is more accessible, repeated specimens can be collected, and it is generally easier to obtain pure preparations of neoplastic cells. Chronic myelocytic leukemia (CML) and Burkitt lymphoma have been extensively studied. The demonstration that CML has a clonal origin has permitted the use of this neoplasm for the study of genetic phenomena such as gene localization and activation.

A. CHRONIC MYELOCYTIC LEUKEMIA(CML)ORIGINAND DEVELOPMENT The electrophoretic variants of G-6-PD appear to be prevalent in populations with a very low incidence of CML. Thus, only three GdA/GdB heterozygotes with CML have been reported (Fialkow et al., 1967). Several questions were explored in that study: 1. Does C M L Have a Clonal Origin?

Although both A and B enzyme types were present in skin fibroblasts in the three patients, lysates of their peripheral blood granulocytes contained only type A enzyme. These findings are compatible with a clonal origin for CML. The fact that all three patients had type A enzyme in the leukemia cells is probably the result of chance alone ( P = 0.125), since there is nothing to suggest that C M L occurs preferentially in type A cells. (For example, CML is more prevalent in Caucasian populations in which Gd" is not found than it is in black populations, and no more than the expected number of black Gd*/Gd" females with C M L was observed in the G-6-PD-CML study.) The possibility that the hemizygous phenotypes in CML are due to selection at some other X-linked locus (as described above in HGPRT heterozygotes) cannot be formally excluded, but studies using other markers make this unlikely (see following). Fialkow and co-workers (1969) reported a CML patient heterozygous a t the 6-phosphogluconate dehydrogenase locus who had a hemizygous phenotype for this autosomal locus in her blood cells. It is most likely that this aberration arose in a single cell from a rare genetic accident such as a deletion (see Section 111,B). The fact that all the CML cells

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had the hemizygous phenotype provides additional support for the theory that C M L has a clonal origin. Earlier studies using a chromosome marker, the Philadelphia' (Ph') chromosome (Nowell and Hungerford, 1960, 1961), also suggested a clonal origin of CML. This abnormal G group chromosome [No. 22 (Caspersson et al., 1970)], which lacks about 40% of its DNA, occurs almost exclusively in CML, where it is seen in almost every dividing bone marrow cell in most patients (Sandberg et al., 1962; Tough et al., 1963; Whang et al., 1963). It has not been reported in lymphocytes. Although these observations provided strong support for single cell origin of CML, i t was impossible to rule out the possibility that the abnormal leukocytes were derived from many parent cells. For example, multicellular origin might have been due to some etiologic agent with affinity for a specific region of DNA on the involved G-group chromosome. However, the evidence derived from studies of G-6-PD make i t appear much more likely that the abnormal cells in clinically evident CML have a single cell origin. One CML patient has been reported to have two abnormal clones (Tough et al., 1961). This man had features of Klinefelter's syndrome, a disorder usually associated with an extra X chromosome (47,XXY). Only cells of this karyotype were found in the skin, but in the blood some cells were 46,XY and others were 47,XXY. Since a proportion of each cell type lacked the Phl chromosome, it appeared that the patient had 46,XY/47,XXY mosaicism prior to the development of CML. Both lines also had cells with the Ph' chromosome, which could therefore have arisen in two separate stem cells ( a 47,XXY and a 46,XY cell). Alternatively, as the authors noted, the Ph' chromosome could have arisen in a stem cell for only one line (e.g., 47,XXY), which subsequently underwent a further divisional error to give rise to the other Ph*-positive cell line. This possibility is not unlikely, since there are data suggesting that cells bearing chromosomal abnormalities are more prone than normal cells to the occurrence of further chromosomal aberrations.

2. In Which T y p e of Cell Does C M L Arise? Both A and B enzymes are found in the erythrocytes of nearly all normal GdA/GdB heterozygotes. In contrast, the red cells of the three studied GdA/GdB heterozygotes with CML were found to have a single enzyme of the same type as that in their white cells. The most likely explanation for this observation is that the C M L clone arises in a single stem cell common to the red cell and granulocyte, but not to the skin fibroblast. This is supported by the demonstration of the Ph' chromosome in erythrocyte precursor cells (Clein and Flemans, 1966; Rastrick et al., 1968) and probably in megakaryocytes as well (Sandberg et al., 1962;

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Trujillo and Ohno, 1964; Whang et al., 1963). The reported absence of the Ph' chromosome in lymphocytes provides support for the suggestion that a t one point in differentiation of the hematopoietic tissues, there are separate stem cells for the myeloid and lymphoid series. Although the isoenzyme (G-6-PD and 6-phosphogluconate dehydrogenase) and cytogenetic (Ph' chromosome) studies indicate that the alteration responsible for the development of CML occurs in a single myeloid stem cell, cytologically this disease is not considered to be a stem cell leukemia. The predominant picture in the bone marrow is an overgrowth of myelocytes and more mature granulocytic cells with little evidence of increased stem cell proliferation. T o reconcile these cytological observations with the genetic data, it Eeems necessary to postulate that in CML the normal stem cell population is replaced by CML stem cells, which in turn give rise to the hyperproliferative myelocytic cells.

3. Does the Abnormal Clone Per'slst during Remission or Do Normal Cells Repopulate the Marrow? Two of the GdA/GdBpatients with CML had chemotherapeutically induced remission during which the single enzyme type persisted in peripheral blood cells. These findings and the observation that the percentage of marrow cells with the Ph' chromosome does not decline during remission provide clear evidence that the abnormal clone persists during remission.

GENE B. CML-UTILIZATIONOF CML CELLSTO STUDY AND ACTIVATION LOCALIZATION Neoplasms with proved single cell origin can be used to study genetic phenomena at the