Advances in Cancer Research Volume 37

ADVANCES IN CANCER RESEARCH VOLUME 37

Contributors to This Volume Kari Alitalo

Jan Klein

J. Michael Bishop

Gilbert Lenoir

Carmia Borek

Arnold J. Levine

Elisabeth Gateff

Zoltan A. Nagy

George Klein

Kenneth Nilsson

Antti Vaheri

ADVANCES IN CANCERRESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

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

Volume 37-1982

ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers

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LIBRARY OF CONGRESS CATALOG CARDNUMBER:52-1 3360 ISBN 0-12-006637-8 PRINTED IN THE UNlTED STATES OF AMERICA 82 83 84 85

9 8 1 6 5 4 3 2 1

CONTENTS CONTRIBUTORS TO VOLUME 37

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ix

Retroviruses and Cancer Genes J . MICHAEL BISHOP I. I1. 111. IV. V. VI . VII . VIII . IX . X. XI . XI1. XI11. XIv.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introducing Retrovirus Oncogenes . . . . . . . . . . . . . . . . . . . . Properties of Retrovirus Oncogenes and Their Products . . . . . . . . . . . The Origin of Retrovirus Oncogenes: Emergence of the Thesis. . . . . . . . The Discovery of c-oncs . . . . . . . . . . . . . . . . . . . . . . . . Characterizing c-oncs . . . . . . . . . . . . . . . . . . . . . . . . . c-oncs Are Cellular Genes . . . . . . . . . . . . . . . . . . . . . . . The Expression of c-oncs . . . . . . . . . . . . . . . . . . . . . . . . Proteins Encoded by C-ORCS . . . . . . . . . . . . . . . . . . . . . . . How Similar Are Viral Oncogenes and c-oncs? . . . . . . . . . . . . . . . Are c-oncs Members of a Multigene Family? . . . . . . . . . . . . . . . How Might Retroviruses Transduce Cellular Genes? . . . . . . . . . . . . What Is the Function of c-oncs in Normal Cells?. . . . . . . . . . . . . . The Paradox of Neoplastic Transformation by Retrovirus Oncogenes . . . . . xv. Does the Homology between Viral Oncogenes and c-oncs Dictate the Host Range of Viral Transformation? . . . . . . . . . . . . . . . . . . . . . . XVI . Do c-oncs Provide a Pathway for Oncogenesis? . . . . . . . . . . . . . . XVII . Conclusion: The Pursuit of Cancer Genes . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 6 7 8 10 12 12 13 14 16 17 20 21 22 23 26 28

Cancer. Genes. and Development: The Drosophila Case ELISABETH GATEFF I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Drosophila Development . . . . . . . . . . . . . . . . . . . . . . . .

111. General Information on the Drosophilu Tumor Mutants . IV. Description of the Drosophila Tumor Mutants . . . . . V. Viruses Found in Drosophila Tumor Cells . . . . . . V I . Retroviral Oncogenes and Their Cellular Counterparts in Species . . . . . . . . . . . . . . . . . . . . . . VII . Transfection and Vertebrate Tumor Genes: A Comparison VIII . Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . V

33 34 . . . . . . . . . . 35 . . . . . . . . . . 37 . . . . . . . . . . 57 Different Animal 62 . . . . . . . . . with Drosophilu . . 66 69 . . . . . . . . . 69 . . . . . . . . .

vi

CONTENTS

Transformation-AssociatedTumor Antigens ARNOLD J . LEVINE I. I1. 111. IV. V VI . VII . VIII .

.

IX.

Introduction . . . . . . . Simian Virus 40 . . . . . . Adenoviruses . . . . . . . Epstein-Barr Virus . . . . Methylcholanthrene-Induced Abelson Virus . . . . . . . Rous Sarcoma Virus . . . . Teratocarcinonias . . . . . Conclusions . . . . . . . . References . . . . . . . .

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. . . . Transformed Cells (Meth A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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. . . . 75 . . . . 81 . . . . 86 . . . . 88 . . . . . 91 . . . . 93 . . . . 96 . . . . 97 . . . . 100 . . . . 104

Pericellular Matrix in Malignant Transformation KARIALITALO AND ANTTI VAHERI I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 I1. Components of the Extracellular Matrix and Their Functions . . . . . . . . 112 111. Pericellular Matrix and the Cellular Phenotype in Vitro . . . . . . . . . . . 123 Iv.

V.

VI . VII . VIII .

Malignant Transformation: Altered Biosynthesis of Matrix Components and Failure to Deposit Them . . . . . . . . . . . . . . . . . . . . . . . . Tumorigenicity. Invasion. and Metastasis . . . . . . . . . . . . . . . . . Proteins of Basement Membranes and Interstitial Matrix as Characteristics of Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell-Matrix Interaction and Anchorage Dependence of Normal Cells. . . . . Molecular Mechanisms of Altered Cell-Matrix Interaction in Rous Sarcoma Virus Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

128 132 138 141 143 146

Radiation Oncogenesis in Cell Culture CARMIA BOREK I. I1. I11. IV.

V

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Transformation in Vitro . . . . . . . . . . . . . . . . . . . . . . Radiation Oncogenesis in Vitro . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

159 160 161 177 220 227

CONTENTS

vii

Mhc Restriction and Ir Genes JAN KLEINAND ZOLTAN A . NAGY I. I1. I11. IV. V VI . VII . VIII . IX.

Mhc through a Keyhole . . . . . . . . . . . . . . . . . . . . . . . . How Was Mhc Restriction Discovered? . . . . . . . . . . . . . . . . . . Mhc Restriction of Cytolytic Responses . . . . . . . . . . . . . . . . . . Mhc Restriction of Regulatory Responses . . . . . . . . . . . . . . . . . Mhc Restriction of DTH and CS Responses . . . . . . . . . . . . . . . . The Puzzle of the Class I and Class I1 Gene Dichotomy . . . . . . . . . . . Is the T-cell Repertoire Individualized? . . . . . . . . . . . . . . . . . . Nature of Nonresponsiveness and the So-called Ir Genes . . . . . . . . . . The Parable of the Blind . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

234 238 242 245 262 265 270 285 309 310

Phenotypic and Cytogenetic Characteristics of Human B-Lymphoid Cell Lines and Their Relevance for the Etiology of Burkitt’s Lymphoma KENNETHNILSSON AND GEORGE KLEIN I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Lymphohlastoid Cell Lines (LCLs). . . . . . . . . . . . . . . . . . . . 111. EBV-Carrying Burkitt’s Lymphoma (BL) Cell Lines . . . . . . . . . . . . IV Basis for Distinction between EBV-Carrying Lymphoblastoid and BL Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. EBV Genome-Negative BL Cell Lines . . . . . . . . . . . . . . . . . . VI . EBV Genome-Negative B-LeukemidLymphoma Cell Lines . . . . . . . . . VII . EBV-Carrying Non.BL. Nonlymphoblastoid Cell Lines Derived from EBV Genome-Negative LeukemidLymphomas . . . . . . . . . . . . . . . . . VIII . The Relationship of EBV-Carrying Lymphoid Cell Lines to Normal B-Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. The Progression in Lymphoblastoid Cell Lines in Vitro and in Vim . . . . . X . The Role of EBV in Progression . . . . . . . . . . . . . . . . . . . . . XI . The Role of Chromosomal Changes in Progression . . . . . . . . . . . . . XI1. General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

319 321 338 346 346 349 352 353 360 362 364 368 371

Translocations Involving Ig Locus-Carrying Chromosomes: A Model for Genetic Transposition in Carcinogenesis GEORGEKLEINAND GILBERT LENOIR Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

381 386

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

389 393

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CONTRIBUTORS TO VOLUME 37 Numbers in parentheses indicate the pages on which the authors' contributions begin.

KARIALITALO,' Department of Virology, University of Helsinki, 00290 Helsinki 29, Finland (111) J. MICHAEL BISHOP,Department of Microbiology and Immunology, University of California, Sun Francisco, California 94143 (1) CARMIA BOREK,Radiological Research Laboratory, Department of Radiology, Cancer Centerllnstitute of Cancer Research, and Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 (159) ELISABETH GATEFF,Biologisches lnstitut 1 (Zoologie), Albert-Ludwigs Universitat, 7800 Freiburg i. Br., Federal Republic of Germany (33) GEORGE KLEIN,Department of Tumor Biology, Karolinska lnstitutet, S-104 01 Stockholm, Sweden (319, 381) JAN KLEIN, Max-Planck-Znstitut f u r Biologie, Abteilung lmmungenetik, 7400 Tubingen 1, Federal Republic of Germany (233) GILBERT LENOIR,International Agency f o r Research on Cancer, F-69372 Lyon, Cedex 2, France (381) ARNOLD J. LEVINE,Department of Microbiology, School of Medicine, State University of New York, Stony Brook, New York 11794 (75) ZOLTAN A. NAGY, Max-Planck-lnstitut f u r Biologie, Abteilung lmmungenetik, 7400 Tubingen 1, Federal Republic of Germany (233) KENNETH NILSSON,The Tumor Biology Group, The Wallenberg Laboratory, University of Uppsala, S-751 22 Uppsala, Sweden (319) ANTTIVAHERI,Department of Virology, University of Helsinki, 00290 Helsinki 29, Finland (111)

'Present address: Department of Microbiology and Immunology, University of California, San Francisco, California 94143.

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RETROVIRUSES AND CANCER GENES J. Michael Bishop Department of Microbiology and Immunology. University of California. San Francisco, California

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

1

11. Introducing Retrovirus Oncogenes. . .............. 111. Properties of Retrovirus Oncogenes a ..................... IV. The Origin of Retrovirus Oncogenes: Emergence of the Thesis.. . . . . . . . . . . . . . .

2 6 7 8 10 12

The Discovery of c-oncs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterizing c-oncs . . . . . . . . . . . . . . . .................. c-oncs Are Cellular Genes ............................................... The Expression of c-oncs . . . . . . Proteins Encoded by c-oncs. .................................... How Similar Are Viral 0 Are c-oncs Members of a Multigene Family? . . . . . . . . . . . . How Might Retroviruses What Is the Function of c-oncs in Normal Cells? . The Paradox of Neoplast Does the Homology between Viral Oncogenes and c-oncs Dictate the Host Range of Viral Transformation? ................................. ............ XVI. Do c-oncs Provide a Pathway for Oncogenesis? . XVII. Conclusion: The Pursuit of Cancer Gen References ......................... .........

V. V1. VII. VIII. IX. X. XI. XII. XIII. XIV. XV.

22 23 28

I. Introduction

The possibility that viruses might be tumorigenic materialized at the turn of this century, with reports that erythroleukemia and fibrosarcomas could be induced in chickens by transmissible agents (Ellermann and Bang, 1908; Rous, 191 1). Skepticism greeted these reports, dissipated only slowly over the ensuing years, and lingers on in the continuing debate about the possible role of viruses in the genesis of human tumors. But these are needless concerns for most experimentalists : the oncogenic potential of many animal viruses is well established, and the use of tumor viruses now dominates efforts to dissect the mechanisms of tumorigenesis. Oncogenic viruses are taxonomically diverse. DNA viruses both large (herpes and adenoviruses) and small (papovaviruses), as well as many (but nor all) members of the large family of retroviruses, can induce tumors in either experimental or natural hosts (Gross, 1970). Two patterns of viral oncogenesis have emerged. Some viruses possess genetic loci (or "oncogenes") whose actions initiate and maintain the neoplastic phenotype of the infected cell. Other viruses are devoid of specific oncogenes and induce tumors by 1 ADVANCES IN CANCER RESEARCH, VOL. 37

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006637-8

2

J . MICHAEL BISHOP

more subtle means, whose particulars we are just beginning to perceive. But both forms of viral oncogenesis are united by the persistence of at least a portion of the viral genome in the host cell, either as an integral part of a host chromosome or as independently replicating units. For the moment, it appears that persistence of the viral genome is a necessary event for viral oncogenesis-to maintain the influence of an oncogene over the host cell, or to sustain the more indirect but equally malicious effects of viruses that induce tumors without benefit of an oncogene. The possibility that transient infection by viruses might trigger an irreversible sequence of events (the “hit-and-run” mechanism) has been posited repeatedly but has gained no substantive experimental support to date. Although several themes appear to unite oncogenesis by diverse viral agents, and although each family of tumor viruses offers important distinctive features to the experimentalist, it is the retroviruses that have provided the most coherent and penetrating view of tumorigenesis presently available to us. Three features of retroviruses account for this sentiment. First, the oncogenes of retroviruses have proved exceptionally accessible to definition and study, and as a consequence, they have provided our first glimpse of enzymatic mechanisms responsible for neoplastic transformation. Second, the diversity of retrovirus oncogenes has provided a rich set of oncogenic agents whose versatility far exceeds that of DNA tumor virus oncogenes, and whose tumorigenic capacities provide separate experimental models for most major forms of malignancy. Third, oncogenes appear not to be indigenous components of retrovirus genomes, but instead have been transduced from normal genetic loci of the vertebrate hosts in which retroviruses replicate. Moreover, we have reasons to believe that the vertebrate genes from which retrovirus oncogenes derive may participate in tumorigenesis induced by agents other than viruses. Thus while tracking the evolutionary origins of oncogenes, retrovirologists have been led well beyond the confines of tumor virology, to confront what may be a final common pathway of oncogenesis. The genesis and status of this confrontation are the main subjects of this essay. II. Introducing Retrovirus Oncogenes

Retrovirus oncogenes display a burgeoning diversity that is largely attributable to nature’s generosity in providing field isolates, but recent years have also witnessed provisional successes in bringing new oncogenes to view by experimental manipulations (Rapp and Todaro, 1978, 1980; Young ef a/., 1981). At least 15 retrovirus oncogenes have been identified (Table 1). Together, they are known by the generic term v-onc (for viral oncogene). Each gene is distinguished by its nucleotide sequence and is designated by a term derived from the name of the virus that bears the gene (hence, v-SYC,

3

RETROVIRUSES AND CANCER GENES

v-my, v-abl, etc.; see the table). Many of these genes are represented in more than one viral isolate, and the topographical details of kindred genes may vary from one isolate to another; a few of the genes have been obtained from more than one host species; and a number share similar oncogenic properties and/or biochemical functions (see later). The replicative unit in all retrovirus genomes is composed of three genes : gag (structural proteins of the virion); pol (reverse transcriptase); and e m (glycoproteins of the viral envelope). An oncogene may be inserted into this unit in at least four distinctive ways (Fig. 1): (1) as an independently expressed

RSV

AMV

MCV

AEV

I

0

I

1

I

2

I

3

I

4

I

5

I

6

I

7

I

8

1

9

KILOBASES

FIG.1. Styles in retroviruses genomes. Genomes of avian retroviruses are used to illustrate typical dispositions of oncogenes. ALV, avian leukosis virus: contains no oncogene; gag encodes structural proteins of the viral core; pol, reverse transcriptase; and e m , the glycoproteins of the viral envelope. RSV, Rous sarcoma virus: the oncogene src is expressed from a spliced subgenomic mRNA. AMV, avian myeloblastosis virus: the oncogene myb is expressed from a spliced subgenomic mRNA. MCV, MC29 virus: the oncogene rnyc is expressed from a genomiclength mRNA that directs uninterrupted translation from gag and myc. AEV, avian erythroblastosis virus: the oncogene erb-A is expressed from a genomic-length mRNA that directs the uninterrupted translation from gag and erb-A; the oncogene erb-B is expressed from a spliced subgenomic mRNA. Stippling denotes regions that do not encode protein; solid vertical lines denote established gene boundaries; and diagonal shading denotes uncertainty in gene boundaries.

J

10

TABLE I RETROVIRUS ONCOGENFS AND THEIRPROGENITORS ~

v-onP

Protypic virus

v-src v-jps v-yes v-rash

Rous sarcoma virus

v-myc v-erb-A v-erb-B v-myb v-re1 v-mos v-ubl v-bus v-rus V+S

v-fms v-sis

Fujinami sarcoma virus Y73 sarcoma virus University of Rochester sarcoma virus 2 Myelocytomatosis virus Avian erythroblastosis virus Avian erythroblastosis virus Avian myeloblastosis virus Reticuloendotheliosis virus Moloney sarcoma virus Abelson leukemia virus BALB sarcoma virus Harvey sarcoma virus Gardner-Arnstein feline sarcoma virus McDonough feline sarcoma virus Simian sarcoma virus

~~~

Probable species of originb

~~~~

Tyrosine phosphorylation'

+ + +

c-one

c-onr expressedd

c-src c-jp9 c-yes c-ros"

Yes ?

Chicken Chicken Chicken Chicken Turkey Mouse Mouse Mouse Rat Cat

c-myc c-erb-A' c-erb-B' c-myb c-re1 c-mos c-ubl c-bus' c-rus" c-fes

Yes Yes Yes Yes Yes

Cat

c: fms

?

Woolly monkey

c-sis

'I

Chicken/quaill Chicken Chicken Chicken

+

? 'I

J

Yes ? Yes Yes

Protein product of c-once pp60'-'" ? > 9

~

The names of viral genes are here treated as generic terms, although in most instances several separate viral isolates are known. The probable species of origin is inferred from the host in which the particular oncovirus first emerged. A plus sign denotes the existence of evidence (not necessarily definitive) for tyrosine phosphorylation by the oncogene product. Expression is defined as either detection of transcription from the c-onc or detection of a protein encoded by the locus. Question marks indicate that suitable analyses have not been completed. Some strains of RSV have been generated experimentally in quail (Wang er al., 1979), but all field isolates of v-src have come from chickens. The c-fps of chicken is apparently related to c-fes of cats (Shibuya et al., 1980). The term v-ros is a provisional designation for a newly identified oncogene in an avian sarcoma virus isolated at the University of Rochester (P. Balduzzi, personal communication). The separate domains of erb (A and B) are represented by similarly separate-but possibly linked-domains in the chicken genome. j All efforts to date have failed to detect transcription of c-mos (Frankel and Fischinger, 1976). Ir Failure to detect transcription from c-mos and features of the gene’s nucleotide sequence (Van Beveren er al., 1981) raise the possibility that c-mos is inactive unless transferred into a retrovirus genome. The c-bas of mice is apparently closely related to the c-ras of rats (S. Aaronson and E. Scolnick, personal communication). Rat DNA contains at least two small, distinct gene families related to v-ras. One family is apparently the source of Harvey and Rasheed v-rus, the other of Kirsten v-ras (DeFeo er al., 1981). a

’

vI

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J . MICHAEL BISHOP

gene that does not impose on either the structure or function of the replicative genes and is expressed from a subgenomic mRNA (v-src of the Rous sarcoma viruses is the sole known example); (2) as an independently expressed gene that replaces part or all of a replicative gene (enu, for example) and is expressed from a subgenomic mRNA (e.g., v-myb of avian myeloblastosis virus); (3) as a fusion between v-onc and a portion of gag that is accompanied by deletions in one or more of the replicative genes (usually pol and portions of gag and env) and is expressed as a polyprotein produced from a genomic-length mRNA (e.g., v-myc of avian myelocytomatosis virus) ; and (4) as two separately expressed v-onc domains-one fused with a portion of gag, the other expressed independently, and the two together replacing portions of replicative genes. In this last instance, the yag-onc protein is produced from a genomic-length mRNA, the second onc protein from a subgenomic mRNA (e.g., erb-A and erb-B of avian erythroblastosis virus). These themes are varied in yet a further way: the same class of v-onc can be fused to gag in some viral isolates and occur as an independently expressed locus in others. With the exception of v-src, the insertion of oncogenes into retrovirus genomes creates genetic defects that preclude the production of virus unless the defective function(s) is provided by a second “helper” virus. The variety of retrovirus oncogene construction is merely tedious at first glance, but in reality it poses an important challenge because all of these configurations are thought to be products of recombination between a replicating retrovirus and the genome of its host cell (see later). The mechanisms that effect this recombination with its remarkable plurality of outcomes may have no precedent in the annals of molecular genetics. Ill. Properties of Retrovirus Oncogenes and Their Products

The oncogenes of retroviruses are genetic luxuries whose actions are highly selective. They are not required for viral replication-indeed they make no known contribution to replication; and their activities may be muted even in cells that sustain vigorous replication, and perhaps chemical expression, of the oncogenes (Graf et al., 1980; Durban and Boettiger, 1981a). This muting underlies one of the cardinal properties of retrovirus oncogenes : the specificity of their pathogenicity. Each oncogene induces tumors in only a limited and characteristic set of tissues; transformation of cells in culture follows the same selective pattern. We cannot at present explain the selectivity of oncogene actions, but the phenomenon has contributed to the view that transformation by retrovirus oncogenes is fundamentally a disturbance of differentiation. According to one prevalent view, oncogenes may act by arresting cellular development within a specific compartment of one or another developmental lineage; tumorigenesis ensues because the immature cells that constitute the compartment continue to divide, as is their nature,

RETROVIRUSES AND CANCER GENES

7

and become a continuously expanding population-a tumor composed of ostensibly normal cells (Graf and Beug, 1978). Other observers have argued that the effects of oncogenes more commonly (or perhaps inevitably) distort the phenotype of susceptible cells to a form that is not representative of a single developmental compartment (Boettiger and Durban, 1979; Durban and Boettiger, 1981b). No matter which view is correct (there appear to be elements of truth in both), the effects of oncogenes on cells, and the selectivity of these effects, point to biochemical functions that in other guises might well direct the course of normal growth and development. Inferences of this sort brook large in present efforts to interpret the evolutionary origins of retrovirus oncogenes (see later). How do oncogenes evoke the myriad changes that accompany neoplastic transformation? Efforts to answer this daunting question have produced at least dim outlines of several potentially important refrains. 1. Some retrovirus-transforming proteins (but not others; see Table I) apparently mediate the phosphorylation of tyrosine in protein substrates (Hunter and Sefton, 1980b). The best-studied example is the 60,000-MW phosphoprotein encoded by v-src (pp60'-"'), but the products of v-fps, v-yes, v-abl, and v-fes also appear to follow suit. Phosphorylation of proteins represents one of the principal devices by which cellular functions are regulated (Rubin and Rosen, 1975) and thus offers an attractive explanation for the pleiotropic effects of retrovirus oncogenes. Moreover, the findings with pp6OV-"*'and other retrovirus-transforming genes have produced unexpected further dividends by alerting cell biologists to the existence of tyrosine phosphorylation. This hitherto unknown reaction is now rapidly emerging as an important regulatory mechanism in normal as well as transformed cells. But efforts to identify cellular proteins that serve as substrates for retrovirus protein kinases have just begun, only a small number of candidate substrates have been sighted, and none of these as yet offers any explanation for the abnormal growth of transformed cells. 2. Several retrovirus-transforming proteins (including pp6OV-"*',pp2 1v-ras, and the product of v-abl) appear to act at the periphery of the cell because they are found attached to the plasma membrane of the transformed cell (Hynes, 1980). This inference is for the moment largely circumstantial, however. The techniques used to locate the transforming proteins have limited resolving power and sensitivity; trace amounts of the proteins in presently unappreciated locations might cause major effects. IV. The Origin of Retrovirus Oncogenes: Emergence of the Thesis

The virus isolated from a chicken sarcoma by Peyton Rous did not spring quickly or easily into view. Rather, an infectious tumorigenic agent was obtained from extracts of tumor tissue only after the original sarcoma had

8

J. MICHAEL BISHOP

been passaged repeatedly from one bird to another (Rous, 1911). It seems possible in retrospect that the original tumor was not the consequence of viral infection; the sarcoma virus that eventually emerged may not have been present in the tissue with which Rous began his work. The isolation of murine sarcoma viruses (Harvey, 1964; Moloney, 1966) and the Abelson murine leukemia virus (Abelson and Rabstein, 1970a,b) decades later raised these issues in a more explicit manner: the new viruses appeared during the passage of leukemia viruses in rodents, as if new capabilities for pathogenesis could be acquired from the host animal. The discovery of endogenous retroviruses in chickens (Robinson, 1978) and mice (Aaronson and Stephenson, 1976), as well as the development of inbred lines of mice whose predisposition to leukemia appeared to involve genetically transmitted retroviruses (Rowe, 1973), added appreciably to these inferences and engendered the “oncogene hypothesis” of Huebner and Todaro (1969). According to this hypothesis, carcinogens of many sorts act by inducing the expression of otherwise cyrptic retrovirus genes already resident in the genome of the target cells. The oncogene hypothesis is no longer regarded as strictly correct, but it served an important heuristic purpose by prompting experimentalists to ask whether normal cellular DNA might contain retrovirus oncogenes. We now know that vertebrate cells do harbor genetic loci homologous to retrovirus oncogenes (designated here by the generic term “c-onc”), but these loci are cellular, not viral genes, and the oncogene hypothesis has been eclipsed by even more sweeping views of the nature of these cellular genes.

V. The Discovery of c-oncs

The search for oncogenes in cellular DNA began with the use of molecular hybridization. The strategy exploited naturally occurring deletions that remove most or all of v-src (but no other viral gene) from the genome of Rous sarcoma virus (Duesberg and Vogt, 1970, 1971 ; Martin and Duesberg, 1972; Lai et al., 1973). Viral RNA bearing this class of deletions could be employed to isolate radioactive DNA (cDNA,,,) that hybridized only with nucleotide sequences encoding (or related to) src (Stehelin et a/., 1976a). The result was a reagent that provided specificity and sensitivity sufficient to detect a single genetic locus among the immense complexity of vertebrate DNA. Similar cDNAs were prepared for replication-defective murine sarcoma viruses (Scolnick et a / . , 1973, 1975; Frankel et a/., 1976), but the genetic definition of these reagents was less rigorous because suitable deletion mutants were not available for isolation of the cDNAs. As a consequence, the experimental strategies had to rely on the assumption that nucleotide sequences not present in the genome of the helper virus must perforce

RETROVIRUSES AND CANCER GENES

9

represent portions of the oncogene-an assumption that proved useful but not inevitably correct (see later). The initial findings with cDNA,,, for Rous sarcoma virus prefigured subsequent conclusions for virtually all-retrovirus oncogenes. Each family of vertebrates examined-including fish, birds, and mammals-displayed evidence of both DNA and RNA related to the src gene (Stehelin et al., 1976a; Spector et al., 1978a,b,c). The DNA related to v-src appeared to occur as only one or very few copies in each haploid portion of vertebrate genomes. Retrovirologists had obtained their first glimpse of the cellular gene we now know as c-src. The mere fact that homologous DNA could be detected across such large phylogenetic distances indicated that the genetic locus or loci in question were highly conserved during the course of evolution. More recent findings have dramatized the extent of this conservation by demonstrating homology with v-src (and several other v-ones) in the DNA of the insect Drosophila (personal communication, R. Weinberg). Conservation of c-src was also explored by evaluating the thermal stability of molecular hybrids formed between cDNA,,, and DNAs from various sources. The results indicated that the nucleotide sequences of c-src might diverge by no more than 10- 15% from fish to chicken genomes on the one hand, from chicken to human genomes on the other (Stehelin et al., 1976b; Spector et al., 1978~).The full implications of these findings were not easily sustained at the outset, largely because no assay was available for the protein product of src; nevertheless, it appeared that vertebrate species possessed a highly conserved and expressed (i.e., transcribed) gene that is closely related to a viral oncogene. The strong evolutionary conservation of this gene, and the fact that it was found to be expressed in every tissue and every species examined, indicated an essential function in cellular metabolism. Early doubts about the veracity of these deductions were obviated by the eventual identification and characterization of a protein encoded by c-src (and known as pp60c~"c).The characteristics of pp6OC-"' vindicate and extend all of the original predictions based on molecular hybridization (see later). Difficulties did arise, however, from the use of a less well-defined cDNA for the oncogene of Harvey/Kirsten murine sarcoma virus (v-ras). Initial results indicated that v-ras was related to (and presumably derived from) nucleotide sequences in the genome of an endogenous retrovirus of rats (Scolnick et al., 1973; Scolnick and Parks, 1074), a troubling deduction because it stood in striking contrast to the mounting evidence that other retrovirus oncogenes are derived from conserved cellular genes. The advent of molecular cloning to the study of retrovirus genomes quickly resolved the apparent anomaly. It now appears that the genome of Harvey/Kirsten sarcoma virus was constructed with three distinct components (Ellis et al., 1980): one derived from the murine helper virus that was used to initiate

10

J . MICHAEL BISHOP

recovery of the sarcoma virus and was isolated together with the sarcoma virus; a second derived in fact from an endogenous virus of rats; and a third, the oncogene proper, derived true to form from a cellular gene of the rat in which the sarcoma virus originally arose. The principles first enunciated for src have since been shown to be widely applicable to retrovirus oncogenes (see table) : homologues of these genes (i.e,, c-oncs) can be found in vertebrate DNA, many (but apparently not all) of which are expressed in phenotypically normal cells. The sole exception at present is the oncogene of the Spleen Focus Forming Virus, which appears to be a recombinant form of the retrovirus env gene rather than the derivative of a cellular gene (Oliff et al., 1980). All of the identified c-oncs are found in more than one vertebrate species, but the extent of evolutionary conservation varies from one c-onc to another: some are readily detectable only in closely related species; others appear to have taken form in primitive vertebrates (or even earlier in evolution) and to have evolved thereafter in concert with speciation. These variations may be only matters of degree, however ; it is now reasonable to suppose that every c-onc represents a genetic lineage that extends throughout the vertebrate phyla and, in an least some instances, farther down the phylogenetic hierarchy. The kinship between retrovirus oncogenes and cellular genes is certain. But how can we distinguish parent from progeny? Phylogenetic patterns provide a clue: in contrast to the evolutionary conservation of the cellular genes, the viral oncogenes are usually restricted to single strains of retroviruses that were isolated from particular species (although not inevitably ; see later. Moreover, the homology between viral oncogene and cellular DNA is greatest for the species in which the oncogene allegedly originated. The most straightforward interpretation of these findings is that retrovirus oncogenes are derived from cellular genes. Widespread acceptance of this scheme, and the remarkable similarity between retrovirus oncogenes and their cellular homologues (to be described later), have engendered a standard nomenclature that is followed here (Coffin et al., 1981): viral oncogenes are denoted by v, as in v-src or v-myc (see earlier, and the table); the cellular progenitors of v-oncs by c, as in c-src or c-myc (see the table). The nomenclature is only a convenience, however, and should not be construed as indicating that homologous viral and cellular genes are necessarily identical in either structure or function. The precise relationship between cellular progenitor and viral progeny has yet to be fully explored for any retrovirus oncogene. VI. Characterizing c-oms

Enumeration of c-oms by molecular hybridization and by mapping with restriction endonucleases has revealed that some may be unique loci within

RETROVIRUSES AND CANCER GENES

11

a given species. Exceptions exist, however, and may eventually prove to be the rule. (a) The DNA of chickens probably contains a second, possibly incomplete locus (a “pseudo-gene”) related to c-src (Parker et al., 1981). (b) The murine retrovirus oncogene v-ras has been isolated in at least three distinctive forms, represents by the Harvey, Kirsten, and Rasheed strains of sarcoma virus (Gross, 1970; Young et al., 1981). The Harvey v-ras may have originated from either of two genetic loci in rats, one with introns, the other without (DeFeo et al., 1981); the Kirsten v-ras apparently derives from yet another rat gene that is related only distantly to the Harvey forms of c-ras (personal communication, E. Scolnick); and the Rasheed v-ras probably emerged from the Harvey C-MS family (personal communication, E. Scolnick). The origin of the Rasheed v-ras came as a surprise because the relationship of the Rasheed oncogene to the other forms of v-YUSoriginally seemed quite distant and was perceived only by serological means, not by tests with molecular hybridization (Young e l al., 1979). The c-oncs behave as classical Mendelian loci. They occupy constant positions within the genomes of particular species (Hughes et a/., 1979a), and they segregate in a predictable fashion when breedings are analyzed with the assistance of structural polymorphisms that have been identified by restriction mapping (D. Spector and B. Vennstrom, personal communications). The loci are recognized by virtue of homology with a viral oncogene, but in most (if not all) instances, the homologous nucleotide sequences do not comprise the entire cellular gene. Three major considerations prompt this statement. First, heteroduplex analysis and restriction mapping have demonstrated that the homology between several viral oncogenes and their C-oncs is interrupted by one or more intervening sequences (or introns) in the cellular locus (Goff et al., 1980; DeFeo et al., 1981; Favera et al., 1981; Franchini et al., 1981; Parker et al., 1981; Shalloway et al., 1981). The c-onc for v-mos provides an interesting exception to this rule: the murine and human forms of c-mos display uninterrupted homology with v-mos in heteroduplex analysis (Oskarsson et al., 1980; and personal communication, G. Vande Woude). The same is true of at least one of the several rat loci representing c-ras (DeFeo et al., 1981). Second, transcription from a number of c-oncs generates RNAs that, even in their mature forms, are appreciably more complex than the homologous viral oncogene (Bishop et al., 1981). For example, the mature form of the RNA produced from c-src contains 3.9 kb (Parker et al., 1981)-a complexity almost threefold greater than that of v-src. Yet both v-src and c-src give rise to a protein of 60,000 MW (Brugge and Erikson, 1977; Collett et al., 1978; Oppermann et al., 1979). It appears that large portions of the mRNA for c-src may not be translated, and that the boundaries of this (or any other) c-onc can only be located by applying the definitions that demarcate a transcriptional unit in eukaryotic DNA (i.e., the sites of initiation and

12

J . MICHAEL BISHOP

polyadenylation). Third, many retrovirus oncogenes have been formed by fusing a portion of a replicative gene (typically, gag; see earlier) to nucleotide sequences of a c-om. It seems unlikely that this fusion always incorporates the entirety of the c-onc locus into the viral genome. in particular, sequences in the 5' domain of the c-onc may be missing from the viral oncogene. VII. C-oncs Are Cellular Genes

The oncogene hypothesis portrayed cellular oncogenes as components of retrovirus genomes-a conceptual predisposition that proved difficult to override. But we are now certain that c-ones are cellular genes, not viral genes in disguise. The conclusion rests on three major points of evidence: (1) the location of c-ones at constant genetic loci in every member of a species (in striking contrast to the diverse distribution and positioning of endogenous retrovirus genes; for example, see Hughes et at., 1979a,c); (2) the presence of intervening sequences within many of the c-oncs-a hallmark of eukaryotic genes and, again, a telling contrast with the organization of retrovirus genes; and (3) the fact that no c-one has been found within or even linked to a complete or defective provirus of an endogenous retrovirus (Hughes et d., 1979a,c; Sheiness r t al., 1980). It seems unlikely that C-OIICS were introduced into vertebrate genomes by infection of ancestral species with retroviruses ; instead, we must explain how complex cellular genes have made their way in part or entirely into the genomes of preexistent retroviruses. VIII. The Expression of c-oncs

The possibility that c-ones might be expressed in phenotypically normal cells emerged from the discovery of RNA homologous to v-src in uninfected fibroblasts of several avian species (Wang et al., 1977; Spector et a/., 1978a,b). Similar findings were made subsequently for other c-oms and other species (Sheiness and Bishop, 1979; Bishop et at., 1981 ; Roussel et al., 1979; Chen, 1980). Some c-onc's may not be expressed, however; for example, a thoroughgoing search has failed to detect transcription from c-mos (Frankel and Fischinger, 1976). Efforts to detect transcription from c-jes were also unsuccessful (Frankel et al., 1979), but a more recent report described a protein (pp92'-jeS) that may be encoded by c+s and that was found in a number of mammalian species (Barbacid et al., 1980). Transcription from c-oms has not been widely studied, but a reasonably coherent set of data are available for four c-oms representative of avian retroviruses : c-src, c-myc, c-erb, and c-myb. Several principles are apparent that may endure even after a greater variety of c-ones has been studied (Bishop et al., 1981).

RETROVIRUSES AND CANCER GENES

13

1. Each of the c-oncs is transcribed in a variety of tissues and in every vertebrate species that has been satisfactorily examined. 2. The c-oms are not coordinately expressed as a group, and the function of each gene may be required only in certain tissues. 3. There is no evidence that transcription from c-oncs is ever coordinated with the expression of endogenous retrovirus genes (Wang et al., 1977; Spector et al., 1978b). 4. The c-oncs give rise to distinctive RNAs whose sizes have so far proved to be identical in various types of cells and even in different species. The constancy of these RNAs among widely divergent species testifies to the selective pressures that have apparently preserved the structure and function of c-oncs. As discussed already, most of the transcripts are appreciably larger than would be required to encode the nucleotide sequences that are shared by the homologous viral and cellular genes. 5. Three of the avian c-oncs give rise to single mature transcripts, in accord with the expectation that each locus represents but one gene. Transcription from c-erb provides a striking contrast, however, because at least four (and perhaps five) different mature RNAs have been identified, and the distribution of these RNAs varies among different tissues. Two of the RNAs are derived from one domain of c-erb, the others from a separate domain; this pattern mirrors the organization of v-erb, which is also composed of two independently expressed domains (v-erb-A and v-erb-B; see Table I, and Sheiness et al., 1981).

IX. Proteins Encoded by c-oncs

RNA transcribed from several c-oms has been found in polyribosomes and is therefore presumably translated into proteins (Spector et al., 1978a; personal communication, Vennstrom). The search for these proteins has not been easy : most cells contain only small amounts of c-onc mRNAs and proteins, and identification of c-onc proteins has so far turned on the development of antisera that react with the product(s) of the corresponding viral oncogenes-an undertaking that is itself unpredictable and technically demanding. Three proteins encoded by c-oncs have been identified to date : pp6OC-'"', a 60,000-MW phosphoprotein specified by c-src (Collett et al., 1978, 1979; Oppermann et al., 1979; Rohrschneider et al., 1979); ~21'-"~',a 21,000-MW protein encoded by the Harvey c-ras (Langbeheim et al., 1980); and p150c-"b', a 150,000-MW protein apparently derived from c-abl and formerly called NCPl5O (Witte et al., 1975b). Both pp6OC-"' and ~21'-*"~ have been extensively studied ; both are remarkably similar to their viral counterparts in

14

J . MICHAEL BISHOP

structure and apparent function (Collett et af., 1978, 1979; Karess et al., 1979; Oppermann et af., 1979; Rohrschneider et af., 1979; Langbeheim et af., 1980; Karess and Hanafusa, 1981); and both are found in a large variety of cells and are distributed across wide phylogenetic distances (Oppermann et al., 1979; Rohrschneider et al., 1979; Langbeheim et a f . , 1980). Much less is known of p150c-ah'(Witte et af., 1979); its size and composition are different from those of any of the proteins encoded by the several variants of v-abl; it has been found in appreciable (but very small) amounts only in thymocytes and other lymphoid cells ; its phylogenetic distribution has not been reported; and nothing is known of its function. Surprisingly, RNA transcribed from c-abf has been found widely distributed among tissues and cells of many different sorts, most of which contain no detectable p150c-"b' (personal communication, D. Baltimore). This discrepancy cannot at present be explained. X. How Similar Are Viral Oncogenes and c-oms?

Assessment of the similarities between c-oncs and their viral derivatives has taken two general forms : comparison of the nucleotide sequences that embody the genes, and comparison of the proteins encoded by the genes. Early studies with molecular hybridization raised the possibility of substantial kinship between c-onc and viral oncogene, but satisfactory tests of the issue awaited the isolation of the genes by molecular cloning on the one hand, identification and characterization of the proteins encoded by the genes on the other. With either or both of these chores now accomplished in several instances, the evidence mounts for remarkable similarity-if not identity-between the viral and cellular forms of oncogenes.

1. Coding sequences shared by viral oncogenes and cellular c-oms have so far been indistinguishable by heteroduplex analysis (Goff et af., 1980; Oskarsson et a f . ,1980; DeFeo et al., 1981; Parker et al., 1981; Favera et af., 198l), although ambiguities arise whenever the cellular locus is punctuated by introns. 2. DNA sequencing has permitted an extensive comparison of v-mos to c-mos (Van Beveren et al., 1981). The first several codons of v-mos are vestiges of the enu gene into which c-mos was inserted. Otherwise, only occasional nucleotide substitutions distinguish v-mos from c-mos. 3. The c-erb locus is extraordinarily complex, extending over at least 40 kb of chicken DNA and containing a minimum of 12 introns (personal communication, B. Vennstrom). When reduced to its exons, however, the locus displays organization similar to that of the homologous viral oncogene: each of the domains of v-erb (A and B; see table) has a separately

RETROVIRUSES AND CANCER GENES

15

expressed counterpart in chicken DNA (c-erb-A and c-erb-B) (personal communication, B. Vennstrom). 4. The viral and cellular forms of pp60"" are remarkably similar (Collett et al., 1978, 1979a,b; Karess et al., 1979; Oppermann et al., 1979; Rohrschneider et al., 1979; Karess and Hanafusa, 1981). They share size, display antigenic cross-reactivities, and yield closely related peptide maps. Both are principally affiliated with the plasma membrane of the cell (Courtneidge et al., 1980). Both are phosphorylated and have similarly disposed phosphoamino acids, with phosphoserine in the proximity of the aminoterminus (Collett et al., 1979a,b) and phosphotyrosine within a carboxyterminal domain (Hunter and Sefton, 1980a; Karess and Hanafusa, 1981). Both are protein kinases that phosphorylate tyrosine in substrate proteins (Collett et al., 1979a,b; Oppermann et al., 1979; Rohrschneider rt al., 1979; Collett et al., 1980; Hunter and Sefton, 1980a,b; Levinson et al., 1980). The two proteins can be distinguished on!y by very subtle criteria: some antisera react with the viral but not the cellular protein-presumably reflecting the fact that the antisera were raised against the viral rather than the cellular protein (Oppermann et al., 1979); the peptide maps of the two proteins differ in some ways (Rohrschneider et al., 1979; Sefton et al., 1980; Karess and Hanafusa, 1981); and the phosphotyrosine may be contained within different tryptic peptides in the two proteins (Karess and Hanafusa, 1981). It also remains possible that the viral and cellular proteins respond differently to controlling influences in the cell, and that the kinase activities of the two proteins have different substrate specificities. Definitive tests of these important potential distinctions are not presently available. 5. The possibility that v-SYCand c-src are functionally similar has received dramatic support from the demonstration that recombination between c-SYC (in chickens) and deletion mutants of v-src can reconstitute a functional oncogene (Hanafusa et al., 1977; Wang et al., 1978, 1979; Karess et al., 1979; Vigne et al., 1980; Karess and Hanafusa, 1981). In the most telling examples, the reconstituted oncogene retains a 3' terminal portion of v-src (-25% of the gene) but is otherwise apparently constructed entirely of nucleotide sequences derived from c-src (Wang et al., 1979; Karess and Hanafusa, 1981).The protein encoded by the reconstituted v-src is so similar to both pp60'-"' and pp6OC-"' that its genetic origins are difficult to discern, although peptide maps suggest that pp60'-"" of the recombinant virus is indeed a hybrid of both cellular and viral origins (Karess and Hanafusa, 1981; Vigne et al., 1980). Even these findings cannot assure us that pp60c--"r' and pp60'-"c are functionally equivalent, however; in every instance, the recombinant viral protein has derived at least 20% of its carboxyterminal domain from the parental virus in the recombination (Wang et al., 1979; Karess and Hanafusa, 1981), and it is the carboxyterminal domain of

16

J . MICHAEL BISHOP

pp6Ov-"*' that bears the protein kinase activity (Levinson et al., 1981; Oppermann et al., 1981). 6. The proteins encoded by Harvey v-rus and c-ras have been compared in considerable detail and appear to be quite similar. They each have a molecular weight of 2 1,000; they react with the same antisera; they yield related peptide maps; and they display the same biochemical function, viz., the capacity to bind guanine nucleotides with high affinity (Scolnick et al., 1979; Shih et al., 1980). They may differ in one regard, however; pp21"-r"s is phosphorylated on threonine residues, whereas phosphorylation of p2 1c-ras has yet to be detected (Shih et al., 1979a,b, 1980; Langbeheim e f al., 1980). 7. Functional similarities between V-ones and c-oncs have been demonstrated most persuasively by work with c-mos and c-rus. Both of these c-ones have been isolated by molecular cloning (c-mos from mouse DNA, c-ras from rat DNA) and coupled to a retrovirus promoter for transcription in order to favor vigorous expression. Some of the cells that receive these hybrid genetic units by transfection become transformed to a neoplastic phenotype (Oskarsson et ul., 1980; DeFeo et al., 1981), as if the c-ones might carry out the same functions as their homologous v-oncs.

-

None of the preceding examples provides a definitive demonstration of identity between viral oncogene and c-onc. But the weight of the evidence now suggests that retrovirus oncogenes encode functions also found in normal vertebrate cells. XI. Are

c-oms Members of a Multigene Family?

We presently know of at least 15 retrovirus oncogenes, each distinguished by its nucleotide sequence, and each with a corresponding oncogene (see Table I). Moreover, ostensibly similar oncogenes may be the products of related but separate cellular loci. For example, the apparently homologous oncogenes of the Harvey and Kirsten murine sarcoma viruses (v-ras), formerly believed to have originated from the same c-one, are now known to be the progeny of two different (albeit related) cellular genes (DeFeo et al., 198 1). The total number of c-oncs is therefore likely to grow as efforts to identify novel isolates of retroviruses continue. On the other hand, the number of these genes may not be inordinately large: the c-ones for src, myc, erb, myb, andfps are each represented at least twice among the handful of independently isolated avian retroviruses; c:fhs is a feline counterpart of cTfjs that appears in two strains of feline sarcoma virus (Shibuya et al., 1980; and v-bus and v-rus are derived from closely related genes in the mouse and rat, respectively (personal communication, S . Aaronson and E.

RETROVIRUSES AND CANCER GENES

17

Scolnick). The reiterative emergence of c-oncs in different viral isolates and from different species suggests that we may have the majority of these genes already in view. Whatever their number, c-oncs might constitute a family of genes whose interrelationships are akin to those found in the multigene families that encode immunoglobulins, histocompatibility antigens, and so forth. This suggestion stems from the fact that all c-oncs, however diverse in structure, give rise to viral genes with the dramatic property of oncogenicity in common. In fact, there are reasons to believe that the apparent structural diversity of c-oncs may obscure common origins and related functions: (a) the nucleotide sequences of v-src, v-mos, and v-fes are very different, yet the amino acid sequences encoded by these genes reveal significant homologies that indicate a common ancestor (Van Beveren et al., 1981 ; personal communication, .I.Stephenson); (b) several different viral oncogenes (and so far as we know, their c-oncs as well) encode tyrosine protein kinases (Collett et al., 1980; Feldman et al., 1980; Levinson et al., 1980; Van de Ven et al., 1980; Witte et al., 1980; Neil et al., 1981), and these enzymes may affect similar sets of cellular proteins (Cooper and Hunter, 1981; personal communication, T. Hunter). Given the apparent functional relationships among the identified c-oncs, it is of interest to know whether these genes might be clustered or linked in the cellular genome. The available data are in conflict. Fractionation of chicken chromosomes by rate-zonal centrifugation has located c-src on one of the smaller macrochromosomes (Padgett et al., 1977; Hughes et al., 1979b),c-myc on one of the two or three largest chromosomes (Sheiness et al., 1980), and c-erb on a chromosome of intermediate size (personal communication, B. Vennstrom). By contrast, hybridization in situ indicated that c-src, c-myc, c-myb, and c-erb are all located on one or another of the chicken microchromosomes (Tereba ef al., 1979; personal communication, A. Tereba). The discrepancies may arise from the fact that the cells used for chromosome fractionation are neoplastic and contain at least one chromosomal translocation. On the other hand, it may not be necessary that all c-oncs be genetically linked. For example, demonstrably related genes (such as a- and B-globin, and genes whose functions are coordinately induced by estrogen) are located on different chromosomes in the chicken (Hughes el al., 1979b). XII. How Might Retroviruses Transduce Cellular Genes?

By what means have c-oncs been acquired by viral genomes? Two competing answers to this question have been proposed.

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J. MICHAEL BISHOP

1. It is possible that each retrovirus arises fully grown from the rearrangement and permutation of cellular genes due to the antics of ancestral transposable elements (Temin, 1980). This account is a restatement of Temin’s original “protovirus hypothesis” (Temin, 1974) and suggests that oncogenes may be present from the inception of certain retrovirus genomes. 2. It is more generally assumed that preexistent retroviruses assimilate c-oncs by recombination (Bishop, 1981). Several lines of evidence conform to (but do not prove) this explanation. First, large (but never complete) deletions in v-src can be repaired by recombination with c-src in chickens (see earlier). It is by no means certain, however, that the mechanism of this recombination provides a general explanation for the transduction of c-oncs. Second, the oncogenes of several murine retroviruses (v-YUS,v-mos, and v-abl) appeared during the passage of leukemia viruses in rodent hosts (Harvey, 1964; Moloney, 1966; Abelson and Rabstein, 1970a,b). We presume, but cannot prove, that here the experimentalist may have reproduced the events that give rise to v-oncs in the wild. Third, several investigators have reported deliberate and apparently successful efforts to transduce c-oncs by infection of cells in tissue culture with retroviruses that do not initially contain oncogenes (Rasheed et al., 1978; Rapp and Todaro, 1978, 1980). These efforts have produced retroviruses with varied and novel oncogenic potentials, but the genetic bases of most of these potentials has yet to be elucidated. The protovirus hypothesis can be sustained only by circumstantial evidence and arguments. But proponents of the recombination model must also confront vexing problems. In particular, suitable homologous regions that could facilitate crossing over between cellular and viral genomes have not been identified, and we need to explain how the introns of c-oncs are removed to generate the uninterrupted coding units of viral oncogenes. Figure 2 illustrates the prevailing model that attempts to meet these strictures. The model posits the chance juxtapositioning of integrated retrovirus DNA and a c-onc locus, followed by a deletion that joins the viral and cellular DNA into a single genetic element. Transcription from this element begins on the retrovirus promoter, proceeds through the c-om, and generates a hybrid RNA from which the introns of the c-onc have been removed. The viral portion of the hybrid RNA permits the formation of heterodimers (retrovirus genomes are diploid) with the genome of a superinfecting (or co-resident) retrovirus and encapsidation of the RNA into virions. Recombination occurs by a form of copy-choice when the virions enter new host cells and initiate reverse transcription from viral RNA. The crossing over is illustrated as “illegitimate”-requiring no homology between the participating nucleic acids-but is otherwise similar to previous models for

-

or

I

C-one

Deletion

Transcription and Splicing AA

Packaging

Reverse Transcription

.ct gag

pol

env

Integration

v-onc

FIG.2. A model for transduction by retroviruses. The scheme begins with an intact retrovirus provirus integrated adjacent to a cellular oncogene. The characteristic terminal redundancies of the provirus (Temin, 1980) are illustrated by black and white boxes. Exons of the c-one locus are denoted by stippled boxes. A deletion fuses the provirus and the c-one into a single genetic unit, in the process eliminating one of the proviral terminal redundancies and other portions of the viral genome; two typical outcomes are illustrated. Transcription from the hybrid unit generates polyadenylated RNA (solid lines), from which the introns of the c-onc have been removed by splicing. The hybrid RNA can form a heterodimer with the genome of another retrovirus in the same cell; the resulting diploid unit can be packaged into virus and transferred into another cell. Infection of a new host cell initiates reverse transcription from viral RNA. Beginning near the 5’ end of the genome and then jumping to the 3’ end to continue chain propagation along the length of the genome (Gilboa ef al., 1979). Copy-choice during reverse transcription can generate a new retrovirus genome contining the c-one.

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J . MICHAEL BISHOP

recombination among retrovirus genomes (Coffin, 1979). Crucial portions of this model have received support recently from work that demonstrated the encapsidation of hybrid RNAs similar to those illustrated by the scheme in Fig. 2, and subsequent illegitimate recombination to bring the parental cellular domain of the RNA into a reconstituted (albeit defective) retrovirus genome (Goldfarb and Weinberg, 1981). Other puzzles remain as well. Is the seizure of c-ones a unique event, or might retroviruses be generalized transducing agents whose acquisition of more prosaic genes is merely less likely to be perceived? Are there selective pressures that favor the transduction of c-ones and their retention by retrovirus genomes? When oncogenes are formed by fusing c-one to a portion of a viral structural gene (as is frequently the case; see earlier), what portion of the cellular locus actually joins the viral genome, and what influence does the hybrid nature of the resulting oncogene have on oncogenicity? (The hybrid genetic structure appears not to be necessary for tumorigenesis: at least two oncogenes-v-myc and v-myb-occur as both hybrid genes and as independently expressed loci not fused with gag.) How are we to interpret the unusual nature of the oncogene for the Spleen Focus Forming Virus? Is it an exception to an otherwise pervasive rule, or does it signify that the origins of retrovirus oncogenes are more diverse and more complex than we presently realize? And what of the oncogenes of DNA tumor viruses? Efforts to trace the origins of these genes to cellular DNA have failed to date (although some uninfected cells allegedly contain a protein that is serologically related to the T antigen of SV40 virus; see Lane and Hoeffler, 1980). It appears that recombination between DNA tumor viruses and their host cells is less facile than that displayed by retroviruses, so we can rationalize the failure of DNA viruses to transduce cellular oncogenes. But to what end have the oncogenes of DNA viruses evolved, and what are the goods of which they are formed? XIII. What Is the Function of c-oncs in Normal Cells?

It has become an article of faith that c-ones serve essential purposes in uninfected cells. Why else would these genes have been conserved over long periods of evolutionary time, and why else would many of their numbers be expressed in both embryonic and adult tissues? The inference is easy to draw, but difficult to explore, and the difficulty lies less with biochemical function than with cellular physiology. Once pp60'-"' was known to be a protein kinase, the demonstration of a similar enzymatic activity associated with pp60""" followed in short order (Collett et al., 1979a,b; Oppermann c't al., 1979; Rohrschneider, ct al., 1979). But how does this enzymatic activity-or the biochemical function of any other c-one, for that matter-

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serve the metabolism of the normal cell? The question is usually answered by reasoning that the actions of viral oncogenes mirror the functions of c-oms. The cell transformed by a retrovirus oncogene divides incessantly. Might the homologous proto-oncogene therefore be a normal effector of cell division? Many (perhaps all) retrovirus oncogenes arrest, reverse, or otherwise disturb the normal course of cellular differentiation (Graf and Beug, 1978; Boettiger and Durban, 1979; Maltzman and Levine, 1981). Might their counterparts in normal cells be regulators of growth and development, and if so, might the lineages in which they are normally active dictate the sorts of cells that are vulnerable to transformation by the homologous viral oncogenes? Experimental data that speak to these issues are sparse and enigmatic.

1 . Because v-src transforms fibroblasts, it is conceivable that expression of the cellular homologue c-src might vary in concert with changes in cell growth. To date, efforts to sustain this expectation have failed. For example, the expression of c-src remained unchanged throughout the course of experiments in which the growth of fibroblasts was first arrested for as long as 2 weeks by serum deprivation and then stimulated by restoration of serum to the growth medium (Spector et al., 1978a). 2. Efforts to discern preferential expression of c-oms in specific tissues have so far failed to yield coherent results. Some loci (such as c-src, c-myc, and c-erb) are active at low or intermediate levels across a broad spectrum of tissues, whereas the activities of others (e.g., c-myb) are more restricted in their distribution (personal communications, T. Gonda and D. Sheiness). In most instances, the distribution of activity does not conform to predictions based on the pathogenicities of the corresponding oncogenes. The most provocative finding at present comes from Scolnick and his colleagues (Scolnick et al., 1981) : primitive hematopoietic cells (pluripotential CFU-S cells)-but not cells of other origins-contain large amounts of ~ 2 1 ' - ~ ~a' , protein whose viral homologue is apparently capable of inducing erythroleukemias as well as sarcomas. XIV. The Paradox of Neoplastic Transformation by Retrovirus Oncogenes

If retrovirus oncogenes embody functions found also in normal vertebrate cells, why do the oncogenes induce abnormal phenotypes in infected cells? Two possible answers come to mind. First, transformation by retroviruses may be a consequence of dosage: the virus may overload cells with otherwise normal gene products; sustained and abundant expression of the genes, rather than anomalous properties of their products, may lie at the root of

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tumorigenesis by v-oncs. Alternatively, viral oncogenes and c-oncs may differ in subtle but important ways. For example, the protein kinase activity of pp6Ov-"*' might have unique substrate specificities that could account for neoplastic transformation by Rous sarcoma virus. Although the extent of resemblance between c-oncs and viral oncogenes has yet to be fully measured, several points of evidence suggest that retrovirus oncogenes transform cells by means of dosage. 1. The dosages of v-onc products are indeed large, when compared with the amounts of c-onc products found in most cells ; fibroblasts transformed by Rous sarcoma virus contain about 100-fold more pp6OvpSrc than pp60""" (Collett et al., 1978; Oppermann et al., 1979), and similar differences have been found for other oncogene and c-onc products (Witte et al., 1979; Langbeheim et al., 1980). The vigor of oncogene expression may be attributable in large measure to the efficacy of the retrovirus promotet for transcription. 2. The relatively large dosages of oncogene products are essential to maintain the neoplastic phenotype. On occasion, presently unidentified events in the infected cell can attenuate the synthesis of retrovirus RNA and, hence, the expression of viral genes. The amounts of oncogene product fall by 10- to 100-fold,and the cell reverts to an ostensibly normal phenotype (MacPherson, 1965; Boettiger, 1974; Deng et al., 1977; Bishop et al., 1979; Prozig et al., 1979). 3. Molecular clones of two c-oncs (c-mos and w a s ) have been linked to transcriptional promoters from the murine leukemia virus genome (see earlier). The chimeric DNAs, bearing no portion of a viral oncogene, can transform fibroblasts to a neoplastic phenotype (Oskarsson et al., 1980; Blair et al., 1981; DeFeo et al., 1981; and personal communications, G. Vande Woude and E. Scolnick). Cells transformed in this manner by c-ras contain relatively large quantities of the gene product, p21'-'"" (DeFeo et al., 1981 ; and personal communication, E. Scolnick), thus sustaining the view that amplification of c-ras expression suffices to induce the neoplastic phenotype. XV. Does the Homology between Viral Oncogenes and c-oms Dictate the Host Range of Viral Transformation?

Neoplastic transformation by retrovirus oncogenes is remarkably specific : pathogenicity for specific tissues is a distinctive property of each strain of retrovirus ; and in cell culture, oncogenes display a similarly predictable and generally limited range of susceptiblecells that correlates well with the actions

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of the oncogene in infected animals (Graf and Beug, 1978). The origins of “target cell specificity,” as this phenomenon is known, are uncertain. It is first of all possible that transformation is merely a reflection of susceptibility to infection by different strains of virus. Inference speaks against this possibility: viral genes that determine the host range of infectivity (such as gag and enu) are shared among families of retroviruses whose spectra of oncogenicities are very different. Moreover, experimental data indicate that cells can be infected by and produce retroviruses bearing oncogenes without necessarily being transformed (Graf et af., 1980; Durban and Boettiger, 1981a,b). These findings have led to the more subtle suggestion that only certain cells are vulnerable to the effects of each oncogene. What factors could determine cellular vulnerability to transformation by v-oncs? We do not know, but it has been suggested that the kinship of viral oncogenes and c-oncs might be responsible : the deleterious effects of oncogene dosage might be restricted to cells in which the homologous c-onc is normally expressed and effective; alternatively, cells in which the c-onc is not usually active might be more vulnerable to the actions of the oncogene. The available (and admittedly provisional) data do not sustain these views. For example, the patterns of c-onc expression among different tissues in no way reflect the spectrum of susceptibility to the homologous viral oncogenes (personal communication, T. Gonda and D. Sheiness). XVI. Do c-oncs Provide a Pathway for Oncogenesis?

The action of viral oncogenes may provide useful analogues for the enzymatic mechanisms that give rise to and sustain the malignant phenotype. But it appears that viruses bearing oncogenes are not usually responsible for tumorigenesis in human beings (Pimentel, 1979). We should therefore look to the cell itself if we are ever to discern common origins of malignancy. In particular, we need to identify the events that spark the onset of oncogenesis, and we must determine whether a particular cellular gene (or set of genes) always mediates progression to and maintenance of the malignant phenotype. We do not know how oncogenesis originates; the matter has elicited great controversy, with some investigators arguing for mutations (Ames, 1979; Epstein and Swartz, 198l), others for chromosomal rearrangements, transpositions of DNA, or even reversible epigenetic events (Rubin, 1980; Cairns, 1981 ; Mintz and Fleischman, 1981). By contrast, the discovery of c-oms may have brought to view genes whose actions can mediate oncogenesis, once the cell has sustained an initiating lesion. We have diverse reasons to suspect the existence of such “cancer genes.”

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1. A number of malignancies have appeared as heritable traits in human pedigrees (Knudson, 1981), and it has even been suggested that each of the roughly 100 types of malignancy will eventually be attributable to abnormalities affecting a specific genetic locus (Knudson, 1979, 1981). 2. Several efforts have been made to enumerate the genetic loci that might mediate neoplastic transformation by chemical carcinogens. In most instances, the results implicate no more than a few dozen or a few hundred genes as potential mediators of chemical carcinogenesis (Parodi and Brambilla, 1977). 3. DNA extracted from some lines of chemically transformed cells and from certain tumors induces neoplastic transformation when transferred into cells in culture (Shih et al., 1979a; Krontiris and Cooper, 1981; Shilo and Weinberg, 1981). Caveats are necessary: the efficiency of transformation is generally quite low; transformation occurs reliably in only a few established lines of recipient cells (particularly mouse NIH-3T3); and only a limited number of transformed cell lines or tumors have so far yielded DNA capable of inducing neoplastic transformation. But the data to suggest that the DNA from at least some forms of neoplastic cells contains stable and heritable changes that are responsible for the malignant phenotype. Moreover, provisional studies with restriction endonucleases indicate that the same domain of DNA may be affected in independent tumors of common type and/or common cause (Shilo and Weinberg, 1981). 4. If first sheared to molecular weights of -0.3-3 x lo6, even DNA from normal cells can transform NIH-3T3 cells at a very low frequency, and the transformed mouse cells in turn yield DNA that can induce transformation at much higher efficiencies (Cooper et al., 1980)-as if the original shearing of normal DNA unleashed a potentially oncogenic gene whose action is now stably established in the transformed mouse cells. Activation of the gene has been attributed to disruption of linkage between the oncogenic gene and a cis-active regulator (Cooper et al., 1980). Are c-oncs among the cancer genes of normal cells? Is the induction of their activity responsible for at least some forms of oncogenesis? Answers to these question may come eventually from surveys of c-onc expression in naturally occurring tumors. At the present, we have a single but immensely provocative clue, derived from an unexpected source-the study of tumorigenesis by avian leukosis virus (ALV). ALV has no oncogene, yet infection of the bursa in young chicks by this virus gives rise to a fatal B-cell lymphoma (Gross, 1970). The course of tumorigenesis is protracted, unlike tumorigenesis by viral oncogenes, and most of the resulting tumors give evidence of having originated from single infected cells-they arise in individual follicles of the bursa (Cooper et id.,

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1978), and they contain proviruses whose sites of integration in cellular DNA are identical in every member of the tumor cell population (Payne et al., 1981a; Nee1 et al., 1981). In many instances, the proviruses are affected by deletions that preclude the expression of viral genes in the tumor cell (Payne et ul., 1981a); thus viral proteins are not required to sustain the lymphomatous phenotype. How then does viral infection induce the tumors? A possible solution to this puzzle began to unfold with the discovery that the leukosis virus DNA is almost always inserted in the vicinity of c-myc, and as a seeming consequence of these insertions, the expression of the c-one appears to have been greatly augmented (Hayward et al., 1981). The apparent activation of c-myc has been attributed to the influence of the nearby ALV provirus, or more specifically, to the promoter for RNA synthesis contained within the provirus. And indeed, in many instances, transcription may initiate within the provirus and then proceed through the c-myc locus, generating RNA that is a composite of nucleotide sequences derived from the provirus and from c-myc (Hayward et al., 1981). But two forms of exceptions have been found that cannot be easily explained (Payne et al., 1981b). In one, the ALV provirus is inserted upstream from c-myc, but the orientation of the provirus is the opposite of that required to promote transcription into the c-myc locus. In the other form of exception, the ALV provirus is located downstream from c-myc; transcription traverses c-myc in the usual direction and then continues into a portion of the provirus. It thus appears that some insertions of viral DNA in the vicinity of c-myc activate expression of the cellular locus by means other than providing a direct promoter for transcription. This nuance should not be allowed to obscure the general significance of the findings with ALV : oncogenesis by retroviruses may not always require viral gene products ; the frequency with which ALV proviruses are found in the vicinity of c-myc implies that the site of insertion figures in the oncogenic mechanism; and it is likely (but by no means proven) that the heightened expression of c-myc provokes and/or sustains the chain of events that eventuate in lymphoid leukosis. Questions remain, however. 1. In a few ALV-induced tumors, viral DNA is not inserted near c-myc, and expression of c-myc has not been induced (Hayward et a/., 1981; and personal communication, W. Hayward). Nevertheless, single (or very few) sites have been used for integration of the ALV provirus in each of these tumors, and it is therefore possible that the insertion of viral DNA has induced the expression of nearby cellular DNA representing a c-one. 2. Is the alleged tumorigenic effect of c-myc limited to cells in the B-lymphocyte lineage? Possibly not. For example, provisional data also implicate

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c-myc in the genesis of renal tumors induced by ALV (personal communication, P. Neiman). It is perhaps significant that these tumors are analogous to the renal carcinomas commonly induced by the action of v-myc (Moscovici et al., 1978). 3. Is c-myc an inevitable participant in the genesis of B-cell (and perhaps renal) tumors, whatever their initiating cause? The induction of c-myc expression has been implicated in lymphomagenesis by another retrovirus that lacks an oncogene, the chicken syncytial virus (CSV) (personal communication, H. J. Kung). Although the CSV genome is not homologous to that of ALV, the similarities of the two viruses are too great to provide a compelling test of the larger issue. 4. The viral oncogene (v-myc) derived from c-myc has never been reported to cause lymphomas (Moscovici et af., 1978). The implication of c-myc in the genesis of B-cell tumors therefore came as a surprise that remains unexplained. Recall, however, that the induction of lymphomas by ALV follows a protracted course of events that may not spring immediately from the effects of a single gene: the roles of c-myc and v-myc in oncogenesis may differ greatly. There is evidence to sustain this deduction (Cooper and Neiman, 1980). DNA from lymphoid tumors induced by ALV elicits the neoplastic phenotype when introduced into mouse fibroblasts by transfection. Contrary to expectations, however, the transformed mouse cells contain neither ALV DNA nor c-myc derived from the lymphoid tumors (personal communication, G. Cooper). Transformation of the fibroblasts must therefore be due to another cancer gene, activated in the infected B cells (perhaps by the effects of c-myc), and more effective than c-myc itself in the transformation of fibroblasts. In this scheme, c-myc can be viewed as the initiator of tumorigenesis, the cancer gene responsible for transformation of fibroblasts as a potential maintenance function. XVII. Conclusion: The Pursuit of Cancer Genes

It now appears likely that normal cells bear the seeds of their own destruction in the form of cancer genes, whose anomalous activities mediate tumorigenesis. The term cancer genes is a convenience, of course, and is viewed by some as a misnomer (Mintz and Fleischman, 1981): the loci in question may be physiologically essential constituents of the cell’s genetic apparatus that become pathogenic only when their structure or control is disturbed by oncogenic agents. The lines of inquiry surveyed in this article have engendered three strategies by which cancer genes might be identified and isolated.

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1. The role of retrovirus c-oncs in tumorigenesis can be explored by testing for their anomalous expression in spontaneously occurring tumors. There are uncertainties in this exercise. How is normal expression of c-oms to be defined? The cells from which many tumors arise are either poorly defined or too rare to be easily studied. Do we have the entire family of c-oncs in hand? It seems unlikely that we do, given the rate at which the repertoire of retrovirus oncogenes has expanded of late (Bishop, 1981), and the indications that even more v-oms can be found if the issue is forced by experimental ingenuity (Rapp and Todaro, 1978, 1980). And how certain can we be that the families of c-oncs and cancer genes are mutually inclusive? It is conceivable, for example, that some cancer genes will elude transduction by retroviruses, or if transduced, will fail to act as v-oms and thus go unrecognized. 2. The findings with ALV reveal for the first time how viruses devoid of oncogenes might cause tumors. The induction of c-onc expression by the integration of ALV DNA is a form of insertional mutagenesis, and other integrative viruses are in principle capable of the same. Potential examples abound: the nephroblastoma retrovirus of chickens (Watts and Smith, 1980); the thymic leukemia (Rowe, 1973) and mammary carcinoma (Bentvelzen and Hilgers, 1980) viruses of mice; the leukemia viruses of cats (Essex, 1975), cows (Burny et al., 1978), and gibbon apes (Gallo and Wong-Staal, 1980); and further afield, leukemogenesis by SV40 virus (Diamandopoulos, 1978), the hepatitis B virus that has been implicated in the genesis of hepatic carcinoma (Szmuness, 1978), and oncogenesis by herpes viruses (Biggs et al., 1972). If any of these sundry viruses acts by means of insertional mutagenesis, the site(s) of its integration in the DNA of the tumor cell may finger a cancer gene (or genes) whose effects are restricted to the tissue(s) in which the virus induces tumors. Virologists are in hot pursuit of these possibilities, hoping to expand the catalogue of cancer genes and to gain insight into the tissue specificity of their actions. 3. DNA-mediated transfection offers a third and versatile strategy whose reach may prove to be broader than the virological approaches just described. Any cancer gene whose action can be perceived in previously normal recipient cells becomes accessible to subsequent isolation by molecular cloning. Informal accounts indicate that first successes with this strategy are in the immediate offing. Success with any or all of these schemes will be heady business, no doubtcause for celebration and optimism. But daunting problems will remain. First, we are not likely to gain the full variety of cancer genes from the present strategies : none of the schemes seems geared to yield recessive

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effectors of oncogenesis, and none seems likely to unveil regulatory loci whose mutation might unleash the activity of an otherwise normal cancer gene. Second, we would be naive if we were to regard a single genetic locus as the full explanation for the genesis of a spontaneously occurring tumor. Most if not all human tumors appear to arise from several successive events (Prehn, 1976). Third, we must invent means by which to distinguish genes that initiate oncogenesis from those that maintain the neoplastic phenotype. The distinction is not necessarily inherent in any of the current strategies for identifying cancer genes (although the immediacy with which transformation follows DNA-mediated transfections appears to betoken maintenance rather than initiating functions). Fourth, the isolation of cancer genes offers no guarantee that we can deduce the mechanisms of their action. Here the lessons of tumor virology-which served so well to launch the search for cancer genes-may again provide seminal clues. Caveats such as these cannot blunt the zest with which cancer genes are now pursued, and they cannot detract from the object lessons the pursuit has produced. The study of viral agents far removed from human concerns appears to have wrought the long-elusive conjunction between tumor virology and human oncology. The issue is not whether viruses might cause human tumors (as perhaps they may), but rather how much viral oncology can teach us of the mechanisms by which human tumors arise. ACKNOWLEDGMENTS 1 thank B. Cook for assistance and R. Swanstrom for conceiving and preparing Fig. 2. Portions of this article will also appear as part of a chapter prepared for a book on RNA tumor viruses, to be published by the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Work in my laboratory is supported by grants from the National Cancer Institute and the American Cancer Society.

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Neel, B. G., Hayward, W. S., Robinson, H. L., Fank, J., and Astrin, S. M. (1981). Cell 23, 323-334. Neil, J. C., Ghysdael, J., and Vogt, P. K. (1981). Virology 109,223-228. 018, A., Linemeyer, D., Ruscetti, S., Lowe, R., Lowy, D. R., and Scolnick, E. (1980). J . Virol 35,924-936. Oppermann, H., Levinson, A. D., Varmus, H. E., Levintow, L., and Bishop, J. M. (1979). Proc. Natl. Acad. Sci. U.S.A. 76, 1804- 1808. Oppermann, H., Levinson, A. D., and Varmus, H. E. (1981). Virology 108,47-70. Oskarsson, M., McClements, W. L., Blair, D. G., Maizel, J. V., and Vande Woude, G. F. (1980). Science 207, 1222-1224. Padgett, T. G., Stubblefield, E., and Varmus, H. E. (1977). Cell 10,649-657. Parker, R. C., Varmus, H . E., and Bishop, J. M. (1981). Proc. Natl. Acad. Sci. U.S.A.,in press. Parodi, S., and Brambilla, G. (1977). Mutat. Res. 47, 53-74. Payne, G. S., Courtneidge, S. A., Crittenden, L. B., Fadley, A. M., Bishop, J. M., and Varmus, H . E . (1981a). Cell23,311-322. Payne, G . S., Bishop, J. M., and Varmus, H. E. (1981b). Nature (London) 295,209-213. Pimentel, F. (1979). Biochim. Biophys. Acta 560, 1669-216. Prozig, K. J., Robbins, K. C., and Aaronson, S . A. (1979). Cell 16,875-884. Prehn, R. T. (1976). Adu. Cancer Res. 23,203-236. Rapp, U. R., and Todaro, G . J. (1978). Science 201,821-823. Rapp, U . R.,and Todaro, G . J. (1980). Proc. Natl. Acad. Sci. U S A . 77,624-628. Rasheed, S . , Gardner, M. R.,and Huebner, R. J. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 2972-2976. Reynolds, F. R., Jr., Van de Ven, W. J. M., and Stephenson, J. R. (1980). J . Biol. Chem. 255, 11040-11048. Robinson, H. L. (1978). Curr. Top. Microbiol. Immunol. 83, 1-36. Rohrschneider, L. R., Eisenman, R. N., and Leitch, G . R. (1979). Proc. Natl. Acad. Sci. U.S.A. 76,4479-4483. Rous, P. (1911). J. Exp. Med. 13,397-411. Roussel, M., Saule, S., Lagrou, C., Rommens, C., Beug, H., Graf, T., and Stehelin, D. (1979). Nature (London) 281,452-455. Rowe, W. P. (1973). Cancer Res. 33,3061-3068. Rubin, C. S., and Rosen, 0. M. (1975). Annu. Rev. Biochem. 44,831-887. Rubin, H. (1980). JNCI J. Natl. Cancer Insr. 64,995-1000. Scolnick, E. M., andParks, W. P. (1974). J. Virol. 13, 1211-1219. Scolnick, E. M., Rands, E., Williams, D., and Parks, W. P. (1973). J . Virol. 12,458-463. Scolnick, E. M., Howk, R. S., Anisowicz, A., Peebles, P. T., Scher, C. D., and Parks, W. P. (1975). Proc. Natl. Acad. Sci. U.S.A. 72,4650-4654. Scolnick, E. M., Papageorge, A. G., and Shih, T. Y.(1979). Proc. Natl. Acad. Sci. U.S.A. 76, 5355-5359. Scolnick, E. M., Weeks, M. O., Shih, T. Y.,Ruscetti, S. K., and Dexter, T. M. (1981). Mol. Cell. Biol. 1,66-74. Sefton, B. M., Hunter, T., and Beemon, K. (1980). Proc. Natl. Acad. Sci.U.S.A.77,2059-2063. Sefton, B. M., Hunter, T., Ball, E. H., and Singer, S. J. (1981). Cell 24, 165-174. Shalloway, D., Zelenetz, A. D., and Cooper, G. M. (1981). Ce1124, 531-542. Sheiness, D., and Bishop, J. M. (1979). J. Virol. 31, 514-521. Sheiness, D. K., Hughes, S. H., Varmus, H. E., Stubblefield, E., and Bishop, J. M. (1980). Virology 105,415-424. Sheiness, D., Vennstrom, D., and Bishop, J. M. (1981). Cell 23, 291-300. Shibuya, M., Hanafusa, T., Hanafusa, H., and Stephenson, J. R. (1980). Proc. Null. Acad. Sci. U.S.A. 77,6536-6540.

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CANCER, GENES, AND DEVELOPMENT: THE Drosophila CASE Elisabeth Gateff Biologisches lnstitut I (Zoologic), Albert-Ludwigs Universitat. Freiburg, Federal Republic of Germany

1. Introduction .

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

11. Drosophilu De .............................................. 111. General Information on the Drosophilu Tumor Mutants . . .

IV. Description of the Drosophilu Tumor Mutants . . . . . A. Embryonic Tumor Mutants .... B . Larval Tumor Mutants ...................... V. Viruses found in Drosophilu Tumor Cells . . . . . . . . . . ........... V1. Retroviral Oncogenes and Their Cellular Counterparts in Different Animal Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V11. Transfection and Vertebrate Tumor Genes: A Comparison with Drosophilu . . . . . VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 34

62 66

I. Introduction

At the outset of this article I wish to express my strong conviction, based on my experience with Drosophila tumors of genetic origin (Gateff, 1978a,b,c), that in all instances cancer has a genetic base and that cancer is a problem of cell differentiation and the maintenance of this state. In Drosophila, 25 of a total of 5000 genes have been found to cause malignant or benign neoplasms in the mutated state. These genes play apparently crucial roles in the differentiation of specific cell types and tissues. Thus in order to understand the primary event(s) leading to malignant neoplastic transformation, we will have to gain insight into the genetics of normal development. The recent revival of interest in genetic factors as causative agents in cancer has been stimulated by findings that on the genome of a wide variety of vertebrates, including humans, homologous sequences to at least a dozen retroviral oncogenes exist (Bishop, 1981;Wong-Staal et al., 1981;Vennstrom and Bishop, 1982; Barnekow et al., 1982). Shilo and Weinberg (1981b) detected on the Drosophila genome, sequences homologous to five acute RNA tumor virus oncogenes. Moreover, evidence that the genomes of different mammals contain genes capable of transforming NIH-3T3 mouse cells stems from transfection studies with DNA from spontaneously, virally, and chemically transformed cells (Shih et al., 1979), and DNA from normal cells (Cooper et al., 1980). 33 Copyright Q 1982 by Academlc Press, Inc. ADVANCES 1N CANCER RESEARCH, VOL. 37

All rights of reproduction in any form reserved. ISBN 0-12-006637-8

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ELlSABETH GATEFF

The genetic base for the melanomas and other malignant tumors in platyfish/swordtail hybrids are by now well established (Anders and Anders, 1978). Finally, it has been known for many years that certain human cancers are associated with gene mutations (Knudson, 1978; Knudson and Meadows, 1978; Lynch, 1976). Based on all these findings, Bishop (1981) proposed that on the genome of vertebrates a family of genes exists whose members control specific differentiation steps during development. However, when mutated or abnormally regulated, these genes lead to malignant neoplastic transformation. The Drosophilu tumor system has shown exactly this for quite some time (Gateff and Schneiderman, 1967, 1969; Gateff, 1978a,b,c). In this system, genes responsible for the proper differentiation of a specific cell type, when mutated, give rise to benign or malignant neoplasms. In the light of the aforementioned exciting developments, it may well turn out that Drosophilu will not remain just a “case” but will help to unravel the function of these developmental genes during normal development and in malignant neoplastic transformation. Maybe the unifying principle in malignant neoplastic transformation can be shown to be a gene family with important, basic functions during development, which for that reason has been conserved throughout evolution in all animal systems from humans down to the sponges. This article describes the different benign and malignant neoplasms found in 25 Drosophilu mutants and discusses these mutants in the light of the new findings just mentioned. II. Drosophila Development

Drosophila development involves one embryonic, three larval, one pupal, and one adult stage. Except for the early part of embryonic life, at all other developmental stages two cell systems exist: (1) terminally differentiated cells incapable of dividing and (2) dividing cells. In view of the well-established fact that only dividing cells express and propagate the neoplastic state, this implies that only the dividing cells can be expected to grow as tumors at any developmental stage. During the first part of embryonic life, all cells divide and thus can all become neoplastically transformed. Thereafter, the majority of cells stop dividing and differentiate into larval structures. The larva grows after hatching exclusively by cell enlargement, polytenization, and polyploidization ; as a result, in larval cells no tumorous growth can ensue. Tumors originating from larval cells proper can be expected during the first half of embryonic life, when the cells determined for larval cell lineages are still dividing.

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In the embryo, in addition to the large majority of differentiating larval cells, small nests of cells are set aside that remain diploid and capable of dividing. These nests of cells represent the primordia for the adult integument, the so-called imaginal discs, the presumptive adult optic centers of the fly in the larval brain, the primordial gonial cells in the female and male gonads, and the blood cells. From this it follows that in the larva, tumorous growth can develop only in the adult primordia as just listed. During adult development in the pupa, oogonia, and spermatogonia, myoblasts, blood cells, and a variety of primordial imaginal cells-from which the various adult organs arise-divide and are candidates for neoplastic transformation. Finally, in the adult only gonial cells and blood cells divide, thus they can be expected to become neoplastic. With this background in mind I will proceed to describe the various tumor mutants. 111. General Information on the Drosophila Tumor Mutants

At present 25 tumor mutants located on three of the four Drosophila chromosomes are known (Table I). They originated either spontaneously or after mutagenesis with ethyl methane sulfonate (Gateff, 1978a,b,c); the latter are indicated in Table I by an asterisk. All mutants are recessive and, with the exception of the gonial cell mutants, lethal. The lethality falls either at the end of embryonic life or at the time of the larval-pupal transformation. Apart from Notch*, which represents a deficiency, all remaining mutants are considered single gene mutations by classical genetic criteria. Future gene cloning and sequencing, however, may show small deletions or insertions of transposable elements within one or the other gene. The individual lethal mutant alleles are propagated in the heterozygous balanced state (for the genetic composition of the balancer chromosomes, see Lindsley and Grell, 1968), whereas the homozygous or hemizygous animals express the tumorous condition. The strains breed through and show full penetrance of the tumorous condition (see later). Depending on the developmental stage at which the tumor becomes obvious, embryonic, larval, and adult tumors can be distinguished (see earlier). Based on the cell type(s) or the tissue(s) affected by the mutation, we recognize in the embryo mixed tumors; in the larva, malignant neuroblastomas, intermediate imaginal disc neoplasms with a compact and invasive mode of growth, and malignant blood cell neoplasms; and in the adult, benign gonial cell neoplasms (Table I). In all mutants, tumorous growth interrupts normal differentiation at a specific developmental stage of a particular cell type(s), which results in unrestrained, mostly lethal neoplastic growth (apart from gonial cell neoplasms; Table I). The following criteria

36

ELISABETH CATEFF

TABLE I Drosophila melanogasier MUTANTS SHOW~NG MALIGNANT AND BENIGN NEOPLASMS Tumor mutants on chromosome number Designation of neoplasm Embryonic tumors Malignant neuroblastorna

Intermediate imaginal disc neoplasm with compact lethal mode of growth

Intermediate imaginal disc neoplasm with invasive, lethal mode of growth Malignant blood cell neoplasm

Benign gonial cell neop1asm

I

I1

DJ"(l)Not~h.1-3.0"; shibire'"- 1-52.2' lerhal( 1 )2,-'*" ; lerhal(l)2269,-'."

111

',

Fourteen alleles of lethal(2) giant larvae; Df(2)lgl net, 2-00'.''; lerhal(2)1542,-c~" lethal(1)2,-'*"; Fourteen alleles of leiha/(I )2269,-'1° ; lethal(2)giantlarvae lethal(1)disc large-1, (two o f them ts'; 1-36'."; lerhal(1)benign Df(2)lgl net, 2-OOj; wing imaginal disc lethal(2)giant neoplasm, 1-34'; disc,-'.'; lerhal lerhal(l)lpr-2, (2)1542,-'.' 1-36.2'*"; shl'"', 1-52.2" lerhal(1)disc large-I, 1-36dJ. lerhal(1)benign imaginal disc neoplasm, 1-34"; lerhal(1)disc. large-2, 1-24.9" lethal( I)malignant lethal(2)malignant blood neoplasm, 1-39"; blood neoplasm,-'*" tumorous-lethal, 1- 34.5/."

benign(2)gonial cell Two alleles of./emale neoplasm, between sterile(l)231,in D f BI 38L-B170RB;,female 13.0 and 48.5'; sterile(l)l621, 1-1 1.7h; female srerile(2) fused, 1-59.5' of Bridges, 2-5" ; ( I 0 alleles) 5 alleles of narrow, 2-83"

lethal(3)giant larvae,-'*"; lethal(3)brain tumoJE-I,-c~" lethal(3)giant larvae,-'*"; lelhal(3)brain tumor's,-c*o

lethal(3)malignant blood neoplasmI , between h-th"; lethal(3)malignanr blood neopIasm-2.-'~" -

' Mohr (1919); Gateff and Schneiderman (1974); * Grigliatti et al. (1973); MacMorrisSwansonandPoodry(l981); Williams(i981);'-, notlocated;d Kisseral. (1978);'Stewart eta/. (1972); Convin and Hanratty (1976); Gans er al. (1975); King (1979); Gollin and King (1981); King er al. (1957); 12 I(2)gl alleles and Df(2) Iglnet Golubovsky and Sokolova (1973); Gateff eral. (1977); Ir Bryant and Schubiger (1971); ' W. P. Hanratty(persona1 communication); Koch andKing(1964);" Kingand Bodenstein(1965)." Mutantsisolated byGateff(l978a,b,c).

'

J

CANCER, GENES, AND DEVELOPMENT

37

were applied to characterize the neoplasms: (1) autonomous and lethal growth in situ as well as after transplantation, (2) loss of the capacity for differentiation, (3) invasiveness, and (4) loss of structure. In some cases the capacity to grow in vitro was used as an additional criterion (Gateff, 1978a). The following section deals with the various tumor mutants.

IV. Description of the Drosophila Tumor Mutants

A. EMBRYONIC TUMORMUTANTS A systematic search for embryonic lethal mutants causing tumorous growth has not yet been undertaken. However, two X-linked mutants exist that show malignant neoplastic growth. These are the Notch' and shibire'"' (shi'"') mutants (Table I). The Notch' mutant, known since 1919, affects the wing morphology in the heterozygous state. In the hemizygous male embryo the ventral ectoderm fails to differentiate (Poulson, 1937, 1940, 1945; for normal embryonic development, see Poulson, 1950; Fullilove and Jacobson, 1978). Instead, neuroblasts are formed exclusively, which differentiate into nervous structures. This mode of growth is nonautonomous when tested in the wild type (Gehring, 1973). However, some implants derived from the anterior portion of older Notch' embryos yielded malignant neoplastic growth after implantation and growth in wild-type female adults (Gateff and Schneiderman, 1974). The lethal growth was propagated for 14 transfer generations before being discontinued. The transplantable tumor consisted of a variety of cells that resembled neuroblasts, glial cells, ganglion-mother cells, and blood cells. Thus it appears that the Notch' tumor originated from undifferentiated cells belonging to two germ layers, the ectoderm (neuroblasts, glial cells) and the mesoderm (blood cells). Lehmann et al. (1981) have studied further mutants that affect early neurogenesis and exhibit phenotypes similar to that of Notch'. It remains to be shown whether they too exhibit tumorous growth. The interest in this locus has extended into the molecular realm. S. Artavanis-Tsakanas (personal communication) has obtained DNA sequences that appear to derive from the Notch' locus. However, the final proof is not yet available. The second embryonic mutant shP' is well known for its various effects on embryonic and larval development (Poodry et al., 1973). Recently, MacMorris-Swanson and Poodry (1 98 l), demonstrated that heat pulses of 4 to 6 hr given between 3.5 and 14 hr of embryonic development resulted in

FIG. 1. Comparisons of wild-type and mutant whole-mount and histological preparations. (a) Whole mounts of fully grown third instar wild-type (A) and l(2)g/4(B) larvae. Note the bloated and transparent appearance of the mutant larva and the region of the tumorous brain and imaginal discs located between the two arrows. (b) Whole mounts of mature wild-type (A) and /(2)gI4 (B) brain (b) and ventral ganglion (vg) with associated imaginal discs (i). Compare the shape of the wild-type imaginal discs with the clumped and shapeless tumorous l(2)ql4 imaginal discs; note further the enlarged and deformed /(2)gp brain hemispheres and the smaller, round wild-type brain hemispheres. (c) Frontal histological section through the two wild-type brain hemispheres (b) and the ventral ganglion (vg). The black lines show the border between the larval portion of the brain (I) and the developing presumptive adult optic centers (aoc). Within the adult optic centers one recognizes the inner and outer formation centers (ifc, ofc), made up of large neuroblasts, and in between the developing outer, middle, and

CANCER, GENES, AND DEVELOPMENT

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disorganized growth and lack of differentiation in the embryo. The heattreated lethal shi‘“’ embryos represent masses of cells that, after transplantation into wild-type female flies, grow into invasive, lethal tumors. The tumor has been subcultured for several transfer generations. However, it is not yet determined which cell types are transformed. Nevertheless, the shP’ mutant shows that between 3.5 and 14 hr of embryonic development the gene is active and responsible for molecular events that apparently have an impact on the differentiation and morphogenesis of one or more cell types or tissues in the embryo. It further shows that the shitsl gene product is, if made at all, irreversibly damaged at the restrictive temperature.

B. LARVAL TUMORMUTANTS

1. Mutants Developing Madignant Neuroblastomas Table I shows seven mutants that, during the last larval instar, develop malignant neuroblastomas in the presumptive adult optic centers of the larval brain. As a result the animals die either before or at the larval-pupal transformation. The mutant larvae can easily be recognized by their larger size and their more or less transparent bodies (Fig. la). Anatomical investigations show an overgrown central nervous system and, in some of the mutants, tumorous imaginal discs as well (Fig. 1 b). The wild-type larval central nervous system consists of two brain hemispheres and a ventral ganglion (Fig. 1b). Associated with the central nervous system are three pairs of imaginal discs, the eye antennae, and the first and second leg discs (Fig. 1b,A). It was mentioned earlier (Section 11) that in the embryo two developmental systems become determined : the larval,

inner optic glomeruli (og, mg, ig), indicated by their respective neuropiles (n) (arrows). f. Fat body; in, integument; 0, oesophagus; rg, ring gland; s, salivary gland. (d) Frontal histological section through the two brain hemispheres (b) and the ventral ganglion (vg) of a l(Z)g14 larva. The brain hemispheres are enlarged, deformed, and have lost the wild-type organization. Tumorous optic neuroblasts (nb) and ganglion-mother cells (gm)are seen in abnormal array. They have invaded the larval portion of the brain where remnants of the neuropile (n) can be recognized. The ventral ganglion, in contrast, shows normal arrangement of the neuropile (n) and the cellular cortex. f, Fat body; s, salivary gland. (e) Wild-type host in the premortal stage which had been injected with a small piece of 4 2 ) g P brain (A). Compare the “bloated phenotype” with the phenotype of the noninjected wild-type control fly (B). (f) Histological cross section through the abdomen of a tumor-bearing wild-type host fly. The tumorous neuroblasts and ganglionmother cells (arrows) have invaded and destroyed the ovaries and all other organs except for the gut (9). The tumorous growth is opposing closely the integument (in). All histological preparations 5 pm; Hematoxylin and eosin.

40

ELlSABETH GATEFF

which differentiates; and the presumptive adult, which remains undifferentiated until later during adult development in the pupa, when it forms the adult structures. Thus in the embryo, 13-20 large neuroblasts, located in the posterolateral region of each brain hemisphere, are destined to give rise to the adult optic centers (Gateff, 1978a; Hofbauer, 1979). After the hatching of the larva from the egg, the optic neuroblasts divide equally, giving rise to new neuroblasts. During the second and third larval instars they divide unequally, producing more of their own, and in addition, small optic ganglion-mother cells. The ganglion-mother cells divide an undetermined number of times before differentiating into optic neurons. (For the development of the adult optic centers in the larval brain, see Hofbauer, 1979; and for the entire nervous system see Kankel er a/., 1980.) At the end of larval life, in the posterolateral region of each brain hemisphere one finds the presumptive adult optic centers showing the inner and outer formation centers, consisting of large neuroblasts, and in between the anlagen of the developing outer, middle, and inner optic glomeruli (Fig. lc). After pupariation and during adult development, they differentiate into the optic centers of the fly. In the mutants, this differentiation process is interrupted at the stage of the optic ganglion-mother cell. The inability of this cell type to differentiate into optic neurons causes a perpetual growth and an accumulation of neuroblasts and ganglion-mother cells. As a result the mutant brains are 1.5 to 2 times larger than their wild-type counterparts (Fig. l b ; compare Fig. lc and d). They show little normal organization (Fig. Id). The mutant neuroblasts and ganglion-mother cells invade the cellular cortex and the neuropile of the larval brain portions and destroy their organization. Because in all mutants the brain tumors originate from optic neuroblasts, they are classified as neuroblastomas. Affected by the invasive growth of the optic neuroblasts and ganglionmother cells are also the neurosecretory cells and their axons. The interrupted neurosecretory axonal pathways cause the accumulation of large amounts of neurosecretory granules in the cells (Akai, 1972). Thus the target cells for the neurohormone, e.g., the prothoracic gland cells in the ring gland, receive no information as to when to release ecdysone, which initiates puparium formation. For this reason in all neuroblastoma mutants, puparium formation is either blocked or delayed (Gateff et al., 1977). The tumors developing in situ in the larval brain show typical characteristics of malignant neoplasms, such as continuous fast growth, lack of differentiation, invasiveness, and lethality to the host. This malignant mode of growth is also observed after transplantation. When small pieces of mutant brains are implanted into the body cavity of wild-type female adult flies they behave autonomously, and grow in 7-14 days in an invasive, lethal

CANCER, GENES, AND DEVELOPMENT

41

way. Figure le compares a tumor-bearing host in the premortal stage exhibiting the typical “bloated syndrome” to an uninjected control. Histological preparations of a similar host show that the tumorous cells have invaded and destroyed most of the host tissues (Fig. If). All these findings show that the described tumor type represents a malignant neuroblastoma. The most thoroughly studied of all the seven mutants listed in Table I is the fetlzal(2)giant larvae (l(2)gl) mutant. More than 78 l(2)gl alleles are known at the present time. Four of these have originated spontaneously or were induced in laboratory strains (Gateff and Schneiderman, 1969; Lindsley and Grell, 1968). The discovery of 78 independent alleles in isolated natural populations from the Soviet Union (Golubovsky and Sokolova, 1973; Golubovsky, 1978) demonstrate that the Drosophila tumors are not solely a laboratory phenomenon. Furthermore, the occurrence of a lethal giant larvae mutation in Drosophila hydei (Z. Srdid and H. Gloor, personal communication; SrdiC and Frei, 1980) indicates that on the genomes of other Drosophila species, and maybe also other dipteran species, homologous genes may exist that are instrumental in the differentiation of the optic neuroblast and that in the mutated state cause malignant neuroblastomas. The expression of the mutant phenotype in 14 l(2)gfalleles and a deficiency were studied by Gateff et al. (1977). This study showed large variations in the expression of the neuroblastoma phenotype, and thus of the severity of the tumorous condition. In one of the alleles no neuroblastomas at all were detected, as revealed by the size of the brain and the inability of brain pieces to grow in a neoplastic way in wild-type adult hosts. In the remaining alleles the growth of the adult optic neuroblasts in the wild-type adult environment varied from extremely fast to slow. The time of action of the l(2)gl and f(2)g14 alleles falls around the middle of embryonic life (Gateff and Schneiderman, 1974). Work with temperaturesensitive (ts) 1(2)91alleles confirmed this finding and established, in addition, a second ts period during larval life (Sokolova and Golubovsky, 1978; T. L. Gough, personal communication). Conditional mutants have been of great value for the study of the time of gene activity and the mode of action of the gene product. A total of four ts tumor mutant alleles have been found : (1) the already discussed embryonic tumor mutant shitsl, (2) two l(2)gl alleles (W. P. Hanratty and P. Adler, personal communication), and (3) a new ts tumor mutant causing malignant neuroblastomas and imaginal disc tumors (E. Gateff, unpublished). The two ts l(2)gl alleles will be discussed in Section IV,B,2 because they cause only imaginal disc tumors. The new ts tumor mutant lethal(3)malignant brain tumor‘”-l [l(3)rnbtfS-1 ; see Table I] is located on the third chromosome. At the permissive temperature (22”C), all animals show wild-type development (Fig. 2A, B, and C). Shift-up to the restrictive temperature (29°C) at the

42

ELISABETH GATEFF

onset of development results in brain and imaginal disc tumor in 100% of the animals, and their death at the time of the larval-pupal transformation (Fig. 2A). Shifts to the restrictive temperature during larval life showed deleterious effects on the imaginal disc and the fertility, but no tumorous growth was encountered (Fig. 2a). Thus the ts period for tumor development falls during embryonic life. To pinpoint this period, a series of shift-up experiments were performed at 2-hr intervals during embryonic life (Fig. 2B). They showed that 2-, 4-,6-, 8-, lo-, and 12-hr-old embryos, when shifted to the restrictive temperature and kept there, develop malignant neuroblastomas at the end of larval life (Fig. 2B). Embryos shifted to the restrictive temperature when 14 to 18 hr old develop into larvae, some of which pupariate but die as prepupae caused by the damage to the imaginal discs. Still older embryos treated the same way either die as larvae, prepupae, or pupae. Similar results were obtained in shift-up experiments during the first and second larval instars. Temperature treatment of late, third instar larvae has no influence on adult development, but renders all adults sterile. The sterility

I

Adult anlmala sterlle -t-----,

/I

22°C

I

I

1

Ji

II

2

4

I

I

/+ x----x Animals die as larvae or prepupae X- / h l m a l s die as larvae prepupae or Pupae

/I

--//

22°C

29oc hr 220c AELO

t I t t t t t t t t ! ! ! /'Z&'J' rF1 6

8

10

12

14

16

18

20

22

24

26

28

In all cases normal adult development

162 Pupariation

FIG.2. Shift-up (arrow u p ; A and B) and shift-down (arrow down; C) experiments, performed with the ts mutant lerhal(3)brain tumofs--I (43)bfs-1).In the shift-up experiments (A and B), adults were allowed to lay eggs for 2 hr at the permissive temperature (22'C), after which the adults were removed. The eggs and larvae, respectively, were shifted to the restrictive temperature a t the indicated hours after egg laying (hr AEL). In the shift-down experiments (C), eggs were collected as in A and B, at 2 hr intervals at the restrictive temperature, and were brought down to the permissive temperature at various hr AEL. (For further details see text.)

CANCER, GENES, AND DEVELOPMENT

43

is caused by tumorous egg chambers (see Section IV,C) and arrest of spermatide differentiation. The determination for tumor development thus falls between 0 and 12 hr of embryonic development. In order to test whether the gene product was made at the restrictive temperature, a series of shift-down experiments were performed during embryonic and larval life (Fig. 2C). These experiments showed that in all shiftdown experiments the mutant effect was abolished, which suggests that at the restrictive temperature the gene product was made, but was not functional. However, at the permissive temperature, the gene product regains its function and normal development ensues. The foregoing experiments allow the following conclusions. (1) The 1(3)mbt"-I gene product acts in various cell types at different developmental stages in a specific manner. (2) The determination for tumor development takes place during the first 12 hr of embryonic life. (3) At the permissive temperature (22"C), the wild-type 1(3)mblr"-1allele synthesizes the gene product during the first 12 hr of embryonic life in the adult optic neuroblasts and the presumptive imaginal disc cells (see Section IV,B,2). (4) At the restrictive temperature (29"C), the 1(3)mbtrS-1gene product is also synthesized, but it is nonfunctional, possibly because of a change in its tertiary structure. (5) The nonfunctional gene product can be rendered functional again by a shift-down to the permissive temperature (22°C) at any time during larval development. (5) The shift-up and shift-down experiments show further that for normal development to proceed, the functional gene product synthesized during the early part of embryonic life has to be present throughout the second part of embryonic and most of larval life. 2. Mutants Developing Imaginal Disc Tumors Twelve imaginal disc tumor mutants have been isolated (Table I). All tumors show intermediate qualities between truly benign and malignant neoplasms. Most of them grow in a compact, lethal manner (Fig. 3c); however, a small number also show invasiveness in situ (Fig. 3d; Table I). The imaginal discs are the primordia for the different parts of the adult integument, such as the head or the thorax with all its appendages. They become determined quite early in the embryo as small groups of 10-50 cells located in distinct regions of the blastoderm. Throughout embryonic life they remain mitotically quiescent and begin to grow only after the larva hatches from the egg. The imaginal discs attain their final size and shape at the end of larval life and differentiate during adult development in the pupa into different adult integumental structures: For further details on wildtype imaginal disc development and pattern formation, consult Gehring (1978), Bryant (1978), and Poodry (1980).

FIG. 3. Whole-mount, histological, and ultrastructural preparations of wild-type and tumorous imaginal discs from mature third instar larvae. (a) Whole mount of wild-type wing (wid), third leg (lid), and haltere imaginal disc (hid), connected to the trachea, t (A) and the /(/)d.ly-2counterparts. which are clumped and fused (B). (b) Wild-type wing imaginal disc showing a folded epithelium and the cells arranged in monolayers. (c) I(l)bwn wing imaginal disc lacking the typical folding pattern of the wild-type wing imaginal discs (compare with b). Instead, the tissue is made u p of regions consisting of small folds and regions with compact cellular arrangement (arrow). (d) /(l)bw,i brain (b)and ventral ganglion complex (vg), showing regions of imaginal disc (i) invasion (arrows) into the neuropile (n) and the cellular cortex. (e) Cross section through the abdomen of a wild-type host fly bearing a compactly growing tumor (midn, arrow), derived from an implanted piece of a l(Z)y/4 imaginal disc. The ovaries

CANCER, GENES, AND DEVELOPMENT

45

The tumorous pattern of growth of the mutant imaginal discs manifests itself during the last larval instar. The mutant imaginal discs are made up of abnormally shaped clumps of cells. (Compare in Fig. 3a the wild-type with the mutant imaginal discs.) Their histology exhibits drastic abnormalities from that of the wild type (Fig. 3b and c). The wild-type imaginal disc epithelium consists of a folded monolayer of columnar cells (Fig. 3b). The apical secretory surfaces of the elongated cells exhibit microvilli (Fig. 30, and the basal surfaces form a basal lamina. Complex junctional complexes connect the cells laterally to each other (Poodry, 1980). The mutant imaginal disc epithelium shows very little of the aforementioned organization. The cells either are clustered or form small folds (Fig. 3c), and show less contacts among each other (Fig. 3g). In some of the imaginal disc tumor mutants the imaginal discs invade the larval central nervous system, where they cause the destruction of the cellular cortex and the neuropile (Table I ; Fig. 3d; Gateff, 1978b,c). A main characteristic of malignant growth in general is the capacity of the tumorous cells to grow autonomously after transplantation into a wild-type host. Small pieces of mutant imaginal discs injected into the body cavity of wild-type flies grow in a fast autonomous lethal fashion. All tumors attain a ball-like shape after 7-14 days of growth., and consist to a large extent of cells in clustered arrangements (see previous discussion; Fig. 3e). Such tumorous imaginal disc tissue sublines grow in the adult for numerous transfer generations. In contrast, pieces of wild-type imaginal discs grow in a regulated way and never cause the death of the adult host. A further property of tumorous imaginal discs is their incapacity to differentiate into cuticular patterns when exposed to the hormones at metamorphosis in a wild-type larva that is ready to pupariate. Here they respond to ecdysone by cessation of cell division; however, none of the complex processes of chitin biosynthesis or the secretion of a cuticular pattern ensues. On the contrary, pieces of wild-type imaginal discs differentiate inevitably into disc-specific cuticular patterns. Therefore, the lack of the capacity to differentiate and the disordered, lethal growth in situ as well as after transplantation into a wild-type host allow the classification of the imaginal disc tumors as lethal benign or lethal invasive tumors (Gateff, 1978a,b,c). (ov) on the tumor side are destroyed. ib,Fat body; g, gut; int, integument. ( f ) Fine-structure preparation of a few wild-type imaginal disc cells (idc) in close lateral contact with each other, showing toward the lumen (I) an apical region with microvilli (mv). Within the cytoplasm, vacuoles (v) and low-opacity droplets (Id)can be observed. In one of the nuclei a few virus-like particles (vlp) are seen. (g) Electron micrograph of portion of an /(2)gI4 imaginal disc, showing in the lumen (I) the tumorous cells (mid) with numerous cytoplasmic processes (cp) and invading blood cells (bc). In the upper left are peripodial membrane-like cells (pml). All histological preparations 5 pm thick and stained with hematoxylin and eosin. The ultrastructure preparations, uranylacetate-osmium tetroxide. (Courtesy of Dr. H. Akai).

46

ELlSABETH GATEFF

The oldest and most thoroughly studied of the eight mutants is the 1(2)gl mutant, with its more than 78 alleles and a deficiency. The 1(2)91and 1(2)g14 alleles were the first to be investigated for their neoplastically growing imaginal discs (Gateff and Schneiderman, 1969, 1974). Of the more than 78 alleles found by Golubovsky and Sokolova (1973) and Golubovsky (1978), Gateff et al. (1977) compared the morphology and developmental capacities of 14 alleles and the deficiency. Just as in the case of the neuroblastoma (see Section IV,B, I), so for the imaginal disc tumors found in the different 1(2)91 alleles, large variations in size, morphology, and the degree of malignancy were found. In some alleles the imaginal discs were very large and proved highly malignant in the transplantation test. In other alleles they were smaller or not present at all. This variation in the expression of the tumorous phenotype in the different alleles seems to point to the possibility that the 1(2)91 locus is a complex one. It may be that most alleles are smaller or larger deletions. Thus, depending on the number of deleted gene sequences, a more or less extreme tumor phenotype may ensue. Gateff and Schneiderman (1969, 1974) determined in transplantation experiments the time of the f(2)yP gene activity around the tenth hour of embryonic life. Anterior portions of l(2)g14 embryos aged 10 hr and older, devoid of the brain-ventral ganglion complex, when implanted into the body cavity of wild-type flies grew tumorous imaginal discs. Corresponding anterior integumental portions from wild-type control embryos showed normal imaginal disc growth. With the help of a ts allele, 1(2)91'"', Hanrathy and Gough (1982) demonstrated that the ts period for the differentiation of cuticular structures extends from larval hatching to the early third instar. In addition, the data suggest a possible ts period during embryonic life. Sokolova and Golubovsky (1978) studied the ts period of two l(2)91heteroallelic combinations. In one of the combinations the ts period was diphasic. At 17°C there is an early embryonic and third larval instar ts period. At 29°C a ts period was found during the first larval stage and the second during the prepupal stage. In the second allelic combination only the third larval stage proved to be sensitive to low temperature. The aforementioned studies indicate that the 1(2)91gene function is required for normal disc development and for the maintenance of the differentiated state throughout embryonic and postembryonic life. W. P. Hanratty and P. Alden (personal communication) studied the developmental capacities of the imaginal discs of the I(2)gl'" allele in a heteroallelic combination with the 1(2)91 deficiency. At the restrictive temperature, imaginal discs of normal as well as of tumorous morphology were found. Brain tumors seem not to occur in this allele. Implanted into wildtype adult hosts, the mutant imaginal discs with tumor morphology proved fully committed to neoplastic growth irrespective of the rearing temperature.

CANCER, GENES, AND DEVELOPMENT

47

Similarly, intact f(2)gPs1imaginal discs with normal morphology subcultured in wild-type hosts at the permissive temperature maintained their normal morphology. At the restrictive temperature, however, they gave rise to lethal neoplasms. Moreover, when mutant discs of normal morphology were cut into two pieces and the pieces were subcultured at the permissive temperature, in contrast to intact imaginal discs, they grew in a lethal neoplastic way. This indicates that the information for neoplastic development is present in both imaginal disc fragments. The f(2)gP' allele shows that the frequency of abnormal imaginal discs is directly related to the length of the exposure to the restrictive temperature. The mutant allele is, however, leaky because the animals, continuously grown at the restrictive temperature, show tumorous imaginal discs in 69.4%, whereas the remainder exhibit normal morphology and developmental capacities. The new autosomal lethal mutant l(3)bt'"-1 (E. Gateff, unpublished ; Table I ; Fig. 2) discussed already in Section IV,B,l is fully penetrant at the restrictive as well as the permissive temperature. It develops imaginal disc tumors in addition to neuroblastomas in the brain, and shows, like the previously discussed 1(2)91'"' allele, imaginal discs with normal and tumorous morphology and developmental capacities. The shift-up and shift-down experiments demonstrate that also for tumor development of the imaginal discs, the ts period falls during the first half of embryonic life. The shift-up and shift-down experiments reveal that in order for normal development to ensue, the gene product-which apparently is synthesized in the optic neuroblasts and the imaginal disc primordial cells during early embryogenesis is needed continuously for the rest of embryonic and larval life, up until the early third instar. Constant exposure to the restrictive temperature renders the gene product nonfunctional, and thus the development of tumors in the brain and the imaginal discs ensues. The f(3)bt"-1 gene product is, however, synthesized not only during early embryogenesis. It must also be synthesized in the neuroblasts and the imaginal disc cells during larval life because shifts-up during large parts of larval life cause lethality in the pupa and during adult development. Anatomical investigation shows morphological abnormalities but no tumors in the imaginal discs and in the optic centers of the brain. For the 1(2)91'"' allele, similar conclusions were drawn (see previous discussion). Maybe in all these mutants the gene product is synthesized at different times during embryonic and postembryonic development, and may be the presence of the gene product in the optic neuroblasts and the imaginal disc cells is an essential prerequisite for all following developmental steps up until the early third larval instar. Gene cloning experiments of the 1(2)gl gene that are now under way in a few laboratories (0.Schmidt, personal communication) should give, in a

FIG.4. Whole mounts and histological preparations of mutant blood cells, hematopoietic organs, and tumor-bearing hosts. (a) Schematic drawing of wild-type (A) and 4l)mbn (B) brain (b) and ventral ganglion (vg) with the dorsal heart vessel (h) and the attached normal (ho) and tumorous hematopoietic organs (tho). Note the enlarged, tumorous, hematopoietic organs in B. (b) Longitudinal histological section through the first hematopoietic lobe of a fully grown wild-type larva exhibiting primordial blood cells. (c) Longitudinal histological section through the first hematopoietic lobe from a mature /(Z)rnbn larva. Note the size difference between the wild-type and mutant hematopoietic lobes and the cells in the /(l)mbn hematopoietic organ. (d) A group of /(l)rnbn blood cells from the hemolymph showing extensive cytoplasmic processes. c, Crystal cell; I, lamellocyte; p, plasmatocyte. (e) Histological section through secondary nests of hematopoiesis (arrows) in the body cavity of a 43)rnbn mature larva consisting of proplasmatocytes. ( f ) Phase contrast of /(3)mbn plasmatocytes @) and giant lamellocytes (I)

CANCER, GENES,

AND DEVELOPMENT

49

few years from now, answers to some of the questions concerned with the role of the genes described here during normal development and in malignant neoplastic transformation. The ts mutant s W 1 (Table I) shows extensive pleiotropic effects on development (Poodry el al., 1973; MacMorris-Swanson and Poodry, 1981). In contrast to the tumor developing in the shitsl embryo after exposure to high temperature (see Section III), tumorous imaginal discs in situ are not observed after restrictive temperature treatment. However, tumorous growth ensues during the first subculture of the eye-antenna1 disc in the wild-type adult abdomen at the restrictive temperature (Williams, 1981). Wild-type imaginal discs, in contrast, never show tumorous growth in a comparable transplantation experiment. The tumorous growth pattern of the implanted disc is established during the first 48 hr of in uivo subculture. Thereafter, the tumorous state is irreversible and restrictive culture conditions are not required for its maintenance during further transfer generations. Apparently in the larva an external factor or factors are present that prevent tumor development, even though the eye-antenna1 disc cells are competent for neoplastic growth, which may not exist in the adult. In this sense the tumorous growth of the imaginal discs in uivo is not autonomous. However, once established, tumorous growth ensues autonomously at the permissive temperature as well.

3. Mutants Developing Hematopoietic Tumors The third type of tumor found in the larva develops in the hematopoietic organs and involves the primary blood cells. Five such mutants are known at present (Table I). A brief outline of the hematopoiesis in the wild-type larva will precede the description of the mutants. The wild-type larval hematopoietic organs, the so-called lymph glands, represent four to seven pairs of lobes located along the dorsal heart vessel (Stark and Marshall, 1930; Gateff, 1977; Fig. 4a). The two blood cell types found in the hemolymph, the plasmatocytes and crystal cells (Rizki, 1957), originate, respectively, from proplasmatocytes and procrystal cells located in the hematopoietic organs (Gateff, 1977; Shrestha, 1979; Shrestha and

from the hemolymph. (g) Longitudinal section through l(3)mbn larva in the premortal stage, filled with millions of blood cells, which have destroyed most of the organs. int, Integument; g, gut. (h) Histological cross section through the abdomen of a wild-type fly that had been injected with a small piece of a l(l)mbrr hematopoietic tumor. The ovary on the left side has been replaced almost completely by tumorous blood cells, whereas the right ovary (ov) is still intact. The only egg chamber (e) on the left is surrounded and invaded by the tumorous blood cells (arrows). f, Fat body; g, gut; int, integument. All histological sections 5 pm thick and stained with hematoxylin and eosin (further details in text).

50

ELISABETH GATEFF

Gateff, 1982a; Rizki et al., 1980). The differentiation of plasmatocytes into plasmatocytes and further into podocytes and lamellocytes is closely related to (1) the formation of the primary lysosomal system and (2) the acquisition of the capacity for melanine biosynthesis (Shrestha, 1979; Shrestha and Gateff, 1982a). Crystal cell maturation, on the other hand, involves the synthesis of the crystalline material, whose molecular nature is not yet established, and crystal formation. While the larva grows, the primordial blood cells divide in the hematopoietic organs. A small number of them are released into the hemolymph at the end of each larval instar. During the larval-pupal transformation, however, all primordial blood cells present in the hematopoietic organs enter the hemolymph, where they play a role in the digestion of the disintegrating larval tissues. Plasmatocytes can take on variable shapes in the hemolymph, which are referred to as podocytes and lamellocytes (Rizki, 1957; Fig. 4d). During defense reactions they phagocytose and encapsulate foreign bodies (Nappi, 1975). The very fragile crystal cells function most probably in hemolymph coagulation but may possess further unknown functions in defense reactions. In all five mutants shown in Table I, the malignantly transformed blood cells are the plasmatocytes, not the crystal cells. Characteristic for the blood tumor mutants are the much enlarged hematopoietic organs (Fig. 4a,b, and c) and the increased blood cell counts in the hemolymph (Gateff, 1978a,b,c; Hanratty and Ryerse, 1981). In contrast to the wild type, where blood cells are released into the hemolymph at particular times and in controlled amounts, in the third instar mutant larvae, blood cells are formed constantly in the hematopoietic organs and are released continuously into the hemolymph. The freed tumorous blood cells invade the organs of the larva, eventually destroying them. This can be seen in Fig. 4g, where in the extreme case of the mutant lethal(3) malignant blood neoplasm-1 (l(3)mbn-I), the body cavity of the larva is entirely filled with blood cells. Most larval organs, such as the muscles, the fat body, and the imaginal discs, have disappeared. Histological sections through l(3)rnbn-1 larvae show secondary nests of hematopoiesis distributed throughout the body cavity (Fig. 4e). The majority of the cells in this mutant are immature and resemble proplasmatocytes. However, 5 to 10% of the free blood cells engage in an abnormal differentiation yielding giant plasmatocytes, podocytes, and lamellocytes (Fig. 4f). Sometimes giant lamellocytes accumulate and melanize, forming melanotic masses. The giant plasmato-, podo-, and lamellocytes show in their cytoplasm enormous amounts of primary and secondary lysosomes, when compared to their wild-type counterparts and the undifferentiated tumorous blood cells (R. Shrestha and E. Gateff, unpublished). Proplasmatocytes are predominantly present within the enlarged l(3)rnbn-1

CANCER, GENES, AND DEVELOPMENT

51

hematopoietic organs. Procrystal and crystal cells are also found but in considerably smaller numbers. The blood tumor mutants designated as f(2)rnbnand f(3)mbn-2(Table I) exhibit a similar tumorous phenotype. Thus it appears that in these mutants the differentiation of proplasmatocytes into plasmato-, podo-, and lamellocytes is largely inhibited or abnormal. As a result proplasmatocytes continue to divide, which causes their considerable accumulation in the hemolymph and the enlargement of the hematopoietic organs. In contrast to the three blood tumor mutants just discussed, in the mutants f(l)mbn(Gateff, 1978a,b,c; Shrestha, 1979; Shrestha and Gateff, 1982b) and Turn’ (Hanratty and Ryerse, 1981;Table I), blood cell differentiation is not impaired. The enlarged mutant hematopoietic organs show, in addition to primordial blood cells (e.g., prohemocytes and proplasmatocytes), also differentiated plasmato-, podo-, and lamellocytes (Shrestha and Gateff, 1982b; compare Figs. 4b and c). During the third larval instar, mature blood cells are continuously released from the hematopoietic organs into the hemolymph, where they invade and destroy the larval tissues. The mutant blood cells exhibit increased amounts of primary and secondary lysosomes and show more phagocytic vacuoles when compared to the wild type (Shrestha and Gateff, 1982b). The malignant qualities of the mutant blood cells can also be demonstrated in the transplantation test. Small pieces of f(l)rnbn or Turn’ hematopoietic organs implanted into the body cavity of wild-type flies grow in an autonomous lethal, invasive fashion for numerous transfer generations (Shrestha and Gateff, 1982b; Hanratty and Ryerse, 1981; Fig. 4h). The I(l)rnbn tumors in uiuo consist predominantly of proplasmatocytes. However, just as in the in siru situation in the hematopoietic organs, one finds differentiated podocytes and lamellocytes interspersed. In contrast to f(l)rnbn,in the Turn’ mutant, extensive melanizations in siru as well as after transplantation into adult hosts can be seen (Hanratty and Ryerse, 1981). This indicates that in this mutant melanin synthesis, a normal repertoire of this blood cell line (see previous discussion), is abnormally regulated. In the wild type, melanization of lamellocytes takes place only in defense reactions after they have encapsulated a foreign body such as a nematode (Nappi, 1975). In this respect the Turn’ mutant resembles melanotic “pseudotumor” mutants in which, for unknown reasons, lamellocytes also encapsulate the fat body or accumulate and subsequently melanize (Sparrow, 1978). Small melanotic “pseudotumors” can also be observed in f(l)rnbn;however, the ability of the lamellocytes to melanize is not expressed after transplantation. The melanotic masses consist of melanized lamellocytes, which are considered dead cells. The capacity for malignant growth after transplantation applies to the last two blood tumor mutants. The

52

ELISABETH GATEFF

tumorous blood cells in the three mutants first discussed do not grow after transplantation in a wild-type adult host. Thus all five blood tumor mutants cause malignant transformation of the plasmatocyte blood cell line, whereas the crystal cells are normal. Furthermore, the individual mutant either affects the capacity for differentiation of the proplasmatocytes into plasmato-, podo-, and lamellocytes (l(2)mbn; I(3)mhn-I; 1(3)rnbn-2), or the mutated genes seem to be involved in the control of the demand for blood cells in the hemolymph and their supply by the hematopoietic organs (I(l)rnhn;Turn'). Dehn (198 1) compared the defense reactions of wild-type I(l)mhn and I(3)rnbn-l blood cells against spores of Aspergillus niger and Methnrrhizium unisopliae. After injection of 30 spores per larva he found a lower mortality rate in the mutants than in the wild-type larvae, which shows that the spores were more efficiently encapsulated by the mutant than by the wild-type lamellocytes. The fact that in both mutants lamellocytes are present in increased amounts may explain this result. Many spores were covered with granulae, pointing to the possibility of a humoral immune response in addition to the encapsulation reaction. Comparing the mortality rates of the two mutants after spore injection, l(l)mbn larvae showed lower mortality rates and thus higher efficiency in rendering the injected spores harmless, than did the I(3)rnbn-l mutant. This finding agrees well with the observation that in I(1)rnbn larvae most free blood cells and many cells in the hematopoietic organs are fully differentiated and apparently functional. In the l(3)nzbn-I mutant, in contrast, the majority of the cells are immature and thus nonfunctional. The availability of large numbers of blood cells in the mutants faciliated the establishment of tumorous blood cell lines in uirro derived from the l(2)rnbn and l(3)rnbn-l mutants. The fine structure of the blood cell lines has been studied by Shrestha (l979), and a variety of properties by Gateff et al. (1980). The cells in uirro show high phagocytic activity during the stationary phase and contain two viruses in addition to the well-known virus-like particle (vlp; see later).

4. Mutants Developing Germ-line Tumors The germ-line precursors of Drosophila, the so-called pole cells, have been already set aside before blastoderm formation. During gastrulation they migrate into the respective female or male gonads, where they become primary oogonia (Mahowald and Kambysellis, 1980) or primary spermatogonia (Lindsley and Tokuyasu, 1980), respectively. During adult development and in the adult, oogonia and spermatogonia differentiate into eggs and sperm, respectively (King, 1970; Mahowald and

53

CANCER, GENES, AND DEVELOPMENT

TABLE I1 MUTANTSCAUSING BENIGNTUMORS IN THE MALEAND FEMALE GERMLINEOF Drosophila melanogaster Designation of mutant Female sterile ( 2 ) of Bridges (,fi(2)E) Tow alleles : female sterile ( 1 ) 231 G ( f i ( 1 )231 G ) and female sterile (1)231 M (js(1)231 M ) Narrow (nw), 5 alleles

Sex

affected

Y ?

0

Fused (,fu), 7 alleles

Y

Female sterile ( 1 ) 1621 (fS(1)1621) lethal(3)brain tumor"-l

P

Benign gonial cell neoplasm (bgcn)

?

9.6

6

Differentiation step affected Early cystocyte division Early cystocyte division

Early cystocyte division Late cystocyte division Late cystocyte division Late cystocyte division Differentiation of cystoblasts into cystocytes and of primary spermatocytes into meiotic spermatocytes Differentiation of primary spermatocytes into meiotic spermatocytes

References Koch and King (1 964) ; King (1970) Cans er al. (1975); King er al. ( 1978); King (1979); King and Buckles (1978); Mohler (1977) King (1970); King and Bodenstein (1965) King er al. (1 957) ; Smith and King (1966); King (1970) Gollin and King (I98 1)

E. Gateff (unpublished) Gateff (1981, 1982)

Lifschytz (1978)

Kambysellis, 1980; Lindsley and Tokuyasu, 1980). Hundreds of sex-specific genes are involved in these differentiation processes (Mohler, 1977; King and Mohler, 1975; Romrell, 1975); mutations in these genes cause either male or female sterility. Genes acting in both the female and the male germ lines are exceedingly rare. In fact, only one such mutant is known (Gateff, 1981, 1982; Table 11). The aforementioned relationship of genes affecting either of the two sexes to genes acting in both sexes is seen also in the mutants causing germ-line tumors. Table I1 shows eight such mutants. Of these, seven affect either the female or the male germ line and only one affects both sexes.

54

ELISABETH GATEFF

Before we proceed with the discussion of the developmental effects of the various tumor mutants on oogenesis, a brief description of wild-type egg maturation should help the reader to understand the steps at which germ-line differentiation is interrupted in the various ovarian tumor mutants. Each of the two Drosophilu ovaries consists of approximately 16 ovarioles. The ovarioles are sausage-like and show distally the germaria and proximally the vitellaria (Fig. 5a). At the distal end of the germaria, two or three oogonia divide and give rise to cystoblasts, which in turn produce in four consecutive mitoses cysts made up of 16 cells. The cells in such cysts are interconnected by a system of ring canals. The cysts become enveloped by follicle cells and enter the vitellaria, where one of the 16 cells develops into the oocyte and the remaining 15 differentiate into nurse cells. For further details on wild-type oogenesis consult King (1970) and Mahowald and Kambysellis (1 980). Table 11 shows the different mutants and the differentiation steps controlled by the various genes. Seven female sterile mutants are listed. Six of them affect cyst formation. In three of the mutants, early cystocyte divisions are abnormal, whereas in the remaining three the late cystocyte divisions are affected. In contrast to the wild type, where cystocyte divisions are incomplete and the two daughter cystocytes remain connected with ring canals, in the mutants a high percentage of the cystocytes show complete cytokinesis, which accounts for the accumulation of free single cystocytes within the egg chambers. However, in some cases cystocytes perform incomplete cytokinesis and remain connected with each other by ring canals. Thus, in addition to the single cystocytes, cysts of two, four, and more cystocytes form. Some of them even differentiate into pseudonurse cells (King, 1970). Young follicles from female sterile mutants implanted into wild-type female hosts grow in the same tumorous fashion as they do in situ (King and Bodenstein, 1965). All ovarian tumors are benign and autonomous. In contrast to the six female sterile mutants just discussed, the recessive mutant benign goniul celZ neoplasm (bgcn) affects an earlier differentiation step, namely, the differentiation of cystoblasts into cystocytes (Gateff, 1981, 1982; Table 11). As a result, cysts and egg chambers never form. The ovaries of newly emerged flies show germaria filled with many single cells (Fig. 5b). Upon rupturing the peritoneal sheath of the germarium in a drop of Ringer's solution, the single cells leave the germarium and in phase contrast closely resemble cystoblasts (Fig. 5c). They exhibit large nuclei with prominent nucleoli and can often be seen in mitosis. As the flies age, they enter the vitellaria, which, in contrast to the wild type, are devoid of follicles (Fig. 5b). Seven days after eclosion the vitellaria contain millions of cystoblastlike cells (Figs. 5d, e, and f). The cells are not connected with each other, and when freed from the vitellaria they show the same phenotype as the cystoblasts in Fig. 5c. Follicle cells are also present. They line the vitellaria and

FIG.5. Histological comparison between the wild-type and bgcn adult gonads. (a) Longitudinal section through two ovarioles from a freshly eclosed wild-type fly. Each ovariole shows a germarium (9) and two young follicles (f) in the vitellarium (v). The ovarioles are surrounded by a peritoneal sheath (t). Each germarium consists of three regions: region 1 contains the mitotically active oogonia ( 0 ) and cystoblasts; region I1 is inhabited by cystocytes (cc) in various stages of cyst formation; region 111 shows a terminal cyst (tc) surrounded by follicle cells (fc). (b) Longitudinal section of two germaria (g) from a freshly eclosed bgcn fly. Note the empty vitellaria (v). delineated by the peritoneal sheath (t) and the germaria (g), filled with loosely arranged cystoblasts. In the posterior region of one of the germaria, follicle cells (fc) can be seen. (c) Phase contrast of the cells derived from a byrn germarium. (d) Whole mount of 7-dayold mutant adult ovary (mov) and 10-hr-old wild-type ovary (wtov). Compare the mutant ovary, devoid of follicles, with the wild-type ovary exhibiting young follicles (arrow). The mutant ovarioles are filled with millions of cystoblast-like cells. (e) Cross section through the abdomen of a 7-day-old bgcn fly showing the two ovaries (ov), each with I5 ovarioles (ovl) embedded in fat body (fb)and surrounding the gut (gt). (f) Detail from (e) showing a few ovarioles in cross section. The ovarioles are lined by a single layer of follicle cells (fc). The entire lumen is filled with tumorous cystoblasts (cb). (g) Longitudinal section through the distal end of a wild-type testis. Various stages of spermatogenesis, such as primary spermatocytes @s) and spermatid bundles (sb) can be observed. (h) Oblique section through bgcn testis showing primary spermatocytes. Histological sections 5 pm; Hematoxylin and eosin.

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ELISABETH GATEFF

are easily distinguishable from the larger cystoblasts by their smaller size and cuboidal shape (Figs. 5b and f). The cells in the germaria of young bgcn flies were tested for their developmental capacity through the implantation of the germaria into the body cavity of wild-type female flies. After 14 days the implants had grown into spheres 40-50 times larger than the original implants. The enlargement was due to millions of cytoblasts growing in them. In order to test whether the female germ line is already determined for abnormal growth earlier in development, gonads from mid-third-instar bgcn larvae (100 hr old) were tested for their mode of growth by implantation into wild-type larvae of comparable age, which were allowed to develop into adults. These experiments showed that the primary gonial cells from the larval gonad develop autonomously in the wild-type host, giving rise to tumors similar to those seen in situ, consisting exclusively of cystoblasts (Gateff, 1982). These experiments indicate that the bgcn gene must act sometime before the third larval instar. The fact that in this mutant both sexes develop tumors strongly indicates that the bgcn gene must function at or before the time of the segregation of the female and male germ lines. After the entry of the primary germ cells into the prospective gonads, separate female and male specific gene sets seem to be turned on that are instrumental either in oogenesis or spermiogenesis. Before discussion the benign tumors developing in the testis of the autosomal bgcn and the x-linked f(455 mutants (Table II), a short compendium of wild-type spermiogenesis should give the reader a basis for comparison with the mutant phenotype. Wild-type spermiogenesis begins at the apical portion of the testis where pregonial cells divide unequally, giving rise to stem cell and primary spermatogonia. The primary spermatogonia produce, in four successive divisions, cysts of 16 primary spermatocytes, which remain in contact with ring canals similar to the ones in cystocyte cysts of the female (King, 1970). After a growth phase, primary spermatocytes become secondary spermatocytes, which undergo meiosis and yield cysts of 64 interconnected spermatids. The spermatids in a cyst engage synchronously in a complex differentiation process ending with mature sperms (Lindsley and Tokuyasu, 1980). Figure 5g demonstrates some of the differentiation stages in wild-type testes. Compared to the female, where six ovarian tumor mutants have been found, in the male only two such mutants are known (Table 11). In both mutants, the differentiation of the male germ line is interrupted at the early primary spermatocyte stage before the onset of the growth phase. As a result, the primary spermatocytes engage in supernumerary cell divisions resulting in cysts of 300 to 400 primary spermatocytes, some of which seem to remain connected by ring canals (H. Akai and E. Gatef€, unpublished

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results). Figure 5h is a section through a bgcn testis that shows exclusively primary spermatocytes (compare with Fig. 5g). The male sterile mutants just discussed reveal two nonallelic developmental genes, both of which, in the mutated state, impair the further differentiation of primary spermatocytes. The resulting continuous mitotic activity of the primary spermatocytes leads to tumorous cysts. The two genes act at different points during development, bgcn functioning in both sexes before the segregation of the male from the female germ lines, and /(1)55 only in the male after the segregation event. In the preceding section, concerned with mutations causing tumors in various larval cell types, we discussed four tumor types involving (1) the adult optic neuroblasts, (2) the imaginal discs, (3) the primordial phagocytic blood cells, and (4)the male and female gonial cells. Except for the l(Z)mbn and Tum' blood tumor mutants, in all the other mutants, differentiation of a particular cell type is arrested. Thus the described mutants are developmental mutants, and the mutated genes that cause the tumorous condition are instrumental in controlling cell-specific differentiation steps. The following sections will relate different aspects of the Drosophilu tumors to recent developments in the molecular biology of retroviruses and the identification of putative vertebrate genes involved primarly in malignant neoplasms. V. Viruses Found in Drosophila Tumor Cells

Seven viruses, belonging to various families, have been found in Drosophilu laboratory and natural populations and cell lines in vitro (Brun and Plus, 1980). Of these, two viruses are present in tumorous blood cells cultured in uitro: (1) a reovirus named F, (Gateff et al., 1980; Haars et a/., 1979) and (2) a picornavirus designated Drosophila C virus (DCV; Plus et al., 1975). In addition, in all tumors whose fine structure has been studied, large numbers of virus-like particles (vlp) have been found primarily in the nucleus and less so in the cytoplasm (reviewed by Gateff, 1978a; see later). The two viruses just named almost certainly have no direct relationship to the neoplastic transformation of the blood cells. However, a study by Plus and Golubovsky (1981) of the resistance to DCV in 15 l(2)gl stocks, derived from isolated, natural populations of the Soviet Union (Golubovsky and Sokolova, 1973; Golubovsky, 1978), should be mentioned. DCV is the most common virus of Drosophila laboratory and natural populations. The above l(2)gl strains were found to be free of DCV. Upon infection with the virus, Plus and Golubovsky encountered considerable variation in the resistance to the virus among the different l(2)gl strains. However, on the whole they demonstrated a higher resistance to the virus in the 42)gl strains

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than in all other wild-type and mutant strains studied. Finally, the resistance to the virus seems to act through some cytoplasmic factor(s). A comparison of the resistance to DCV with the degree of malignancy in the different l(2)gl alleles (Gateff et al., 1977) revealed no correlations. In physiologically stressed cells, such as aged cells, cells treated with carcinogens or X rays, or mutant cells, virus-like particles (ulps) were regularly found (for review see Gateff, 1978a). They are especially common in tumorous cells in situ, in subculture in uiuo and in uirro (Akai et d., 1967; Shrestha, 1979; Shrestha and Gateff, 1982b). Vlps are round to oval with a diameter of 37 nm, showing a single envelope and a core that is often empty. They occur singly, in groups, or in crystalline array, primarily in the nucleus, and in older or dying cells, also in the cytoplasm. Two recent papers report on particles of 36- and 40-nm size, which resemble ulps morphologically. The first paper, by Scott et al. (1980), describes a new type of virus designated HPS-1. The virus contains a single segment of double-stranded RNA and shows two coat proteins (1 20,000 and 200,000 daltons). It bands at 1.41 gm/cm3 in CsCl. The particle was isolated from the supernatant of Schneider cell line 2, cultured in Dulbecco modified Eagle’s medium supplemented with 10% calf serum and 0.5% lactalbumin hydrolysate. The origin of this particle and its relation to the ulp will have to be demonstrated. The second paper by Heine et al. (1980), shows a 40-nm particle in the cytoplasm of Schneider cell line 1 cells. The particle is found in a subcellular fraction banding at 1.22 gm/ml in sucrose. This fraction was associated with reverse transcriptase activity, high-molecular-weight polyadenylated RNA, and the above particle. The authors concluded that the Drosophilu cells in vitro contain retroviruses, similar to but not identical to A-type particles found in mammalian and avian cells. This conclusion awaits further substantiation. Thus the existence of retroviruses in Drosophilu is not yet established. However, evidence is accumulating that suggests a relationship between the transposable elements of eukaryotes and the integrated retroviral proviruses of mammals and birds (Finnegan et al., 1978; Shimotohno er al., 1980; Sutcliff et al., 1980; van Beveren et ul., 1980; Dhar et al., 1980; Gafner and Phillippsen, 1980; Levis er ul., 1980; Dunsmuir et al., 1980; Scherer and Pirrotta, 1981; Scherer er al., 1982; see also article by Spradling and Rubin, 1981). Copia and other copia-like transposable elements in Drosophilu represent families of moderately repetitive gene sequences with an average length of 5 to 8.5 kb, repeated 10 to 100 times per genome, which code for abundant polyadenylated mRNAs (Table 111). The mRNAs are found predominantly in the nucleus and only a small proportion, as expected, in the cytoplasm as

TABLE 111 CHARACTERISTICS OF SIX Copia-LIKE TRANSP~SABLE ELEMENTS FOUND ON Internal segment

Direct terminal repeat

2

1 (1)DNA sequence of internal segment in kb

Designation of transposable element

5.0"*b

Copia 412 29 7 mdglk mdg3 8104

7.0" 6.5b*f 7.28 5.5h,' 8.7'

(2)Number of base pairs in direct terminal repeat 276' 481' 412/ 30O-40Og 269h*J 429'

3 (3,4)Number of base pairs in terminal inverted repeat 13/17 8/10' 3f ND 15/1gh,' 3'

THE

Drosophila GENOME

Host DNA

4

5 (5)Number of base pairs in flanking sequences at point of insertion" 5d

4' 4f ND 5 5'

Mean number Percentage and classes of POlY(A) mRNA 3% 5 kb, 2 kb 0.6 ND 0.7-0.8 0.8 8 and 0.8-1.2

of hybridization sites on the giant chromosomesh

Copies per haploid genome

Percentage of total DNA

35

20-60'

ND'

30 30 20-30 20-30

40b 30b 20-308 15hj 80-95'

ND ND 0.7 0.3 0.5

+ chromocenter

100

a Finnegan et al. (1978);b Potter ef al. (1979); ' Levis et al. (1980); Dunsmuir et al. (1980); ' A. A. Bayev, K. Will, and D. J. Finnegan (personal communication); M. W. Young and G. M. Rubin (personal communication); Ilyin et al. (1980b); Bayev er al. (1980); Scherer et al. (1982); j Ilyin et al. (1980a); ' The fragments described in earlier publications as Dm225 and Dm234 are parts of the complete mdgl element; ND, no data; Columns 1-4 and 7 from Spradling and Rubin (1981); different for each insertion site; " the number and distribution of hybridization sites vary widely among the different wild-type and mutant stocks (Strobe1 er al., 1979; Ilyin et al., 1978; Ananiev et al., 1978; Young, 1979). f

'

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ELISABETH GATEFF

RNP particles (for review see Spradling and Rubin, 1981). Approximately 30 families of copia-like sequences have been detected (Rubin et a/., 1981). They comprise about half of the middle repetitive DNA sequences (6%). The members of a family show similar nucleotide sequences. Between the families, on the other hand, very little sequence homology exists. Six copiiilike elements have been cloned, sequenced, and analyzed by in situ hybridization to giant salivary gland chromosomes (Table Ill; Rubin et id., 1981). The hybridization sites are dispersed throughout the genome. Furthermore, the distribution pattern varies from stock to stock, even after short periods of separate rearing, which suggests that the elements are mobile within the genome. Characteristic for all six transposable elements are direct terminal repeats, which are flanked by short inverted repeats. A further peculiarity exists in the site of insertion of the transposon into the host genome, where a few base pairs become duplicated to give rise to flanking direct repeats (Table 111). In these features copia-like elements resemble bacterial transposons (Kleckner, 1977; Nevers and Saedler, 1977; Calos and Miller, 1980), the transposable element Tyl of yeast (Cameron et a/., 1979), and the integrated retroviral genomes (Bishop, 1978; Shimotohno et a/., 1980). The mature DNA of retroviral proviruses in the nucleus of virus-infected cells has been shown to represent covalently closed circles (Rotenberg et al., 1977; Shank and Varmus, 1978; Clayman et al., 1979). Small circular DNA has been isolated from Drosophib embryos and cells cultured in oitro (Stanfield and Helinski, 1976; Stanfield and Lengyel, 1979). The analogy between retroviral proviruses and the transposable copiu element is carried even further by the finding in in oitro cultured cells of copia DNA, in covalently closed circles containing either one or two terminal direct repeats (Flavell and Ish-Horowicz, 198l), and which resembles closely integrated proviruses of vertebrate retroviruses. Recently, Gehring and Par0 (1980) found a copin element associated with a large transposone (TE) (more than 200 kb), carrying the genes whiteapricot, roughest, and uerticuls, which encompasses four bands on the polytene chromosomes. This TE transposes with a frequency of and has been mapped in genetic experiments to more than 100 positions scattered over the genome (Ising and Ramel, 1976). Because copia is a movable element, it presumably causes the observed transpositions of the three aforementioned genes. One of the effects of transposition in bacteria is the induction of mutations (Starlinger and Saedler, 1976; Kleckner, 1977; Calos and Miller, 1980). lsing and Ramel (1976) reported that many transpositions of the aforementioned TE induce nonallelic, recessive-lethal mutations that persist after

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the loss of the TE from the particular site. The present author has investigated some of these lethal stocks such as TE 41 and TE 42 for putative tumorous growth, with negative results. However, two recently reported transpositions of the above TE to the aouter most tip of the left arm of the second chromosome, such as TE 75 (Ising and Block, 1981) and TE 133 (G. Ising, personal communication), showed in crosses to various /(2)g/ alleles in the heterozygous condition the typical 1(2)91 tumor phenotype (E. Gateff, G. Ising, and 0. Schmidt, unpublished results). The results indicate that the two TEs have inserted either into the l(2)gllocus or into its regulatory region. This fortunate coincidence creates a realistic possibility of cloning the /(2)91 genomic sequences with the help of the above TE. In another case of high transposition frequency of the white locus, Rasmuson et al. (1980) found that their transposon shows a DNA segment that is homologous to the intercalary heterochromatic DNA, cloned in the plasmid mdgl (Table 111). Also here, the observed transpositions may be caused by the mdgl transposable element. M. M. Green (personal communication) observed recently increased mutation rates on the 1(2)g/locus as a result of the insertion of a transposable MR element on the left arm of the second chromosome (for discussion of MR see Green, 1980). The accumulating data on transposable elements in Drosophih are highly significant in relation to their mutagenic activities (Green, 1980). Future cloning experiments of one or the other tumor gene described here may reveal an insertion of a transposon into the gene sequence, thus proving it responsible for the mutant effect. Nevertheless, the main question is whether copia-like sequences are involved in regulating normal development. The fact that the copy number and the points of insertion of the different copia-like elements vary considerably from stock to stock (Ilyin et a/., 1978) and also from individual to individual (Strobe1 et a/., 1979) makes it unlikely that they have some important role in normal development. It seems more likely that they are evolutionary ballast that obeys the cellular control mechanisms. In stress conditions, however, in which the control processes in the cells become sloppier, they may be transcribed and translated at higher rates and thus may enhance the pathological condition. In this respect it would be of prime interest to affirm their putative retroviral nature and their possible relation to the previously discussed vlps. I suggest, on purely speculative grounds, that the ulps are the visible products of the various copia-like elements. In the wild type, ulps are not assembled because their transcription and translation are regulated. In pathological states, however, particles are made.

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VI. Retroviral Oncogenes and Their Cellular Counterparts in Different Animal Species

Genetic factors have been considered by many as causative agents in the induction of malignant growth (Knudson, 1978; Knudson and Meadows, 1978; Lynch, 1976; Anders and Anders, 1978; Gateff, 1978a,b,c; Ponder, 1980; Arrighi et af., 1981; and others). However, it was not until recently that this view gained optimistic approval. The finding that directed the spotlight toward the genome as the initiation site of malignant growth came from molecular and genetic studies on retroviral oncogenes (v-om) and from transfection experiments with DNA from normal and tumorous tissues. Oncogenes are essential for malignant neoplastic transformation, but they play no role in viral structure and replication. Because oncogenes are not a constitutive part of retroviruses, it was important to learn where oncogenes come from. Several hypotheses suggested that oncogenes may be transduced cellular genes (Hieger, 1961; Huebner and Todaro, 1969). Indeed, in the 1960s a number of novel defective murine sarcoma and acute leukemia viruses were isolated from animal tumors inoculated with murine leukemia virus (MuLV). This was the first indication that the V-ORCS may be transduced cellular genes (Harvey, 1964; Moloney, 1966; Kirsten and Mayer, 1967; Abelson and Rabstein, 1970). Molecular hybridization experiments provided further compelling evidence for this possibility. By now about 20 independent isolates of oncogenic retroviruses have proved to contain sequences homologous to genomic host sequences (Vennstrom and Bishop, 1982). Moreover, the cellular sequences incorporated into the viral genome are expressed in cells transformed by that virus. The best-studied viral oncogene, the src gene of Rous sarcoma virus (RSV), shows complete conservation in all avian species investigated (Spector et a/., 1978). At least part of the gene has been detected in a variety of vertebrates other than birds (Stehelin er af., 1976a,b; Hanafusa er af., 1977; Spector er al., 1978; Wang et af., 1980; Oskarsson er al., 1980). For other retroviruses, such as the myelocytomatosis virus (MC29V), the myeloblastosis virus (AMV) and the erythroblastosis virus (AEV), homologous genomic sequences have also been demonstrated in birds and other vertebrates (Sheiness and Bishop, 1979; Roussel er af., 1979). Less well conserved in evolution are the corresponding c-onc sequences to the viral oncogenes of Moloney murine sarcoma virus (MSV), Harvey sarcoma virus (HaSV), and feline sarcoma virus (FeSV), because they may exist only in mammals (Fishinger, 1980). Sheiness and Bishop (1979) examined the possibility of a cellular genetic element in the avian myelocytomatosis virus (MC29V) responsible for

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tumorigenesis. The phylogenetic origin of the v-om of MC29V, containing 1500 nucleotides, was analyzed by hybridizing the v-onc to genomic DNA of birds and other vertebrate species such as cow, mouse, and salmon. In all of them, nucleotide sequences related to v-onc DNA were found. Chickens for instance, the natural host of the virus, harbor at least 90% of the 1500 nucleotide-long viral DNA sequence in their genome. According to their evolutionary divergence from chickens, the other species showed correspondingly decreased sequence complementarity with the viral oncogene. A single, constant locus of the c-MCV oncogene is located on each chicken genome. The locus shows no linkage with c-src (Sheiness et al., 1980). The replication-defective avian leukemia viruses (ALV), which require helper viruses for propagation in cultured cells, are highly tumorigenic. Roussel et al. (1979) found three new oncogenes in them and demonstrated cellular homologues. Depending on the target cells they transform, they were designated as v-erb (erythroblasts), v-mac (macrophages), and v-myb (myeloblasts). Vennstrom and Bishop (1982) studied, by restriction endonuclease mapping and heteroduplex analysis, the structure of the cellular genomic locus corresponding to the AEV oncogene, c-erb, and c-erb, . They found that the cellular homologue to the v-erb, consists of approximately 750 nucleotides with three introns, and the c-erb, of 2000 nucleotides and 11 introns. The flanking genomic sequences were unrelated to the nononcogenic part of the AEV. With the help of immunological methods, Baltimore et al. (1979) identified a protein in mouse cells that appears to be the cellular counterpart to the viral P120 transforming protein of Abelson murine leukemia virus (Ab-MuLV). A single and constant locus on the human genome, homologous to the transforming gene v-sis of simian sarcoma virus, was described recently by Dalla Favera et al. (1 98 1). Of particular interest for this discussion are the spontaneous melanomas, occurring with high frequency in hybrids between the teleosteans platyfish (Xiphophorus maculatus) and swordtail (Xiphophorus helleri), which are a model system for the study of genetic factors in cancer (Gordon, 1958; Anders and Anders, 1978). Fish from wild populations growing for many generations in the laboratory never develop melanomas or other tumors, and are insensitive to carcinogens. The hybrids here discussed, however, show spontanously malignant tumors of all sorts and are susceptible to carcinogens (Anders and Anders, 1978).The genetic information responsible for the malignant neoplastic growth of pigment cells is mediated by a gene designated as “tumor gene” (Tu),which is repressed in wild-type animals by a system of regulator genes (R).In the hybrids, however, regulation of the Tu fails, and instead of normal differentiation malignant growth ensues.

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In this system, Barnekow et al. (1982) detected the expression of endogenous pp6OSrcphosphoprotein (Collett and Erikson, 1978; Erikson er ul., 1980) and found associated kinase activity in brain extracts from different Xiphophorus species, hybrids, malignant, and nonmalignant tissues. Thus the conclusion was drawn that on the Xiphophorus genome a cellular src gene (c-src) is present that codes for the 60K phosphoprotein with kinase activity. Moreover, the expression of c-src, as identified by the pp60"-""associated kinase activity in melanomas of different genotypes and degree of tumorigenicity, were compared (Schartl et al., 1982). That study showed a correlation between the expression of c-src and Tu. Therefore, cells containing Tu express more pp60'-"c than cells lacking Tu. A correlation was also found between an elevated pp60"-"*'-associated kinase activity and the degree of Tu expression, which may indicate that Tu and c-src are regulated coordinately. Schwab (1981) reported the existence of v-src homologous sequences on the genome of Xiphophorus fish hybrids as detected in hybridization experiments. Further, he has been able to transform NIH-3T3 mouse cells in uirro by transfecting with c-src genomic DNA (see later). All the previous data demonstrate that animal genomes contain a group of evolutionarily highly conserved genes homologous to the different oncogenes. The conservation of these genes may have been necessitated by their fundamental role in specific cell types engaged in particular developmental programs (see later). Drosophila exhibits genomic sequences homologous to the v-src, v-ras, v-myc, vTfes,and v-ah1 oncogenes (Shilo and Weinberg, 1981b). Some of the Drosophila tumor genes may turn out to represent the corresponding homologous sequences to one or the other viral oncogene. All Drosophila tumor mutants represent developmental mutants. Therefore, the wild-type gene product must control some key differentiation step, as judged by the devastating effects caused to the animals by the mutant gene. Similarly, the various c-onc appear to interfere with particular pathways of differentiation of the infected target cells (Graf rt ul., 1980; see later) and resemble in this respect Drosophila tumor genes. The state of differentiation of macrophages is differentially affected by acute leukemia viruses, such as avian myeloblastosis virus (AMV) and avian cytomatosis virus (MC29V), whereas RSV fails to induce functional alterations in the macrophages, despite the production of infectious virus (Durban and Boettiger, 1981). Thus retroviral oncogenes show target-cell specificity in uiuo as well as in uitro. Infection of cells, determined for the erythrocyte, macrophage, or myelocyte differentiation pathways, with the corresponding avian erythroblastosis virus (AEV), MC29V, respectively, AMV causes acute leukemias. Graf ef ul. (1980) showed that the different viruses replicate

CANCER, GENES, AND DEVELOPMENT

65

and produce the respective transformation proteins, not only in their target cells but also in hematopoietic nontarget cells. There, however, they do not cause malignant transformation. These results support the notion that the respective transforming proteins block the differentiation of their target cells by competitively inhibiting the action of homologous cellular differentiation proteins. Chen et nl. (1981), on the other hand, demonstrated that in the case of the feline sarcoma virus (FeSV), cells of embryonal layers other than the mesoderm could also be transformed, and they expressed antigenically indistinguishable transformation-associated polyproteins designated as gag-x. Thus alternative pathways of neoplastic transformation must also exist. A number of nonallelic Drosophilu genes were found to induce, in the mutated state in a particular cell type, one and the same tumor phenotype. For instance, six independent mutant alleles, representing different loci, cause malignant neuroblastoma (see Section IV,B, 1 ; Table I), which implies that the six wild-type gene products control six developmental steps leading from an optic neuroblast to a fully differentiated optic neuron. A mutation in any one of these six genes results in a neuroblastoma. The situation is analogous in the case of the different retroviral oncogenes. The various v-ones causing sarcomas differ in their nucleotide sequence and thus in their function. For instance, the src genes of both the Fuginanii sarcoma virus and RSV cause sarcomas in chicken fibroblasts despite their differing genomic structure (Bishop, 1982). Thus, as in Drosophilu (see earlier), different cellular homologues to retroviral oncogenes also seem to be involved in the control of specific differentiation steps in one and the same cell type. Experiments using temperature-sensitive RSV showed that the transformation as well as the maintenance of the transformed state of fibroblasts depends on the continuously functioning v-src gene product pp60"" present in excessive amounts (for review, see Bishop, 1982). In Drosophila, in the case of the l(2)gl alleles and the l(2)gl deficiency, the mechanism seems to be different. Here, the absence of a gene product appears to be responsible for the malignant transformation and not its excessive synthesis, because the deficiency of the gene locus shows the most extreme tumor phenotype. Similarly, in the case of the 1(3)mbt'"-l mutant the lack of the functional gene product from the beginning of embryonic life up until the middle of the third larval instar is responsible for the brain and imaginal disc tumors. Retroviral studies suggest that functional cellular genes can be subject to different regulation, depending on whether they are in the cellular or the

66

ELISABETH GATEFF

viral genome. The excessive production of a cellular protein by the viral gene causes malignant transformation (Hunter and Sefton, 1980). This new, exciting, and simple concept of cancer development will have to allow for other types of genetic mechanisms as well, because there are also slow-transforming RNA tumor viruses that do not possess oncogenes, but despite this cause malignant neoplastic transformation. Nee1 ef al. (1981) demonstrated for the avian and murine leukemia viruses that they activate a cellular gene through the integration of the provirus adjacent to the c-myc, the cellular counterpart of the MC29 viral oncogene. The transcription of the gene begins at the proviral promoter, which results in enhanced gene expression and malignant neoplastic transformation (Hayward et al., 1981). The studies just discussed provide compelling support for the existence in animals of phylogenetically highly conserved genes that are causally related to the malignant neoplastic transformation of cells. However, because most cancers are not of viral origin, the question arises whether the postulated cellular oncogenes are primarily involved also in cancers lacking viruses. The approach taken by a group of investigators and its relation to the Drosophila tumor system is discussed next.

VII. Transfection and Vertebrate Tumor Genes: A Comparison with Drosophila

The coordination of gene activities in a cell resulting in what we call development is still largely obscure. For instance, during Drosophifaoogenesis, between 200 and 600 genes must be involved (King and Mohler, 1975). Most of these genes transcribe, however, only very few mRNA molecules per cell and are thus not yet amenable for investigation. These genes are, however, the gears that act time and tissue specifically, and drive the cells into their final differentiation. Attention is beginning to focus on this large gene family, some of which, when abnormally regulated or mutated, cause a halt of differentiation and continuous malignant growth (see Section IV). The study of this kind of genes in a complex genetic system, such as that of humans or of any other animal that does not possess a well-known genetic makeup, meets with immense difficulties. Nevertheless, a method is available for probing genes of the rare mRNA type (Weinberg, 1981). The method of gene transfer or transfection allows the study of genes in a cellular environment away from the regulatory mechanisms of the host cells in which they normally function. The combination of this method with an in uirro transfection focus assay has given further support for the notion that the genome of vertebrates contains

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potential transforming genes (Anderson et al., 1979; Cooper et al., 1980; Shih et al., 1979; Cooper and Neiman, 1980; Krontiris and Cooper, 1981 ; Shih et al., 1981; Shilo and Weinberg, 1981a; Lane et al., 1981; for review see Weinberg, 1981; Willecke and Schafer, 1982). Transformation by transfection of NIH-3T3 mouse cells with genomic DNA is assumed to result from the activation of a cellular gene with transforming potential. This may occur, for instance, if sequences needed for the regulation of a cellular transforming gene are removed by the shearing of the DNA, and the gene is thus expressed differently (Cooper et al., 1980; Krup-Kovalinkova and van den Berg, 1980). Blair et al. (1981) showed data that agree with the foregoing interpretation. Clones containing long terminal repeats (LTR) from Moloney sarcoma virus (M-MSV) provirus to c-mos, the cellular homologue of the M-MSV specific sequence v-mos, transform cells as efficiently in the transfection focus assay as cloned v-mos together with the LTR of the virus. The experiments suggest that LTR-like elements on the genome may activate cell sequences whose abnormal expression leads to malignant transformation. Transfection with high-molecular-weight DNA from three human cell lines shows that in each case different DNA sequences are responsible for the transformation of the individual tumor cell lines (Murray et al., 1981). Similar results were obtained by Lane er al. (1981) with high-molecularweight DNA from ( a ) mammary tumor virus-induced mouse mammary carcinomas, (b) carcinogen-induced mouse mammary tumors, and (c) a spontaneous human mammary tumor cell line. All but one tumor DNA exhibited high transformation efficiency. The transfection studies just mentioned, performed with the DNA of a variety of mouse, chicken, and human tumors that had been induced either by carcinogens or by viruses, suggest that during transfection, tissue-specific cellular genes become abnormally regulated. At the moment only speculations can be made concerning the role of these genes in the cells. A reasonable suggestion, which has been expressed by all investigators in the field, is that this family of genes is involved in growth control and/or the establishment of the differentiated state and its maintenance. The Drosophila tumor genes have shown just this for more than 10 years. All Drosophila genes causing tumors in the mutated state are developmental genes acting specifically in one or, at the most, two cell types in which they surely must have important functions during normal development (Gateff, 1978a,b,c). In relation to the above transfection studies, the mutagenic effect of exogenous DNA in Drosophila deserves to be mentioned. DNA of different viral and nonviral origin induces increased rates of germ-line mutations when fed to male flies. These mutations are restricted to a small number of

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specific loci (for review see Gershenson, 1980). Moreover, the mutation spectrum is different from that induced by conventional mutagens, most of which cause gross genomic rearrangements. Exogenous DNA, in contrast, shows exclusively single gene mutations or at the most small deletions that cannot be detected on the giant chromosomes. The visible and recessive-lethal mutations involve only a few specific loci. For example, on chromosome I1 only about 12 loci were mutated, each of which showed a number of alleles, whereas mutagens induce hundreds of mostly nonallelic recessive-lethal mutations. Transfection with Drosophila DNA, on the other hand, showed many nonallelic lethal mutations. The mutants induced by exogenous DNA are often unstable, frequently reverting to the wild type. Reversions taking place in the germ line produce wild-type flies, whereas reversions in somatic cells give rise to mosaic animals. It was further found that only high-molecular-weight DNA is mutagenic and that premeiotic germ cells are more susceptible to the foreign DNA than postmeiotic germ cells (Gershenson, 1980). Transfection of mutant Drosophilu embryos with wild-type DNA yielded repair of the mutant phenotype (Fox and Yoon, 1966). The genetic analysis of 3 of the 51 “transformed” stocks showed that the introduced wild-type allele inserted itself on the chromosome at the proper site without the excision of the mutant allele (Fox et a/., 1971). Furthermore, DNA from the aforementioned transformed stocks induced second-step “transformation” at a frequency resembling that of the original transformation (Fox et al., 1975). The transfection studies with exogenous and homologous DNA in Drosophilu show, in parallel with the above transfection experiments, a high degree of specificity of the mutagenic effect. Unfortunately, the cause for the developmental arrest in the different lethal alleles was not investigated. Another similarity seems to exist between the specific behavior of exogenous DNA in the vertebrate and Drosophila transfection studies just mentioned. In both, no species and tissue barriers seem to exist. Shih et a/. (1981) demonstrated, for instance, that DNAs obtained from human, rabbit, and mouse bladder carcinoma and mouse glioma lines are able to induce transformation of NIH-3T3 mouse fibroblasts in uitro upon transfection. Thus the transforming genes not only act across tissues, but they also seem to overcome the species barrier. The situation is similar in the case of the DNAs of two human bladder carcinoma cell lines, which induce transformation in mouse fibroblasts (Krontiris and Cooper, 1981). Nevertheless, one discrepancy arises when one compares these observations to the tissue specificity of the Drosophika genes (see Section 1V) and of the c-mar, c-erb, and c-myc oncogenes described by Beug rt al. (1979), Graf er a/. (1980), and Durban and Boettiger (1981). Thus caution will have

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to prevail in drawing conclusions before the molecular basis of the observed phenomena is unveiled. VIII. Concluding Remarks

Among the approximately 5000 genes on the Drosophila genome, 25 are presently known to be causally involved in the development of malignant and benign neoplasms (Table I). The wild-type alleles of these genes control particular pathways of development in specific tissues or cells at particular times (see Section VI). The cells and tissues involved include the adult optic neuroblasts, the imaginal discs, and the primordial blood cells in the larva, the male and female gonial cells in the adult, and neuroblasts, blood cells, and other not yet identified cell types in the embryo. The mutations originated either spontaneously or after treatment of animals from wild-type laboratory populations with mutagens. However, the phenomenon is not solely a laboratory one; more than 78 l(2)gl alleles have been found in natural Drosophila populations. Moreover, at least one other Drosophila species, D. hydei, exhibits a homologous Z(2)gl locus, which in the mutated state shows a similar tumor phenotype. Normal and ts alleles have been isolated. Thus Drosophila demonstrates unequivocally that single mutated genes cause malignant or benign neoplastic transformation of specific cell types by interfering with their differentiation. Two research fields, (1) retroviral oncogenes and (2) transfection with DNA from normal and tumorous tissues, have provided compelling evidence that this may be true for higher animals as well (see Sections V, VI, VII, and VIII). ACKNOWLEDGMENTS The skilled technical assistance of D. Willer and I. Brillowski are gratefully acknowledged. I am also grateful to Professor K. Sander for providing the research facilities. The research was generously supported by the Deutsche Forschungsgemeinschaft, SFB 46. M y thanks to S. Chambers for typing the manuscript.

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Shilo, B.-Z., and Weinberg, R. A. (1981a). Nature (London) 289,607-609. Shilo, B.-Z., and Weinberg, R. A. (1981b). Proc. Natl. Arad. Sci. U.S.A. 75,6789-6792. Shimotohno, K., Mizutani, S., and Ternin, H. M. (1980). Nature (London) 285,550-554. Shrestha, R. (1979). Doctoral Thesis, Fakultat fur Biologie, Albert-Ludwigs-Universitat, Freiburg i. Br., FRG. Shrestha, R., and Gateff, E. (1982a). Develop. Growth Dtfler. 24,64-82. Shrestha, R., and Gateff, E. (1982b). Develop. Growth Dtfler. 24, 83-98. Smith, P. A., and King, R. C. (1966). JNCI, J . Narl. Cancer Inst. 36,455-463. Sokolova, K., and Golubovsky, M. D. (1978). Drosophila Inform. Sew. 53, 195-196. Sparrow, J. C. (1978). In “The Genetics and Biology of Drosophila,” (M. Ashburner and T. R. F. Wright, eds.), Vol. 2B, pp. 277-307. Academic Press, New York. Spector, D . H., Varmus, H. E., and Bishop, J. M. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 4102-4106. Spradling, A. C., and Rubin, G . M.(1981). Annu. Rev. Gener. IS, 219-251. Srdid, Z., and Frei, H. (1980). Differenriation 17, 187-192. Stanfield, S., and Helinski, D . R. (1976). Ce/l9,333-345. Stanfield, S., and Lengyel, J. A. (1979). Proc. Narl. Acad. Sci. U.S.A. 76,6142-6146. Stark, M. B., and Marshall, A. K. (1930). J . Am. Inst.Homeopatho1. 23, 1204-1206. Starlinger, P., and Saedler, H. (1976). Curr. Top. Microbiol. Immunol. 75, 111-152. Stehelin, D., Guntaka, R. V., Varrnus, H. E., and Bishop, J. M. (1976a). J . Mol. Biol. 101, 349-365. Stehelin, D., Varmus, H. E., Bishop, J. M., and Vogt, P. K . (1976b). Nature (London) 260, 170- 173. Stewart, M., Murphy, C., and Fristrorn, J. (1972). Develop. Eiol. 27,71-83. Strobel, E., Dunsmuir, P., and Rubin, G . M.(1979). Cell 17,429-439. Sutcliffe, J. G., Shinnick, T. M., Verma, 1. M., and Lerner, R. A. (1980). Proc. Narl. Acad. Sci. U.S.A. 77,3302-3306. van Beveren, C., Goddard, J. G., Berns, A., and Verma, 1. M. (1980). Proc. Natl. Acad. Sci. U.S.A. 77,3307-331 1. Vennstrom, B., and Bishop, J. M. (1982). Cell, submitted. Wang, L.-H., Snyder, P., Hanafusa, T., and Hanafusa, H. (1980). J. Virol. 35,52-64. Weinberg, R. A. (1981). Eiochim. Biophys. Acra 651,25-35. Willecke, K . , and Schafer, R. (1982). In “Somatic Cell Genetics” (C. T . Caskey, ed.), Plenum, New York (in press). Williams, J. M. (1981). Drosophila Inform. Serv. 56, 158-161. Wong-Staal, F., Dalla Favera, R., Franchini, G., Gelmann, E. P., and Gallo, R. C. (1981). Science 213,226-228. Young, M. W. (1979). Pror. Narl. Acad. Sci. U.S.A. 76,6274-6278.

TRANSFORMATION-ASSOCIATEDTUMOR ANTIGENS Arnold J. Levine Department of Microbiology, School of Medicine, State Universityof New York. Stony Brook, New York

I. Introduction .............................. .... A. Why Do Tumors Express Tumor Antigens? ............................. B. Are These Tumor Antigens Useful in Sorting out the Primary Events of Tumorigenesis? . . . . . . . . . . .............. ..................... C. Tumor Antigens as Diagnostic Tools D. Viral and Cellular Associated Tumor ................. 11. Simian Virus 40 . . . . . . . . . . . . . . ...........................

B. Small t-Antigen-Associated Proteins, 56K and 32K ....................... 111. Adenovimses. ............................................... IV. Epstein-Barr Virus . . . . . ................................ ells (Meth A) ......... V. Methylcholanthrene-Indu VI. Abelson Virus . . . . . . ............................... VlI. Rous Sarcoma Virus ................................ VIII. Teratocarcinomas ................................... IX. Conclusions. ................................ A. A Classification of Tumor Antigens ....................................

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

References . . . . . . .

75

15 80 80 80 81 82 84 86 88 91 93 96 97 100

100 103 104

1. Introduction

A. WHYDo TUMORS EXPRESS TUMORANTIGENS? Tumor antigens are experimentally defined as chemical moieties, associated with a tumor in an animal, that elicit an immune response in the host. The tumor antigen may reside on the cell surface and result in transplantation rejection of the tumor, in which case it may be termed a tumorspecific transplantation antigen (TSTA). Alternatively, the tumor antigen may be localized intracellularly and induce an immune response by virtue of cell death or tissue necrosis. Some tumor antigens will induce antibody production in animals bearing a syngeneic tumor graft or even with an autochthonous tumor graft. There are, however, several different reasons a 15 ADVANCES IN CANCER RESEARCH, VOL. 37

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006637-8

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tumor antigen may elicit an immune response in spite of the fact that the tumor tissue is syngeneic with the host :

1. Alteration of a normal molecule on the surjace of the cell can produce a new antigenic determinant. Some types of transformed cells, like human adenocarcinomas, appear to contain glycolipids that lack the terminal sugar residue (glucosamine) found in the gangliosides of normal cells (Tal, 1965; Hakomori, 1971; Burger, 1971). In this case tumorigenic cells contain lower levels of the glycosyl transferases responsible for the addition of terminal sugars to glycolipids of the membrane (Grimes, 1970), and so the altered terminal sugar groups of these glycolipids become antigenic (Hakomori and Murakami, 1968). The appearance of Forsman-like antigens in some transformed cells can be explained in this way (Robertson and Black, 1969). Similarly, the increased levels of proteases (Unkeless et al., 1973; Ossowski et al., 1973) or glycosidases detected in transformed cells (Warren, 1969) may expose to the immune system antigenic determinants that previously were buried in the cell membrane (Uhlenbruck et al., 1970; Burger, 1969). Thus glycoproteins or glycolipids, which are altered because the tumorigenic cell has decreased or enhanced enzymatic activities (Levine, 1973), can then serve as tumor antigens. 2. Several types of tumors have been shown to express proteins or glycoproteins that are predominantly found in,fetal tissue. The increased expression of alpha fetoprotein in humans or animals with hepatomas (Abelev et a/., 1979) or of carcinoembryonic antigen (Neville and Laurence, 1974) in some colon carcinomas is presently utilized in diagnostic tests in humans. Serum from multiparous females can react with some tumor antigens (Coggin and Ambrose, 1979), or immunization of animals with stage-specific fetal tissues can activate a cell-mediated immunity, resulting in tumor rejection upon subsequent challenge of that animal (Coggin and Ambrose, 1979). Antigens which are expressed solely or predominantly durung fetal development may indeed be recognized as foreign by a mature immune system and therefore qualify as tumor antigens. The debate over whether to classify these antigens as reexpression of stage-specific fetal gene markers or an antigens associated more correctly with cell proliferation in either the fetus or adult animal, still continues (Hirai, 1979; Stillman and Sell, 1979). The available evidence suggests that adult liver can synthesize high levels of alpha fetoprotein even in the absence of DNA synthesis (i.e., growth) (Watanabe et a f . , 1975). On the other hand, the so-called embryonic form of thymidine kinase (Bull et al., 1974) clearly is related to the requirement for cell replication and is regulated by or with the cell cycle or cell growth (Postel and Levine, 1975). A number of isozymes such as glucose-ATP phosphotransferase, alkaline phosphatase, pyruvate kinase, and aldolase have both specificity of tissue

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distribution and adult versus fetal forms (Stillman and Sell, 1979). As such, they certainly qualify as tumor-specific enzyme markers and could under the appropriate conditions be tumor antigens. 3. Increased concentrations of normal cellular protein in transformed cells : Recently a wide variety of transformed cell lines have been shown to express high levels of a cellular protein termed p53 (53,000 MW) (Lane and Crawford, 1979; Linzer and Levine, 1979; DeLeo et al., 1979) which is found in much lower levels in nontransformed cells in culture (Linzer et al., 1979). Syngeneic mice hyperimmunized with a chemically transformed cell line (Meth A) produce antibodies to p53 (DeLeo et al., 1979). Similarly, BALB/c mice bearing tumors derived from an injection of SV40-transformed BALB/c-3T3 cells also produce anti-p53 antibodies (Linzer and Levine, 1979). Both the Meth A- and SV40-transformed BALB cell lines contain higher levels of p53 (10- to 100-fold higher) than normal cells (DeLeo et al., 1979; Linzer and Levine, 1979). This brings up the possibility that increased levels of a normal cellular protein can now act as an antigen and result in antibody production. In the case of SV40-transformed cells, the p53 protein is physically complexed with a viral protein, the SV40 large T antigen (Lane and Crawford, 1979; Linzer and Levine, 1979). 4. It is possible that the viral-cellular protein multimeric complex presents new antigenic determinants to the immune system that result in antibody production against the p53 antigenic determinants. This carrier-haptene (viral-cellular protein complex)-like concept could also result in the creation of a new tumor antigen (Linzer and Levine, 1979). Alternatively, increased levels of a cellular protein normally found in low levels might also trigger an immune response. 5. Perhaps the best understood case of why tumors present new antigens to the immune system is when the tumor cells contain new or rearranged genetic injormation, peculiar to the tumor cell. This phenomenon happens under normal circumstances, as immunoglobulin V and C genes create new idiotypic determinants which then may be recognized as foreign by B or T cells. Indeed this type of network theory of the immune system has been suggested to play a role in normal regulation of antibody production (Jerne, 1971). The rearrangement of genetic information, mediated by insertion elements (IS elements), has been suggested as a major cause of human cancer, possibly promoted by carcinogens or environmental factors (Cairns, 1981). The movement of a genetic element could either create a new cellular gene product or activate the synthesis of a gene product normally not produced in an adult animal. In the best documented case, with avian long latency leukemia viruses, a virus or a part of a virus (IS element-like) can activate new cellular genes by virtue of its integration into the cellular genome in the vicinity of that cellular gene (Nee1 et al., 1981; Payne et al., 1981). This is

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presently believed to be the mechanism underlying the causes of long latency leukemias caused by virus infection in chickens (Nee1 et a/., 1981 ; Payne et al., 1981). The clearest case of why a new tumor antigen is expressed in a tumor results from the acquisition of new genetic information introduced by viruses. The virus may carry a copy of genetic information either identical or closely related to a normal cellular gene, as is the case with some of the RNA tumor viruses (Tooze, 1981). Alternatively, the virus may introduce new genetic information into a tumorigenic cell that is not detectably related to any genes in the cellular genome, as is the case with the DNA tumor viruses (Tooze, 1981). Rous sarcoma virus contains a gene (pp60v-src)that is closely related, but not identical, to the cellular gene (pp6OcpSrc) from which it likely was derived. The viral or v-src gene product has a number of peptides which differ from the cellular or c-src protein (Karess and Hanafusa, 1981). 6. Some chickens bearing sarcomas induced by this virus produce antibodies to pp60v-src,perhaps because of these direrences in the viral and cellular src proteins. However, it is clear that antibodies are not only made to the differences between viral and cellular src gene products in such animals. Newborn rabbits injected with the appropriate Rous sarcoma virus strain develop tumors and make antibodies against the pp60 chicken v-src gene product (Brugge and Erikson, 1977). These tumor-bearing sera (TBR sera) not only recognize the viral pp60'-"" but react with cellular chicken or more important rabbit c-src found in normal cells (Collett et al., 1978). Thus the rabbit produces antibodies to antigenic determinants common to its own pp60"-"", perhaps because a variation of its own cellular protein was presented to the immune system. In this context, the levels of pp60'-src in a tumor cell are much higher than that found in a normal cell (pp60c-src) (Collett et al., 1978), and these enhanced levels may also play a role, as appears to be the case with the p53 protein. 7. The D N A tumor viruses like simian virus 40 (SV40), adenovirus, or Epstein-Barr virus (EBV) introduce a new genetic information into the tumor cells and express viral proteins that are clearly and classically foreign antigens to the immune system of the host. These tumor antigens, found on both the cell surface (TSTA) (Tevethia and Tevethia, 1977) and intracellularly, can result in transplant rejection that is specific to the virus but not individual tumors or even the species of the tumor host cell. These are the most clearly understood examples of why tumors express foreign antigens and the host then mounts an immune response. This brief summary demonstrates that the origins of tumor antigens peculiar to tumor tissue may be quite diverse: (1) alterations of normal cellular components as a result of changes in the tumor cell physiology; (2)

TABLE I CLASSIFICATION OF TUMOR ANTIGENS' Tumor antigen classes Unique to a tumor

Transforming agent SV40 (2.5 kb early region genes)

Unique to a class of tumors

Specific to a transforming agent

1. > p53 levels 2. T-p53 complex

-

1. >p53 levels 2. Elb-58K-p53 complex

-

42K,28K 2. Elb*-58K,15K 1. EBNA, 48K, 65-72K 2. LDMA* (in cirro)

1. > p53, levels 2. 48K-p53 complex

1. p120*

1. >p53 levels

1. pp60'-s'c

1. >p53 levels

1. Large T antigen*

-

Common to many tumors

2. Small t antigen 3. t-56K-36K complex Adenovirus (2.9 kb early region genes Ela, Elb)

-

EBV (10% of viral genome expressed in transformed cells)

-

A k l s o n virus (I 5.5 kb, derived from the cellular genome)

-

RSV ( 21.8 kb, derived from the cellular genome)

-

1. Meth A*

Methylcholanthrene carcinogen (Meth A) Teratocarcinoma (genetic predisposition)

-

1. Ela-54K,52K,48K,

Developmental antigen* (B cells)

1. Gt-l,2,(B6)* (EC cells)

-

1. >p53 levels

-

1. > p53 levels

~

A summary of the tumor antigens derived from the seven different systems considered in the text. The tumor antigens are classified (Section IX, A) in one of four categories and the antigens involved or responsible for tumor transplantation rejection are indicated by an asterisk (*). These TSTAs can be localized in any of the three restricted classes of tumor antigens. To date there are no good examples of TSTA activities common to all tumors. A-placed in a box does not imply that an antigen is absent. Indeed SV40-transformed cells have been shown to express stage-specific fetal antigens (Coggin and Ambrose, 1979). In this case, the-indicates the poorly characterized nature of these fetal antigens. and therefore the unclear position of these antigens in this classification. For Abelson virus-transformed cells both the developmental antigen and p120 are on the cell surface and one or both of these antigens may be responsible for tumor transplant rejection.

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reexpression of fetal- or growth-related proteins in an adult animal; (3) increased levels of a normal cellular protein in tumor tissue; (4) physical association of normal cellular protein and a foreign protein in a complex resulting in antibody to the normal cellular protein (carrier-haptene-like) ; ( 5 ) presentation of a slightly altered cellular protein (pp60v-src)resulting in antibody production to the normal cellular protein (pp6OcpS");(6) rearrangement of the genetic information in tumor cells by IS elements or viruses (e.g., avian leukosis virus, ALV) could result in a new antigen being produced or the reexpression of a cellular protein not normally found in the adult animal ; (7) The acquisition of foreign genetic information (DNA viruses) in tumor cells leading to the expression of viral (foreign) proteins that trigger an immune response (see Table).

B. ARETHESETUMOR ANTIGENS USEFUL IN SORTING OUT THE PRIMARY EVENTS OF TUMORIGENESIS? One can see from this discussion that tumor antigens may be a fingerprint or a reflection of what is happening at the genomic level in a tumorigenic cell. Antibodies produced in tumor-bearing animals can identify a virus present in tumor cells, localizing new cellular genes which are turned on because of genetic rearrangements or proliferation of that cell. Altered carbohydrate structures of cellular glycolipids or glycoproteins may reflect the physiological changes of a tumor cell, which may then be useful in understanding control of cell division. It is equally true, however, that some of these antigens may be irrelevant to either the primary or secondary causes of the tumor or even the control of tumor growth. Sorting out the important variables involved in tumor antigen expression becomes one of the essential tasks of present-day studies. C. TUMORANTIGENS AS DIAGNOSTIC TOOLS Whether tumor-specific antigens reflect primary events in tumor formation or not, they can be useful in therapy or diagnosis of the tumor. Monoclonal antibodies to tumor-specific antigens have been used in attempted therapy (Sears et al., 1982a) diagnosis (Koprowski er al., 1981), or for localizing metastatic lesions. Only further studies will determine if these approaches will have practical significance in tumor management at the clinical level (Herlyn et al., 1982; Sears et al., 1982b). D. VIRALAND CELLULAR ASSOCIATED TUMOR ANTIGENS At the basic science level the study of tumor antigens is leaving the descriptive phase of characterizing proteins or glycolipids expressed in tu-

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81

morigenic cells. Antibodies generated by tumor-bearing animals are being used to identify functional properties of tumor antigens like pp60"-"c and its role as a protein kinase (Collett and Erikson, 1978; Levinson et al., 1978) and not just an antigen. As these tumor antigens are better characterized, they have been found to be associated with cellular or viral proteins. These complexes of viral and cellular proteins detected in cell extracts presumably reflect functional activities or interactions in a tumorigenic cell. The isolation, characterization, and functional significance of these protein complexes is under intensive study at this time. This article will focus on cellular proteins that have been found to be associated with viral tumor antigens in cell extracts. The biochemical and functional properties of the viral proteins have been, in some cases, well characterized by the availability of viral mutants. We can say with some confidence that these viral proteins play a primary role in cellular transformation (see table). As such, the cellular proteins that interact with these viral tumor antigens may well play a central role in transformation. In addition, this article will include two examples of nonviral transformed cells: methylcholanthrene-induced transformation and teratocarcinomas. These examples are included for two reasons: (1) these tumors and transformed cells share a cellular tumor antigen (p53) in common with the viral transformed cells; and (2) they represent examples of tumor-specific transplantation antigens (Meth A antigen, Gt-1 antigen) detected by transplantation rejectien methods. This class of antigen, for which antibody is usually not available as an experimental tool, nevertheless provides new insights into the approaches and results of tumor immunology. Thus tumor antigens, detected by both the humoral and cellular responses of the immune system, will be discussed in this article (see table). II. Simian Virus 40

Simian virus 40 (SV40) encodes the genetic information for two gene products expressed at early times after productive infection or in transformed cells (see table). The gene A product, or large tumor antigen (T antigen) is about 94,000 MW (94K) in SDS-polyacrylamide gels (Tegtmeyer, 1974). The second protein, or small t antigen, has a molecular weight of 17,000 (17K) (Prives et al., 1977; Crawford et al., 1978) and shares common amino acid sequences with large T antigen at its N-terminal end (Paucha et al., 1978). The nucleotide sequences that contain the information for the small t antigen are found beteen 0.67 and 0.54 map units on the genome (Berk and Sharp, 1978), whereas the genetic information for the large T antigen is located in two noncontinuous segments of the SV40 genome between 0.67-0.59 and 0.54-0.14 map units (Berk and Sharp, 1978). Temperaturesensitive and deletion mutants in the large and small tumor antigen genes (Tegtmeyer et al., 1975; Rundell et al., 1977; Shenk et al., 1976) have been

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utilized to demonstrate that both proteins are required for a fully transformed phenotype (Tegtmeyer, 1975; Brugge and Butel, 1975; Kimura and Itagaki, 1975; Martin and Chou, 1975; Osborn and Weber, 1975; Sleigh et al., 1978). Viral DNA derived from restriction enzyme fragments of the genome located between 0.14 and 0.72 map units will transform cells in culture (van der Eb, 1979), indicating that the early viral genes (T and t antigens) are sufficient for the production of a fully transformed cell phenotype. A. LARGET-ANTIGEN-ASSOCIATED PROTEIN, p53 Animals bearing SV40-induced tumors produce antibodies to both the large and small tumor antigens (Black et al., 1963; Tegtmeyer, 1974; Prives et al., 1977), which have been utilized to detect these proteins by immunoprecipitation reactions. When SV40-transformed mouse cells were labeled with [35S]methionineand the soluble proteins were incubated with antisera from animals bearing SV4O-induced tumors, three proteins were reproducibly immunoprecipitated : the large T antigen (94K), small t antigen (17K), and a cellular protein, termed p53 (53K) (Linzer and Levine, 1979; Lane and Crawford, 1979). It was shown that p53 is a cellular protein because the same protein was detected in embryonal carcinoma cells not infected with SV40 (Linzer and Levine, 1979). Nontransformed cells in culture (primary or 3T3 cells) contained lower levels (1/10-1/100th) of p53 than detected in the homologous transformed cell line (Linzer et al., 1979). Indeed a wide variety of murine-transformed cells were found to express high levels of p53 (De Leo et al., 1979; Maltzman et al., 1981) independent of the transforming agent. Homologous proteins related to p53 were detected in SV40-transformed cells derived from human, monkey, mouse, rat, and hamster cells (Linzer and Levine, 1979; Kress et al., 1979; Melero et al., 1979; Smith et al., 1979; Simmons et al., 1980; Gurney et al., 1980), and in human cells derived from a wide range of human tumors (Crawford et a]., 1981; Dippold et al., 1981). Thus p53 is a cellular protein expressed in high levels in transformed cells, independent of the transforming agent, and a homologous protein can be detected in many different species of host cells (see table). In SV40-transformed cells, the cellular p53 tumor antigen is physically complexed with the SV40 large tumor antigen (Lane and Crawford, 1979; Linzer and Levine, 1979). Monoclonal antibodies directed against the SV40 large T antigen, and which do not cross-react with p53, immunoprecipitate the large T antigen and coimmunoprecipitate the 'associated p53 protein (Linzer and Levine, 1979). Conversely, monoclonal antibodies to p53 immunoprecipitate that protein and coimmunoprecipitate the viral large T antigen (Gurney et al., 1980; Maltzman et al., 1981). The T antigen-p53

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complex sediments rapidly and in a heterogeneous fashion (McCormick and Harlow, 1980), suggesting a high-molecular-weight, multimeric mixture of at least the SV40 T antigen and p53 proteins. In transformed mouse cells most but not all of the SV40 T antigen can be found in association with p53, while all of the cellular p53 molecules reside in this complex (McCormick and Harlow, 1980; Oren et al., 1981). Because serum from animals bearing SV4O-induced tumors contain antibody to the SV40 large T antigen (Tegtmeyer, 1974), it remained possible that the tumor-bearing animals produced antibody to the SV40 large T antigen, which then coimmunoprecipitated the associated p53 protein. In this case no antibody would be made against p53 itself. That this was not the case was demonstrated by utilizing sera from animals bearing SV40-induced tumors to immunoprecipitate p53 from embryonal carcinoma cells which do not contain the SV40 T antigen (Linzer and Levine, 1979). In spite of one report to the contrary (Melero et al., 1979), the SV40 T antigen and p53 have no detectable cross-reacting antigenic determinants. Thus many animals bearing SV40-induced tumors produce antibodies to a normal cellular protein, p53 (Linzer et al., 1979). The possible reasons for this have been discussed in the previous section (1,A). Nontransformed cells as well as transformed cells express p53. The difference between these cells is that most transformed cell lines examined to date (but not all transformed cell lines) express 10- to 100-fold higher levels of p53 than their corresponding nontransformed cell counterpart (Linzer et al., 1979; Oren et af., 1981). In SV40-transformed cells the higher levels of p53 detected require a functional viral large T antigen (Linzer et al., 1979). SV40ts.4 mutants, defective in the large T antigen, regulate, in a temperaturedependent fashion, both the initial increase in the levels of p53 after virus infection and the maintenance of high levels of p53 in transformed cells (Linzer et al., 1979). Thus the SV40 large T antigen, known to be required for maintaining the transformed state (Tegtmeyer, 1975), also regulates the levels of a p53 cellular protein in these cells. Because many different transformed cell lines express high levels of p53 the mechanisms regulating the level of this protein in SV40-transformed cells are of some interest. SV40 T antigen could regulate the amount of p53 produced at the transcription level of the p53 gene, RNA processing, mRNA stability, translation of p53 mRNA, or even at post translational steps such as protein modification or stability of p53 protein (turnover). To investigate these possibilities the levels of p53 mRNA in SV4O-transformed (SV3T3 cells) and nontransformed cells (3T3 cells) were compared. Messenger RNA from 3T3 cells and SV40-transformed 3T3 cells (SVT2) were translated in a reticulocyte in uitro translation extract. Translation of p53 mRNAs was specifically measured by immunoprecipitation of the radioactively labeled p53 protein synthesized in uitro. The peptide maps of p53,

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ARNOLD J . LEVINE

synthesized in v i m or in vitro using mRNA from the same cell line, were similar (Oren et a/., 1981). The levels of the p53 immunoprecipitated protein synthesized in oitro were proportional to the amount of mRNA (over 1-10 pg) added to the reticulocyte reaction mixture. Therefore, the levels of p53 synthesized in vitro accurately measure the level of translatable p53 mRNA in a preparation extracted from different cell lines. When p53 mRNA levels in nontransformed 3T3 cells and SV40-transformed 3T3 cells were compared in this way, both cell types had about equal amounts of translatable p53 mRNA (Oren et ul., 1981). This was in spite of the fact that SV3T3 cells contain as much as 100-fold more p53 protein than the 3T3 cells when the protein was labeled with [3sS]methionine in vivo (in cell culture) (Linzer et al., 1979; Oren et al., 1981). The reason for this appears to be that the levels of p53 in 3T3 and SV3T3 are regulated at a posttranslational step. When 3T3 and SV3T3 cells were pulse labeled with [35S]methionineand then the cell culture was placed in unlabeled medium (chase period), the half-life of p53 protein was found to be 20-60 min in 3T3 cells and more than 22 hr in SV3T3 cells (Oren ef a/., 1981). Thus it appears that the rate of synthesis (and mRNA levels) of p53 is similar in 3T3 and SV3T3 cells, but p53 protein stability differs dramatically in these two cell lines, leading to differing levels of p53. Because antibody to p53 is used to detect and measure the levels of this protein in all these experiments, it remains possible that the p53 protein is not actually degraded with time, but is altered so as not to react with the antibody, or fails to be extracted from cells and would no longer be detected by this assay (Oren et al., 1981). There are at least two possible reasons that p53 stability (or reactivity) is enhanced in SV3T3 cells compared to 3T3 cells : (1) there is some suggestion that posttranslational protein modifications of the p53 protein from SV3T3 cells differ from those of other cells (Maltzman et a/., 1981) and this could alter the protein turnover of p53 in SV3T3 cells; or ( 2 ) SV40 T antigen, which regulates the levels of p53 in a transformed cell (Linzer el a/., 1979), is physically associated with p53 in SV3T3 cells and this protein complex could alter the stability or turnover of p53 in transformed cells (Oren et ul., 1981).

B.

SMALL t-ANTIGEN-ASSOCIATED PROTEINS,

56K

AND

32K

Deletion mutants in the SV40 small t-antigen gene have been employed to study the functions of this protein. Although the small t antigen is not required for virus replication in cell culture (Shenk et al., 1976), it is essential for efficient transformation of growth-restricted cells (Bouck et a/., 1978; Sleigh et al., 1978; Martin et a/., 1979). Microinjection of small t antigen into cells in culture results in the disorganization of actin cables in these

TRANSFORMATION-ASSOCIATED TUMOR ANTIGENS

85

cells (Graessmann et al., 1980), and this observation is consistent with the predominantly cytoplasmic location of small t antigen in infected and transformed cells. The SV40 small t antigen in both productivity infected and transformed cells is physically associated with two cellular proteins of 56,000 and 32,000 MW (Yang et al., 1979). If unlabeled small t antigen is added to an extract of [3 'Slmethionine-labeled uninfected cell proteins, and antibody to the small t antigen is employed to immunoprecipitate it, the labeled 56,000 (56K) and 32,000 (32K) MW proteins associate in v i m with the small t antigen and coimmunoprecipitate along with it (Yang et al., 1979). The same small t-antigen-56K-32K complex is detected in SV40-infected and -transformed cell extracts (Yang et al., 1979). Small t-antigen deletion mutants fail to produce a small t-56K-32K complex and these two cellular proteins are not detected by this immunoprecipitation assay (Yang et al., 1979). These experiments also tend to eliminate a role for the large T antigen, which is not altered by these small t-antigen deletion mutants, in associating with the 56K-32K proteins. The 56K cellular protein complexed with small t antigen is most likely different from the cellular p53 antigen that is associated with SV40 large T antigen. The small t-antigen deletion mutants fail to produce a t-56K-32K complex but have a normal large T-p53 complex (Linzer et al., 1979). In addition, p53 is a nuclear antigen, in both normal and SV40-transformed cells, whereas the 56K and 32K proteins have been localized in the cytoplasm (Rundell and Yang, 1981). Finally, antisera to the 56K and 36K antigens failed to immunoprecipitate p53 from embryonal carcinoma or chemically transformed cell lines (Rundell and Yang, 1981). Thus it is likely that both the SV40 large T and small t antigens associate with different cellular proteins and this presumably reflects a functional interaction. The small t antigens from the related papovaviruses, BK and polyoma, also form complexes with the cellular 56K and 32K proteins (Rundell et d., 1981). Virtually all of the antisera from animals bearing SV40-induced tumors contain antibody directed against the large T antigen. A smaller subset of these sera will immunoprecipitate the small t antigen. Some tumor sera also contains antibodies directed against the cellular p53 antigenic determinants (Linzer and Levine, 1979), and in rare cases antisera will contain antibodies that immunoprecipitate the 56K and 32K proteins from uninfected cell extracts (Rundell et al., 1981). Antibodies may be made against these cellular proteins because (a) they are complexed with viral proteins and therefore act as a stronger immunogen, (b) the cellular protein (p53) is present in higher than normal concentrations in tumor cells, (c) the proteins in normal cells and tumor cells may differ (modification for example) in some as yet

86

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undetected manner, or (d) abnormal tumor tissue necrosis exposes these proteins or fragments of these proteins to the immune system in a form not normally observed. Ill. Adenoviruses

The human adenovirus DNA sequences derived from the left-hand 8- 12% of the viral genome contain the genetic information that is sufficient for transformation of cells in culture (Graham et al., 1975; Sambrook et al., 1975; Flint et al., 1976). This portion of the viral genome has been subdivided into two regions, termed Ela and Elb, based on the two discrete transcriptional units encoded therein (Berget e f al., 1977; Chow et al., 1977) and two genetic complementation groups defined by host range mutants (Harrison et al., 1977; Jones and Shenk, 1979; Ross et al., 1981b). The E l a transcripts produce at least five proteins with molecular weights of 54,000, 52,000, 48,000,42,000, and 28,000 (Harter and Lewis, 1978).The E l b transcripts encode the information for a 58,000 and a 15,000 MW set of proteins (Gilead et al., 1976; Levinson and Levine, 1977a,b; Schrier et al., 1979; Ross et al., 1980a,b). Some or all of the Ela gene products are required to turn on the E l b gene products and other early adenovirus genes in a positive fashion (Berk et al., 1979; Jones and Shenk 1979; Ross et al., 1980a). One or more of the E l b gene products are required for viral DNA replication (Jones and Shenk, 1979), and modulate down the levels of adenovirus early proteins produced by the E2 and E3 viral genes (Ross et al., 1980a). Both Ela and Elb viral gene products are required for efficient transformation of cells in culture (Graham et al., 1978; Jones and Shenk, 1979) and are expressed in transformed and tumorigenic cells (Ross eta/., 1980b). For this reason, these proteins have been termed the adenovirus tumor antigens (see table). In adenovirus-transformed mouse, rat, and hamster cells, the El b-58K protein is physically associated with the same cellular p53 protein that is found in the SV40 T antigen-p53 complex (Sarnow et al., 1982). Several monoclonal antibodies to the p53 protein (Gurney et al., 1980; Coffman and Weissman, 1981) immunoprecipitate p53 and coimmunoprecipitate the adenovirus El b-58K protein. Conversely, monoclonal antibodies to the El b-58K protein immunoprecipitate that protein and coimmunoprecipitate p53 in that complex (Sarnow, unpublished results). Partial peptide maps of the murine p53 proteins that were complexed with either the SV40 large T antigen or the adenovirus El b-58K protein, demonstrated that these p53 proteins were either closely related or identical (Sarnow et al., 1982). Thus, based on both immunological and chemical (peptide maps) evidence, the SV40 large T antigen and the adenovirus El b-58K protein are both physically associated with the same cellular protein, p53, in their respective transformed murine cell lines (Sarnow et al., 1982).

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The adenovirus El b-58K-p53 complex and the SV40 large T-antigen-p53 complex share several properties. Both complexes are heterogenous in their size and mass (sedimentation rate is 25-30 S) and appear to be composed of multimeric combinations of at least these two proteins (McCormick and Harlow, 1980; Sarnow et al., 1982). In both the SV40- and adenovirustransformed cells, all of the p53 detected in these cells is in the form of a high-molecular-weightcomplex, whereas a variable proportion of the SV40 and adenovirus T antigens have been detected in a free or noncomplexed form (McCormick and Harlow, 1980; Sarnow et al., 1982). In the case of SV40-infected and -transformed cells, the increased levels and stability of p53 may be due to its physical association with T antigen (Oren et al., 1981), and so no free form of p53 protein would be expected. The enhanced levels of p53 in the adenovirus-transformed mouse cells, when compared to untransformed 3T3 cells, could also be due to the association of p53 with the Elb-58K viral tumor antigen. This has yet to be clearly demonstrated, however. The SV40 large T antigen and the adenovirus Elb-58K protein are both modified by the addition of phosphate to these proteins (Tegtmeyer, 1975; Levinson and Levine, 1977a,b). The phosphorylation of these two proteins is heterogeneous with some molecules containing more moles of phosphate per molecule than others (Tegtmeyer et al., 1977; Walter and Flory, 1979; Levinson and Levine, 1977a,b). The SV40 large T antigen associated with p53 appears to be more heavily phosphorylated than the free T-antigen species detected in the cell (Fanning et al., 1981). On the other hand, the adenovirus Elb-58K protein associated with p53 appears to contain less phosphate per molecule than the free or noncomplexed form of this E l b protein (Sarnow et al., 1982). These results suggest that posttranslational modifications of the viral T antigens could play a role in the association of viral proteins with cellular proteins and presumably influence their function. The association of SV40 and adenovirus tumor antigens with the cellular p53 protein is dramatically influenced by the species of host cell employed for these studies. In mouse, rat, or hamster cells the T antigen-p53 complexes are very stable and are readily demonstrated. In adenovirus-infected or -transformed human cells, most or all of the Elb-58K protein is not complexed to p53 (Sarnow et al., 1982). In SV40-infectedor -transformed human or monkey cells the T antigen-p53 complex either dissociates readily in uitro or higher levels of free T antigen and p53 are detected by immunoprecipitation of these soluble extracts (Gurney et al., 1980; Levine, unpublished results). In SV40-transformed human cells (SVSO), the stability of the T-antigen-p53 complex in crude extracts is significantly weaker than in SV40-transformed mouse cells (Levine, unpublished results), even though in freshly prepared lysates virtually all of the p53 is complexed to T antigen

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in both human and mouse cells. It appears likely then that the T-antigen-p53 complexes from human and monkey cell extracts have a lower stability than when the same viral T antigen is present in murine or rat cells. Because SV40 and adenoviruses have evolved for their replication in monkey and human cells respectively, the evolutionary significant host is of primate origin. This suggests that the low-stability, association-dissociation reaction, is a more significant one and reflects some function. It also remains possible that the unusually tight association of T antigen with p53 in mouse or rat cells leads to an abnormal situation, i.e., transformation rather than replication. This type of reasoning could explain why a lytic virus for one species can induce tumors in a different organism. The SV40 large T antigen and the adenovirus Elb-58K protein have several functions in common. Both proteins are involved in viral DNA replication (Tegtmeyer, 1972; Jones and Shenk, 1979), and both proteins can modulate early viral gene expression (Tegtmeyer, 1975; Ross et al., 1980a). In addition, the SV40 large T antigen can stimulate cellular DNA synthesis (Tjian et al., 1978) and modulate up cellular transcripts in infected or transformed cells (T. Schutzbank, R. Robinson, and A. Levine, unpublished results). The viral T-antigen-p53 complexes observed in transformed cells could act as intermediates in the DNA replication and/or gene regulation process reflecting the phenotypes of these viral tumor antigens. IV. Epstein-Barr Virus

The virion DNA obtained from different strains of Epstein-Barr viruses (EBV) or different cell lines transformed by EBV, is heterogeneous with regard to the number of repeated DNA sequences, deletions, or insertions and restriction enzyme polymorphisms (Raab-Traub et al., 1978;Dambaugh et al., 1980). For that reason, the major features of EBV DNA will be outlined with emphasis placed on describing the properties of the B95-8 virus isolate which has been extensively studied (Dambaugh et al., 1980; Skare and Strominger, 1980; Kintner and Sugden, 1979). The genome from this virus is about 115 x lo6 daltons (Raab-Traub et al., 1978; Dambaugh et al., 1980),linear, double-stranded DNA. Accordingly, molecular weight map coordinates from 0 to 115 have been assigned to identify regions of the genome. Both ends of the genome contain a variable number of direct tandem repeats (TRor terminal repeats) of about 500 base pairs each (Given et al., 1979), located at 0-1 map units and at 114-1 15 map units. About 10 x lo6 daltons from one end (10-26 map units) is located a second variable number of tandem internal repeats (IRor internal repeats). The I, region contains a 3000 base pair repeated sequence which is distinct in its polynucleotide sequence from the TR repeats (Dambaugh et al., 1980). These I , sequences

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then divide the remainder of the viral genome into two regions, a 9 x lo6dalton short unique DNA region ( U s , 1-10 map units) and a 80 x lo6dalton long unique region (U,, from about 26 or 28 to 110 or 114 map units). The B95-8 DNA differs from several other EBV isolates (P3HR1, W91, and AG876) in that it contains a 9 x 106-dalton (1 1,000 base pair) deletion in the UL region (at about 93-95 map units) (Dambaugh et al., 1980). This deleted DNA in B95-8 contains some nucleotide homology with B95-8 DNA located near the ZR region at about 28 map units (Dambaugh et al., 1980). Thus the UL regions of some EBV isolates contain regions of homology at 28 and 94 map units. It should be recalled that the TR and IRsequences can vary in size and so the length and map coordinates given herein are approximate. In transformed lymphocytes the EBV DNA is present in a circular episoma1form (Lindahl ef al., 1976) in copy numbers varying from 2 to 200 per cell (zur Hausen et al., 1970; Lindahl et al., 1974; Nonoyama and Pagano, 1973). Similarly, cell lines developed from biopsies of nasopharyngeal carcinomas contain circular EBV episomal DNA (Kaschka-Dierich et al., 1976). In lymphocytes transformed by EBV (IB-4 cell line), about 30% of the EBV circular genome is transcribed to produce detectable levels of RNA (King et al., 1980). Of this, only 10% of the EBV genome produces RNA species that are polyribosome associated (van Santen et al., 1981). At least six polyadenylated cytoplasmic RNAs have been identified and derive from (1) the ZR-UL region (5-30 map units), (2) the TR region between 110 and 03 map units of the circular genome (possibly UL-TR-US sequences), and (3) a U, transcript at 63-66 map units. Less abundant RNAs have also been detected from additional regions of the genome (van Santen et al., 1981). Similar transcriptional patterns have been described in several Burkitt tumor cell lines (King et al., 1981). Two low-molecular-weight RNAs (180 nucleotides long), called EBER-1, 2, which are detected as ribonucleoprotein particles in the cytoplasm (Lerner et al., 1981), have been mapped to the Us segment of the genome (van Santen et al., 1981;Lerner et al., 1981). Based on these studies, it is clear that only a small segment (about 10%) of the EBV genome encodes information expressed in the transformed or tumorigenic cell. Employing antisera from patients with either Burkitt’s lymphoma or infectious mononucleosis, several EBV-associated antigens have been described. An EBV nuclear antigen, termed EBNA (Pope et al., 1969;Reedman and Klein, 1973), is invariably associated with transformed cells, Burkitt’s lymphoma, and nasopharyngeal carcinoma. An EBNA species has been purified utilizing an acid-fixed nuclear binding assay (Luka et al., 1977), and the native antigen appears to be a high-molecular-weight (170,000-230,000 MW) basic protein derived from monomeric subunits of 48,000 MW (Luka et al., 1978; Baron and Strominger, 1978). When EBNA is purified utilizing

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mild procedures, the 48,000 MW subunit copurifies with a 53,000 MW protein (Luka et al., 1980). The 48,000 MW protein was only detected in EBVpositive cell lines, whereas the 53,000 MW protein was detected in a number of transformed cell lines with or without EBV present (Luka et al., 1980). The 53,000 MW protein, found in an oligomericcomplex with the 48,000MW EBNA protein, was shown to be similar to the p53 cellular antigen derived from the Meth A murine cell line (Jornvall et af., 1982). The Meth A-p53 and the SV40 T-antigen-p53 associated proteins are known to be similar or identical from their peptide maps (Maltzman et al., 1981). Thus it appears that the 48,000 MW EBNA can also be associated (and copurify) with a p53 protein, as is the case with SV40 and adenovirus tumor antigens. Recently, Strnad et al. (1981) have utilized human sera to identify an EBNA antigen by fluoroimmunoelectrophoresis and radioimmunoelectrophoresis. They detected a 65,000 MW antigen in Burkitt’s lymphoma cells but not in EBV-negative B-cell lines. The partially purified 65,000 MW antigen was an effective immunoadsorbant for EBNA antibodies in human sera (Stmad et al., 1981). Different EBV-positive cell lines showed some molecular weight heterogeneity (65,000-73,000 MW) for this antigen (Strnad et al., 1981). The relationship of this 65,000 MW EBNA to the 48,000 MW protein purified by Luka et al. (1980) is at present unclear. The 48,000 MW EBNA could be a proteolytic breakdown product of the larger EBNA. Alternatively, because EBNA was originally defined and detected using polyclonal human antisera, EBNA could well be a large number of different proteins. When monoclonal antibodies to human p53( R. Thomas, C. Sullivan, and A. Levine, unpublished results) are used to immunoprecipitate p53 from an EBV-transformed lymphocyte cell line (Raji), the p53 protein is detected along with another protein of 65,000 MW (L. Kaplan and A. Levine, unpublished results). At present it is not clear whether this second protein is coimmunoprecipitated in a complex with p53 or the monoclonal antibody reacts directly with it. The molecular weight of this protein (65,000), found in the anti-pS3 immunoprecipitate, agrees very well with the EBNA antigen detected in these same cells (Raji) by Stmad et al. (1981). Taken together, all of these results (Luka et al., 1980; Jornvell et al., 1982; Strnad et al., 1981 ; L. Kaplan and A. Levine, unpublished) suggest that EBNA moieties may be physically complexed, in an oligomeric form, with the same p53 antigen associated with the SV40 and adenovirus tumor antigens. Other EBV-associated antigens, such as the early antigen (Henle et al., 1970; Gergely et al., 1971), viral capsid antigen (VCA) (zur Hausen, 1976), membrane antigens (Klein et al., 1968), lymphocyte-detected membrane antigen (Svedmyr and Jondal, 1975), .and an 81,000 MW DNA-binding protein (Strnad et al., 1981), all have been reported. Like EBNA, all of these

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antigens were detected using polyclonal human sera or T lymphocytes. As such it is difficult to determine how many different proteins these antigens represent or, in all cases, whether or not the EBV genome encodes the genetic information for these antigens. Although antigens like EBNA are clearly correlated with the presence of the EBV genome, and have not been reported in the absence of the viral genome, no formal proof has been presented to date to show that EBV DNA encodes these antigens. The main reason for this ambiguity is that specific antisera to defined antigenic moieties of EBV proteins is lacking and the apparent levels of both viral transcripts (van Santen et al., 1981) and antigens (Strnad et al., 1981) are quite low in EBVtransformed cells. In addition, no reliable EBV mutants are available to correlate genetic alterations with the viral encoded proteins or functions. V. Methylcholanthrene-InducedTransformed Cells (Meth A)

Murine sarcomas, produced by some polycyclic hydrocarbons like methylcholanthrene, often express tumor-specific transplantation antigens (Klein et al., 1960; Old et al., 1962).A remarkable feature of these chemically transformed cells is that each independently derived tumorigenic cell line expresses an antigen unique to that cell line as measured by tumor transplantation immunity tests in syngeneic mice. For example, two independently derived methylcholanthrene-inducedBALB/c sarcomas, Meth A and CMS4, contain cell surface antigens that provide transplant immunity only when a BALBjc mouse is immunized with the homologous tumor (De Leo et al., 1977, 1978). Cytotoxic antisera prepared against Meth A cells reacted with the Meth A cell line but not 19 other BALB/c sarcomas (De Leo et al., 1977, 1978). Similarly, cytotoxic antibodies directed against the CMS4 sarcoma cell line reacted with only 2 of 20 BALB/c sarcomas. The Meth A and CMS4 antigens were not related to Moloney leukemia virus antigens and were not detected in a wide variety of normal or transformed cell types (De Leo et al., 1977, 1978). Thus chemically transformed cells express a cell surface antigen which is unique or specific to the cell line of origin. These unique antigens are readily identified by tumor transplantation tests in syngeneic mice or in some cases by cytotoxic antibodies (De Leo et al., 1977, 1978). Chemically transformed cell lines such as Meth A also express a nuclear antigen (p53) that, in contrast to the unique antigen, is common to a wide variety of transformed cell lines (De Leo et al., 1979). BALB/c mice immunized with the BALB/c Meth A sarcoma produce antibodies to a p53 antigen. The peptide map of the methionine-containing tryptic peptides of the Meth A p53 and the p53 associated with SV40 T antigen were very similar or identical (Maltzman et al., 1981). Monoclonal antibodies prepared against p53 detected (by immunofluorescence) this protein in 13 diverse murine-

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transformed cell lines but not in normal mouse fibroblasts, 3T3 cells, bone marrow cells, thymus cells, or embryo cells (Dippold et al., 1981). This monoclonal antibody cross-reacted with the homologous human p53 antigen, providing positive immunofluorescence in melanoma, renal cancer, breast cancer, and Burkitt’s lymphoma cell lines. Some normal human (but not the mouse) cell cultures were also positive by immunofluorescence (with this monoclonal antibody) for the presence of p53. Primary human kidney epithelium cultures showed positive nuclear immunofluorescence that declined in intensity over serial passage in culture (Dippold et al., 1981). Similarly, primary human fetal brain cells and primary human skin fibroblasts contained lower levels of p53, which were no longer detectable by the second passage in cell culture (Dippold et al., 1981). Dippold et al. (1981) interpret these results in support of the possibility that p53 in normal cells is related to active cell division and may be involved in the regulation of cell division. Milner and Milner (198 1) have come to a similar conclusion. Four hours after mitogenic stimulation of mouse spleen lymphocytes, increased levels of p53 were synthesized. This led to the suggestion that p53 is involved in the Go to G, transition or mitogenic transition (Milner and Milner, 1981) of lymphocytes. The fact that these studies, with normal human kidney cell cultures, were carried out with an immunofluorescent assay and a monoclonal antibody to p53 (Dippold et al., 1981) presents some possible problems. First, p53 is present and detectable in normal or primary mouse cultures at 1 to 10%of the levels observed in murine-transformed cells (Linzer et al., 1979). The fluorescent antibody test is difficult to quantitate, and so comparisons of normal and transformed cells by this test do not usually provide good quantitative results. Second, monoclonal antibodies can cross-react with unrelated proteins that may fortuitously share an antigenic determinant with the immunogen. For that reason, it would be useful to provide additional data (molecular weight of the protein, for example), which is not a feature of the fluorescent antibody assay. In spite of these difficulties, it may well be that primary normal mouse and human cell cultures differ in p53 levels and the high levels in human cells are a reflection of cell growth or cell cycle declining in subsequent culture passages. When p53 from either Meth A cells or SV40-transformed cells is immunoprecipitated with a monoclonal antibody (Dippold et al., 1981), the immunoprecipitates contain an associated phosphotransferase activity (Jay et ul., 1981). This associated protein kinase phosphorylates p53 in immunoprecipitates from Meth A extracts, and p53 and SV40 T antigen in immunoprecipitates from SV40-transformed cell extracts (Jay et al., 1981). The phosphorylation of these proteins is at serine and threonine residues (Jay el al., 1981). This result is potentially attractive in light of the reports that

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SV40 large T antigen (Tjian and Robbins, 1979; Griffin et al., 1979) and the adenovirus tumor antigens (Lassam et al., 1979) have an associated protein kinase activity in immunoprecipitated reaction mixtures. In the case of SV40 it has been possible to separate the SV40 T-antigen immunological activity from the protein kinase activity by conventional biochemical purifications of these proteins (Tjian and Robbins, 1979; Gracherio and Hager, 1979). If p53 was indeed a phosphotransferase itself, or was tightly associated with a protein kinase activity, this would explain the SV40 and adenovirus results. However, immunoprecipitates could well contain contaminating protein kinases, and the control experiments employed to eliminate contaminating enzyme activities do not totally rule out this possibility. Although p53 might have an associated protein kinase activity (Jay et al., 1981), better criteria will be required to prove that p53 itself has an intrinsic phosphotransferase activity. The presence of high levels of p53 in chemically transformed cells brings up some interesting questions. In nontransformed murine 3T3 cells, p53 is synthesized and then degraded rapidly with a half-life of 20-60 min (Oren et al., 1981). In SV40-transformed cells, the viral large T antigen both regulates the levels of p53 in transformed cells (Linzer et al., 1979) and is physically complexed with p53 (Lane and Crawford, 1979; Linzer and Levine 1979). The high levels of p53 in SV40-transformed cells could result from the inability of p53 to be degraded or turned over (Oren et al., 1981), because it is in a multimeric T-antigen-p53 complex. The half-life of p53 and T antigen in SV40-transformed cells is greater than 22 hr (Oren et al., 1981). If this explanation is correct, then what regulates the high levels of p53 in Meth A-transformed cells, which have no viral tumor antigens? Is p53 in Meth A cells associated with a cellular protein that stabilizes it and prevents rapid protein turnover? The search for p53-associated proteins in nonviral transformed cells, or in normal cells at specific stages of the cell cycle, has just begun. If successful, it may lead to clues concerning the function of p53 in normal and transformed cells. VI. Abelson Virus

Abelson murine leukemia virus (A-MuLV) is a defective retrovirus which induces an acute nonthymic lymphoproliferative disease in mice (Abelson and Rabstein, 1970). The virus can transform some fibroblast cell lines in culture (Scher and Siegler, 1975) and bone marrow-derived lymphoid cells (Rosenberg et al., 1975; Rosenberg and Baltimore, 1976). These Abelson virus-transformed lymphocytes resemble immature B cells and in some cases produce immunoglobulin chains (Premkumar et al., 1975). The Abelson-transformed lymphocytes express three different tumor antigens :

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(1) a viral encoded protein called p120 (Reynolds et al., 1978; Witte et al., 1978); (2) a differentiation-specific antigen (Risser et al., 1978); and (3) the same p53 cellular antigen observed in a large number of transformed cell lines (Rotter et al., 1980). The defective genome A-MuLV encodes the genetic information for a 120,000-160,000 (p120) MW protein consisting of an N-terminal portion of the viral gag protein linked to a unique gene segment derived from the host (mouse) genome (Reynolds et al., 1978; Witte et al., 1979). Thus the Abelsontransforming protein is a fusion product between the viral gag genes and a portion of a cellular gene. Some deletion mutants in this protein fail to transform cells, suggesting that the p120, virus encoded protein is important in the transformation process (Rosenberg and Witte, 1980). Abelson virus was first isolated from BALB/c mice (Abelson and Rabstein, 1970), and so the murine genomic contribution to this virus is from a BALB/c genetic background. Abelson virus-transformed lymphocytes from C57BL/6 mice (Ll-2-MuLV tumor cells) produce antibodies in C57BL/6 mice against the p120 gag fusion product of the virus (Witte et al., 1979). These antibodies react with both the viral gag component and the host cell protein component (Witte et al., 1979). With this transformed cell line, in C57BL/6 mice, the tumor regresses and a strong p120 sera is obtained. The reason anti-p120 antibodies are directed against the cellular antigenic determinants in pl20 could be (1) the possible allogeneic differences between the BALB/c genetic material in the virus and the similar C57BL/6 gene, (2) the increased levels of p120 in A-MuLV lymphocytes compared to normal lymphocytes, (3) The anti-gag antibody response acting to enhance the immune response against the cellular antigeneic determinants, (4) the tumor rejection enhancing the humoral response, or (5) a combination of these factors. The p120 antigen has been localized both at the cell surface (Rotter et al., 1981) and associated with the detergent-insoluble cellular matrix fraction of A-MuLV transformed cells (Boss et al., 1981). A second, and most likely distinct, cell surface antigen found on A-MuLVtransformed lymphocytes appears to be a differentiation-specific antigen (Risser et al., 1978). C57BL/6 mice, immunized with A-MuLV-transformed C57BL/6 lymphocytes, produce antibodies that react with a cell surface antigen on A-MuLV lymphocytes and a subset of normal BALB/c bone marrow and fetal liver lymphocytes. This developmental antigen was not detected in C57BL/6 normal bone marrow or fetal liver cells, and so these antigenic determinants also behave as alloantigens in the BALB/c and C57BL/6 mice (Risser et al., 1978). The expression of this developmental antigen in normal cells correlates with the leukemogenic target cells (preB cells) of this virus (Rosenberg and Baltimore, 1976). In addition, the developmental alloantigen has been mapped by genetic crosses at or near

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the Av-2 locus which determines the host range (susceptibility) for A-MuLV leukemogenesis (Risser et al., 1978). The virus’ ability to induce leukemogenic changes in a limited set of pre-B cells that may express this developmental antigen and the genetic determination of susceptibility to this virus (Au-2)-both of which map at or near the gene for this differentiation antigen -suggests an important role of this tumor antigen in leukemogenesis. The third tumor antigen expressed at high levels in Abelson-transformed lymphocytes is the p53 protein (Rotter et al., 1980). BALB/c mice immunized with syngencic BALB/c A-MuLV-transformed lymphocytes produce hightiter anti-p53 antibodies. The p53 antigen from A-MuLV-transformed cells is similar or identical (by immunological criteria and peptide maps) to the p53 antigen associated with the SV40 large T antigen (Rotter et al., 1981). A monoclonal antibody to p53 used in these studies, RA3-2C2 (Coffman and Weissman, 198I), immunoprecipitated p53, and no detectable associated proteins, from A-MuLV-transformed cells. The RA3-2C2 monoclonal antibody also detected a cell surface antigen on a subset of normal mouse B lymphocytes (Coffman and Weissman, 1981) and on the cell surface of A-MuLV-transformed pre-B lymphocytes (Rotter et a[., 1981). The binding of the RA3-2C2 monoclonal antibody to the cell surface of some normal and A-MuLV-transformed lymphocytes appears to be due to a fortuitous crossreaction between an antigenic determinant on p53 and a B-cell developmental or differentiation antigen (D. Baltimore, personal communication). Other monoclonal antibodies to different p53 antigenic determinants (Gurney et al., 1980) do not recognize these cell surface antigens (D. Baltimore, personal communication), and so it appears that p53 in Abelson-transformed lymphocytes has an intracellular location as determined by cell fractionation studies (Rotter et al., 1981). These studies provide a good example of the potential problems of utiliqing monoclonal antibodies for immunofluorescent studies. Without a second criterion, like the molecular weight of the antigen under study, cross-reactions, which may or may not be significant, can provide false positives (see Section V). The Abelson-encoded p120 protein has an associated protein kinase activity in immunoprecipitates, transferring a phosphate moiety to tyrosine residues of the acceptor protein (Witte et al., 1980). P53 is a phosphoprotein but contains only phosphoserine residues and no detectable phosphotyrosine (Rotter et al., 1981), and it is therefore unlikely that the p53 antigen is a substrate for the A-MuLV kinase. Under the conditions employed to test the p120 protein for kinase activity, the p53 protein failed to act as a protein kinase when immunoprecipitated from Abelson-transformed cells (Rotter et al., 1980). This is not in agreement with the report of a p53-associated protein kinase detected in other cell lines (Meth A, SV40 transformed) (Jay et al., 1981).

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VII. Rous Sarcoma Virus

Rous sarcoma virus (RSV) is a nondefective avian retrovirus. The virus genome is composed of three genes-gag, pol, and enu, required for replication and production of virions-and a fourth gene, pp60'-"*', required for transformation but not virus production. The pp6OV-"' gene was likely derived from a normal chicken cell gene (Stehelin et al., 1976), pp6OcpSrc, but is not identical to that cellular gene (Beemon et al., 1979). Pp60'-"*' is a phosphoprotein of 60,000 MW with both phosphoserine (within N-terminal half) and phosphotryosine (within C-terminal half) residues. The majority of this protein appears to be bound to the inside (cytoplasmic) portion of the plasma membrane (Courtneidge et al., 1980; Krueger et al., 1980). Pp60'-"r' has protein kinase activity, as first detected using immunoprecipitates of this protein. In this reaction the rabbit heavy chain of the immune immunoglobulin was phosphorylated at a tyrosine residue after the addition of labeled ATP (Collett and Erikson, 1978; Levinson et al., 1978; Hunter and Sefton, 1980). Purified pp60'-"' retains this protein kinase activity for a number of substrates tested (Erikson et al., 1979; Levinson et al., 1980). When radiolabeled pp6OV-"" is immunoprecipitated from infected and transformed chicken cells by antibodies directed against pp6OvpSrc, two other proteins are coimmunoprecipitated with pp60'-"' by virtue of the fact that they are associated with this transforming protein (Hunter and Sefton, 1980; Brugge et al., 1981; Opperman et al., 1981a). The molecular weights of these cellular proteins are 90,000 (90K) and 50,000 (50K), and both proteins are phosphorylated. The 90K protein contains only phosphoserine, whereas the 50K protein has both phosphoserine and phosphotyrosine residues when isolated from the transformed cell. In the uninfected cell the 50K protein contains only phosphoserine residues, suggesting that this protein is phosphorylated at a tyrosine residue upon transformation with RSV (Brugge and Darrow, 1982). The association of phosphotyrosine residues in the 50K protein with RSV infection is confused by the observation that temperature-sensitive mutants in the src gene result in phosphorylation of 50K at tyrosine residues even at the nonpermissive temperature (Brugge et al., 1981). Though these apparent contradictory results are not fully explained to date, it remains possible that a temperature-sensitive src gene product could be suppressed in its phenotype while complexed with these (50K and 90K) cellular proteins. The pp6OV-"*'-50K-90K complex represents about 5% of the intracellular pp6OV-"' detected in the transformed cell (Brugge et al., 1981). This complex sediments with an apparent molecular weight of about 200,000, while free pp6OV-"' (95%) sediments as a monomer (Brugge et a/., 1981). Pp60'-"' is first synthesized on free ribosomes (not associated with the rough endoplasmic reticulum) and is then phosphorylated

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at its serine residue. Shortly after synthesis the pp60'-"' is detected as the pp60'-"*'-50K-90K complex for a brief period. The pp60'-"' monomer then leaves the complex and moves to the membrane (Brugge et a/., 1981). Thus the temperature-sensitive pp60'-"" mutants could still phosphorylate the 50K protein in the pp60'-""-50K-90Kcomplex (suppression of the mutation by complex formation) and then act as a temperature-sensitive mutation in a monomer form at the membrane. The 50K protein is present at low concentrations in both normal and transformed cells, and comparisons of 50K protein from avian and mammalian sources (both bound to pp60'-"') show little or no evolutionary conservation of the peptides derived from this protein. By contrast, the 90K protein is present in high concentrations in normal and transformed cells as expected for a structural protein. In addition, the peptide maps of the 90K protein from avian and mammalian sources are very similar. Curiously, the synthesis of the 90K is greatly stimulated by elevated temperatures (it is a heat shock protein) (Opperman et al., 1981b) or other noxious agents. Like the 50K protein, the 90K protein has a cytoplasmic localization in the cell (Brugge, unpublished results). Based on its electrophoretic mobility in SDS-polyacrylamide gels, partial peptide maps, and localization in the cell, the 50K protein does not appear to be identical with the p53 antigen that is associated with the SV40 T antigen (D. Linzer, J. Brugge, and A. Levine, unpublished results). Although the function of the pp60'-""-50K-90K complex is not yet clear, this complex has also been detected in defective avian retrovirus-transformed cells, such as Fujinami sarcoma virus and Y73 virus (J. S. Brugge, personal communication). The transforming gene products of these viruses are protein kinases, but are chemically unrelated to the RSV pp60'-"'. VIII. Teratocarcinornas

Teratocarcinomas are tumors derived from developmentally immature pluripotent cells (Stevens, 1975; Pierce, 1975). The tumors are composed of malignant stem cells, called embryonal carcinoma cells (EC cells), and a variety of benign cell and tissue types that are differentiated from the stem cell population (Kleinsmith and Pierce, 1964; Kahn and Ephrusii, 1970; Rosenthal et al., 1970; Mintz and Illmensee, 1975). The origin of these tumors has been extensively studied in the mouse, where defined inbred strains develop either testicular (Stevens and Little, 1954) or ovarian (Stevens, 1975) teratomas with high frequency. Thus there is a genetic predisposition for the production of these tumors. Transplantable teratocarcinomas (Stevens, 1958, 1970) and cloned EC cell lines (Bernstine et al., 1973) have been utilized to demonstrate two

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classes of tumor antigens associated with these tumors. Tumor transplantation rejection between allogeneic mouse strains has identified three genetic loci that encode the information for teratocarcinoma transplant rejection (Avner et al., 1978; Siegler et al., 1979; Shedlovsky et al., 1981 ; Levine and Teresky, 1981). At least some of these antigens appear to be limited to teratocarcinomas (Levine and Teresky, 1981). The second antigen detected at high levels in EC cell lines is the p53 antigen (Linzer and Levine, 1979) common to many tumors and transformed cells. Teratocarcinomas or EC cell lines derived from 129/sv mice produce tumors in syngeneic 129/sv mice but are rejected in a variety of allogeneic mice (BALB/c, for example) (Avner et al., 1978; Siegler et al., 1979). The tumor rejection was not due to differences at the H-2 locus and the basis for tumor rejection was lymphocyte mediated (Avner et al., 1978; Siegler et al., 1979). Genetic crosses between tumor-susceptible mice and tumor-rejecting mice permitted the identification of three teratocarcinoma transplantation loci called Gt-1, Gt-2, and Gt-(B6) (Shedlovsky et a/., 1981; Levine and Teresky, 1981). Gt-1 was localized about 4 centimorgans from the H-2 locus between the Tlocus and H-2K region of this chromosome (Siegler et al., 1979; Shedlovsky et al., 1981 ;Levine and Teresky 1981).Gt-2 has also been mapped to chromosome 17, about 17 centimorgans on the H-2D side of this locus (Shedlovsky et al., 1981). Gt-(B6) has not been mapped to a linkage group. It appears likely that these Gt loci encode transplantation rejection antigens expressed on EC cells. These stem cells, like the early mouse embryo, do not contain detectable H-2 antigens (Forman and Vitetta, 1975; Isa, 1976). When EC cells differentiate they acquire the H-2 antigens (Artz and Jacob, 1974; Nicholas et al., 1975; Stein et al., 1975) and are thought to lose the Gt antigens (Levine and Teresky, 1981). These observations, the strength of the Gt transplantation rejection antigens, and the proximity of Gt-1 and 2 to the H-2 locus, has led to the suggestion that the Gt loci are the embryonic analog of the major histocompatibility locus in mice (Shedlovsky et al., 1981). If this explanation is correct, the Gt antigens would be fetal antigens and are recognized as foreign by virtue of their restricted gene expression in early embryos. In addition, these tumor antigens would be restricted to developmentally immature stem cells in a tumor. Embryonal carcinoma cell lines often express very high levels of the same p53 antigen found to be associated with the SV40 large tumor antigen (Linzer and Levine, 1979). In SV40-transformed cells, p53 levels are regulated by the SV40 T antigen (Linzer et al., 1979), which is found in a multimeric protein complex with p53 (Lane and Crawford 1979; Linzer and Levine, 1979). In nontransformed 3T3 cells, p53 is synthesized and degraded rapidly (leading to low levels), whereas p53 in SV40-transformed cells has a much longer half-life and is quite stable (Oren et al., 1981). This has led to the hypothesis

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that p53 protein is stabilized, avoiding proteolytic degradation by virtue of its association with SV40 T antigen or a T antigen-induced protein modification of p53 (Oren et al., 1981). If this explanation is correct, then it affords little help in understanding why p53 levels are also high in cells transformed by other agents, with no viral T antigen present. To address this problem, Oren et al. (1982) have examined the regulation of p53 levels in an EC cell line called F9 (Bernstine et al., 1973) and its progeny cells induced to differentiate by treatment of F9 cells with retinoic acid (Strickland and Mahdavi, 1978; Strickland et al., 1980). The F9 EC cells are fully transformed and highly tumorigenic. When treated with retinoic acid and dibutyryl cyclic AMP, these cells differentiate into benign and nontransformed endoderm-like cells. Accompanying this reversion of the malignant or transformed state is a 5- to 10-fold decrease in the steady-state levels of p53 antigen (Oren et al., 1982). If these endoderm cells are infected with SV40, the viral T antigen is synthesized and the p53 levels increase in the endoderm cells (Oren et al., 1982). Pulse-chase experiments, to determine the stability of p53 protein in EC cells and endoderm-like cells, indicated that in both cell types the half-life of p53 was the same, about 3.5 hr. This was in dramatic contrast to nontransfoimed 3T3 cells (20-60 min half-life of p53) and SV-40-transformed cells (a half-life of more than 22 hr) (Oren et al., 1981, 1982). Thus the stability of the p53 protein in F9 and its progeny endoderm cells was similar in spite of the fact that F9 cells contain 5- to 10-fold higher levels of p53. When total cytoplasmic RNA extracted from either F9 and endoderm cells was employed in a reticulocyte in vitro protein translation system, the RNA from endoderm cells synthesized 6- to 10-fold less p53 in vitro than comparable RNA concentrations derived from F9 cells (Oren et al., 1982). Over a wide range of mRNA concentrations (dose-response experiment), comparable levels of RNA from F9 cells always translated 6- to 10-fold more p53 in vitro, than the mRNA from endoderm cells. Thus it appears that the levels of p53-translatable mRNAs decline upon differentiation of F9 cells to endoderm cells, and this may account for the decreased levels of p53 in these nontransformed cells (Oren et al., 1982). These results are in contranst to the nontransformed 3T3-transformed SV3T3 cell system,where both cell types have roughly equal levels of p53-translatable mRNA but protein turnover regulates the intracellular levels of p53 (Oren et al., 1981). Thus, depending on the system under study, p53 levels may be regulated by the amount of p53-translatable mRNA or a posttranslation event. The decline of p53 levels during differentiation of F9 cells in culture may be analogous to the detection of p53 at defined stages of embryogenesis in the mouse (Mora et al., 1980). Mora et al. (1980) have shown that p53 levels decrease as mouse development proceeds. It is difficult, however, based on

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the studies published to date, to quantitate the relative levels of p53 found in stage-specific embryos versus the levels of this antigen in normal and transformed cells. For example, using immunofluorescence and immunoprecipitation of [35S]methionine-labeled proteins, Dippold et al. (1981) failed to detect p53 in mouse embryo cell cultures. Steady-state levels of p53, measured by a radioimmune assay, and utilizing cell lines, embryonic tissue, normal and tumor tissue, will be required before quantitative results permit definitive conclusions as to the relative levels of expression of p53 in embryonic tissue. IX. Conclusions

A. A CLASSIFICATION OF TUMOR ANTIGENS Based on the examples of different tumors examined in this article, a useful classification of tumor antigens can be proposed. Four distinct classes of tumor-specific antigens may be recognized: (1) Antigens that are unique to a particular tumor have been detected in sarcomas and transformed cell lines produced by chemical carcinogens (e.g., Meth A, CMS4) (DeLeo et al., 1977, 1978). (2) Antigens that are unique to, or at least preferentially expressed in, a class or specific type of tumor. Examples of this category of tumor antigen are the Gt-1 and 2 antigens of teratocarcinomas (Shedlovsky et al., 1981 ; Levine and Teresky, 1981), the differentiation-specific antigen of Abelson-transformed lymphocytes (Risser et al., 1978), carcinoembryonic antigen in colon carcinomas, and alpha fetoprotein detected in hepatocellular carcinomas (Abelev et al., 1979). (3) Antigens that are specific for the transforming agent employed to produce the tumor. The DNA and RNA tumor viruses provide the best examples of this category of tumor antigen. (4) Antigens that are common to many different tumors such as p53, which is expressed in high levels in transformed cells of all the examples considered in this article (Table I). 1. Antigens Unique to a Tumor Antigens that are unique to a particular tumor, like Meth A, could arise in a number of ways. If exposure of cells to carcinogens results in genetic rearrangements, via insertion elements or recombination of genomic DNA, that could give rise to a large number of unique antigens much like the immunoglobulin V gene-C gene genetic rearrangements. It is even possible that V gene idiotypes are themselves the unique antigenic moieties of carcinogen-transformed cells. A second source of gene families that encode

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membrane antigens would be the glycoprotein structural genes of endogenous RNA retroviruses of the mouse. Recombination between these genes could produce a cell surface antigen with multiple antigenic specificities. Although the nature of the Meth A-like antigens remains to be identified, the proposed mechanisms, like the immunoglobulin system, could generate considerable diversity. Because immunoglobulins are also B-cell surface receptors that trigger cell division upon antigenic stimulation, tumor antigens with similar functions are attractive candidates for Meth A-like antigens. There are, however, other ways to generate antigenic diversity and no evidence that these tumor-specific antigens play a causal or primary role in tumorigenesis. 2. Antigens Unique to a Class o j ’ Tumors

Antigens that are unique to the class or type of tumor under study are usually fetal or developmental antigens. The tissue specificity of alpha fetoprotein, carcinoembryonic antigen, or the Abelson developmental specific antigen reflect this fact. The close genetic linkage between A-MuLV susceptibility (Au-2locus) in mice and the locus responsible for the expression of this pre-B cell antigen (Risser et al., 1978) suggests that this tumor antigen could play a role in the causation or rejection of A-MuLV tumors. This class of tumor antigen is presumably responsible for the rejection of some tumors by animals previously immunized with fetal tissue (Coggin and Ambrose, 1979). At the minimum, however, these antigens have been useful in the diagnosis and classification of tumors. Antigens that are unique to a class or type of tumor may also be a reflection of the immature nature of the tumorigenic target cells for viruses or carcinogens. In this case the “fetal antigen” or “developmental antigen” of the tumor results from the selection and growth of a few target cells in the host. If this hypothesis is correct, then the tumor antigen identifies the rare cell susceptible to carcinogens or viruses, and the normal developmental progression of these cells has been stopped. Pp60-”“ is an example of a viral tumor antigen that can arrest the development of myoblasts (Kaighn et al., 1966), retinal cells (Pessac and Calothy, 1974), epithelial cells (Ephrussi and Temin, 1960), chondroblasts (Pacifici et al., 1977), and melanoblasts (Boettiger et al., 1977). Indeed developmental maturity could preclude the tumorigenic or malignant state, as is the case with teratocarcinomas (Pierce, 1975). 3. Antigens Unique to the Transforming Agent

Viral transformation or tumorigenesis provides the clearest example for both the origin of tumor antigens and their essential role in the transfor-

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mation or tumorigenic process. It is well established that the SV40 large and small tumor antigens, some of the adenovirus tumor antigens, RSV pp6OV-'"', and A-MuLV p120, are required for cellular transformation and probably tumorigenesis (see Table I). The persistence of the viral genome in a tumor and the expression of these viral proteins in the transformed cells reflect the role of these viral proteins in maintaining the transformed phenotype or the tumor. As such, the mechanisms that underlie viral carcinogenesis are among the best understood at this time. In addition, it has become clear that the DNA and RNA tumor viruses differ from each other in both the origin of their " o m genes" and in the mechanism of action of the ''onc gene products" (tumor antigens). RNA tumor viruses acquire their onc genes, like the pp60'-"*' or A-MuLV p120 (Stelelin et al., 1976), from the cellular genome. Pp6OV-"' and pl20 are protein kinases that phosphorylate acceptor proteins at specific tyrosine residues (Collett and Erikson, 1978; Levinson et al., 1978; Witte et a/., 1980). The SV40- and adenovirus-transforming genes do not appear to share nucleotide sequence homology with cellular DNA. When protein kinase activities have been detected in association with SV40 or adenovirus tumor antigens, these kinase activities (Lassam et al., 1979; Griffin et a/., 1979) phosphorylate serine residues on the acceptor protein, and these activities may well result from contamination of the tumor antigen preparation (Tjian and Robbins, 1978; Gracherio and Hager, 1979). Thus the protein kinases of some RNA tumor viruses like RSV pp6OV-"' would be expected to act by modifying proteins in the cell, and that ultimately gives rise to the transformed phenotype. In this case, an inappropriately high concentration of pp6OV-"', protein kinase in the cell, yields an aberrant phenotype because most or all cells already have low levels of pp60"-"" (Collett et al., 1978). One the other hand the SV40 large T antigen is responsible for the initiation of viral DNA replication (Tegtmeyer, 1972) and the modulation down of viral gene expression (Tegtmeyer, 1975). These regulatory events are mediated by the binding of SV40 T antigen to virusspecific nucleotide sequences at or near the origin of viral DNA replication (Shortle et a/., 1979). The SV40 large T antigen is also involved in the stimulation of cellular DNA synthesis (Tjian et al., 1978) and the modulation of cellular gene expression (Postel and Levine, 1975), possibly by acting directly on cellular DNA sequences. If the action of pp6OV-"*'and SV40 T antigen result in similar alterations of cellular gene expression (transformation), the mechanisms by which these two proteins regulate the transformed phenotype appear to be quite different. These differences in the modes of action of SV40 T antigen and p60'-"' are further emphasized by the nuclear (SV40 T antigen) versus plasma membrane (pp60'-"') localization of these two tumor antigens in the transformed cell.

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4. Antigens Common to Many Tumors P53, the tumor antigen common to many different transformed cells, is detected in elevated levels in cells transformed by both the RNA and DNA tumor viruses. In spite of this similarity, the interactions of p53 with the DNA and RNA virus tumor antigens point out another fundamental difference in the mode of action of these viral proteins. The tumor antigens of the DNA viruses, like SV40 T antigen, adenovirus Elb-58K protein, and EBV EBNA, are found in a physical association or complex with p53 in the cell nucleus. In contrast, RSV pp6Ov-"*' (N. Reich, J . Brugge, and A . Levine, unpublished results) and A-MuLV p 120 (D. Baltimore, personal communication) are not detectably associated with p53 in viral transformed cells. Although p53 could be involved in the pathway of events leading to transformation in all cells, the interactions of p53 with DNA or RNA virus tumor antigens clearly differ. Consistent with this is the observation that p53 is phosphorylated at serine and not tyrosine residues, even in Abelson virustransformed cells.

B. WHATIs THE FUNCTION OF ~ 5 3 ? What is the function of p53 in normal and transformed cells? There are some indications that p53 in normal cells is regulated by or with the cell cycle (N. Reich, M. Oren, and A. Levine, unpublished results), and its levels may be related to growth of cells in culture (Dippold et al., 1981; Milner and Milner, 1981). Based on the observation that p53 is physically associated with SV40 T antigen, one could reasonably postulate that p53 participates in one or more of the functions of SV40 T antigen. Basically, SV40 T antigen is involved in two processes: (1) viral and cellular DNA replication, and (2) viral and cellular gene regulation. In the case of viral-mediated gene regulation and DNA replication, T antigen acts via recognition of specific nucleotide sequences and DNA binding. It is of some interest that SV40 Tp53 protein complexes also bind to the same or similar nucleotide sequences in SV40 DNA (Reich and Levine, 1982). Based on this reasoning, p53 could well be one of several cellular proteins that form a DNA replication or initiation complex and/or protein complexes involved in transcriptional regulation in the normal and transformed cell. These speculations are consistent with the role of p53 in cell cycle or growth control. At present there are two observations suggesting that p53 plays a central role in important cellular functions : 1. The p53 protein has been conserved in its primary sequence over evolutionary time scales. Both immunological (monoclonal antibodies) and

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chemical (peptide maps) analysis of the p53 proteins from human, monkey, mouse, rat, rabbit, and hamster show common antigenic determinants and peptides in these proteins (Gurney et af., 1980; Simmons et al., 1980). This suggests a strong selective pressure on function and an important role of p53 in cellular processes. 2. The cellular p53 protein binds to or associates with a number of diverse viral proteins, i.e., SV40 T antigen, adenovirus, Elb-58K protein, and EBV EBNA. These viruses have evolved to optimize either their replication in a cell or attain a functional equilibrium within their host. In evolving, the viruses must utilize normal cellular functions in DNA replication and gene regulation for their own advantages. The role of both the SV40 large T antigen and the adenovirus Elb-58K protein in viral DNA replication and viral gene regulation should reflect these interactions with cellular proteins like p53. At this juncture, enough is known about the p53 protein to suggest that it may play a central role in transformation as well as normal cellular growth processes. Only further studies will detail its function and hopefully lead to new insights in the areas of gene regulation, DNA replication, and the control of cell division.

ACKNOWLEDGMENTS The author thanks D. Baltimore, J . Brugge, L. Kaplan, N. Reich, R. Risser, and P. Sarnow for information prior to publication, useful discussions, or critical reading of this manuscript. The able technical assistance o f G . Urban in typing this manuscript is deeply appreciated. During the preparation of this work the author’s laboratory was supported by grants from the American Cancer Society, MV47B, and the National Cancer Institute, CA28146 and CA28127.

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PERICELLULAR MATRIX IN MALIGNANT TRANSFORMATION Kari Alitalo’ and Antti Vaheri Department of Virology. University of Helsinki, Helsinki. Finland

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

................. A. Matrix Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Proteoglycans . . . . . . . . . . . . . Pericellular Matrix and the Cellular Phenotype in Vitro ............

11. Components of the Extracellular Matrix and Their Functions

111.

11I 112

123

IV . Malignant Transformation : Altered Biosynthesis of Matrix Components and Failure to Deposit Them ...... . . . . . . . . . . . . . . 128 V. Tumorigenicity, Invasion, and M VI. Proteins of Basement Membranes and Interstitial Matrix as Characteristics of Tumor Cells ................................ VII. Cell-Matrix Interaction and Anc VIII. Molecular Mechanisms in Altered Cell-Matrix Interaction in Rous Sarcoma Virus Transformation ..................... 143 References ............................................... 146

I. Introduction

Adherent cells in uiuo are intimately surrounded by extracellular matrix, with very few possible exceptions, such as the terminally differentiated keratinocytes and neuronal cells. In differentiated tissues, the matrix is classified as interstitial connective tissue matrix and basement membranes between similar and dissimilar cells, respectively. The principal function of the matrix is to give mechanical support and to anchor cells in tissue typespecific structures, but it may also have other duties, such as that of a selective permeability barrier. The composition of the matrix surrounding cells, the pericellular matrix, is closely dependent on the cell type and the degree of its differentiation. The matrix phenotype is largely retained even in cell culture, which has greatly facilitated studies on the biosynthesis of matrix components and on their function at the cellular level. It is becoming increasingly evident that altered or defective cell surface-matrix interactions may be salient features of the malignant phenotype. Failure to maintain an intact basal lamina may be invoIved in the neoplastic disorganization of

* Present address : Department of Microbiology and Immunology, University of California, San Francisco, California 94143. 111 ADVANCES I N CANCER RESEARCH, VOL. 37

Copyright 0 1982 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-006637-8

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tissue architecture and development of invasive tumors. In this article we first describe some of the properties of the major defined matrix components, and then consider their role for the cell phenotype. The main emphasis will be on the pericellular matrix components that seem to be involved in cell adhesion, and less on the extracellular matrix material. Special attention will also be given to recent results on epithelial cells, proteolysis of basal laminae, and interstitial matrix, and to the matrix components that are characteristic of some tumor cells. There is now good evidence that certain cellular genes (“oncogenes”) cause cancer when expressed in excess or as altered protein products by oncogenic viruses (see Bishop, this volume). The most thoroughly studied is the avian Rous sarcoma virus (RSV), a retrovirus which causes sarcomas in uiuo and transforms a variety of cells in culture through activity of the protein product of its viral oncogene v-src. Interesting new data, discussed in the last section of this article, suggest that the cellular homolog of the viral gene product, pp60‘-”“, serves highly conserved functions in the cell periphery, and apparently at the sites where the cells are anchored to extracellular matrices. Rous sarcoma virus-induced sarcomas may thus represent virally promoted overexpression of this gene, c-src, or alterations in the properties of its virus-encoded form, v-src, which result in profound perturbations in the cell-matrix interaction.

II. Components of the Extracellular Matrix and Their Functions A. MATRIX PROTEINS

The extracellular matrix contains collagenous and noncollagenous glycoproteins and glycosaminoglycans (Table I). Collagens, fibronectin, and laminin are the major matrix proteins identified so far and are found in association with a variety of different cellular phenotypes. 1. Collagen

Collagen (reviews by Prockop et al., 1979; Bornstein and Sage, 1980), the most abundant protein in the vertebrate body, constitutes more than a third of the total protein in many adult organisms. The triple helix of collagen is resistant to a variety of proteases, due to the ordering of successive amino acid sequences in its primary structure of the form (X-Y-Gly), (n = 338 for type I collagen), where the variables in position Y often are hydroxylated proline residues. The temperature of dissociation of the collagen triple helix

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is a function of the degree of its hydroxylation. Underhydroxylated collagen polypeptides, produced in the absence of known cofactors for peptidyl hydroxylases, sodium ascorbate, or ferrous ions, are not stable at body temperature, and such collagen is secreted from cells only at a low rate (see Prockop et al., 1979). Recent evidence indicates that intracellular cyclic AMP levels also regulate the amounts of collagen degraded before secretion (Baum et al., 1980). The other hydroxylated amino acid found in collagen, hydroxylysine, serves as a template for the addition of carbohydrate into collagen polypeptides and for covalent crosslinking of collagen fibrils. On the basis of protein chemical analysis, at least five different collagen types have been defined in man (Table I). They differ in their primary structure and, therefore, are apparently products of different genes. Whereas the interstitial collagen (types 1-111) fibers display a periodicity of 60-70 nm, noninterstitial collagens (types IV and V) do not. As implied by the nomenclature, the collagen types differ in their distribution in tissues (see Gay and Miller, 1978; Bornstein and Sage, 1980). This is PISO reflected in their relatively specific expression by a given cellular phenotype. Interstitial collagen types have been thoroughly characterized due to the ease of their isolation from tissues and from fibroblasts cultures. Basement membrane (pro)collagen may be best studied in cell or tissue culture without the conventional pepsin treatment used to obtain interstitial collagens from tissues, because the long triple helix of type IV collagen is interrupted by frequent discontinuities, making it sensitive to pepsin digestion. A soluble biosynthetic precursor, procollagen, is secreted by collagenproducing cells (Fessler and Fessler, 1978). Biosynthesis of collagen is characterized by multiple co- and posttranslational modifications (Table 11). Coordinate action of at least eight enzymes is needed to complete the pathway to a collagen fibril (Table 11). In the case of interstitial procollagens, the mostly nontriple-helical propeptides at both ends of the molecule are cleaved off after secretion in the extracellular space by specific enzymes: the procollagen aminoproteases and carboxyproteases (see Fessler and Fessler, 1978; Prockop et al., 1979). Further enzymatic processing of the maturing collagen fibrils involves covalent cross-linking of lysyl and hydroxylysyl residues by lysyl oxidase (Siegel, 1979). Another type of crosslinking of collagen to fibronectin is catalyzed by plasma transglutaminase (factor XI&) in vitro (Mosher et af., 1980). 2. Fibronectin Fibronectins (Mosesson and Amrani, 1980; Mosher, 1980 ; Pearlstein et al., 1980; Vaheri et al., 1980; Ruoslahti et al., 1981; Yamada, 1982) are

TABLE I MAJORDEFINEDMATRIXCOMPONENTS' Type

Chain composition

Interstitial collagen types I

--

111

P

Basement membrane collagens IV

At least 2 chain subtypes: al(1V) a2(IV) a3(IV)?

Other types of collagen V

EC collagen (Sage er af., 1981a; Alitalo er a/., 1982e)

a l , n2, a3 al(V)3 a W 2 a2W) a2(V), ?

?

Distribution

Distinctive features

Skin, bone, dentin, tendon, and corneab

Presence of a2 chain, < 10 hydroxylysines per chain, 0.1% carbohydrate

Cartilage: vitreous body, notochord As type I, prominent in fetal skin: arteries, amniotic membrane, not in bone or tendon

1% carbohydrate, > 10 hydroxylysines

per chain Presence of cysteine, high levels of hydroxyproline, glycine, histidine

Basement membranes (lamina densa)

High 3-hydroxyproline, > 20 hydroxylysines per chain, low alanine and arginine

Associated with membranous structures? (Roll er a/., 1980; Martinez-Hernandez et al., 1982)

Slightly larger than al(I), 0.3% 3-hydroxyproline

Basal laminae?

Secreted in a nontriple-helical form?

Other types of proteins 140K glycoprotein (Carter, 1981)’ Fibronectin

2 x 200.000

Laminin

Subunits 200,000-220,000 400,OOO-440,000 158,000

Entactin Tropoelastin-elastin

vI

140,000

Proteoglycans Sulfated proteoglycans Hyaluronic acid

72,000

Interstitial matrix, between cells and basement membranes, body fluids Basement membranes (lamina rara)

Contains hydroxyproline and hydroxylysine (W. Carter, personal communication) Interactions

Ordered polypeptide structures (or-chain, p-sheet), disulfide knot

Endothelial and epithelial cell surfaces in basement membranes Elastic fibers (Uitto, 1979)

Sulfated glycoprotein

Matrix and body fluids

See Roden (1980), Lindahl and Hook (1978)

High content of alanine, valine, glycine; contains hydroxyproline, crosslinking

’For other references see text, Bornstein and Sage (1980) and Sage (1981). A higher content of type I trimer and type 111 collagens is found in embryonic and fetal tissues. On the basis of amino acid sequence heterogeneity, nonallelic subtypes have been postulated to exist (Butler er al., 1977). Minor collagen types have been found in cartilage (Burgeson and Hollister, 1979; Reese and Mayne, 1981). May be indentical with the polypeptides described by Lehto el al. (1980) and/or Knudsen er 01. (1981),

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KARI ALITALO A N D ANTTl VAHERl

TABLE I1 THEBIOSYNTHESIS OF COLLAGEN Biosynthetic step Transcription Processing to mRNA Translation Cleavage of prepropeptides Hydroxylation of prolyl and lysyl residues Glycosylation of hydroxylysyl residues

Chain association and disulfide bonding Regulated and controlled intracellular degradation Triple helix formation Secretion and deposition of procollagen into the extracellular matrix Conversion of procollagen into collagen Crosslinking

Collagen-specific enzymes involved

Prolyl 3-hydroxylase, 4-hydroxylase, and lysyl hydroxylase" Hydroxylysyl galactosyltransferase, galactosyldroxylysyl glucosyltransferase (see Kivirikko and Myllyll, 1979, 1981) ?

Aminoproteases, carboxyproteases (distinct procollagen types) Lysyl oxidase, transglutaminase?

Enzymes are present also in collagen nonproducing cells such as in lymphocytic cells (Chen-Kiang el a / . , 1978), and in macrophages (Myllyla and Seppa, 1979). The smaller enzymatically inactive subunit is present in large amounts in tissues (Chen-Kiang et al., 1977).

antigenically and biochemically defined glycoproteins, characteristically present in interstitial matrices, between cells and many basement membranes, and in plasma and other body fluids. In contrast to collagen, fibronectin possesses only domains of higher order structure, and no posttranslational modifications or enzymes specific for fibronectin are known. The fibronectin molecule is a dimer, apparently a heterodimer (Kurkinen et al., 1980b; Sekiguchi et al., 1981a), of a and subunits ( M , = 230,000 and 210,000) linked near the carboxyl termini by disulfide bonds (Fig. 1). Fibronectin is visualized in electron microscopy as a two-stranded molecule with an arm length of 61 nm (Engel et al., 1982; Erickson ef al,, 1981). These arms are joined at one end in a rather rigid angle of -70" and contain distinct bent and rigid regions that may correspond to the functional domains (Fig. 1). Asparagine-linked carbohydrate chains, 5-8 per fibronectin subunit, are concentrated in the collagen-binding domain and seem to protect the molecule from proteolysis. Biosynthetic experiments have demonstrated both sulfate (Dunham and Hynes, 1978) and phosphate (Teng and Rifkin, 1979; Ali and Hunter, 1981) in fibronectin. Purified fibronectin is characterized by its multiple interactions (Table III), tendency to polymerize

PERICELLULAR MATRIX IN MALIGNANT TRANSFORMATION CATHEPSIN G PLASMIN TRYPSIN CATHEPSIN D

NH2

!

J

II

II

I

NH 2

-30 -70

kd -'

117

CATHEPSIN G PLASMIN TRYPSlN THROMBIN

I

I

I

I

I

I

I I

I

kd

-S.AUREUS -COLLAGEN BINDING AND BINDING -POLYAMINE -COLLAGEN BINDING X LINKING -FIBRIN BINDING AND X LINKING -HEPARIN BINDING

-CELL -HEPARIN BINDING BINDING

FIG. 1. Proposed structural model of fibronectin indicating proteinase-susceptible regions and binding domains.

(Vuento et al., 1980), and sensitivity to proteolysis (cf. Vartio et al., 1981). These properties and biological data suggest a more dynamic role for fibronectin than for collagen in connective tissues. Soluble fibronectin can be purified by virtue of its specific affinity to denatured collagen (gelatin) (Engvall and Ruoslahti, 1977; Dessau et ul., 1978; Vuento and Vaheri, 1979). The purified protein has sedimentation properties of an elongated globular protein (Mosesson and Umfleet, 1970; Alexander et al., 1979) and is slowly polymerized spontaneously (Vuento et al., 1980)and rapidly in the presence of heparin (Jilek and Hormann, 1979) or polyamines into a filamentous form (Vuento et al., 1980). Although fibronectin antigen can be solubilized from tissues by bacterial collagenase (Bray, 1978), it is not clear whether binding of fibronectin to collagen fibrils, as observed in uitro (Kleinman et al., 1981b), actually occurs in uiuo. As fibronectin promotes the attachment of fibroblastic cells to collagen substrates (Klebe, 1974; Pearlstein, 1976), a role for it in adhesion of cells to collagen also in uiuo has been proposed. The adhesion of some cells to fibrin is mediated by fibronectin (Grinnell et al., 1980) which may be covalently linked to the clot by plasma transglutaminase (Mosher, 1980). Therefore, fibronectin may also function as a temporary organizing matrix in wound healing before collagen is laid down. Mediation of cell-substrate interaction may also be involved in the stimulatory effects of fibronectin on cell migration (Ali and Hynes, 1978; Gauss-Muller et ul. 1980; Postlethwaite et al., 1981) and on cell growth in defined media (Orly and Sato, 1979; Rizzino and Crowley, 1980).The ability of fibronectin to associate with both

118

KARI ALITALO AND ANTTl VAHERI

TABLE 111 INTERACTIONS OF FIBRONECTIN Interaction Binding of fibronectin to: Fibronectin Collagen, especially gelatin Asymmetric acetylcholinesterase (Emmerling el a/., 1981) Clq complement component (Menzel et at., 1981; Ruoslahti et al., 1981) Polyamines Glycosaminoglycans Fibrin (see Sekiguchi et a/., 1981) Cell surface Sialylated glycolipids Certain bacteria, actin, and DNA C-reactive protein' Susceptibility of fibronectin to: Disulfide bonding Proteinases

Transglutaminase (factor XIII)

Event where possibly significant

Assembly into filamentous polymers Matrix formation and opsonization ?Its attachment to cell surfaces Opsonization ?

?

Cell-matrix interaction and matrix formation Attachment site for cells in fibronectin-fibrin clots in the beginning of wound healing Cell adhesion Cellular receptor for fibronectin Opsonization Acute phase reactions? Matrix stabilization Degradation of fibronectin matrix in cell invasion; generation of biologically active fibronectin peptides Matrix stabilization

Binding is observed when one of the proteins is in a solid phase (Salonen and Vaheri, 1982). For other references see text and reviews (Mosher, 1980; Vaheri et a/., 1980; Ruoslahti et a/., 1981; Yamada, 1982).

the phagocytosed particle and the phagocytic cell may be essential for its function as a nonspecific opsonin (Mosher, 1980; van de Water et al., 1981 ; Villiger et al., 1981). The principal binding domains of fibronectin-collagen interaction have been identified on both proteins, but little is known about the fibronectin binding to the cell surfaces. A 47,000 MW glycoprotein (Hughes et al., 1981) and sialylated glycolipids (Kleinman et al., 1979; Yamada et al., 1982) as well as sulfated proteoglycans (Perkins et al., 1979; Hedman et al., 1982b) have been proposed to be involved in fibronectin binding. The latter may participate in regulation of the deposition of matrix fibers because it has been shown that sulfated polysaccharides induce the precipitation of fibronectin (Stathakis and Mosesson, 1977) and promote formation of fibronectin-collagen complexes (Jilek and Hormann, 1979; Johansson and Hook, 1980; Ruoslahti and Engvall, 1980; Del Rosso et al., 1982). However, a major cell-binding site (Pierschbacher et al., 1981, 1982) is separate from the heparin-binding sites (Hayashi et al., 1980; Sekiguchi and Hakomori,

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119

TABLE IV ATTACHMENT PROTEINS I N CELL-MATRIX INTERACTION' Cell Fibroblast

Substratum

Chondrocyte

Interstitial collagens (types 1-111) Collagen type 11

Epithelial cell

Collagen type IV

Muscle cell

Collagen type V

Mediator molecule Fibronectin ( M , = 440,000) Chondronectin ( M , = 180,000) (Hewitt er a/., 1980). cartilage proteoglycan (Hewitt era/., 1981b) Laminin ( M , = 850,000) (Terranova et a/., 1980) Apparently no exogenous mediator molecules (Grotendorst er a/., 1981)

For a review, see Kleinman et al. (1981a).

1980; Yamada et al., 1980; Smith and Furcht, 1982) identified in fibronectin (Fig. 1). According to Yamada et al. (1980), fibronectin will bind also to hyaluronic acid, whereas Laterra et al. (1980) and Del Rosso et al. (1982), using other techniques, find no binding but provide evidence suggesting that hyaluronic acid above a critical concentration interferes negatively in fibronectin-collagen binding. Not all cells depend on fibronectin for their adhesion (see Table IV). Adhesion of chondrocytes to type 11collagen is stimulated by another protein present in serum, called chondronectin (Hewitt et al., 1980). Epidermal cells attach to type IV collagen in the absence of serum factors or exogenous fibronectin (Murray et al., 1979), probably through laminin (Terranova et al., 1980). Rat hepatocytes may utilize either fibronectin (Hook et nl., 1977) or laminin (Carlsson et al., 1981 ; Johansson et al., 1982) as adhesion proteins, and do not need any exogenous mediator molecules for attachment to either native or denatured collagen (Rubin et al., 1981).The attachment of smooth muscle cells to interstitial collagens is stimulated by fibronectin (Gold and Pearlstein, 1980), but it occurs with collagen type V through an intrinsic cell surface glycoconjugate (Grotendorst et al., 1981). Cell surface fibronectin purified from the pericellular matrix of chick embryo fibroblasts is found to possess about 50-fold higher activity as compare to adult chicken plasma fibronectin in restoring a nontransformed phenotype to transformed cells (Yamada and Kennedy, 1979) that have a greatly reduced fibronectin matrix. The reason for the differences in the biological properties and solubility of the two fibronectins has not been settled. Amino acid analyses show no differences between the two forms. The hydrodynamic and spectroscopic properties, as well as denaturation transition curves of the two forms, closely resemble each other (Alexander et al., 1978, 1979; Colonna et al., 1978). It has been shown that purified soluble fibronectin can be deposited in a fibrillar form (Vuento et al., 1980), and that

120

KARl ALITALO AND ANTTl VAHERI

serum or plasma fibronectin can be incorporated into the matrix in cell cultures (Hayman and Ruoslahti, 1979) or even in vivo (Oh et al., 1981). It is possible that glycosaminoglycans copurifying with urea-extracted cell surface fibronectin (Perkins et al., 1980), or the differences in carbohydrate composition (Carter and Hakomori, 1979; Takasaki et al., 1979; Fukuda et al., 1982), may account for the differences found in biological properties. Interestingly, Hayashi and Yamada (1982) in an extensive study report differences in domain structures between chicken plasma and chick embryo fibroblast matrix fibronectins, and Atherton and Hynes (198 1) report a monoclonal antibody raised against fibronectin isolated from culture medium of NIL8 hamster fibroblasts that reacts with NIL8 fibronectin, but only poorly with hamster plasma fibronectin. Certainly the designation “cell surface fibronectin” (as defined by surface labeling) is misleading in the case of adherent cells, as the antigen at most appears as a peripheral membrane protein and is predominantly a pericellular matrix protein (Hedman et al., 1978, 1979). In vivo the soluble form of fibronectin has been found in all extracellular fluids studied. The relative protein concentrations suggest local synthesis by cells surrounding a particular anatomical compartment. Insoluble fibronectin is detected in loose connective tissues, often distributed in a fibrillar pattern, and in association with most basement membranes (Linder et al., 1978 ;Stenman and Vaheri, 1978). Immunoelectron microscopy has localized fibronectin to the peripheral lamina rara of basal laminae (Couchman et al., 1979; Oberley et al., 1979). Fibronectin is abundant in newly formed connective tissues, such as in granulation tissue (Kurkinen et al., 1980a; Weiss and Reddi, 1980) or developing avian corneal stroma (Kurkinen et al., 1979), but is not detected in mature matrices, such as cornea, tendon, cartilage, bone, and enamel, or in differentiated nervous tissue (see Vaheri et al., 1980). As already mentioned, fibronectin can promote cell migration in vitro (Ali and Hynes, 1978), and it appears that fibronectin is present in embryos at times and places where cell migration is occurring (see Hynes, 1981).

3. Laminin

-

Laminin is a large basement membrane-specific glycoprotein ( M , 900,000) originally isolated from mouse tumors that produced great amounts of matrix material (Chung et al., 1979; Robey, 1979; Timpl et al., 1979). In electron microscopy, laminin is visualized as a cross with one long and three short arms (Engel et al., 1982) that correspond to the heavy and light chains ( M , = 440,000-200,000) seen in electrophoresis after reduction of disulfide bonds. Pepsin releases an antigenic large disulfide-rich fragment P1 (Mr = 290,000) localized by electron microscopy to the region where the short arms

PERICELLULAR MATRIX IN MALIGNANT TRANSFORMATION

121

intersect (Engel et al., 1982; Timpl and Martin, 1981). Laminin contains about 12-15% carbohydrate, is rich in sialic acid (4-6%) (Chung et al., 1979; Timpl et af., 1979), and binds to heparan sulfate (Sakashita et al., 1980; Del Rosso et al., 1981). Interestingly, both laminin and heparan sulfate-rich proteoglycans (Kanwar and Farquhar, 1979 ;Hassell et al., 1980) have been located to lamina rara (Foidart et al., 1980), between basement membrane collagen and epithelial cells (Leivo, 1982). In the early mouse embryo, laminin is the first matrix protein detected by immunofluorescence. It is found already in the compacted preimplantation morula stage; fibronectin and type IV collagen are first found in the inner cell mass of the blastocyst during differentiation of the endoderm and first basement membranes, and interstitial (pro) collagen only during differentiation of the mesoderm several days later (Leivo et al., 1980). Laminin has been used as a marker for the detection of basement membrane material in differentiating tubulogenic mesenchyme in the embryonic mouse kidney (Ekblom et al., 1980), in odontoblast differentiation (Thesleff et af., 1981), and in vascular sprouts during bone formation (Foidart and Reddi, 1980). Laminin is a major component of Reichert’s membrane of the mouse embryo (Leivo et al., 1980) and seems to be the major protein secreted by parietal endodermal cells attached to Reichert’s membrane (Hogan et al., 1980), as well as by a variety of parietal endodermal and teratocarcinoma-derived cell lines in culture (Chung et al., 1979; Sakashita and Ruoslahti, 1980; Cooper et al., 1981 ; Leivo et al., 1982). A striking induction of laminin synthesis is obtained when F9 embryonal carcinoma cells are induced to differentiate to parietal endoderm cells by treatment with retinoic acid and dibutyryl cyclic AMP (Strickland et al., 1980). A 150,000 MW glycoprotein, immunoprecipitated by antilaminin from culture medium and possibly identical to entactin, is synthesized by normal or F9-derived parietal endodermal cells (Hogan et al., 1980; Cooper et al., 1981), but does not apparently accumulate in significant amounts in the culture medium of the parietal endodermal cell lines or in the extracellular matrix of the lamin-producing EngelbrechtHolm-Schwarm tumor (Timpl et al., 1979). 4. Entactin A sulfated glycoprotein, entactin, was isolated by Chung and collaborators from extracellular basement membrane-like matrix elaborated by cultures of a mouse embryonal carcinoma-derived cell line, M1536-B3 (Chung et al., 1977a,b, 1979; Bender et al., 1981;Carlin et al., 1981). In suspension cultures, these cells initially form small aggregates, which enlarge to hollow, one to two cell layer-thick spheres. When cells are detached from such spheres by cytochalasin B treatment, membranous sacs are obtained, composed mainly of laminin (Chung et al., 1979) and smaller amounts of entactin (Carlin et al.,

122

KARl ALITALO A N D ANTTI VAHERI

1981). Entactin ( M , = 158,000)was purified by preparative polyacryamide gel electrophoresis from membranes solubilized with sodium dodecyl sulfate and a reducing agent (Carlin et al., 1981). Antisera to entactin localize it to basement membranes in several tissues ;predominant staining is observed on the surface of epithelial and endothelial cells in kidney glomeruli, suggesting a role for entactin in the interaction of epithelial cells with extracellular basement membrane matrix (Bender et al., 1981; Carlin et al., 1981). Entactin solubilized from glomeruli has the same molecular weight as the membrane sac antigen from embryonal carcinoma cultures, suggesting that a similar but obviously disulfide-complexed molecule is a constituent of normal basement membranes (Carlin et al., 1981).

5 . Chondronectin In studies on the adhesion of chondrocytes to cartilage-specific collagen type 11, Hewitt et al. (1980) found that serum contained a factor with an adhesion-promoting activity. The factor was named chondronectin and was purified subsequently from serum. Chondronectin is a glycoprotein of 180,000 apparent molecular weight composed of disuffide-linked subunits with M , = 70,000 each (Hewitt et al., 1981a). Antibodies to chondronectin inhibit attachment of chondrocytes to type I1 collagen and stain pericellular regions of chondrocytes in immunofluorescence of both tissues and cell cultures. On a weight basis, chondronectin is 20-50 times as active as fibronectin in cell attachment assays, but its circulating serum levels are estimated to be almost 100-fold lower than those of fibronectin (Hewitt et al., 1981a). Both cartilage proteoglycan and chondronectin are required for attachment of chondrocytes to type I1 collagen (Hewitt et al., 1981b). B. PROTEOGLYCANS Proteoglycans (reviews Lindahl and Hook, 1978; RodCn, 1980) are polysaccharides that are major components of most connective tissues. Due to their large size and polyanionic nature, they are involved in determining hydration and elasticity of tissues, as well as their permeability properties, foremost of basement membranes. Recent cell culture studies have implied important roles for a special class of proteoglycans at the cell surface (Culp et al., 1980; Kjellen et al., 1980, 1981). Proteoglycans contain chains of repeated disaccharide units covalently linked to a protein core. Chondroitin sulfate proteoglycans are linked to hyaluronic acid chains by a special protein domain, where the link protein attaches and increases the stability of the interaction. According to the monomer composition, glycosidic linkages and sulfation of the constituent

PERICELLULAR MATRIX IN MALIGNANT TRANSFORMATION

123

glycosaminoglycan chains, seven different types of proteoglycans are distinguished. Six of these glycosaminoglycansare composed of alternating uronic acid and hexosamine residues. Only one, hyaluronic acid, lacks sulfate (review Lindahl and Hook, 1978). Heparan sulfate is associated with various cell surfaces (Kraemer, 1971; Rollins and Culp, 1979) and is a major component of basement membranes (Kanwar and Farquhar, 1979).Heparan sulfate is also enriched at sites of cell contact with growth substrata in culture (Rollins and Culp, 1979). New evidence indicates that two classes of heparan sulfate are present on the cell surface. One class is displaced by heparin from its cell surface receptors (Vogel and Dolde, 1979; Kjellen et al., 1980), the other being apparently intercalated to the lipid bilayer through its core protein (Kjellen et al., 1981). This kind of membrane-intercalated proteoheparan sulfate may represent a secretory intermediate to heparan sulfate, but may also account for some of the cell surface receptors of laminin and fibronectin, which both bind to heparin. Indeed, heparan sulfate is the major component that binds to fibronectin in isolated cell-substratum adhesion sites (Laterra et al., 1980). Also, as pointed out by KjellCn et al. (1981), the cell surface proteoheparan sulfate molecule could mediate cell-cell contacts by anchorage of the core protein in the plasma membrane of one cell and binding of the polysaccharide part to its receptors on another cell. Antibodies raised to chondroitinaseor alkali-treated chondroitin sulfate proteoglycan have been used (Hedman et al., 1982b;Oldberg et al., 1982) in immunochemical demonstration of this species in the pericellular matrix of cultured human fibroblasts and at the surface of rat yolk sac tumor cells. Evidence has recently been obtained for the presence of covalently linked polypeptide also in hyaluronic acid produced by RSV-transformed chick embryo fibroblasts (Mikuni-Takagaki and Toole, 1981). The hyaluronate in the culture medium may be processed from the parent molecule by proteolytic scission (Mikuni-Takagaki and Toole, 1981). The relative importance of such proteoglycans in determining both cell-cell and cell-surface interactions is as yet difficult to assess. 111. Pericellular Matrix and the Cellular Phenotype in Virro

Most individual cells in uiuo are in contact with extracellular matrix material ; the exceptions include blood cells and certain terminally differentiated cells such as epidermal keratinocytes. Even the proliferation of hematopoietic stem cells may require an extracellular matrix (Hendrix et al., 1981; Lanotte et al., 1981). In culture conditions adherent cells, perhaps all of them, synthesize matrix glycoproteins or glycosaminoglycans. Even in adherent monocytes, upon differentiation to macrophages, induction of fibronectin synthesis has been detected (Alitalo et al., 1980a). Many cells

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KARl ALITALO A N D ANTTl VAHERI

also produce collagen (Green and Goldberg, 1965; Langness and Udenfriend, 1974). Changes in collagen types and other matrix proteins occur during embryogenesis in many tissues (reviews Wartiovaara and Vaheri, 1980). Furthermore, the matrix material produced by a given differentiated cell, at least in primary culture, often has a composition similar to that found in its pericellular matrix in the tissue of origin, i.e., it represents the differentiated phenotype of the cells as found in v i v a The pericellular matrix in human skin fibroblast cultures consists of fibronectin and procollagen types I and 111 (Bornstein and Ash, 1977; Vaheri et al., 1978b; Hedman et al., 1979). Moreover, some cultured fibroblasts also produce types IV and V collagen (Alitalo, 1980; Fessler et al., 1981) and deposit them in their pericellular matrix (Alitalo et al., 1980b). Extraction of the cell layers with detergents, as introduced by Hedman et al. (1979), leaves the pericellular matrix structure intact and attached to the substratum, amenable for analysis. Both sulfated and nonsulfated proteoglycans are found as constituents of such a matrix (Hedman et d.,1979, 1982a,b) . Most of the fibronectin synthesized by adherent cells (Fig. 2) is secreted into the culture medium, and only a small proportion (5530% ) is deposited in the pericellular matrix (see Vaheri and Mosher, 1978). Fibronectin must interact with a substratum to show the cell adhesion-promoting activity (Pearlstein, 1978). Possibly a conformational change upon adsorption of fibronectin to the culture substratum exposes binding domains. Matrix fibronectin is rapidly polymerized after secretion and subsequently disulfidebonded to multimers (McConnell et al., 1978; Choi and Hynes, 1979). EXTRACELLULAR MATRIX INTERSTITIAL COLLAGEN TYPES 1-111 PROTEOGLYCAN FIBRONECTIN ELASTIN

BASEMENT MEMBRANE COLLAGEN TYPES IV-VI LAMININ PROTEOGLYCAN FBRONECTIN YCRETlON OF SOLUBLE FIBRONECTIN

CROSS- LINKING

GAG HYDROLASES-

-3-T -

CELL MIGRATION

FIG. 2. Major identified components of the extracellular matrix and involvement of fibronectin in cell-matrix interactions.

PERICELLULAR MATRIX IN MALIGNANT TRANSFORMATION

125

Fibronectin fibrillogenesis may be independent of collagenous matrix components, as suggested by the spontaneous polymerization of soluble fibronectin (Vuento et af., 1980) as well as by the resistance of the pericellular fibronectin-containing structures to bacterial collagenase (Vaheri et al., 1978b). When cell-free fibroblast matrices are treated with thrombin or a M, 10,000 peptide fraction isolated from sarcoma cell cultures (KeskiOja and Todaro, 1980; Keski-Oja et a)., 1981), fibronectin and procollagens are released but collagenase treatment of the matrix does not release fibronectin. Inhibitors of prolyl hydroxylase-such as a&-dipyridyl, an iron chelator-that slow down the secretion of procollagen, do not interfere with the secretion or deposition of fibronectin (our unpublished data). In the early fibroblast matrix, fibronectin, interstitial procollagens, heparan, and part of chondroitin sulfates codistribute in immunofluorescence microscopy (Vaheri et af., 1978b; Hedman et af., 1982b). Matrix-derived fibronectin substituted with a photoactivable heterobifunctional cross-linker was found to cross-link to sulfated glycosaminoglycans when added to hamster fibroblast cultures (Perkins et af., 1979), suggesting a close topographical neighborhood. Fibronectin matrix is stabilized in part by disulfide bonding (Hynes and Destree, 1977; Keski-Oja et af., 1977; Choi and Hynes, 1979). Solubilization of fibronectin from the matrix is facilitated by denaturation prior to disulfide reduction (our unpublished results; Carter and Hakomori, 1981). Soluble forms of fibroblast surface fibronectin, and plasma fibronectin, contain free sulfhydryl groups (Wagner and Hynes, 1979; Mosher, 1980; Sekiguchi and Hakomori, 1980; Smith et af., 1982). The degree of procollagen processing in cell culture is dependent on 60th species and cell type as well as on the collagen type (Taubman and Goldberg, 1976; Goldberg, 1977). In human fibroblast cultures, for example, mainly procollagen is deposited in the matrix before propeptide proteinases have cleaved it to collagen (Hedman et al., 1979), whereas in cultures of human amniotic epithelial cells that mostly produce type Ill procollagen (Alitalo et af., 1980b), the partly processed pNa(II1) molecules are deposited into periodic fibrils and subsequently cleaved to the collagen form (Hedman et af.,1982a). Evidence at present indicates that type IV collagen is deposited in its intact procollagen form in tissues (see Sage, 1981). The procollagen fibrils may also be covalently crosslinked. Lathyrogens, which inhibit lysyl oxidase, reportedly increase the proportion of soluble procollagen in fibroblast culture media (Layman et al., 1971; Bissell, 1981); they are used routinely in studies on procollagen. Transglutaminase-mediated crosslinking of fibronectin to itself or to collagen (Keski-Oja et al., 1976; Mosher et al., 1980) apparently does not occur under ordinary cell culture conditions.

-

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KARI ALITALO AND ANTTI VAHERI

TABLE V PERICELLULAR MATRIX PROTEINS PRODUCED BY CULTURES OF HUMAN A N D CHICKCELLS~ Culture Human Fibroblasts

Smooth muscle cells Endothelial cells

Epithelial cells

Organ

Skin Lung Gingiva Human uterus Aorta Umbilical vein Capillary Glomeruli Amniotic fluid

Amnion Epidermal cells Chick Embryo fibroblasts

Chondrocytes

Myoblasts Organ cultures

Body wall Tendon Cornea Sternum

Skeletal muscle Corneal epithelium Neural retina Cartilage Blood vessels Cranial bones

Fibronectin

(Pro)collagen types

+ + +

I, 111, v I, 111, IV, (V) (Alitalo, 1980) I, 111, v 111, I 111, IV, V, EC (review Sage, 1981)

NDh

+ -L

ND

+

+ k

+ + +

ND

1v

I, I11 IV (Killen and Striker, 1979) I,, IV (Crouch and Bornstein, 1980; Crouch era/., 1978, 1979) 111, (IV, V) (Alitalo er a/., 1980b) IV, unknown (Alitalo ct al., 1982~) 1, 111, (V) 1, 111, V (Herrmann et al., 1980) 1. 111, V (Conrad et a/., 1980)

11 or I, 11, and minor components (phenotype labile, reviewed by Bornstein and Sage, 1980) 1, 111, V (Sasse er al., 1981) I, I1 (Linsenmayer el al., 1977) 11, V (Linsenmayer and Little, 1978) I1 Ill, (I, V) (Fessler and Fessler, 1978) I

‘“Stable” species in cell culture (see Ponten, 1976). (For other references see text; also Vaheri and Alitalo, 1981, Bornstein and Sage, 1980.) ND, Not determined.

Table V lists the pericellular matrix glycoproteins in certain cell cultures studied to date. Cultured fibroblast cell strains have in general a relatively stable pattern of procollagen isotype production (Hance and Crystal, 1977). Some fibroblastic cells, however, such as chick embryo fibroblasts from tendon (Herrmann et al., 1980) or cornea (Conrad et al., 1980), synthesize procollagen type I11 only when released from their tissue matrices and passaged in vitro in cell culture. Also, smooth muscle cells and chondrocytes (Mayne et al., 1976; Burke et ul., 1977) show a phenotypic convergence

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toward fibroblast-like cells with common features of cell shape and pattern of fibronectin and procollagen production (see Table V). It has not been established in all cases whether the fibroblastic phenotype is selected among the cells in the primary culture or whether a true derepression of synthetic programs occurs in vitro. The differentiation program of chondrocytes, myoblasts, and certain epithelial cells is affected by the extracellular matrix they are in contact with in culture (Hauschka and Konigsberg, 1966; de la Haba et a/., 1975; Furcht et af., 1978; Chiquet et al., 1979; West et af., 1979; Salomon et al., 1981). Primary avian tendon cells in appropriate ascorbate-supplemented culture conditions show synthesis of extraordinarily large amounts of collagen (Schwarz and Bissell, 1977; Schwarz et al., 1978). They have been used as a source of procollagen in biosynthetic studies on type I collagen. Moreover, they may be one of the best of the reported cells for studies on matrix glycoprotein metabolism in virus transformation, as they are derived from a single organ containing mainly fibroblasts. When mixed fibroblasts and other cells (e.g., from chick embryo body wall) are used instead, there is no clear in uiuo reference point to define the state of differentiation of control cells. As discussed later, some cell types have a labile phenotype in uirro. Cultured rodent cells, so commonly used, can undergo spontaneous “transformation-immortalization” and show also genetically unstable behavior upon prolonged cultivation in uitro (Ponten, 1976). Rodent cell lines usually show reduced amounts of both procollagen synthesis (Peterkofsky and Prather, 1974) and pericellular fibronectin matrix in vitro (our unpublished data), possibly reflecting their degree of dedifferentiation or transformation (see Green and Goldberg, 1965). Contrary to rodent cells, which have a relatively short life span in uioo, the genetic control in human cells in culture is considered tighter. Spontaneous establishment of human cell lines has not been reported [for carcinogeninduced transformation of human cells, see Kakunaga (1977) and Milo and DiPaolo (1978)], and remarkably few cells have a truly labile phenotype in vitro. Even transformation of human cells by simian virus 40 seems to lead to relatively small changes in gene expression by the transformed progeny (Williams et al., 1977), and the transformed cells do not induce progressive tumors (Stiles et al., 1975; Kahn and Shin, 1979; Martin, 1981). Schwartz (1978), among others (Gospodarowicz et al., 1978; McAuslan and Reilly, 1979; Mueller et af.,1980), has reported on a phenotypic change, called sprouting, observed in cultures of bovine aortic endothelial cells. Hereby some cells in the pavement-like strict endothelial cell monolayer acquire fibroblastic morphology and migrate under the monolayer. Instead of producing mainly type 111 procollagen (Sage et a/., 1979), these cells begin

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KARI ALITALO A N D ANTTI VAHERI

to produce both types I and 111 (Cotta-Pereira eta/., 1980). A phenomenon analogous to sprouting in uitro has been described iii uivo in the process of tumor angiogenesis studied by Folkman ( 1974; Folkman and Haudenschild, 1980). Epithelial and endothelial cell and tissue cultures have been quite useful in characterization of basement membrane components (reviews by Kefalides et d., 1979; Bornstein and Sage, 1980; Timpl and Martin, 1981), as conventional chemical analyses of the isolated basement membranes have yielded variable results. For example, basement membrane proteins of human amniotic epithelial cells (Alitalo et al., 1980b; Crouch et ul., 1980), bovine aortic endothelial cells (Sage et al., 1981a,b), and human epidermal keratinocytes (Alitalo el ul., 1982c) have been analyzed in culture. From these studies it appears that normal epithelial cells, in comparison tp fibroblasts, deposit a larger proportion of their matrix proteins, and that little or no processing of procollagen type IV takes place in culture conditions. h uiuo in stratifying epithelia, only the basal cells are involved in the synthesis of the abutting basal lamina. In culture, similarly, the synthesis of basal lamina proteins takes place in the noncornified, relatively undifferentiated cells (Alitalo et ul., 1982a,c). Even in cells from nonstratified epithelia, synthesis and degradation of fibronectin and of basement membrane collagen have been shown to be under complex regulation by hormones and extracellular matrix (Chen et ul., 1977; Furcht et al., 1977; Marceau et ul., 1980; Salomon et ai., 1981). It seems possible that the widely acknowledged instability of the phenotypic expression of cultured cells could be due to their inability, when maintained on plastic, to produce a normal basal lamina (Gospodarowicz et al., 1982). If this should be the case, providing the cells with an artificial substratum closely resembling that produced in uiuo should stabilize their phenotypic expression.

IV. Malignant Transformation: Altered Biosynthesis of Matrix Components and Failure to Deposit Them

Studies in t i c 0 have clearly documented the diversity and clonal evolution of tumor cell populations (Ponten, 1976; Poste and Fidler, 1980; Poste et ul., 1981). The ability to invade and metastasize, the distinguishing feature of the malignant phenotype, is a combination of properties rather than a phenomenon explained by any unifying concept. Regardless of whether neoplasms are monoclonal or multiclonal in origin, by the time they can be diagnosed clinically, they can be heterogeneous and contain subpopulations of cells with a wide range of phenotypic characteristics (Poste and Fidler, 1980; Smets, 1980). The metastatic cells acquire an increasing genetic

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instability during tumor progression (Cifone and Fidler, 1981). In addition, the invasive and metastatic cells are subject to host selection (Nowell, 1976; Klein, 1979). Many models for aspects of metastatic behavior, such as attachement and infiltration by transformed cells and subsequent angiogenesis of the chick chorioallantoic membrane (Scher, 1976), or by tumor cells of human amniotic basement membrane (Liotta et al., 1980a), and vascular endothelial cell monolayers (Kramer and Nicolson, 1979 ; Kramer et al., 1981), depict the stages of infiltration but are still difficult to analyze quantitatively (review, Mareel, 1980). A major advantage in such models is, however, that the complex immunology involved in the tumor-host cell relationship can be avoided. Nevertheless, it may be impossible to find models with properties relevant for all malignant phenotypes. The term malignant transformation is used to denote various phenomena during the progression of a normal cell into the malignant phenotype. In some contexts it is taken to mean a one-step process conferring many properties associated with malignancy to cells in culture by integration and activity of transforming viral genes (such as the src gene of avian sarcoma viruses) in the host genome. In other contexts, as in chemical carcinogenesis, transformation appears as a continuous process during which the phenotype progresses over many divisions, through morphological transformation and anchorage independence to a tumorigenic state (Smets, 1980). Whether it be integration of a viral genome or some kind of DNA damage, neoplastic transformation is basically a genetic event, which may include gene amplifications (Klein, 1981; Pall, 1981; Varshavsky, 1981). The maintenance of the transformed phenotype then appears to be dependent on the activity of the transforming gene(s). In the following, we review some approaches to understand the relationship of the malignant phenotype and the connective tissue matrix. A great interest in fibronectin dates back to 1973, when it was first found that a polypeptide was greatly reduced from the surface of virally transformed fibroblastic cells as compared to normal cells (see Vaheri and Mosher, 1978). It was also shown that a reduction of collagen production occurred in virus-transformed fibroblasts (Levinson et al., 1975). The role of the transforming viral genes in the loss of the pericellular matrix (Gahmberg et al., 1974; Vaheri and Ruoslahti, 1974; Hynes and Wyke, 1975; Adams et al., 1977; Arbogast et al., 1977) was established using virus mutants temperature sensitive for transformation. It is now known that the transforming src gene of Rous sarcoma virus encodes a tyrosine-specific protein kinase, and the relevant cellular substrates for this unique enzymatic activity are being keenly sought and studied. Loss of the pericellular matrix in RSV transformation of chick fibroblasts may be partly explained by the 5-fold reduction in the biosynthesis of both

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fibronectin and procollagen due to a corresponding reduction in the copy number of their messenger RNAs (Adams et al., 1977, 1979; Howard et al., 1978; Rowe et al., 1978; Fagan et al., 1979, 1981 ;Sandmeyer and Bornstein, 1979; Sandmeyer et al., 1981; Parker and Fitschen, 1980). The biosynthetic defect has been traced to the nuclear level, probably involving a reduction in the transcription of collagen (Sandmeyer et al., 1981). It should bementioned that several of the phenotypic changes upon transformation by avian tumor viruses, including the decrease in tropomyosin, have been attributed to loss of transcriptional controls (Groudine and Weintraub, 1980; Hendricks and Weintraub, 1981). Not only the quantity but also the quality of the matrix proteins is altered. In transformed chick fibroblast cultures probably even less of the procollagen propeptide cleavages are carried out than in cultures of normal cells (Arbogast et al., 1977). In addition, in RSV-transformed chick embryo fibroblast cultures there is also a quantitatively altered phosphorylation and an increased degradation of fibronectin (Olden and Yamada, 1977; Teng and Rifkin, 1979; Ali and Hunter, 1981). It is also clear that malignantly transformed mesenchyme-derived cells have a reduced capacity to deposit the fibronectin they synthesize (Vaheri and Ruoslahti, 1975; Vaheri et al., 1978b). The reduction in the amount of matrix fibronectin upon transformation has been observed consistently using a variety of methods in both viral, spontaneous, and chemical transformation. Exceptions have been rare (see Vaheri and Mosher, 1978). Notably, Parry et al. (1979) observed a reduction in procollagen synthesis but found no change in the synthesis of fibronectin by RSV-transformed avian tendon cells when compared with uninfected normal cells. Nevertheless, pericellular fibronectin was greatly reduced in the cell layer (Parry et ul., 1979). A further decrease in collagen synthesis was observed when the virustransformed tendon fibroblasts were treated with a potent tumor promoter, 12-0-tetradecanoyl-phorbol13-acetate (TPA) (Bissell et al., 1979). The decrease effect is similar to that found when TPA is applied to normal cells (Blumberg et al., 1976). In RSV-infected chick chondrocyte cultures, only biosynthesis of cartilage-specific collagen type 11 and proteoglycan are decreased, whereas fibronectin production is increased (Pacifici et al., 1977; Yoshimura et al., 1981). In contrast, the avian osteopetrosis retrovirus causes a manyfold increase in collagen production of the infected cells, possibly through a mechanism of promoter insertion (R. Franklin, personal communication). The effect of Kirsten murine sarcoma virus transformation on BALB-3T3 cells is an increase in type 111 relative to type I collagen and a large increase in the production of noncollagenous protein (Bateman and Peterkofsky, 1981).

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Complex posttranslational modifications (Table 11) make collagen biosynthesis sensitive to many perturbations. We have recently studied the enzymatic processing of procollagen in cultures of human sarcoma cells (Myllyla et al., 1981)and found an increase in procollagen modifications as compared to normal cells. The initial effect of virus transformation of chick embryo cells was reminiscent of acute tissue injury in effecting a rise in prolyl hydroxylase activity. In virus-transformed cells, however, all the enzyme activities of prolyl and lysyl hydroxylation and hydroxylysyl glycosylation are decreased, as is collagen biosynthesis. Although several differences are found between fibronectins from normal and transformed cells, mainly in the cell-specific patterns of glycosylation (Pande et al., 1981; Wagner et al., 1981), these do not seem to result in defective functions of the protein. Neither the differences nor the often decreased biosynthesis or increased proteolytic degradation of fibronectin of transformed cells seem to be sufficient explanations for the failure of transformed cells to deposit fibronectin in a pericellular matrix form. The major defect is considered to be on the malignant cell surfaces. Quantitative proportions of different glycosaminoglycans are changed in transformed cells. The overall tendency is a shift from complex sulfated copolymeric glycosaminoglycans, e.g., heparan sulfate, to hyaluronic acid production (see Kramer, 1979; RodCn, 1980; Chiarugi, 1982; Stanley et al., 1982). Certain virus-transformed cell lines have been found to synthesize heparan sulfate with reduced degree of sulfation (Winterbourne and Mora, 1981) and of self-associating properties (Fransson et al., 1982). As already discussed, strongly sulfated glycosaminoglycans,especially heparan sulfate, interact with fibronectin and laminin and are candidate receptor molecules for these proteins in matrix assembly at the cell surface. Recent studies on RSV transformation of rat cells show loss of the pericellular matrix containing fibronectin, laminin (Hayman et al., 198l), procollagen, and heparan sulfate proteoglycan (Alitalo et al., 1982b). To date, no differences between the laminin produced by normal versus transformed cells have been described. Altered patterns of glycosylation of cell surface proteins and of glycolipids in transformed cells have also been offered to explain the loss of cell surface matrix (see Vaheri, 1978; Warren et al., 1978; Atkinson and Hakimi, 1980). Moreover, cytoskeletal structures are disturbed in transformed cells (Nicolson, 1976a,b; Ball and Singer, 1981). Experimental disruption of the microfilament bundles by cytochalasin B causes partial loss of the fibronectin matrix from the surface of normal cells (Ali and Hynes, 1977; Kurkinen et al., 1978). On the other hand, addition of matrix-derived fibronectin restores a more normal morphology, microfilament bundles, and reduces

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surface microvilli concomitant with enhanced spreading of the cells (Yamada et al., 1976a,b; Ali et al., 1977; Willingham rt al., 1977). An interaction of the cytoskeletal microfilament bundles and the exoskeletal fibronectincontaining matrix has been inferred from double-label immunofluorescence studies (Mautner and Hynes, 1977; Heggeness ei al., 1978; Hynes and Destree, 1978) and immunoelectron microscopic data (Hedman et ul., 1978; Singer, 1979). In these studies a transmembrane linkage between actin stress fibers and fibronectin fibers was postulated (review Hynes et al., 1982). However, a direct interaction between actin and fibronectin (Keski-Oja et al., 1980) probably can occur only after damage to the plasma membranes. Proteins that have been localized to focal adhesion plaques, the sites where actin microfilament bundles terminate at the plasma membrane, are potentially important in mediating actin-fibronectin interaction, as discussed in detail in the last section of this article. The loss of pericellular fibronectin is not generally regarded as instrumental for stimulation of growth in normal cells. On the contrary, added fibronectin seems to be necessary for growth in many cell cultures, probably owing to the effect of fibronectin on cell attachment and spreading (Grinnell, 1978; Couchman et al., 1982), the latter permissively affecting growth control (Folkman and Greenspan, 1975; Willingham et al., 1977; Folkman and Moscona, 1978). Growth-promoting effects of the feeder cell layers may be partly explained by the pericellular matrix they provide. Some permissive control can also be exerted by substrate-bound (pro)collagen, because a reduction in collagen hydroxylation and secretion by a proline analog cishydroxyproline, detaches normal cells from the growth substratum (Kao et al., 1979; Liotta et al., 1978; Vembu rt al., 1979). Transformed cells, especially the tumorigenic ones, possess a higher degree of autonomy and can mostly be grown without solid support. Interestingly, tumor cells plated on an isolated pericellular matrix, instead of plastic, assume a flattened, nonoverlapping morphology as well as more migratory activity (Vaheri et al., 1978a; Gospodarowicz et ul., 1980; Vlodavsky et al., 1980). The proliferative response of capillary endothelial cells to tumor angiogenesis factor also requires a collagen substratum (Schor et ul., 1979). Several lines of evidence show that direct proteolysis cannot explain the loss of matrix proteins in culture. The subject of proteolysis will be discussed below. V. Turnorigenicity, Invasion, and Metastasis

It has been suggested that lack of fibronectin would liberate the metastasizing cells from their pericellular matrix. Specific experiments testing the

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effect of such a single parameter cannot be performed in ciuo, but the correlation between the two seems incomplete in a large variety of cells (Coll et al., 1977; Der and Stanbridge, 1978; Kahn and Shin, 1979; Neri et al., 1979, 1981). It may be argued that most of these studies have used immunofluorescence detection that may have picked up heterologous fibronectin from serum, and the presence of extracellular fibronectin antigenic determinants does not imply the presence of functional pericellular fibronectin molecules. An additional complication in these studies is exemplified by Smith et al. (1979), who observed that human epithelial cell lines derived from nonmalignant tissues or from primary carcinomas deposited fibronectin in culture, whereas cell lines grown from metastatic carcinomas did not, but that upon subculture, two of the latter cell lines became fibronectin-positive. In assays of tumorigenicity of an in citro cell line, one should thus reisolate the tumorigenic cells and show that the parameters one attempts to assign as their correlates also apply to the population grown and selected in cico (Smets, 1980).The use of rodent cells is another shortcoming in such studies. Phenotypically normal anchorage-dependent cells, such as 3T3 cells, may be highly tumorigenic when implanted while attached to glass beads (Boone, 1975). Anchorage dependence may thus apply in cico only until intrinsically tumorigenic cells are selected. Even rodent host cells can be transformed in uivo by implanting tumor cells (Huebner et al., 1979) or foreign particles of defined physical and chemical characteristics. Sarcomas of various histopathological types develop in the connective tissue capsule surrounding the particle (Brand, 1975). The loss of the fibronectin-containing matrix in transformation may in fact be a cell culture artifact, as Stenman and Vaheri (1981) found, studying sections of human tumors. Whereas carcinoma and melanoma cells had no pericellular fibronectin in uivo, in various sarcomas the individual tumor cells were surrounded by fibronectin. The role of basal laminae in the neoplastic disorganization of tissue architecture has been emphasized (Ingber et a/., 1981). Complex and incompletely known mechanisms regulate the integrity of basal laminae. Basal lamina is in many cases synthesized, at least in part, by cells that are destined to invade it, although underlying mesenchyme may also provide stabilizing components (David and Bernfield, 1981; Brownell et al., 1981). In considering the process of invasion, one has to explain the penetration by tumor cells of interstitial matrix and basal laminae. Although it has not been proven that a breakdown of the matrix must occur in invasion, there is evidence that a major mechanism promoting the invasive function of cancer cells is the enzymatic hydrolysis of connective tissue components (review Liotta et al., 1981a). Triple helical collagen is very resistant to most pro-

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teinases. However, after an initial cleavage by specific collagenases, the triple helical conformation of collagen undergoes changes that make it sensitive to a variety of proteinases. Considerable specificity has been found in animal collagenases. The latent collagenase produced by fibroblasts, which is activated by trypsin, for example, works best on collagen types I and 111,poorly on type I1 (collagen or cartilage), and not at all on collagen types IV and V (Welgus et al., 1981; Harris and Vater, 1982). Increased amounts of latent collagenase of interstitial specificity are produced by phorbol ester-stimulated endothelial cells (Moscatelli et al., 1980). It may be that this activity is manifested in the process of tumor angiogenesis, beginning with a focal disappearance of adjacent basement membrane and sprouting of endothelial cells from preexisting vessels to the interstitium toward the tumor (Haudenschild, 1980).This process apparently starts the development of an abundant capillary supply to the tumor mass, which then can enter an exponential phase of growth (Folkman and Greenspan, 1975). Gross et al. (198 1) observed a complete block of neovascularization and three-dimensional tumor growth concomitant with inhibition of tumor collagenase production by medroxyprogesterone and dexamethasone. Other experiments from the same laboratory suggest that certain tumor cells stimulate cocultured normal fibroblasts to produce collagenase (Biswas and Gross, 1981) and plasminogen activator activity, probably in response to angiogenic factors that the tumor cells secrete (Gross and Rifkin, 1981). Liotta and others (1979) showed the existence in murine tumor tissue of a metalloprotease with a specificity for basement membrane collagen. Subsequently, in murine melanoma cell lines with graded metastatic potential, it was demonstrated that this enzyme activity correlated with the ability of the cells to metastasize (Liotta et al., 1980b). Disruption of basement membrane is also associated with granulocyte extravasation at sites of inflammation and may mainly occur by means of neutrophil elastase (review Werb et al., 1981). The neutrophil enzyme apparently cleaves type IV collagen (Mainardi et al., 1980a; Uitto et al., 1980), laminin (Ott et al., 1981), fibronectin (McDonald et al., 1979), and type I11 collagen (Gadek et al., 1980),which is a component of the subendothelium. Fibronectin is also quite sensitive to cleavage by cathepsin G (Vartio et al., 1981), another major neutrophil proteinase active at neutral pH. The distribution and functional role of type V collagen are poorly known. This collagen type may be associated preferentially with cell surfaces (Gay et al., 1981), though it also seems to exist adjacent to certain basement membranes (Roll et al., 1980; Sano e f al., 1981 ; Martinez-Hernandez et al., 1982). As for type IV collagen, a latent trypsin-activated metalloproteinase that cleaves type V collagen has been shown to be secreted by certain leiomyo-

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sarcoma and reticulum cell sarcoma cells in culture (Liotta et al., 1981c), and a related enzyme is produced by macrophages (Mainardi et al., 1980b). Activation of the coagulation system commonly occurs in association with neoplasia and there is evidence implicating local production of thrombin in the vicinity of invasive tumors (review Edgington, 1980). Several effects on matrix proteins have been reported for thrombin, but whether they have any relevance in invasion or even in cell migration or repair processes is unclear. Thrombin will in low concentrations stimulate the release of pericellular fibronectin from cultured fibroblasts (Mosher and Vaheri, 1978) and from cell-free fibroblast matrices (Keski-Oja et al., 1981). In somewhat higher concentrations thrombin will cleave fibronectin (Furie and Rifkin, 1980), laminin selectively at its M, = 400,000 chain (Liotta et al., 1981b), and, interestingly, type V collagen (Sage et al., 1981b). Fischer discovered in 1947 that tumor cells but not normal cells lysed blood clots. The molecular mechanism for this, the production of plasminogen activator by malignant cells, was later discovered by Reich and co-workers (Reich, 1975). Secretion of the activator(s) is by no means restricted to transformed cells, but is now considered part of a wide-range physiological regulatory mechanism. This proenzyme system has quantitatively an extraordinary capacity that cannot be neglected in studies of proteolysis in the extracellular matrix. Both fibronectin (Jilek and Hormann, 1977b; Balian et af., 1979) and laminin (Liotta et al., 1981b; Ott et al., 1981) have defined sensitive cleavage sites for plasmin (Fig. 1). Plasmin as well as other serine esterases can also activate latent collagenases with interstitial (Stricklin et al., 1977; Moscatelli et al., 1980; Paranpje et al., 1980) or basement membrane specificity (Liotta et al., 1981a). There is evidence (Quigley, 1979), albeit indirect, that plasminogen activator itself may act on substrates other than plasminogen and that these effects may be involved in virus-induced morphological cell transformation. More recently, Keski-Oja and Vaheri (1982) found that when cell-free preparations of pericellular fibroblast matrices are exposed to purified urokinase, cleavage of a 66,000 MW polypeptide and a concomitant generation of a 62,000 MW polypeptide were detected. The matrix-associated protein has not been identified, but interestinglythe same cleavage is brought about by a peptide fraction secreted from cultured sarcoma cells (Keski-Oja and Todaro 1980), and by thrombin (Keski-Oja et al., 1981), which, as mentioned before, release proteins from the matrix. Of obvious potential significancefor tumor biology is the finding (De Petro et al., 1981) that proteolytic fragments of fibronectin produced by digestion with plasmin or cathepsin G promote morphological cell transformation in fibroblast cultures infected with RSV. All activity in cathepsin G digest

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resides in the gelatin-binding fragments ( M , = 30,000), which are active in nanomolar concentrations. Moreover, a similar transformation-promoting activity detectable in plasma .cryoprecipitates of most cancer patients and very rarely in normal individuals (Mignatti et al., 1980) also binds to gelatin (Barlati et al., 1982). It thus seems possible that fibronectin-degradation products may have an active role in the transformation process and may serve as tumor markers. The latter possibility is supported by the findings that elevated levels of some proteins recognized by fibronectin antibodies have been detected in tumor patient sera (Parsons et a/., 1979; Todd et ul., 1980). Fibronectin may be visualized as a series of active domains with intervening proteinase-susceptible regions (Fig. 1). The proteinase-released fragments may have feedback regulatory or interfering effects on the functions of the intact molecule, and quantitation of the fragments might serve as markers for cancer and other pathological processes characterized by proteolytic events. In considering the mechanism of its effect, it is of interest that the gelatin-binding fragment of fibronectin binds to type I collagen (Engvall et a/., 1981a) in a temperature-dependent way; that it interacts with a new variant of basement membrane collagen (Engvall et al., 1982) and with polyamines (Vartio, 1982); that M , = 60,000 gelatinbinding fragments apparently bind intact fibronectin (Ehrismann et al., 1981); and that Fab’ to such fragments inhibit both fibronectin and collagen 1982). It may deposition in cultures of human fibroblasts (McDonald et d,, also be relevant for tumor biology that proteolytic fragments of fibronectin modulate phagocytic functions of monocyte/macrophages (Czop et d., 1981 ; Ehrlich et al., 1981) and are chemotactically active (Postlethwaite ef al., 1981). There is some evidence that pericellular proteolysis of normal plasminogen activator-producing cells may be regulated by proteinase inhibitors. Several years ago (Mosher et al., 1977) found that various types of human embryonal cells synthesize and secrete a,-macroglobulin (aZM), known as a plasma proteinase inhibitor with a broad spectrum of activity. These included adherent cells grown from lung, heart, and kidney, but not cells from embryonal skin, for example. There seems to be a close correlation between the capacity to secrete a,M and plasminogen activator in the case of normal cells. For example, human peripheral blood monocytes, which d o not appear to synthesize or2M or plasminogen activator, secreted both after differentiation in culture conditions (Hovi et a/., 1977, 1981; Saksela et a/., 1982). All malignantly transformed cells that we have examined have been negative for a2M production (Saksela and Vaheri, 1982). This has led to the hypothesis that if a normal cell produces plasminogen activator, production or endocytosis of a2M, a high-molecular-weight, slowly diffusing proteinase

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inhibitor, is required for normal cell surface integrity and interactions with the pericellular matrix. Observations on placental cells support this hypothesis. Prominent immunoperoxidase staining for a,M has been observed in uiuo in the syncytiotrophoblasts of normal human placentas and of hydatidiform moles, but not in the invasive moles or in choriocarcinomas. Studies on cultured normal and malignant trophoblasts showed that only the former endocytosed detectable amounts of cc,M from the culture medium (Saksela et af., 1981). It has been suggested that due to its unique hydrodynamic properties, hyaluronate exerts a swelling pressure that can cause alterations in the size of intercellular spaces and separation of cell and connective tissue layers, thus opening avenues for cell migration (review Toole, 1976). During tissue development, hyaluronic acid is enriched at sites through which cells migrate such as cornea, sclerotome, neural crest, heart, and primary mesenchyme. In newt limb regeneration, chick embryo sclerotomal cells, and chick embryo limb mesoderm, hyaluronate synthesized during migration and proliferation is degraded by hyaluronidase after the proliferative and morphogenetic phase (review, Lash and Burger, 1977). As has already been mentioned, heparan sulfate may stabilize fibronectin-adhesion sites in cell attachment (Culp et al., 1980). Because malignant cells, as discussed before, in general have less heparan sulfate on their surface and produce greater amounts of hyaluronic acid, this could allow more freedom for them to move into neighboring host tissues. The mechanism was proposed for local invasion, i.e., in continuity with the original tumor mass, rather than for metastasis. In agreement with this theory, Toole and co-workers (1979) found the hyaluronate of the invasive rabbit V2 carcinoma (which also produces collagenase; ref. Biswas et al., 1978),elevated at the tissue interface between the tumor mass and host tissue totaling four to five times that of the tumor in nude mice, in which it is noninvasive. Recent results of Winterbourne and Mora (1981) also show that cells selected for high tumorigenicity synthesize heparan sulfate with a reduced degree of sulfation. Tentative evidence has been presented (Kramer et af., 1981) for production by metastatic murine B16 melanoma cells of a glycosidase capable of cleaving specifically glycosaminoglycans at the heparan sulfate-rich intrachain sites. Metastatic tumor cells tranverse organ boundaries and capillary walls to initiate growth, often at specific sites. In metastatic spread, tumor cells encounter the extracellular matrix material lying beneath epithelial and endothelial cells. The metastatic potential of B16 melanoma cells may correlate with cell surface alterations (Rieber and Rieber, 1981) and with preferential adherence to the subendothelial matrix (Kramer ef af., 1980), a process which may be partly mediated by fibronectin (Nicolson et al., 1981). Certain metastatic cells attach more rapidly to type IV than to type I collagen,

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whereas nonmetastatic cells prefer type I collagen (Murray et al., 1980). Laminin, but not fibronectin, increases cell attachment to type IV collagen (Vlodavsky and Gospodarowicz, 1981 ; Terranova et al., 1981), suggesting that tumor cells which utilize laminin preferentially may have a selective advantage in forming metastases. VI. Proteins of Basement Membranes and Interstitial Matrix as Characteristics of Tumor Cells

The emergence of human tumors is a relatively slow process during which there is a clonal evolution of phenotypically heterogeneous tumor cell populations (Nowell, 1976; Smets, 1980). In spite of this divergence, even after malignant transformation, cells retain much of the characteristics of their tissues of origin and this property forms the basis for their classification, which is currently based mainly on light microscopic criteria including organization of the tumor cells into a tissue type-specific architecture and their relationships to surrounding interstitial connective tissue structures and to basement membranes. The establishment of human tumor cells for in uitro growth has been a rare event (Ponten, 1976), and in many cases it is not known how well the in uitro-selected cells represent the corresponding tumor cells in uiuo. Therefore, we have found it of interest to study the matrix glycoproteins of human tumor cell lines. The results show a characteristic profile of secreted matrix proteins for each cell line (Table VI). The patterns obtained for tumors of mesenchymal and of epithelial origin can be broadly grouped on the basis of their secreted interstitial and basement membrane collagen types, respectively. Tumor cells of mesenchymal origin-fibrosarcomas, a leiomyosarcoma, and a rhabdomyosarcoma-produced interstitial collagen types I and/or 111 and fibronectin, in agreement with their tissues of origin (Table VI). Osteosarcoma cells produced types I and 111 procollagen and small but detectable amounts of fibronectin. In a previous study by Stem et al. (1980), other osteosarcoma cells were found to synthesize predominantly type I procollagen, whereas the major procollagen of Ewing sarcoma cells, for example, was found to be type 111. Here it is interesting to note that in mature bone no fibronectin or type 111 collagen is present (Thesleff et al., 1979; Gay and Miller, 1978). Another tumor cell line, originating from a rhabdomyosarcoma, produced only type V procollagen (Alitalo et al., 1982f).Two fibrosarcoma cell lines produced matrix glycoproteins typical of fibroblastic cells, but a human sarcoma cell line (HT-1080) derived from a fibrosarcoma (Rasheed et al., 1974) showed exclusive production of basement membrane proteins, type IV collagen, fibronectin, and laminin (Alitalo et af., 1980~).

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We therefore suggest that either the HT-1080 tumor cell line is not representative of the original tumor, a poorly differentiated fibrosarcoma, or that the histopathological diagnosis was incorrect. Of the carcinomas studied by us, only an alveolar cell carcinoma from lung produced fibronectin. The lack of fibronectin in human carcinomas in cico is a conspicuous finding recently corroborated in an immunohistological study of human solid tumors (Stenman and Vaheri, 1981). Several normal and some malignant human epithelial cells do produce fibronectin in culture (Crouch et af., 1978; Smith eral., 1979; Alitalo et af.,1980b, 1982a,c; TaylorPapadimitriou et af., 1981). There are remarkably few examples of a change in cell type-specific collagens upon malignant transformation (see Vaheri and Alitalo, 1981). Several tumors that show enough histopathological patterns for classification also secrete matrix glycoproteins specific for their supposed tissues of origin. This has been the case in chondrosarcomas (Smith et al., 1975; Breitkreutz ef al., 1979; Lanzer et af., 1981), a leiomyoma (Chung and Miller, 1974), and the RD rhabdomyosarcoma cell line (Krieg et ul., 1979). Although dedifferentiation occurs as evidenced by production of type 111 collagen by osteosarcoma cells and some type IV collagen by sarcoma cells, the exclusive production of basement membrane collagen by carcinoma cells may serve as a marker for tumors of ectodermal origin. It thus appears that immunochemical identification of matrix proteins, as well as of intermediate-sized filaments as proposed by Gabbiani et ul. (1981), may serve as an adjunct in the classification of human tumors. Antibodies to matrix proteins are becoming generally available and may be applied on routine histopathological sections. Besides their usefulness in classification, the antibodies may also be useful in detection of basement membrane disintegration and of micrometastasis in lymph nodes by carcinoma cells (Liotta et af., 1979; Albrechtsen et al., 1981). Alternatively, when feasible, polypeptide analysis of cultured tumor cells would aid in the classification. In particular, the polymorphic soft tissue sarcomas are currently classified according to schemes that provide an array of terms and eponyms without a thorough knowledge of the histogenesis of these tumors (Stem, 1981). Analysis of their pericellular matrix proteins may uncover aspects of the biology of these tumors. On the other hand, malignant cell lines provide unique biosynthetic systems for studies of those matrix proteins that are difficult to obtain from tissues, due either to their presence only in small amounts or to the difficulties in their solubilization without fragmentation. Thus, for example, advances were made in studies of the structure of type IV collagen from tumor cells rather than from basal laminae (Tryggvason et al., 1980; Alitalo et al.,

TABLE VI MATRIXGLYCOPROTEIN OF CERTAINHUMAN TUMORCELLLINES'

Cell line and its presumed origin Mesenchymal 8387 A9733 A3048 MG63 RD A204 Epithelial A43 1 HT-29 Bowes A549 BeWo HeLa Other TuWi HT-1080 251MG a

Gel electrophoretic analysis of collagen types

Fi brosarcoma Fibrosarcoma' Leiomyosarcoma Osteosarcoma Rhabdomyosarcoma Rhabdomyosarcoma

I, 111, (Ivy 1.111, (Iv) I, 111, (IV) I, 111, (IV) 111, IV, (V) V

Epidermoid carcinoma of vulva Colon carcinoma Melanoma Alveolar cell carcinoma of lung Choriocarcinoma (Adeno)carcinoma of uterine cervix

IV

Wilms' tumor Fibrosarcoma? Astrocytoma

IV

v, IV -, (WJ

Intracellular Fibronectin

Laminin

+ + + fd +

Reference

Krieg er al. (1979) Alitalo er 01. (1982f)

-

+ ND'

+

+ f Iv IV Novel type (VI)

Reference, Alitalo er al. (1981).

' Parentheses indicate small amounts detected. Isolated from soft agar colonies of the original tumor. f , Weakly positive. ND, Not done. Some clones positive (H. Sage, personal communication).

+ + +

Alitalo et al. (1980~) Alitalo et al. (1982e)

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198Oc), and a novel, possibly largely nonhelical procollagen was identified in cultures of human astrocytoma cells (Alitalo et al., 1982e). Similarly, the structure and biosynthesis of basal lamina heparan sulfate proteoglycan have been elucidated in tumor tissue and cell cultures (Hassell e t a / . , 1980; Oohira ef al., 1981), and in antibodies prepared against the isolated molecules.

VII. Cell-Matrix Interaction and Anchorage Dependence of Normal Cells

A fundamental but not absolute requirement for in oitro growth of normal nonhematopoietic cells and maybe even hematopoietic stem cells (Lanotte et al., 1981) is their anchorage to a substratum. There is evidence to suggest that this requirement is not a property of early embryonal cells, but is manifested during the process of cellular differentiation (Nakano and Ts’o, 1981). On the other hand, transformed cells in general have lost anchorage dependence together with the density-dependent inhibition of growth (MacPherson and Montagnier, 1964; Todaro et al., 1964; Dulbecco, 1970; Shin et a/., 1975). Chondrocytes (Honvitz & Dorfman, 1970) and aortic endothelial cells (Laug et al., 1980) are among the few karyotypically stable nontransformed cells that can be grown in soft agar in the usual culture conditions. Recently, however, normal human fibroblasts have been grown in methyl cellulose in regular medium supplemented with 20”/, serum and hydrocortisone (Peehl and Stanbridge, 1981). Although the long-held criterion of anchorage independence as the best in citro correlate of tumorigenicity fails, one still is left with abundant data on the relative autonomy of growth of tumor cells also in semisolid media. In an effort to understand the anchorage dependence and the densitydependent inhibition of cell growth, Folkman and Moscona (1978) designed experiments to study correlation between cell shape and growth rate in nontransformed cells. In these studies growth substrata were coated with increasing concentrations of poly(2-hydroxyethylmethylacrylate), to which the cells or fibronectin do not bind (Klebe et al., 1981). A series of cultures with graded cell shapes due to differential spreading was obtained and both thymidine incorporation and number of divisions correlated with cell height. Growth rate showed a direct correlation with the degree of cell spreading in such cultures. In a series of studies, Penman and collaborators (Benecke et al., 1978, 1980; Ben-Ze’ev et a/., 1980) have addressed themselves to the question of what happens in the cell when it is suspended in anchorage-independent conditions. Placing anchorage-dependent fibroblasts in suspension culture was found to have profound effects on macromolecular metabolism. Protein synthesis declines slowly but extensively as messenger RNA molecules are

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withdrawn from translation (Benecke et al., 1978). Protein synthesis resumes rapidly when suspended cells reattach; recovery requires only cell contact with an external surface and is shape-insensitive. In contrast, nuclear events, such as DNA and rRNA synthesis, and mRNA processing, recover more slowly, require extensive cell spreading, and therefore appear to be responsive to cell shape (Ben-Ze’ev et af., 1980). It is most interesting that the selected spontaneously immortalized aneuploid mouse fibroblasts show a progressive loss of such shape-responsive metabolic control functions with an increasingly transformed phenotype (Wittelsberger et al., 1981;Vasiliev and Gelfand, 1982). Cell attachment and spreading to the culture substratum have been extensively studied in the past (review, Grinnell, 1978). Following the bridging of electrostatic repulsion by cytoplasmic microextensions, adhesion to a protein-coated substratum appears to occur through ligand-receptor-like interactions (see, e.g., Rauvala and Hakomori, 1981), which may involve redistribution of several low-affinity receptors (Grinnell, 1978; Rubin et al., 1981).Concomitant with this, there is a general reorganization of the microfilaments. The closest and probably the strongest adhesion of well-spread fibroblasts to a solid surface occurs through the specialized areas at the ventral cell surfaces called focal adhesion plaques or focal contacts (Curtis, 1964; Abercrombie and Dunn, 1975), which form at the cell periphery only after initial adhesion and considerable spreading has taken place. Actin microfilament bundles terminate at these adhesion plaques (Abercrombie et al., 1971;Abercrombie and Dunn, 1975; Izzard and Lochner, 1976,1980; Heath and Dunn, 1978; Wehland et af.,1979). The first focal contacts close to the cell margin are probably devoid of the attachment protein, fibronectin (Birchmeier et al., 1980; Chen and Singer, 1980; Fox et al., 1980). In fact, exogenous fibronectin is removed from cell-substratum contacts upon cell spreading (Avnur and Geiger, 1981a). However, in stationary cultures arrested in growth in low serum concentrations, fibronectin is found in the focal adhesion plaques (Singer and Paradiso, 1981). Other matrix molecules possibly associated with the focal contacts include heparan sulfate and, in some cells, laminin (Alitalo et al., 1982d; see Culp et a!., 1980). In addition, vinculin, a 130,000-MW intracellular protein, is specifically localized within the adhesion plaques (Geiger, 1979; Burridge and Feramisco, 1980). There is evidence from in uitro studies that vinculin and a-actinin, the latter being possibly located somewhat more distant from the membrane (Geiger et ul., 1980,1981), have opposing effects on the formation of the filament networks. Whereas cross-linking and increased viscosity of the fibers is observed with a-actinin, vinculin causes actin fibers to bundle, thus decreasing their viscosity (Jokusch and Isenberg, 1981). Also, receilt studies by Wilkins and Lin (1 982) have identified approximately one high-affinity vinculin-binding site for every 1500-2000 actin monomers in actin filaments, a property

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consistent with the proposed function of vinculin as a linkage protein between actin filaments and the plasma membrane. Focal contacts found in cultures supplemented with higher concentrations of serum often lack fibronectin but retain vinculin (Singer and Paradiso, 1981; Singer, 1981). Thus focal contacts to the substratum may behave as dynamic structures subject to modulation by the growth state of the cell and apparently also by the cell cycle.

VIII. Molecular Mechanisms of Altered Cell-Matrix interaction in Rous Sarcoma Virus Transformation

There is extensive evidence to indicate that retrovirus-induced tumors result from perturbations in the structure or expression of normal cellular genes (see Bishop, this volume). These viruses act by promoting the expression of potential oncogenes of the host, either by inserting a provirus copy in its vicinity (slowly transforming or lymphatic leukemia viruses) or by carrying in their genome a modified cellular oncogene under proviral control of expression (rapidly transforming retroviruses). Several retrovirus oncogenes have been found to code for distinct tyrosine protein kinases. Since the discovery of peptidyl tyrosine phosphorylation by tumor virologists, certain polypeptide growth hormones were shown to stimulate tyrosine phosphorylation of specific membrane proteins through their receptors at the plasma membrane. These include epidermal growth factor (EGF) (Ushiro and Cohen, 1980), platelet-derived growth factor (Ek et ul., 1982), and, interestingly, also the transforming growth factors (Reynolds et al., 1981) secreted by various retrovirus-transformed and tumor cells in culture that initiate anchorage-independent growth of untransformed cells that will not otherwise multiply in soft agar (Todaro et al., 1980). The dose of EGF needed for the cellular proliferation is strongly affected by the tissue culture substratum (Gill and Lazar, 1981). Also, the effect of certain growth factors may be enhanced by supplying cultures with specific substrate-adhesion proteins or by growing cells on prepared extracellular matrices or feeder layers instead of plastic (Gospodarowicz et al., 1980). Indeed, both substrates bind certain growth factors in a biologically active form (Smith et a / . , 1982). The transforming RSV oncogene-coded protein, a MW 60,000 phosphoprotein, pp60"' (see Bishop and Varmus, 1982), has been shown to catalyze phosphorylation of tyrosine residues in several proteins in uitro (review Hunter et al., 1981), and in RSV-transformed cells several possible substrates for pp60""ave been identified (review Sefton et al., 1982).Transformation of avian sarcoma virus-infected cells may result from provirus-controlled and

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enzyme-amplified increase in phosphorylation of some substrates, or also from a changed substrate-specificity or cellular regulation of the slightly altered virus-coded enzyme (Smart et al., 1981). The pp60"" antigen and its kinase activity are specifically associated with both the inner face of plasma membrane and the cytoskeleton (Willingham et al., 1979; Burr et al., 1980; Courtneidge et al., 1980; Krueger rt al., 1980; Levinson et ul., 1981), as do several other proteins (Avnur and Geiger, 1981b; Ben-Ze'ev et al., 1979; Boss et ul., 1981 ; Hughes and August, 1982). In many substrate-attached transformed cells, the protein is enriched under membrane ruffles and intercellular junctions (Willingham et al., 1979), and colocalizes with vinculin to the focal adhesions present in transformed cells (Rohrschneider, 1980; Shriver and Rohrschneider, 1981; see Fig. 3). Because vinculin in transformed cells contains about 10-fold more phosphotyrosine than in normal cells (see Sefton et al., 1982), the colocalization of vinculin with pp60"" in the focal adhesion plaques, as well as the vinculincontaining rosette-like structures appearing in transforming cells (DavidPfeuty and Singer, 1980), have led to the suggestion that ~ ~ 6 kinase 0 " ~ phosphorylates vinculin at the focal adhesions (see Rohrschneider et al., 1982). Thus the activity of pp6OSrcin the adhesion plaques could account for several phenotypic changes that occur upon transformation, like dissociation of microfilament bundles (Weber et al., 1974); Edelman and Yahara, 1976), partial retraction of intermediate filaments (Ball and Singer, 198I), rapid formation of the rosette-like structures on the cell surface (DavidPfeuty and Singer, 1980; Boschek et al., 1981),loss ofpericellular fibronectin, decreased adhesiveness, and rounding up of the cells. The morphological changes can be reproduced in transformed cells that have been enucleated (Beug et al., 1978). Recent experiments by Rohrschneider et al. (1982) show a correlation between the presence of pp60"" in the adhesion plaques and loss of pericellular fibronectin in a series of RSV mutants. Because the enzymatic activity or substrate specificity of pp6OSrcis apparently not altered in all these mutants, the results suggest additional functions for the viral and cellular src proteins. The parameter that best correlates with anchorage-independent growth of cells infected with these mutants is the amount of phosphotyrosine recovered in vinculin of the adhesion plaques. However, the increased phosphorylation of vinculin is not sufficient for disruption of actin stress fibers in such cells (Rohrschneider et al., 1982).So, as has been shown earlier, loss of growth control can occur independently of loss of the actin microfilament bundles (Willingham et al., 1977; Fujita et al., 1981). Tumorigenic properties of the transformed cells correlate with the membrane association of certain mutant pp60"'" proteins (Krueger et a/., 1982) and with a defective cytoskeletal architecture (Leavitt et al., 1982).

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FIG.3. Immunofluorescence (A) and interference reflection microscopy (B) demonstrates pp60”‘ antigen in the substrate adhesion sites (dark areas in B) in SR-RSV-transformed NRK cells. (Figures kindly provided by Dr. L. Rohrschneider.)

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Interestingly, pp60"' and enzymes of other classes of transforming kinases (Cooper and Hunter, 1981a) share substrates with the EGF-stimulated tyrosine-specific protein kinase of the EGF receptor-kinase complex. Both phosphorylate anti-pp60"' IgG (Chinkers and Cohen, 1981; Kudlow P t ul., 1981), a synthetic tridecapeptide corresponding to the sequence of the reported site of tyrosine phosphorylation in pp6OSrc(Pike et al., 1981) and in cell cultures through a shared pathway, a 36,000 MW protein (Cooper and Hunter, 1981b; Erikson et al., 1981b; Hunter and Cooper, 1981) which becomes rapidly phosphorylated upon transformation by RSV (Radke and Martin, 1979; Erikson et al., 1981a) and is possibly partially associated with the cellular framework (Cheng and Chen, 1981; Burr et al., 1981). These findings lead us to reinforce the postulation that for normal cells the interaction with the pericellular matrix substratum may provide regulatory signals, while the autonomous growth of tumor cells may be uncoupled from this requirement, by mechanisms such as production of transforming growth factors or by transforming kinase-catalyzed reactions occurring at the cell surface.

ACKNOWLEDGMENTS Original contributions from this laboratory were supported by The Academy of Finland, The National Institutes of Health, NCI (CA 24 605), The Finnish Cancer Foundation and a Fogarty Fellowship (F05 TW03138). We thank Klaus Hedman, Jorma Keski-Oja, Gary Ramsay, Helene Sage, and Tapio Vartio for comments on the manuscript and several colleagues who provided us with preprints of their recent work.

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RADIATION ONCOGENESIS IN CELL CULTURE Carmia Borek Radiological Research Laboratory, Department of Radiology, Cancer Centerllnstitute of Cancer Research. and Department of Pathology, College of Physicians and Surgeons, Columbia University, New York. New York

.............. ...... ........................................................... 111. Ce ation in Vitro . . . . . ......................... A. sformation Studies . . . . . B. Initiation and Phenotypic Expression of Transformation .................... C. Criteria for Transformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Fo

11. Int

nsfonnation . . . . . . . . . . . V. Discussion References .....................................

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

159 159 161 161 167 169 177 177 202 206 220 227

1. Foreword

The scope of this article is limited to the oncogenic effects of radiation with emphasis on ionizing radiation. Yet because methods in radiationinduced oncogenesis are similar to those employing other oncogenic agents, one must sometimes generalize beyond the scope. Also, because some aspects of the phenotypic nature of radiation-transformed cells may be similar to those of cells transformed by other means, again one must expand the horizon. Although basic aspects of radiobiology and radiation physics are well described elsewhere (Lea, 1947; Bacq and Alexander, 1955; Rossi, 1964; Rossi and Kellerer, 1972; Kellerer and Rossi, 1972, 1975; Hall, 1978), some facts are worth mentioning. Radiation is termed “ionizing” when it possesses sufficient energy to remove electrons from their orbits in atoms constituting the irradiated material. This leads to a breaking of chemical bonds and results in permanent changes. In most cases ionization occurs through electrically charged particles, which may be high-speed electrons or nuclear components such as protons or CI particles. These are directly ionizing radiations. They may originate from external or internal sources. They can also be generated inside the irradiated matter by indirectly ionizing radiations. The latter 159 ADVANCES IN CANCER RESEARCH, VOL. 37

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006637-8

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include electromagnetic quanta (or photons) such as X rays, y rays, and electrically neutral particles such as neutrons. Different types of ionizing radiation can be characterized by the rate of energy deposition along their tracks (linear energy transfer or LET). Thus low-energy radiations such as X rays or y rays are of sparsely ionizing or low-LET radiation, whereas a particles, neutrons, and heavy ions are high-LET radiation, releasing densely clustered ionization along their tracks. These patterns of ionization account for differences in the biological effects of these radiations. Comparison of various radiations in producing a particular effect is defined as the relative biological effectiveness (RBE) and is expressed as the ratio of the absorbed doses required to produce the same biological effect. Ionizations result in short-lived (lo-"' sec) ion pairs which go on to produce free radicals of a somewhat longer life (1 0- ' sec). The amount of ionizing radiation can be expressed in terms of the exposure, the unit of which is the roentgen (R), or in terms of the absorbed dose the unit of which is the rad or the Gray (1 Gy = 100 rad). The exposure of water or soft tissue to 1 roentgen of X rays or y rays results in an absorbed dose of approximately 1 rad. II. Introduction

The oncogenic potential of X rays in humans was realized within the first decade after their discovery by Roentgen in 1895 (Brown, 1936), and confirmed in later years through epidemiological data, the largest single source being from Hiroshima and Nagasaki (Rossi and Kellerer, 1974; BEIR, 1972; UNSCEAR, 1977). Most experimental studies in radiation carcinogenesis have utilized animals (Fry and Ainsworth, 1977; Ullrich C I a/.. 1977; Upton, 1964, 1975; Bond r i d., 1960; Shellabarger rt ul., 1969; Kaplan, 1967; Storer, 1975). Though these systems have yielded important quantitative data, they have their limitations in studies concerned with the effects of low doses of radiation where inordinately large numbers of animals are required, and in the investigation of cellular and molecular events in radiation oncogenesis. The development of cell culture systems has made it possible to study the effects of radiation under defined conditions where complex homeostatic mechanisms d o not prevail. Using in oirro systems free of host-mediated influences affords the opportunity to assess qualitatively and quantitatively dose-related and time-dependent interactions of radiation with single cells. The modifying influences of agents present in the cellular milieu on the genetic levels as well as on gene expression can be studied, and the role of cell-cell interaction in the process of neoplastic development can be evaluated as well.

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111. Cell Transformation m V/tro

A most significant contribution, which served as a foundation for the study of radiation oncogenesis in uitro, was the development of the clonal assay by Puck and Marcus (Puck and Marcus, 1956; Puck o t al., 1956; Puck, 1958) and their demonstration of a dose-related effect of radiation on the survival of single cells. These findings made it possible in later years to evaluate which of these surviving cells has been transformed into a neoplastic state following exposure to radiation, and to determine the frequency of transformation. Thus in 1966 Borek and Sachs first reported the direct oncogenic effect of X rays by exposing diploid hamster embryo cells to 300 rad of X rays and transforming a fraction of them into cells which differed morphologically from untreated controls (Fig. 1). The transformed cells gave rise to tumors upon injection into hamsters, whereas untreated cells showed no spontaneous transformation (Borek and Sachs, 1966a, 1967). The work indicated that in order to fix radiation transformation as a hereditary property, cell divisions must take place soon after exposure, and that subsequent additional replications are required for expression of the neoplastic state (Borek and Sachs, 1966a, 1967). The work also indicated that there exists among cells a differential physiologic and genetic competence to be transformed and that surface-mediated cell recognition was modified in culture upon transformation (Borek and Sachs, 1966b). Transformation of mammalian cells in uitro by radiation was later approached in mouse cell systems (Terzaghi and Little, 1976a; Little, 1979; Han and Elkind, 1979a; Miller and Hall, 1978) and in human cells (Borek, 1980), making it possible to evaluate the effects of radiation on cells across the lines of various species. A. CULTURE SYSTEMS CURRENTLY USED IN RADIATION TRANSFORMATION STUDIES Cell systems currently used in radiation transformation studies are composed of fibroblast-like cells, where morphological criteria serve well in quantitative assays of transformation. Because in the human the preponderance of carcinomas over sarcomas is unequivocal, there is a constant and urgent need to develop epithelial cultures to study transformation. A number of epithelial cell systems have been developed and used in studies on chemically induced transformation (reviewed in Borek, 1979a, 1980a; Heidelberger, 1973, 1975; Weinstein ez a/., 1979; Upton, 1982), but so far not applied to studies in radiation carcinogenesis. There is a particular uniformity in fibroblast-like cells that does not exist in epithelial cells, whose

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FIG. I . A progression of neoplastic transformations in uitro in mass cultures of hamster embryo cells following exposure to 300 rad of X rays (Borek and Sachs, 1966). (A) Appearance of single transformed fusifom cells 18 days after exposure on a background of flat untransformed cells. (B) The same culture 25 days after irradiation. (C) The same culture 58 days later. Note that the transformed cells dominate the culture. By this time the controls degenerated. Phase x340.

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nature and susceptibility to radiation transformation may depend on the particular differentiated qualities and on the source and age of tissue from which they are derived. Human as well as animal data have clearly indicated that, in radiation carcinogenesis, latency, age dependence, and specific organ susceptibility determine the frequency of cancer. A further difficulty with epithelial cells arises from the fact that criteria for early stages in the neoplastic state of epithelial cells are expressed phenotypically in a less consistent manner than in fibroblasts (see Table I). TABLE 1 CHARACTERISTICS OF FIBROBLASTS AND EPITHELIAL CELLSMALIGNANTLY TRANSFORMED in Virro DISTINGUISHING THEM FROM UNTRANSFORMED PARENTAL CELLS Property

Fibroblasts

Morphology (light microscopy)

Pleomorphic. refractile crisscross orientation; irregular growth pattern

Topography (scanning electron microscope)

Increase in surface features

Cell density

Increased saturation density, multilayering, loss of density-dependent inhibition of growth

Serum requirement for growth

Decreased in rodent cells; less pronounced characteristic in transformed human cells, because normal human cells can grow in lower serum levels Yes

Altered cell surface glycoproteins and glycolipids Agglutinability by low concentrations of lectins Increased protease production Changes in cytoskeleton Growth in agar Tumorigenicity

Epithelial cells Often not dramatically different from normal, somewhat more pleomorphic in some cases (e.g., liver) Inconsistent changes; sometimes an increase in microvilli Inconsistent; depending on the cell line and the tissue of origin; in some cell lines piling up of cells, in others maintenance of monolayer growth pattern Low as in the normal (in liver cells); has not been sufficiently studied in a variety of systems

Yes

Yes

Yes

Yes

Inconsistent

Pronounced Yes Yes

Inconsistent Yes Yes

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Among the fibroblast lines there are two main cell systems used in radiogenic transformation studies: primary cultures and cell strains, and established cell lines. 1. Primary Cultures and Cell Strains

Primary cells are freshly derived from animal or human tissue. They are direct descendents of the cells in situ and consist of diploid cells. These cultures have a finite life span that differs from one cell type to another and is related to the longevity of the species from which they originate. The primary cultures used most commonly in radiation transformation studies are those of mixed cell populations derived from hamster embryo cells (Figs. 1,2A, B, and 3) (Borek and Sachs, 1966a ; Borek and Hall, I973 ;

FIG. 2. Morphological criteria for transformation. (A and B) Clonal assay in hamster embryo cell cultures; (C and D) focus assay in mouse embryo lOT4 cells. (A) Normal hamster embryo cell clone, 14 days old. (B) A 14-day-old clone of hamster cells transformed in uitro by 300 rad of X rays. ( C ) A monolayer of untransformed 1OTi cells 6 weeks in culture. (D) A type 111 focus of 10TS cells transformed by 300 rad of X rays, 6 weeks in culture. Note the flatness of the normal cells and their regular orientation. In contrast, the transformed cells form multilayers and display a lack of orientation that is clearly distinguishable at the periphery.

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DiPaolo et al., 1976). The advantage of these cultures lies in the fact that they are composed of normal diploid cells. They senesce upon continuous subculture, allowing “immortal” transformants to emerge against a background of dying cells, thus confirming in vitro their distinct transformed state. Cell survival and cell transformation can be scored simultaneously in the same dishes, and the rate of spontaneous transformation in these cells is less than Expression time for transformation is 8-10 days, a relatively short period, and the cells can be cryopreserved (Pienta, 1977; Pienta et al., 1979). Transformed colonies are identifiable by dense multilayered cells, random cellular arrangement, and haphazard cell-cell orientation accentuated at the colony edge (Fig. 2A,B). Normal counterparts are usually flat, with an organized cell-to-cell orientation. Because of the mixed population of cells there exist untransformed cells which may possess a higher cell density than the usual flat colonies. These, however, do not exhibit the randomness at the colony edge just described. Human primary cultures used in transformation studies are fibroblasts derived from adult human skin (Kakunaga, 1978; Borek, 1980), human embryos (Sutherland et al., 1980), or foreskin (Milo and DiPaolo, 1978; Silinskas et al., 1981). The assay is a focus assay in which the loss of cell

Hamster embryo

i

i

Normal

lhnsformed

Transformed cioneo identified morphologically

FIG.3. Assay for hamster embryo cells transformed in uirro by radiation. Hamster embryos in midterm gestation are removed, minced, and trypsinized progressively with 0.25% trypsin. After removal of the trypsin by centrifugation, cells are suspended in complete medium and seeded as single cells on feeder layers (Puck rt al., 1956). They are exposed to radiation 24 hr later. After 8-10 days in incubation, cultures are fixed and stained. Transformed colonies are distinguished morphologically from controls.

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density inhibition among the transformed fibroblasts renders them capable of proliferating over the untransformed sheets of cells, thus forming distinct recognizable foci (described later). 2. Established Cell Lines Established cell lines possess an unlimited life span. They represent cell populations which originated as primary cultures. Following a continuous and meticulously timed regime of subculturing resulted in the emergence of a selected population had undergone a “crisis,” enabling the cells to grow indefinitely at a constant rate. The karyotype of these cells is usually characterized by various chromosomal rearrangements and heteroploidity. Often these cell lines are cloned and the cloned cells are further propagated into large populations. Examples of cell lines that have been used extensively in transformation studies are the BALB-3T3 cell line developed by Todaro and Green (1963) and further cloned (Kakunaga, 1973) to establish susceptible and nonsusceptible cell lines (Kakunaga and Crow, 1980), and the C3H lOTq clone 8 cell line developed in Heidelberger’s laboratory by Reznikoff et al. (1973a) (Fig. 2C,D). Both cell lines originated from mouse embryos. They are transformable by a variety of oncogenic agents and used extensively in radiation transformation studies, in particular the lOT4 cells (Figs. 2 and 4). The advantage of these systems lies in the fact that they are “im-400 VIABLE CELLS

CELLS STAIN CALCULATE P.E. and S.F.

FIG.4. Protocol for experiments with IOTt cells. Cells are seeded at two cell concentrations into 50 cmz petri dishes. To assess the plating efficiency (P.E.) and cell suriving fraction (S.F.), a sufficient number of cells are seeded so that an estimated number of about 50 cells survive the subsequent treatment; these are incubated for 2 weeks, after which they are fixed and stained and the number of discreet colonies per dish counted. For the assessment of transformation, cells are seeded so that an estimated 400 reproductively viable cells survive the subsequent treatment. Cells are allowed to attach by overnight incubation at 37°C before being irradiated, following which they are incubated for 6 weeks, with the growth medium changed weekly. (Reproduced from Hall and Miller, 1982.)

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mortal,” so one can continuously utilize particular cell passages by maintaining “banks” of frozen cells. The disadvantages are that the cells are not diploid, and if not treated meticulously as originally described they can give rise at high passage to spontaneous transformants. Transformation assay is a focus assay, thus survival must be scored in separate sets of dishes (Fig. 4). In the C3H lOTi system three types of transformed foci are identifiable (Reznikoff et al., 1973b; Terzaghi and Little, 1976a): types I, 11, and 111. Their morphology can be related to their oncogenic potential, type 111 being the most malignant (Fig. 2). 3. In Utero-In Vitro Systems Here exposure to radiation or chemicals is carried out in utero and the tissues are cultured and assayed for transformation in vitro (Borek et al., 1977).

B.

INITIATION AND PHENOTYPIC

EXPRESSION OF TRANSFORMATION

One of the basic conundrums in cancer research evolves from our inability at the present time to distinguish unequivocally primary events associated with initiation of neoplastic transformation from those that function as secondary events. Although we aim to identify the process of initiation and consequently hope to modulate it, we are faced with the fact that at present we determine the occurrence of initiation by its phenotypic expression. Thus although radiation carcinogenesis was recognized some 85 years ago, we are still relatively ignorant of the mechanisms involved and must judge the events determining neoplastic transformation by a variety of phenomena associated with the neoplastic phenotype. These phenomena appear to be similar irrespective of the initiating oncogenic agent, whether it is a virus whose contribution is the introduction of new genetic material, a chemical carcinogen forming adducts with cellular DNA, or radiation, whose initiating action on the cell is established and over within a fraction of a second. We therefore strive to define various steps within the processes of transformation and try to associate cellular and molecular events with each step.

I . Sequence of Events in Transjormation in Vitro (a) Initiation, i.e., exposure of cultured cells to the carcinogen. (b) Fixation of the transformed state requiring cell replication within hours after initiation (Borek and Sachs, 1966a, 1967, 1968; Reznikoff et al., 1973b; Terzaghi and Little, 1976a; Kakunaga, 1974, 1975; Little 1979). (c) Expression of the transformed state of a single cell requiring several cell replications, depending on the cell type. The results are the growth of a

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focus or a colony (depending on the assay), which in fibroblasts and some epithelial cells are morphologically distinct from control (Borek and Sachs, 1966a, 1967, 1968; Reznikoff et al., 1973b; Kakunaga, 1974, 1975; Little 1979) (Fig. 2). It should be added that there is little information on the neoplastic characteristic of exposed fibroblasts, which do not differ morphologically from the normal. So far, the assessment of the transformed state in fibroblasts has consistently adhered to the premise that the earliest observable phenotypic change in the process of transformation is morphological. 2. Methods Used for Transformation Studies

(a) Exposure of mass cultures to radiation or other oncogenic agents and continuous subculturing for several weeks or months until transformed cells are selected out and form foci. Periodic clonings are made to assess the frequency of transformation (Borek and Sachs, 1966a, 1967). (b) Treatment of mass cultures and cloning out at various periods of time after exposure. Transformation can then be scored in a colony or focus assay (Borek and Sachs, 1966a, 1967; Han and Elkind 1979a,b). (c) Treatment of single cells seeded at a low-density clonal level sometimes on feeder cells (Puck et af., 1956). Each cell is then allowed to proliferate into a distinct clone. Cultures are fixed and stained. Transformed clones are distinguished morphologically from normal by high cell density and random cell orientation. This method is used routinely in quantitative studies and has been applied in the hamster system (Borek and Hall, 1973, 1974; Borek et af.,1978), where incubation time is 8-10 days (Fig. 2). (d) Treatment of single cells seeded at low density. Cells are allowed to proliferate to high density. Transformed cells form foci clearly distinct against a background of flat density-inhibited cells. The characteristics of the foci are high cell density as well as randomness of cell organization, especially in the peripheral area of the focus invading the surrounding area. Incubation time for the appearance of these foci ranges from 4 to 8 weeks depending on the cells employed. The method is widely used, for the 3T3 and lOTi transformation assays (4 and 6 weeks incubation time, respectively) (Reznikoff et al., 1973b; Terzaghi and Little, 1976a; Miller and Jall, 1978a; Borek et al., 1979; Little, 1979) (Figs. 2 and 3) and can be applied in human transformation studies where incubation is approximately 8 weeks (Borek, 1980). In all these transformation methods, especially in the treatment of single cells, an important prerequisite is knowledge of the cytotoxic effect of the oncogenic agent on the particular cells used. Thus a dose-response curve for cell survival is essential to evaluate the surviving fractions following exposure to a specified radiation (Borek and Hall, 1973; Terzaghi and Little,

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1976a; Miller et al., 1979; Han and Elkind, 1979a). Another factor of importance that can be applied from knowledge of the survival curve is the requirement for a particular number of viable cells. Transformation rate is decreased if the number of surviving cells exceeds a certain range, such as 200 to 400 viable cells in the 10T; system (Terzaghi and Little, 1976a; Reznikoff et al., 1973b) and approximately 10 to 30% viability in human cell transformation (Borek, 1980). Though the mechanisms underlying this effect are not understood, one could suggest an inhibitory action of normal cells on growth of transformed cells (Borek and Sachs, 1966b), the effects of initial events associated with the interaction of radiation with living matter (e.g., free radicals), which may be less effective under conditions of high cell density, or the modifying effect of cell-cell interaction at later stages of expression where intercellular contacts are established (Borek et al., 1969, 1977). Quantitative analysis in the hamster cell system, where transformation frequency is established from among the survivors in a single plate, can be dealt with statistically in several ways (Borek rt al., 1978). In the 10Ti cells, quantitation of transformation is different because scoring of survivors and transformants is carried out separately. These have been discussed in detail elsewhere (Reznikoff et al., 1973a,b; Terzaghi and Little, 1976a; Kennedy et al., 1980; Fernandez et al., 1980; Rossi et al., 1982; Hall et al., 1982). For optimal quantitative analysis it is important to have no more than one transformed focus per plate because the lower adhesiveness of the transformed cells may lead to cell migration from a focus, forming secondary foci and introducing an error. Another factor that must be considered when comparing results from various laboratories is the method of irradiation. In some cases mass cultures are exposed to radiation and then trypsinized and cloned, thus introducing a variable of cellular plating efficiency ujier radiation. In other cases cells are cloned and then irradiated, thus plating efficiency is established prior to irradiation. Although this method is more cumbersome (requiring the irradiation of single cells contained in hundreds of plates), it eliminates the need for trypsinization after treatment, a procedure that introduces variables in the 10Ti system because the frequency of transformation greatly differs depending on the time (in hours) post irradiation at which cells are dissociated and cloned out (R. C. Miller and E. J. Hall, personal communication).

C. CRITERIA FOR TRANSFORMATION The establishment of criteria for transformation is based on the characterization of transformed cells as compared to their untransformed parental cells. These studies utilize mass cultures of mixed cell populations as well as

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cultures propagated from single isolated clones. Most of the characteristics associated with the transformed state of fibroblasts hold true for epithelial cells, though in the latter case less consistency is observed (Table I). Although we would like to evaluate early changes associated with transformation, we are limited by the low frequency of the event. Thus in order to be certain that we are indeed dealing with transformed populations, we must utilize cells that have expressed their transformed state by morphological alterations. The difficulties become evident. Critical evaluations of transformation are necessary at early stages following exposure and expression, yet for some of the assays, such as biochemical analysis, growth in agar, or tumorigenicity, large populations are necessary. These require extensive propagation of the cells in uitro and the inevitable introduction of variables associated with continuous culture, such as karyotypic instability. Thus although the acquisition of phenotypic characteristics associated with neoplastic transformation has been considered a multistage process in rodent cells (Barret and T’so, 1978), the inability to evaluate some of these expressions until a late stage when further propagation is achieved complicates the issue. 1. D N A and Chromosomes

Because with ionizing radiation no specific DNA-repair enzymes have been identified, in mammalian cells, exploration of the effects of radiation on transformation at the level of DNA damage and repair, and at a chromosomal level, is a study of associated phenomena. This is especially true because the frequency of transformation is low, so the relationship of chromosomal changes or DNA damage and repair, carried out on parallel cultures, can be inferred but not conclusively stated. This holds true for initiation by radiation, where DNA damage and repair (Painter and Cleaver, 1969;Painter and Young, 1972; Painter, 1978) and sister chromatid exchanges (SCE) (Perry and Evans, 1975; Little et al., 1979; Miller et al., 1981 ; Geard et al., 1982) have been investigated, as well as in studies on the modulation of transformation by a variety of agents (Little et al., 1979; Kinsella and Radman, 1978; Miller et al., 1981; Geard et al., 1982). Observing a morphologically changed colony or a foclss prepares the investigator to explore with more certainty the relationship between the karyotypic alteration and the phenotypic expression of the cells, though admittedly one can evaluate the neoplastic nature of these cells only later when progressive culture, and most probably karyotypic changes, have taken place. Another critical factor in studying chromosomal changes associated with transformation is the starting material. The utilization of

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171

diploid cultures in the study may differ from the study of heteroploid cell lines where chromosomal imbalance has already taken its course. Subtle chromosomal alterations following exposure to oncogenic agents may denote genetic rearrangements and instability (Bloch-Schacter and Sachs, 1976; Kinsella and Radman, 1978; Schimke et al., 1981), which may be associated with transformation. Specific genes may play a role in determining susceptibility to neoplastic transformation (Knudson, 1981). However, minimal changes in chromosome number (cells remaining neardiploid) and no changes in banding patterns were observed in diploid hamster or human cells transformed in uitro. These studies were carried out on cells within several passages after initiation and expression of transformation, i.e., at early stages after having expressed other criteria associated with their transformed state (Borek et al., 1977,1978; DiPaolo and Donovan, 1973; Borek, 1980; Silinskas et al., 1981).

2. Loss of"'Contact Inhibition" and Changes in Cell Topography The most obvious phenotypic changes observed in transformed fibroblasts are mediated via the cell surface. A loss of contact inhibition of movement (Abercrombie, 1966) and replication (Todaro et al., 1964), reduced densitydependent inhibition (Stoker and Rubin, 1967), irregular growth patterns, and ability to grow in multilayers (Berwald and Sachs, 1963; Borek and Sachs, 1966a,b; Todaro et al., 1964; Reznikoff et al., 1973b; Terzaghi and Little, 1976a) are all features that characterize the transformed nature of fibroblasts derived from solid tissue and differentiate them from normal counterparts grown under the same conditions. These morphological differences, so distinct in dense populations of cells, are not apparent at low density, when cells are not in contact with one another (Borek and Fenoglio, 1976). Changes at a single-cell level can be found when cell topography is evaluated using scanning electron microscopy (Borek and Fenoglio, 1976). At early stages, following initiation, within 8 days after exposure to radiation and expression of transformation the relatively smooth and simple surface of normal hamster fibroblasts acquires a variety of excrescences and a marked cellular pleomorphism (Fig. 5). These topographic changes, comprising ruffles and blebs, are present on the transformed cell surfaces throughout the cell cycle and remain as an integral part of the transformed cell after many years of culture. The normal parental cells exhibited these complex features only during mitosis (Borek and Fenoglio, 1976), thus affirming other observations indicating that a variety of membrane-associated properties characteristic of the neoplastic state are found in normal cells in mitosis (see Borek, 1979a).

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FIG.5. (A) A normal cell ending mitosis in a high-density culture. The curved daughter cells are flattening out to resume contact with their neighbors. Surface features are observed mainly near the edges of the dividing cell. The interphase cells are completely flat and smooth. SEM x 1500. (B) A high-density culture of X ray transformed cells in interphase and mitosis (possibly telophase). Note the extensive surface feature of blebs and microvilli on both the interphase and mitotic cell. SEM x 7475. (C) A normal mitotic cell (possibly cytokinesis) in a low-density culture. Note the extensive blebs and microvilli on the mitotic cell against the background of the flat, smooth interphase cells. The surface of the normal cell in mitosis is indistinguishable from that of a transformed cell. SEM x 5850. (D) An X-ray-transformed cell in late mitosis (possibly cytokinesis) in a low-density culture. Note the surface, which is rich in blebs and microvilli and is indistinguishable from the normal mitotic cell in (C). SEM x 8125. (Reproduced from Borek and Fenoglio, 1976.)

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3. Decreased Serum and Calcium Dependence

Once rodent fibroblasts are initiated by radiation or other oncogenic agents, and undergo several replications to express their transformed state, their dependence on nutrients decreases and they are capable of proliferating well in medium containing low serum concentrations, as low as 1% serum, whereas their untransformed counterparts remain essentially in the nonproliferating state (Borek et al., 1977; Borek, 1 9 7 9 ~Terzaghi ; et ul., 1976a). This feature, which serves well in selecting out low-frequency transformants from a background of normal cells, holds true for rodent cells but not necessarily for human fibroblasts, where some normal cells as well as the transformants grow at low serum concentrations (Borek, 1980). Low calcium dependence, namely the ability to proliferate in medium containing less than 0.5% calcium, seems to be a feature common to transformed fibroblasts and epithelial cells of rodent origin (Swierenga et al., 1978), as well as transformed human fibroblasts (Borek, 1980). In all cases it has served as a selective feature for transformants, because normal cells die within days after exposure to maintenance medium containing a low calcium concentration, while the transformed cells thrive and can form distinct foci (Borek, 1980). 4. Membrane Structural Changes The cell membrane is a complex dynamic organelle which is thought to exert control over a variety of cellular patterns of behavior (Puck, 1977, 1979; Nielson and Puck, 1980). This control system may function by the coordination of interacting molecules of both surface receptors and submembranous fibrillar elements. This control appears to be transmembranous in nature, inextricably related to the structure of the cell membrane and to the molecular features of its surface receptors. These are glycoproteins, some of which traverse the matrix of the membrane as integral membrane proteins. Their hydrophilic portions project into the cytoplasm, where a number of interactions with cytoplasmic components such as cytoskeletal elements take place and can be affected by antimitotic drugs, hormones, and cyclic AMP (Puck, 1977). Upon neoplastic transformation the cell membrane undergoes a variety of structural and functional changes. Neoantigens are observed (Embelton and Heidelberger, 1975) and glycoproteins decrease, disappear, or are no longer completely glycosylated (Gahnberg and Hakomori, 1973). Similarly, sialoglycolipids (gangliosides), a major group of membrane glycolipids, are incompletely glycosylated (Gahnberg and Hakomori, 1973), showing a reduction in higher gangliosides (Brady et al., 1969; Borek e f al., 1977) (Fig. 6). The enzyme Naf/KfATPase, an Na-transport membrane-associated en-

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CARMIA BOREK

FIG. 6. Thin-layer chromatogram of gangliosides from normal and X-ray-transformed hamster embryo cells. (Lane 1) Gangliosides in normal control; (Lane 2) gangliosides standards; (Lane 3) gangliosides in X-ray-transformed cells. Note that in the transformed cells, gangliosides higher than GM, are absent. (Reproduced from Borek e t a / . , 1977.)

zyme, is altered (Borek and Guernsey, 1981) (Table 11). Intercellular communication is modified (Borek et al., 1969) and cytoskeletal units are modified with the appearance of new elements (Leavitt and Kakunaga, 1980; Vanderkerckhove et al., 1980; Hamada et al., 1981). Some fibroblasts and epithelial cells acquire an enhanced tendency to undergo agglutination following exposure to low levels of plant lectins (Inbar and Sachs, 1969; Borek et ul., 1973), in contrast to normal counterparts, which exhibit this property in mitosis or following trypsinization. This agglutinability could be inhibited by some protease inhibitors and

175

RADIATION ONCOGENESIS IN CELL CULTURE

TABLE I1 MEMBRANE ENZYME ACTIVITIES I N NORMAL AND TRANSFORMED FIBROBLASTS".*

C3H lOT4 X ray-transformed C3H 1OTj Hamster embryo (HE) X ray-transformed HE

N a + , K+-ATPase

Mg2+ ATPase

5'-Nucleotidase

0.68 0.19 1.08 k 0.17'

1.12 k 0.18 1.03 k 0.15

0.54 k 0.07 0.60 k 0.05

0.59 5 0.9 1.02 0.14'

1.91 k 0.29 1.59 k 0.21

5.65 k 1.10 15.25 k 2.61'

Modified from Borek and Guernsey (1981). Mean k SE (pnol Pi/hr/mg protein). p i0.5. Significantly different from normal cells.

enhanced by high levels of retinol (Borek et al., 1973). The recovery of the membrane following trypsinization of the normal cells and their loss of agglutinability takes place within a period of approximately 6 hr, the same time scale required for restoration of ionic intercellular communication following trypsinization (Borek et al., 1969).

5 . Proteolytic Enzymes Produced by Transformed Cells The observation that a variety of surface properties characteristic of transformed cells can be mimicked in normal cells following trypsin treatment encouraged a major research effort to evaluate whether the process of neoplastic transformation is associated with an increased release of cellular proteases and whether protease inhibitors would modify transformation. Although the idea of increased protease activity (fibrinolysis) by neoplastic cells has been known since the early 1900s, when cells were grown on plasma clots, the availability of modern techniques with radioactive labeled components has made it possible to analyze the processes involved (Unkeless et al., 1973; Wigler and Weinstein, 1976). The amount of proteases produced by transformed fibroblasts depends on the origin of the cells studied and must be related to the production of these proteases by the untransformed counterparts. Such proteases include the plasminogen activator (Unkeless et af., 1973; Jones et al., 1975), which can be identified at a clonal level using an overlay agar method (Jones et al., 1979, as well as other proteases, including a series of acid hydrolases (Borek, 1976). Thus, although radiation-transformed hamster embryo cells exhibit an increased level of plasminogen activator (Borek et al., 1977) and a serine protease MIF factor (Borek, 1979a), an examination of 15 different acid hydrolases indicated that only acid phosphatases were elevated in the radiation-transformed cells (Borek et al., 1977).

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6. Growth in Agur Agar suspension assay as a selective assay for transformed cells was first applied to virally transfrormed cells (MacPherson and Montagnier, 1964). The underlying premise is the observation that normal cells derived from solid tissues cannot proliferate in suspension or in semisolid medium such as agar or methylcellulose. Thus the acquisition of the ability to grow in semisolid medium following exposure to oncogenic agents, including radiation (Borek and Sachs, 1966a; Borek and Hall, 1973, 1974; Lloyd et d., 1979) (Fig. 7), has been associated with the neoplastic state of the transformed cells for both rodent fibroblasts and epithelial cells (for review see Borek, 1979a), as well as for human cells transformed in uitro (Borek, 1980; Kakunaga, 1978; Sutherland rt al., 1980; Silinkas et a / . , 1981 ; Milo and DiPaolo, Andrews and Borek, 1982). Though growth in agar is an accepted criterion for transformation that is considered to be closely associated with the malignant potential of cells, there are instances where transformed cells did not grow in agar yet gave rise to tumors (Borek and Sachs, 1966a), as well as other transformed cells of both rodent and human origin that grew in agar yet did not give rise to tumors (Borek, 1980; Sutherland et a/., 1980; Silinskas ef a/., 1981). Alternately, by manipulating growth conditions and fortifying the semisolid medium with high serum and other hormonal factors, it is possible to observe colony formation by normal human fibroblasts, though these cells are unable to form tumors in the animal (Peehl and Stanbridge, 1981).

FIG.7. The most reliable criteria for the neoplastic state of cells transformed in uirro. A cell population derived from normal solid tissue is transformed in uiiro, in this case by 300 rad of X radiation, and is capable of growing in agar (B). It is potentially capable of inducing a tumor in the appropriate host, either a sarcoma (A) if the original cell population is composed of fibroblasts, or a carcinoma (C) if the transformed cells were epithelial. x 175. (Reproduced from Borek, 1979a.)

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Thus growth in agar or on agar (Borek, 1980) can be used as a selective criterion for transformation when the employed conditions are minimal, i.e., they do not favor the growth of normal counterparts. To evaluate the neoplastic nature of the cells in an unequivocal way the inoculation of transformed cells into appropriate hosts should be carried out in conjunction with growth in agar. It is of interest to note in this context that within the limited experience of human cell transformation in uitro, it seems that the ability of transformed human fibroblasts to grow in agar appears concomitantly with focus formation (Borek, 1980; Andrews and Borek, 1982), and that often focus formation in culture can be circumvented (Silinskas et al., 1981; Sutherland et al., 1980; Milo and Di Paolo, 1978; Andrews and Borek, 1982; Zimmerman and Little, 1981). In contrast, rodent diploid cells such as hamster cells, have been reported to grow in agar following transformation only at a later stage after morphological exhibition of transformation. As mentioned before, this point has yet to be clarified because experiments carried out with the diploid rodent cells differed in protocol from those with the human cells. Furthermore, cell cycle time of the hamster cells is markedly shorter than that of the human cells, accounting in part for different patterns and temporal events associated with neoplastic progression. 7. Tumorigenicity The ultimate and unequivocal demonstration of malignancy is the induction of tumors in syngeneic inbred hosts or in immunosuppressed animals. In the case of human cell transformation the animal of choice is the immunologically crippled nude mouse nu/nu (Kakunaga, 1978 ; Milo and DiPaolo, 1978; Borek, 1980; Sutherland et al., 1980; Silinskas et al., 1981; Maher et al., 1982). Because radiation transformation studies utilize fibroblasts, the injection of transformed cells gives rise to sarcomas with different degrees of differentiation. It is of interest to note that when hamsters were irradiated in utero and the embryos cultured in uitro, the transformed lines that arose were capable of inducing both sarcomas and carcinomas (Borek et al., 1977) (Fig. 7), indicating that though the neoplastic epithelial cells were not visible in culture, they developed in the host as epithelial tumors.

IV. Radiation Oncogenesis in Vitro

A. IONIZING RADIATION Though the various criteria for transformation have been dealt with in the foregoing section, the following will summarize these criteria as they have been utilized by various investigators in radiation oncogenesis studies.

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C A W I A BOREK

1. Formation of multilayered clones or foci under conditions where normal cells d o not display this pattern (Borek and Sachs, 1966a,b, 1967; Borek and Hall, 1973, 1974; Borek e f al., 1977, 1978; Borek, 1980; Terzaghi and Little, 1976a; Kennedy et al., 1978; Miller and Hall, 1978; Little, 1979; Han and Elkind, 1979a; Lloyd et al., 1979; Yang and Tobias, 1980). 2. Alteration in cell topography as evaluated by scanning electron microscopy (Borek and Fenoglio, 1976). 3. Chromosomal analysis (Borek and Hall, 1973; Borek et ul., 1977, 1978; Borek, 1980; Miller et al., 1981 ; Nagasawa and Little, 1979). 4. Proliferation of transformed cells in low-calcium medium (Borek, 1980). 5 . Proliferation of transformed cells in medium containing low serum concentration (Borek and Hall, 1973; Borek et ul., 1977, 1978; Borek, 1980; Terzaghi and Little, 1976a). 6. Agglutination of transformants but not of normal cells by low concentrations of plant lectins (Borek and Hall, 1973; Borek et al., 1978; Borek, 1980). 7. Alterations in ganglioside pattern (Brady rt al., 1972; Borek et a/., 1977). 8. Increased proteolytic activity in transformed cells (Borek et al., 1977; Borek, 1979a). 9. Changes in intercellular communication (Borek et al., 1969). 10. Growth in soft agar under conditions where normal cells do not grow (Borek and Sachs, 1966a; Borek and Hall, 1973; Borek et al., 1977, 1978; Borek, 1980; Lloyd et al., 1979). 11. Tumor formation upon injection into appropriate hosts (Borek and Sachs, 1966a; Borek and Hall, 1973; Borek ef al., 1977, 1978; Borek, 1980; Terzaghi and Little, 1976a; Han and Elkind, 1979a).

1. The Oncogenic Efects oj'Low-LET Radiation

a. Single Dose Effects. i. Neoplastic Transjormation of Diploid Cell Strains. The successful induction of neoplastic cell transformation in vitro by X rays was first reported by Borek and Sachs (1966a). Short-term primary cultures derived from midterm golden hamster embryos were used as the source of normal cells. Mass cultures were irradiated with 300 rad and subcultured at low density 3 days later onto rat feeder layers. Further progressive subculturing without feeders of both irradiated cells and controls resulted in senescence in control cultures, and a gradual enhancement of mitotic rate in the X-irradiated cells. Within 3 to 4 weeks after exposure, foci of fusiform cells began to pile up and overtake the culture. Quantitative evaluation of this transformation event was carried out by irradiating mass cultures and

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cloning them out immediately. Results showed a 0.7-0.8% transformation in the treated cultures and no observable transformants in the controls (c It is of interest to note that transformability of the embryonic cells by X rays declined with cell passage in vitro, and at passage 3 to 4 no transformation was observed following irradiation with 300 rad (Borek and Sachs, 1967) (Fig, 8). The ability of the transformed cells to grow in agar was tested, as well as their ability to form tumors in 6-week-old hamsters. Some of the d l s did not proliferate in agar yet gave rise to tumors in the animals. Some of the tumors, identified as fibrosarcomas, regressed (Borek and Sachs, 1%6a); upon subsequent injection of cells, progressive tumor growth leading to the death of the animal was observed (Borek, 1967). Subsequent studies to ascertain conditions and requirements for X-rayinduced transformation indicated that one to two cell replications were required for the fixation of the transformed state (Borek, 1967). Replication had to take place within 24 hr after exposure of log-phase cultures to X rays. Irradiation of mass cultures and trypsinization soon there after gave similar results. Inability to divide resulted in a loss of fixation of the transformed state (Borek, 1967, 1968). This was indicated by maintaining cultures at

No. d parroges of morr cultures before X irrodiotion

FIG.8. Plot showing percentage transformation in primary cells and in cells passaged in cultue. Cells taken straight from the animal and from subsequent passages as mass cultures were seeded for cloning and irradiated 1 day after seeding. The colonies were grown in EM with fetal calf serum, and the mass cultures. which were passaged every 3 days, were grown in EM with nonfetal calf serum. Cells from the different passages gave cloning efficiencies of 3.1-4%. (From Borek and Sachs, 1967.)

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CARMIA BOREK

plateau phase in liquid holding for 24 to 72 hr post irradiation. Transformation rate following trypsinization of these cultures declined progressively. The frequency of transformation was inversely proportional to the length of time of maintenance in plateau phase (Fig. 9). Loss of fixation was inhibited by maintaining the plateau-phase cells at 25°C for 24 to 72 hr. When cells were cloned after liquid holding at low temperature, transformation frequency was fully restored (Borek and Sachs, 1967). This suggested that repair mechanisms were involved in the loss of fixation and that maintenance at low temperatures, where repair was slowed, prevented this loss. Further experiments indicated that expression of transformation could occur within 2 days after treatment. Irradiation of cloned cells at different stages of growth resulted in partial clonal transformation whose expression was related to the number of days in culture (Borek and Sachs, 1967). Once cells were

.-nY

C

0

P

F c

c

. I

e

c

No of days oiler X Irrodiolion of conflurnl cultures when c e l l s ploled for cloning

FIG.9. Transformation in confluent cultures cloned at daily intervals after irradiation. Confluent primary cultures grown in EM with nonfetal call serum were irradiated at 3 or 4 days after seeding, and the cells cloned at daily intervals with fetal or nonfetal serum. Each point represents an average of at least three experiments. Circles, percentage of transformed colonies; triangles, percentage cloning efficiency; open circles and triangles, cells cloned in fetal calf serum; solid circles and triangles, cells cloned in nonfetal calf serum. (Reproduced from Borek and Sachs, 1967.)

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181

transformed by X rays, they acquired specific surface properties that were characteristic of every transformed line individually derived. Thus cells transformed at different times by X rays were incapable of proliferating over other X ray-transformed cell lines as well as on various chemically and virally transformed cell lines (Borek and Sachs, 1966b). Surface-mediated changes following radiogenic transformation were also indicated by the loss of intercellular communication in some X ray-transformed hamster embryo lines, as measured by ionic movement via permeable membrane junctions (Borek et al., 1969) and in modification of cellular gangliosides (Brady et al., 1972; Borek et al., 1977). The first dose-response relationship for radiation-induced in uitro transformation was shown by Borek and Hall (1973) (Fig. 10) using the hamster clonal cell system. Single cells seeded onto syngeneic feeder cells were exposed 24 hr later to X-ray doses ranging from 1 to 600 rad. The results shown in Fig. 10 indicated that a transformation incidence per survivor was evident at doses as low as 1 rad and that the frequency rose with doses from 1 rad up to a plateau of 150 to 300 rad. At higher doses radiation toxicity appeared to be a competing factor in these asynchronous mixed cell populations and the transformation rate declined. Recent experiments using the same system indicate that cell transformation can be detected at doses as low as 0.3 rad

X-ray absorbed dose (rad)

FIG. 10. Incidence of hamster embryo cell transformation following exposure in uitro to X irradiation. The broken line is drawn by eye to the data points; the full line has a slope of + 1 and passes through the error bars of each datum point. (Reproduced from Borek and Hall, 1973.)

182

CARMIA BOREK 10’

J 10’1

10

DOSE

100

1C 0

(rrd)

FIG.11. Dose-response relationship of transformation of hamster embryo cells by 300 kVp X rays and 6oCo rays. Note that RBE is inversely proportional to the dose. (Borek and Hall, 1982).

of X rays Borek and Hall, 1982; see Fig. 22. Other data indicate that at low doses the effectiveness of X rays in producing transformation is about twice as high as that induced by “Co y rays (Borek et ul., 1982) (Fig. 11). This could be predicted on microdosimetric grounds and is important for its implications in radiation protection. When transformed clones were isolated and propagated, they were tested for their agglutinability by 20 pg/ml of concanavalin A (Con A), for their ability to grow in 0.33% agar, and for their malignant potential in animals. Positive results served as an affirmation of the neoplastic potential of the isolated cells (Borek and Hall, 1973). ii. Transformation Of’Established Cell Lines. Utilizing the C3H 10T; mouse embryo cell line (Reznikoff et al., 1973a) and following the transformation assay described for chemically induced transformation (Reznikoff et ul., 1973b), Terzaghi and Little (1976a) carried out a study showing that these cells are transformable by X rays. A dose response for transformation indicated that transformation rate per viable cell rose exponentially from 100 rad up to 400 rad and remained constant at higher doses (Fig. 12). Similar dose-response curves were reported by Han and Elkind (l979a, 1981a,b), Yang and Tobias (1980), Miller and Hall (1978), Miller et ul. (1 979) (Figs. 13and 14), though interlaboratory differences in transformation frequencies at the various doses may exist. A dose response for y ray-induced transformation in IOT;, evaluating transformation per exposed cells, is seen

RADIATION ONCOGENESIS IN CELL CULTURE

183

a

a

k!

F

I

ol -@@

200 400 600 800 1000 1200 1400 DOSE (rod)

FIG.12. X radiation-induced oncogenic transformation in exponentially growing 1OTj cells in v i m . Each point represents data from 20 to 100 dishes. At low doses with low associated transformation frequencies, more dishes were necessary in order to obtain a total number of transformants comparable to that observed at higher doses. (Reproduced from Terzaghi and Little, 1976.)

in Fig. 13 (Han and Elkind, 1981a,b). It may be noted at this point that a variability in transformation in the 10T; cells can vary with batches of serum (Terasima rt al., 1982). Similar to the hamster embryo systems, fixation of X-ray-induced transformation in the lOTi cells required replication soon after exposure to radiation and further replications for expression of the transformed state (Terzaghi and Little, 1976a). Liquid holding in plateau phase resulted here too in a loss of fixation, and a reduced transformation rate was seen when cells were kept in a nondividing state for 24 hr or more after irradiation (Terzaghi and Little, 1975). Here, however, if cells were removed from liquid holding at 12 hr after exposure, transformation rate increased. The lOT; cells transformed by X rays exhibited three different types of foci distinguished by their morphology, similar to those seen following treatment with chemical carcinogens (Reznikoff rt ul., 1973b), and these corresponded to different degrees of malignancy (Terzaghi and Little, 1976a).

184

CARMIA BOREK

0

200

400

600

800

no0

1200

Hm

Hvx)

lam

DOSE (rod)

FIG.13. Survival and neoplastic transformation of C3H lOT$ cells by "Co y rays delivered at 100 rad/min. Transformation is expressed per exposed cell. Bars, SE of the pooled data from three to five experiments; Do,dose required to reduce survival by l/e along the terminal straightline portions of the curves; P.E., plating efficiency; N,multiplicity. (Reproduced from Han ei al.. 1980.)

It was also observed that high cell density at treatment time resulted in a lower transformation frequency. Another cloned cell line, the BALB/3T3 A31 (Kakunaga, 1973), has also been transformed by X ray (Little, 1979) (Fig. 15). Showing a dose-response curve similar in shape to that of the lOT;, this line requires a shorter time for expression of transformation. Here, too, the effect of cell density on transformation frequently was observed. Although the BALB/3T3 line could serve as an additional system to study radiation oncogenesis, several drawbacks were observed. Under certain conditions transformation rates altered from one experiment to another and a background of spontaneous transformation was observed. The complexity of this system may be in part explained by Kakunaga's finding that there exists in this cell line a range of cells whose competence to transformation by UV and perhaps by other agents ranges from highly susceptible cells to those that show resistance (Kakunaga, 1980). b. Split Dose Eflkcts. As already seen, the ability to transform cells directly in oitro by X rays has made possible detailed quantitative assessment of radiation effects over a wide range of doses. A logical extension of these studies has been to investigate the influence of the temporal distribution of

RADIATION ONCOGENESIS IN CELL CULTURE

185

FIG. 14. Dose-response relationships for the number of transformants per surviving C3H IOTr cell following single (.---a) or split doses (0-0) of 300 kVp X rays. In the case of split doses, the radiation was delivered in two equal exposures separated by 5 hr. (Reproduced from Miller and Hall, 1978.)

radiation on transformation, in order to evaluate the oncogenic potential of of radiation delivered as protracted doses or at low dose rates, the types of exposure that are of continuous concern to humans. Some unexpected information was obtained. Borek and Hall in 1974 first reported with the hamster embryo cell system that fractionation of an X-ray dose results in an elevated transformation incidence (Fig. 16). In these experiments it was shown that two doses of 25 rad separated by 5 hr produced more transformation than a single dose of 50 rad, and that two doses of 37.5 rad were much more effective than a single exposure of 75 rad. Splitting the dose resulted in a 70% enhancement in transformation rate as compared to the same dose delivered as a single exposure; but fractionation also produced a sparing effect on cell survival, indicating that cellular mechanisms involved in repair for survival differ from those associated with cell transformation. Elkind and Han (1979a) reported a similar observation, though they interpreted the observation differently. Experiments carried out by Terzaghi and Little (1976b), using the 10TS cells and studying the effects of split doses

J J

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300 '

400

500

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FIG.15. Dose-response curve for X-ray-induced transformation. Data points are from four separate experiments. Vertical bars join the data points for three experiments; the dashed line was drawn by eye through these data. Points with arrows indicate transformation not detectable, thus less than indicated value. (Reproduced from Little, 1979.)

FIG. 16. Incidence of cell transformation in cloned cultures of hamster embryo cells. Four replicate experiments were performed in which the effect of a single dose of 50 rad ( 0 )was compared with that of two doses of 25 rad (0). The results of the individual experiments are shown, together with the pooled data; vertical bars are standard deviations. The consequence o f a single dose of 75 rad (m) compared with two doses of 37.5 rad (0) are shown. (Reproduced from Borek and Hall, 1974.)

RADIATION ONCOGENESIS IN CELL CULTURE

187

at a higher dose range of 150 to 800 rad, found that splitting the dose resulted in a reduced transformation rate. Similar results were seen by Han and Elkind (1979a). Miller and Hall (1978), utilizing the lOTf cells and spanning a dose range from 25 to 800 rad, reported that although splitting the dose at the low-dose range resulted in enhanced transformation, a sparing effect on transformation was observed when doses above 150 rad were split into two equal fractions (Fig. 14). Moreover, dividing the dose of 100 rad into two to four fractions over 5 hr resulted in a progressive enhancement of transformation. Borek, extending the split doses studies in the hamster system to a higher dose level (Borek, 1979b), reported that in this system too, at doses higher than 200 rad a lower transformation frequency was observed when the doses were split into two fractions. It may be of interest at this point to elaborate on the complex structure of the dose-response curves for X rayinduced transformation clearly seen in the lOTi cells (Fig. 14) and suggested from the hamster embryo data (Fig. 7). The shape of the curve is complex (Fig. 14). Above 100 rad the data are consistent with a slope of two, implying that transformation may exhibit a quadratic dependence on dose. Below 30 rad the data are consistent with a slope of unity, implying that transformation may be directly proportional to dose. Between 30 and 100 rad the curve is shallow and transformation does not vary with dose over this range. The experiments comparing single and split dose in the cases of both the hamster embryo (Borek and Hall, 1974; Borek, 1979b) and the 10T; (Miller and Hall, 1979) systems were designed in such a way that single and split does were always compared within the same experiment. The pattern which emerges is interesting (Figs. 14, 16, and 17). At doses above about 150-200 rad, fractionation leads to a reduction in transformation, whereas between 30 and 100 rad fractionation enhances the incidence of transformation. These results emanate directly from the changing slope of the dose response for single exposure, so it is assumed that the two exposures 5 hr apart are totally independent and d o not interact with one another. One can derive the relationship for the split doses from the single exposure. The results of Borek and Hall (1974), Miller and Hall (1979b), and Borek (1979b) are illustrated in Fig. 17, where the data are presented in terms of ratio of the transformation incidences produced by split to single doses. The agreement between the results obtained with the two different systems using different techniques is quite remarkable. These data, as well as similar observations in the 3T3 cells (Little, 1979) (Fig. 18), indicate that in three different cell systems the oncogenic effects of split doses of radiation differ with dose range. They clearly show that the use of linear extrapolation from high to low dose levels may lead to cancer risk estimates that are neither conservative nor prudent depending on the distribution of the dose in time.

188

CARMIA BOREK

FIG. 17. Comparison of the data presented in this article for C3H IOTf cells (U) with the data of Borek and Hall (1974) and Borek (1979) for fresh explants of hamster embryo cells ( 0 ) . The ratio of the transformation frequencies for split and single doses is plotted as a function of total dose. A ratio in excess of unity implies that fractionation enhances transformation; a ratio of less than unity implies that fractionation results in a reduction of the transformation rate. For both cell systems the crossover point between the enhancing and sparing effect of fractionation occurs at about 1.5-2.0 Gy. (Reproduced from Miller rt a/., 1979.)

FIG. 18. Transformation frequency induced by single ( O - - - - o )or split ( x x ) X-ray exposure with 5 hr between fractions. Results are mean of three separate experiments k SE. The differences between the two exposure groups are statistically significant for the 25-, 50-, and 300-rad points as determined by a paired I test. (See Table 11). (Reproduced from Little, 1979.) ~

RADIATION ONCOGENESIS IN CELL CULTURE

189

FIG. 19. Transformation frequency of C3H 1OTi cells induced by acute (A) and low (B to D) dose-rate exposures of boCoy rays. Bars, SE of the pooled data from two to four different experiments; ---, induction curves for acute exposures from (A). (Reproduced from Han et a / . , 1980.)

The oncogenic effects of y rays delivered at various dose rates have recently been pursued using the lOTi cells. The results are conflicting. Han and Elkind (1979a) and Han et al. (1980) reported a dose-dependent transformation frequency and a lower transformation at low dose rates (Fig. 19), whereas Hall and Miller (1982) find that when 100 rad are distributed over a 6-hr period, transformation is significantly higher as compared to the same dose delivered over 10 min (Fig. 20). Clearly, further experimentation is necessary to evaluate the effectiveness of low dose rates. It should be noted that the two groups differed in the handling of the cells and in their respective experimental protocols. In the Han and Elkind experiments, mass cultures of cells were irradiated at different dose rates and trypsinized and cloned after irradiation. Hall and Miller cloned cells 24 hr prior to irradiation, so exposures to various dose rates were carried out on single cells. 2. Hamster Cells Initiation in Utero and Assay in Vitro The available data on the subject of in utero carcinogenesis are sparse (Storer, 1975). Experiments conducted by Borek et al. (1977) set out to compare transformation incidence in hamster embryo cells exposed in utero to 300 rad and then cultured in oitro immediately with cells of embryos

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CARMIA BOREK

D O S E (Gy)

FIG.20. Pooled data from three large self-contained experiments for the incidence of transformation produced by 1 Gy of 6oCo y rays delivered in an acute exposure (10 min) or in a protracted exposure over 6 hr. The open circles represent earlier data for single acute doses of 300 kVp X rays. (Reproduced from Hall and Miller, 1982.)

cultured in uitro and later exposed to the same dose of radiation. The results indicated that transformation incidence induced in utero was 10-fold lower than that induced in uitro, thus closer to the frequency of oncogenesis in uivo. .One cannot discount modifying effects of various cell populations and some differences in plating efficiency. However, these striking differences in incidence support some other factors that may be involved, such as host mediation, repair and loss of fixation at high density, inhibition of expression by cell-cell interaction, or other influences exerted by the tissue-specific organization present in viuo. When cells are transformed in uitro they are devoid of tissue-specific arrangements and are able to replicate under conditions where normally they may remain in a nonreplicating state. Thus the in uitro situation may yield an exaggerated rate of transformation because fixation of transformation can be carried out with ease in log-phase cultures. A number of transformed cell lines were developed from these in utero experiments and studied for a variety of properties presented in Table 111. The most striking finding was that injection of the mixed populations of transformed cells into hamsters yielded carcinomas as well as sarcomas (Fig. 7). Although these epithelial transformed embryonic cells went undetected in culture, because of their unaltered morphology they proliferated in the animal to form carcinomas.

TABLE 111 OF HAMSTER 5 CELLSTRANSFORMED in Utero BY X IRRADIATION" CHARACTERISTICS

Characteristic Karyotype Growth in culture Saturation density no. of cells/cm-2 In 10% serum In 1% serum Doubling time In 10% serum In 1% serum Morphology-scanning electron microscope Agglutinability by Con A (20 p g / d and WGA (50 pg/ml-') Plating efficiency In 10% serum In 1% serum Efficiency in 0.33% agar Plasminogen activator Lysosomal hydrolases : phosphatase Production of MIF Gangliosides Presence of radiation leukemia virus Tumorigenicity (invasive malignant sarcomas and carcinomas) a

From Borek et a / . (1977).

Normal (secondary culture)

Transformed (4th to 30th passage after isolation)

Tumor (1st to 10th passage) after reculturing

Diploid Monolayers

Near diploid Multilayers

Aneuploid Multilayers

7.6 x 104 Minimal growth

1.8 x 105 105

1.5 x 105 105

16 2 hr 90 & 5 hr Flat and regular surface features except during mitosis -

14 f 2 hr 24 f 2 hr Pleomorphc with complex surface features throughout the cell cycle

16 & 2 hr 25 f 2 hr Pleomorphic with complex surface features throughout the cell cycle

2.75 f 1% 0.05 f 0.05% 0 Low Low Absent Presence of full range of gangliosides None

52.5 & 5.5% 5.5 f 2.5% 10% High High High Absence of gangliosides higher than GM3

49.5 f'6.5% 6.0 f 2.5% 30% High High High Absence of gangliosides higher than GM3

+

+

-

29/30

+

-

Transplantable 12/12

192

CARMIA BOREK

3. The Oncogenic Eflects of' High-LET Radiution As an oncogenic agent high-LET radiation is more effective than lowLET radiation. It is also more cytotoxic. The effectiveness of cell transformation is matched by the enhanced killing effect. There have been few reports on the oncogenic potential of high-LET radiation, and of these most of the available information is on the effectiveness of neutrons, a type of radiation that is not only used in therapy but is also a product of nuclear energy. Using the hamster embryo clonal system, Borek (1976) and Borek et al. (1978) reported the induction of cell transformation in vitro following exposure to 430 keV neutrons in the dose range of 0.1 to 150 rad. A 430-keV van de Grdaff accelerator was used, where protons accelerated onto a tritium source produced the spectrum of neutrons. The angle of the cells with respect to the source determined the neutron energy they received (Borek et ul., 1978). The results indicated that whereas neutrons were much more efficient than X rays in producing cell transformation, they were also more effective in cell killing (Figs. 21 and 22). Transformation was observed with neutron doses as low as 0.1 rad. It increased as a function of the dose to 150 rad, indicating a much higher frequency than that observed for x rays and resulting in an inability to evaluate the RBE (ratio of doses which produce the same biological effect) at that dose level. However, when transformation

c

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4

1 1 1 1 1 1 1 1 1 1 1 ( I I 200 400 600 000 1000 1200 Dose (rod)

Fa.21. Cell survival curves following irradiation by 430-keV neutrons (0-0) and by 250-kVp X rays ( 0 - - -0). - (Reproduced from Borek er al., 1978.)

+ 10-

6

6

10-I loo. DOSE/Cy

lo1

FIG. 22. (A) Dose-response curves for cell transformation by X rays

(@----a), neutrons

(0-O), and argon ions (*). The data points plotted are the means of replicate experiments, and the error bars represent the SE or the error expected from the number of clones counted, whichever was larger (Borek et al., 1978). (B) Dose response curves for transformation of hamster embryo cells by X rays (+@) and 430-keV neutrons (0-0).Here additional data have been obtained for X-ray-induced transformation going down to dose of 0.3 rad. The data points are the means of replicate experiments and the error bars indicate 95% confidence intervals for the estimated values. The curves were obtained by fitting the data with splines (De Boor, 1978) in the least-squares sense. (Analysis by Dr. M. Zaider.)

194

CARMIA BOREK

Absorbed dose (rod)

FIG.23. Dose-response relationships for transformation by neutrons (0-0)

or X rays

(o----o) wherein the number of transformed clones per initial cell at risk is plotted as a function of dose (Borek e t a / . , 1978).

FIG.24. (A) A 12-day-old normal clone of hamster embryo cells. Giemsa x 25. (B) A 12-dayold clone of cells transformed by 1 rad of 430 keV neutrons. Note the dense multilayers in the transformed and the random cell orientation at the periphery. Giemsa x 25. (Reproduced from Borek L'I a / . , 1978.)

195

RADIATION ONCOGENESIS IN CELL CULTURE

frequency is expressed as the number of transformants per exposed cell (Fig. 23), an evaluation more closely related to the in vivo situation, both neutrons and X-ray curves rise to the same peak value. Thus whereas the RBE for survival varies and is inversely proportional to the square root of the neutron dose, RBE for transformation per exposed cell does not reflect this wide variation. Qualitatively, the hamster cells transformed by neutrons did not differ from those transformed by X rays. A representative picture of a clone transformed by 1 rad of neutrons is presented in Fig. 24. The multilayered center and the periphery with randomly oriented cells are clearly visible, in contrast to the flat and well-oriented control. Other properties of the neutrontransformed cells are presented in Table IV. Transformation of the mouse 10Ti cells by single doses of 0.85 MeV fission neutrons as compared to X rays was reported by Han and Elkind (1979a) (Fig. 25). The results were qualitatively similar to those reported by TABLE IV CHARACTERISTICS OF CLONED HAMSTER CELLSTRANSFORMED in Virro BY NEUTRONS'~~ ~~

Characteristic Karyotype Growth in culture Saturation density No. of cells/cm2, in 10% serum No. of cells/cm2, in 1% serum Doubling time (in 10% serum) Morphology (scanning electron microscopy)

Agglutinability by Con. A (20 jcg/ml) Plating efficiency In 10% serum In 1% serum Plasminogen activator Production of macrophage migration inhibitory-like factor Growth in semisolid agar Presence of C-type virus particles Tumorigenicit y

Normal (secondary cultures)

Transformed (6th to 35th passage)

Diploid Monolayers

Near diploid Multilayers

8 x lo4 Minimal growth 16 2 hr Flat and regular surface features except in mitosis

2.2 x 105 1.3 x 105 15 f 2 hr Pleomorphic with complex surface features throughout the cell cycle 4

3 f 1% 0.06 0.05% Low Absent

65 f 5.5% 7.1 2.2% High High

+ +

From Borek er al. (1978). This table represents a study of transformed cells propagated from a transformed clone. Similar properties were found in all other clones tested, with the exception of one clone which differed by becoming malignant at a later stage. a

196

CARMIA BOREK I

1

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1

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I

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0

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0.2

0.4

0.6

0.8

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1.2

1.4

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FIG. 25. Transformation frequency of C3H 10Ti cells induced by 50 kVp X rays (0) and . was determined from the ratio of the X-ray to neutron doses fission (Janus) neutrons ( 0 ) RBE at the frequency levels shown. Bars, S.E. of the pooled data from three to five different experiments. (Reproduced from Han and Elkind, 1979a.)

Borek (1976) and Borek et al. (1978) using the hamster system. Fractionation of the neutron dose spaced by an interval longer than 6 hr resulted in a reduced transformation rate (Han and Elkind, 1979a). The effect of low-energy tl particles in transforming the 1OTi cells was evaluated by Lloyd et al. (1979). Transformation frequency per survivor increased as the cube of the dose, peaking at an cr-particle fluence of 1.5 x 10’ to 2.5 x lo7 particles (205-342 rad). Maximum transformation frequency reached 4%. No parallel experiments were carried out with X rays; thus RBE for a could not be determined. Data on the effects of other types of heavy ions on cell transformation are limited. Borek et al. (1978), using the hamster cell system, studied the transforming effects of argon ions, a type of radiation that has been considered for therapy. Cells were irradiated at the Bragg peak with high-energy argon ions (429 meV/amu) at doses of 1 and 10 rad, resulting in transformation

RADIATION ONCOGENESIS IN CELL CULTURE

197

frequency of 0.26% and 0.70% respectively, a rate similar to that observed following exposure to 430 keV neutrons (Fig. 22). Cell transformation by a 600-MeV/amu ion beam has been investigated in the 10T; cells (Yang and Tobias, 1980). 4. Human Cell Transformation by X Rays In 1980 Borek demonstrated the transformation of human skin fibroblasts by 400-rad X rays into cells which progressed in vitro to malignancy and were able to grow in agar and give rise to tumors when injected into nude mice (Borek, 1980). The cells used were a strain of diploid fibroblasts, the K D strain, previously used for studies in chemical transformation (Kakunaga, 1978). Early passage cells were used and their diploid nature was ascertained by chromosome G-banding analysis. Their doubling time is 30-32 hr. Survival curves indicated that survival fraction following a dose of 400 rad was close to 12% of the total population (Fig. 26). No shoulder was observed, indicating the different response of the human cells compared to that of the hamster (Borek and Hall, 1973). The protocol used for transformation was a combination of two methods employed for chemically induced human cell transformation, that of Kakunaga (1978), the focus assay; and that of Milo and DiPaolo (1978), where cells were synchronized prior to exposure to the oncogenic agent. The method used is illustrated in Fig. 27 and can be summarized briefly as follows. Cells at concentrations of 2.6 x lo5 were seeded into 75-mm2 flasks and 2 days later medium was replaced by another medium containing 0.1% serum for a period of 24 hr, whereby cell proliferation was greatly reduced. Twenty-four hours later the medium was replaced by complete medium (11% serum), which contained either j3-estradiol 1 pg/ml or the protease inhibitor antipain (6 pg/ml). Cells were then irradiated and subcultured 10 hr later, after which they were subcultured again upon reaching confluency . The foregoing experimental protocol took advantage of the following : The cells were quiescent by serum deprivation, and thus entered a synchronous wave of DNA synthesis when released from quiescence by medium change. Treatment of the cells at this point enabled the capturing of cells entering S phase. Within 60 to 80 days after treatment, foci appeared in treated cultures that were clearly distinguishable from the untreated controls (Fig. 28). They grew progressively when medium was changed to a lowcalcium medium; the transformed morphology of the foci was enhanced (Fig, 28), while the normal cells died within 24 hr. These clearly distinguishable foci were isolated and propagated in uitro. Chromosome G banding

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CARMIA BOREK

0

2

4

X-RAY

6

0

1

0

DOSE (Gy)

FIG.26. Cell survival of KD cells following irradiation by X rays, from a Siernan's 300 kVp constant potential generator with an added filter of 0.2 mm copper. (Reproduced from Borek, 1980.)

indicated a near diploid range of chromosomes (46-49), saturation density was 2-fold compared to the normal, and the transformed but not the normal were agglutinable by 25 pg/ml of Con A. When seeded into 0.33% agar or grown on agar (0.5% concentration), the KD-transformed cells formed colonies. Cloning efficiency in agar was 2-3%. Neither the unirradiated nor the irradiated but untransformed cells formed colonies in this semisolid medium. The ultimate proof of the neoplastic nature of the cells transformed in uitro is their ability to form tumors in an appropriate host. Swiss nu/nu mice were injected intradermally with 5 x lo6 cells (suspended in 1 ml medium) into the subscapular region of animals X ray irradiated 24 hr earlier with 450 rad, or unirradiated.

RADIATION ONCOGENESIS IN CELL CULTURE

-84 hr

CELLS PLATED

-36 hr

-12 hr

ANTIPAIN CHANGE OR TO MEDIUM fl-ESTRADIOL SUPPLEMENTED ADDED WITH IN 0.1 % COMPLETE SERUM MEDIUM

0

199

+12 hr”

SUBCULTURE

SCORE FOR TRANS FORMED FOCI

FIG. 27. Experimental design for the induction of human cell transformation in v i m by 4 Gy ofX rays ( I Gy = 100 rad). Cells were trypsinized (0.25% trypsin), suspended in complete medium, and plated in tissue culture flasks (75 cm2) (Falcon) at 2.6 x lo5 cells. Two days later, the medium was replaced with MEM containing 0.1% serum for 24 hr, whereby cell proliferation was greatly reduced. After 24 hr medium was again exchanged, this time for complete medium (11% serum) containing either p-estradiol (Sigma) ( 1 pg/ml) or the protease inhibitor antipain (6 pg/ml). Experimental cultures were irradiated 12 hr later with 4 Gy or X rays as previously described. All cultures were divided into two parts 10-12 hr after irradiation and reseeded in fresh complete MEM without antipain or estradiol. At near confluency, one of the two flasks was subcultured into 12 flasks, the other being left undisturbed. In each experiment, when these 12 flasks reached confluency, one flask was subcultured 1 : 10 and thus defined as “continuously passaged,” and the remaining 11 flasks were left a high density. (Reproduced from Borek, 1980.)

Whereas the five transformed lines tested formed colonies in agar, three gave rise within 6 weeks both in the irradiated and unirradiated mice to tumors that have been characterized as noninvasive fibrosarcomas. Cells cultured from the tumors possessed a human karyotype which will be described in detail elsewhere. None of the unirradiated or the irradiated but untransformed cells (seven samples of each) gave rise to tumors in nude mice. Cultures that were treated, i.e., irradiated but not allowed to replicate more than four or five times before reaching confluency, did not exhibit transformation. This indicated that, as in the rodent cells (Borek and Sachs, 1967; Terzaghi and Little, 1976a), replication is required following radiation for the fixation and expression of the transformed state. At the present time quantitative assays for human cell transformation are not firmly established, though a number of laboratories have utilized growth in agar as an assay (Sutherland et al., 1980, 1981 ; Silinskas et al., 1981 ;Milo and DiPaolo, 1978; Andrews and Borek, 1982; Zimmerman and Little, 1981 ; Maher et al., 1982). The correlation between the malignant

200

CARMIA BOREK

FIG.28. (A) Human KD cells in irradiated but untransformed cultures. Phase x 58. (B) Human KD cells transformed in uitro by 4 Gy X rays. Note the crisscross pattern of the cells. (C) The same area as in B 24 hr after incubation in low calcium. Phase x 58. (D) X-ray-transformed KD cells growing in 0.33% agar. x 12. (Reproduced from Borek, 1980.)

nature of the cells and their ability to grow in agar is clearly not established. On the one hand, as reported, normal human fibroblasts can give rise to colonies in semisolid medium (Peehl and Stanbridge), and on the other hand fibroblasts that showed anchorage independence could not form tumors in nude mice (Sutherland er al., 1980). Another problem with using agar as the sole endpoint for analysis of dose-response relationships in transformation studies is the inability to evaluate plating efficiency in agar. For example, do the number of clones growing in agar reflect the total number of cells that have undergone transformation or do they reflect only a fraction of transformants, those with a higher plating efficiency in this semisolid medium and an ability to proliferate under those conditions? Growth efficiency in agar can be enhanced by using a high-quality serum, by washing the agar multiple times, and in addition (T. Kakunaga, personal communication), by adding pyruvate or modifying incubator conditions. Though the quantitative aspects of X-ray-induced human transformation

RADIATION ONCOGENESIS IN CELL CULTURE

20 1

are currently not precise, a number of observations using the KD cells can be mentioned. The incidence of transformation in the human cells appears to be lower than that observed in the rodent cells, given the same dose of X rays. Whereas in the human cells the frequency per treated cells is closer to at 400 rad, a frequency associated with mutational events, rodent cells show an incidence closer to at that dose level. The number of doublings required for the expression of the transformed state of the human cells was approximately 10-1 3, similar to that observed in some rodent cells. It must be stressed that future experiments may indicate that the quantitative aspect of these endpoints may vary with the source of the human cells studied. Genetic and physiologic differences, as well as organ- and tissue-related variability in response may play important roles in the assay. Other observations on the transformed human cells can be stressed. (a) Initial loss of contact inhibition is not as striking as that seen in rodent cells. (b) In contrast to rodent cells, the ability to proliferate in medixm with low serum (1%) is not confined to the transformed cells (Borek, 1980); our normal KD cells as well as the transformed cells proliferated in medium containing low serum. (c) The transformed state is associated with membrane changes and, as in rodent cells, agglutinability by plant lectins can be used as a distinguishing probe. (d) Surface topography in the X-ray-transformed human cells is altered but not as dramatically as in the rodent cells. Microvilli found in abundance on rodent cells (Borek and Fenoglio, 1976) were not as abundant in transformed human cells (C. Borek, unpublished). We cannot yet assess the roles played by growth inhibition and release from this arrest and exposure in late G I in the process of X-ray-induced transformation. Recent experiments indicate that though these steps are not essential for observing transformation, without growth arrest transformation incidence is decreased and latency in appearance of transformed foci is lengthened (as long as 4 months). At present we are ignorant of the molecular mechanisms underlying the effects of growth inhibition and release from this arrest in the process of transformation. A tempting thought is provoked by reports of Woodcock and Cooper (1979) and Schimke el al. (1981). The work indicates that following an inhibition of DNA replication begins at the same origins (Woodcock and Cooper, 1979). Quiescence by serum deprivation and refeeding (Borek 1980) could lead to similar events. These may include disproportionate DNA replication and gene amplification (Schimke et al., 1981), which may play a role in potentiating the induction of radiogenic transformation and enhancing its frequency. Other studies (Namba et al., 1978) utilized high doses of 6oCoy ray, delivered in multiple fractions to WI-38 fibroblasts. Exposure under these conditions resulted in marked and prominent chromosome aberrations in these cells. Transformed populations were observed 150 days after exposure.

202

CARMIA BOREK

B. NONIONIZING RADIATION Of the nonionizing radiations, ultraviolet (UV) is most notorious for its oncogenic properties (Setlow, 1974; Setlow and Hart, 1974), though transformation by fluorescent light in the mouse 1OTj cells has been reported (Kennedy et al., 1978). 1. UV-Induced Oncogenic Transformation in Rodent and Human Cells

The effects of UV light are mediated via electron excitation and are exerted on cellular DNA with the resulting formation of thymidine dimers (Setlow 1966, Setlow et al., 1969; Setlow and Hart, 1974; Cleaver, 1969; Cleaver and Bootsma 1975). Mechanisms of UV-induced DNA damage and repair are quite different from those associated with ionizing radiation (Regan and Setlow, 1974; Painter and Cleaver, 1969; Cerutti, 1976). Yet, similarly to ionizing radiation, the association between DNA damage and repair, and in uitro transformation is so far largely inferential. An association in uivo has been shown (Setlow and Hart, 1974). In rodent cells UVinduced transformation in uitro has been studied with the hamster cell system (DiPaolo and Donovan, 1976; Doniger et al., 1982) (Fig. 29), the C3H 1OTi cell line (Chan and Little, 1976; Mondal and Heidelberger, 1976), and the BALB/3T3 clone A31 cells (Little, 1979; Kakunaga, 1980). Ultraviolet transformation has also been shown to be enhanced by X rays but not by chemicals (DiPaolo and Donovan, 1976), and by TPA (Mondal and Heidelberger, 1976). The data from the various laboratories indicate that the frequency of UV-induced transformation is dose dependent. Cell susceptibility to UV-induced transformation may vary within the cell population (Kakunaga, 1980). Action spectra for UV-induced transformation in rodent cells and its correlation with dimer production are reported using the hamster system and a morphological assay (Doniger et al., 1981), though this morphology has been correlated with tumorigenicity (DiPaolo and Donovan, 1976). Using wavelengths between 240 and 313 nm, the most effective wavelengths in producing transformation were 265 and 270 nm. Relative sensitivities per quantum for transformation, pyrimidine dimer production and toxicity appeared the same at each of the wavelengths tested, thus implying that DNA may be the target for these processes (Doniger et al., 1981). Data from various groups on the effectiveness of UV irradiation in transforming rodent fibroblasts into morphologically identifiable colonies are presented in Table V, where they are compared to data on transformation by ionizing radiation. Transformation in rodent cells has been largely based on morphological changes, growth in agar, and tumorigenicity (DiPaolo and Donovan, 1976).

RADIATION ONCOGENESIS IN CELL CULTURE

'

O

203

E

ERGSIMM

FIG.29. Transformation of hamster cells after exposure to various doses of UV radiation. Results expressed as transformed colonies/total colonies counted x 100. (Reproduced from DiPaolo and Donovan, 1979.)

Human Cells. Transformation of human cells by UV into anchorageindependent cells which did not give rise to tumors has been induced in embryonic cells (Sutherland et al., 1980, 1981). Cell susceptibility to transformation by UV decreased with cell passage in uitro in the human embryo cells similar to that observed in experiments utilizing hamster embryo cells (Borek and Sachs, 1967). Transformation by UV radiation has also been reported in human foreskin cells, in both fibroblasts (McCloskey and Milo 1977) and epidermal cells (Milo et al., 1981) and in adult skin cells (Maher et al., 1982; Andrews and Borek, 1982). Growth in agar was reported and with the epidermal cells tumorigenicity was assessed in chick embryonic skin (Milo et al., 1981). The procedure for UV cell transformation, which generally followed that for chemical transformation (Milo and DiPaolo, 1978), was modified in the case of the epidermal cells, for it was found that the program for fixation and expression of transformation in the epidermal cells differed from that found in the fibroblasts (Milo et al., 1981).

TABLE V TRANSFORMATION OF RODENT CELLSin Vitro

Radiation

Dose

Dose rate

RADIATION

BY

Lowest and highest rate of transformation per survivor

RBE

Cell system

X ray (250 or 50 kVp) X ray (210 kVp)

300 rad

60 or 280 rad/min

7 x 10-’-8

1-600 rad

4.25 or 70.6 rad/min

10-4-6

X Ray (300 kVp)

0.3 rad

4.25 rad/min

10-5

X Ray (100 kVp)

50- 1200 rad

83.5 rad/min

3 x 10-5-3

x 10-3

1

Hamster embryo Hamster embryo Hamster embryo C3H 1OT+

X Ray (50 kVp)

50-1200 rad

-

8 x 10-5-3

x lo-’

1

C3H 1OT*

X Ray (300 kVp)

100-1000 rad

32 or 180 rad/min

7 x 10-5-2

x 10-3

1

C3H 1OT+

X Ray (100 kVp) Neutron (430 keV)

10-400 rad 0.1-150 rad

78 rad/min 10-80 rad/hr

10-4-3 x 10-3 7.6 x 10-5-3.2 x lo-’

1

Fission neutron

25-60 rad

10.3-37.8 radimin

10-4-6

3T3 A31 Hamster embryo C3H 10Ti

c( Particle (5.6 MeV) Argon ion (429 MeV/amu) Iron particles

205-342 rad 1 and 10 rad

-

14 rad/sec

x

1 1

x lo-’

-

-4 x 10-2 2.7 x 10-3-7.0

1 rad/min

-5 x 10-4--6

10-90 rad

-

uv

7.5-60 erg/mm2

0.76 erg/mm2/sec

8 x 10-’-7

uv

25-300 erg/mm‘

4.5 erg/mm2/sec

10-4-10-

uv

1 .O-7.55/m2 75 erg/mmz

0.38 J/mz/sec

10-5-6 x 1.6 x 10-3-10-5

uv

-

1

x

2.6-10 -

lo-’

-

x lo-’

-

x

x lo-’ 3

6-10

-

-

C3H 10Ti Hamster embryo C3H 1OT$ Fetal hamster C3H lOTi 3T3 A31 3T3 A31

Reference Borek and Sachs (1966) Borek and Hall (1973) Borek and Hall (1 982) Terzaghi and Little (1976) Han and Elkind (1 979) Miller and Hall (1 979) Little (1979) Borek et a/.(1978) Han and Elkind (1979) Lloyd et al. (1979) Borek et al. (1978) Yang and Tobias (1980) DiPaolo and Donovan ( 1976) Chan and Little (1976) Little (1979) Kakanaga (1980)

RADIATION ONCOGENESIS IN CELL CULTURE

205

More recently, adult skin fibroblasts from patients with xeroderma pigmentosum (Setlow et al., 1969; Cleaver, 1969) and Bloom syndrome cells (Chagani et al., 1974) have been transformed in vitro by UV light into anchorage-independent cells (Fig. 30) (Andrews and Borek, 1982). The source of UV light was a UVB, a sunlamp type of irradiation, of relevance to human skin carcinogenesis. Single cells were exposed to wavelengths of 280 and 320 nm. Both morphological foci as well as ability to grow in agar were assessed. It appears, as mentioned earlier, that here too in human cells transformed by UV, ability to grow in agar was exhibited concomitantly with the appearance of foci comprising piled-up, randomly oriented cells that were clearly distinguishable morphologically from the flat, orderly oriented control cells. Transformation of XP cells by UVA has also been reported (Maher et al., 1982). A higher rate of transformation into anchorage-independent cells was observed in the XP cells compared to the normal skin fibroblasts.

FIG.30. (A) A focus of Bloom syndrome fibroblasts transformed in uitro by UVB. (B) An anchorage-independent colony of UVB-transformed Bloom syndrome fibroblasts growing in agar. (Andrews and Borek, 1982.)

206

CARMIA BOREK

C. COCARCINOGENS AND MODULATORS OF RADIOGENIC TRANSFORMATION In recent years it has become increasingly clear that environmental factors including diet play a crucial role in determining cancer incidence in humans (Higginson, 1979; Pet0 et al., 1981;Miller and Miller, 1979; Weinstein rful., 1979; Upton, 1981; Borek, 1981a; Nagao and Sugimura, 1978; Cairns, 1981). Although radiation is a weak oncogenic agent compared to some chemicals, it is the most universal. Thus, in assessing the effects of radiation from the point of view of cancer risk to humans (Beir, 1972; Storer, 1975; Upton, 1975; UNSCEAR, 1977; Sinclair, 1981), one cannot exclude the possibility that a multitude of genetic, physiological, and environmental factors influence cancer incidence initiated by radiation. The difficulty lies in identifying the carcinogens that act in an additive or synergistic manner. Once recognized, measures may be sought to alter exposure to these agents and to find ways to modify their effects and interactions. Under defined conditions in uitro we can evaluate some of these interactions at a cellular level, although admittedly the absence of host-mediated effects or intact tissue organization give us a somewhat slanted view. Within the context of the accepted concept of cancer development (Farber, 1973; Berenblum, 1975; Pitot and Sirica, 1980), one thinks of neoplastic transformation as comprising the early phases of initiation and a later stage of expression by which we recognize initiation. Agents that modify expresssion can interact at early or at later stages, serving as promoters. Although initiation by radiation is irreversible, it can sometimes be prevented (Guernsey et al., 1980, 1981; Borek, 1981). Expression or promotion can be reversed (Borek et al., 1979; Miller et al., 1981; Kennedy and Little, 1978; Borek, 1981a,b.) 1. Enhancement of' Transformation The enhancement of viral transformation following a preexposure of cells to radiation was the first type of interaction observed (Stoker, 1963; Pollack and Todaro, 1968) and is reviewed elsewhere (Yang and Tobias, 1980). a. Chemicals. Preexposure of hamster embryo cells to X-ray doses of 150 to 250 rad rendered them more responsive to transformation by benzo[a]pyrene as well as UV light (DiPaolo and Donovan, 1976; DiPaolo, 1976). Pretreatment with UV light did not have an enhancing effect. Maximum enhancement was found when radiation was administered 48 hr prior to the carcinogens, and it was directly related to the absorbed radiation. A synergistic interaction was reported (Borek and Ong, 1981) between X rays and the food pyrolysate product 3-amino- 1-methyl-SH-pyrido(4,3-b)indol(TrpP-2). Isolated in Japan from broiled meat and fish, foods widely consumed there, this compound has been shown to be a mutagen and a carcinogen

RADIATION ONCOGENESIS IN CELL CULTURE

207

in uiuo and in uitro (Nagao and Sugimura, 1978). Preexposure of hamster embryo cells to 50 or 150 rad of X rays followed by treatment with Trp-P-2 resulted in an enhanced transformation that was dose-dependent, indicating a synergistic interaction at 150 rad. The study of the mechanisms of action between these two agents is complex because both X rays and Trp-P-2 damage DNA and the exposure of the irradiated cells to Trp-P-2 took place within the period of fixation of radiation transformation. The interaction, however, is of interest. Though we are clearly aware that these are studies in uitro, that the compound is in concentrated form, and that we are not exposed daily to doses of 50 or 150 rad, the fact that these two agents interact is of importance; it further compounds the interpretation of some of the data from Hiroshima and Nagasaki, the largest source of evidence on radiation carcinogenesis. b. Promoters. Enhancing agents can also be part of the family of tumor promoters. The most widely studied in conjunction with radiation transformation has been the phorbol ester derivative 12-0-tetradecanoyl-phorbol 13-acetate (TPA) (Hecker, 1971). Promoters in themselves are considered noncarcinogens and TPA has been considered a classic promoter. However, recent work suggests that TPA possesses some initiating action (Kinsella and Radman, 1978; Emerit and Cerutti, 1981; Han and Elkind, 1981a,b). The effects of TPA are pleotropic (Mondal and Heidelberger, 1980). In studies on radiation transformation, TPA shows an interaction with chromosomes as well as an effect on membrane-associated enzymes within the same cell system (Borek et al., 1981 ; Borek 1981a). The enhancement of X-rayinduced transformation by TPA has been studied in detail (Kennedy et al., 1978, 1980, 1980a; Borek, 1981a; Miller et as., 1981; Borek et al., 1981). TPA exhibits a temporal enhancement of transformation consistent with the idea of promotion, namely that its effectiveness can be observed at various stages after initiation (Mondal et al., 1976; Weinstein et a[., 1979; Kennedy et al., 1978). More recently (Han and Elkind, 1981), studies reported that TPA enhanced both X-ray- and fission neutron-induced transformation in the 1OT; cells. The data indicated that TPA alone acted as a weak initiator and produced a 10-fold transformation incidence above background. Enhancement of transformation was greater after low radiation, compared to its effect after high doses of either radiation. The RBE for transformation following TPA treatment increased with level of effect. Thus TPA potentiation of X-ray-induced transformation in the low-dose region made the combined treatment as effective as neutron action alone. The effect of TPA acting in consort with radiation is also seen in the experiments by Kennedy and Little (1980), where the yield of transformants following treatment of 100-rad X ray followed by TPA is similar to that obtained by exposing cells to 400-600 rad of radiation alone. In view of the probability that TPA is a

208

CARMIA BOREK

weak initiator, the interaction between X ray and TPA as cocarcinogenesis rather than promotion cannot be ruled out. It is worth mentioning at this point that in conjunction with these last experiments (Kennedy and Little 1980), a series of studies was carried out with the lOT4 cells (Kennedy et al., 1980), in which cells were irradiated, grown to confluency, then resuspended, diluted, and plated at different cell densities. Cultures were then allowed to grow until confluency, after which 4 additional weeks were allowed for focus formation. The results indicated that the absolute yield of foci was constant over a wide range of dilutions and similar to that found in cultures which were not diluted. These results were interpreted in terms of a two-step transformation hypothesis : the first step occurring at or soon after radiation and being of high probability followed by a second, low probability event occurring after confluence has been reached. That is, in this cell system initiation is not a rare event, and exposure to X rays results in an initial functional change that enhances the probability of subsequent transformation under conditions of confluence. This interesting observation clearly complicates the interpretation of a wide volume of data based on studies in this cell system, including the results obtained with TPA, and calls for further studies with other systems. More recently this interpretation has been challenged (Rossi et al., 1982; Hall et al., 1982) as a result of experiments in which cultures were trypsinized and plated into triplets of dishes at various times after irradiation. By 1 week after irradiation there was a perfect correlation between triplets in that either all three dishes contained transformed foci or all three contained none. This was interpreted as evidence that the commitment to transformation occurs early after irradiation and well before confluence is reached ; the experimental data totally rule out the hypothesis of Kennedy and Little (1980). Furthermore, the number of foci/dish did not increase with time between irradiation and replating as rapidly as would be expected, and indicated the possibility of a strong suppression of the development of transformed foci by the presence of other foci. c. Interferon andg-Estradiol. Other agents reported to enhance radiogenic transformation include interferon, studied in the 1OT; cells (Brouty-Boyce and Little, 1977), an agent that is an inhibitor of cell growth. The hormone /I-estradiol potentiated X-ray-induced transformation in human cells (Borek, 1980) but in itself did not transform the human cells. In the C3H 10T* cells P-estradiol acted itself as a carcinogen as well as interacting with radiation to enhance transformation (Kennedy and Weichselbaum, 1981). These data alert one to the fact that in the process of evaluating the additive or synergistic interaction between agents to enhance carcinogenesis, we must be cognizant of the strain and species from which the cells are derived and to their response in vivo. For example, tumor incidence in the C3H

RADIATION ONCOGENESIS IN CELL CULTURE

209

mouse strain is particularly sensitive to the action of steroids. (LaCassagne, 1938). d . The Protease Inhibitor Antipain, u Dual Action. Antipain (AP) (Umezawa et al., 1979) is a protease inhibitor that has been shown to have anticarcinogenic activity (Troll, 1976). Its action in vitro on influencing radiationinduced transformation has been dual (Borek et al., 1979; Geard et al., 1982). Antipain potentiated X-ray-induced transformation in human (Borek, (1980), hamster, and 10Ti mouse cells, when added to the cells prior to radiation. It reduces X-ray -induced transformation when added after irradiation (Kennedy and Little, 1978; Borek et al., 1979; Geard et al., 1982). These dual actions are exerted without any effect on cell survival. The enhancing effects of AP are more pronounced if AP is removed after irradiation (Gerard et al., 1982), as compared to adding it before irradiation and keeping it on for the duration of the experiment, whereby its protective effect reduces the enhancing actions. The dual activity of AP (Table VI) was not reflected in chromosomal alterations as measured by sister chromatid analysis (Geaard et al., 1982). Antipain has been shown by Kinsella and Radman (1980) to modify chromosomal aberation but not SCE. Antipain had no effect on DNA damage or replication (Borek and Cleaver, 1981) (Figs. 31 and 32). Other inhibitory actions by AP were shown in its ability to inhibit in 1OT4 cells the enhancement of X ray-induced transformation by P-estradiol as well as the carcinogenic effect of the hormone. Another protease inhibitor, leupeptin, had the same effect on inhibiting transformation but not on DNA repair (Borek and Cleaver, 1981). Clearly, the mechanisms of the protease inhibitor AP call for more inquiry. One can only speculate that the two diametrically opposed actions of AP on transformation are mediated via different mechanisms; some are associated with the direct cellular interactions with radiation at which time cascading events could occur, whereas in later events its inhibitory activity, acting in a temporal fashion (Borek et al., 1979), may be mediated via an effect on specific cellular proteases.

2. Inhibition oj'Radiogenic Transjormation Although we try to identify agents that act as cocarcinogens or promoters, we aim at finding compounds or conditions that may inhibit the progression of transformation and, even better, prevent initiation. a . Retinoids. Within the last decade analogs of vitamin A (retinoids) have been shown to modulate malignancy both in laboratory animals and in the clinic (Spron et al., 1976; Lotan, 1980; Pet0 et al., 1981). Their effectiveness in inhibiting X-ray-induced transformation in uitro was first shown

210

CARMIA BOREK

TABLE VI CELLTRANSFORMATION in Virro AFTER TREATMENT WITH X RAYSAND ANTIPAIN" Hamster embryo

Treatment

Surviving fraction

Transformation incidence ( x 103)

Antipain added before X irradiation with 300 rad 24 hr I .oo 0 Control Antipain 0.94 0 X irradiation 0.60 6.6 & 1.0 X irradiation and 0.55 12.3 f 1.2 antipain Antipain added after X irradiation with 300 rad 10 min Control 1.oo 0 Antipain 1.96 0 X irradiation 0.53 8.4 & 0.7 X irradiation and 0.44 3.7 0.5 antipain 24 hr Control Antipain X irradiation X irradiation and antipain 48 hr Control Antipain X irradiation X irradiation and antipain

1.oo

0.92 0.60 0.62

1.oo

0.89 0.65 0.62

0 0

6.5 f 0.7 5.9 _+ 1.2

C3H lOTi

Surviving fraction

Transformation incidence ( x 104)

1 .oo 0.98 0.41 0.38

0 0 4.9 k 0.6 7.4 k 1 . 1

1.oo

0 0 3.1 k 0.4 1.5 k 0.4

0.94 0.38 0.35

1.oo

0.96 0.40 0.41

0

0 * 0.1 1.0 f 0.3 1.1

0 0

6.4 f 1.3 5.9 1.2

*

Data from Borek el al. (1979).

using the 1OTicells (Harisiadis et af.,1978), indicating that these compounds could act in uitro and that their action was not limited to an effect on neoplastic epithelial cells, as previously observed in uiuo. The ability of retinoids to inhibit not only X-ray-induced transformation but also the enhancement of this transformation by TPA in both 1OTi and hamster embryo cells was reported more recently (Miller et af., 1981; Borek, 1981a). The effect was striking. Two analogs, p-all-trans retinoic acid (RA) and trimethyl methoxyphenyl analog of N-ethyl retinamide (TMMP-ERA), were used. Retinoids

21 1

RADIATION ONCOGENESIS IN CELL CULTURE 1 .!

2 Y v)

I I-

2

?;

1.(

1 n U

0 Y

I-

dw 2

0.1

I-

4Y K

I

1

1 3

I 2

HOURS AFTER EXPOSURE

FIG. 31. Relative rate of DNA synthesis in human fibroblasts exposed to 1.7 mM antipain for 30 min (B),13 J/m2 of 254 nm UV light (O),or 2 mM hydroxyurea for 30 min (a) and pulse-labeled for 15 min with 10pCi/ml [3H]dThd (60 Ci/mmol) at various times after exposure.

1

2

3

HOURS AFTER EXPOSURE

FIG. 32. Relative rate of DNA synthesis in human fibroblasts exposed for 30 min to S9 mix (a),1.7 mM antipain plus 10% serum (A),1.7 m M antipain plus S9 mix (A), or 30 pg/ml benzypyrene plus S9 mix ( V ) and pulse-labeled for 15 min with 10 pCi/ml [3H]dThd (60 Ci/ nmol) at various times after exposure.

212

CARMiA BOREK

were present in the medium at the time of irradiation and TPA was added after irradiation to some of the experimental plates. The retinoids were kept in contact with the cells for 4 days only and thereafter removed with an exchange of medium. TPA was maintained for 2 weeks (hamster) or 6 weeks (10Ti) for the total length of the experiment. Thus inhibitory action of the retinoids had to be exercised within 4 days to override the enhancing effect of TPA. As seen in Table VII, the retinoids did indeed inhibit both X-ray transformation and TPA enhancement of this transformation, thus indicating that their action on radiogenic transformation takes place within a short time and is irreversible. Studies to evaluate the mechanisms of action indicated that the inhibitory action of retinoids on transformation and on TPA action was not reflected in an inhibition of sister chromatid exchanges (SCE) (Miller et al., 1981 ; Borek, 1981) (Figs. 33 and 34). Radiation enhanced SCE about 2-fold; TPA causes a slight enhancement of SCE, but so did the retinoids. The retinoid inhibited the small enhancement of SCE by TPA. When added after irradiation, TPA slightly enhanced the X ray-induced SCE, but so did the retinoids to the same degree. When retinoids were present along with X ray and TPA, a combination that resulted in inhibition of transformation, the highest degree of SCE enhancement was observed (Figs. 33 and 34). Thus the retinoids were not exercising inhibition at the chromosomal levels. Their action was reflected at the membrane level on the membrane-associated Na-transport enzyme Na+ , K+-ATPase, but not on Mg2+ATPase or on 5'-nucleotidase (Borek, 1981, 1981a). TPA enhanced the level of the enzyme; retinoids decreased it. When cells were exposed to both agents concomitantly the Na', K+-ATPase returned to control level.

TPA

AND

TABLE VII RETINOID MODIFICATION OF X RAY-INDUCED TRANSFORMATION IN HAMSTER EM8RYO AND C3H 1oTt CELLS Hamster embryo

Treatment ~~

Control TPA (0.16 bm) Retinoid (7.1 pm) Retinoid, TPA X Rays X Rays, TPA X Rays, Retinoid X Rays, Retinoid, TPA

Surviving fraction 1 .oo

0.70 0.62 0.89 0.42 0.53 0.38 0.41

C3H IOTj

Mean rate of transformation +_ SE

Surviving fraction

0 0 0 0 6.99 f. 1.65 12.52 +_ 1.81 2.94 +_ 0.79 2.41 k 0.85

0.93 0.75 0.67 0.32 0.52 0.30 0.36

1 .oo

Mean rate of transformation SE

+

0 0 0 0

+

8.78 1.29 16.15 1.59 4.37 f 0.93 2.46 0.54

213

RADIATION ONCOGENESIS IN CELL CULTURE 20 19

18 0.8

11 16 15

0.1

I4

0.6

Sister 0.5 Chromatid Exchanges o,4 per Chromosome o,3 0.2 0.1 0

!1

Control M i i d 3 6

PA

13 12 11 10 9

Transformants p@f 10.000 Surviving Cells

1 ' 6

' 5

h

4

. 3 2

. 1 ' 0

Control Retinid

I

FIG.33. Comparison of the effects of X rays, retinoid TMMP-ERA, and TPA on transformation induction (right side) and sister chromatid exchanges (left side) in mouse C3H IOTt clone 8 cells. Standard errors of the mean are indicated. For the SCE studies cells were scored up to 20 hr after irradiation; transformation incidence was assessed at 5 weeks after irradiation with TPA in continuous cell contact and retinoid present for 4 days only. (Reproduced from Miller er a/., 198I .)

Thus the retinoids appear to exercise their effect at the level of gene expression. Once cells are transformed and exhibit a neoplastic phen,otype, their membrane Na+, K+-ATPase changes (Borek, 1981a). Its activity is no longer modulated by either retinoids or TPA. Although the mechanisms of action of the retinoids are not clear, the data lend support to the notion that these compounds are effective suppressors of carcinogen-induced neoplastic progression. They are agents that act on expression of oncogenesis. They increase cell adhesion to the plates and also alter the cellular morphology into fusiform elongated cells. Although we considered their action on TPA as an effect on a promoter, we cannot rule out their action on X ray and TPA as an inhibition of cocarcinogenesis. A clear inhibition of the synergistic interaction between two oncogenic agents has been reported (Borek, 1981a). Retinyl acetate was found to inhibit in hamster embryo cells the oncogenic effect of tryptophan pyrolysate (Trp-P-2), to suppress radiation transformation, and also to inhibit markedly

214

CARMIA BOREK

FIG.34. Sister chromatid exchanges in chromosome properties of C3H IOTj cells. (Courtesy of Dr. C. R. Geard.)

the synergistic interaction between Trp-P-2 and radiation. The inhibitory action by retinoids was exercised within 4 days in an irreversible manner. The retinoid inhibition of synergism between X rays and these pyrolysates is of significance. The potential role in carcinogenesis of food products presents a complex dilemma in terms of assessing the risk to the general public (Miller and Miller, 1979), and any agent that may decrease the effectiveness of the carcinogenic action and inhibit synergism and cocarcinogenesis offers hope in prevention, especially because retinoids are derivatives of a natural product, vitamin A. b. Selenium. Selenium, a micronutrient and a ubiquitous element in nature, has long been considered a chemopreventive agent, though it does

RADIATION ONCOGENESIS IN CELL CULTURE

215

possess some carcinogenic action under certain conditions (Griffin, 1979). One of its major functions may be related to its role as a cofactor in glutathione peroxidase (Griffin, 1979). A recent report (Borek, 1981a) indicates that selenium inhibits in 10Ti cells both X-ray-induced transformation and also that induced by pyrolysate products and benzo[a]pyrene. Its effectiveness on radiogenic transformation indicates that its inhibitory action follows, in part, a path other than the one previously assumed in relation to chemical carcinogenesis; namely, inactivation of the carcinogen. A more plausible suggestion is that its inhibitory action on radiation transformation is via an interference with the availability of free oxygen radicals, which are closely associated with the action of radiation (Alexander and Lett, 1968) and may be mediating, in part, the oncogenic action of X rays (Borek and Troll, 1982). c. Free Rudical Scavengers. The generation of reactive oxygen species in living systems exposed to radiation has long been recognized, as well as the protective action by radicals scavengers (Bacq and Alexander, 1955; Alexander and Lett, 1968). More recently, tumor promoters have been shown to produce free radicals. These include TPA (Goldstein et al., 1981) and teleocydine (Fujiki et al., 1979). Utilizing hamster embryo cells, the effects of superoxide dismutase (SOD) and catalase on X-ray-induced transformation (300 rad) and its enhancement by TPA were evaluated (Borek and Troll, 1982). SOD and catalase were added to the cells at seeding, or were combined during irradiation and removed immediately after exposure or at the end of the experiment. TPA was added after irradiation, and SOD and catalase were kept on (alone or in combination) for the full course of the experiment. The results indicate that SOD, and to a lesser extent catalase, inhibited both X-ray induced transformation (inhibition of 50% by SOD) and the TPA-enhancing effect, while having a less marked effect in enhancing cell survival. Their effectiveness on lowering X-ray transformation was similar to that exhibited by 8-all-truns-retinoic acid and antipain. The results suggest that X-ray-induced transformation and TPA action may be mediated in part via the action of free radicals. Superoxide dismutase which was not detected in the serum used in these experiments) will convert 0; to H,O,, thus preventing it from forming toxic species such as HO; and singlet oxygen (McCord and Fridovich, 1969). The prevailing H,O,, which is a substrate for many toxic peroxidases, is then converted by catalase. The lower effectiveness of catalase in inhibiting transformation may be due to the finding that the hamster embryo cells contain a high level of this enzyme, sufficient to remove the toxic H,O, . Though we are relatively ignorant of the mechanisms by which free radicals may influence neoplastic transformation, one could speculate that their effect may involve membrane lipid peroxidation (Wendel and Fenerstein,

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in press), which may result in a cascade of events (Puck, 1977, 1979), with cell death and transformation being some of the consequences. It is interesting in this context to note that SOD has been shown to inhibit TPA-induced chromosomal aberrations (Emerit and Cerutti, 1981), indicating that in certain systems TPA may indeed possess an X-ray-like effect. 3. The Role of Thyroid Hormone in Radiation Transformation Little is known about the cellular and molecular mechanisms involved in the process of initiation and the physiological requirements for the induction of transformation. Recent reports indicate that thyroid hormones play a crucial role in the process of initiation of X-ray-induced transformation (Guernsey et al., 1980, 1981; Borek et af., 1981a,b). Experiments conducted using hamster embryo cells as well as the 1OTi cells indicate that though removal of thyroid hormone from serum used in the medium does not modify cell growth rate (Fig. 35) or cell survival (Fig. 36), the hypothyroid

10'

I

2

3

4

5

6

7

8

J

9

Days

FIG.35. Growth curve of C3H lOT4 cells grown in medium containing resin-treated serum (hypothyroid, 0 )or in the same medium supplemented with lo-' M triiodothyronine (T3)(0). Note the similar growth rate.

RADIATION ONCOGENESIS IN CELL CULTURE

217

i

0

2

4 6 8 1 X-ray dose (Gy)

0

FIG.36. X-Ray survival data for C3H IOT* grown and maintained in medium with regular serum (m); medium supplemented with resin-treated serum, devoid of thyroid hormone (a); or medium supplemented with resin treated serum to which 10- 'pg triiodothyronine was added, hyperthroid conditions (0) (1 Gy = 100 cad).

10,

CJH/lOT

0

2.2

Cells

220 2 . 2 0

X-Ray Dose (Gy) (2 T j )

FIG.37. Histogram relating the inhibition of radiation-induced transformation in hamster (HE) and IOT4 cells irradiated under hypothyroid conditions (-T3), as compared to those treated in euthyroid and hyperthyroid ( fT,) media (numerical data from Guernsey er al., 1980).Note that no transformation is observed under hypothyroid conditions.

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conditions completely inhibit the initiation of transformation by radiation (Guernsey et ul., 1980, 1981; Borek et al., 1981a) as well as by chemicals (Borek et al., 1981b). In the experiments, cells grown in medium containing resin-treated serum, which selectively removes triiodothyronine (T,) and thyroxine (T4) (Samuels et ul., 1979), were considered hypothyroid ( -T3). Others grown in medium with untreated serum were euthyroid, and cells grown in medium containing hypothyroid serum fortified with lo-’ M T, were hyperthyroid (+ T,). When both types of cells were irradiated (300 or 400 rad) under these three conditions, no transformation was observed in the hypothyroid cells whereas euthyroid and hyperthyroid cells showed neoplastic transformation (Table VIII; Fig. 37). When T, was added to the hypothyroid medium prior to irradiation, transformation was observed. A dose-related role of T, in the transformation process was illustrated by the fact that when T, was added to the hypothyroid milieu, prior to irradiation at doses of 10- l 2 A4 to lo-’ M , transformation incidence was T, dosedependent (Fig. 38). The action of T, was specific to the active hormone and was not mimicked by reverse T,, an inactive isomer. The involvement of thyroid hormone in initiation of transformation was further indicated by the fact that maximimum transformation was achieved when T, was added at least 12 hr prior to irradiation (Fig. 39). If added at the time of radiation, transformation was dramatically decreased, and if added after radiation no transformation was observed. The activity of T, when added prior to irradiation was exercised within 24 hr and its removal after radiation did not reduce transformation.

H

(0-12

10-11

10-10

10-9

10-6

10-7

1, (MI

FIG. 38. The effect of various T, concentrations on transformation rate ( O - - - -0) and Na/K ATPase activity (A-A). (Reproduced from Guernsey er al., 1981.)

TABLE VIII THEEFFECTOF THYROID HORMONE ON X-RAY-IRRADIATION-INDUCED CELLTRANSFORMATION in Virro OF C3H lOT4 MOUSECELLSAND HAMSTER EMBRYO CELLSIN CULTURE".^ Serum treatment (T3 condition)

Cells C3H/lOT+

Untreated FBS Untreated FBS Resin-treated FBS/(-T3 - T4) Resin-treated FBS/( -T3 - T4) Resin-treated FBS + T3/(+T3) Resin-treated FBS T3/(+T3)

0 3 0 3 0 3

Untreated FBS Untreated FBS Resin-treated FBS/(-T3 - T4) Resin-treated FBS/( -T3 - T4) Resin-treated FBS + T3/(+T3) Resin-treated FBS + T3/(+T3)

0 2.2 0 2.2 0 2.2

+

HE

X rays (GY)

Total surviving colonies

No. of colonies transformed

Transformation frequency

23,313 27,737 23,154 33,747 15,884 21,040

0 24 0 0 0 16

0 8.65 x 10-4 0 0 0 7.60 x 10-4

2800 4800 2400 1500 2600 3600

0 3.33 x 10-3 0 0 0 4.17 x 1 0 - ~

Values given are totals of three separate experiments with C3H 1OT* cells and two separate experiments with hamster embryo cells. Stock A4 T3 in 50% n-propanol was diluted in media with 10% resin-treated fetal bovine serum (FBS) to give a final concentration of lo-’ M T3 (designated T3). Media without thyroid hormone was prepared with 10% resin-treated FBS and an amount of diluent equal to that added in the +T3 media. Stock cultures of C3H 1OT$ cells were maintained in Eagle’s basal medium + 10% heat-inactivated FBS; hamster embryo cells in Dulbecco’s modified Eagle’s medium +lo”/, FBS. Both cultures contained penicillin (50 U d-’) and streptomycin (50 pg m1-l). All cells were maintained at 37°C with 5% CO, in air throughout the experiment. One week before seeding, stock cultures were placed in the experimental culture media, with and without T3 (as described in the text), and maintained in these conditions for the duration of the experiment. Twenty-four hours after seeding, the cells were irradiated with X rays at room temperature (2.2 Gy for HE and 3 Gy for C3H 1OT4 cells), at a dose rate of 0.322 Gy min-’, thereafter receiving weekly media changes. After an appropriate incubation period (6 weeks for C3H lOT4 and 2 weeks for HE), the cells were fixed and stained with Giemsa and scored for transformation. Both type I1 and I11 foci were scored in C3H 10Ti experiments. From Guernsey er al., 1980.

+

220

CARMIA BOREK

C3H/ 10T3 Cells

R d l mlc TrA L n a t i o n

Fmauencv

f T3 TIME COURSE (4 6,) FIG.39. Time course relating exposure of cells at different times prior to and after irradiation to media devoid ofT, ( -T3)as well as supplemented with T3(+T3). Maximum transformation is observed when T3is added 12 hr prior to irradiation, and no transformation if added after irradiation. (Numerical data from Guernsey et al., 1981.)

Exposure of the cells to cyclohexamide at 100 ng/ml concurrently with T, (i.e., 12 hr before and removed 24 hr after irradiation) inhibited transformation entirely, indicating a requirement for protein synthesis. As seen in Fig. 38, there was close similarity in the concentration dependence of the induction of N a + , K+-ATPase and of transformation. In view of the evidence indicating that T, regulates Na', K+-ATPase activity in inducing de nouo synthesis of subunits (Lo and Edelman, 1976), these results, as well as the time course data and the observations on the inhibitory effects of cyclohexamide, raise the possibility that T, induces the synthesos of host proteins that are involved in the initiation of neoplastic transformation. Thus, in contrast to modulating agents, e.g., retinoids, which affect later steps in neoplastic development, namely, expression and promotion, thyroid hormones are crucial in the initial steps of induction of transformation, perhaps in making the cell competent for events involved in initiation. V. Discussion

Radiation is a fact of life. It occurs in nature and pervades the environment. Although a relatively weak carcinogen and mutagen compared to some chemicals, it is the most ubiquitous and measurable at low doses.

RADIATION ONCOGENESIS IN CELL CULTURE

22 1

In recent years public concern has focused on the potential biological hazards of low-dose radiation within the range of 0.1 to 1 rad. This is the dose level involved in public exposure from nuclear installations as well as from medical diagnostic X rays. We cannot discount the effect of low doses in the initiation of an event, which may later be amplified. Any carcinogens given at the right dose to a competent and specific target cell may serve as effective initiators and promoters. There is a need to develop suitable systems to assess directly the oncogenic potential of low-dose radiation. Epidemiologic studies do not lend themselves to evaluation of the carcinogenic effects of low doses of radiation. The contributions from epidemiology have been through extrapolation from incidents where limited numbers of individuals received high doses of radiation delivered in most cases as single acute exposures. In practice, public exposure comprises multiple small doses. Animal systems, in which the induction of tumors by radiation serves to assess the oncogenic action of X rays, are limited to studies in the low-dose range. Cell cultures offer the best systems for evaluating the varied biological effects of radiation at a cellular level and investigating the mechanisms involved. Indeed, knowledge of basic aspects of cellular radiobiology was acquired within the last 3 decades. Clonal assays were developed, as well as survival curves, with the intriguing observation that some cells require “company” provided by irradiated feeder cells (Puck and Marcus, 1956; Puck et al., 1956; Puck, 1958). Later, work carried out on radiation-induced sublethal damage in mammalian cells showed that the half-life for repair of this damage, as shown with split dose experiments, was approximately 1 hr (Elkind and Sutton, 1960). However, the use of cell culture to study radiation oncogenesis came later (Borek and Sachs, 1966a,b), and with it the opportunity to study cellular and molecular mechanisms associated with radiation oncogenesis as well as quantitative cancer risk estimates related to radiation quality, dose, dose rates, and cocarcinogenesis. Transformation in citro was suggested as early as 1925 by Alexis Carrel, who stated that “the best method of ascertaining the properties that characterize a malignant tissue would be to transform in uitro a strain of cells of a known type into cells capable of producing sarcomas or carcinomas and to study the changes undergone by the strain.” The successful study of the direct oncogenic action of radiation on cells in culture was achieved in 1966 by Borek and Sachs, who showed that the exposure of mixed cultures of hamster embryo cells to 300 rad of X rays resulted in the neoplastic transformation of a defined low fraction of treated cells (Fig. 1). At the same time, the unirradiated as well as the treated but not transformed cells, senesced and died. N o spontaneous transformation

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CARMIA BOREK

was observed (> lo6), indicating that the transforming effect resulted directly from the interaction of radiation with the exposed cells rather than from host effects present in vivo. Although cell cultures offer defined systems that afford the opportunity to evaluate various aspects of transformation, they must be recognized as such; and their response to a variety of agents and conditions suggests rather than definitely establishes a condition comparable to that in vivo. “Suggestion” may vary among cell strains and lines, including cells from human origin where the genetic makeup of the donor cannot be excluded. We use in oitro cultures as simplified systems, yet these cells are derived of proliferating and nonproliferating tissues, “forced” to grow freely in uitro. Though cell strains from freshly explanted cultures such as the hamster or human senesce in vitro, cell lines such as the 10Tj are populations of selected cells that are no longer subject to the control of “time clocks” and finite life spans. Thus the radiation transformation process in the lOT4 (Fernadez et a/., 1980; Kennedy et al., 1980a,b; Rossi rt al., 1982; Hall et al., 1982) may differ from that observed in a cell strain like the hamster, consisting of normal diploid cells (Borek and Sachs, 1966a, 1967; Borek and Hall, 1973).Normal embryos explanted in culture give rise to cell populations in which some are competent to undergo induction by radiation (Borek and Sachs, 1967). The frequency of cells in this state of competence may vary not only with the cell population explanted but also with the donor, because there exist differences in transformability among cells of different embryos (Borek, unpublished). The frequency of cells sensitive to initiation by X rays decreases with passage in vitro (Borek and Sachs, 1967) and this also holds true for cells from human embryos following UV treatment (Sutherland et al., 1980). This is in contrast to the situation in the lOT4 and the 3T3 lines in which progressive culture in vitro may sometimes result in spontaneous transformation. The clonal assay in the hamster cell system (Borek and Sachs, 1966a,b; Borek and Hall, 1973)does not require the lengthy cell-to-cell contact needed in the lOTi cells suggested to be a major factor in determining the frequency of transformation in 10Ti (Kennedy rt al., 1980). When hamster cells maintained under an identical environment are irradiated at various stages of clonal growth, only part of the clone may be transformed (Borek and Sachs, 1967), indicating a complex relationship related to genetic competence, sensitivity to transformation, and physiological environment. Transformation by radiation can be compounded by viral transformation of these cells (Borek and Sachs, 1966b). This additional alteration modifies their surface properties and their interaction with parental X-ray-transformed cells as well as with normal cells which clearly inhibit the proliferation of the transformed (Borek and Sachs, 1966b). Change in cellular environment does

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not modify the inhibition of the transformed cells by the normal ones, indicating that it is an actual contact phenomenon. The success of a new field is measured by the unique and sometimes unexpected information contributed. We can therefore assess the success of cell cultures in studies related to radiation oncogenesis by the current acquisition of information unobtainable via other biological systems. Some of the followingfindings illustrate that, indeed, the in vitro systems have been useful and hold promise for the future. 1. A number of assay systems based on rodent fibroblasts offer excellent tools for estimating quantitatively the incidence of radiation-induced oncogenic transformation under conditions where high and low toxicity prevail (Borek and Hall, 1973; Borek et a/., 1978; Terzaghi and Little, 1976a; Little, 1979; Han and Elkind, 1979a, 1980; Lloyd et al., 1979). 2. Using these systems it is possible to obtain dose-response relationships over a wide range of doses and with a level of precision that cannot be rivaled by epidemiological studies of carcinogenesis in humans. (a) High-LET radiation is more oncogenic than X rays (Borek et ul., 1978; Han and Elkind, 1979a; Lloyd et a/., 1979; Yang and Tobias, 1980), but is also more toxic. (b) Transformation can be detected at doses as low as 0.3 rad of X rays. (c) The RBE for y rays as compared to X rays at low-dose level is 0.5, which has important implications in medical radiation (Borek et ul., 1982). (d) The incidence of neutron-induced transformation peaks higher than X-ray transformation when assayed per cell survivor; but when evaluated on this basis of cells at risk, which is more relevant to the in uivo situation, X rays and neutrons rise to the same peak value (Borek et ul., 1978; Han and Elkind, 1979a). 3. Assessment of transformation frequency can be made with in citro assay systems at doses that are relevant to public health concern for exposure to medical radiation, with no extrapolation. 4. Dose-response relationships established in uirro indicate that the data are poorly fitted by a simple linear relationship between dose end incidence. A linear extrapolation from data derived at high doses does not accurately predict transformation at low doses (Miller et al., 1979, Hall et al., 1982; Borek, 1979). 5 . Data from several in uitro transformation systems indicate that fractionation of an X-ray dose leads to an enhanced transformation for total doses less than about 150 rad. A linear extrapolation from single acute large doses therefore does not lead to cancer risk estimates that are either conservative or prudent for low doses as delivered as a series of fractions (Borek and Hall, 1974; Terzaghi and Little, 1976b; Miller and Hall, 1978b; Miller et al., 1979; Borek, 1979b; Han and Elkind 1979a).

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6. The transforming effects of UV light are dose-dependent in rodent cells (DiPaolo and Donovan, 1976; Chan and Little, 1976; Little, 1979; Mondal and Heidelberger, 1976) and in human cells (Sutherland et a/., 1980; Andrews and Borek, 1982; Maher et al., 1982). 7. Human fibroblasts can be transformed in uitro by X rays (Borek, 1980) and UV (Sutherland et al., 1980; Milo et al., 1981; Andrews and Borek, 1982; Maher et al., 1982). The temporal process of transformation is different (Borek, 1980; Andrews and Borek, 1982; Maher et al., 1982; McCloskey and Milo, 1977; Milo et al., 1981) from that seen in rodent cells, as are also some aspects of the phenotypic expression of the cells. Potential to grow in agar appears concomitantly with morphological changes, and the frequency of human cell transformation by X rays is lower than that of rodent cells given the same dose of radiation (Borek, 1980, 1981~). 8. Agents that interact with radiation to enhance radiation transformation have been identified. These include TPA, chemicals, pyrolysate products, Antipain added before radiation, and estradiol (Borek r t al., 1979, 1981 ; Borek, 1980, 1981a; Kennedy and Little, 1980; Miller et al., 1981; Borek and Ong, 1981 ; Han and Elkind, 1981). 9. We can successfully identify agents that suppress radiation-induced carcinogenesis and its cocarcinogenic interactions with other agents. These include the protease inhibitors antipain (added after radiation), leupeptin, (Kennedy ef ul., 1978; Borek et al., 1979; retinoids (Miller et al., 1981 ; Borek et a/., 1981 ), and selenium (Borek, 1981a). 10. We can test conditions which will modify cellular competence for transformation. Thus, thyroid hormones were found to be essential for the initiations of transformation (Guernsey et al., 1980, 1981; Borek et ul., 1982a,b), while a hypothyroid state in uitro inhibited both X-ray and chemically induced transformation. 1 1. Other agents that inhibit radiation-induced transformation and its enhancement by TPA are SOD and catalase, agents that scavenge free radicals, suggesting that X-ray-induced oncogenesis may be mediated in part via the effect of free radicals. The cellular content of catalase and SOD may determine to some extent cellular response to radiation (Borek and Troll, 1982; Borek, 1982). 12. It has been possible to evaluate underlying cellular and molecular mechanisms which regulate the effect of modulating compounds on radiation t.ransformation. Thus the action of retinoids is not mediated by the type of damage inflicted on DNA, which can be monitored by SCE analysis (Borek, 1981a; Miller et al., 1981), but is mediated at the level of gene expression at the membrane level, expressed by altered adhesion and morphology and by changed levels of the Na-transport enzyme Na+/K+ATPase (Borek c r al., 1981 ; Borek, 1981a). The molecular effect of AP, which has diametrically

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225

opposed action on transformation depending on its temporal interaction with the cells being irradiated, does not alter DNA damage and replication. (Borek and Cleaver, 1981). Antipain does not alter SCE in a direction parallel to its effect on transformation (Geard et ul., 1982). Its effectiveness in the inhibition of carcinogen-induced chromosomal aberrations rather than SCE (Kinsella and Radman, 1978, 1980) suggests that perhaps aberrations are a more responsive assay to assess agents that modulate the action of carcinogens. 13. The surface expression of a variety of cellular features associated with the neoplastic state of X-ray-induced transformation can be identified within a week after exposure to radiation, thus allowing the identification of early transformants (Borek and Fenoglio, 1976). Such surface changes and other cytoskeletal modifications which may be associated could conceivably be intricately related to a cascade of events affecting the genetic apparatus (Puck, 1979). In chemically transformed cells a cytoskeletal element has been identified as a mutational product associated with transformation (Hamada et al., 1981). 14. The role of chromosomal fine structure changes and instability associated with radiation-induced transformation can only be inferred, though it is compelling. Diploid strains of hamster and human origin remain near diploid or diploid even after transformation has taken place and other phenotypic changes have been expressed (Borek et ul., 1977, 1980), but near diploid is perhaps sufficient for instability. No detailed studies are available on fine structure chromosomal changes directly associated with transformation of diploid cells. Inference has been suggested with SCE changes in the heteroploid IOT; cells (Nagasawa and Little, 1979). However, in a culture exposed to radiation where the frequency of transformation is low, how is one to know which of the cells will ultimately transform? Given the suggestion that in the 1OTi cells transformation may be unrelated to the direct actual dose of radiation administered (Kennedy et al., 1980), chromosomal changes are rather hard to interpret. With diploid cells one is hampered in such studied by the limited life span of the cells. Chromosomal assays of bona fide transformed cells do not reflect the course of events. 15. The suggestions of DNA damage repair and misrepair in the process of radiation transformation are again inferential. No DNA-repair enzyme has been identified in mammalian cells for ionizing radiation. Studies on UVinduced transformation in XP cells (Andrews and Borek, 1982; Maher et al., 1982) and on cells that are defective in DNA repair (Setlow et al., 1969; Cleaver, 1969) may bring us closer to the understanding of the exact role of DNA in transformation. 16. In the course of evaluating the role of repair in transformation it is worth stressing that the variety of studies described here clearly indicate that

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CARMIA BOREK

mechanisms associated with repair or cell survival probably differ from those responsible for cell transformation. The increased survival associated with splitting the X-ray doses is paralleled by higher transformation at low doses and lower transformation when the higher doses are split (Borek and Hall, 1974; Borek, 1979b; Miller et al., 1979). It has been suggested that subtransformation damage done by X rays is repaired more slowly than sublethal X-ray damage (Elkind and Han, 1979). Although X rays are mutagenic (Cox and Masson, 1976) and some of the cell systems lend themselves to concurrent studies on both transformation and mutagenesis (Landolph and Heidelberger, 1979; Huberman et al., 1976; Barrett et al., 1981), transformation cannot be equated with mutagenesis (Barret et al., 1981). The underlying mechanisms associated within radiation transformation are still unclear. Normal cells contain various inherent growth factors which can serve as internal promoters and may vary from one cell type to another (Roberts et a/., 1981). Chromosomal disturbances which may be amplified in diseased cells (Wolf el a / . , 1977) may be subtle in the course of transformation. Events such as imbalance (Bloch-Stacher and Sachs, 1976), disproportional DNA replication and gene amplification (Schimke et ul., 1981), or involvement of specific genes (Kundson, 1981; Klein, 1982) following exposure to radiation cannot be excluded from playing a part in oncogenesis. Thus there is much to learn, in order to synthesize the information from various biomedical disciplines with our knowledge of radiation biology (Rossi, 1964) and to develop new techniques. Some important goals for the future in studies on in virro transformation in radiation must be (1) to develop quantitative systems using differential epithelial cells derived from various organs. This would enable studies related to organ and cell susceptibility and to radiation carcinogenesis (Upton, 1964; Storer, 1975), to age-related sensitivity and to latency; (2) to refine the human cell-transformation assay in fibroblasts for quantitative assessment of radiation incidence in human cells, a task that is difficult because of the lower frequency of human cell transformation by radiation compared to rodent cells; (3) to expand studies on the progression of transformation; (4) to find markers that may be related to early stages of neoplastic conversion ; and (5) to develop human epithelial systems for transformation studies. It is of prime importance to assess how frequency, latency, expression, and mechanisms of transformation in human cells are related to those observed in the rodent systems. Most of our current data are from rodent cells and we are at a loss as to which of these cell systems matches most closely the human situation.

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In terms of studying the mechanisms underlying radiation-induced transformation, we are limited by techniques and knowledge of cellular and biochemical products that may be modified following radiation. New techniques to study gene transfection (Shilo and Weinberg, 1981 ; Robins et ul., 1981) could help us evaluate genetic mechanisms associated with transformation, as well as establish the cellular physiological milieu required to provide a competent state for neoplastic conversion.

ACKNOWLEDGMENTS This investigation was supported by Contract DE-AC02-78EV04733 from the Department of Energy and by Grant N o . CA 12536 to the Radiological Research Laboratory/Department of Radiology, and by Grant No. CA 13696 to the Cancer Center/Institute of Cancer Research. awarded by the National Cancer Institute, DHHS.

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Mhc RESTRICTION AND Ir GENES’

Jan Klein and Zoltan A. Nagy Max-Planck-lnstitut fur Biologie, Abtetlung Immungenettk,Tubingen. Federal Republic of Germany

B. Cytolytic Responses to Mhc Alloantigens . . . . . . . . . . . . .

B. Contact Sensitivity. VII. Is the T-cell Repertoire Individualized A. Statement of the Problem . . . . . . . . B. Testing a Hypothesis C. Tolling the Bells for t .................... D. On Joining the Head of a Man to the Body of a Horse: T-cell Receptor Models .............................................. VIII. Nature of Nonresponsiveness and the So-called Ir Genes. ....... A. Definition of Nonresponsiveness ....................................... B. Manifestation and Methods of Assaying Nonresponsiveness . . C. Evidence That a Failure in the Mhc-Restriction Mechanism Is Responsible for Nonresponsiv D. The Is Genes ............... F. Selection of Mhc Molecules for the Context of Antigen Recognition G. The Cause of Nonresponsiveness. ...................................... IX. The Parable of the Blind . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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In this article we discuss the most recent information in a form that we hope will be comprehensible to nonimmunologists. We believe that a review should not be an anonymous heap of haphazardly gathered data but rather a position paper that divulges the authors’ views on the data; it should be an opportunity for the authors to take a stand. One should not be afraid of overstating certain points if this is necessary to provoke consideration of alternative views to those held by the majority. 233 ADVANCES I N CANCER RESEARCH,

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Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in m y form reserved. ISBN 0-12-006637-8

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I. Mhc through a Keyhole

Some day, when electron microscopists have refined their scanning techniques to the degree that they can see individual molecules, the view that will open to them when they focus their lenses on the lymphocyte membrane will probably resemble a weed-overgrown wheat field. The lipid molecules of the membrane bilayer will look like wheat stalks growing at regular intervals and the scattering of proteins and glycoprotein molecules in this bilayer will appear as different kinds of weeds swaying in the wind among the wheat ears. The first two “weeds” that future electron microscopists will want to identify undoubtedly will be the molecules encoded in the genes of the major histocompatibility complex (Mhc) and the antigenrecognizing T-cell receptors. For although not quantitatively dominant in the lymphocyte membrane, the Mhc molecules and the receptors will arouse electron microscopists’ curiosity as the molecules that have been leading so many people around by their noses for so long. Like weeds, the Mhc molecules have their roots in the ground (the cytoplasm), sprout through the rows of stalks (the membrane), and flower above the spikes (the membrane surface). As far as is known, there are two kinds of Mhc molecules, prosaically referred to as class I and class 11. The class I molecules are glycoproteins with a molecular weight of some 44,000, which associate noncovalently with a small polypeptide, &-microglobulin, encoded in a gene that is not part of the Mhc. The class I1 molecules are dimers consisting of two noncovalently associated glycoprotein chains, ct (MW 32,000) and fl (MW 28,000), both encoded in the Mhc; there are reports that a third, invariant chain might somehow be associated with the ct and p chains, but whether the gene coding for.this chain is located in the Mhc is not known. In the mouse, the animal to which this article will be restricted, the Mhc is called H-2 and is known to consist of at least three class I loci (K, D,and L ) and at least four class ZZloci ( A , and A,, the products of which come together in the membrane as A,A, dimers or the A molecule; and E, and E,, the products of which form the membrane EaEpdimers or the E molecule-see Fig. 1 ; for reviews see Klein, 1975, 1979, 1981). Additional loci in the H-2

n

n

FIG.1. Genetic organization of the H-2 complex. Brackets indicate that the order of loci in these brackets is not known.

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complex have also been identified, the class membership of which is uncertain. At the telomeric (noncentromeric) end of H-2, there is a cluster of loci (Qa-1 through Qa-5 and Tla), the products of which resemble the class I molecules, but it is uncertain whether these molecules also have the same function as the K, D, and L molecules. In the region of the class ZI loci (the I1 region) there appear to be two other loci, J and C,the products of which have not yet been identified. It is unlikely that all the H-2 loci have already been identified. The work of Ivanyi and Demant (1981) suggests that there may be a cluster of thus far unidentified loci near the D locus, and a similar situation may also occur near the K locus. Work using recombinant DNA methods suggests that there may be some 15 or 20 class Zloci in the H-2 complex (Kvist et al., 1981 ; Steinmetz et af. 1981 ; Cami et af., 1981). If this estimate is correct, we have thus far identified about one-half of the existing class Zloci. No estimate has yet been made in regard to class IZloci but here the situation might be similar to that of the class Z loci. However, neither classical genetic methods nor the methods of molecular genetics support the postulate of a large number of H-2 loci made by the proponents of alien H-2 specificities(a claim that a cell homozygous for one H-2 haplotype may aberrantly express H-2 molecules normally encoded in a different H-2 haplotype; cf. Parmiani et af., 1979). In fact, DNA cloning suggests that most of the approximately 20 class Z loci are nonfunctional pseudogenes that cannot be expressed in the form of membrane proteins. On the H-2 map the K locus is separated from the D, L loci by a chromosomal segment estimated to be lo6 nucleotides long. It is within this segment that the class ZZ loci are located. The A, and A , loci are close to each other and may even be adjacent, whereas the E, and E, loci are separated by at least one ( J ) and possibly more loci. Some of the inbred strains and some wild mice carry a mutation (or mutations) that causes aberrant expression of the E molecules in the plasma membrane-and that has caused great confusion in the minds of immunogeneticists attempting to map various traits in the I1 region. The molecular consequences of this mutation are as follows (Jones et al., 1978, 1981). In nonmutant mice the E, and E, chains are synthesized and processed separately in the cytoplasm and only join each other at the time of their incorporation into the membrane. The Ea chain can apparently be inserted into the membrane without the fl chain, although in a much reduced quantity (as in H - Y P 5 ) The . fl chain, on the other hand, appears to be strictly dependent on the association with Ea for membrane integration. If E, fails to associate with E,, no E molecules can be detected on the cell surface-a situation occurring in H-2 haplotypes b and s. In these two haplotypes, the E, chains

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are found in the cytoplasm but not in the membrane. A somewhat different defect occurs in cells carrying the H-2 haplotypesfand q ; these cells do not seem to synthesize either the a or the fl chains. The defect in the H-2’ and H-2s haplotypes seems to be associated with the a chain; the /3 chain appears to be normal. Hence, when one genetically combines, say, the E i locus with an E, locus derived from a nondefective haplotype, say H-2d, the E j chains associate with the E,d chains and the E);E,d molecules are inserted into the membrane. One can combine the nondefective genes either in a trans configuration (as in the H-2b/H-2d heterozygote) or in a cis configuration (as in an E ; / E t recombinant strain carrying the two loci on the same chromosome). Genetically, the reconstitution of the standard phenotype (the expression of E molecules on the cell surface) appears to be a classic case of complementation, in which a defect is corrected by the combination of two genes. Because crossing over between the E, and E, loci is not excessively rare (remember that they are separated by other loci) and the E-affecting mutation is carried by almost one-half of the H-2 haplotypes present in the classical inbred strains, phenotypic complementation between E, and E, loci had repeatedly been observed before its molecular basis became known. However, with no knowledge of the molecular mechanisms, this complementation appeared an extremely puzzling phenomenon ; we shall return to this phenomenon in the section on Zr genes. Although, as already mentioned, Mhc molecules are not quantitatively well represented on the membrane, they are nevertheless immunologically highly “visible.” Using once more the metaphor of the wheat field: among the weeds in the field the Mhc molecules are the red poppies. This high “visibility” is reflected in that the Mhc can stimulate every conceivable form of immune response : the Mhc molecules easily induce antibody formation (when in 1936 Peter Gorer injected erythrocytes of one mouse strain into another strain, the first antibodies he obtained were specific for H-2 molecules, despite the fact that erythrocytes express only negligible amounts of class I molecules); they stimulate T cells in mixed lymphocyte culture as no other membrane molecules do ; they are superb targets in cell-mediated lymphocytotoxicity; they initiate such violent allograft rejection that Snell and his colleagues felt compelled to separate the Mhc molecules from other transplantation antigens and consider them the sole representatives of the “major” category, contrasting them with many minor histocompatibility antigens (Counce et al., 1956); and they elicit graft-versus-host reactions, immunological tolerance, delayed-type hypersensitivity reactions, and other responses. In fact, it was because they stimulate the immune system of an

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H-2-disparate individual so well that for almost a quarter of a century immunologists thought that this stimulation must be their natural function. Why are the H-2 molecules such good antigens in so many immunological assays? Part of the answer undoubtedly lies in their spectacular polymorphism. They occur in so many forms that, whenever one randomly chooses two individuals and they are not members of the same inbred strain or the same family, the chances are that these individuals will differ in at least some of their H-2 molecules. In contrast, molecules such as glyoxalase (Me0 et al., 1977) or superoxide dismutase (Szymura et al., 1981), which as far as we know are identical in virtually all mice, cannot be expected to stimulate immune reactions in mice. Another part of the answer is probably in the degree in which the primary structure of two H-2 molecules differs: not infrequently, of the 350 amino acids composing the class I polypeptide chain, some 40 or 50 amino acids are different when two strains are compared (Nathenson et al., 1981)-a staggering number not found in any other molecule with the possible exception of certain immunoglobulins (Strosberg, 1977). Hence, to the immune system of a mouse X, an H-2 molecule of a mouse Y must appear far more foreign than, say, a hemoglobin molecule, which differs from that of the host in only one amino acid. Still another part of the answer might be in the location of the H-2 molecules. All that we know today about specific immune responses suggests that they are initiated on the surface of the interacting cells. Hence a molecule that is an integral part of the membrane has certain advantages in participating in these reactions. Because, however, many cell surface molecules do not benefit from this advantage (as, for example, the minor histocompatibility antigens, which are notoriously poor stimulators of immunological responses), there must be more to antigenicity than having a conspicuous place on the membrane. Perhaps even all these factors together do not fully explain the extraordinary antigenicity of H-2 molecules. Perhaps there is something in the structure of these molecules themselves that makes them so highly antigenic perhaps in the way the amino acids are assembled in polypeptide chains, in the way the chains are folded, or in the manner in which the molecules take up their position on the cell surface. The ability to elicit strong allogeneic immune responses has cast a long shadow on H-2 studies, a shadow that until recently has obscured the true function of the H-2 complex. It is only now that we are slowly emerging from this shadow and are beginning to grasp the extent of Mhc involvement in immunity. What we see when we step out into the sunshine dazzles us. We are beginning to realize that we might have been wrong in thinking for over half a century that immunoglobulins are the most important molecules

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in immunity. It might be that, in fact, this epithet rightly belongs to the Mhc molecules.

II. How Was Mhc Restriction Discovered?

It has only been 10 years since the first hint about the true function of the H-2 complex was obtained, but the details of how the Mhc function was elucidated are already beginning to become somewhat fuzzy. It might be useful, therefore, to refresh our memories about how the discovery came about. In 1972, Kindred and Shreffler published a four-page communication in which they established, for the first time, the H-2 involvement in cooperation between T and B lymphocytes in antibody production. The authors inoculated athymic nu/nu H-2d/H-2dmice, known to be deficient in T cells, with thymocytes and, at the same time, with an antigen (sheep red blood cells or bacteriophage T4). After two additional weekly antigen injections, they tested the sera of the injected mice for the presence of specific antibodies 3 weeks after the first injection. They observed that when the thymocytes came from various H-2d strains, the injected mice responded well to the antigens; if the thymocyte donors were of some other H-2 haplotype, no response would be elicited. Because they did not find any evidence that the inoculated allogeneic thymocytes were rejected, they concluded that the cooperation between T and B cells was controlled by the H-2 complex. This short communication was the proverbial small stone that set off the avalanche-an avalanche which still echoes through the immunological valleys. The discovery was quickly confirmed and extended by Katz and his co-workers (1973a), and the genes responsible for the cooperation were mapped in the I1 region (Katz et al., 1973b). But then things took a wrong turn. The control was interpreted as a requirement for identity of H-2 loci on the cooperating cells, and because the possibility that this requirement might be the result of like-like interaction was quickly ruled out, the postulate was made that mysterious, unidentified interaction loci existed in the ZZ region of the H-2 complex. As has often happened in modern times, immunologists refused to consider the simplest solution to the problem (in this case, that the cooperation was controlled by already-known loci) and instead made fanciful hypotheses based on mostly irreproducible data which reflected reality very little. Controversy arose because only some investigators could find evidence for the identity requirement in T cell-B cell cooperation, and it seemed for a while that the studies would end up in a blind alley. Fortunately these speculations were soon brought down to earth by a series of discoveries based on a different system from that used in the T cell-B cell collaboration studies.

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The first communication in this series was published by Zinkernagel and Doherty in 1974. These investigators infected various mouse strains intracerebrally with lymphocytic choriomeningitis virus (LCMV), killed the infected mice 7 days later, and tested the spleen (T) cells for their ability to lyse ‘Cr-labeled, LCMV-infected H-2k fibroblasts (Zinkernagel and Doherty, 1974a). They observed that T cells from all H-2k strains lysed the H-2k targets, whereas T cells from other strains did not. On the surface, this observation was nothing but confirmation, in the cytolytic system, of what Kindred and Shreffler had described for antibody formation. However, this communication was followed a few months later by another in which Zinkernagel and Doherty presented their finding in a completely different light than others had considered the data on T cell-B cell collaboration before (Zinkernagel and Doherty, 1974b). Instead of following the mirage of hypothetical interacting structures, the authors put forward the idea that the precursors of the T cells responsible for target-cell lysis might have been stimulated by the simultaneous recognition of two molecules : an antigen encoded in the viral genome but expressed on the host cell surface and the host’s own Mhc molecule. Once “primed” by the particular combination of antigen plus Mhc, the progeny of this initial precursor cell then go on recognizing this particular combination of the two elements and lyse only target cells carrying it. If either the antigen or the Mhc molecules are changed, the primed cells are unable to recognize the new combination. To put it differently, if we designate the viral antigen by a capital letter and the Mhc molecule by a small letter, then T cells stimulated by A + a will react only with target cells carrying the A + a combination and not with cells carrying the A + b or B + a combination. It appeared as if the T cell had dual specificity, simultaneously recognizing self (Mhc molecule) and nonself (foreign antigen), or as if the specificity of the T cell for the foreign antigen were restricted by the Mhc molecules. Thus the concept of dual recognition or Mhc restriction was born. The concept was not completely new-it had been hinted at before by Lawrence (1959)-but its exposition by Zinkernagel and Doherty and its application to the recognition of viral antigens was highly original and provided the much needed alternative to the concept of interacting structures. Although more than a year after the publication of the second Zinkernagel-Doherty article, Doherty complained that most immunologists did not seem to grasp the significanceof the concept (P. Doherty, personal communication), the concept-applied to cytolytic T cellsgained acceptance relatively rapidly, mainly because its generality became apparent almost instantaneously. Independently of Zinkernagel and Doherty, Shearer and his colleagues (Shearer, 1974; Shearer et al., 1975) and Forman (1975) demonstrated that the specificity of T cells recognizing cellsurface molecules to which another, small molecule (hapten) had been

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artificially attached, was also restricted by Mhc molecules. Almost simultaneously with them, Simpson and her colleagues (Gordon et al., 1975) and Bevan (1975a,b) published evidence that similar restriction applied to the recognition of minor histocompatibility antigens. Because we shall often be referring to experiments using the hapten-modified proteins and minor H antigens as targets, we shall now briefly state the principle of these two experimental systems. In the former system lymphocytes are incubated with the hapten, most frequently 2,4,6-trinitrobenzene sulfonate (TNBS), for about 10 min, and then washed and cocultured with syngeneic, untreated T lymphocytes for about 5 days. TNBS is a nitrophenyl derivative with three nitro groups (NO,) attached to a benzene ring. Nitrophenyls bind to proteins by interacting with the eNH, group of lysine, the a-NH, group of the terminal amino acid, or the SH group of cysteine. When incubated with lymphocytes the trinitrophenyl (TNP) of the TNBS attaches nonselectively to the many cell-surface proteins, among others also to class I Mhc molecules. The TNP attachment modifies these proteins so they are no longer regarded as self by the syngeneic, untreated lymphocytes, and they stimulate these cells to differentiate into cytolytic effector cells. Which of the many modified proteins are the actual stimuli-whether the H-2 or some other molecules-is not known. Unclear also is what exactly the lymphocytes recognize on the modified protein, in addition to the TNP group. That more than the hapten alone is recognized is indicated by studies in which the TNP group was separated from the protein by a tripeptide spacer: such proteins are then no longer recognized by TNP-stimulated lymphocytes (Rehn et al., 1976). Lymphocyte activation by TNP-modified proteins is determined by harvesting the effector cells after 5 days of culture and exposing them to TNPmodified 'Cr-labeled, mitogen-stimulated blast cells : the effector cells lyse the labeled target blasts, thus releasing the intracellularly stored radioactive label. In all cases tested, the T cells recognize not only the modified protein but also the class I molecules present on the stimulatory cell. When target cells carrying a different hapten [even one so similar as dinitrophenyl, (DNP)] or a different H-2 molecule are presented to the differentiated effector cells, they are not lysed. In the second system, allogeneic spleen or lymph node cells are injected into mice; after several weeks the recipients' spleens are removed and the spleen cells restimulated in uitro with lymphocytes derived from the immunizing strain. After 4-5 days of coculture, the generated effector cells are tested for the lysis of mitogen-stimulated, 'Cr-labeled spleen cells. The responder-stimulator combinations are such that the two strains share the Mhc and differ in one or more minor histocompatibility loci. For example, BALB/c anti-BlO.D2 effector cells lyse B10.D2, B6.C-H-2d,and DBA/2 but

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not BlO.BR, B6, or BALB/c cells; that is, they lyse cells carrying the H-T‘ haplotype and minor H antigens controlled by genes present in the C57BL/10 (or B10) strain (Bevan, 1975a). The reaction is directed against these unidentified minor histocompatibility antigens, which are recognized by the T cells in the context of the H-2d molecules. One of the minor histocompatibility antigens has been studied in particularly great detail, namely the H-Y antigen controlled by a locus presumably located on the Y chromosome. In 1976-1977 it seemed logical to some of us to extend the Mhc-restriction concept to the interaction between macrophages and T cells and between T cells and B cells, and to explain the control of the immune response by the Mhc genes on this principle (Doherty and Zinkernagel, 1975; Klein, 1977). Yet this explanation encountered considerable resistance and, to a certain degree, continues to do so. The specter of Ig-like Mhc genes coding for the T-cell receptor was too deeply rooted in immunologists’ minds to be cut out by Occam’s razor. This residual distrust notwithstanding, there can now hardly be any doubt that the concept of Mhc restriction applies to all principal kinds of T cells : to those that develop the potential to kill other cells (cytolytic T cells) as well as to those that regulate other cells (helper and suppressor T cells). In recent years there have been attempts to give the phrase Mhc restriction an additional meaning, namely that encompassing the purported influence of the Mhc on the T-cell repertoire (see Section VII). In this article, however, we shall use the term only in its original meaning: the property of a T cell to recognize an antigen together with an Mhc molecule and, once stimulated by a particular antigen-Mhc combination, to be restimulated only by this combination. Some authors have objected to the phrase Mhc restriction as being incorrect (Mitchison, 1981). However, we find nothing wrong with it as long as its meaning is defined as above: a T cell, which otherwise could be specific for an antigen only, is restricted in its specificity by the Mhc. Here we shall also speak of “context of recognition” and of an antigen being recognized “in the context of Mhc,” by which we mean that the antigen is recognized together with an Mhc molecule. In the following two sections we will consider two principal kinds of T-cell response-cytolytic, in which the target of the response is killed, and regulatory, in which the action of the target cell is either amplified or suppressed. We will then deal with one type of response (delayed-type hypersensitivity or DTH) that apparently combines the elements of both the cytolytic and the regulatory responses, and we shall close this part of the article with a discussion on what determines whether a response will be of the cytolytic or the regulatory type. For each response, we will always consider two situations: a physiological one in which the cell recognizes a foreign antigen

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(nonself) and the responder’s own Mhc molecule (self); and a nonphysiological one in which the cell reacts against an Mhc molecule of another individual (against an Mhc alloantigen). The latter situation occurs only when it is artificially set up by an experimenter. For completeness, we shall also mention a third situation in which a T cell recognizes a foreign antigen and an allogeneic Mhc molecule. This situation is also an experimenter’s invention but an important one, as we shall see in Section VII. Ill. Mhc Restriction of Cytolytic Responses

A. RESPONSES TO FOREIGN ANTIGENS RECOGNIZED IN THE CONTEXT OF SELFMhc 1. The Question of the Antigen-Presenting Cell

One question we have to ask at the outset of every discussion on T-cell responses is: in what form does the T cell recognize the antigen? Antigens recognized by cytolytic T cells (Tc cells) include viral antigens lodged in the plasma membrane and encoded in an intracellularly located viral genome, minor H antigens, hapten-modified cell-surface proteins, and tumorspecific antigens (for references and reviews see Zinkernagel and Doherty, 1979)-in other words, molecules displayed on the cell surface. Nobody has ever succeeded in stimulating or inhibiting cytolytic lymphocytes with soluble antigens : Location in the plasma membrane, therefore, appears to be a sine qua non for the generation and function of Tc cells. The next question one has to ask is: if the antigen must be cell-bound, are there any special antigen-presenting cells that display the antigen for recognition by the cytolytic lymphocytes? This question can thus far be answered only partially and only in the case of the cytolytic lymphocyte reacting with hapten-modified cell-surface proteins. Pettinelli and her colleagues ( 1 979) have demonstrated that primary in vitro response to TNP-modified syngeneic cells can be obtained by the stimulation of the responder cells with T cells, B cells, or macrophages (all TNP-modified). There was no evidence in their experiments that the TNP-modified proteins had been reprocessed and redisplayed by some other cells (e.g., when the responders were stimulated by the TNP-modified T cells one could argue that the T-cell membrane fragments were picked up by macrophages and the TNP-modified proteins then presented to the T lymphocytes by these cells). Although macrophages were needed for an efficient response to occur, it made no difference whether they were syngeneic or allogeneic with the responding cells. Most likely the

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macrophages were not necessary to present the antigen but rather to perform some nonspecific function such as release of factors facilitating T-cell growth (see Section IV,A,2). One can therefore tentatively conclude that the modified proteins can be presented to Tc precursor cells by lymphocytes without the need for special processing before presentation. In the primary cytolytic responses to viral and minor histocompatibility antigens, the question of the antigen-presenting cell cannot be answered. In virtually all the tests used to study these responses, the cytolytic lymphocytes had been generated in uiuo (and then merely restimulated in uitro)-that is, under conditions in which it is almost impossible to find out on what cell the cytolytic lymphocyte precursors first encountered the antigen. Although in vitro primary responses to certain viral antigens have been reported (Blanden et al., 1977; Schrader and Edelman, 1977; Jung et al., 1978), they either were extremely weak or occurred under conditions in which natural sensitization against the virus in viuo could not be excluded. However, the fact that for in uitro restimulation and target-cell lysis one can use T-cell blasts, B-cell blasts, macrophages, or fibroblast lines suggests that in these responses, too, no special antigen-presenting cell is needed. 2. Mhc Restriction All the known cytolytic responses to foreign antigens are restricted by class Z Mhc loci (the K,D , and L loci in the mouse), which means that the Tc cells recognize the antigen in the context of the Mhc molecules on the antigen-presenting cells.’ There is only one report of cytolytic cells being generated against TNP-modified proteins and possibly restricted by class I1 molecules (Wagner et al., 1977), but even in this study the great majority of the Tc cells were restricted by class I molecules. Attempts to generate class 11-restricted Tc cells in other experimental systems have invariably failed (Z. A. Nagy, unpublished data). The simplest explanation for this failure of class I1 molecules to provide the context of recognition for Tc cells might seem to be that class I molecules themselves are somehow involved in the lytic process and that class I1 molecules are unable to mediate cell lysis. However, this explanation is invalidated by the finding that, as we shall see shortly, the recognition of allogeneic class I1 molecules by Tc cells does lead to cell lysis. Another possibility is that the cells presenting foreign antigens to Tc cells do not express class I1 molecules, but this explanation is again contradicted by experimental data demonstrating that both B lymphocytes In this article we use the term antigen-presenting cell (APC) to mean any cell-be it a lymphocyte or a macrophage-that can stimulate a T cell specifically. The presented antigen may or may not be processed before it is displayed on the membrane.

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and macrophages, which are rich in class I1 molecules, nevertheless elicit class I-restricted cytolytic responses (Pettinelli et al., 1979). We shall consider other explanations in Section VII.

B. CYTOLYTIC RESPONSES TO Mhc ALLOANTIGENS 1. The Antigen-Presenting Cell In these responses, the Mhc molecules themselves act as foreign antigens. In the usual experimental setup, spleen and lymph node cells of one strain (responding cells) are mixed with X-irradiated (or otherwise inactivated) spleen or lymph node cells of another strain (stimulating cells), and after 4 to 6 days of coculture in oitro the generated cytolytic cells are tested for their ability to lyse ‘Cr-labeled T-cell blasts, B-cell blasts, or appropriate tumor cells sharing at least some of the Mhc molecules with the stimulator strain. Removal of macrophages from the mixed culture usually precludes the generation of Tc cells (Wagner et al., 1972; MacDonald et al., 1973; Davidson, 1977), but the lesion can be corrected by the addition to the culture of macrophages of either stimulating- or responding-strain origin. Macrophages thus play an essential but nonspecific role in the induction of cytolytic cells-apparently in optimizing the tissue culture conditions. They can be replaced in this role by soluble factors (see also Section IV,A,2). 2 . Mhc Restriction Originally some investigators claimed that only class I alloantigens can serve as stimulators and targets of the cytolytic response. Our laboratory was the first to demonstrate that class 11 molecules can fulfill this function as well (Klein et ul., 1974), and a similar observation was made independently, a year later, by Wagner and his co-workers (1975). However, from the beginning, the idea of class I1 molecules stimulating cytolytic responses has been vehemently resisted, so that even today, when the experimental data can no longer be disputed, there is a tendency among some investigators to regard this stimulation as some kind of weird exception. Apparently some immunologists subscribe to Erasmus Darwin’s thesis that ”if the facts won’t fit in, why so much the worse for the facts is my feeling.” But this thesis has no place in immunology. Investigators who question the significance of class 11-specificcytolytic responses simply have not gotten the facts straight : they should once in a while leave their ivory towers of sterile speculations and dirty their hands in an experiment. They would then realize that such responses are not only reproducible but that they occur under normal

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conditions, in primary cultures, and are quite strong. Hence, any hypothesis of T-cell activation that does not account for activation of Tc cells by class I1 molecules must be considered incomplete. We have demonstrated that both the A and the E molecules stimulate allogeneic cell-mediated lymphocytotoxicity (CML), both primary and secondary (Klein et al., 1976, 1977; JuretiC et al., 1981a,b). There is no evidence that the CML against either class I or class I1 alloantigens requires the recognition of any other molecules than the targets of the response (Klein et al., 1977). In other words, the CML to allogeneic Mhc molecules is not restricted by the responder-type Mhc or any other molecule. However, this finding does not mean that the dual recognition observed in responses to other foreign antigens does not apply to the Mhc alloantigens. It may well be that when a T lymphocyte responds to an alloantigen, it recognizes by one receptor site determinants shared by the Mhc molecules of the responding and stimulating cells, and by the other site determinants distinguishing the Mhc molecules of these two molecules (Klein and Nagy, 1981).

IV. Mhc Restriction of Regulatory Responses

We divide the regulatory responses into helper-type responses, in which activated T lymphocytes (Th cells) amplify the effect of other cells (B cells or other T cells); and suppressor-type responses, in which the activated T lymphocytes (Ts cells) inhibit the action of other cells (most often of other T cells).

A. HELPER-TYPE RESPONSES The two situations in which the amplifying effect of Th cells has clearly been demonstrated are the B-cell (antibody) responses and the cytolytic responses. 1. B-Cell Responses Although a B cell can bind an antigen, it is not activated by this binding unless it receives another signal (a kick in the butt, so to speak) ; this other signal is usually provided by a Th cell. But a Th cell cannot be activated by a free antigen either: the antigen must be presented to it by another cell, together with this cell's Mhc molecules. a. Identity of' the Antigen-Presenting Cell. We are still much in the dark about the identity of the cell that presents the antigen to the Th lymphocyte. Most would agree that this antigen-presenting cell (APC) is some kind of

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macrophage, but the definition of a macrophage is so imprecise that this identification does not help much. There are probably many functional subsets of macrophages, and probably only some of these have the ability to present antigen to T cells. There is now some evidence identifying the antigen-presenting macrophages with the so-called dendritic cells, which probably also go under the alias of Langerhans cells. Dendritic cells (Steinman and Nussenzweig, 1980) are characterized by long cytoplasmic processes which continually elongate, retract, and reorient themselves and so give the cell a variety of shapes. Both the cytoplasm and the large, contorted refractile nucleus stain weakly with basic dyes. In some respects the cells resemble splenic reticular cells, yet they differ from these in that they do not synthesize collagen. Dentritic cells are present in the spleen, where they comprise about 1% of the total nucleated cell population, and in smaller numbers in lymph nodes and Peyer’s patches. They are absent from bone marrow, thymus, liver, intestine, and the peritoneal cavity, but all these areas contain other cell types that might be related to dendritic cells. The dendritic cells carry class I1 molecules on their surface and stimulate strongly mixed lymphocyte reaction. Their antigen-presenting function is not at all certain, nor is it established that they are the only kind of cell capable of presenting antigen to Th lymphocytes. However, these APCs, whatever their identity, may be the only cells that can truly process the antigen and present it to the Th cell in this processed form-they feed the Th cell with a spoon, so to speak. What does this processing consist of? Here, we are sorry to say, our ignorance in this area of immunological research is almost scandalous. Here is the very beginning of an immune response and all one can state about it is that the antigen is somehow picked up by the macrophage-like APC, taken up, broken down into fragments inside the cell, and the fragments then “displayed’ on the cell surface. But precisely how all these events happenand some immunologists even doubt that they do happen-is not known. This ignorance is made worse by the sneaking feeling that most of the time until now we might have been asking the wrong cells for the answers. Those who do not find satisfaction in a one-sentence summary should consult other articles in which they will find the same conclusions stated in as many words (Unanue, 1972). In the rest of the text we shall refer to the cells presenting antigen to Th lymphocytes simply as macrophages. b. Interaction between Th Cells and Macrophages. The Th cell that directly communicates with the macrophage bears the Lyt-1 ‘Lyt-2- phenotype and has a great propensity to proliferate when stimulated. Because of their proliferative potential the Th cells are relatively easily assayed for in culture, usually by measuring the incorporation of [3H]thymidine into the DNA of DNA-synthesizing cells.3

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Their need to recognize Mhc molecules together with the antigen was first demonstrated by Rosenthal and Shevach in 1973. These authors immunized inbred guinea pigs with purified protein derivative (PPD) and then cocultured T cells from these immune animals with macrophages that had been previously exposed to PPD for 60 min in culture (in laboratory jargon, the macrophage had been “pulsed” with the antigen). After some 48 to 72 hr of culture, they measured T-cell activation by adding [3H]thymidine to the culture for a few hours and determining how much of the radioactive label was incorporated into the DNA of the proliferating cells. They discovered that when the antigen-pulsed macrophages came from the same strain as the T cells, the proliferative response was high; when they came from an Mhcdisparate strain the response was only slightly higher than in the control containing macrophages that were not exposed to the antigen. Undoubtedly influenced by the claims of Mhc-identity requirement in T cell-B cell collaboration, the authors concluded from this observation that Mhc identity was also necessary for collaboration between T cells and macrophages. However, one should note that the experiment was complicated by the presence of alloreactive cells : when cells from Mhc-disparate strains are mixed, the T cells are stimulated by Mhc alloantigens carried by the macrophages and this reaction could mask any response to the immunizing antigen. But the most important point about the experiment is that the T cells were primed, that is, preselected in terms of Mhc recognition. They were stimulated by the antigen (PPD) in the context of one set of Mhc molecules, and then asked to respond to the same antigen presented to them in the context of another set of Mhc molecules, and this they, of course, refused to do. Hence, the experiment does not prove that only Mhc-compatible T cells and macrophages collaborate; it does prove, however, that the recognition of the antigen on macrophages by Th cells occurs in the context of the macrophages’ Mhc molecules. The question that remains is, can Mhc-incompatible macrophages and T cells collaborate? A number of researchers have tried to answer this question and most of them reached the same wrong conclusion as Rosenthal and Shevach (Shevach and Rosenthal, 1973 ; Erb and Feldman, 1975 ;Yano et ul., 1977; McDougal and Cort, 1978, to name just a few). They all used T cells primed in one context and asked them to collaborate with macrophages in another Mhc context, and, when no collaboration occurred, the authors

This statement is not meant to imply that other T lymphocytes do not proliferate upon encountering an antigen. However, the majority of cells proliferating in response to macrophagepresented antigen probably are Th lymphocytes (see also Section IV,A,3).

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thought they had proved the Mhc-compatibility requirement for Tmacrophage interaction. There were, however, a few exceptions. For example, Kapp and her colleagues (1 973) separated spleen cells from unprimed mice into glassadherent (macrophages) and nonadherent (T B cells) fractions, combined fractions obtained from different strains, added antigen [ e g , sheep red blood cells (SRBC)] to the culture, and after incubation for 5 days determined antibody response in terms of the number of plaque-forming cells (PFC). So the assay required not only macrophage-T cell collaboration but also T cell-B cell collaboration to occur for positive results, but by varying the source of macrophages the authors were able to draw conclusions about macrophage-T cell collaboration exclusively. They discovered that in terms of anti-SRBC response it made no difference whether they mixed B10 (H-2") macrophages with SJL (H-2') T cells or with B10 T cells, and vice versa. This experiment should have allowed one to conclude that Mhcdisparate T cells and macrophages do collaborate, but unfortunately it is again complicated by the fact that H-2" T cells can respond to H-2¯ophages, so the response the authors observed could have been a mixed lymphocyte reaction against H-2s rather than against H-2" plus antigen. Thus the experiment must be considered inconclusive with respect t o the collaboration of T cells with allogeneic macrophages. A similar objection can be raised against all other experiments in which Mhc-disparate T cells and macrophages were found to collaborate (e.g., Cosenza and Leserman, 1972; Katz and Unanue, 1973). All these experiments are therefore similarly inconclusive. Yet the question of the histocompatibility requirement in T-macrophage interaction can now be answered. in our laboratory we have obtained data clearly demonstrating that collaboration between T cells and allogeneic macrophages does occur, apparently to the same degree as between syngeneic cells (Ishii et ul., 1981a). To avoid the complication of the mixed lymphocyte reaction against alloantigens, y e used a trick discovered originally by Djordjevic and Szybalski in 1960. The trick is based on the observation that incorporation of 5-bromo-2-deoxyuridine (BUdR), an analog of thymidine, into the DNA of dividing cells causes DNA damage when the analog breaks down upon irradiation with ultraviolet light. The damage eventually leads to cell death and thus to selective killing of dividing cells (Zoschke and Bach, 1970). We thus stimulated unprimedT lymphocytes of the H-2' type with macrophages of the H-2Ytype, after 3 days removed dividing cells (responding to the H-2Yalloantigens) by the BUdR and light treatment, and then stimulated the remaining cells with H-2Ymacrophages and antigen using either the synthetic polypeptide p ~ l y ( G l u ~ ~ A l a(GA) ")

+

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or the natural protein lactate dehydrogenase (LDH,). After 3 days of culture, we let the stimulated T cells "rest" for 4 days (culturing them in the absence of antigen), then restimulated them for another 3 days with H-2Y macrophages pulsed with the same antigen, and measured their proliferative response by [ 3H]thymidine incorporation. In all the allogeneic combinations tested-and there were some 40 of them-the T cells responded normally to the antigen in the context of the allogeneic Mhc. The response was specific for the antigen (exposure to another antigen failed to restimulate the cells) and for the allogeneic Mhc molecules (the same antigen failed to restimulate the T cells if presented by either H-2' or H-2" macrophages). The conclusion we draw from this experiment is, therefore, that Th cells can uecogniie antigen in the context of'allogeneic Mhc molecules and Mhc compatibility is not required for successjul collaboration between T cells and macrophages. However, in both the syngeneic and the allogeneic T-macrophage interactions the context for antigen recognition is provided by the class I1 Mhc molecules ; interaction restricted by class 1 molecules has never been observed. c. Interaction between Th Cells and B Lymphocytes. Once activated by the antigen presented to them by the macrophage in the context of the macrophage Mhc, the Th cells then presumably help to activate B lymphocytes that have bound the same antigen via their immunoglobulin receptor. The nature of this help is not known and we refer the reader interested in the various explanations proposed for the mechanism of T-B collaboration to other reviews (Katz, 1977). Here we shall attempt to answer only one question : is this interaction Mhc restricted? Many experiments have searched for the answer to this question, most of them based on the following protocol. One set of animals is immunized with a high-molecular-weight antigen-a carrier, for example, keyhole limpet hemocyanin (KLH)whereas another set is immunized with a low-molecular-weight substance (a hapten) conjugated to another carrier, for example, T N P coupled to bovine serum albumin. Animals of the first set are then used as a source of carrier-primed T cells and animals of the second set as a source of haptenprimed B cells. The T and B cells are then mixed, either in uitro or injected into an irradiated, third set of animals, restimulated with the hapten coupled to the T-cell priming carrier (e.g., TNP-KLH), and the T-B collaboration is measured by the production of hapten-specific antibodies by the primed B cells. By varying the source of T cells or B cells one can determine whether the Mhc of the collaborating cells matters or not. Unfortunately the answers to this question are divided: some authors find that T cells primed in the context of one Mhc do not collaborate with B cells primed in the context of another Mhc (Katz et al., 1973c; Sprent, 1978a,b; Swierkosz et al., 1978;

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Yamashita and Shevach, 1978; Kappler and Marrack, 1977), whereas others have come to just the opposite conclusion, namely that T-B collaboration is not Mhc restricted (McDougal and Cort, 1978; Erb ef al., 1979; Singer et al., 1979, 1980; Shih er al., 1980). As an example of the evidence against Mhc restriction, we shall describe one of the experiments done by Singer and his colleagues (1979). These authors prepared purified populations of macrophages, T cells, and B cells, mixed them in uitro, cultured them for several days with TNP-KLH, and then determined the number of PFCs formed. They observed that the mixture of BI0.A macrophages, (BIO.A x B 1O)F, T cells, and B I0 B cells gave as good a response as a mixture of B 10 macrophages, F, T cells, and B10 B cells. So it seemed that F, T cells activated by KLH in the context of H-2" were able to help B cells expressing the H-2" haplotype-in other words, that they were unrestricted. One point should, however, be noted about the experiments interpreted as arguing against the Mhc restriction of T-B collaboration, namely that in virtually all of them the B cell-stimulating hapten was TNP. This hapten was once considered a polyclonal activator and, although this view was later revised, one must keep in mind that T N P has a tendency for "nonspecific" B-cell activation. A solution to the controversy has recently been proposed by Singer and his colleagues (198 l), based on their demonstration that there are two B-cell types, one carrying the Lyb-5 antigenic marker, and the other Lyb-5 negative. The Lyb-5- cell, probably a precursor of the Lyb-5+ cell, interacts with Th cells in an Mhc-restricted fashion, whereas the Lyb-5' cell appears to be unrestricted. The authors propose that the different conditions used in the various experiments designed to test T-B collaboration favor either the Lyb-5- or the Lyb-5' cells and that, depending on the predominance of one or the other cell type, one does or does not observe Mhc restriction. Singer and his colleagues refer to the interaction of the Th cells with the Lyb-5+ B cells as Mhc-unrestricted, but because this interaction might be mediated by nonspecific soluble factors and the T-cell receptor may not participate in it, such a designation is not quite correct. This Th cell may be (and probably is) Mhc-restricted in the same way as other Th cells, but by its ability to turn on B cells nonspecifically gives the impression of being independent of Mhc in its action. So, when one separates the grain from the chaff, one finds no convincing evidence in the immunological literature for the existence of Th cells that can recognize a carrier without simultaneously recognizing Mhc molecules. The reported instances of seemingly unrestricted T-B collaboration must be attributed either to faulty experimental designs or to poor execution and control of experiments. The molecules that restrict the specificity of T-B collaboration are encoded in class I/ loci; no evidence has been found of class I-restricted collaboration.

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However, B cells can be nonspecifically triggered by T cells stimulated by class I alloantigens. The first example of such a triggering was provided by Kettman and his colleagues (1977), who cocultured stimulating and responding cells differing at the H-2K locus only, and demonstrated that the supernatant of such cultures, when added to a culture of T cell-depleted splenocytes, helped B lymphocytes to produce SRBC-specific antibody. This observation was later confirmed and extended by Panfili and Dutton (1978) and Swain and Panfili (1979). The T cells responding to class I alloantigenic differences and producing this T cell-replacing factor have the Lyt- 1 'Lyt-2' phenotype (Swain and Panfili, 1979). d. Idiotype-SpeciJ7:c Th Cells. Recent studies in several laboratories (Woodland and Cantor, 1978; Hetzelberger and Eichmann, 1978; Eichmann et al., 1978; Adorini et al., 1979) have demonstrated that in addition to the antigen-(carrier-) specific Th cell (AgTh), there also exists a lymphocyte with specificity for the idiotype of the immunoglobulin receptor on the B-cell surface (i.e., for the unique antigenic determinant of the V region in the immunoglobulin molecule). The idiotype-specific cell (IdTh) can be removed from a suspension of carrier-primed T cells by incubating this suspension in a dish the surface of which is coated with immunoglobulins expressing the particular idiotype. The suspension from which the idiotype-specific Th cells have been removed then poorly amplifies the B-cell response to a hapten ; addition of the removed adherent cells to the suspension restores its original effect on B cells. Because removal of the carrier-specific Th cells similarly decreases the effectiveness of the Th-cell population, the authors of these experiments have concluded that both the carrier-specific Th and the idiotype-specific Th cells are needed for efficient antibody production by B cells. Whether the two Th cells act on a B cell in the same or in different stages of differentiation is not clear. Unclear also is what the IdTh cell recognizes in addition to the immunoglobulin idiotype. Because the cell is not retained on antigen-coated dishes, one assumes that it is not antigen (carrier) specific ; and because the cell is retained by the immunoglobulin molecules alone, one concludes that it is not Mhc restricted. However, this latter point cannot be considered as definitely established, for the depletion by the immunoglobulin-coated dishes is rather inefficient, suggesting that the cells may bind best to idiotype determinants on the cell surface, in association with other membrane components (Woodland and Cantor, 1978). One must also ask how the IdTh cell is triggered if it does not recognize the immunizing antigen. The usual answer to this question is that the triggering occurs as a result of a network perturbation, but this answer is so vague that it almost amounts to no answer at all. Some immunologists consider the discovery of the ldTh cell to be the first

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peek into the world of complex cellular interactions characterizing the immune system. There may be, so they argue, other Th cells which communicate not only with B cells but also with one another. There may be Th cells, as postulated by Sercarcz and Metzger (1980), for every step in the B-cell-differentiation pathway, and other cells locked into complex network (Jerne, 1974) or circuit (Herzenberg et al., 1980) interactions. The recent discovery of immunoglobulin allotype-specific Th cells (Snodgrass e f al., 1981a,b) can be interpreted as pointing in this direction. On the other hand, there is virtually no evidence that the ldTh cells play a role in physiological T-B collaboration in v i m Until such evidence is provided, one can argue that the reported requirement for cooperative action of AgTh and ldTh cells on B cells is an experimental artifact. One may also want to consider the possibility that the ldTh cells are in fact specific for another carrier mimicked by the idiotype of a given B-cell receptor. If so, they could be expected t o be Mhc restricted in the same manner as other Th cells are, but this restriction could be obscured when the cells cross-react with the idiotype. Obviously, there are still many questions that require an answer.

2. Th Cells Participating in Cytolytic Responses An amplification of cytolytic responses by noncytolytic T cells has been observed in all the cytolytic systems thus far studied: cytolytic responses to viral antigens, minor histocompatibility antigens, modified membrane proteins, and cell-mediated lympholysis (CML) against alloantigens. a. Cells Helping Tc Lymphocytes to Respond to Vim1 und Other Antigens. One of the first experiments presumably demonstrating Th-cell involvement in CML to non-Mhc antigens was done by Zinkernagel and his colleagues (1978b). These authors observed that Tc cells carrying class I and class I1 genes of type u, and differentiating in an environment that purportedly presents the antigen (vaccinia virus) in the context of type a class 1 and type b class I1 molecules, did not kill target cells infected with vaccinia virus and carrying type a class I and IZ genes. In this situation, so the authors argued, the Tc and Th lymphocytes were activated by the viral antigen presented to them in the context of type u class 1 and type b class I1 molecules, respectively. However, the activated Th cells could not help the Tc cells, because to do so they would have to recognize type b class I1 molecules on them and these the T cells did not possess. So, because of lack of help, the Tc cells were ineffective in lysing the appropriate Mhc-matched target cells. A similar observation was also made by von Boehmer and Haas (1979) on Tc cells directed against the H-Y antigen. Bennink and Doherty (1978), on the other hand, using vaccinia virus as did Zinkernagel and his colleagues,

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were able t o generate virus-specific, class I-restricted Tc cells in class 11 gene-incompatible animals. The discrepancy in the data remains unexplained ; it would be easy to blame it on some differences in the experimental protocol but such an explanation would still not answer the question whether the interaction between the Tc and Th cells is Mhc restricted. At any rate, the experiments described by Zinkernagel and his colleagues and by von Boehmer and Haas provide only indirect evidence for the involvement of Th cells in cytolytic responses to non-Mhc antigens. Direct evidence for such involvement has been provided for Tc responses against modified membrane proteins by Finberg et al. (1979), Cooley and SchmittVerhulst (1979), and Wagner et al. (1980a), and for response to viruses by Ashman and Mullbacher (1979), Pfizenmaier et al. ( 1 980), and Wagner et ul. (1980a). As an example of these studies we shall consider the work of Finberg and his colleagues. These authors injected TNP-modified syngeneic cells subcutaneously into mice, 7 days later X-irradiated (600 R) spleen cells from these mice, and added them to a culture of syngeneic-responding and TNPconjugated stimulating cells. After 5 days of culture, they compared the lytic ability of the generated Tc cells with cytolytic cells generated in cultures without the irradiated cells. They observed that the inclusion of the primed cells augmented the in uitro response 4 to 10 times. The augmentation was antigen specific because the added cells enhanced anti-TNP but not, for example, anti-DNP responses. The authors concluded that spleens of in uiuo primed mice contain a population of radioresistant, antigen-specific Th cells capable of augmenting Tc-cell responses. Although these studies provided convincing evidence for the existence of the Th cells in cytolytic responses, they still did not answer the question of whether the Th cells and the Tc cells interacted in an Mhc-restricted manner. In fact, this question cannot be answered by mixing syngeneic cells; and mixing allogeneic or semiallogeneic (F, ) cells (Cooley and Schmitt-Verhulst, 1979) opens the experiment to the criticism that the mixed lymphocyte reaction (MLR) to the alloantigens may obscure the Th-Tc cell interaction. To avoid the allogeneic effect, Wagner and his colleagues (1980a) separated the Tc from the Th cells by a cell-impermeable membrane. They demonstrated that, in such a culture, interaction between the two cell types still occurs, apparently mediated by a soluble factor referred to as T-cell growth factor (TCGF) or interleukin-2 (IL-2). Furthermore, although Th cells stimulated by one antigen Mhc molecule combination can be restimulated only by this combination, the produced factor helps almost any Tc cell so that, for example, Th cells stimulated by TNP-modified proteins help not only TNP- but also virus- or alloantigen-stimulated Tc cells, and vice versa. The generated factor is also unrestricted in its action : when produced by Th cells of one Mhc type, it acts on Tc cells of not only the same but also of other

+

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types. The function of IL-2 is apparently to provide a second nonspecific signal to the Tc cell, the first signal being the recognition of an antigen in the context of a class I Mhc molecule. With these data in mind, one can explain the lack of help in the experiment of Zinkernagel et a / . (1978b) and von Boehmer and Haas (1979) by postulating, for example, that the Th cells failed to recognize the viral or the H-Y antigen in the context of the particular class 11 molecule (i.e., they were genetic nonresponders). Alternatively, in the experiment of Bennink and Doherty (1978), the observed help might have been provided via nonspecific stimulation of the Th cells (IL-2 can also be generated in a mixed culture of allogeneic cells or in a culture of mitogen-stimulated lymphocytes; mitogenic substances may be present in some cultures and not in others). It is possible, of course, that in addition to the nonspecific factor, Tc and Th cells may also interact in a specific and Mhc-restricted way. b. Helper Efects in Allogeneic C M L . Eijsvoogel and his colleagues reported in 1972 (paper published in 1973) that disparity at an Mhc locus distinct from the then known class 1 loci was a prerequisite for in oitro induction o f CML in human mixed-lymphocyte culture. A similar observation was also made using mouse mixed-lymphocyte culture, and the prerequisite loci were identified to be of the class /I type (Schendel ef ul., 1973; Alter and Bach, 1974; Schendel and Bach, 1974; Cantor and Boyse, 1975b; Wagner et al., 1975). The conclusion reached from these studies was that the Tc precursors recognize class I alloantigens but their differentiation into effector cells depends on Th-cell stimulation by class I1 alloantigens : the Tc cells cannot be stimulated by class I1 alloantigens and the Th cells cannot be stimulated by class I antigens. This conclusion is contradicted by the finding that in H-2-mutant, class 11-identical strain combinations lymphocytes directed against class I alloantigens can be stimulated (Berke and Amos, 1973; Forman and Klein, 1974, and others), and that class I1 alloantigens can also stimulate cytolytic lymphocyte responses (Klein et al., 1974; Wagner et al., 1975; Juretik et al., 1981a,b). The most likely explanation of these contradictory data is similar to that offered for the antiviral cytolytic response: the Th cells stimulated by allogeneic class 11 molecules in mixed lymphocyte culture produce IL-2, which then acts on the precursors of the cytolytic lymphocytes and facilitates their differentiation into effector cells (Hardt et ul., 1981). This facilitation is totally nonspecific in that there is no correlation between the class I1 molecules stimulating the Th cells and the class I molecules stimulating the Tc precursors. In fact, any stimulus that turns on Th cells, not just class I1 alloantigens, may lead to 1L-2 production. The culture may or may not contain such nonspecific stimulators of Th cells depending on the constitution of the medium and, in particular, on the

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supplements added to the medium. If it does, a low level of "background" Th-cell stimulation occurs, producing enough IL-2 for the Tc precursors to differentiate into effector cells; if it does not, the effector cells may be generated only when an additional stimulus in the form of class I1 disparity is included in the experimental conditions. Thus it may happen that some laboratories get class I-specific CML in the absence of class I1 disparity and others do not. 3. T-cell Prolijeration in Response to Alloantigen Stimulation In a culture of responding and inactivated allogeneic stimulating cells, a large proportion (1 - 10%) of the stimulating small lymphocytes transform into blasts and proliferate. Maximal proliferation in this primary MLR occurs between 4 and 6 days after cell mixing, at which time it can be measured by the degree of [3H]thymidine incorporation into the DNA of the growing and dividing cells. The stimulus for lymphocyte activation in MLR is provided by alloantigens encoded in the Mhc and, to a lesser degree, by alloantigens encoded in the Mls locus located on chromosome 1 (the Mhc is located on chromosome 17). Of the Mhc alloantigens, the strongest stimulators of MLR are the class I1 A and E molecules (Bach et al., 1972; Meo et al., 1973); somewhat weaker stimuli are delivered by class I K and D alloantigens (Klein et al., 1972). Stimulation of MLR by J (Okuda et al., 1978) and C (Okuda and David, 1978) alloantigens has also been reported. The proliferating cells are T lymphocytes which bear the Lyt-1 'Lyt-2phenotype when stimulated by class I1 alloantigens (Cantor and Boyse, 1975a) and the Lyt-1'Lyt-2' phenotype when stimulated by class I alloantigens (Wettstein et af., 1978). The true identity of these cells in terms of receptor specificity is not known; however, because it is inconceivable that an organism would possess a large number of cells that it never uses, the best bet is that the original specificity of the alloreactive lymphocytes was for Mhcs + X and that they cross-react with MhcnS(s = self, ns = nonself, X = foreign antigen, not encoded in the Mhc). Supporting this crossreactivity hypothesis, first formulated by Zaleski and Klein (1978), are recent findings on the specificity of cloned T-cell lines. For example, Sredni and Schwartz (1980) obtained a T-cell clone from a culture of BI0.A (H-2") cells stimulated by dinitrophenyl ovalbumin (DNP-OVA) and demonstrated that this clone can be restimulated not only by H-2" + DNP-OVA but also by H-2s cells (i.e., by allogeneic cells without the antigen). We postulate, therefore, that the majority of T cells proliferating in MLR are Th lymphocytes which the organism usually uses for the recognition of soluble foreign antigens presented in the context of Mhc'.

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The debate about the identity of the stimulating cells does not seem to have come to an end as yet. For a while it seemed as if lymphocytes (mainly B cells), macrophages, and epidermal cells were able to initiate the MLR (for a review, see Klein, 1975), but now it appears that in all the past experiments the cell preparations were contaminated by macrophages and that it was this cell-or more precisely the class II-positive dendritic cell-that was responsible for the activation of the responding lymphocytes (Steinman and Witmer, 1978; Ahmann et a/., 1979; Minami ef al., 1980; Minami and Shreffler, 1981). Only in secondary MLR, where the requirements for T-cell activation are somewhat relaxed, can lymphocytes assume the role of stimulating cells (Minami and Shreffler, 1981). In primary MLR, class 11positive dendritic cells are needed even in cases where the stimulus is provided by class I alloantigens (Minami and Shreffler, 1981). The dependence on antigen presentation by dendritic cells further supports the notion that the cells responding in MLR are mostly Th lymphocytes. As in the case of the cytolytic responses, in anti-Mhc MLR there is no evidence that the responding T cell recognizes anything in addition to the Mhc molecule. Mixed lymphocyte reaction against the Mls alloantigens has been reported to be Mhc-restricted by some authors (Peck et ul., 1977), but others dispute this finding (Molnar-Kimber and Sprent, 1980) and are probably right.

B. THESUPPRESSOR T-CELLPATHWAYS Reviewing the literature on suppressor T lymphocytes (Ts cells) is like peering through the plastic window of someone else's running washing machine: all one can see is a whirl of amorphous linen with only here and there the glimpse of a sock or a pair of underpants. It takes great power of imagination to visualize how the outfit, put together at the end of the washing cycle, will look. Although Ts cells participate in most if not all responses, one is normally not aware of their action, because their physiological function is not to abolish an immune response but rather to forestall an excessive one that could harm the individual. One learns about their presence in two principal situations: when a genetic lesion occurs leading to an exaggerated suppression or when the experimental conditions are manipulated so as to favor suppression over help. The former situation occurs with certain antigens in individuals carrying certain haplotypes. For example, mice of the H-2" haplotype are unresponsive to the synthetic polypeptide poly(Cilu60Ala30Tyr'0) (GAT), not because they lack Th cells capable of recognizing this antigen but because the antigen in these mice preferentially stimulates Ts cells (Kapp et a/., 1975). However, rather than relying on a genetic defect, one can obtain exaggerated suppression at will by artificially steering the response in this

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direction. The conditions favoring suppression are determined empirically ; they are created, for example, by the administration of a large antigen dose without adjuvant over a period of several weeks (a protocol used, for example, by Tada and his colleagues; reviewed by Tada and Okumura, 1979) or stimulation of isolated Ts cells in vitro (a protocol used by Gershon, Cantor, and their colleagues ; reviewed by Gershon, 1980). Despite considerable efforts to elucidate the Mhc’s role in the activation of Ts cells, this role is still poorly understood. Much of the information obtained thus far comes from the study of soluble factors (TsF) released by most Ts cells upon stimulation. Although none of these factors has been isolated in a pure form and fully characterized biochemically, and although much confusion exists as to which factor is which and which factor is doing what, “factorology” is slowly gaining respectability. The main reason behind the interest in factors is, of course, that it is much easier to work with supernatants than with cells. One passes a supernatant through a column of beads coated with an antigen, and if the suppressor activity is removed by this procedure, one concludes that the factor is antigen specific. One adds to the supernatant antibodies specific for an idiotype or antibodies specific for the J molecule, and when the removal of the resulting precipitate abolishes the activity of the supernatant one concludes that the factor carries the idiotypic or the J determinants. In still another experiment, if the supernatant obtained from cells of one H-2 haplotype or of one Igh-V type is capable of acting on cells carrying another H-2 haplotype or another Igh-V type, one concludes that the factor is not Mhc or Igh-V restricted. One must bear in mind, however, that it is still not certain whether the known suppressor factors are the physiological messengers of signals between cells. At least some of them might be experimental artifacts-for example, products of cell shedding-whereas in physiological situations the cells may primarily rely on direct cell-to-cell interaction. Unfortunately, we know next to nothing about these interactions. At least three Mhc molecules have been implicated in the suppressor pathway: J, E, and C . 1. J-Restricted Ts Cells?

Several experimental systems have been claimed to involve the J molecule. Although the details of the individual systems vary considerably, the systems have enough in common to allow the construction of a “consensus pathway” (Fig. 2 ;for review and references, see Germain and Benacerraf, 1981) and to assume that what individual investigators see in their different systems are fragments of this common pathway. Examples of responses using at least part of this common pathway are antibody responses to GAT (Kapp et al.,

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/

SUPPRESSOR PATH WAY

I

I

CONTRASUPPRESSOR PATH WAY

!

Ag

Ag

I I I I

I

0

I I I

I I

Ly t - It 2;

It, Id ,'

I

Lyt - I-:2 I+,&Ag? I

I

(Y Ag

I

I

I

-0 Suppiession

Tcs3

Lyt-1'2:

I+,(rAg?, UId?

Lyt-1-2:

J'nJ-, (YAg

Lyt-1'2-,

I*

L y t -1+2+, I+

FIG.2. Putative consensus pathway of suppression and contrasuppression.

1974),p o l y ( G 1 ~ ~ ~ T(GT; y r ~DebrC ~ ) et al., 1975a,b), or KLH (Takemori and Tada, 1975); contact sensitivity to DNP (Claman et al., 1977); and delayedtype hypersensitivity to 4-hydroxy-3-nitrophenyl (NP; Bach et al., 1978). The pathway consists of a minimum of three interacting cells designated Ts,, Ts,, and Ts,. The cells differ in their Lyt phenotype, expression or nonexpression of the J molecule, and ability to recognize the antigen, idiotype, and an Mhc molecule. The cells interact in such a way that the first one in the sequence induces the second cell, which then in turn induces the third cell. The interaction occurs via soluble suppressor factors (TsF, ,TsF,, and TsF,); whether it can also occur by direct cell-to-cell interaction is uncertain. The first known cell in the pathway is Ts,, also referred to as Ts inducer or Tsi because it is involved in the induction phase of the immune response. The cell is presumably activated by an antigen, hence it must have an antigenspecific receptor. This receptor appears to express the same idiotype as antibodies specific for the particular antigen. The cell has the Lyt-1 'Lyt-2phenotype and in this respect resembles the Th cell; however, it differs from the latter in that it expresses the J alloantigen. Upon induction, the Ts, cell releases a factor, TsF,, which then acts on the Ts, cell. The factor is able to

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bind antigen specifically in some systems (e.g., GAT), but appears to act independently of the antigen in other systems ( e g , NP). The Ts, cell, also referred to as the Ts acceptor, has the Lyt-1 +Lyt-2+J+phenotype; whether it carries antigen-specific or idiotype-specific receptors is uncertain. Unclear also is what the Ts, cell does: some investigators believe that it produces a factor, TsF,, which then acts on the Ts, cell, whereas others favor the notion that Ts, itself differentiates into Ts,. The last cell in the pathway, Ts, or the Ts effector, has the Lyt-1 -Lyt-2+ phenotype; opinions differ as to whether it is J + or J-, and whether it carries an antigen-specific receptor. The mature Ts, cell produces a factor, TsF,, which then mediates suppression by presumably acting on Th cells. The nature of the suppressory mechanism is not known. Gershon and his collagues (1981) have recently described a “contrasuppressor pathway” which appears to be a mirror image of the suppressor pathway (Fig. 2). It, too, consists of three interacting cells which we designate Tcs,, Tcs,, and Tcs,. The phenotypes of these cells and their presumed modes of interaction are depicted in Fig. 2. The major difference between the suppressor and contrasuppressor pathways is that the ultimate target of the former is the Th and of the latter the Ts cell (hence the designation contrusuppressor pathway). However, the authors have not excluded the possibility that the contrasuppressor pathway is nothing but the suppressor pathway running in reverse or that it represents a mere feedback mechanism somewhere along the suppressor pathway. There are two principal unanswered questions regarding the consensus suppressor pathway: What is the nature of the antigen-presenting cell and what do the individual Ts cells recognize? Concerning the first question, the only thing that is known is that suppression is relatively independent of macrophages. This conclusion is supported by at least three observations. First, depletion of macrophages in primary in uitro responses leads preferentially to induction of Ts cells, whereas Th cells are preferentially induced in cultures containing macrophages (Ishizaka and Adachi, 1976; Feldman and Kontiainen, 1976; Pierres and Germain, 1978). Second, Ts cells, like B cells, recognize conformational antigenic determinants whereas Th cells recognize determinants that are more directly related to the primary structure of the antigen. This observation might be related to the need for antigen processing and presentation by macrophages in the induction of Th but not of Ts cells (Endres and Grey, 1980). Third, Ts cells can react with conventional, unprocessed antigen in the absence of macrophages and they can bind antigens directly in uifro;Th cells, in contrast, need to interact with antigen on macrophages (Okumura et al., 1977; Taniguchi and Miller, 1977). I t could be, therefore, that Ts cells recognize either soluble antigen (like B cells)

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or antigen presented by other lymphocytes (like Tc cells). However, one should emphasize that none of these observations excludes the possibility of macrophage-like cells presenting the antigen to Ts cells. As far as the receptor specificity of the Ts cells is concerned, none of the evidence presented thus far is worth two straws. This or that Ts cell has been claimed to be antigen-specific or idiotype-specific and J-restricted or Igh-V restricted, but the claims either are not convincing or are contradicted by other data. In particular, the role of the J molecule in restricting the specificity of Ts cells is nothing but a myth. We have referred to the Ts cells in the title of this section as possibly J-restricted but, in fact, there is not one indisputable set of data supporting this designation. The J locus is poorly defined to begin with, and all one can say about the J alloantigen is that it is present on some Ts cells and Ts factors. However, this observation does not prove that the J molecule functions as a restriction element in T-cell recognition. We are not saying that the J molecule does not have this function, but we question the evidence for it and we consider the problem of the Ts receptor's specificity as completely open in virtually all the suppressor systems, with the exception of the one described in the following section. 2 . E-Restricted Ts Cells? Suppressor T cells restricted by E molecules were recently discovered in our laboratory during a study of the genetically controlled response to lactate dehydrogenase B (LDH,) and to allotypic determinants of the mouse IgG molecules (Baxevanis et al., 1981a,b). In strains expressing the A, chains controlled by alleles 6, d,,f, k, p , q, r, s, u, u, w13, and w16, immunization with LDH, (or IgG) results in priming of Th cells, as evidenced by the proliferation of these cells upon restimulation in uitro. In strains expressing the A, chain controlled by other alleles (e.g., j or w l 7 ) , LDH,specific Th cells cannot be activated, and so we conclude that responsiveness or nonresponsiveness to LDH, is controlled by the A , locus of the H-2 complex. This conclusion is further supported by the observation that the restimulation of the LDH,-specific Th cells in uitro can be blocked by addition to the culture of monoclonal antibodies reacting with the A molecule of the antigen-presenting cell. Apparently the basis for the control is that the antigen (LDH,) is presented to Th cells by macrophage-like cells in the context of the A molecule and that only some combinations of LDH, and A molecules find Th cells with corresponding receptors in the immunized recipients (see Section VIII,C,3). However, there is yet another level of restriction imposed on the responsiveness to LDH, : in strains carrying the E: allele no anti-LDH, response occurs even if the immunized mouse carries responder-type AB allele. In

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these strains, the immunization activates Ts cells which recognize LDH, in the context of the EjE; molecule (the tl chains seem to play no specific role in the activation), and these cells then suppress proliferation of the LDH,specific Th cells. The addition to the culture of E-specific monoclonal antibodies prevents activation of the Ts cells and allows responsiveness to occur. Similarly, repeated inoculation of E-specific antibodies into LDH,immunized mice also blocks the generation of Ts cells and permits responsiveness even in Ej-bearing strains. Another allele, El;, also promotes the generation of Ts cells but not as strongly as the Ej allele: in strains carrying the Ef; allele (and the respondertype A , allele), partial responsiveness occurs that can be augmented by blocking the E molecule on the antigen-presenting cell with E-specific monoclonal antibody. All other Ep alleles tested have no effect on the response to LDH, or IgG. The effector of this suppressor pathway is an Lyt-1 +Lyt-2+T lymphocyte. For generation of the effector Ts cell, the antigen must be presented to the precursor cell by antigen-presenting cells in the context of the EkE; or E;Ei molecules. Thus both the precursor and the effector Ts cells are restricted in their specificity by the E, locus. How the E,-restricted effector Ts cells interact with Ap-restricted Th cells is not clear; the simplest explanation would be that this interaction is of a nonspecific type-for example, via a nonspecific factor-but evidence for such a factor is still lacking. Recent studies (Baxevanis et al., 1981b) indicate that the E-restricted suppressor pathway contains at least one other cell which is needed for the maturation of the precursor cell into the Ts effector. This Ts-inducer cell is of the Lyt-l' Lyt-2- type and is apparently not restricted by either the E, or the A, molecules; whether it is at all Mhc restricted remains to be determined. The Ts inducer is apparently antigen specific and it somehow (it is not clear how) interacts with the precursor of the Ts effector. We could thus far find no evidence for the involvement of other cells in this suppressor pathway. The relationship between the E-restricted and other suppressor pathways is not known. Because, however, the context of antigen recognition by Ts cells has not been determined in these other systems, the possibility exists that at least some of them are also E-restricted. 3. C-Restricted Ts Cells?

The C locus was originally defined serologically as a locus coding for the class I1 determinant Ia.6, defined by an H-2h2anti-H-2h4serum (David et al., 1974). However, later Cullen and her co-workers (1980) were unable to reproduce the Ia.6-specific antibody and they suggested that the C locus may not exist. But in the most recent report Sandrin and McKenzie (1981) were

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able to produce another H-2h2 anti-H-2h4 serum and so the locus may exist after all. The claim of C-restricted Ts cells has been made by Rich and Rich (1976). These authors injected allogeneic lymphocytes into the footpads of recipient mice; a few days later they obtained spleen cells from the immunized recipients and cocultured these cells with mitomycin C-treated donor-type spleen cells. Twenty-four hours later they harvested the supernatants from these cultures and demonstrated that a factor in these supernatants suppressed MLR. The interaction of the suppressor factor with the MLRresponding cells was restricted by the C locus (Rich and Rich, 1976), and the factor could be removed from the supernatant by anti-C sera (Rich et al., 1979). The postulate of C-locus participation in suppression also explains some of the complexities of the genetic control of Ts-cell generation upon stimulation with GT (reviewed by Dorf, 1978). However, the evidence for the existence of a C locus and its involvement in immune suppression must be considered as tentative at best. Before we close this section on Ts cells, we should mention one other point, namely that these cells-like the Tc and Th cells-can also be activated by Mhc alloantigens of both the class I and class I1 type (see, for example Brondz et al., 1980; Swain and Dutton, 1977a,b). This activation requires the specific recognition of the Mhc alloantigen but apparently of no other molecule; we presume that, again, as in the case of Tc and Th cells, the allogeneically stimulated Ts cell mistakes the alloantigen for the Mhcs + X molecular complex.

V. Mhc Restriction of DTH and CS Responses

A. DELAYED-TYPE HYPERSENSITIVITY To induce delayed-type hypersensitivity (DTH) experimentally one injects the antigen (e.g., fowl gammaglobulin, KLH, GAT, SRBC, or a virus) emulsified in complete Freund’s adjuvant subcutaneously into a mouse or a guinea pig; 2 to 3 weeks later one injects the same antigen in physiological salt solution intradermally at a suitable site on the body (the animal’s back in the guinea pig, the footpad or ear pinna in the mouse). The second injection induces an inflammatory reaction in the skin at the injection site and, as a result, the skin thickens within 48 hr. The inflammatory response is initiated by T cells sensitized to the antigen by the first injection: upon a second encounter with the antigen the sensitized T cells in the skin release lymphokines, which then attract mononuclear inflammatory cells to the injection site (for a review see Turk, 1980). For sensitization to occur, the

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263

antigen must first be processed by macrophages and then presented to the T lymphocytes; for restimulation the antigen is apparently picked up by the Langerhans cells in the skin and presented to the sensitized T cells by them. T o determine the Mhc restriction of the DTH reaction, the sensitized lymphocytes are adoptively transferred into nonimmunized animals, which are then challenged intradermally with the antigen. By varying the strains used one can determine what genetic homology between the first and the second host is needed for the transfer to be successful. When studies of this kind were carried out, different answers were obtained for different antigens. In their original study, Miller and his co-workers (1975, 1976) observed that class II locus homology was sufficient to transfer DTH to protein antigens; they concluded, therefore, that the DTH reaction was restricted by class I1 Mhc molecules (see also Weinberger et al., 1979; Leung et al., 1980). In contrast, Zinkernagel (1976) found DTH to lymphocytic choriomeningitis virus to be class I locus restricted; Nash et al. (1981) found DTH to herpes simplex virus to be class I I locus restricted, and Weiner et al. (1980) found DTH to a reovirus to be class landclass II locus restricted. The resolution of these contradictory findings is probably in the proposal of Miller and his co-workers (1976) that there are at least two distinct T cells capable of inducing inflammatory reactions, one resembling the Th cell and the other resembling the Tc cell. Depending on the nature of the antigen used, one preferentially stimulates the former or the latter cells. Because the Th-like cells recognize soluble, macrophage-processed antigens in the context of class I1 molecules, the DTH induction by soluble proteins is class I1 locus restricted. On the other hand, because the Tc-like cells recognize viral, unprocessed antigens in the context of class I molecules, DTH to viral antigens is class I locus restricted. Antigens that stimulate both the Th-like and the Tc-like cells then appear as both class I and class I I locus restricted. Recent evidence indicates that the Th cells sensitized during the DTH reaction to soluble proteins not only are Th-like, but in fact are identical with the Th cells. In addition to having the same Lyt-1 'Lyt-2- phenotype (Miller et al., 1975, 1976) and displaying the same Mhc restriction of their specificity (Vadas et al., 1977), the cells have the same antigen specificity. Bianchi and his colleagues (1981) obtained several Th clones by repeated stimulation with antigens and then demonstrated that the clones induced DTH in nonimmune animals upon a challenge with the same antigen. Similar evidence is not yet available for the Tc-like cells but it would be surprising if these, too, did not prove identical with true Tc cells. As in all other T-cell responses, the Mhc molecules can also become the target of a DTH reaction when recipient-donor combinations differing at Mhc loci are used for the induction. To induce DTH to alloantigens, Smith and Miller (1979) sensitized cyclophosphamide-treated recipient mice (the

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JAN KLEIN AND ZOLTAN A. NAGY

cyclophosphamide treatment delays rejection of the injected cells) by subcutaneously inoculating them with viable allogeneic spleen cells. Six days after the sensitization, they injected the same recipients intradermally with viable spleen cells from the same donor and measured immigration of inflammatory cells into the skin 24 hr later. They found that both class I and class I1 Mhc alloantigens as well as minor H antigens induced DTH (of the class I alloantigens tested those encoded in the K locus induced DTH whereas those encoded in the D locus did not, but this difference was probably a result of allelic rather than locus variation in the strength of the response). There was no evidence of DTH to Mhc alloantigens being restricted by any other genes, whereas the adoptive transfer of DTH to minor H antigens was restricted by the Mhc loci: no evidence of macrophage processing could be obtained in the responses to Mhc alloantigens; in contrast, the presentation of minor H antigens apparently occurred with the help of macrophage-like antigen-presenting cells.

B. CONTACT SENSITIVITY To induce contact sensitivity (CS), the animal’s skin is painted with a solution of a hapten salt such as 2,4-dinitrofluorobenzene (DNFB; Miller et al., 1976) or 4-hydroxy-3-nitrophenyl acetyl-O-succinimide (NP-O-Su; Sunday et al., 1980) on days 0 and 1. Five days later one challenges the skin with a dilute solution of the same hapten and measures skin swelling on day 6. Contact sensitivity is a form of DTH believed to be induced as follows. As the hapten salt permeates through the skin, the hapten couples to cell-surface proteins of the different cells, including the Langerhans cells. The haptenmodified self proteins are then recognized together with Mhc molecules of the Langerhans cells, by the receptors on T cells, and the latter are thereby stimulated. The rest of the reaction then follows a similar course to that occurring in DTH. The Mhc restriction of the CS reaction can be determined, like that of the DTH reaction, by an adoptive transfer of the sensitized T cells into a naive host, Such determination reveals that CS can be transferred between strains of mice sharing either class I or class II genes (Miller et al., 1976; Sunday et al., 1980). Sunday and Dorf (1981) observed that the NP-specific footpad swelling can be elicited with N P conjugated to a heterologous protein carrier only if the NP-O-Su-primed donor cells are compatible with the recipient at the class II loci, and that, in contrast, N P coupled directly to syngeneic cells can elicit CS in a class I-restricted manner. These studies indicate that in the CS most of the time the challenge activates two kinds of T cells: Th-like cells recognizing the modified proteins in the context of class I1 molecules and

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Tc-like cells specific for the antigen and class I molecules. The identity of these two cell types with Th and Tc cells remains to be established but appears likely.

VI. The Puzzle of the Class I and Class I1 Gene Dichotomy

Table I summarizes the current knowledge about the specificity of T cells as discussed in the preceding section. Two generalizations can be made based on the information contained in this table. The first is that whenever T lymphocytes recognize an antigen, they also recognize self Mhc molecules simultaneously. There seem to be two exceptions to this rule: the recognition of Mhc alloantigens and the recognition of receptor idiotypes. In the former case, T cells recognize-as far as one can tell-the foreign Mhc molecule alone, without simultaneous recognition of self Mhc molecules. However, we would like to argue that the foreign Mhc molecule also bears elements of selfness (i.e., determinants shared by the self and the nonself Mhc molecules), so there is no need to involve self Mhc molecules in this recognition (Klein and Nagy, 1981). The T cell may treat the shared portions of the Mhc molecule as self and the nonshared portions as nonself. At any rate, the recognition of alloantigens is an artifact that has no physiological significance. As for the second exception, the lack of Mhc restriction in the recognition of Igh-V idiotypes is not as firmly established as it is sometimes presented or as one would like it to be. However, even if it is real, one important question still remains, namely, what is the physiological function of the idiotype-recognizing T cells? Are they part of a vast network of interacting cells or are they inconsequential artifacts, just like the alloreactive T cells? There is no definitive answer to this question, but even if the idiotypespecific T cells had an important physiological function in vivo, their apparent lack of Mhc restriction would not necessarily mean that the cells are truly independent of the Mhc in their recognition specificity. We would like to argue, for example, that there is considerable, if not total, overlap between antigenic determinants on Mhc molecules and idiotypic determinants (Klein, 1980) and would interpret the recognition of idiotypes by T cells as another case of “mistaken identity” : to a T cell the idiotypic determinant of a receptor molecule may look like an Mhc determinant. If so, then recognition of an idiotypic determinant in one situation could mean recognition of an Mhc determinant in another situation. We believe, therefore, that the two exceptions may in the end turn out to confirm the rule and that once all the principal facts have been obtained we may reach the conclusion that all T cells are Mhc-restricted in their specificity.

TABLE I PROPEXTIESOF T CELLSINVOLVED

IN THE VARIOUSFORMS OF I m w

RESPONSE'

Lyt phenotype'

Type of response Cytolytic (Tc)

Receptor specificity*

Immature cell

Mature cell

pKTY.1

1 +2+

1+2+

Self I1 + X Allo I + X Allo I1 + X

Help for B cells (AgW

Antigen processing required

Targetd

LY

No

Ly, M, other

I

APCd

Mhc restriction' of effector-target interaction

1-2+ Existence uncertain Existence demonstrated but properties not determined Not demonstrated

I Allo I I

1+2+

1-2+

LY

No

Ly, M, other

I

Allo I1

1+2+

1-2+

Ly?

No?

B

I1

1+2-

M

Yes

B

I1

Not demonstrated ? 1 +2-

M

Yes

B?

II?

? ?

M? M?

No? No?

B B

None? None?

?

M

Yes

Tc

I1

Self I

+X

pEiGT-1 Allo I + X Allo 11 X

+

Not demonstrated but may exist ?

Help for B cells (IdTh)

Id + ? Allotype

Help for Tc cells

Self I + X Self11 + X

Not demonstrated

+X +X

Not demonstrated Not demonstrated

Allo I Allo I1

+?

? ? ?

unspecrnea proirreraring cells

qy,mmr

I\ 0

i~one

Ly, M?

No?

None

1 +2-

None?

No?

TSZ

1+2+ 1+2+ 1+2+ 1+2+ Properties not determined

1-2' 1-2' 1-2+ 1-2+

Ts, Ts, TM

Ts3 Th? Th

?

No No Yes No?

1+2+? 1+2+? 1+2+ 1+2+?

1 +2-

? ?

? ?

Tcs, Tcs, Tcs, Th(Ts, ?)

1 2

AIIO I

V M L d

?

Self I

+X

I Self11 + x +X A110 I1 + X Allo I

Not demonstrated but likely to exist

]

1+2+?

X

+?

+

Id J ? X + ? II(E) X A110 I

+

A110 II(C) Contrasuppression induction Ucsi ) Tcs, Tcs,

X +? X + ? Id + J? X + ?

1+2-

Not demonstrated but likely to exist Not demonstrated but likely to exist

A110 I A110 I1 Suppression induction (Ts,)

1+2-

1 +2?

1+21+ 2 + 1 +2-

,

Tcs Tcs,

No No

None?

?

None None J? None?

For references see text. Self I or 11, individual's own class I or class I1 Mhc molecules; allo, allogenic Mhc molecules; X, foreign antigen; Id, idiotype of the receptor. Boxed symbols indicate dominant types of recognition in the particular type of response. 1 + 2 + ,Lyt-1 +Lyt-2+; 1-2+ represents Lyt-l-Lyt-2+ ; etc. APC, antigen-presenting cell; Ly, lymphocyte; M, macrophage. ' I, class 1 Mhc; 11, class I1 Mhc molecule.

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JAN KLEIN AND ZOLTAN A. NAGY

The second generalization following from Table 1 is that the two principal types of T-cell response, cytolytic and regulatory, are neatly distinguished also according to the context of recognition, with cytolytic T cells recognizing foreign antigens mostly in the context of class 1 molecules and with regulatory T cells being restricted in their specificity mostly by class I1 molecules. Although there are important exceptions to this rule, the dichotomy is very clear and very puzzling. What could be the reason for this correlation between the type of response and the context of recognition? There are three levels at which the foundation for this correlation could be laid down : the level of the antigen, the level of the antigen-presenting cell, and the level of’ the T cell itself. If the cause of the correlation were at the antigen level, one would have to argue that there are two groups of antigen and that antigens of one group have some properties in common but at the same time differ from all the antigens in the second group. When one considers the extreme diversity of antigens recognized by T cells, from synthetic polypeptides to the various natural proteins, one is tempted to dismiss the presumed two groups as a physical impossibility. However; there are in fact, two clearly differentiated categories of antigen : soluble and cell-bound. Indeed the restriction specificity follows rather precisely the division line between these two groups of antigens. T-cell responses to soluble antigens are almost exclusively restricted by class I1 Mhc molecules, whereas the cytolytic responses to cell-bound antigens occur almost exclusively in the context of class I Mhc molecules. So it may very well be that the context of recognition is determined by the physical form of the antigen-cellular versus acellular. However, one can also argue that really it is not so much the form of the antigen that decides the context of recognition but the cell that presents the antigen to the T cell-which brings us to the second level of decision making. There are unfortunately still many uncertainties about the identity of the APC in the different types of T-cell response; however, a certain trend can clearly be seen in Table I. In general, antigens presented to T cells by lymphocytes or other cells that are not capable of antigen processing are recognized in the context of class I molecules, whereas antigens processed by the subset of class 11-positive macrophages or by some special antigenprocessing cell are, in general, recognized in the context of class I1 Mhc molecules. Furthermore, because it is the soluble antigen that is most likely to be picked up by a macrophage for processing, whereas cell-bound antigens normally escape processing, there is a clear correlation between the form of the antigen, the need for processing, the type of antigen-presenting cell, and the Mhc context of recognition.

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The third possibility is that the decision of whether an antigen will be recognized in the context of class I or class 11 molecules is made by the T cells themselves. As Table I shows, there is also certain correlation between the context of recognition and the responding T-cell subset as defined by the Lyt markers: Lyt-1 -Lyt-2+ cells are predominantly of the cytolytic or the suppressor-effector type, whereas Lyt-1 ' Lyt-2- lymphocytes tend to become helper cells. One could, therefore, argue that the correlation arises because the Lyt-1 'Lyt-2- cells have different receptor repertoires from the Lyt-1 -Lyt-2+ cells, the former having a repertoire centered around class I1 molecules and the latter around class I Mhc molecules. However, this argument is clumsy for several reasons. First, it presupposes that the context of recognition is determined somatically : at some point during ontogeny something must decide that the Lyt-1 'Lyt-2- cells will recognize an antigen in the context of class 11 molecules and the Lyt-1-Lyt-2' cells will recognize an antigen in the context of class I molecules. Although several immunologists believe that such somatic influencing of the T-cell repertoire does occur, in the following section we will argue againsr this possibility. Second, if both the Lyt-l' Lyt-2- and the Lyt-1 -Lyt-2+ lymphocytes begin, as some immunologists believe they d o (Cantor and Boyse, 1977), as Lyt-1 'Lyt-2' cells, then the decision about the context of recognition must be made when they are still in an immature precursor form because this, presumably, is the time that they encounter the antigen. How then does the link between the Mhc context of recognition and the Lyt phenotype, as well as the function of the maturing T cell, arise? Third, the repertoire hypothesis does not explain the observed correlation between the Mhc context of recognition and the form of antigen (soluble versus cell-bound) as well as the correlation between the context of recognition and the apparent need for antigen processing. We propose that the most likely explanation of the recognition dichotomy is a combination of the first and the second possibilities just listed. We believe that the antigen repertoires of class 1- and class 11-restricted T cells are largely overlapping and that the decision about the context of recognition is made during antigen presentation. A T cell cannot recognize a soluble, acellular antigen; such an antigen must first be processed and displayed on the membrane of a macrophage-like cell. For unknown reasons, antigen-processing macrophages insert the processed antigen almost always next to class I1 molecules so only T cells capable of recognizing the antigen in the context of class I1 molecules are activated by this form of antigen. The interaction between the T cell and the macrophage also determines the direction of the T cells' differentiation-that is, it steers it toward helper function and hence toward inactivation of the Lyt-2 gene. In contrast, antigens already present in the membrane are recognized by T cells directly without

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JAN KLElN AND ZOLTAN A. NAGY

the need for the macrophage-mediated, antigen-processing step, and the interaction between the T cell and the antigen-presenting cell steers the former in its differentiation course toward the inactivation of the Lyt-1 gene and the expression of the lytic functions. How critical the inactivation of the Lyt genes is for the effector function of the mature T cells is unclear. There are, however, T cells in which neither the Lyt-2 nor the Lyt-2 gene has been inactivated, yet some can function as cytolytic effectors whereas others become involved in helper functions. Whether these Lyt-1 Lyt-2’ effectors are the maturation products of another differentiation pathway or whether the immature cell (in terms of Lyt gene expression) can already perform effector functions is unclear. Unanswered also are all the questions pertaining to the expression of effector potential in the T cells that do not show any obvious sign of Mhc restriction. +

VII. Is the T-cell Repertoire Individualized?

A. STATEMENT OF THE PROBLEM The conclusion reached in the preceding section was that most if not all T lymphocytes recognize foreign antigens (X) together with cell-surface Mhc molecules. However, the Mhc is polymorphic so the class I and class 11 molecules of different individuals differ, usually substantially so (Klein and Figueroa, 1981). One can therefore ask: If an individual A recognizes antigen X in the context of its own Mhca molecules, can it recognize the antigen only in this context or is it in principle able to recognize the antigen also in the context of Mhcb carried by an individual B? Moreoever, if so, does an individual A ever use the Mhcb-restricted T cells! The main reason such a question comes up at all is a paper published in 1971 by Niels Jerne. In this paper Jerne put forward a hypothesis according to which the lymphocyte receptor repertoires of different individuals are different. According to Jerne, when the progenitors of lymphocytes enter a primary lymphoid organ-the thymus in the case of the T cells-, they display their receptors and match them against this organ’s Mhc molecules. Because, according to Jerne, the germ line-encoded receptors are specific for the Mhc molecules of a given species, all receptors recognizing the individual’s own Mhc molecules find a matching partner and are retained in the thymus, whereas receptors for Mhc molecules of other individuals (i-e.,for Mhc alloantigens) are allowed to emigrate into the periphery. The thymus thus acts, in Jerne’s words, as “a column through which entering stem cells, whilst proliferating, descend toward the exit with a velocity that is restricted by their degree of stickiness to the histocompatibility antigens in the column”

Mhc

RESTRICTION AND

Ir

GENES

27 1

(Jerne, 1971). However, the proliferating cells mutate their receptors so that their descendants are less and less able to interact with the Mhc molecules until they too eventually become “nonsticky” and leave the thymus. But now they have gained, via the mutational process, the ability to recognize foreign antigens. Knowing what we know today, we would say the cells retain some affinity for Mhc and gain affinity for foreign antigens and so become Mhc restricted. In 1971 Jerne already had a reputation as a sort of immunological prophet and so immunologists immediately listened to what he said. The implications of this speculation are clear-cut : the thymus is manipulating the T-cell receptor repertoire. So when Mhc restriction was discovered 3 years later and the discovery had been digested, it did not seem at all odd to ask whether T cells that come out of the thymus can recognize foreign antigens only in the context of the individual’s own Mhc molecules. But it may be that attempts to answer this question have actually steered immunology into another cul-de-sac.

B. TESTING A HYPOTHESIS One can only admire the ingenuity and sophistication with which immunologists have attempted to answer this question. The result, however, appears all the more frustrating: not only have different methods provided different answers, but the same methods have also often provided different answers in the hands of different investigators. Here we shall first consider the various methods and the answers provided by them, and then give our own answers. 1. Radiation Chimeras The use of chimeras-animals composed of tissues derived from different individuals-seemed to be the most straightforward approach to testing the T-cell repertoire. Alas, the “simple” model quickly turned the ground under immunologists’ feet into a slushy swamp with Jack-o’-lanterns and all. If chimeras proved anything, it is that with their help one can prove everything. Yet we cannot simply abandon the mess; eventually someone will have to clear it up. To produce a radiation chimera one irradiates a mouse or some other animal with a dose high enough to kill all lymphoid (hemopoietic) cells and stem stells, but not so high as to cause serious damage to most other somatic tissues; but achieving this goal amounts, as the Czechs say, to feeding the wolf while saving the goat. Many of the contradictions in chimeric research probably stem from the failure to achieve this ideal situation. The irradiated

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JAN KLElN AND ZOLTAN A. NAGY

individual most then be inoculated with new stem cells-contained in the bone marrow or fetal liver cell suspension-from another individual. The donor-cell inoculum must not contain T lymphocytes, otherwise they would attack and kill the host. It took immunologists some time to realize this fact and so most of the early experiments on radiation chimeras in which T cells were not removed are suspect (reviewed by Zinkernagel and Doherty, 1979). In 1975 von Boehmer and his colleagues introduced the practice of treating the bone marrow suspension, before it is inoculated into the irradiated host, with anti-T lymphocyte serum and complement to kill mature T cells (von Boehmer et al., 1975a,b), and this practice is now used routinely in all chimera studies. The injected bone marrow or fetal liver suspension contains stem cells from which all blood and lymphoid cells differentiate. During this differentiation the donor-derived lymphocytes become tolerant of the host, but this tolerance is quite fragile: the lymphocytes do not acutely attack the host but the tolerance easily breaks down, particularly when the cells are taken out of the host (Ada rt af.,1981). It is thus important to realize that, although the chimera may appear outwardly helathy, it is not a normal animal. Depending on the type of genetic disparity between the donor and the host and the type of tissue used for transplantation, we shall recognize five types of radiation chimeras: F, + PA, thymus chimeras, PA + F, , fully allogeneic chimeras, and PA PB + F, . a . F, +PA Chimeras. In this instance one irradiates adult mice of one parental strain PAwith a supralethal dose of X or y rays (for mice the dose is 925 to 1000 rad, depending on the strain), and a few hours later injects into these animals F, T-cell-depleted bone marrow or fetal liver cells. When fully established, such chimeras contain only lymphocytes of donor origin as determined by H-2 typing or chromosome analysis. Then, 6 to 24 weeks after the irradiation, one immunizes the chimeras by inoculating them with a virus or with cells carrying minor histocompatibility antigens, cells modified with a hapten or tumor cells. If immunizing with a virus, one kills the chimeras 6 days after the inoculation, incubated the spleen cells for a few hours with virus-infected, 'Cr-labeled target cells (fibroblasts or macrophages), and then measures the release of radioactivity from these cells. This type of assay tests the presence of immune Tc cells, but the technique can also be modified to test for the presence of Th cells by asking these cells to provide help for antibody production by B cells. Doing this kind of experiment and testing the effector cells on various targets leads invariably to the finding that the spleen cells kill (or provide help to) F, and PA but not PB cells. The experiment has been repeated may times measuring Tc responses to unidentified minor H alloantigens (Bevan, 1977), Tc responses to various viruses (Zinkernagel et al., 1978a,b), Tc

+

Mhc

RESTRICTION AND

Ir

GENES

273

responses to the H-Y antigen (von Boehmer et al., 1978a,b), Tc responses to TNP-modified cellular proteins (Billings et al., 1978a,b), and Th responses to a variety of antigens (Sprent, 1978c,d; Waldman et al., 1978, 1979; Kappler and Marrack, 1978; Katz ef al., 1978), and the results were always similar (a rare event in immunology). The response to the PA parent was almost always significantly higher than that to the PBparental cells. The only discrepancy among these experiments was in the degree of anti-P, response : some investigators did not find any reactivity against PBcells at all (Zinkernagel ef al., 1978a,b for Tc cells; Sprent, 1978c,d; Kappler and Marrack, 1978; Singer et al., 1979, for Th cells), whereas other found preferential response to PAbut some response also to P, cells (Bevan, 1977; Blanden and Andrew, 1979; Matzinger and Mirkwood, 1978, for Tc-cell responses; Bevan and Fink, 1978; Waldman et al., 1978; Katz et al., 1978, for Th-cell responses). However, even authors who clearly did not want to find any predilection for the one parent, did find such predilection (Katz et al., 1978). The discrepancy can be explained by postulating differences in the degree to which the bone marrow is contaminated with T cells. If the transferred donor cells are not completely rid of mature or maturing T cells, the residual T cells may then mount an anti-P, X response (Zinkernagel and Doherty, 1979). This finding means that T cells that are genetically Mhc"/Mhch and normally recognize antigen X in the context of both Mhc" and Mhcb molecules, when they have differentiated in an Mhc"/Mhc" environment, recognize the antigen only or preferentially in the context of Mhc". This tailoring of the T-cell repertoire by the environment is referred to as Mhc restriction, but this term is confusing because its original meaning is as defined in Section 111 (i.e., a T cell that has encountered an antigen together with a certain Mhc molecule can be restimulated only by a combination of the same antigen and the same Mhc molecule). To avoid misunderstanding, in this article we shall use the term Mhc restriction as originally defined and will designate the purported tailoring of the T-cell receptor repertoire by the environment as Mhc individualization, for, as we shall see shortly, an individual is thought to tailor the repertoire to its own needs. The question is then what causes the Mhc individualization of the T-cell receptor repertoire? One obvious possibility is that the emergence of antiMhcb + X T lymphocytes is checked by suppressor cells (after all, most of the body is of the Mhc" type and so, to the body, Mhcb molecules represent foreignness). However, all attempts to prove the presence of suppressor cells in the radiation chimeras have failed (Fink and Bevan, 1978; Bevan and Fink, 1978; Zinkernagel and Althage, 1979). In one set of experiments, for example, normal F, spleen cells were admixed at various ratios to spleen cells from F1 + PA chimeras and the mixture was transferred to irradiated immunized F, recipients. In this experimental setup a response to the X

+

274

JAN KLElN AND ZOLTAN A. NAGY

antigen in the context of Mhcb did occur, indicating that cells from the chimera failed to suppress the responsiveness of cells from nonchimeric mice. In another set of experiments, F, mice were irradiated sublethally (500 rad), given an inoculum of spleen cells from chimeric mice, and then tested for response at various times after inoculation. The rationale of this experiment was as follows. The low dose of irradiation should allow some host T-cell progenitors to survive but the activity of T cells generated from these progenitors should be suppressed by the chimera-derived cells if the chimera contains suppressor T cells. Yet there was no sign of such suppression : the mice also responded to the antigen in the context of Mhcbmolecules. If suppression is not responsible for the observed Mhc individualization, then what is? It seemed as if some tissue or organ in the host not destroyed by the irradiation determined in what Mhc context the recognition occurred. It seemed natural to assume that this organ was the thymus. b. Thymus Chimeras. To test the influence of the thymus one thymectomizes adult mice and, after they have recovered from the operation, irradiates them with a supralethal dose of X or y rays and then reconstitutes their lymphohemopoietic system with T-cell-depleted syngeneic bone marrow. One week after the irradiation one transplants into the host, either subcutaneously or under the kidney capsule, irradiated (800 rad) thymic lobes of a newborn mouse (the irradiation of the thymus is intended to kill all lymphoid cells and keep alive only the radioresistant epithelial tissue). After this procedure the chimeras are handled in the same way as the ones in the preceding section. In the thymus chimeras, the lymphohemopoietic system and the rest of the body are genetically identical but the thymus epithelium is different. If the thymus tailors the T-cell repertoire, these chimeric mice should respond differently from normal mice of the same genetic constitution as the host and the bone marrow donor. At first, the results were unambiguous and they seemed to validate what had been expected: Mhc"/Mhc" stem cells maturing in an aa thymus borne by an ab host produced T cells that responded to antigens preferentially in the context of Mhc" molecules and this observation applied to Tc cells responding to viral antigens (Zinkernagel et ul., 1978a), Tc cells responding to minor H alloantigens (Fink and Bevan, 1978), Th cells (Waldman ef al., 1979), and TDTHcells (Miller et al., 1979). Again there was a discrepancy in the degree of preference for the thymus Mhc type, with Zinkernagel and colleagues (1978a) finding the response to be almost absolutely restricted and Fink and Bevan (1978) finding it merely predilected toward the recognition in the context of the thymus Mhc type. Although Katz and his co-workers (1979) concluded that they found "only marginal or sometimes no thymic influence on preference of helper T cell activity" for the thymic

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RESTRICTION AND

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Mhc type, in fact their data show a substantial thymic influence. The real contradiction has come in the form of more recent data from Wagner's laboratory (Wagner et al., 1980b; Stockinger et al., 1980). Wagner and his colleagues modified the experimental protocol somewhat and used F, irradiated hosts which had been reconstituted with PAstem cells and grafted with PB thymic epithelium. In this situation the stem cells are fully allogeneic with the thymus graft, while both the stem cells and the thymus are semiallogeneic with the host. Furthermore, the authors did not test the spleen cells of the virus-infected chimeric mice immediately after killing the mice, but instead restimulated the cells in uitro and only then tested them on virus-infected PA and PB target cells. The response was variable and clearly influenced by the genotype of the restimulating cells. Some chimeras produced cells that, when restimulated with PA + X, responded to PA X and not to PB X, and vice versa; cells from other chimeras, when restimulated with PA X (the Mhc of the stem cell donor), responded strongly to PA X and weakly to PB + X (PBcarrying the Mhc of the thymus donor); and there were also some chimeras whose cells did not respond at all to restimulation with PB X and responded only to restimulation with PA + X (lysing only PA X and not PB + X cells). So if anything, in these experiments there is a tendency for the T cells to recognize the antigen in the context of the stem cell Mhc rather than the thymus Mhc. Wagner and his colleagues conclude from these data that although the thymus in a normal mouse does individualize the T-cell repertoire, it does not individualize it completely; that is, although the majority of the T cells coming out of the thymus are able to recognize X in the context of the thymus Mhc, a minority of cells entering the periphery are able to recognize antigens in the context of Mhc molecules not expressed in the thymus. The authors explain the preference of the chimeras for the stem cell Mhc molecules by the postulate that syngeneic cells interact better-a postulate which, as we discussed in Section 111, is most likely wrong. It is important to realize, however, that Mhc" lymphocytes must first learn how to coexist with Mhcbcells in chimeras and that this learning might have a profound effect on their subsequent behavior upon antigen restimulation. We shall come back to this point later. At any rate, the contradiction between the two sets of experimental data remains unresolved. c. PA -P Fl Chimeras. In this situation the radiation chimeras consist of Mhc" lymphoid cells, but the thymus epithelium and the rest of the body are Mhca/Mhcb. If the Mhc-individualization hypothesis were correct, one would expect that the a-type T cells coming out of the a / b thymus would be able to recognize the antigen in the context of either Mhc" or Mhcb molecules. This expectation was borne out by one set of experiments reported by

+

+

+ +

+ +

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JAN KLEIN AND ZOLTAN A. NAGY

several investigators (for Tc responses by Zinkernagel, 1976; von Boehmer rt al., 1976; Pfizenmaier et al., 1976; von Boehmer and Sprent, 1976). However, subsequent experiments generated a different set of data : in these the spleen cells of PA+ F, chimeras responded to Mhc" X but not to Mhcb X stimulation (demonstrated for Tc responses by Zinkernagel et al., 1978b; and for Th responses by Sprent, 1978a; Erb et ul., 1978, 1979; Katz et a/., 1978). This discrepancy between the two sets of experiments was explained by Zinkernagel and his co-workers (1978b) as being caused by the survival (because of a lower radiation dose) in the first experimental group of host lymphoreticular cells, which then presented the antigen to the T cells in the Mhc" as well as Mhcb context. Supporting this interpretation was the following experiment (Zinkernagel rt al., 1978b). Spleen cells from completely reconstituted PA -+ F, chimeras were transferred to a second group of virus-infected F, recipients that were irradiated only with a dose of 850 rad so as to allow some host APCs to survive. Spleen cells of these second hosts were then tested for specificity 6 days after the adoptive transfer. The transferred T cells were found to react with both Mhca X and Mhcb X targets under conditions in which the spleen cells of the original chimera reacted only with Mhc" X and not with Mhcb X targets. Why then do the PA + F, chimeric cells recognize the antigen only in the context of Mhc" when the thymus carries both Mhcn and Mhcb molecules'? The explanation offered by Zinkernagel and his colleagues (1978b) is this: if the dose is high enough the radiation kills not only all host lymphocytes and their stem cells but also all (APCs) and their precursors. However, stem cells of APCs are contained in the transferred bone marrow, so in the PA + F, chimera all the APCs are of the donor origin and can present the antigen only in the context of the Mhc" molecules. There is thus a principal difference between F, PAand PA+ F, chimeras : in the former there are only T cells capable of recognizing the antigen in the context of Mhc", whereas T cells recognizing the antigen in the context of Mhcb are missing. In contrast, in the PA + F, chimeras both T-cell types are present (as evidenced by the adoptive-transfer experiments), but there are no cells in these mice that could present the antigen in the context of the Mhcb molecules. d. AIlogeneic Chimeras. Here chimerism is established by inoculating T-cell-depleted bone marrow of strain A into irradiated hosts of an Mhcdisparate strain B (Zinkernagel ef al., 1978a; Matzinger and Mirkwood, 1978; Onoe et al., 1980) or, as was already described under Thymus Chimeras (Section Vll,A,l,b), by inoculating PA bone marrow cells into F, hosts carrying a PB thymus graft (Wagner et a/., 1980b; Stockinger et al., 1980). The former type of chimeras are extremely difficult to produce because most of the mice die, presumably because for a time the hosts are not immunol-

+

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+

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+

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Mhc RESTRICTIONAND Zr

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ogically competent (Zinkernagel et al., 1978a). One can only admire the confidence of those investigators who use such obviously abnormal mice in an attempt to answer questions about the nature of immune responsiveness. It is not surprising that there has been controversy surrounding allogeneic chimeras almost from the beginning. Zinkernagel and his colleagues (1978a) found these to be unable to respond immunologically to virus infection and this observation was confirmed by Wagner et al. (1980b) as far as the primary response was concerned. On the other hand, Matzinger and Mirkwood (1978), Onoe et al. (1980), Wagner et al. (1980b), and Stockinger et al. (1980) all found allogeneic chimeras to be immunologically competent when tested for the secondary response. This discrepancy can perhaps be blamed on the immunization and testing protocol; but there are other discrepancies as well. Matzinger and Mirkwood (1978) produced allogeneic chimeras by injecting BALB.B (H-2’) bone marrow cells into irradiated BALB/c (H-2“) hosts; after the reconstitution was complete, they primed T cells of these chimeras by injecting into them D l .C cells (H-2d but carrying other minor H antigens than the BALB strains). Then they restimulated the primed cells in uitro with (B10 x BI0.D2)Fl cells (H-2b/H-2dbut different background genes from both BALB and D1, some presumably shared with D l ) and demonstrated that the cytolytic lymphocytes killed B10.D2 (H-2d) but not B10, BlO.BR, and (BALB/c x BALB.B)F, targets. However, when they restimulated the chimeric cells with BALB.K (H-2k), the generated cytotoxic lymphocytes killed B1O.BR (H-2k) and no other tested targets; similarly, after restimulation with B10 (H-2’) only BlO cells were killed, and so on. The authors then concluded that “strong H-2-restricted cytotoxic activity against minor antigens was detected and the specificity of the restriction could be to the H-2 haplotype of the donor or the host depending on cells used for priming and boosting.” However, it should be noted that the system used by these authors was extremely complex. Not only was the physiological status of the mice highly suspect, but also the antigens used for immunization and testing were totally undefined. There were also complications that cast doubt on the interpretation of the data. For example, when the chimeras were primed with minor histocompatibility antigens in the context of H - 2 (presumably) and restimulated with minor H antigens in the context of H-24, killing of H-24 cells resulted, suggesting that the reaction was not directed solely against minor H antigens. It is possible that the H-2’ T cells were not fully tolerant of H-2” and that at least some of the observed reactions might have represented a residual response to H-2 alloantigens. It is thus not clear what the results actually demonstrate. e. Other Types of Chimeras. Chimeras obtained by injecting into F, mice a mixture of PA and PB cells (von Boehmer et al., 1975 ) or by injecting

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JAN KLEIN AND ZOLTAN A. NAGY

Mhca/Mhcbcells into Mhca/Mhc‘ mice (Zinkernagel et al., 1978a) have also been produced and tested. However, these merely confirmed some of the observations obtained with the other types of chimeras and so we shall not discuss them further here. f: Evaluation. If there is one unambiguous conclusion that can be made from the experiments on radiation chimeras, it is that the chimeras cannot tell us whether or not individualization of the T-cell repertoire occurs. There are simply too many unknowns hidden in the experimental system to make it useful in this kind of study. We do not know what kinds of host cells survive the treatment; we cannot exclude the persistence of a small number of host lymphoreticular cells; we are uncertain about the origin of the antigenpresenting cells and the entire mode of antigen presentation; we are completely in the dark about the adjustments that the allogeneic cells must undergo to coexist in a single individual and so on. All these factors may vary from experiment to experiment and thus generate contradictory data. In fact, we come to the conclusion later in this section that radiation chimeras probably provide a distorted reflection of the true situation in normal mice. The hypothesis that T cells learn the Mhc context of antigen recognition in the thymus comes out of the radiation chimera studies battered but in one piece. However, to keep it in one piece one has to make additional assumptions which, although not impossible, detract greatly from the elegance and simplicity of the original hypothesis.

2. Neonatally Tolerant Mice In this experimental setup one inoculates into newborn mice of strain PA bone marrow and spleen cells of an adult F, hybrid (the F, +PA combination is chosen to prevent genetic graft-versus-host reaction) and, when the mice grow up, one checks whether tolerance of P, alloantigens has been achieved by placing a PBskin graft on the treated PAmice. One then exposes the mice that tolerate the graft to the selected antigen X (e.g., infects them with a virus or inoculates them with TNP-modified cells) and asks in what Mhc context the mice will recognize the antigen. The answer to this question varies depending on the antigen. Forman and his co-workers (1977) reported that spleen cells from tolerant mice respond to TNP-modified cells of both PA and PB origin, whereas Zinkernagel and his colleagues (1977, 1978a,b,c) observed no response of the tolerant cells to various viruses when the cells were tested against P, targets. However, one could argue that in the experiments of Zinkernagel and his colleagues the T cells did not respond because they simply had no opportunity to be sensitized against the viral antigen in the context of Mhcb (there were no detectable F, cells in the tolerant mice). In contrast, in the experiments of Forman and his colleagues, the antigen

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RESTRICTION AND

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279

was injected together with antigen-presenting cells possessing Mhcb molecules and hence the tolerant mice had a chance to encounter the Mhcb + X molecular combination. To test this possibility Zinkernagel and his coworkers (1978a) transferred spleen cells from the tolerant animals to sublethally irradiated, virus-infected Fl hybrids in which the cells should have had plenty of opportunity to encounter the antigen in the context of Mhcb molecules; however, even after this adoptive transfer the cells remained unresponsive to Mhcb + X target cells. The contradictory findings remain unexplained but it should be noted that later Forman and his co-workers (1979), by slightly changing the experimental conditions, also could not find any response of tolerant T cells to TNP-modified PBcells. Furthermore, Kindred (1975a) reported that T cells of strain A and tolerant of strain B do not collaborate with B cells of congenitally athymic B-strain mice. To interpret all these negative data correctly one would have to know what happens in newborn mice when one suddenly floods them with allogeneic cells, but unfortunately, even after many years of research, we still have no notion of the mechanism of tolerance induction. The individualization hypothesis, of course, has no problem with the negative data: the thymus in the neonatal mice remains of the Mhc"/Mhc" type, so it is expected to release only Mhc"-restricted T cells. 3. Congenitully Athymic Mice

Mice homozygous for the mutation nu/nu (meaning "nude") lack a functional thymus in addition to having a defect in hair growth. In a 14-dayold nujnu embryo, an abnormal epithelial thymus anlage forms, but the rudiment is never seeded by lymphocytes and, as a result, adult nujnu mice lack mature T cells. However, nu/nu mice do possess T-cell progenitors, some of them differentiated to the state where they express the Thy-1 T-cell marker (reviewed by Kindred, 1978). Congenital athymy spares the experimenter the trouble of removing the thymus; by grafting genetically disparate thymic epithelium under the kidney capsule of the nujnu mouse and allowing the mouse's stem cells to differentiate in it, one can ask the same questions that were asked of the radiation chimeras. However, Kindred (Kindred and Loor, 1974; Kindred, 1975a,b, 1977a,b, 1980) and Zinkernagel and his co-workers (Zinkernagel et ul., 1979a,b, 1980) documented convincingly that there are certain limitations to the types of questions one can ask of a thymus-grafted nujnu mouse. The main limitation is that a nujnu mouse of strain A becomes immunocompetent when reconstituted with unirradiated fetal or newborn H-2-incompatible strain B thymus grafts but remains immunoincompetent when reconstituted with an irradiated thymus graft from an adult B mouse. Except for this latter situation, one can treat nujnu mice similarly to the

280

JAN KLEIN A N D ZOLTAN A. NAGY

irradiation chimeras: 8 to 10 weeks after thymus grafting, one can infect the mice with a virus, and 6 days after the infection, test the spleen cells for their capacity to lyse virus-infected target cells. Experiments of this sort have demonstrated that spleen cells of Mhc"/Mhcbnu/nu mice bearing an MAC"/ Mhc" thymus graft recognize viral antigens in the context of Mhca but not in the context of Mhcb molecules (Zinkernagel et al., 1979a,b)-a result predicted by the individualization hypothesis. Similar results have also been obtained for Th cells (Kindred, 1975b, 1977a,b; 1980; Kappler and Marrack, 1978). However, in the reverse situation, Mhc"/Mhc" nu/nu mice grafted with Mhc"/Mhchthymus also recognize viral antigens only in the context of Mhc" molecules and not in the context of the Mhcb molecules (Zinkernagel et al., 1980). This result is not expected; on the contrary, it contradicts the individualization hypothesis because one would expect the Mhc"/Mhc" thymus to disgorge both Mhc"- and Mhc'-restricted T cells. To save the individualization hypothesis, Zinkernagel and his colleagues (1 980) had to postulate that "thymic selection alone is not sufficient to promote T cells to mature and express the restriction specificity for the H-2 expressed in thymus" and that "T cells have to be exposed to lymphohemopoietic cells with the H-2 of the thymus" to complete their differentiation; this purported second maturation step might occur either in the thymus or postthymically, in the periphery. Although this post hoc postulate saves the individualization hypothesis, it is unlikely that the hypothesis will ever fully recover from the heavy blow delivered by nude mice.

4. Evidence from Negative-Selection Experiments When circulating T cells encounter an antigen in viuo, within 1 day they disappear from the central lymph (which is present, for example, in the thoracic duct) and settle down in the lymphoid tissues where they proliferate extensively (Ford and Atkins, 1971; Sprent et al., 1971). After about 1 day the progeny of the activated cells begin to reenter the circulation in expanded numbers. Because the temporary removal from circulation concerns only T cells that have encountered an antigen, this observation gives an experimenter the opportunity to select out these cells negatively : all one has to do is obtain thoracic duct lymphocytes at the time when the antigenactivated T cells are sequestered in the lymphoid organ-that is, on the second day after antigen injection. The lymphocytes thus obtained then contain T cells responsive to all the possible antigens except the ones used for the immunization of the mice. This "negative-selection" protocol has been used to determine whether T cells of strain A can recognize an antigen in the context of Mhcb molecules. For example, Wilson and his colleagues (1977) injected CBA lymph node

Mhc

RESTRICTION AND

I!’ GENES

28 1

cells into irradiated (900 rad) (CBA x B6)F1 mice and then collected the lymph of these mice over a period of 8-30 hr. As the cells circulated through the hosts, they responded to the B6 alloantigens of the F, hybrid, the activated cells were sequestered into the lymphoid organs, and the collected lymph was thus free of anti-B6 (H-2”)cells. The collected cells, stimulated with TNP-modified B6 cells, were then shown to yield good response to B6-TNP targets while remaining unreactive to unmodified B6 targets. The authors concluded, therefore, that “lymphocyte populations negatively selected to a particular Mhc haplotype to the extent that they no longer react to that haplotype by any parameter yet studied can nevertheless generate killer cells against chemically modified allogeneic target cells expressing the selecting haplotype” (Wilson et ul., 1977). Similar results were obtained by Sprent (1978a,b,c) for Th cells and by Doherty and Bennink (1979) for Tc cells responding to viruses. These results, too, argue against the individualization hypothesis, although one can again counterargue that anti-Mhcb + X cells in an Mhc“/Mhc“ mouse are the product of thymus “leakage” during the selection for Mhc”-restricted cells or the result of some as yet not understood form ofcross-reactivity between anti-Mhcb + X with Mhc” + Y.

5 . In Vitro Selection Experiments In this experimental system one mixes, in culture, T cells of strain A with macrophages of strain B, removes the alloreactive cells by the BUdR and light treatment described in Section IV,A, and then stimulates and restimulates the remaining T cells with B strain macrophages and antigen. In one of the first experiments of this sort, Thomas and Shevach (1977) observed that T cells of strain 13 guinea pigs depleted of cells reacting with strain 2 alloantigens responded to TNP-modified strain 2 macrophages ; the authors concluded, therefore, that strain 13 T cells are capable of responding to TNP-modified proteins in the context of strain 2 Mhc molecules. Unfortunately, when similar experiments were done in mice known to differ at both class I and class I1 loci, just the opposite result was obtained: Mhc” T cells were found not to respond to TNP-modified proteins presented to them by Mhcb cells (Shearer and Schmitt-Verhulst, 1977; Janeway et ul., 1978). However, in this case, one must bear in mind that some of the antigens stimulate proliferating T cells poorly, and hence it is doubtful that the BUdR and light treatment has any effect on the precursors of the class I-specific Tc cells. All it might do is remove alloreactive T cells recognizing class I1 alloantigens. Hence, what the authors attributed to a lack of recognition of TNP-modified proteins in the context of allogeneic class I molecules might, in fact, have been lack of nonspecific help brought upon the culture by the depletion of alloreactive proliferating T cells. Another complication-

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which applies to both experiments-is that TNP probably also modifies Mhc molecules themselves and may make syngeneic Mhc molecules look like alloantigens. Therefore, TNP-modified proteins are not suitable for finding out about the restrictions placed on the T-cell repertoire of a given individual. However, experiments using helper T cells and conventional antigens demonstrate very clearly that T cells can recognize antigen in the context of allogeneic Mhc molecules. We already mentioned in Section IV,A the experiments of Ishii and his colleagues (1981a,b, 1982), which convincingly lead to this conclusion. These experiments, performed in a large number of strain combinations and with several antigens, reveal that a normal unmanipulated Mhc" individual contains T cells specifically recognizing antigen X in the context of Mhcb, Mhc", Mhcd (and so on) molecules and that these cells are distinct from those recognizing X in the context of Mhc" (syngeneic) molecules. Stockinger and his colleagues (1 980) have reached a similar conclusion using a somewhat different approach. They removed spleen cells from a normal strain A mouse and incubated them for 1 hr on a monolayer of Mhcb spleen cells. During this incubation, T cells recognizing Mhcb alloantigens bind to the monolayer and are thus depleted from the suspension when the nonadherent cells are harvested at the end of the incubation period. The suspension of Mhc' cells depleted of anti-Mhcbcells could be stimulated by viral antigens in the context of Mhcb molecules. To determine the frequency of the anti-Mhcb + X cells in an A mouse, the authors used the limiting dilution test : they depleted unimmunized spleen cells of alloreactive T cells, then sensitized the former-under limiting dilution conditions-against Mhcb + X and determined the proportion of nonresponding cultures. They reached the conclusion that there is a 6-fold excess in an A mouse of antiMhc" + X cells over anti-Mhcb X cells. Hence on the surface of it there still seems to be hope for the Mhc-individualization hypothesis : although the thymus (or some other tissue) may not individualize the T-cell repertoire completely, it may be responsible for a predilection of T cells to recognize an antigen in the context of self Mhc molecules. However, this consolation diminishes when one considers that there are not two but perhaps a hundred Mhc alleles (Klein and Figueroa, 1981). So there may be an excess of antiMhc" + X T cells over anti-Mhcb + X T cells, but this same individual must also contain anti-Mhc' + X, anti-Mhcd + X (and soon) T cells. A logical conclusion of this argument is that the T-cell repertoire is actually predilected toward recognition of antigens in the context of allogeneic Mhc moleculues. This conclusion may seem absurd but this does not mean that it is wrong. Anyway, the absurdity can be taken from this argument by postulating that at least some of the anti-MhcbT cells in an Mhc" individual may

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also possess specificity for Mhc" from X.

+ Y , where Y is another antigen, different

C. TOLLING THE BELLSFOR

INDIVIDUALIZATION HYPOTHESIS'?

THE

In Table I1 we summarize the main conclusions reached using the various approaches described in the preceding sections. Going through the table one might be tempted simply to tally the results and take a majority vote. However, this would be the wrong thing to do because the different data do not have the same weight. The one conclusion that stands out very clearly from the table and from the discussion is that manipulated (irradiated, thymectomized, neonatally treated, congenitally athymic) animals tell us a different story from unmanipulated animals. Experiments on manipulated animals produce data suggesting that the T-cell repertoire might sometimes be skewed toward the recognition of foreign antigens in the context of self Mhc and that the skewing could occur sometimes in the thymus and sometimes postthymically or even prethymically (Wagner et al., 1981). In contrast, the data generated in the unmanipulated animals (spleen cells of normal mice tested in uitro) clearly indicate that in the periphery there is a large pool of cells perfectly capable of recognizing foreign antigens in the context of allogeneic Mhc molecules. Perhaps others may feel differently but, when given the choice of believing data on manipulated or unmanipulated animals, without hesitation, we go for the latter. An interesting question is why the manipulated animals show skewing. Clearly it must have something to do with the manipulation and with the way an individual manages to tolerate foreign cells in its body. What exactly happens in the manipulated animals that leads to skewing of the T-cell repertoire must be determined by future experimentation. As a substitute for the individualization hypothesis we suggest the following. Stem cells emerge from the bone marrow with a set of genes coding for a complete or almost complete repertoire of T-cell receptors (the completeness is of about the same degree as that of the B-cell receptors). The receptors have dual specificity, one for foreign antigens and another for Mhc molecules, but in principle there is no difference between the two; in fact, they might be interchangeable. The passage of the receptor-displaying cells through the thymus leads to functional inactivation or deletion of selfreactive clones; otherwise the T-cell repertoire remains intact and contains T cells recognizing foreign antigens in the context of self Mhc as well as T cells recognizing foreign antigens in the context of foreign Mhc molecules. Originally, the quantitative representation of the T cells restricted by the different Mhc molecules (syngeneic versus the different allogeneic molecules)

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TABLE I1 CONCLUSIONS REACHED ABOUT INDIVIDUALIZATION OF T-CELLREPERTOIRE I N THE VARIOUS ASSAYS USEDTO STUDYTHISPROBLEM" ~

Response in the context of Experimental system

Stem cells"

Thymus'

Radiation chimeras

Hosth U

b ulb alh ril b

1";

U'

ajh

b' alb'

Ulb a/b

Allogeneic chimeras

{d

b

b

Semiallogeneic chimeras

{

a/c

Neonatally tolerant mice

ab', aa

a

nulnu mice

a a

a' b' a/ b' a'

Thymus chimeras

a/&

ab'

0

nib alb alb

b' a

+ h'

6

U

+ (or f) + +

- (or + )

-

+ +

- (or k)

+

+ - (or +) +

- (or f) - (or f)

+ + v

+

-

a

+

-

a a a alb a/b alb

+ + + + +

+ +

or t

-

" For references see text.

' a, b, c-Mhc

haplotype of the cell, organ, or animal. Grafted. Variable, mostly uninterpretable response influenced by priming and boosting.

is approximately the same, but later it may change because of antigenic stimulation and expansion of clones recognizing self Mhc molecules. Whatever the solution to the problem posed at the outset of this section might be, when we look back on the heroic 1970s in a few years, we may decide that Jerne and all those who followed his lead (including, for a while,

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the senior author of this article) had tried to outsmart Mother Nature. And who knows, at one of the next Immunology Congresses, we might even see the lady grinning cunningly and saying: “It was a nice try by these fellows Jerne and Zinkernagel, but having it their way just would not work.”

D. ON JOINING THE HEADOF A MANTO THE BODYOF A HORSE: THET-CELLRECEPTOR MODELS At this point, we should discuss the nature of the T-cell receptor, but we will refrain from doing so for this topic is more than adequately covered in the immunological literature. It seems that every possible solution to the T-cell receptor problem has been proposed by someone or another. Thus we have one-receptor, two-receptor. and three-receptor models ; we have altered self and dual recognition; factors, acceptors, and receptors; mutational diversification of one receptor while keeping the other receptors constant; mutant-breeding organs ; receptors with different affinities; negative selection and positive selection acting on diversifying receptors; all kinds of signals passing via the receptors and no signals passing, and so on, and so forth. All this reminds us of a paragraph we found in David Hume‘s writings : Nothing is more free than the imagination of man . . . it has unlimited power of mixing, compounding, separating, and dividing. . . ideas in all varieties of fiction and vision. . . . We can, in our conception, join the head of a man to the body of a horse, but it is not in our power to believe that such an animal has ever really existed (David Hume, 1748).

Yes, every possible solution to the T-cell receptor problem has been put forward-except the correct one.

VIII. Nature of Nonresponsiveness and the So-Called Ir Genes

A. DEFINITION OF NONRESPONSIVENESS Most combinations of a given antigen and a given Mhc molecule stimulate T-cell response but some-particularly those in which the antigen carries only a few determinants-do not. When the latter situation occurs, the animal or the isolated T cells either do not respond at all to a given antigen or they respond much less vigorously than other animals or T cells do to the same antigen. There are thus individuals and inbred strains that are responders (high responders) to a given antigen and others that are nonresponders (low responders). When the antigen happens to be encoded in a virus or some other intracellular parasite, the responder individual is referred

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to as resistant and the nonresponder as susceptible. When one crosses a responder and a nonresponder individual and backcrosses the F, hybrid to the nonresponder parent, one often finds that the F, hybrid and some 50% of the backcross individuals are responders although often not as good responders as the responder parent. Nonresponsiveness thus behaves as if controlled by a single Mendelian locus, and linkage studies demonstrate that this locus maps in the Mhc (McDevitt and Chinitz, 1969). One can distinguish two principal kinds of nonresponsiveness. In the first kind, nonresponsiveness occurs because no responding T cells are activated. The genes controlling this kind of nonresponsiveness are referred to as immune-response or Ir genes. In the second kind of nonresponsiveness, responding T cells are activated by the antigen but their activity is blocked by suppressor T cells. Genes controlling this kind of nonresponsiveness are referred to as immune-suppression or Is genes. The only difference between the two kinds of nonresponsiveness is that Ts cells can be demonstrated in the latter but not in the former kind. In an individual A, antigen X is recognized in the context of Mhca molecules; if an individual B fails to mount responsiveness to the same antigen in the context of its own Mhcb molecules, the nonresponsiveness appears to be caused by the Mhc” allele, and the Ir gene appears to be identical with one of the Mhc genes. It is for this reason that all the different Ir (and Is) genes map in the Mhc region. Of course, before all this was known, Ir genes seemed to be discrete entities mapping in the Mhc region but possibly separate from genes coding for the serologically detectable Mhc molecules. Because they seemed to have been responsible for the specific recognition or nonrecognition of the various antigens, they were even believed at one time to code for antigen-specific T-cell receptors. Now we know, however, that the separateness of the Ir genes from genes coding for the Mhc molecules was an illusion caused by the fact that a given antigen is recognized by T cells in the context of some Mhc molecules but not in the context of other Mhc molecules. Under these circumstances the term “Ir gene” has outlived its usefulness; it is also misleading because in the minds of some investigators it still stands for some mysterious genetic entity coding for some as yet unidentified product. Because there are two classes of Mhc molecules, there are also two classes of nonresponsiveness. In the first class, T cells fail to be activated by a combination of an antigen X and a class I molecule, in the other T cells fail to respond to a particular combination of X and class 11 molecules. Further, because class I and class I1 molecules provide the context for Tc and Th cells, respectively, one class of nonresponsiveness affects primarily cytolytic responses, and the other class, regulatory responses. Historically, the second

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class of nonresponsiveness was discovered first, through the effects of Th cells on antibody production.

B. MANIFESTATIONS AND METHODS OF ASSAYING NONRESPONSIVENESS There are as many ways of spotting a nonresponder individual as there are methods of measuring immune response, but immunologists use only four of these ways frequently. 1. The T Cell-Prolijerution Assay

This assay was not the original method of detecting nonresponsiveness, but because it reflects events that are closest to those responsible for nonresponsiveness itself, we shall discuss it first. As far as we can find out, it was first used in humans for the in uitro stimulation of lymphocytes with purified protein derivative (Pearmain et al., 1963). Later it was adapted to all the major species used in immunological research; it was adapted to the mouse by Schwartz and his colleagues (1975). We have already described the principle of this assay in Section IV,A. I t consists of coculturing T cells for a few days with macrophages and soluble antigen (or alternatively T cells with antigen-pulsed macrophages) and measuring proliferation of the stimulated cells. The T cells recognize the antigen presented to them by the macrophages together with class I1 molecules and are thereby stimulated. Because the presentation occurs mostly or exclusively in the context of class I1 molecules, the stimulated cells belong mostly or exclusively to the Th category. The T cell-proliferation assay thus measures helper-type responses, and the nonresponsiveness detected by this assay therefore maps in the class ZZ Mhc region. The nonresponders, as determined by this assay, are usually true nonresponders-that is, their T cells display only a background level of proliferation; the responders, on the other hand, may display a considerable variation in the degree of proliferation. 2. The Antibody Response A T cell which fails to be activated by a given combination of antigen and Mhc molecules (presented by the macrophage) also fails to provide help to its ultimate target, the B cell. Consequently, the B cell is not activated, although it might have bound the antigen, and fails to differentiate into an antibody-secreting plasma cell. The net result is a failure to produce antibodies against a given antigen. Although in this case the end effect is rather remote from the initial defect of interaction, it too provides a good measure of responsiveness. The responders produce antibodies, the nonresponders

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do not, and hence it is only necessary to test sera of immunized animals or count plaques produced by antibody-secreting cells in an agar layer. However, the distinction between the responder and the nonresponder is in this case less sharp than when determined by the T-cell-proliferation assay, and only rarely do the nonresponder individuals completely fail to produce antibodies. More often one distinguishes high responders, intermediate responders, and low responders, all defined arbitrarily. The antibody assay was one of the first methods used for the detection of nonresponders. Like the T cell-proliferation assay, it measures primarily Th responses, that is, responses occurring in the context of class 11 molecules. 3. The Cytolytic Assuy

Responses in which the antigen is recognized in the context of class 1 molecules are mostly of the cytolytic type and hence can be measured by one of the assays used for the detection of Tc cells. The most commonly used assay is based on coculturing of responding and stimulating lymphocytes in oitro for a few days and then testing the ability of the activated cytolytic cells to kill appropriate 'Cr-labeled target cells. As was discussed earlier in this article, this assay can be used to detect virally encoded antigens, histocompatibility antigens (major and minor), membrane proteins modified by haptens, and tumor-specific antigens. Because, however, cytolytic responses often depend on a cooperative action of Th cells (activated by antigen and class I1 molecules), nonresponsiveness to all these antigens need not always map into the class I regions: if the primary lesion is at the interaction step between Th cells and macrophages, it can map into the class I / region, or it can map simultaneously in the class I and class 11 regions, when lesions occur in the interaction between a Th cell and a macrophage or between a Tc cell and the antigen-presenting lymphocyte.

4. Measuring Susceptibility and Resistance A lesion affecting cytolytic responses to intracellular parasites may eventually result in increased susceptibility of the individual to the particular parasite. So, by measuring susceptibility or resistance of individuals or inbred strains to various intracellular parasites, one can also detect the nonresponsiveness lesions. However, because the cause of death is far removed from a failure to generate appropriate Tc cells, and many other genes may function along the way, the correspondence between the phenotype and the genotype is often not as straightforward as it may be in other responses. Nevertheless, the gene for susceptibility to a virus was the first Ir gene placed in the Mhc region (Lilly, 1966).

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c. EVIDENCE THATA FAILURE IN THE MhC-RESTRICTION MECHANISM IS RESPONSIBLE FOR NONRESPONSIVENESS Although the relationship between Mhc restriction and the control of immune responsiveness was already recognized in 1975- 1977 (Doherty and Zinkernagel, 1975; Klein, 1977), in 1981 this relationship has still not fully sunk into immunologists' minds. It may therefore be worthwhile to mention briefly some of the evidence for the link between the two phenomena, as it has been generated in our laboratory. 1. Nonresponsiveness Controlled by a Single Locus An example of this unigenic type of control is the response to a random copolymer poly (G1u4'Ala6') or GA. When one tests the proliferative T-cell response to this antigen, one finds that some mouse strains are responders and others nonresponders (Baxevanis et al., 1980) and that the strain distribution pattern of responsiveness matches almost exactly that obtained by typing for antibody responses (Merryman and Maurer, 1976). The latter observation indicates that both assays do indeed measure an effect with the same cause. When one then reaches into the treasury of H-2 recombinant strains, one can demonstrate that the responsiveness to GA is controlled by a single Mendelian, codominant gene (the F, hybrids between a responder and a nonresponder parent are responders), which maps into the so-called A region of the H-2 complex, consisting of the A , , A , , and Eflloci. On retesting the T-cell-proliferative response in the presence of monoclonal antibodies specific for class I or class I1 molecules, one finds the response to be inhibited by antibodies reacting with the A (= A,A,) molecules but no other antibodies. One then concludes from such an experiment that presumably the T cells recognize GA, presented to them by the macrophages, in the context of the A molecule, and when the A molecule is coated with antibodies, the T cells do not have access to it and hence fail to respond. The concordance of the strain distribution pattern of responsiveness as determined by the T-cellproliferation assay and the antibody assay, the concordance of genetic mapping in these two tests to the region coding for the A molecule, and the blocking of the response by A-specific monoclonal antibodies strongly argue that the ir-GA phenotype is a reflection of the fact that the anti-GA response is restricted by the genes coding for the A molecule. Antibody blocking of T-macrophage interaction has previously been accomplished by others (e.g., Shevach et al., 1972; Schwartz et al., 1976, 1978a), but in these experiments polyclonal, polyspecific antibodies were used, so one could always argue that the inhibition obtained was not caused by antibodies against class I1 molecules but by some unknown contaminating antibody-for example, an antibody to the hypothetical Ir-gene

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product presumably distinct from the serologically detectable Mhc molecules. The use of monoclonal antibodies rules out this possibility. 2. Nonresponsiveness Controlled by Two Complementing Loci Another antigen, the copolymer poly(Glu5' L y ~ ~ ~ T yor r ' ~GLT, ) also divides mouse strains into responders and nonresponders when tested in the T cell-proliferation assay (Baxevanis et al., 1980) or by the antibody assay (Merryman and Maurer, 1975). As in the case of GA, the strain distribution patterns of the anti-GLT response determined by the two assays match perfectly and the responsiveness is controlled by the H-2 complex. However, in contrast to GA, the anti-GLT response is controlled by two closely linked loci in the H-2 complex. Furthermore, the anti-GLT responsiveness is characterized by an unusual feature : F, hybrids between two nonresponder strains are often responders, and so are H-2 recombinants derived from two nonresponder strains (Dorf and Benacerraf, 1975). Formally one can explain these two observations by postulating that the two loci (let us call them simply 1 and 2 ) complement each other. If one nonresponder strain has the constitution 1'2- and the other 1-2+, then an Fl hybrid carries one "+" allele at each of the two loci and hence is a responder because the loci complement each other in a trans configuration, 1 '2-/ 1 - 2 + , and because one + allele at each locus is sufficient for the response to occur. If crossing over occurs in a 1+2-/1-2+ heterozygote and separates the two loci, one obtains a 1'2'' recombinant which is a responder because it carries the complementing loci in a cis configuration. Genetic mapping of the anti-GLT response suggested that one of the Ir-GLTloci was located in the A region (AaA,EB)and the other in the E (E,) region. By doing antibody-blocking studies, we discovered that the anti-GLT response was inhibited only by E-molecule-specific monoclonal antibodies and not by antibodies specific for the A or the K and D molecules (Baxevanis et al., 1980). This finding then explains the duality of the genetic control: this duality is a reflection of the fact that the E molecule is put together by two loci, E, in the A region and E, in the E region, and that crossing over occurs between these two loci with a measurable frequency. Although the A molecule is also put together from the products of two loci, the anti-GA response appears monogenetically controlled because thus far no recombinant has been found separating the A , and A , loci. But why then does the complementation occur in the anti-GLT response? The explanation lies in the finding of Jones and her colleagues (1978) that we mentioned in Section I. Let us consider a situation in which one nonresponder is of the EYE: type and the other is of the E1;E; type. The former is a nonresponder because it carries a nonresponder allele at the EDlocus (indicated

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29 1

by the superscript nr); the other is a nonresponder because it does not express the complete E molecule necessary to provide the context for the recognition of GLT. In an F, hybrid, however, the E; combines with the E,' chain and the hybrid EiE; molecule then provides the necessary recognition context. Similarly, in a recombinant between the E, and E, loci, the EjET loci are placed in the same chromosome, and again EiE; molecules are produced. The genetics of the anti-GLT response indicates that responsiveness versus nonresponsiveness is determined by the E, chain of the E molecule. Yet, the antibody that in our studies blocked the response is directed against the E, chain. This observation means that the blocking occurs via steric hindrance on a molecule in which the chains are intimately intertwined. Results similar to ours were obtained by Lerner et al. (1980) for a polymer of Glu, Ly, and Phe (GL0), and somewhat later by Nepom et al. (1981) for the same antigen. The findings of these two groups are contradicted by earlier experiments of Schwartz et al. (1978a) in which the authors demonstrated inhibition of anti-GL0 proliferative response by two antibodies, one specific for molecules encoded in the A region and the other specific for C-region products. These earlier data, however, must be in error and are a good example of the inherent difficulty in interpreting blocking achieved by polyspecific and polyclonal antibodies. 3. Nonresponsiveness Controlled by the So-called B Region The data discussed thus far demonstrate that whenever a T-cell response is controlled by the A loci it can be inhibited by monoclonal antibodies specific for the A molecule, and whenever it is controlled by the E loci it can be inhibited by E-specific antibodies. There are, however, several cases in which this rule seemingly breaks down: the response to the lactate dehydrogenase B (LDH,, Melchers et al., 1973) and to the myeloma protein MOPC173 (Lieberman et al., 1972) is controlled by a gene that has formally been mapped into a region between A ( A r nA, , , E D )and J , the so-called B region. Because attempts to produce antibodies specific for the products of this hypothetical region have failed, it appears that we have here a case in which the Ir gene does not have any corresponding serologically detectable product. As it turns out, the elucidation of this exception provides one of the strongest pieces of evidence for the function of Ir genes via the Mhc-restriction mechanism. Our studies (Baxevanis et al., 1981a) demonstrate that the T-cell response to antigens such as LDH, and myeloma protein is the result of an interplay between two loci, one involved in stimulation and the other in suppression of the response. The antigen presented by macrophages is recognized by Th cells in the context of the A molecule and the proliferation

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of these cells can be correspondingly blocked by the addition to the culture of A-specific antibodies. As with other responses, here too there are A molecules that do and others that do not provide the context for LDH, or myeloma protein recognition (in other words, there are responder and nonresponder alleles at one of the A loci, most likely the A , locus). A complication arises, however, when the responder allele at the A , locus combines with n g alleles " certain alleles at the E, locus. Thus far two such L ' ~ ~ m p l i ~ a t i E, have been identified, Ek and E l . When a mouse carries one of these alleles (and of course, also the E,' allele necessary for the expression of the E molecule on the cell surface), the E molecule then also provides the context for antigen recognition but instead of stimulating Th cells, the combination of EkpE; + LDH, or E);E; LDH, stimulates Ts cells, which then act, as we discussed in Section IV, on the Th cells and block their proliferation. The suppression is complete if the context is provided by the EfE; molecules and only partial if it is provided by the EjE; molecules. How the illusion of the B region arises in such a situation is depicted in Fig. 3. Here, the H-2' haplotype is a responder to LDH, because A : is a responder allele and there is no E molecule expressed that could activate Ts cells (the E: allele is of the nonexpressor type); and the H-2" haplotype is a nonresponder because, although it carries a responder-type A ; allele needed for the stimulation of Th cells, it also possesses the E; allele responsible for the simultaneous stimulation of Ts cells. From the H-2"/H-2" heterozygotes two intra-H-2 recombinants have been derived, H - P and H-Zh4.The H-2i5 recombinant carries the A ; and Ef; alleles in combination with the expressor E," allele. The EBE): molecules, together with LDH,,

+

H-2'

-

H-Zb

FIG.3. The illusion of the B locus in the H-2 complex. This figure shows how the origin of the indicated recombinants can be interpreted; the correct interpretation is given in the text. BLRand BHRindicates low-responder and high-responder alleles, respectively, as far as response to LDH, or a myeloma protein is concerned.

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stimulate Ts cells, which partially suppress the proliferation of Th cells generated in response to the LbA) + LDH, combination: Because of incomplete suppression, H-2" appears as an intermediate responder or, in the antibody response, even as a high responder. The H-2h4 haplotype, on the other hand, carries the responder A5 allele and a nonexpressor allele at the E, locus. Because it does not express any E molecules on the cell surface, the responsiveness of the Th cells to the ABAk, LDH, combination occurs undisturbed and the H-2h4 mice behave as LDH, high responders in both T-cell proliferation and the antibody assay. Of course, when the first mapping studies on the Zr-LDH, gene were done, all these peculiarities were not known and so at that time it seemed logical to postulate a new B region in the manner shown in Fig. 3. However, now that we know about the nonexpressor E, alleles and the generation of Ep-restricted Ts cells, the postulate is no longer necessary. It is clear that no B region exists, and the correspondence between serologically detectable Mhc molecules, restriction elements, and Zr genes is now complete. All the conclusions reached in the study of the LDH, response apply also to the anti-MOPC173 response (Baxevanis et al., 1981a); so the genetic control of the latter response can also be explained by interacting A and E genes without the need to invoke a B region. There are, however, three antigens-staphylococcal nuclease (Nase), H-Y, and oxazolone-the response to which has been mapped into the B region and which we have not studied as yet. Might these responses require a separate B region? The response to the staphylococcal nuclease is quite complex (Lozner et al., 1974; Berzofsky et al., 1977a,b; Schwartz et al., 1978b; Fathman et al., 1977; Pisetsky and Sachs, 1977; Nadler et al., 1981). If the immunizing dose is large enough, T cells from all the mouse strains thus far tested respond to the antigen, and it is only by comparing the level of responsiveness with the level of responsiveness against an unrelated antigen that one detects strain differences; there is a profound effect of genetic background on the response, in addition to the control exerted by the H-2 complex; some haplotypes (e.g., H - 2 4 ) give highly variable responses, even in the antibody response; some haplotypes (e.g., H-2") that are normally of the nonresponder type become responders when the immunization protocol is changed ;and there is evidence that the response is directed to at least two separate antigenic determinants. A combination of these factors may easily create the illusion of a region separate from A and E, and we feel, therefore, that judgment on the genetic control of anti-Nase response should be suspended until we know more about the cellular interactions taking place during this control. The mapping of the anti-H-Y response to the B region is based on the work of Hurme and his colleagues (1978a, b). These authors observed that H-2b, H-2h4,H-2', and H-2'' female mice do, whereas H-2" and H-2h2mice

+

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do not reject syngeneic make skin grafts. This situation is so remarkably similar to that found in the anti-LDH, and anti-MOPC173 responses that we would be quite surprised if it were not subject to similar control mechanisms. At any rate, one should check the grafted mice for the presence of E-restricted Ts cells before continuing to insist that the anti-H-Y response defines a separate B region. The assignment of the gene controlling the anti-oxazolone response to the B region is based on the study of delayed-type hypersensitivity to this antigen (Fachet and Ando, 1977). As we understand it, the mapping data are now doubtful and should not be interpreted as requiring a B region. In summary then, there is at present no convincing evidence that the B region as originally defined by Lieberman, Melchers, and their colleagues does exist, and there is convincing evidence that in the situations that were supposed to define it, the region is an illusion. Under these circumstances we believe it is not justified to keep the B region in our current maps of the H - 2 complex. D. THEIs GENES In many class 11-molecule-restrictedresponses one can demonstrate, when using the usual methods of priming with antigen in complete Freund's adjuvant and boosting with antigen in saline, only activation of Th cells; in some, however, one detects also activated Ts cells. The genes controlling the nonresponsiveness caused by the activation of Ts cells are then referred to as immune-suppression or Is genes. In this respect, the anti-LDH, and antiMOPC173 responses discussed already can be regarded as controlled by an Is rather than by an Zr gene, but the difference between the two terms is more semantic than real. One of the first examples of Is control of a response to an antigen was that observed upon immunization of mice with the random terpolymer p ~ l y ( G l u ~ ~ A l a ~ ~ or T y GAT r ' ~ ) (Martin et al., 1971; Merryman and Maurer, 1972; Gershon et ul., 1973). Mice carrying H-2 haplotypes a , b, d, and k are responders to GAT, whereas mice carrying haplotypes p, q, and s are nonresponders. However, all mice, whether carrying responder or nonresponder haplotypes, become responders to GAT if they are immunized with GAT complexed to the carrier methylated bovine serum albumin (GATMBSA). In contrast, nonresponder mice immunized first with GAT and then with GAT-MBSA remain unresponsive to GAT. Further studies have indicated that GAT bound to nonresponder macrophages can stimulate responder-type T cells but that in the reverse situation, GAT-pulsed responder macrophages cannot stimulate nonresponder-type T cells (Kapp et ul., 1973). This finding suggests that the lesion in the nonresponder

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mice is at the T-cell rather than the macrophage level, a conclusion which we believe applies to most nonresponder situations (see Section VII1,G). The conclusion reached from subsequent studies (Benacerraf et al., 1974; Kapp et al., 1975) was that responder as well as nonresponder mice possess Th cells capable of recognizing GAT, but that nonresponder mice, when exposed to soluble GAT, generate Ts cells which prevent the anti-GAT Th response from occurring; GAT-specific Th cells can be stimulated in nonresponder mice by GAT bound to MBSA or to macrophages. The Mhc context in which the Th and the Ts cells recognize GAT is not known, although there is some evidence suggesting that the gene controlling the anti-GAT Th proliferation maps in the A region. The crucial unanswered question is what provides the context for the recognition of GAT by Ts cells. It cannot be the E molecule because H-24 and H-2' cells do not express it ;so it must be the A molecule, class I molecules, or some as yet unidentified molecule, possibly controlled by the hypothetical C region. Another example of Is-controlled nonresponsiveness is provided by mice immunized with p o l y ( G l ~ ~ ~ Tory rGT ~ ~(Debre ) et al., 1975a,b, 1976). All inbred mice tested are nonresponders to this antigen ; the only responders found are some random-bred Swiss mice. However, all inbred mice respond to G T if they are preimmunized with GT-MBSA. The immunization with GT prevents the response to subsequent challenge of GT-MBSA but only in certain strains (those carrying H-2 haplotypes d , j ; k , and s ;GT-pretreated H-2" and H-24 mice remain responders to GT-MBSA). In these strains the G T immunization induces Ts cells, which then suppress Th cells normally stimulated by GT-MBSA. Genetic studies suggest that responsiveness and nonresponsiveness are determined by the interaction of two loci, one mapping in the A region and the other in the still hypothetical C region. The C-region involvement is indicated, for example, by the fact that H-2k mice develop GT-specific Ts cells, whereas H-2" mice do not; H-Zk and H-2" mice are presumably identical in the whole segment of the H-2 map from K to E,-hence the Ts-controlling locus cannot lie in this region. Other H-2 recombinants suggest that the D locus is not involved in Ts-cell stimulation either, and so, by elimination, one is left with the segment between E, and D. However, it is also possible that some as yet unidentified manner of cell interaction creates only an illusion of a locus mapping in the C region and that in actuality the Ts-cell activation is controlled by some of the already known loci. Clearly there are many uncertainties regarding the genetic control of Ts-cell activation, but most of these can be removed by sensibly planned experiments. Hence, because it is likely that we will soon know the answers to most of the unanswered questions, we will refrain from trying to provide them here through speculation. One can state already, however, that there is

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no evidence in any of these responses for the recognition of antigens in the context of the J molecule. The function of the J molecule as a restriction element remains, therefore, in doubt. E. NONRESPONSIVENESS I N CYTOLYTIC RESPONSES Most of what we have discussed thus far concerned regulatory responses to soluble antigens-responses restricted by class I1 molecules. There is, however, a whole group of responses for which the context of recognition is provided by class 1 molecules, and it is this group that will concern us now. As was already mentioned, in the cytolytic responses one can envision that two principal types of nonresponsiveness occur. In one type nonresponsiveness occurs because a particular combination of antigen and class I molecules fails to generate cytolytic cells. In the other type, nonresponsiveness results from a failure of Th cells to recognize antigen and a particular class 11 molecule, and the lack of help then prevents the responsiveness of the cytolytic cells, which are otherwise perfectly capable of recognizing the antigen in the context of class I molecules. Thus, in the first type, the lesion is primary, affecting Tc cells directly, whereas in the second type the lesion is secondary, affecting Tc cells indirectly. The first clear-cut evidence for the former type of lesion was provided by Zinkernagel and his colleagues (1978d). These authors observed that mice can generate Tc cells capable of recognizing vaccinia virus-encoded antigens in the context of Kk,Kd, Kb, Db, and Ddmolecules, but that no Tc cells can be detected that recognize these antigens in the context of the Dk molecule. A similar observation applies to the Sendai virus, which is not closely related to the vaccinia virus (vaccinia is a poxvirus and Sendai a paramyxovirus); other viruses (e.g., lymphocytic choriomeningitis virus) can be recognized in the context of the Dk molecule. Why the Dkmolecule fails to provide the recognition context for two unrelated viruses remains an unexplained observation. However, Dk is not the only class I molecule linked to nonresponsiveness: mice are also unable to respond at all or respond poorly to influenza A virus presented in the context of the Kb molecule (Doherty rt a/., 1978); SV40 presented in the context of Kd, Dk, and Kq molecules (Pfizenmaier e t a / . , 1980); Sindbis, Bebaru, and Semliki Forest viruses presented in the context of most K and D molecules except Dk(Mullbacher and Blanden, 1979 ); Friend virus presented in the context of Kd and Dd molecules (Blank et al., 1976); and murine sarcoma virus presented in the context of Kband Dd molecules (Gomard el a/., 1977). In all these cases, the lesion appears to affect Tc cells directly and thus closely resembles the lesions associated with nonresponsiveness to soluble antigens presented in the context of class 11 molecules. The only difference between the class 11- and class I-restricted

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responses, in this respect, is that in the former responsiveness is dominant (an R x NR F, hybrid is a responder), whereas in the latter it is recessive (an R x NR F, hybrid is a nonresponder). The reasons for this difference are unclear. Purportive evidence for Th-cell lesions indirectly affecting cytolytic reactions has come from the study of the anti-H-Y responses (Gordon et al., 1975; Simpson and Gordon, 1977; Hurme et al., 1977, 1978a; Matsunaga and Simpson, 1978; von Boehmer et al., 1977, 1978b). Of all the unrelated H-2 haplotypes ( d , f , k , p , q, r , and s), only the H-2 haplotype is associated with responsiveness to the H-Y antigen as measured by the cell-mediated lymphocytotoxicity (CML) assay of spleen cells from females preimmunized against male cells. In female mice carying the H-2b haplotype, cytolytic cells can be generated recognizing the H-Y antigen in the context of the Db molecule (as evidenced by the fact that H-2b killer cells lyse H-2h2 or K k D bmale cells) but not in the context of Kb (the same cells do not lyse H-2i5 or K"Dd male cells). Although H-2" mice do not generate anti-H-Y CML responses, H - 2 l H - 2 " heterozygotes generate cytolytic cells recognizing H-Y + Kk molecules, and H-2k/H-2" heterozygotes generate killer cells responding to H-Y presented in the context of Dk molecules. These results suggest that H-2" and H-Zk mice have T cells capable of recogKk and H-Y Dk, respectively, but that these are normally nizing H-Y not stimulated because of a lesion elsewhere in the cytolytic pathway. By typing various H-2 recombinants the gene controlling this lesion has been mapped in the A region and the suggestion has been made that the lesion results from a failure of H-2k Th cells to recognize H-Y in the context of the Ak molecule. According to this interpretation, in the H-2k/H-2hheterozygotes, Th cells are stimulated by H-Y + Ab and the activated cells then help in the generation of Kk-or Dk-restricted, H-Y-specific Tc cells. (There is a controversy over the question of whether haplotypes other than H-2h can bring about anti-H-Y responses when combined in H-2 heterozygotes : von Boehmer's group maintains that they cannot, whereas Simpson and her colleagues claim that they can.) However, this interpretation of the anti-H-Y responsiveness has recently been challenged by Miillbacher and Brenan (Miillbacher and Brenan, 1980; Mullbacher rt al., 1981 ; Brenan and Miillbacher, 198l), who have demonstrated that subcutaneous hind footpad immunization of nonresponder H-2k mice with syngeneic male cells changes them to responders. Using monoclonal antibodies the authors could further demonstrate that H-Zk mice immunized in this manner generate three kinds of T cells in response to H-Y: Tc cells that can be blocked by Kk-specific antibodies, Th cells that can be blocked by Ak-specific antibodies, and Th cells that can be blocked by Ek-specific antibodies. These results argue against a defect in the generation of Th cells in H-2k mice and call for a

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reexamination of the question of why these mice do not respond to H-Y upon intraperitoneal immunization of females with male cells. Evidence for a Th-cell defect in cytolytic responses against viruses has been reported by Blanden et ul. (1975) but it, too, is equivocal. We must conclude, therefore, that though nonresponsiveness caused by a lesion in Th cells probably does occur in some cytolytic systems, convincing proof of such nonresponsiveness has yet to be provided. The cytolytic responses, however, display new kinds of nonresponsiveness that could not be predicted from the existing knowledge about nonresponsiveness in class 11-restricted responses. One example of this kind has been described by Shearer and his colleagues (Shearer, 1974; Schmitt-Verhulst and Shearer, 1976; Schmitt-Verhulst et ul., 1976) in one of the first welldocumented reports of genetically controlled nonresponsiveness in cytolytic responses. These authors observed that H-2' ( K k D d )mice generate cytolytic cells specific for TNP-modified proteins and Kkmolecules but do not mount any detectable D"-restricted response. The latter response can, however, be generated in H-Zd ( K d D d )H-,?i5 , (K"D"),and H-2' (Pod) mice. Thus, in this case, Dd molecules can provide the context for the recognition of TNPmodified proteins in some H-2 haplotypes but not in others. The decision as to whether they do or do not provide the recognition context is controlled by two loci, one mapping in the K and the other in the A region. The nature of this control is not understood. Another example of a novel kind of nonresponsiveness has been provided by Zinkernagel and his colleagues (1978d). These authors observed that the response to vaccinia virus in the context of the Db molecule was influenced by the allele at the K locus: when the allele was b, q, or s the response was strong but when it was k , the response was weak or nonexistent. This K - D interaction resembles that observed by Shearer and his colleagues but without the complicating effect of the class I1 loci. No sound explanation for the interaction is available and most likely will be impossible to put forward until we know more about the cellular interactions during the induction of cytolytic responses.

F. SELECTION OF Mhc MOLECULES FOR OF ANTIGEN RECOGNITION

THE

CONTEXT

During every Mhc-restricted T-cell response two choices must be made concerning the context of recognition. The first choice is whether the response will use class I or class I1 molecules for the context of recognition; we discussed this choice earlier in this article and came to the conclusion that the most likely deciding factor is the antigen-presenting cell. This first

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choice of Mhc class is also linked to the decision as to what the function of the stimulated T cells will be, and we suggested that the decision might also be made by the antigen-presenting cells. In each Mhc class, however, there are at least two molecules that can provide the recognition context, and so a second choice must be made, namely whether to use K or D in the case of class I molecules and A or E in the case of class I1 molecules. The rules governing the intraclass choice are apparently different in the two classes and so we shall discuss them separately. 1. Class I Molecules The general rule is that in most cytolytic responses both class I molecules are used simultaneously for the recognition context. For example, H-2" (KkDd) mice infected with vaccinia virus generate two populations of virusspecific cytolytic cells, one population restricted by the Kkand the other by the Ddmolecule (Zinkernagel et al., 1978d). However, because certain class I alleles are of the nonresponder type and also for other as yet poorly understood reasons, K- and D-restricted Tc cells need not be generated in every mouse. For example, as we already discussed, vaccinia virus-infected H-2k (KkDk ) mice generate only Kk-restricted vaccinia-specific Tc cells because Dk is a nonresponder allele; however, other D alleles (e.g., D d ) are perfectly capable of providing the context of recognition of vaccinia virus antigens. Although there are unfortunately very few data concerning the responder status of the individual K and D alleles, as far as the individual viruses are concerned, one very conspicuous finding is already emerging from the limited studies, namely that mice carrying nonresponder alleles at both the K and D loci are relatively rare. To this day virtually the only proven example of such total nonresponsiveness are the inbred strains which, with the exception of H-Zk strains, do not respond to the alpha viruses such as the Sindbis, Bebaru, and Semliki Forest virus (Mullbacher and Blanden, 1979 ). However, as pointed out by Doherty (1980), these are all viruses derived from the tropics, whereas inbred strains almost certainly derive from wild mice living in temperate regions. Inbred strains thus have no reason to be resistant to these viruses. For all other viruses thus far tested in some detail (ectromelia, vaccinia, lymphocytic choriomeningitis virus), each mouse always carries a responder allele at either the K or the D locus and hence is phenotypically of the responder type. This general lack of mice that are nonresponsive to viruses is in sharp contrast to the relative abundance of nonresponders to at least certain soluble antigens (see later). One reason for this difference is undoubtedly the fact that the same virus uses both K and D as restriction molecules, whereas a given soluble antigen, as we shall see shortly, uses either the A or the E molecules but not both for

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the context of recognition. However, this reason may not be the full explanation for the relative scarcity of mice that are genetically nonresponsive to viruses. The main reason could be that viruses exert strong selective pressure on the types and combinations of alleles maintained in the wild mouse populations from which inbred strains derive. In this context it is interesting to recall the reported asymmetrical frequencies of reciprocal intra-H-2 heterozygous mice. Could it be that the underrepresented class of recombinants has inherited nonresponder alleles to some life-threatening viruses at both the K and D loci? This proposition can be tested by intentionally attempting to produce H-2 recombinants with nonresponder K and D alleles to, say, vaccinia or ectromelia virus. Can such recombinants be obtained, and if so, will they be seriously handicapped by their double nonresponsiveness ? The possibility that viruses and other pathogens determine to a large degree what K or D alleles and what combinations of these alleles are maintained in the population opens a new chapter in the study of the biology of the Mhc region. For the first time, one will have the opportunity to relate structure with function, and to find a plausible explanation for the extraordinary polymorphism of the H-2 loci (Klein and Figueroa, 1981). Clearly, classical inbred strains will not be of much value in the study of H-2 population biology; such a study will have to be carried out on wild mouse populations. The first step in this study should be the determination of the distribution, frequencies, and combinations of K and D nonresponder alleles among wild mice, with nonresponders affecting various biologically important parasites. This step is not only feasible, it is also long overdue. 2 . Class I1 Molecules Responses using class I1 molecules for the context of recognition have a choice between the A (A,A,) and E (E,E,) molecules. Although one could deduce from the Zr gene-mapping studies that both molecules are used-A molecules in some responses and E molecules in others-no direct proof of this contention existed and no indication was available about the constancy of the recognition context. Is a given antigen always recognized in the context of either A or E, or can the context of responsiveness oscillate between these two molecules as it does between K and D in the class I-restricted responses? To answer this question, we determined the context of recognition in responses to three antigens-GA, GLT, and LDH,-testing some 20 alleles at the A locus and about the same number at the E, locus [we were able to test this large number of alleles only because we had extracted them from wild mouse populations (Ishii et al., 1981b)l. The test was based on the T-cellproliferation assay and blocking of this proliferation by monoclonal class

THEA

AND

TABLE 111 E MOLECULE AS RESTRICTION ELEMENTS IN T-CELLRESPONSES' T-cell proliferative response to

GA

GLT

LDH,

Molecule (chains) Restricted by

Restricted by

E (EP

A (A& b d f

b d None

j

j

k

k P

P

r

None r

S

S

U

U V

9

V

w3 w4 w13 w13 w15 w16 w16 w17 w23? b

w3 w4

w13 w13 w15 w16 w16 w 17 or none w23 or none w27

Responder status R R R NR R NR NR NR NR NR NR NR R R NR R NR NR R NR R

A

E

Responder status R R R NR NR R R R R R R R

*

R R

*

NR R NR NR R

A

+ + + + + + + + + + +* + +* -

+

-

+

E

Restricted by Responder status

A

E

R R NR R

NR NR R R R NR NR R R R R NR NR NR R R NR

a R, responder; NR, nonresponder; + or -, Th cells do (+) or do not (-) recognize the antigen together with the indicated molecule; S, recognition of the antigen together with this molecule stimulates Ts cells; 0, indicated molecule is not expressed on the cell surface; *, not tested.

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II-specific antibodies : responses blocked by A-specific antibodies were regarded as A-restricted and those blocked by E-specific antibodies were concluded to be E-restricted. The results of this testing are summarized in Table 111. The anti-GA response, when it occurred, was always restricted by the A molecule; not a single case of recognition in the context of the E molecule could be found. Of the 20 tested A , alleles, 9 or less than half were of the responder type, the others being of the nonresponder type. The anti-GLT response was, with two exceptions, restricted by the E molecule (of the 19 E, alleles tested, 8 were of the responder type). In two cases, the response occurred in the context of the A molecule, and both cases occurred in H-2 haplotypes that do not express a functional E molecule. Finally, in the anti-LDH, responses, Th cells were always restricted by the A molecule (14 responder alleles out of 18 tested); no switching to E restriction was found. The Ts cells, on the other hand, were E-restricted but only 2 of 18 alleles tested were of the responder type (i.e., they provided the context for LDH, recognition by these cells). These studies indicate that the class I1 context of antigen recognition is remarkably constant: the response to a given antigen is either A- or Erestricted and it stays this way for most of the alleles tested. The only time it switches is when the allele carries a mutation preventing the expression of functional class I1 molecules, although the absence of one molecule does not automatically mean that the other molecule then takes over (the H-2 haplotypes 6, f ; and s do not express functional E molecules and they confer nonresponsiveness to GLT, indicating that the A molecule is unable to provide the appropriate context for the recognition of the GLT antigenic determinants). So one can say that antigenic determinants recognized in the context of class I1 molecules are not only remarkably “monogamic” but also faithful to the molecules in the company of which they have chosen to be seen; they choose a new partner only when they have been left “widowed.” This “monogamy” is in striking contrast to the “bigamy” of antigens recognized in the context of class I molecules. To a viral antigen, for example, it makes apparently no difference whether its partner is the K or the D molecule, as long as it is encoded by a responder allele. What could be the reason for this difference between class I- and class II-restricted responses? The first answer that comes to mind is that K molecules are much more closely related to D molecules than A molecules are to E molecules (discussed by Klein, 1982). In fact, the K and D molecules are so close to each other that for a while it appeared as if there were no characteristic features of K in comparison to D (no “K-ness” or “D-ness,” see Silver and Hood, 1976). Although this notion is probably wrong and although all the K molecules may have an amino acid sequence in common in which they differ from the D molecules (Klein, 1982), the fact is nevertheless that there is

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some 70-80% sequence homology between K and D molecules and that this degree of homology is very close to that between two K or two D alleles. In contrast, no homology in the primary structure has yet been established between the A, and E, chains, although admittedly little sequence information is available for these chains. Hence, A, is likely to be less homologous to E, than K is to D (if for the K and D molecules only the amount of information were available that now exists for the A, and E, chains, homology between the two former chains would already be apparent). One could therefore argue that the mechanism, whatever it might be, that selects the context of antigen recognition has a hard time telling K from D molecules and hence uses both “promiscuously,” whereas it easily distinguishes A from E and hence consistently uses one or the other but not both for a given antigen. It is fair to say, however, that there is still a possibility that the observed difference is more seeming than real. One could argue, for example, that antigenic determinants recognized in the context of class I molecules are also monogamic, but because viruses, minor H antigens, and haptenmodified proteins are antigenically complex, there are always two or more determinants recognized simultaneously, some consistently in the context of K molecules and others consistently in the context of D molecules; and that the impression of promiscuity arises from not being able to distinguish the “married couples.” Some support in favor of this notion comes from recent observations that class 11-restricted responses to complex antigens, such as SRBC or KLH, also use both A and E as restriction molecules (Sprent and Alpert, 1981; Shigeta and Fathman, 1981). In fact, the reason Ir control was originally discovered for simple antigens and many complex antigens do not appear to be under such control, is that total nonresponsiveness to complex antigens is relatively rare because there is always at least one determinant to which a given individual can respond. In other words, the lack of Ir-gene control of responses to complex antigens does not mean that the Mhc-restriction rules do not apply to these antigens; it simply stems from the complexity of the response and masking of nonresponsiveness to one determinant by responsiveness to another. These arguments will have to be dealt with by doing the proper experiments and analyzing the response to, for example, viruses at the level of the individual determinants, and determining the context of recognition of these determinants. What then determines whether a given antigen will be presented to the T cell in partnership with the A or the E molecule? We would like to suggest that his decision is part of the antigen processing by the macrophage. Assuming that the macrophage experts are right and the antigen is broken down into fragments which are then inserted into the membrane, one can envision the existence of two basic processing channels dealing with crudely

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different fragments (the difference could be in size, charge, or some other parameter that does not require a high degree of specificity for distinction. One can speculate further that one of the channels places one kind of fragment in the vicinity of the membrane-bound A molecules and the other in the vicinity of E molecules. If one of the channels is not functioning properly, the processing may occur through the other channel, thus leading to a switch of the recognition context of the type already described. There is no reason to suppose that similar channels exist also in lymphocytes, and if the antigens here are distributed randomly in the membrane then they also occur randomly sometimes near the K and sometimes near the D molecules. This random distribution of antigens could be another reason for the “bigamy” of antigenic determinants recognized in the context of class I molecules. There is, however, one point that should be mentioned before one gets carried away with this sort of speculation. There are unfortunately not enough T-cell-proliferation and antibody-blocking data available for calculation of the frequencies with which the A and E molecules are used for recognition of the different antigens. One can nevertheless get some notion of these frequencies from the fr gene-mapping studies, which include many TABLE IV TO VARIOUS ANTIGENS AS PREDICTED THECONTEXT OF RESPONSE BY Ir GENE-MAPPING STUDIES“

Antigen

Context of recognition

Antigen

Context of recognition

~

GLA’ GLA” GLA~O

GLA4’ GLAbo GLT’ GLT” GL0 GL Leu GAT4 GATIO ~ 6 0 ~ 4 0 ~ 9 0 ~ 1 0

A A A A A Eh Eb E E? A A A ?

~ 5 0 ~ 5 0

?

(T,G)-A-L (T-A-G-GIy), (T-G- A-Gl y)“

A A A

Horse spleen ferritin Keyhole limpet hemocyanin DNP-ovomucoid DNP- bovine gamma globulin Insulin (bovine) Myoglobin Lysozyme (hen) Thyroglobulin (mouse) Acetylcholine receptor Cytochrome C (pigeon) Collagen, type I Collagen, soluble Sheep red blood cell Myeloma protein IgA Myeloma protein IgG Lactate dehydrogenase B Staphylococcal nuclease

Compiled from various sources, too numerous to be listed here. But H-24is a high responder.

A A and E A A A A and E? A and E? A and E? A A and E? A A A and E A A(Th) and E(Ts) A(Th) and E(Ts) A(Th) and E(Ts)?

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diverse antigens. When one makes certain assumptions (e.g., that the response occurs in the context of the A molecule if strains that do not express the E molecule function as responders to a particular antigen), one can very tentatively assign the recognition context for several antigens. This compilation of data (Table IV) suggests that most of the antigens use the A molecule for the context of recognition; there are only a few responses that are E-restricted. This asymmetry might, of course, be a reflection of the type of antigen studied or of some other bias but if it were to prove to be real, one should begin to wonder whether the E molecule is not preferentially used for some other purpose-generation of Ts cells, for example.

G. THECAUSEOF NONRESPONSIVENESS Now, at last, we come to the most basic but also the most difficult of all the questions we have asked: How does nonresponsiveness arise and what causes it? Before we attempt to answer this question, however, we must answer another question : which cell is responsible for the nonresponsiveness lesion? Here we have only three choices, B cells, T cells, and macrophagesthe holy trinity of immunology. B cells were considered in the past as a likely candidate (Mozes, 1976) but they can now be safely excluded: because nonresponsiveness is detected also by the T-cell-proliferation assay in a culture that does not contain any B cells, the B cell is out of the running. The choice is thus between the macrophage and the T cell. From scanning current immunological literature one gets the impression that virtually every immunologist believes the macrophage to be the guilty party. Here we shall argue that this belief is wrong, but before we do we shall first mention what experimental evidence has convinced immunologists of the macrophage’s guilt. There is in fact only one piece of such evidence that can be interpreted as placing the nonresponder lesion into the macrophage, evidence first reported by Shevach and Rosenthal ( I 973). These authors immunized guinea pigs of strains 2 and 13 with DNP-GL or with GT and demonstrated, using the T-cell-proliferation assay, that strain 2 is a responder to DNP-GL and a nonresponder to GT, whereas the opposite is true for strain 13. They then immunized (2 x 13)F, hybrids with one or the other antigen and restimulated the F, T cells in v i m with antigen-pulsed strain 2 or strain 13 macrophages. They observed that the F, T cells responded to DNP-GL when it was presented to them by strain 2 macrophages and did not respond when it was presented by strain 13 macrophages, and again the opposite was true for GT. In other words, F, T cells respond to an antigen presented by responder macrophages but do not respond to an antigen presented by nonresponder macrophages.

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This line of investigation was then carried further by Rosenthal and his colleagues (1977,1980) using a different antigen, namely pork insulin, which has the advantage that the antigenic determinants recognized by T cells are known. Pork insulin consists of two chains, A and B, each carrying one antigenic site: the site on the A chain involves amino acids 69 through 75; that on the B chain involves amino acids 1 through 16. One can demonstrate the existence of the two determinants by immunizing animals with fragments consisting of these amino acids instead of the whole insulin molecule. As it turns out, guinea pigs of both strains respond to the whole molecule of pork insulin, but only strain 13 animals respond to the B( 1- 16) fragment. When an F, hybrid is immunized with the B(1-16) fragment and its T cells are then stimulated in uitro with B( 1- 16)-pulsed macrophages or with pork insulinpulsed macrophages, they respond only when the antigen is presented to them by strain 13 macrophages. T cells from F, hybrids immunized with pork insulin respond, of course, to pork insulin-pulsed strain 2 and 13 macrophages but, again, only to B(l-l6)-pulsed strain 13 macrophages. The authors conclude from this experiment that the F, T cells can respond to A-chain determinants presented by strain 2 macrophages and to B-chain determinants presented by strain 13 macrophages. Objectively viewed, these experiments can be interpreted in two different ways. According to one interpretation, the nonresponder animals have T cells capable of recognizing the antigen and the Mhc”‘ molecules but the macrophages fail to present the antigen properly, and it is for this reason that the F, hybrid responds to the antigen presented by the responder macrophages but does not respond to the same antigen presented by nonresponder macrophages. One possible reason for the failure of presentation could be that to be presented the antigen must physically associate with the Mhc molecules and in the nonresponders such association does not occur. In an extreme form of this hypothesis, the Mhc molecules can be viewed as receptors for antigens, and nonresponsiveness can be considered the result of a failure of a particular antigen to find a matching receptor in a given Mhc molecule (Benacerraf, 1978; McDevitt, 1980). However, such a hypothesis requires a high degree of specificity of the antigen-Mhc interaction and a large number of Mhc genes for the generation of a decent receptor repertoire, and there is no evidence for either of these two suppositions. Another possibility is that the macrophage selects the fragment and hence the antigenic determinant that it will display on its surface. In the example just described, strain 2 macrophages display the A-chain fragment and determinants, whereas strain 13 macrophages display the B-chain determinants. Where macrophages get this incredible sophistication and specificity in handling the antigen, the “determinant-selection’’ hypothesis does not explain. Further, it is also not clear why nonresponsiveness should be controlled by the Mhc;

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Ir

307

GENES

the macrophage would have to possess a complex machinery for the determinant selection, which would have nothing to do with the serologically detectable Mhc molecules, and this machinery would have to be genetically controlled by an as yet unknown set of loci. In fact, in the determinantselection hypothesis, the Mhc molecules appear to be superfluous and to have no function. An alternative explanation of the Fl hybrid experiments presupposes that there is nothing wrong with the macrophages in nonresponder animals but that these animals lack functional T cells capable of recognizing the particular antigen-Mhc molecular combination. The F, hybrid then inherits this T-cell lesion from the nonresponder parent and consequently it has Tcells capable of recognizing antigen plus Mhc' but no T cells for the antigen plus Mhc"' combination (where r and nr are alleles at the Mhc loci carried by the responder and nonresponder strains, respectively). Because the nonresponder macrophages present the antigen plus Mhc"' combinations for which the F, hybrids lack T cells, the hybrid cannot respond to such antigen presentation. The determinant-selection experiment is simply explained thus: the responder and the nonresponder macrophages each present both the A-chain and the B-chain determinants on their surfaces, but because the F, hybrid does not have T cells for the recognition of A M h d 3 and of B + Mhc2 (where A and B are the corresponding insulin fragments, and 2 and 13 Mhc alleles of strains 2 and 13, respectively), it does not respond to A presented by strain 13 macrophages and to B presented by strain 2 macrophages. We emphasize that there is nothing in these experiments that would favor one interpretation over the other and it is difficult to understand why so many immunologists have so uncritically accepted the view that there is something wrong with macrophages rather than T cells in the nonresponder animal. The confusion possibly stems from the fact that in the F, hybrid experiment one keeps the T cells constant and, by varying the macrophage, one gets responsiveness or nonresponsiveness. Superficially such a result may give the impression that the macrophage is the culprit, but we have explained already why this impression is false. To swing the argument one way or the other, one must do the reciprocal experiment-that is, to keep the macrophages constant and very the T cells. If the nonresponder macrophages were not able to present the antigen, no matter what T cells they were given as partners, then indeed one would have to argue that the macrophage is the site of the lesion. It is obviously not possible to do such an experiment in a syngeneic combination but we have done it in several allogeneic T-macrophage combinations (Ishii et af., 1981a). We have asked nonresponder macrophages to present an antigen to T cells derived from different responder strains (responsiveness and nonresponsiveness always relate in this case to

+

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JAN KLEIN AND ZOLTAN A. NAGY

the same antigen) differing from the macrophage donor at their Mhc loci. To prevent response to Mhc alloantigens we removed the alloreactive T cells prior to the main experiment by BUdR and light treatment. In all the combinations we tested (and there were more than 15) the responder T cells responded well to the antigen presented by the nonresponder macrophages. We will mention just one example. Strains B10 and B1O.P are responder and nonresponder to GA, respectively. But a B10 T-cell suspension, depleted of alloreactive cells, responds strongly to GA presented by the nonresponder BIO.P macrophage. Hence there cannot be anything intrinsically wrong with the nonresponder macrophages ;they are perfectly capable of presenting GA to T cells capable of recognizing this antigen. These experiments demonstrate that the lesion is not in the macrophage and hence, by inference, it must be in the T cell. Most likely the nonresponder animals lack functional T cells reactive with a given antigen presented in the context of Mhc"' molecules. Thus we come to the final question: why d o nonresponder animals lack functional T cells for any of the antigen Mhc"' combinations? To answer this question we tested some 40 different allogeneic T cell-macrophage combinations for their responsiveness to G A and GLT. The combinations were as follows : responder T cells-allogeneic responder macrophages ; responder T cells-allogeneic nonresponder macrophages ; nonresponder T cells-allogeneic responder macrophages ; and nonresponder T cellsallogeneic nonresponder macrophages. In all these combinations a response to the tested antigen was always obtained (Ishii et al., 1982). Thus in these allogeneic combinations we did not find a single case of a nonresponder phenotype. Clearly nonresponsiveness is the hallmark of syngeneic T cellmacrophage interaction; it either does not occur at all or it is a relatively rare phenomenon in allogeneic interactions. One can also restate these findings as follows : Unmanipulated animals contain T cells specific for antigen and Mhcs (s for self) and T cells specific for the same antigen and MhcnS(ns for nonself = allogeneic Mhc molecules). The repertoire centered around the Mhss molecules has blind spots, so each individual lacks T cells specific for certain antigens and Mhcs molecules ; the repertoire centered around the various Mhc"" molecules does not have these blind spots-it is complete. Because an individual is normally never exposed to Mhc"" molecules, whereas throughout its life it is exposed to Mhcs molecules, one is tempted to conclude that the blind spots arise because of the exposure to Mhcs molecules-that they are induced by this exposure. We can then answer the question posed at the outset of this section thus: nonresponsiveness arises because certain T-cell clones are functionally deleted from the repertoire as a result of an encounter with Mhcs molecules. We emphasize that this deletion concerns only certain clones; the overwhelming majority of T-cell clones remain unaffected. We have no information about the mechanism and location of the

+

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postulated T-cell clone deletion (or inactivation). We presume that it is somehow linked to the induction of self-tolerance and that it occurs in the thymus. An obvious possibility is that a T cell has two receptors or receptor sites, one normally used for the antigen and the other for the Mhc”molecules. If the repertoires of these two receptors were partially or completely overlapping, situations would arise where an antigenic determinant would mimic determinants of an Mhc” molecule. In such cases both receptors on a single T cell would react with the Mhc“ molecule and such a reaction would result in the inactivation of the T cell during ontogeny. In all other cases only one of the two receptors on each T cell would be specific for the Mhc” molecule and such a T cell would be allowed to enter the pool of antigen-reactive lymphocytes. We emphasize that these are mere speculations; the fact is, however, that Mhc”-centered repertoires do and MhcnS-centeredrepertoires do not have blind spots, and that from now on any hypothesis of T-cell function must deal with this fact. IX. The Parable of the Blind

More than 500 years ago, as the Western World began to emerge from the nightmarish darkness of the Middle Ages, a great era in painting began not only south but also north of the Alps. The southern and the northern

FIG.4. The Parable ojthe Blind, Brueghel.

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JAN KLEIN AND ZOLTAN A. NAGY

Renaissance presented quite different views of the world. While in the sundrenched South painters depicted the world as an unreal oasis of harmony, timelessness, and peace ;for the northern painters the world remained a bleak place to live in. In the paintings of Hieronymus Bosch and Pieter Brueghel the Elder in particular, life has a cruel, grotesque, apocalyptic quality-very much like in the real world. To this day, Bosch’s and Brueghel’s paintings are mirrors reflecting, in a distorted and exaggerated way, our weaknesses and follies. These mirrors also reflect what goes on in science, and so we think it fitting to close this discussion of Mhc restriction with a reproduction of one of Brueghel’s paintings : The Parable of’the Blind, sometimes also called the Blind Leading the Blind (Fig. 4).

ACKNOWLEDGMENTS We thank Ms. Rosemary Franklin and Ms. Karina Masur for help in preparing this manuscript. The work was supported in part by a grant from the Deutsche Forschungsgemeinschaft.

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Strosberg, A. D. (1977). Immunogenetics 4,499-513. Sunday, M. E., and Dorf, M. E. (1981). J. Immunol. 127,766-768. Sunday, M. E., Benacerraf, B., and Dorf, M. E. (1980). J. Exp. Med. 152, 1554-1562. Swain, S. L., and Dutton, R. W. (1977a). J . Immunol. 118,2262-2268. Swain, S . L., and Dutton, R. W. (1977b). J. Immunol. 119, 1179-1186. Swain, S. L., and Panfili, P. R. (1979). J. Immunol. 122,383-391. Swierkosz, J. E., Rock, K., Marrack, P., and Kappler, J. W. (1978). J . Exp. Med. 147,554-570. Szymura, J., Wabl, M., and Klein, J. (1981). Immunoyenerics 14,231-240. Tada, T., and Okumura, T. (1979). Adu. Immunol. 28, 1-87. Takemori, T., and Tada, T. (1974). J . Exp. Med. 142, 1241-1253. Taniguchi, M., and Miller, J. F. A. P. (1977). J . Exp. Med. 146, 1450-1454. Thomas, D. W., and Shevach, E. M. (1977). Proc. Nail. Acad. Sci. U.S.A. 74, 2104-2108. Turk, J. L. (1980). “Delayed Hypersensitivity,” 3rd ed., Elsevier, Amsterdam. Unanue, E. R. (1972). Adu. Immunol. 15,95-165. Urso, S., and Gengozian, N. (1974). J . Immunol. 113, 1770-1779. Vadas, M. A., Miller, J. F. A. P., Whitelaw, A. M., and Gamble, J. R. (1977). Immunoyenetics 4, 137-153. von Boehmer, H., and Haas, W. (1979). J . Exp. Med. 150, 1134-1 142. von Boehmer, H., and Sprent, J. (1976). Transplant. Rev. 29,3-23. von Boehmer, H., Sprent, J., and Nabholz, M. (1975a). J. Exp. Med. 141,322-334. von Boehmer, H., Hudson, L., and Sprent, J. (1975b). J . Exp. Med. 142,989-997. von Boehmer, H., Sprent, J., and Haas, W. (1976). ColdSpriny Harbor Symp. Quant. Biol. 41, 539-545. von Boehmer, H., Fathman, G. C., and Haas, W. (1977). Eur. J . Immunol. 7,443-447. von Boehmer, H., Haas, W., and Pohlit, H. (1978a). J . Exp. Med. 147, 1291-1295. von Boehmer, H., Haas, W., and Jerne, N. K. (1978b). Proc. Natl. Acad. Sci. U.S.A. 75,24392442. Wagner, H., Feldmann, M., Boyle, W., and Schrader, J. W. (1972). J . Exp. Med. 136,331-343. Wagner, H., Gotze, D., Ptschelinzew, L., and Rollinghoff, M. (1975). J . Exp. Med. 142, 14771487. Wagner, H., Starzinski-Powitz, A,, Jung, H., and Rollinghoff, M. (1977). J . Immunol. 119, 1365-1 368. Wagner, H., Rollinghoff, M., Bzenmaier, K.,Hardt, C., Johnscher, G. (1980a). J. Immunol. 124,1058-1067. Wagner, H., Rollinghoff, M.,Rodt, H., and Thierfelder, S. (1980b). Eur. J . Immunol. 10,521525. Wagner, H., Hardt, C., Bartlett, R., Stockinger, H., Rollinghoff, M., Rodt, H., and Pfizenmaier, K. (1981). J . Exp. Med. 153, 1517-1532. Waldman, H., Pope, H., Brent, L., and Bighouse, K.(1978). Nature (London) 274, 166- 168. Waldman, H., Pope, H., Bettles, C., and Davies, A. J. S. (1979). Nature(London)277,137-138. Weinberger, J. Z., Greene, M. I., Benacerraf, B., and Dorf. M. E. (1979). J. Exp. Med. 149, 1336- 1348. Weiner, H. L., Greene, M. I., and Fields, B. N. (1980). J . Immunol. 125,278 Wettstein, P. J., Bailey, D. W., Mobraaten, L. E., Klein, J.. and Frelinger, J. A. (1978). J . Exp. Med. 147, 1395-1404. Wilson, D. B., Fischer-Lindahl. K., Wilson, D. H., and Sprent, J . (1977). J . Exp. Med. 146, 361-367. Woodland, R., and Cantor, H. (1978). Eur. J . Immunol. 8,600-606. Yamashita,V.,and Shevach, E. M. (1978). J . Exp. Med. 148, 1171-1185. Yano, A., Schwartz, R. H., and Paul, W. E. (1977). J . Exp. Med. 146,828-843.

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PHENOTYPIC AND CYTOGENETIC CHARACTERISTICS OF HUMAN B-LYMPHOID CELL LINES AND THEIR RELEVANCE FOR THE ETIOLOGY OF BURKITT’S LYMPHOMA

Kenneth Nilsson The Tumor Biology Group. The Wallenberg Laboratory, University of Uppsala. Uppsala. Sweden

George Klein Department of Tumor Biology, Karolinska Institutet. Stockholm. Sweden

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Lymphoblastoid Cell Lines (LCL A. The Target Cell of EBV I B. Early Events during EBV C. “Spontaneous” Establish D. Phenotypic Characteristics of LCLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

319

326

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

331 338 . . . . . . . . . . . . . 339

nes .........................

111. EBV-Carrying

...................... IV. Basis for Distinction between EBV-Carrying Lymphoblastoid ......................... and BL Cell Lines V. EBV-Genome-Negative BL Cell Lines ..................................... VI. EBV Genome-Negative B-Leukemia/Lymphoma Cell Lines . . . . . . . . . . . . . . . . . . . VII. EBV-Carrying non-BL, Nonlymphoblastoid Cell Lines Derived from EBV Genome-Negative Leukemia/Lymphomas ............................. VIII. The Relationship of EBV-Carrying Lymphoid Cell Lines to Normal B-Cell Differentiation .. ..... A. Normal B-Cell Di ......................... B. The Stage of Differentiation of LCL and BL Cells ........................ IX. The Progression in Lymphoblastoid Cell Lines in Virro and in Viuo. . . . . . . . . . . . . X. The Role of EBV in Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. The Role of Chromosomal Changes in Progression . . . . . . . . . . . . . . . . XII. General Discussion ..................................... References ...................................

346 346 349 352 353 353 355 360 362 368

I. Introduction

During the last 15 years intensive studies have been performed to clarify the role of Epstein-Barr virus (EBV), a lymphotrophic DNA virus, in the pathogenesis of Burkitt’s lymphoma (BL). 319 ADVANCES IN CANCER RESEARCH, VOL. 37

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-006637-8

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The original suggestion by Dennis Burkitt that BL was caused by a transmissible agent, possibly a virus (Burkitt, I962a,b), has been strongly supported by seroepidemiological studies, in particular the recent prospective West Nile study (de The et al., 1978; de The, 1979). Early experimental work with BL in oitro led to the discovery of EBV as a new human herpes virus (Epstein et al., 1964) subsequently found almost invariably present in African BL biopsy cells and derived cell lines but absent in most cases from nonendemic areas (Anderson et al., 1976; Ziegler et al., 1976). EBV was soon identified as the causative agent of infectious mononucleosis (IM) (Henle et al., 1968; Niedermann et al., 1968) and as a most powerful transforming (immortalizing) agent for human B lymphocytes in vitro (Henle et af., 1967; Pope et al., 1968; Diehl et al., 1969; Gerber et al., 1969; Miller el al., 1969; Baumal et al., 1971; Chang et al., 1971a; Nilsson et al., 1971). In the early 1970s several hypotheses were put forward to explain the role of EBV in the development of BL. One attractive theory, designated as the “co-factor hypothesis” (Klein, 1971), suggested that EBV converts normal lymphocytes into BL cells by a one-step process. Such cells would be present in all EBV-seropositive individuals, but under strict control of host mechanisms, probably immunological in nature. As a result EBV would not cause tumors except under the extraordinary circumstances of the highendemic areas of Africa and New Guinea, where these control mechanisms would have become deranged, perhaps by the onslaught of malaria or other parasitic diseases. This article summarizes recent experimental studies that suggest a different role of EBV in the pathogenesis of BL. Particular emphasis will be placed on the distinct phenotypic and cytogenetic differences that have been reported to exist between EBV-transformed normal B lymphocytes (lymphoblastoid lines) and BL cells. They show that the in vitro transformation of B cells cannot be regarded as the full equivalent of the malignant transformation of the BL precursor cell in uivo. We shall also discuss the secondary abnormal progression of lymphoblastoid cells during continuous growth in vitro, including the acquisition of cytogenetic abnormalities, the potential for tumor formation in nude mice, and high clonability in agarose. Finally, these results will be discussed in relation to the recent seroepidemiological data concerning the relationship of EBV to the development of BL and the specific chromosomal translocations in BL cells. We shall put forward the view that ( I ) a BL clone develops in one or several steps from B lymphocytes that have acquired preneoplastic characteristics due to EBV transformation, that (2) the fully malignant (tumorigenic) phenotype develops only when the lymphoblastoid cell acquires a specific chromosomal translocation-i.e., the typical reciprocal 8 ; 14 translocation

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found in most BL cases, or its variants t(2; 8) or t(8; 22)-and (3) we shall discuss the hypothesis that the translocations act by the permanent switch-on of an oncogene located in the distal segment of chromosome 8, following its transposition into the immediate neighborhood of a functionally active Ig locus.

II. Lymphoblastoid Cell Lines (LCLs)

A. THETARGETCELLOF EBV INFECTION Only human and some nonhuman primate B lymphocytes can be infected by EBV under experimental conditions. This extraordinary host restriction is determined at the receptor level. Non-B cells of all examined species and even B cells of nonprimate origin lack EBV receptors. This selectivity is due to the fact that the virus uses a differentiated B-cell receptor as its receptor (Yefenof et al., 1976; Jondal et al., 1976). The EBV receptor is either identical with or located in the very close neighborhood of the C3d receptor of the B lymphocyte. This association may also explain why the virus preferentially infects IgM-producing B lymphocytes, although both IgG- and IgA-producing B cells can also be infected, but less regularly. It is known that the switch from surface IgM to IgG is frequently associated with the disappearance of the C3 receptor (Coda1 and Funderud, 1982). Artificial implantation of B lymphocytes with membranes from EBV receptor-positive cells increases the number of EBV-infectable IgG producers (Tsukuda et al., 1982). The only cell type of non-B-lymphocyte derivation that was found to carry EBV in viuo so far is the epithelial tumor cell of poorly differentiated or anaplastic nasopharyngeal carcinoma (NPC). It is not clear how the NPC cell or its precursor become infected (for review see Klein, 1979a). Fusion with an EBV-carrying lymphocyte has been considered as one possibility. It has not been excluded experimentally, however, that at least some epithelial cells may have EBV receptors. Recently, Ben Bassat et al. (1982) found that a human amnion line adsorbs EBV and responds with EBV-induced antigen synthesis in a minority of the cells. Nonnatural host cells that lack EBV receptors have been recently subjected to “receptor bypass” experiments. Four different methods were used : microcapillary injection of virus particles (Graessman et al., 1980); receptor “transplantation” by fusing coreconstituted cell membrane/Sendai virus envelope vesicles with various target cells, followed by exposure to EBV (Volsky et al., 1980); reconstitution of de-enveloped EBV particles with Sendai virus envelopes (Shapiro et al., 1981); and transfection of cultured fibroblasts or epithelial cells into EBV DNA (Miller et al., 1981 ; Stoerker

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et al., 1981). The results were quite surprising. All four methods showed

that a variety of target cells of both human and murine origin that were noninfectable under normal conditions could be infected after bypassing the receptor block, with EBV-antigen synthesis as the result. Susceptible targets included human T lymphocytes and derived leukemic cell lines, murine fibroblasts, murine T and B lymphocytes, and a variety of established mouse and human cell lines. Successfully infected cells of most types responded with the synthesis of early antigen (EA) and viral capsid antigen (VCA), i.e., the antigens associated with the lytic cycle. There was little or no detectable induction of the nuclear antigen, EBNA, the first vital product known to appear after the natural B-cell infection and the only viral or virally induced component regularly associated with transformation. The findings were particularly striking with normal mouse lymphocytes, in view of the strong contrast they provide in comparison with the human lymphocyte system. In human B cells, transforming EBV (B95-8 or B substrain) induces only EBNA, but no detectable EA or VCA synthesis. EBNA synthesis is followed by polyclonal activation of the B cells, DNA synthesis, mitosis, and cell immortalization (Miller and Yale, 1971; Rosen et al., 1977; Einhorn and Ernberg, 1978). The abortively lytic, nontransforming P3HR- 1 virus variant does not stimulate DNA synthesis and fails to induce EBNA or any other detectable EBV antigen (Menezes et al., 1975a) Its only known effect on the normal human B-cell target is the impairment of its viability. The effect of the two EBV substrains on receptor-implanted mouse lymphocytes was quite different (Volsky et al., 1981). B95-8 virus induced no EBNA at all, but did induce EA and VCA. P3HR-1 virus induced EBNA in a small proportion of the cells and induced EA and VCA in a much larger fraction of the cell population. Infectious EBV could be readily recovered from the P3HR-1 virus-infected mouse lymphocytes. Its biological characteristics corresponded to ordinary P3HR-1 virus: it was capable of inducing EA in superinfected Raji cells, a distinctive property of this substrain. Neither one of the two virus strains has been able to transform mouse lymphocytes into permanent lines so far, although the infected cultures survived several weeks (6-9), longer than the uninfected cultures. Arguing by analogy with the SV40 large T antigen, it has been widely presumed that EBNA synthesis must be a part of the early stage of the normal viral cycle. There is no experimental evidence in support of this idea, however (although the reasons for this could be technical). Meanwhile, one may consider an alternative possibility suggested by the aforementioned results. EBNA and EA may signal two alternative pathways of interaction between EBV and its host cell. Conceivably, EBNA synthesis may cause or at least occur concomitantly with a suppression of the viral cycle, which is in itself a prerequisite for immortalization. The appearance of EA without

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detectable EBNA would, on the other hand, signal the direct entry of the cell into the lytic cycle. The natural host cell, the human B lymphocyte, is obviously geared to the former pathway. All known inducers of the viral cycle in latently EBV-infected cells are capable of interfering with the expression of differentiation markers (Gerber, 1972; Hampar et al., 1972; Klein and Dombos, 1973; Tovey et al., 1978; zur Hausen et al., 1979). In somatic hybridization experiments between EBV-carrying B-cell lines and other cell types, virus-producer status and inducibility behave as differentiated B-cell properties (Zeuthen and Klein, 1981). It is therefore likely that the differentiation of the B cell makes it specially adapted to respond with EBNA, cellular DNA synthesis, suppression of the viral cycle, and transformation, rather than lytic infection. It is intriguing to speculate that the EBNA-associated cellular 53K protein (Luka et al., 1980) may play a key role in the scenario. This protein is similar to or identical with the transformation-associated 53K proteins described in other systems. In the SV40 system it forms a complex (Jornvall et al., 1982) with large T antigen (Lane and Crawford, 1979). The SV40 large T-53K complex is stabilized within the nucleus, whereas for 53K it is broken down rapidly; this may be a clue to the understanding of the role played by SV40 large T in transformation (Linzer et al., 1979). Could it be that B lymphocytes respond to EBV infection with the synthesis of the appropriate 53K, required for the production of the EBNA complex, which may in turn act as the suppressor of the viral cycle? The potential value of the scheme lies in the fact that it is available for experimental testing. In an equally speculative vein, it is conceivable that EBV has evolved from an originally highly lytic herpes virus that could infect many different cell types. The present EBV (and the related simian EBV-like viruses) may represent host-range mutants that were selectively favored due to their restriction to the B lymphocyte at the receptor level. The moderate nonlytic interaction with the B lymphocyte leads to a stable latent system that includes protection against reinfection. These two attributes may have increased the survival value of the virus. The immortalization of the target cell and the relative freezing of its differentiation that follows from this interaction (see Section VIII) will have selected the host species for efficient surveillance mechanisms, keeping the entire system under tight control. B. EARLYEVENTS DURING EBV TRANSFORMATION The earliest recognizable event that follows the infection of the human B lymphocyte with live transforming EBV is the appearance of the EBNA (Engblom and Ernberg, 1981). It is followed by the induction of host-cell RNA, protein, and DNA synthesis, in this order. The process is often com-

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pared to polyclonal B-cell activation (Bird and Britton, 1979). This analogy is quite valid, although there are differences in details, compared to the action of the commonly used B-cell mitogens. The latter usually require macrophage or T-cell help. In contrast, activation of the B cell by EBV is an “autonomous” interaction : it occurs readily with purified B-cell populations, and does not require accessory cells. Also, although mitogen activation is triggered by the interaction between the receptor and the ligand on the cell surface, activation by EBV can only occur with live but not with inactivated lines (Gerber, 1972; Robinson and Miller, 1975; Engblom and Ernberg, 1981). Compared to B-cell mitogen activation, still another difference lies in the uneven distribution of EBV receptors on different B-cell subclasses, leading to the asymmetric of predominance of IgM producers in the EBVactivated lymphocyte population, as already discussed in the previous section. Pokeweed mitogen (PWM) and other B-cell mitogens induce a more egalitarian activation of IgM and IgG producers (Bird and Britton, 1979). Recently, Tsukuda e f al. (1982) “transplanted” EBV receptors from a transformed B-cell line to normal human B-lymphocyte populations. On subsequent infection with transforming (B95-8) virus, the ratio between IgM and IgG plaque-forming cells shifted in favor of the IgG procedures. This made the picture more like mitogen activation and confirmed the notion that the asymmetry of the unmanipulated population is determined at the receptor level. Interestingly, the absolute number of activated JgM-producing cells increased as well, suggesting that not all IgM-producing B cells express EBV receptors. EBV activation also amplifies immunoglobulin (and, in the case of specific antigen-binding cells, antibody) synthesis and induces secretion in B lymphocytes (see Section VIII) (Rostn et al., 1977; Bird et al., 1981). This fact has been exploited to establish specific antibody-producing human lymphoblastoid lines, by combining the selection of antigen-binding B cells with subsequent EBV immortalization (Steinitz et af., 1977; Steinitz and Klein, 1980). It is also possible to select EBV-transformed lines that produce specific subclasses of immunoglobulin (e.g., IgA) (Steinitz and Klein, 1980) or rheumatoid factor (Steinitz et al., 1980). This method provides an important alternative to the hybridoma technique for the production of monoclonal antibodies of human origin. The early cellular DNA synthesis, induced by transforming virus, corresponds to a normal S phase. It can be followed by cell division and transformation into a permanent line. The question whether abortive transformation occurs and the frequency at which EBV-infected cells grow into permanent lines has not been explored in detail. The synthesis of EA is not “early” in the same sense as EBNA. Originally

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defined by immunofluorescence (Henle et al., 1970), it is now recognized that EA is a complex of at least nine different polypeptides (Kallin et af., 1979; Mueller-Lantzsch et al., 1979; Kawanishi et al., 1981). Its appearance signals the irreversible entry of the cell into the lytic cycle that leads inevitably to cell death. Combined immunofluorescence and radioautography have shown (Gergely et af., 1971) that the synthesis of EA is paralleled by a progressive inhibition of host-cell macromolecular synthesis. At least five components of the EA complex are DNA binding proteins (Roubal et al., 1980). Recent findings suggest that one of them, a 152K polypeptide, is required for subsequent viral DNA replication, which is in itself a prerequisite for late VCA synthesis (Kallin and Klein, 1982). The unique position of EBNA as the single known transformation-related protein is at least suggestive of the possibility that EBNA may be responsible for transformation. This is also supported by the finding (Klein et al., 1980) that microinjected EBNA stimulated DNA synthesis in contact-inhibited cells in analogy with the effect of microinjected SV40 large T antigen (Graessman et al., 1981). In conclusion, the sequence of early events after the infection of B lymphocytes with transforming EBV can be described as EBNA synthesis followed by cellular RNA, protein, and DNA synthesis, mitosis, and immortalization. The picture is strongly dominated by the role played by EBV as a polyclonal B-cell activator; IgM-producer lymphocytes are affected preferentially, with only a moderate representation of IgG and IgA producers. This is likely to be related to the distribution of EBV receptors on lymphocytes of the different Ig-producer subclasses. Receptor transplantation permits the more widespread activation of other minority Ig-producer subclasses. The critical events that lead to immortalization have not been analyzed in detail. Although it is clear that EBNA synthesis is an important prerequisite, it is not known what proportion of the EBNA-positive, blast-transformed B lymphocytes actually grow into immortal clones. It cannot be excluded that a certain proportion may differentiate terminally, and that some cells may abort the viral genome.

c. “SPONTANEOUS” ESTABLISHMENT OF LCLS Immunoglobulin-producing cell lines with a typical lymphoblastoid morphology and other distinctive characteristic properties can be established in vitro not only by experimental infection of B lymphocytes by EBV as described earlier, but also “spontaneously” after explantation of blood lymphocytes, bone marrow, or lymphoid tissue from any EBV-seropositive individual (Gerber et al., 1969; Chang et al., 1971b; Nilsson et al., 1971). This spontaneous establishment of a permanent LCL, which normally in-

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volves a lag phase of 30-90 days, was termed “lymphoblastoid transformation” by Benyesh-Melnick et al. (1963) in her original description of the phenomenon, as she assumed that it represented a phenotypic “transformation” of fibroblastic cells to malignant lymphoblasts. When the causative role of EBV in IM (Henle et al., 1967; Henle et al., 1968; Niedermann et af., 1968) and the capability of EBV to immortalize lymphocytes in uitro had been demonstrated (Pope et al., 1968; Diehl et al., 1969; Gerber e f al., 1969; Miller et al., 1969; Baumal et al., 1971; Chang et al., 1971a,b; Nilsson et al., 1971), it was suggested that the observed spontaneous lymphoblastoid transformation of Benyesh-Melnick represented a selection process in uitro of precursor B cells immortalized by EBV in uiuo (Sinkovics et al., 1967; Nilsson, 1971a; Klein, 1973). However, this view had to be revised once more when Rickinson, Epstein, and co-workers showed that the vast majority of the LCL cells were B lymphocytes which became immortalized by EBV in vifro and not in uivo (Rickinson ef al., 1974, 1975, 1977). Using cocultivation experiments they demonstrated that B lymphocytes latently infected by EBV in vivo underwent a lytic EBV-producer cycle after explantation in uitro, and that the released virus transformed normal B cells to immortal LCLs. Because the “spontaneous” establishment of an LCL also represents an in uitro infection of B lymphocytes by this two-step process, it is not surprising that the LCLs, regardless of derivation (blood, bone marrow, lymph nodes, spleen, tonsils) and method of establishment (spontaneous, cocultivation, EBV infection), are polyclonal. This has been convincingly demonstrated using synthesis of Ig isotypes (Glade and Chessin, 1968a; Bechet et al., 1972; Nilsson, 1971a) and glucose-6-phosphate dehydrogenase (G-6-PD) as markers (Bkchet et al., 1974). As will be discussed (Section IX), a clonal evolution takes place in LCL during continuous passage in uitro, and within about a year they have all become monoclonal (Bechet et al., 1974).

D. PHENOTYPIC CHARACTERISTICS OF LCLs The phenotypic properties of LCLs when examined during early in uitro passage seem to be basically identical regardless of the method by which they have been established (Table I). Neither have any systematic differences between LCLs established from different sources (blood, bone marrow, etc.) been described. This phenotypic constancy between different lines contrasts with the apparent morphological diversity (heterogeneity) within each cell line (Nilsson and PontCn, 1975). With prolonged passage ( > 6 months) in vitro, however, secondary changes of the phenotype may occur (Section IX).The following description of the major typical characteristics of LCL therefore concerns cell lines passaged in uitro for less than 3 months.

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LYMPHOMA

TABLE I PROPERTIESOF NEWLYESTABLISHED HUMANEBV-CARRYING LYMPHOID CELLLINES" Characteristic Morphology Cell type (diameter in microns, Itm) Diversity Between lines Within lines Ultrastructure (TEM)

Surface morphology (SEM)

Motility Time-lapse cinematography

Location of actin Growth characteristics Efficiency of establishment Length of lag phase during establishment (days)b Attachment to feeder cells

Growth in suspension Dependence on rich medium for maintenance in suspension Population-doubling time (hr) Maximal cell density (cells/ml) Colony formation in agarose

Surface characteristics Lymphocyte markers (% positive cells) SRBC C3 (EAC) Fc (EA) Fc (aggregated Ig) Ig

LCLS

BL lines

Lymphoblastoid (1 2- 13)

Lymphoblastoid (10-1 1)

None Prominent Moderate development of endoplasmic reticulum and Golgi apparatus Long villi often with asymmetric (uropod) location

Present Slight Sparsely developed Golgi apparatus and endoplasmic reticulum ; fat droplets Thin, short villi covering the entire surface

High-motilit y, translocation Surface villi; uropod

Low-motility, translocation

High 30- 100

High 0-30

Strong and rapid, most cells Large, dense clumps

Loose and slow, only some cells Single cells or usually small, loose clumps Yes or no

Yes

Diffuse, submembranous

24-48 1-1.2 x lo6 Yes, low cloning efficiency; microscopic colonies

18-30 1-3 x lo6 Yes or no; often high cloning efficiency; sometimes macroscopic colonies

0 60-95

0 0-95 0-60 2-3 70-100

0-5

2+ 70- 100

+

(continued)

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KENNETH NILSSON A N D GEORGE KLEIN

TABLE I (Continued) Characteristic

LCLS

BL lines

Surface glycoprotein pattern

B-blast-like

Lectin agglutinability HLA-A.B,C, antigens ,O,-Microglobulin No. molecules/cell Secretion (ng/ 10‘ cellsi24 hr) HLA-DR antigens 38.13 Antigen Common ALL antigen

3f Present Present 1O6 200-250 Present Absent Absent

B-cell-like; B-lymphoma pattern (gp 87/85, gp 71/69) 4+ Present (one exception) Present (one exception) Variable, 0-106 Variable, 0-80 Present Present (exceptions) Variable

Functionul markers Ig, surface Molecules/cell Ig, secretion (pg/ 10‘ cells/24 hr) Interferon production Phagocytosis

IgG, IgM, IgA. IgD 2 104 1-3 Yes Yes

IgM (single exceptions) 8 x lo4, never IgD 0 (exceptions) Yes Yes

Turnorigenicity (subcutaneously in nude mice)

No

Mostly, but not always

Kuryotype

Normal diploid

Aneuploid, t(8; 14) or t(2;8). or t(8;22)

Sensitivity to NK cells

Low

Variable. but usually high

a

For references see Nilsson (1979) and this article. “Spontaneous” establishment with the Spongostan grid culture method (Nilsson, 1971a).

A detailed review on the nature of established LCL has been published (Nilsson, 1979). This description will therefore be limited to biological properties of LCL cells relevant to the subject of this article.

1. Morphology The most easily recognizable and distinctive feature of an LCL is its morphology. The characteristic morphology has been studied not only by the use of light microscopy and various staining techniques, but also by scanning and transmission electron microscopy (for a review, see Nilsson, 1979). The major morphological hallmarks are summarized in Table I and illustrated by Figs. 1 and 2. The very active motility of individual cells within an LCL, as demonstrated by time-lapse cinematography (Clarkson et a/., 1967; Nilsson et al., 1970a; Nilsson, 1971b; Nilsson and Pontkn, 1975; Fagraeus et al., 1975), explains at least most of the morphological heterogeneity typical of LCL. During

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FIG. 1 . Prototype LCL growing on a fibroblast monolayer. Note typical clumps, the morphological pleomorphism, and the peripolesis in the feeder cells of isolated cells. Inverted microscope x 350.

the cell cycle, individual LCL cells exhibit a pronounced flexibility with respect to cell shape. A single cell growing on a fibroblast feeder layer may within a few hours present with a number of interchangeable cell shapes. At any time the most common cell within an LCL is “hand-mirror’’ shaped, but round and elongated cells are also common. That this morphological heterogeneity is mainly due to the flexibility with respect to cell shape of individual LCL cells and not merely to the polyclonal state of the cell line is further emphasized by the demonstration that the same heterogeneity was encountered in cloned LCLs (Nilsson, 1971~).However, it cannot be excluded that at least shortly after establishment, the pleomorphism is to some extent the result of an ongoing B-cell-plasma cell differentiation (Section VIII). In suspension most LCL cells adhere to each other to form characteristic clusters which can be dispersed only by vigorous pipetting. In these clumps the cells in the periphery are hand-mirror shaped and have long, thin,

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FIG. 2. Transmission electron micrograph of an LCL cell (U-296). Note the moderately developed endoplasmic reticulum and Gold apparatus. (From Nilsson, 1978.)

contractile surface villi recognizable by the inverted microscope and demonstrable by immunofluorescenceusing anti-actin antibodies (Fagraeus et al., 1975; Nilsson and Pontkn, 1975). Cell clumps of this appearance are almost unique to LCLs. Only in rare BL and T-leukemia cell lines may cell clusters with some similarity to those of LCLs be found. The general morphology of the EBV-transformed B cell conforms best with that of normal mitogen-activated B and T blasts (Douglas et a!., 1969). The cell is larger (mean diameter 12 pm) than a normal B cell (Nilsson, 1971b). Its cytochemical staining properties are also similar to those of activated lymphocytes. The cells stain intensively with methyl-green-pyronin and acridine orange, indicating an active RNA and protein synthesis (Nilsson, 1971~).Some periodic acid Schiff (PAS) activity and acid phosphatase, P-glucuronidase, and some esterases can also be demonstrated. The profile of cytochemical stains, however, varies considerably depending on the culture conditions (Sundstrom and Nilsson, 1977; Sundstrom et al., 1978) and can therefore not be used as indicative of any biological heterogeneity among LCLs as claimed by some authors (Karpas et al., 1977). The ultrastructural studies confirm that the morphology of LCL cells best corresponds to that of activated normal B cells which have undergone

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further differentiation along the B-lymphocyte-differentiation lineage (Section VIII). Thus the cells have numerous mitochondria and a comparatively well-developed Golgi apparatus, and they contain some stretches of rough endoplasmic reticulum in addition to the free ribosomes and the polyribosomes (Fig. 2). 2. Cell Surface Properties The immunological markers of LCL cells are summarized in Table I. Like normal B cells and B blasts, they express Ig and Fcy, C3, and EBV receptors. The frequency of Fcy receptor-positive cells and the density of Fcy receptors, as quantitated by '251-labeled aggregated IgG, seems to be lower than on normal B cells (Huber el al., 1976). The LCL cells express qualitatively unaltered products of the major histocompatibility complex (MHC ; HLA-A, B, C ; HLA-DR ; p,-microglobulin) (Bernoco et al., 1969; Papermaster et al., 1969; Rogentine and Gerber, 1969, 1970). The extra HLA specificities described in early studies (Bernoco et al., 1969; Ferrone et al., 1971;Dick et al., 1972; Lindblom and Nilsson, 1973; Pious et al., 1974) have later been explained by the presence of HLA-DR antibodies in some of the anti-HLA-A, B, C antisera employed (Bodmer et al., 1975; Dick et al., 1975). The surface expression of HLA-A,B,C is increased as compared to normal lymphocytes (McCune et al., 1975; Welsh et al., 1977). The amount of HLA per cell was found to be similar to (Welsh et al., 1977), or higher than on PHA- or pokeweed mitogen (PWM)-stimulated lymphocytes (McCune et al., 1975), and thus considerably higher (10- to 30-fold) than on peripheral T and B cells. Some aspects of the general composition of the surface membrane of LCL cells, as compared to normal and mitogen-activated B cells, have been studied. EBV transformation seems to be followed by an increased concanavalin A (Con A) induced agglutinability (DeSalle et al., 1972; Glimelius et al., 1975; Ben-Bassat et al., 1976), an increased amount of fucosyl glycopeptides (van Beek et al., 1979, 1981), and a decreased lateral motility of membrane glycoproteins (Ben-Bassat et al., 1976). Using the galactoseoxidase catalyzed tritiated sodium-borohydride surface-labeling technique, it could also be demonstrated that the LCL expressed a number of new major surface glycoproteins as compared to various normal blood cells including B lymphocytes (Anderson et al., 1977; Nilsson et al., 1977a; Anderson and Gahmberg, 1978; Gahmberg et al., 1978) (Fig. 3). However, the pattern of labeled surface glycoproteins was similar to that of B cells from blood stimulated by PWM and B cells from spleen activated by lipopolysaccharide (LPS) of Escherichia coli (Nilsson et al., 1977a). It cannot be decided as yet to what extent the various differences in surface characteristics between the EBV-carrying LCL cell and the normal B cell

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FIG. 3. Polyacrylamide gel electrophoresis patterns of surface glycoproteins of lymphoid cells labeled after treatment with neuraminidase and galactose-oxidase. (A) spleen B cells; (B) blood B blasts; (C) spleen B blasts; (D) BI line Ramos; (E) LL line U-698; (F) LL line U-715; (G) LB line U-61M; (H) LB line U-974; (I) BI line P3; (J) BL line Daudi; (K) BL line Raji; (L) myeloma line 8226; (M) blood T cells; (N) acute lymphoblastic leukemia line Molt 4. The apparent molecular weights of some relevant glycoproteins are indicated. (From Gahrnberg e? a/.. 1978.)

are related to the presence of the EBV genome per se in the former cell type, or are the result of the change in gene expression that would be expected to follow the shift in differentiation stage that B cells seem to undergo when transformed by EBV (Section VIII). However, the comparative phenotypic studies on clonal B-cell populations, represented by the EBV genomenegative Ramos and BJAB and their EBV-converted sublines, may be relevant in this context. After EBV infection the examined changes of the cell surface membrane were the same as in the EBV-transformed peripheral B cells, i.e., an increased Con A-induced agglutinability, an impaired lateral diffusion (capping), and a decreased Fc- but increased C3-receptor expression (Yefenof et af., 1977b; Jonsson et a]., 1980; Jonsson and Klein, 1981). However, as the simultaneous changes in morphology and surface Ig expression (IgM IgM IgD) were suggestive of an induced further differentiation along the B-cell differentiation lineage, one cannot but conclude that these studies have not clarified whether the surface changes were directly or indirectly (via induced differentiation) the result of EBV infection. --+

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3. Functional Properties The most prominent function of the LCL cell is its capacity for Ig production. Without exception, newly established lines synthesize secretory and surface-bound Ig. Consonant with the polyclonal derivation of the LCLs, all heavy-chain classes, except IgE, and the two light-chain types have been identified shortly after their establishment. However, with time a gradual selection of one clone will occur, and the synthesis of Ig is usually monoclonal after 1-2 years of continuous cultivation (Nilsson, 1971a; BechCt et al., 1974). Particularly in LCLs derived by EBV transformation in uitro of fetal lymphocytes, but also in cultures derived from adult lymphocytes, a predominance of IgM production is found (Section 11). However, in early studies the most common class of Ig produced was IgG (Tanigaki et al., 1966; Finegold et al., 1967; Nilsson et al., 1968; Takahashi et al., 1969a; Nilsson, 1971a), or IgG together with IgM (Litwin et al., 1973). In fact the relative frequency of Ig classes identified in the LCL corresponded roughly to the distribution of the Ig classes in normal serum (Nilsson, 1971c). The reason(s) for the discrepancy between the early and later studies have not been clarified. Possible explanations, however, are (a) that differences in selective pressure during the establishment and the early passages may have yielded LCLs with differences in predominant Ig isotype and (b) that IgM, which is mainly surface bound, may have escaped detections in several early studies where assays for Ig secreted to the medium and not immunofluorescence techniques were used. LCLs usually produce complete Ig molecules both for expression at the surface membrane and for secretion (Tanigaki et a/., 1966; Wakefield et al., 1967; McConahey et al., 1971; Litwin et al., 1974; Nilsson and Ponten, 1975; Gordon et al., 1977). However, in some lines a secretion of free light chains has been demonstrated in addition to the intact Ig molecules (Nilsson, 1971~).Exceptional cell lines may lose the capacity to produce complete Ig molecules during prolonged passage in uitro (Tanigaki et al., 1966; Yount et al., 1976). In such lines exclusive production of light or of heavy chains is found. All cells within an LCL synthesize Ig as demonstrated by cloning experiments and subsequent analyses of surface Ig by immunofluorescence (Hinuma and Grace, 1967; Takahashi et a/., 1969b; Bloom et a/., 1971 ; Nilsson, 1971c; Litwin and Lin, 1976). Individual cells usually produce only one type of Ig, but may occasionally synthesize two heavy chains (usually 1' and p) but only one type of light chain. The rate of Ig secretion in LCL during optimal growth varies between 1 and 3pg/106 cells/24 hr (Fahey and Finegold, 1967; Matsuoka et al.,

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1968; Nilsson and Ponten, 1975). The mean amount of surface Ig per cell in an asynchronously growing LCL seems to be in the order of 2 x lo4 molecules (McConahey et al., 1971). The synthesis, assembly, and export of Ig molecules in LCL cells take place during separate phases of the cell cycle. The synthesis was found to be maximal in late G, and early S phase (Buell and Fohey, 1969; Lerner and Hodgke, 1971), and in addition in late G 2 (Watanabe et al., 1973), whereas the intracellular accumulation and secretion occurred slightly later and peaked in late S and G 2 phases. Some LCLs have been found to produce Ig with antibody activity (Evans et al., 1974; Steel et al., 1974; Joss et af., 1976), demonstrating that the induction of Ig secretion after EBV transformation corresponds to that in normal B lymphocytes after antigen or mitogen stimulation. As mentioned before, this fact has been utilized in the production of human monoclonal antibodies. Thus several laboratories have succeeded in immortalizing B lymphocytes, usually preselected for their production of specific antibody (anti-SRBC, anti-hapten, anti-tetanus toxoid, anti-chlamydia) by EBV (Luzzati et al., 1977; Steinitz et al., 1977, 1979a,b; Zurawski et al., 1978; Kozbor and Roder, 1981) and rheumatoid factor (Steinitz et a/., 1980). After a series of successive subcloning steps of the established LCL cells, it has been possible to isolate clones with a stable, high rate of production of specific antibody (Rosen et al., 1982). In conclusion, LCL cells synthesize complete Ig molecules predominantly of the IgM class. The Ig is both secreted and expressed on the plasma membrane and is usually produced at a rate which corresponds to 1 :3- 1 :5 of that observed in human myeloma lines. With respect to these aspects of the Ig synthesis, LCL cells appear to correspond to antigen-stimulated B blasts within the normal B-lymphocyte-differentiation lineage (Section VIII). Other functional properties (Table I) documented in LCLs are the production of various kinds of lymphokines and interferon. Macrophageinhibition factor (MIF) (Glade et al., 1970; Granger et al., 1970; Papagiorgio et a/., 1972; Tsuschimoto et al., 1972; Tubergen et al., 1972), leukocyte migration inhibitory factor (LIF) (Ashorn et af., 1982),lymphoblastogenesisinhibition factor(s) (Smith et al., 1970; Hersh and Drewinko, 1974; Han et al., 1975), and lymphotoxin having the capacity to destroy allogenic fibroblasts (Granger et al., 1970; Lisafeld et af., 1980), are examples of such putative mediators of cell-mediated immunity. Both MIF and LIF produced by LCLs have biochemical and biological properties indistinguishable from the corresponding prototype factors produced by sensitized guinea pig macrophages (Tubergen et al., 1972). Interferon may be produced spontaneously at a low rate and more actively after induction with Sendai virus (Deinhardt and Burnside, 1967; McCombs and Bengesh-Melnick, 1967;

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Kasel et al., 1968; Zajac et al., 1969; Haase et al., 1970; Minnefor et al., 1970; Adams et al., 1975). Finally, LCL cells have some phagocytic activity (Kammermeyer et al., 1968; Sundstrom, 1977). This, together with the capacity to produce MIF, interferon, C3 (Glade and Chessin, 1968b), and the adherent growth on feeder cells (and in old lines also on plastic surfaces), demonstrate that LCL cells have some properties commonly ascribed to cells of the monocytemacrophage cell series. 4. Growth Characteristics

With the exception of BL cell lines, LCLs are the most easily established type of hematopoietic cell line. This is exemplified by the fact that LCL cells, but not the neoplastic cells, often become established at a high frequency in most non-BL leukemia, and myeloma (Nilsson and PontCn, 1975). In mixing experiments the growth advantages of autologous LCL cells over malignant hematopoietic cells have been demonstrated (Nilsson et al., 1970b; Nilsson, 1971b). In the absence of feeder cells the growth of LCL cells is dependent on a rich medium (e.g., RPMI 1640, F-10, F-12), but when such cells are present in the culture they may, like BL cells, grow in simpler media (e.g., Eagle’s MEM). LCLs can be grown in low serum concentrations, and may survive and even proliferate in serum-free F-10 for months (Nilsson, 1971~).However, for optimal growth 5-10% fetal or newborn calf serum is required. Lymphoblastoid cell lines are maintained as nonstirred suspension cultures. The proliferation mainly takes place in cells in the periphery of the typical cell clumps (Levy et al., 1968).This probably explains why suspension cultures with continuous stirring (e.g., Spinner cultures), in which the cells are prevented from forming clusters, will be inferior to the stationary suspension cultures. The population-doubling time at logarithmic growth is usually around 30 hr. Proliferation and viability cannot be maintained in LCL cultures at cell densities < lo4 cells/ml. The maximal cell density is 1.2-1.5 x lo6 cells/ml. LCL cells cannot be easily cloned by single-cell dilutions in microwells or by colony formation in agarose (Nilsson and PontCn, 1975; Nilsson et al., 1977b). The very low efficiency of colony formation may, however, be increased by the use of feeder cells (Nilsson, 1971~). It is likely that the ease by which LCL cells can be maintained in vitro is the result of their EBV-carrier state. This assumption is based mainly on the observations that the EBV genome-negative BL cell lines Ramos and BJAB became less strict in their nutritional (serum) requirements, and less sensitive to cell crowding after EBV conversion (Steinitz and Klein, 1975, 1976, 1977).

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The virus-negative cell lines did not grow in 10% dialyzed fetal calf serum unless reconstituted with the dialysate, whereas the EBV-converted sublines proliferated well in the dialyzed serum. These EBV-associated changes are reminiscent of the decreased serum dependence-probably exogenous growth factor(s)-characteristic for DNA (polyoma, SV40) and RNA (e.g., Rous sarcoma virus, RSV) virus-transformed monolayer cultures (Temin, 1968; Jainchill and Todaro, 1970). The same growth-promoting effect of the EBV genome has also been demonstrated in chronic lymphocytic leukemia (CLL) cells, which could be maintained as continuous cell lines after infection by EBV, although they never become spontaneously established as cell lines in vitro (Karende et al., 1980). E. CYTOGENETIC STUDIES ON LYMPHOBLASTOID CELLLINES Most chromosomal analyses on LCLs were performed prior to the introduction of the banding techniques (for a review see Nilsson, 1979) and before it had been clarified that secondary cytogenetic changes took place in LCLs during continuous culture (Nilsson and PontCn, 1975). The early reports, therefore, contained results on both newly established and old cell lines, and the conclusions about the karyotypic characteristics of LCLs were consequently quite confusing for a long time. However, with the introduction of the improved staining techniques, several reports could document that newly established LCLs had normal diploid karyotypes. LCLs examined so far include lines established spontaneously from blood of patients with acute IM (Jarvis et al., 1974; Steel et al., 1977, 1980) or with leukemia (Hellriegel et al., 1977; Steel et al., 1977, 1980); from lymph nodes of patients with Hodgkin’s disease (Zech ef al., 1976; Hellriegel et al., 1977); and lines established from peripheral blood lymphocytes by EBV infection in uirro (B95-8 and X-35-BL viruses) or by cocultivation with X-irradiated EBV-producing lymphoid cell lines (Jarvis et al., 1974; Zech et al., 1976; Hellriegel et al., 1977; Steel et al., 1977, 1980; Giovanella et al., 1979). A minimum of 100 recently established LCLs have been examined in several laboratories, and without exception they had a normal diploid karyotype. It seems safe to conclude, therefore, that the EBV infection of a B cell, although conferring to the cell one neoplastic feature (immortality), is not followed by any chromosomal alteration whatsoever. Secondary chromosomal changes may, however, be detected in polyclonal LCLs as early as after 2-3 months of cultivation (Giovanella et al., 1979). After 1 year of continuous cultivation most (80%) of the lines will be aneuploid (Steel et al., 1977, 1980). The secondarily altered LCLs usually remain near diploid, but major rearrangements and a change in the chro-

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mosome mode to hyper- or hypodiploidy, and even to tetraploidy, have been recorded after years of cultivation (Huang et al., 1969; Steel, 1971; Steel et af., 1977, 1980; Nilsson and Ponten, 1975; Venaut et al., 1978). Both gains and losses occur, the former more frequently than the latter (Steel et af., 1977, 1980). These gains were nonrandom with common trisomies for chromosomes 3, 7, 8, 9, 12, and 21. IN NUDEMICE F. TUMORIGENICITY

Recently established polyclonal and euploid LCLs do not form tumors in the subcutaneous space in athymic nude mice (Diehl et af., 1977; Nilsson et al., 1977b). This absence of growth potential in uiuo correlated with a nonexistent or very low capacity for colony formation in agarose in uitro (Nilsson 1977b). Furthermore, polyclonal diploid LCLs may grow progressively as polyclonal diploid tumors in nude mice when inoculated intracerebrally (Giovanella et af., 1979; Schaadt et al., 1979). Only one of 51 tested LCLs failed to form a tumor. However, old aneuploid LCLs and BL cell lines, as well as other hematopoietic malignant cell lines, could grow autonomously both subcutaneously and intracerebrally in the nude mice, and in agarose in uitro (Nilsson et al., 1977b). The previously reported findings that LCLs from normal individuals can be tumorigenic in immunosuppressed or newborn hamsters, rats, or mice (Christofinis, 1969; Adams et al., 1970, 1973; Imamura et al., 1970) were therefore most probably due to the use of old, chromosomally altered cell lines in these tests. Alternative explanations may be that different animal species and inoculation routes were used. Taken together, the recent nude mice studies suggest (a) that EBV infection of a normal B lymphocyte will not give to the cell the same neoplastic potential as found for BL cells (capacity for subcutaneous growth) but will lead to a preneoplastic state (growth intracerebrally), and (b) that tumorigenicity subcutaneously correlates with another common marker for malignancyaneuploidy. The results also demonstrate (c) that the nude mice system, when used for assessment of malignancy, is complex and that perhaps only tumor formation subcutaneously is acceptable as a criterion on malignancy. The mechanisms by which the growth of LCLs are restricted subcutaneously in the nude mouse are largely unknown. The simultaneous acquisition of potential for growth subcutaneously and in agarose in uitro of old LCLs suggests that microenvironmental factors play a role. Some circumstances would indicate, however, that the growth subcutaneously is also restricted by immunological host mechanisms. First, LCLs may grow intraperitoneally or subcutaneously in nude mice under

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naturally (newborn) or experimentally induced (antilymphocyte serum or X-irradiation treatment) immunosuppressive conditions (Giovanella et al., 1979; Watanabe et al., 1980). Second, LCLs grow in the brain, which is well known as an immunologically privileged site (Medawar, 1948; Greene, 1953). In humans this is reflected by the fact that neoplastic lymphoproliferation during iatrogenic or genetically determined immunosuppression is particularly prone to occur in the brain (Schneck and Penn, 1971; Penn, 1978). The nude mice have a powerful natural killer cell (NK) system (Kiessling et al., 1975; Herbermann et al., 1975) and an intact potential for antibody formation. The possibility that NK cells were involved in the prevention of growth of LCL cells subcutaneously was investigated (McCormick et al., 1981). It was not possible, however, to find any difference in sensitivity to NK cell-induced lysis in vitro between LCL and BL cells, although only the latter cell type was tumorigenic. The lack of NK-cell sensitivity of LCL in the nude mouse, as shown by these experiments, confirms the previous experiments with mouse (Haller et al., 1977) and human (Jondal et al., 1978; Pattengale et al., 1981) NK cells, suggesting that LCLs are insensitive to NK lysis. Attempts were also made to investigate the importance of the B-lymphocyte part of the immune system by comparing the take incidence subcutaneously in nu/nu athymic Swiss mice with that in athymic and asplenic NFR and NIH-2 mice (Giovanella et al., 1980). However, as the nature of the B-cell defect in the asplenic, athymic mice is unclear and the number of LCLs tested was limited, one cannot but conclude that the nature of immunological restriction of LCL cell growth subcutaneously is essentially unknown. Ill. EBV-Carrying Burkitt's Lymphoma (BL) Cell Lines

Burkitt's lymphoma cell lines usually become established in vitro with a sonsiderably shorter lag phase (2-4 weeks) than spontaneously established LCLs (2-3 months) (Nadkarni et al., 1969; Nilsson, 1977). The frequency of success (50-75%) is higher than for any other human tumor, a fact that may be due to the presence of the EBV genome (Section 11). Cell lines obtained from BL biopsies originate almost without exception from the tumor cells, as demonstrated by comparative studies on biopsy cells and derived cell lines using immunoglobulin isotype, G-6-PD isoenzymes, and chromosomes as markers (Fialkow et al., 1970, 1971, 1973; Bechet et al., 1974; Zech et al., 1976). In the exceptional cases, cell lines with LCL phenotype were established, but then only when using grid organ cultures with feeder cells, and after the long lag phase typical of the spontaneous establishment of LCLs (Nilsson and PontCn, 1975; Nilsson, 1979).

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A. PHENOTYPIC CHARACTERISTICS Burkitt’s lymphoma cell lines have a common basic phenotype (Table I), which in many respects is similar to that of EBV-negative B-lymphoma lines (Sections V and VI). Many of the distinct differences between the basic lymphoma and LCL phenotypes most probably reflect the general difference in stage of differentiation (Section VIII). On careful examination, however, the phenotype of each BL line is unique, underlining the commonly described individuality of human tumors. The phenotypic stability of BL lines has not been systematically studied, but the chromosomal evolution described in BL lines (Steel et al., 1977, 1980) suggests that secondary changes may occur, although perhaps to a lesser degree than in LCLs. The following description of the BL properties therefore concerns mainly those of recently established lines. 1. Morphology The cytology of BL biopsy cells is quite typical. The BL cell is usually described as an immature lymphocyte containing numerous fat vacuoles (Epstein and Barr, 1964, 1965; Pulvertaft, 1964a,b; Epstein et al., 1965a,b,c; 1966a,b; Stewart et al., 1965; O’Conor and Robson, 1965; Rabson et al., 1966; Pope et al., 1967; Nilsson and PontCn, 1975). However, the current FAB classification recognizes an acute B leukemia (L3) in which the malignant cells are cytologically indistinguishable from the classical BL (Flandrin et al., 1975; Ganick and Finlay, 1980) and may furthermore have a BLtype chromosome translocation t(8q; 14q+) (Berger et al., 1979a; Mitelman et al., 1979). With the exception of a slight increase in volume, the BL cells seem to retain their basic morphology when established as BL cell lines. Burkitt’s lymphoma cells are usually uniformly round (Fig. 4), but in exceptional lines cells may have an elongated or pear-shaped form. As compared to LCL cells, the BL cells are somewhat smaller (mean diameter 10-1 1 pm) and the flexibility with respect to cell shape is limited, explaining the morphological homogeneity of BL cell lines. The BL cells are comparatively immobile. Time-lapse cinematography has revealed that the motility is confined only to the cell surface, leading to only minor modifications of the cell shape (Nilsson and Ponten, 1975; Fagraeus et nl., 1975). No translocational movements were found. This comparatively restricted mobility is also reflected by the staining pattern with anti-actin antibodies (Fagraeus et al., 1975). At the ultrastructural level (Fig. 5), the difference in maturation as compared to LCL cells is evident (Epstein and Achong, 1965; Epstein et al., 1966; Pope et al., 1967; Moore et al., 1968; Hammond, 1970). The ultra-

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FIG.4. BL cells seeded onto a fibroblast monolayer. Note the uniform monotonous morphology, absence of cell clusters, and absence of peripoletic activity on the feeder cells. Inverted microscope x 300.

structural features of BL cells compare best to those of resting or slightly immature B cells. The nucleocytoplasmic ratio is high ; the numerous ribosomes are present either in a free state or as polyribosomes. Rough endoplasmic reticulum is only infrequently developed, i.e., in the Seraphina line (Klein et al., 1975). The mitochondria and the Golgi apparatus are developed only to a limited extent.

2. Cell Surface Characteristics The marker profile of BL cell lines is summarized in Table I and demonstrates again the far greater heterogeneity within the group of BL lines than encountered among LCLs. As previously stressed, this heterogeneity is probably due not only to the neoplastic state per se but also to the variability in differentiation stage. BL cells usually express easily detectable surface Ig but in some lines (producing cytoplasmic p chains) surface Ig is undetectable. Similarly, most

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FIG.5. Transmission electron micrograph of a BL cell showing the high nucleocytoplasmic ratio, the poor development of Golgi apparatus, the absence of endoplasmic reticulum, and the numerous polyribosomes. (From Nilsson, 1978.)

BL lines express C3 and low levels of Fc receptors but some lines are negative (Huber et al., 1976; Nilsson, 1979). The surface expression of MHC antigens in BL cell lines is quantitatively more variable than in LCLs (Ostberg et al., 1975; Welsh et al., 1977). Except in one line (Daudi), HLA-A,B,C and &-microglobulin (&p) can be demonstrated (Nilsson et al., 1973; Bodmer et al., 1975). HLA-DR is expressed on BL cell lines including the HLA and / ? - i t negative Daudi (Bodmer et al., 1975; Dick et al., 1975; Trowbridge et al., 1977). The lack of surface HLA-A,B,C on Daudi cells is not due to gene defects, as “DaudiHLA” was detectable on Daudi-BL cell hybrids (Ber et al., 1978) and demonstrated intracellularly by biosynthetic methodologies (Ploegh et al., 1979; Sege et al., 1981). It has been suggested that the absence of &i, due to a deletion of part of chromosome 15 (Goodfellow et al., 1975; Zech et a/., 1976), is responsible for the failure in the processing of intracellular HLAA,B,C to become exposed at the plasma membrane (Sege et al., 1981). Some qualitative differences in the surface glycoprotein composition have been demonstrated between LCL and BL cells. Using the galactose-oxidase catalyzed tritiated sodium-borohydride surface-labeling method, it was

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shown that the pattern of major surface glycoproteins of BL cells was distinct from that of LCL cells and mitogen-stimulated normal lymphoblasts, but had some resemblance to that of peripheral blood lymphocytes (Fig. 3). Furthermore, and more importantly, four species of surface glycoproteins, present as double bands on the gels, were detected (gp85/87, gp69/71) (Nilsson et al., 1977a; Gahmberg et al., 1978). The gp69/71 bands have never been found on normal resting or mitogen (LPS, PWM)-stimulated B and T lymphocytes, other normal hematopoietic cells, leukemic non-T, non-B cells, leukemic T cells, myeloid leukemia, erythroleukemia, myeloma, and histiocytic lymphoma of the B-cell type (Andersson et al., 1977, 1979; Nilsson et al., 1977a, 1981; Andersson and Gahmberg, 1978; Gahmberg et al., 1980). However, they were also detectable in EBV genome-negative B lymphomas (U-698, U-715) and in the EBV genome-negative BL cell lines Ramos and DG-75 (Nilsson et al., 1977a). The EBV genome-negative BL line BJAB expressed only a gp71 band (Nilsson et al., 1977a). Interestingly, after EBV conversion the complete gp 69/71 appeared in the BJAB cells (Koide et al., 1982). The nature of the gp 69/71 molecules is unknown. First, they do not seem to represent C virus-coded glycoproteins as suggested by their lack of reactivity with a panel of anti-gp70 sera (Nilsson, unpublished). Second, they may be normal, differentiation-linked surface structures, as the phenotype of all examined B lymphomas was similar. Finally, they may represent malignancy-associated glycoproteins. The latter possibility has not been explored as yet in detail by tumorigenicity testing in the nude mouse system, but is suggested by the fact that BJAB after conversion by EBV not only expressed both gp69 and gp71 but also increased its tumorigenic potential (Koide et al., 1981). The expression of a few other surface structures has been studied in BL and LCL cells: two are defined by cellular reactivities and two by antibodies. The target structure for EBV-specific T-killer cells (LYDMA) is present on both cell types, whereas the NK target structures appear to be present preferentially on BL cells (Jondal et al., 1978; Pattengale e f al., 1981). The monoclonal antibody 38 : 13 reacts with most BL cell lines, but not with LCL cells, with the possible exceptions of some long-established lines (Wiels et nl., 1981; Klein et al., 1982). Antibodies against the common CALLantigen-a IOOK glycoprotein (Greaves e f al., 1975; Greaves and Janossy, 1978)-react with a large fraction of BL lines (Minowada et al., 1982), but have never been found on LCL cells. Other membrane properties have been found to be similar in LCLs and BL cell lines. In both cell types the cap formation, after exposure to fluoresceinated Con A, is reduced as compared to normal lymphocytes (Ben-Bassat et al., 1976), and the Con A-induced agglutinability (De Salle et al., 1972;

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Glimelius et al., 1975; Ben-Bassat et al., 1976) and the amount of fucosyl glycopeptides are increased (van Beek et al., 1979,1981). All these alterations may be ascribed to the presence of EBV genome in both types of cell lines, as it has been demonstrated that they will appear in EBV-genome-negative BL cell lines (BJAB, Ramos) (Yefenof and Klein, 1976; Yefenof et nl., 1977a,b) and normal B lymphocytes after EBV conversion (van Beek e f d., 1981).

3. Functional Characteristics Most, if not all, BL cell lines seem to produce Ig (Finegold et nl., 1967, 1968; Klein et al., 1967, 1968; Osunkoya et a/., 1968; Nadkarni et nl., 1969; Sherr et nl., 1971; van Furth et nl., 1972; BCchet et nl., 1974; Nilsson and PontCn, 1975). In the most extensive early studies a variable fraction (2050%) of the lines were reported negative (Finegold et a/., 1967; van Furth et nl., 1972). It has been shown, however, the BL cell lines may synthesize cytoplasmic chains not expressed at the cell surface or secreted to the medium (Preud’homme et al., 1978). Therefore, the true frequency of nonproducing BL cell lines seems to be unknown. Such lines, probably frozen at a differentiation stage similar to that of pre-B cells, would not have been detected in these studies. With only a few exceptions, the Ig produced is of the IgM class expressed at the surface membrane, in Daudi cells shown to be in monomeric form (Eskeland and Klein, 1971), with the extra sequence of amino acids present terminally in the Fc part of the heavy chains, typical of membrane-bound Ig (Williams et al., 1978; McCune et al., 1980; Singer and Williamson, 1980). Only in one of the exceptional lines (Seraphina) has the production of secretory IgG been reported (Klein et al., 1975). The type of Ig production (cytoplasmic 11 chains, expression of surfacebound IgM, or secretion of IgG) probably reflects that BL cell lines represent B cells frozen at different positions along the B lymphocyte-differentiation lineage (see Section VIII). The rate of Ig synthesis is lower in BL than in LCL cells, whereas the expression of Ig at the surface is higher, a fact that again probably reflects differentiation-linked properties. With respect to other functional properties (e.g., interferon, MIF, C3, and lymphotoxin production and phagocytosis), no consistent differences between BL and LCL cells have been documented (Table I). 4. Grow th Properties The heterogeneity of BL lines is also evidenced by the variability in their growth characteristics (Nilsson, 1979). Most BL lines grow in simple media

344

KENNETH NILSSON AND GEORGE KLEIN

like MEM, whereas exceptional lines require richer media like RPMI 1640, and even feeder cells for maintenance (Nilsson and Ponten, 1975). The population doubling time is generally shorter and the maximum cell density higher than in LCLs. Burkitt’s lymphoma cells grow as single cells or in small loose clusters or short chains. They will attach to feeder cells but slowly (days), in contrast to the rapid attachment of LCL cells (hours). Burkitt’s lymphoma cells usually grow with a high cloning efficiency in agar or agarose, often with formation of macroscopically visible colonies (Moore, 1972; Nilsson and Pontkn, 1975; Nilsson et al., 1977b). In conclusion, the general morphology of BL cell lines is distinct from that of LCLs. However, as some morphological heterogeneity (probably reflecting differences in differentiation stage) is encountered among BL lines, the distinction between the two types of EBV-carrying cell lines may be difficult on morphological grounds only (e.g., Seraphina and Maku). The morphology of the vast majority of BL lines conforms best to that of immature and mature blood B lymphocytes. The data on growth properties, surface characteristics, and Ig production confirm that there is heterogeneity among BL lines, that the phenotypes of various BL lines conform with that of normal B lymphocytes of several distinct differentiation stages, and that BL cells are biologically different from EBV-immortalized normal lymphocytes (LCLs). B. CYTOGENETIC STUDIES All BL lines when examined by banding techniques have been found aneuploid (for articles see Nilsson, 1979; Mitelman, 1981). In the prebanding era they were reported to be near diploid or even diploid. The chromosome 14q+ marker was first described by Manolov and Manolova (1972). The nature of the 14q+ marker was later demonstrated to be a 8q- ; 14q+ translocation (Zech et al., 1976), suggested to be reciprocal by Manolova et al. (1979). The specificity of this translocation in BL has been indicated by the finding of constant breakpoints on the two chromosomes involved (q24; q32). Subsequently the marker has been an almost consistent finding in both endemic and nonendemic BL biopsy cells (15/16; Manolov and Manolova, 1972; Zech et al., 1976), as well as in derived cell lines (28/31: Manolov and Manolova, 1972; Petit et al., 1972; Jarvis et al., 1974; Zech et al., 1976; Kaiser-McCaw et al., 1977; Manolova et al., 1979; Mitelman, 1981). In fact all biopsies with unquestionable histopathology and probably all authentic cell lines in these studies seem to have a BL-type translocation. Recently, the findings of variant translocations, t(2; 8), t(8; 22) in African as well as non-African BL patients and cell lines, show that the t(8; 14),

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345

although characteristic and highly frequent, is not specific for BL (Berger et al., 1979b; Miyoshi et al., 1979; Van den Berghe et al., 1979; Lenoir et al., 1982). It may be significant that the breakpoint on chromosome 8 (band q23-4) in all cases is identical to the breakpoint characteristic of the t(8; 14). The importance of these findings to the development of BL will be discussed in Section XII. A 14q+ marker has been identified in a number of other lymphoid and even some nonlymphoid neoplasms (for a review see Mitelman, 1981). Except in some acute B-lymphocytic leukemias (considered a leukemic form of BL), the chromosome piece translocated to chromosome 14 was, however, derived from chromosomes other than No. 8. In addition to these translocations, other chromosome abnormalities have been described. Among the occurring numerical changes, trisomy 7 is found in addition to random gains and losses and unidentifiable marker chromosomes (Steel et al., 1977, 1980; Zech et al., 1976). The trisomy 7 may be significant, as it is also frequent in non-BL B-lymphoma cell lines (Zech et al., 1976). Other reported deviations from normalcy have been minor numerical anomalies and deletions (Steel et al., 1980; Zech et al., 1976). The loss of a region on the long arm of one chromosome 15 has been of particular interest, as it was associated in the Daudi line with the absence of, and in Namalwa with reduction of P,-microglobulin production (Nilsson et al., 1973; Zech et al., 1976; Zeuthen et al., 1977). Some chromosomal evolution occurs in BL cell lines during prolonged cultivation in uitro, although it may be less pronounced than in LCLs (Steel et al., 1980). The expression of the t(8; 14) and the trisomy 7 (if present) appear to be stable abnormalities (Steel et al., 1980). IN NUDEMICE C. TUMORIGENICITY

Unlike LCL cells, but in accordance with their neoplastic derivation, BL cells will in most cases be tumorigenic subcutaneously in nude mice (Diehl et al., 1977; Nilsson et al., 1977b). This confirmed earlier work, using other test systems for heterotransplantation (Imamura et al., 1970; Levin et al., 1969; Southam et al., 1969a,b), that BL cells usually were tumorigenic. The few nontumorigenic cases included not only recently established cell lines but also some that were several years old, indicating that capacity for tumor formation might be a more stable property in BL cell lines than in LCLs (Nilsson et al., 1977b). As in the case of LCL, the capacity for growth in uiuo correlated with the efficiency of colony formation in agar or agarose in uitro (Nilsson et al., 1977b). It has not been clarified whether the tumorigenicity correlates also with any of the specific chromosomal trans-

346

KENNETH NILSSON AND GEORGE KLEIN

locations associated with BL cells. Neither has the importance of the surface glycoproteins associated with B-lymphoma cell lines (gp69/71) for the tumorigenic potential been examined. IV. Basis for Distinction between EBV-Carrying Lyrnphoblastoid and BL Cell Lines

As described, recently established LCL and BL cell lines have several distinct biological properties (Table 11). Basically, some phenotypic heterogeneity is encountered among BL lines, whereas within the group of LCLs only limited variation is found between individual cell lines. This is particularly pronounced for the morphological aspects of the two types of EBV-carrying cell lines. For LCLs the morphological diversity is pronounced within, but virtually absent between the lines. This is in contrast to the BL cell lines, where the morphological diversity is evident between, but only minimal within the lines. Morphology stands out as the easiest and best single parameter for distinguishing between LCLs and BL cell lines. Other important defining features are surface glycoprotein pattern and mode of Ig production (membrane-bound or secretory Ig, Ig class), which, like the morphology, most probably reflects the difference in differentiation stage rather than a difference between normal and neoplastic cells. V. EBV Genome-Negative BL Cell Lines

EBV genome-negative cell lines have mostly been established from nonAfrican BL (Klein et al., 1975; Epstein et al., 1976; Ben-Bassat et al., 1977; Miyoshi et al., 1977b; Magrath et al., 1980; Lenoir et al., 1982). The rare EBV-negative African BLs are represented by the BJAB and Abana cell lines (Menezes et al., 1975b; Magrath et a/., 1980a,b). The number of cell lines of this category as compared to EBV-positive BL cell lines is still limited, and only a few have been investigated extensively enough with respect to phenotypic properties to allow but a few safe conclusions about possible general differences or similarities as compared to the EBV-carrying LCLs and BL lines. Another important point of concern, when making an attempt to evaluate the phenotypic characteristics of the EBV genome-negative cell lines in comparison with the EBV-carrying BL cell lines, is the precision in the diagnosis of the original tumor at least in two cases, the BJAB and the DG-75 (Menezes et al., 1975; Ben-Bassat et al., 1977). The features of a collection of EBV-negative BL lines as described in the original articles are summarized in Table 111. The lines recently reported by

B-CELLLINESAND

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TABLE 11 DEFINING FEATURES OF EBV-CARRYING LYMPHOBLASTOID AND BL CELL LINES' Characteristic Clonality Karyotype Tumorigenicityb Morphology Cytology Diversity Growth Surface glycoprotein pattern Immunoglobulin production

LCLS

Polyclonal Normal diploid Negative

Monoclonal Aneuploid Positive, rarely negative

Typical hand-mirror shaped lymphoblasts Within but not between lines Mainly in clumps gp160, gpl15, B blast-like Surface Ig Secretion

Uniformly round lymphoblasts

All Ig classes a

BL cell lines

Between lines but only slight within lines Mainly as single cells gp210, gp85187, gp69171 Surface Ig (Exceptionally, secretion, exceptionally cytoplasmic p chains) IgM (exceptionally IgG)

Examined within 6 months after their establishment.

* Tested subcutaneously in athymic nude mice.

Lenoir et al. (1982) have been examined with respect to chromosomes and Ig production and will be discussed only in the text. It seems unquestionable that the extent of phenotypic heterogeneity within the group of EBV-negative BL lines is at least as pronounced as among the conventional EBV-positive BL lines. Another similarity is that they represent the same different stages of B-lymphoid differentiation (Table V) as the EBV-carrying BL cell lines (Section VIII). Thus one line described by Lenoir et al. (1982) have a pre-B-cell phenotype. All tested lines in Table I11 express CALLantigen and, in addition, surface IgM, which suggests that they correspond to cells at a stage intermediate between pre-B cells and B cells. The CA 46 and ST 486 cell lines of Table 111, finally, seem to represent a more advanced stage within the B cell-differentiation lineage, as two heavy Ig chains including y and a chains are expressed. One may, however, also note a few differences between the EBV genomepositive and -negative BL lines, some of which may be ascribed to the presence of EBV genome (Section 111,C). The process of establishment of the EBV genome-negative BL lines seems often to be slow. Sometimes even feeder cells were required for successful establishment (SU-AmB-1). The growth in low serum concentrations was poor in the two lines tested. Furthermore the EBV and C3 receptors are not regularly expressed. Finally, Fc-

TABLE I11 CHARACTERISTICS OF EBV GENOME-NEGATIVE BL LINFS Cell line Characteristic

BJAB"

RAMOSb

SU-AmB-1'

JBLd

Establishment

Slow Poor

Feeder cells used NT9

Slow

Growth in low serum concentration Lymphocyte surface markers Surface Ig Fc receptor (aggregated Ig) Fc receptor (EA) C receptor (EAC) EBV receptor (EAC) SRBC receptor (E) Common ALL (CALL)antigenh Tumorigenicity in nude mice KaVotYpe 14q+ Marker BL-Type translocation BL (EBV+) surface glycoprotein pattern

No lag phase Poor

a

Menezes et a/. (1975b).

Epstein et a/. (1976a,b); Kaplaa et a/. (1979). Miyoshi et al. (1975). ' Ben-Bassat et al. (1977). I Magrath et al. (1980a,b). NT, Not tested. From Minowada et al. (1982). Tumorigenic in hamsters.

CA46I

ST486'

Abanaf

No lag phase NT

Slow

NT

No lag phase NT

P

P

P

P

P,Y+

NT

(+I

(+I

NT

NT -

+

(+I

+ +

+ + + + +

NT NT

-

+ +

(+I

* Klein ef al. (1975).

DG-75'

+ + -

+

NT NT

NT

-

+'

NT NT

+

+

+

+-

+

NT

NT

NT

NT

NT

B-CELLLINES AND

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receptor expression and the presence of a BL-type translocation may be less frequently associated with the EBV genome-negative BL. VI. EBV Genorne-Negative B-Leukernia/Lyrnphoma Cell Lines

In recent years a limited number of EBV genome-negative cell lines with B-cell characteristics have been derived from acute leukemia and nonHodgkin’s lymphoma (Table IV). These lines have all been derived from the malignant cell population as evidenced by their monoclonality, aneuploidy, and common potential for growth subcutaneously in nude mice. The frequency of establishment is low ( < 10%) except for the large cell lymphomas when special tissue culture techniques were employed (Nilsson, 1977). Thus the only non-tumor-derived type of immortal human cell line seems to be the LCL, representing EBV-transformed B cells. T cells may be another example because when stimulated by interleukin-2 (11-2), they will grow for an extended period of time (months to years), but at least one report claims that such cells have a finite life span (Walford et al., 1981). Pre-B leukemia/lymphoma cell lines have been reported from seven patients (Minowada et al., 1977a, 1982; Lazarus et al., 1978; Hurwitz et al., 1979; Smith et al., 1981). Most lines have been.established without an apparent lag phase. However, the growth was comparatively slow during the first few weeks. The growth rate is generally slower than that of EBV-carrying cell lines. The terminal cell density as reported for a few of the lines appears to be equivalent to that of BL lines. Pre-B leukemia cells have a non-T, non-B surface phenotype. The only positive markers are the CALL and HLA-DR antigens. A characteristic feature is also the presence of a small amount of ,u heavy Ig chains but no light chains in the cytoplasm. Like other immature lymphoid cells, these leukemias express a high terminal deoxynucleotidyl transferase (tdt) activity. All pre-B-leukemia lines are aneuploid, but the presence of BL-type translocation or a chromosome 14q+ marker has not been demonstrated (Minowada et al., 1982). This accords with the finding that fresh pre-Bleukemia cells with one exception (Kaneko et al., 1980), were aneuploid but did not have any marker involving chromosome 14 (Mitelman, 1981). The capacity for colony formation in agarose and the tumorigenic potential in nude mice have not been studied in these lines. B-ALL cell lines have been reported from three laboratories (Hiraki et al., 1977; Minowada et al., 1977b; Roos et al., 1982a,b). Like other leukemia/ lymphoma lines, the three B-ALL cell lines became established with a very short lag phase. Proliferation was noted shortly after explantation of the leukemic cells. The B-ALL lines grow slightly slower than BL lines, but will reach the same terminal cell density.

TABLE IV OF EBV GENOME-NEGATIVE B-LEUKEMIA/LYMPHOMA CELLLINES' CHARACTERISTICS Type of cell line

Characteristic Establishment Population doubling time (days) Surface markers SRBC receptor Fc receptor C3 receptor Surface Ig HLA-DR antigen CALLantigen tdt Kavotype 1%' Marker BL-Type translocation a

For references see text. NT. Not tested.

he-B leukemia Continuous growth, short lag phase 2-4

B-ALL Continuous growth, short lag phase 2-3

k f

+ +

Lymphocytic lymphoma

Large cell lymphoma

Undifferentiated lymphoma

Lag phase

Continuous growth, lag phase 2; NTh

Continuous growth, lag phase NT

2-4

+ NT NT NT

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All lines express surface Ig and HLA-DR antigens. In the case of Balm-1, Ig chains were expressed in addition to p and K chains. The expression of Fc and C3 receptor varies, whereas no line expresses cALL antigen. In the two examined cell lines a chromosome 14q + marker was identified in Balm-1, whereas in MN-60 a BL-type translocation t(8; 14) was detected (Minowada et al., 1977b; Roos et al., 1982a). The capacity to form tumors in nude mice was tested in the case of MN-60 cells and positive growth was noted (Roos et al., 1982b). Cell lines from patients with lymphocytic lvmphoma (a total of eight) have usually been established from pleural effusions (Gallmeier et al., 1977: Lok et al., 1979; Smith and Rosen, 1979; Magrath et al., 1980a). The two exceptional lymph node-derived cell lines were obtained from patients with unusually therapy-resistant and fatal lymphomas (Klein et af., 1974; Nilsson and Sundstrom, 1974). All but one line became established only after a lag phase of several weeks. The population doubling time is longer than for the BL cell lines. All lines express surface Ig. The expression of other surface markers varies. It is notable that one line (U-698) is cALL antigen-positive. Six of seven examined cell lines has a 14q+ marker. In two of the lines chromosome 8 was the source of the translocated fragment (Magrath et d., 1980a). The specific breaking points have not been reported in these lines. however. Some of the lymphocytic lymphoma lines have been tested for tumorigenicity subcutaneously in nude mice and for colony formation in agarose, and found positive (Nilsson et af.. 1977b). Nine large cell (“histiocytic” ) lymphoma cell lines were shown to express surface Ig (Epstein et af., 1978; Kaplan et al., 1979; Nilsson et af., 1982). As for most other non-BL leukemia/lymphoma cell lines, the expression of Fc and C3 receptors varies. N o line is CALL-positive (Nilsson et al., 1981). All lines formed tumors in the nude mouse but in the 3/4 cases the test route was intracranial inoculation. In three of the seven lines tested a 14q+ marker, but no BL-type translocation was detected. From undzfferentiated lymphoma only two cell lines have been established (Magrath et al., 1980a). The growth properties of these lines have not been reported. Neither has the surface phenotype been extensively studied. However, both lines express surface Ig, but no Fc or C3 receptors were detected. Both lines have a 14q+ marker. In one of them a t(8; 14) was identified. The tumorigenic potential has not been tested. Taken together the studies on EBV genome-negative B leukemia and non-BL B-lymphoma cell lines show that they, as a group, are distinguishable from EBV-carrying BL cell lines and LCLs. Individual B-ALL and lymphocytic lymphoma lines, however, may be hard to distinguish also from the

352

KENNETH NILSSON AND GEORGE KLEIN

EBV-negative type of BL lines, as such lines may have similar morphology, growth properties, surface markers, and chromosome alterations. This is not unexpected because the cytology of B-ALL and the histopathology of some poorly differentiated lymphocytic lymphomas are sometimes very similar to that of BL. As for other leukemia/lymphoma cell lines, but in contrast to LCLs, the individuality of the cell lines is marked. With respect to surface markers, the B leukemia and non-BL, B lymphoma are generally either more immature (pre-B leukemias) or represent more advanced differentiation stages (lymphocytic lymphomas, large cell lymphomas) than most BL (see Section VIII).

VII. EBV-Carrying Non-BL, Nonlymphoblastoid Cell Lines Derived from EBV Genome-Negative Leukemia/Lymphomas

Recently, a few EBV-positive cell lines derived from EBV genome-negative hematopoietic neoplasms have been reported to have phenotypic and cytogenetic properties identical to the tumor biopsy cells in uiuo, and to be distinct from that of LCLs. These cell lines were derived from one lymphocytic lymphoma (S9’; zur Hausen et al., 1972; Nilsson et al., 1975), one B-ALL (Balm-2; Minowada et al., 1977), one hairy cell leukemia (Anderson et af., 1981), and four American BLs (MS 115, DW 6, AG 876, KK 124; Magrath et af., 1980a,b), respectively. As the comparative studies on biopsy cells and thd derived cell lines appear to exclude cross-contaminations, there seem to be a few possible explanations for this unexpected EBV positivity in apparently neoplastic cell lines established from EBV-negative tumor biopsy cells. 1. The lines represent secondarily altered LCLs. This is a far-fetched explanation because the lines have the same chromosomal alterations as the tumor cells in uiuo, including a 14q+ marker [which, so far, has been reported to be present in only two old LCLs (Manolov et al., 1981)]. 2. The lines originated from a small minority of EBV-carrying tumor cells which escaped detection in the EBNA or hybridization tests. This is also unlikely because these tumors, as judged by the karyotypic analyses and the marker studies, were no exceptions to the rule that human leukemia/ lymphomas are monoclonal. 3. The in uivo tumor cells carried EBV receptors and became infected in vitro by EBV liberated from normal B cells presented among the explanted tumor cells. This possibility cannot be excluded because it has been demonstrated that both EBV genome-negative BL cell lines and fresh chronic lymphocytic leukemia cells may become EBV-infected in uitro by exposure

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to exogeneous EBV (Clements et al., 1975; Klein et al., 1975; Fresen and zur Hausen, 1976; Karande et al., 1980). It is thus possible that these cell lines represent rare cases of hematopoietic tumors which have picked up EBV in vitro, and probably like the fresh CLL cells, acquired the potential for in vitro proliferation. It is, however, interesting that no infection seems to have occurred in vivo, although at least in the case of the American BL patients the patients were EBV-sero-positive (Magrath ef al., 1980a,b). VIII. The Relationship of EBV-Carrying Lymphoid Cell Lines to Normal B-Cell Differentiation

A. NORMAL B-CELLDIFFERENTIATION The present knowledge about the differentiation of cells within the B-cell lineage stems from studies on the ontogeny of Ig-producing cells, from studies on mitogen and antigen activation of B cells, and from phenotypic studies on B leukemia/lymphomas which appear to represent clonal expansions of B-lymphoid cells frozen at various differentiation stages (Katz, 1977; Serrou and Rosenfeld, 1978; Greaves et al., 1977). Compared to T-cell differentiation, B-cell differentiation is but poorly understood, however, and only a few differentiation stages can be defined by reliable markers. The following description of the various stages of B-cell differentiation will therefore by necessity be schematic. Cells with the capacity for Ig synthesis, the hallmark of a B cell, appear first in the human liver at the seventh week of gestation. Such cells contain cytoplasmic IgM. About 2 weeks later surface(s) IgM-positive cells can be detected in the liver and subsequently in the bone marrow, blood, spleen, and lymph nodes. By the twelfth week of gestation IgD-, IgA-, and IgGexpression cells will be demonstrable in the lymphoid organs (Gathings et nl., 1977). The experimental evidence defines a few different categories of cells within the B cell-differentiation lineage (Fig. 6, Tables V and VI), but other intermediate stages most probably also exist. The B-lymphoid stem cell (BLSC) develops in the bone marrow from a stem cell (LSC) common to B- and T-lymphoid cells, and seeds into the circulation and peripheral lymphoid organs. The LSC seems to be derived from the pluripotent stem cell (PSC), as suggested by studies of the Ph chromosome marker representation in patients with chronic myeloid leukemia (CML) (Fialkow et al., 1978). The extent of recruitment of LSC from the PSC is unknown. The characteristics of the normal BLSC are

354 CELL

KENNETH NILSSON AND GEORGE KLEIN

BLSC

- -3 PRE-B

B

MB

TISSUE

B-BLAST

-

pc

Bone marrow Spleen, lymph node,other periph.tissue

STIMULI

Microenvi rormental

------

Influence o f antigen ---PROLIFERATION

------

-----

FIG. 6. Scheme of B-cell development. For abbreviations see text. (From Biberfeld and Nilsson, 1980.)

essentially unknown. However, studies on its possible malignant counterpart-the acute non-T, non-B leukemias-suggest that its essential features (Table V) are an immature lymphoid morphology, the expression of HLA-DR and cALL antigen but not Fc, C3, EBV receptors, the synthesis of high amounts of tdt, and no capacity for Ig synthesis, although the Ig genes may become rearranged already at this differentiation stage (Korsmeyer, 1981).The BLSC has a high proliferative capacity which is dependent entirely on undefined microenvironmental influences and not on antigenic stimuli (Fig. 6). The pre-B cell is a relatively large cell with immature cytological features, which multiply independently of specific antigenic stimuli mainly in the bone marrow (Fig. 6 ) . Transfer experiments and in uitro studies indicate that this cell type matures to medium-sized cytologically mature lymphocytes in the bone marrow and also at extramedullary sites (Melchers et al., 1975; Raff et al., 1976; Gathings et al., 1977). The most characteristic feature of the pre-B cell is its capacity to synthesize p chains. These are not detectable at the surface membrane but are found in the cytoplasm and may even be actively secreted. Other useful markers of the pre-B cell are the expression of cALL and the HLA-DR antigens, and the high tdt activity. The virgin B cell, constituting the third category of B cells, develops mainly in extramedullary sites but also in the bone marrow (Nossal et al., 1977). The hallmark of this cell type is its Ig receptors, which are of the IgM or IgM IgD isotype. This cell circulates in the peripheral blood and constitutes the “peripheral blood lymphocyte,” with its well-known morphological and surface marker characteristics (Table V). The B cell is immunologically competent and may, by use of its Ig receptors and most often with macrophage and T help, respond to specific antigenic stimuli by

+

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proliferation and stepwise maturation to an antibody-secreting cell or develop into a memory B cell (MB) (Fig. 6). The B cell appears to be the target cell for EBV (see Section I1,A) and will respond to immunologically nonspecific stimulation (e.g., polyclonal B-cell activators). The B blasts (secondary B cells) arise upon further differentiation of the B cell. The morphology of the B blasts is quite characteristic both at the light and electron microscopic level and reflects the specialization of this cell type for a moderate rate of Ig secretion (Table V). The B blasts produce all classes of Ig and may have a simultaneous expression at the surface of p, 6 or p, y or p, a in addition to IC or 1, light chains. The B blast represents a transitional cell during the stepwise maturation toward the terminally differentiated plasma cell. The lymphoplasmablast represents a morphologically distinct cell type representing an intermediate stage of differentiation between the B blast and the plasma cell (Table V). The plasma cell (pc) represents the end stage in the B-cell-differentiation lineage. The pc has a characteristic morphology, secretes Ig at a high rate (Tables V and VI), and is unable to proliferate but for a limited number of divisions. The pc does not express any of the cell surface markers characteristic of the pre-B cell, B cell, and B blast, except for HLA-DR expression in a fraction of them.

B. THESTAGE OF DIFFERENTIATION OF LCL AND BL CELLS Studies on leukemia/lymphomas have demonstrated that the phenotype of tumor cells corresponds closely to that of the various types of normal B cells. It has therefore been suggested that they often represent clonal expansions of B cells frozen at a particular stage of differentiation. The relationship of the B-cell tumors to the normal B-lymphoid-differentiation lineage is illustrated in Tables V and VI. It should be noted that the phenotype of BL usually corresponds to the pre-B-cell-B-cell-differentiation stages, as evidenced by its morphology, surface markers, and functional characteristics. Most notably, many BL lines express CALL antigen and IgM, but never IgD, and do not secrete Ig. Seraphina represents the only BL line in which morphology, Ig isotype (IgG), and function (Ig secretion) suggest that the stage of differentiation is more advanced than that of the B cell. No difference has been found between EBV-negative and -positive BL with respect to differentiation stage. The LCL type of EBV-carrying B-lymphoid cell line closely corresponds to the B-blast stage of lymphoid differentiation. The morphology as well as surface markers, surface glycoproteins, and functional properties agree that it has a B-blast phenotype. This demonstrates that EBV turns on the

TABLE V HUMANB-LYMPHOID CELLLINES: MORPHOLOGICAL AND FUNCTIONAL DIFFEREN~ATION AND RELATIONSHIP TO THE B-CELLDIFFERENTIATION LINEAGE

NORMAL

Differentiation stage of representativecell lines

Poorly d i f f . lymph. lymphoma

Poorly d i f f . l y m p h . lymphoma

I

I

U-698

Pre-B- ALL 'Nalm-1, 16, LAZ1221

U-715,

Balm-5

Large c e l l B lvmmohwna SU-DHL-4.5,7

,

SKW-4

IB-ALL Balm-l,2, BALL11

,Biopsy CLL cells

, CI L . EBV c o n v e r t e d '

Corinna 11, Sarahl I # 60

LCL I

Raji

B u r k i t t ' s 1 vmphoma Daudi NK 9 Seraphi na

I

Myeloma

'U-266, RPMI 8226'

Normal B-cell lineage Morphology

Mu 1 t i potent

stem c e l l

Lymphoid stem c e l l

Pre-B c e l l

B lymphocyte

B blast

Lymphoplasrna blast

Differentiation markers

vl

I .

Surface Ig IntracellularIg Secreted Ig Ig isotype Fc receptor C3 receptor EBV receptor HLA-DR antigen CALLantigen

-

+" +" 'h

+

++ ++ Y. P3 a,6,

-h

? ?

-h

?

+ +

Only p-heavy but no light chains. Indirect evidence from studies on pre-B leukemia cells.

+

Plasma c e l l

TABLE VI DIFFERENTIATION WITH REGARD TO MORPHOLDGY AND Ig

PRODUCTION WITHIN THE

B-CELLLINEAGE?

c SECR. Ig Corresponding type of in oirro cell line

Ig production Surface (molecules/cell) Secretion (pg/ I O6 cellsi24 hr) Percentage of total protein synthesis a

From Nilsson (1978).

Pre-B leukemia

B lymphoma

0

8 x 10"

2

-k

0

1-3

10-20

3-10 (50)

20-30

1-2

B lymphoblastoid

SECR. Ig

104

Myeloma

(+)

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differentiation program of the B cell at the infection. However, although EBV, like some mitogens and specific antigenic signals, acts as a polyclonal B-cell activator, certain differences have been found (Section 11,B). First, EBV seems not to activate the B cells by surface membrane signals, because UV-inactivated virus is inactive. Second, the differentiation induced by EBV in the B cells does not seem to proceed stepwise to the terminal plasma cell stage but is “arrested” at the B-blast stage, perhaps because the cell is forced to divide continuously. It cannot, however, be excluded that at least early after infection a fraction of the B cells will terminally differentiate and reach the nonproliferative B-blast stage. The latter B blasts will be selected for during the continued growth in uitro and will eventually constitute the polyclonal LCLs. This possibility was indeed suggested by recent experiments showing that the fraction of EBV-transformed B cells having a high secretory capacity (plaque-forming cells) could not be passaged further in uitro to become established LCLs (Bird et af.,1981). Lymphoblastoid cell lines do not seem to be irreversibly frozen at the B-blast stage of differentiation, because they may be induced to further maturation as suggested by an increased capacity for Ig secretion after exposure to human T-cell factors (Kishimoto et al., 1978) and phorbol esters (Ralph and Kishimoto, 1981). The differentiation-inducing capacity of EBV after infection of B-cell targets has also been suggested by the comparative studies of the EBV genome-negative BL lines (BJAB, Ramos) and their EBV-converted sublines (Section 11,C). Apart from the growth-promoting effect of EBV and the alterations of the surface membrane, certain morphological differences and changes of the Ig isotype were found. The morphology and expression of actin filaments became similar to that of the LCL cells in several of the EBV-converted Ramos sublines (K. Nilsson and A. Fagraeus, unpublished observations). Furthermore, IgD and insulin receptors appeared at the cell surface, whereas the 38 : 13 antigen specific for BL cells often diminished (Spira et al., 1981a). A similar differentiation-inducing effect of EBV has been documented in chronic lymphocytic leukemia (CLL) of the B-cell type (Karande ef a/., 1980). In two cases it was possible to establish authentic tumor cell lines in addition to LCLs by addition of exogeneous EBV. In comparative studies the phenotype of the fresh CLL cells was compared with that of the autologous LCL and the EBV-transformed tumor line. In both cases the phenotype of the CLL tumor line was distinct from that of the fresh CLL cells but similar, if not identical, to that of the LCLs. It thus seems as if the introduction of the EBV genome into the CLL cells has led to further differentiation and differentiation arrest at the B-blast stage. As will be detailed in Section XII, we interpret the present epidemiological

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and experimental data on the association of EBV to BL to mean that BL develops from EBV-transformed B cells via a stepwise process in which the appearance of a t(8; 14) chromosomal abnormality plays a decisive role for the transformed B cell in its acquisition of the full malignant BL phenotype. If this reasoning is correct, it also follows that the translocation of the chromosome 8 fragment is followed by a dedifferentiation event, as BL cells represent less differentiated B cells than LCL cells. Although an abnormal clonal progression in LCLs in vitro and in uiuo has been noted (Section IX), it is notable that the acquisition of a tumorigenic potential was never paralleled by an altered differentiation stage and by acquisition of a BL-specific translocation. IX. The Progression in Lymphoblastoid Cell Lines in Vitro and in Vivo

Whether the phenotype of LCLs should be regarded as normal or neoplastic has been a matter of dispute for a long time, especially so before it was realized that secondary chromosomal and associated phenotypic changes occurred during prolonged cultivation in vitro (Table VII).The fact that the LCLs were immortal was taken as evidence that they represented malignant cells because otherwise this would violate the concept, derived from studies on fibroblasts, that normal human cells have a finite life span in uitro. TABLE VII SECONDARY PHENOTYPIC ALTERATIONS IN LYMPHOBLASTOID CELL LINES(LCLS) DURING LONG-TERM CULTIVATION Type of alteration Acquisition of a tumorigenic potential subcutaneously in nude mice Capacity for growth in agarose Growth in low serum concentrations Growth in Eagle’s medium Shortened doubling time Increased frequency of surface-attached cells Morphological changes Change in cytochemical profile (cytoplasmic fat) Increased expression of Fc receptors Increased Con A-induced agglutinability Increased expression of HL-A and j 2 - p increased or decreased secretion of j z - p Unbalanced Ig synthesis Surface expression Synthesis

Reference Nilsson er a/. (1977b) Nilsson et al. (1977b) Nilsson (1979) Nilsson (1979) Nilsson (1977) Nilsson (1979) Nilsson and Ponten (1975) Sundstrom and Nilsson (1977) Huber et al. (1976) Glimelius e/ al. (1975) Welsh er al. (1977) Nilsson et al. (1973) Nilsson (1979)

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TABLE VIII SUMMARY OF “NONMALIGNANT” PROPERTIES OF RECENTLY ESTABLISHED LCLs Characteristic Polyclonality Euploidy Absence of capacity for colony formation in agarose Lack of tumorigenic potential subcutaneously in nude mice Lack of tumorigenic potential at autologous inoculation Lack of sensitivity to NK cells Absence of gp69/71 typical of B-lymphoma cells

Reference Bechtt et al. (1974) Jarvis et al. (1974); Zech ei al. (1976) Nilsson ei al. (1977b) Diehl ei a/. (1 977); Nilsson et al. (1977b) Moore and Gerner (1970) Jondal et al. (1978) ; Pattengale el al. (1982) Nilsson ei al. (1977a)

However, the series of comparative studies on LCL and BL cells, already described, have led to a less rigid conception of the biological nature of recently established LCLs. Table VIII summarizes selected “nonmalignant” properties of LCLs. These characteristics strongly argue against the idea that LCLs should represent B cells having undergone a full malignant transformation after EBV infection. However, Table IX gives some arguments for the assumption that LCLs may indeed represent premalignant cells. As already mentioned, polyclonal diploid nontumorigenic LCLs may, however, when continuously passaged in uitro become monoclonal, aneuploid, and tumorigenic subcutaneously in nude mice. These phenotypic and cytogenetic changes (Table VII) have suggested to us that BLs may indeed originate from preexisting LCL cells haveing undergone the necessary stepwise neoplastic changes. The chromosomal changes that LCLs undergo in vitro have never been shown to involve a BL-specific translocation [t(8; 14), t(2; 8) t(8; 22)]. However, the translocation is probably a rare event and in uitro selection TABLE IX SUMMARY OF “PREMALIGNANT” PROPERTIES OF RECENTLY ESTABLISHED LCLs Characteristic

Reference

Capacity for infinite growth in uiiro Tumorigenicity intracerebrally in adult or intraperitoneally in newborn nude mice Increased Con A-induced agglutinability

Nilsson (1979) Giovanella et al. (1979); Schaadt ei a/. (1979) De Salle et al. (1972); Glimelius e? al. (1975) van Beek et al. (1979, 1981)

Increased expression of cell surface fucosyl glycopeptides

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must be different from in uiuo selection. This probably explains why LCLs never evolve in uitro to cell clones with a full BL phenotype. In order to apply a different and perhaps more relevant pressure to select clones with a BL or at least lymphoma phenotype from LCLs, such lines were inoculated intracerebrally into nude mice (Giovanella et al., 1979). In contrast to the conventional subcutaneous route, the brain tissue will accept the growth of diploid polyclonal LCL cells (Giovanella et al., 1979; Schaadt et al., 1979), and it was thus possible to do an in uiuo cultivation. The progressively growing LCLs could then be recultivated and tested for clonal evolution by analyses of the Ig isotypes and for chromosomal changes by conventional banding techniques. The results showed that a clonal restriction sometimes occurred during the intracerebral passage and that an aneuploid clone sometimeswas selected. However, as for aneuploid LCL clones selected in uitro, the phenotype of these in uiuo-selected clones was not that of BL cells but they had retained the basic morphological function and surface features of polyclonal diploid LCL cells. We can thus conclude that LCL may acquire a full malignant phenotype, as judged by a number of criteria, by continuous cultivation in uitro of intracerebral passage in nude mice. However, in neither instance did the selected abnormal clone display a BL phenotype or have the BL-specific cytogenetic abnormalities. X. The Role of EBV in Progression

Does the virus play any role in determining the difference between the immortalized diploid LCL cell and the BL cell? There is no adequate information on this point. Clearly, the virus does not interfere with the maintenance of strict euploidy in the EBV-transformed LCL of normal derivation over periods of several months (Zech et al., 1976). In normal immunocompetent individuals, corresponding in uiuo-transformed cells appear to be Fully responsive to some form of restrictive control. Because BL cells carry the 8; 14 translocation or the variant 2; 8 or 8 ; 22 translocation that arises independently of the virus (i.e., is present in both the EBV-carrying and the EBV-negative forms of BL), it would seem that EBV itself is not involved in the progression from LCL to BL. There are some other observations that suggest, however, that EBV may nevertheless facilitate certain progressional steps. Experimentally, these effects can be approached by superinfecting established EBV-sensitive lymphoma lines of B-cell origin, rather than normal B lymphocytes, with EBV. By using this approach, permanently EBV DNAand EBNA-positive sublines were established from two EBV-negative lymphoma lines, Ramos and BJAB (Klein et al., 1975; Fresen and zur

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Hausen, 1976). Comparisons between the original EBV-negative lines and their EBV-converted sublines gave unexpected clues concerning the phenotypic changes that can be induced by the viral genome. They included decreased serum dependence, increased resistance to saturation conditions, independence of a dialyzable serum factor, decreased lateral mobility (capping) of surface moieties, increased lectin agglutin ability, and an increased ability to activate the alternate complement pathway (Yefenof et al., 1977a,b, 1978; Steinitz and Klein, 1975, 1976, 1977; McConnel et al., 1978). Some of these changes are strongly reminiscent of the well-known transformation-associated changes in monolayer cultures transformed by oncogenic RNA or DNA viruses. This suggests a certain unity between widely different transforming viruses. Also, it seems that EBV can “push” already immortalized cells to proceed further on their pathway of progression. This conclusion is also supported by the finding that the gp69/71 double glycoprotein band, regularly expressed on the surface of BL-derived cell lines, but not on normal lymphoblastoid cell lines, can be induced in the EBV-negative BJAB line by EBV superinfection (Koide et al., 1981). EBV conversion also increases the agarose clonability of the Ramos and the BJAB line (Montagnier and Gruest, 1979; Zerbini and Ernberg, 1982). The findings on the BJAB line deserve some special comments. BJAB is an atypical African lymphoma (Klein et al., 1974; Menezes et al., 1975b). The tumor was diagnosed as BL, but with a number of atypical features that made the diagnosis doubtful. In contrast to the usual form of African BL, EBV DNA- and EBNA-positive in 97% of the cases, BJAB cells contain no detectable EBV genomes. In contrast to both EBV-positive and EBVnegative BL cases, BJAB cells do not contain a 14q + marker, nor do they have any of the alternative (variant) chromosome 8-derived translocation markers. Alone among the BLs tested, it fails to express the gp69/71 double band, has low agarose clonability, and will not consistently grow in nude mice. EBV conversion was able to shift all these properties, except the chromosomal markers, in a direction that is typical for the usual BL line. Although EBV can thus clearly promote the progression of already established lymphoma lines to states of higher independence, cells with BL-like phenotypes have not yet been isolated from EBV-transformed lymphoblastoid cell lines of normal origin. If the theory (see Section XI) that attributes the origin of a BL clone to the specific 8; 14 translocation (or to 2; 8 and 8; 22 variants, respectively) that occurs by chance in an EBVtransformed normal B cell is correct, it may be expected that the same course of events should be reproducible in an in uitro system. So far, this has not been possible. However, if the specific translocation is a very rare event, as one would expect, it would not appear under ordinary in uitro culture conditions, due to the lack of an appropriate selective pressure, as discussed

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elsewhere in this article. In contrast, the in uiuo organism may represent a highly selective system for the autonomous cell clone, created by the translocation, according to our hypothesis. The rarity of the specific translocation would be also in line with the fact that the incidence of BL is relatively low, even in the most highly endemic areas. It is important to design new selective systems in uitro that will permit the isolation of the specific translocation-carrying clones. Agarose clonability may provide a potentially useful selective method. Another alternative may be provided by the recently developed monoclonal antibody of Wiels et al. (1981). It reacts with most (but not all) BL lines but not with lymphoblastoid lines of normal origin. We have recently observed that a small minority of the cells in certain LCL gave a brilliant reaction after exposure to this antibody. It is not yet known whether they represent stable variant or transient phenotypic changes. If the former is true it may provide a new way to select BL-type cells from normal LCL populations (Klein et al., 1982). XI. The Role of Chromosomal Changes in Progression

As already mentioned, LCLs are diploid as a rule, and remain diploid immediately after transformation and during several months of serial propagation. Subsequently, they tend to change to aneuploidy without any demonstrably specific patterns. Concurrently, they may increase their agarose clonability and tumorigenicity in nude mice (Nilsson et al., 1977b). Burkitt’s lymphoma cells are never diploid. Between 80 and 90% of the cases with a properly controlled BL diagnosis carry a specific chromosomal marker, first described as the 14q+ marker, and later identified as a reciprocal 8; 14 translocation. The remaining 10-20% contain one of the two variant translocations, 2; 8 or 8; 22 (see later). The BL-associated 14q+ marker differs from 14q+ markers found in other hematopoietic neoplasms in several respects. In BL, the extra band attached to the distal end of the long arm of one chromosome 14 is always derived from chromosome 8. In other lymphomas, the translocated piece can be derived from various chromosomes and the breakpoint on chromosome 14 is also variable. The same applies to occasional 14q + markers found in a minority of the cells in certain LCL lines. In contrast, the typical BL translocation regularly involves the same bands, 8q24 and 14q32 (for articles see Rowley, 1980; Mitelman 1981). The only nonBurkitt’s lymphoma that was found to contain the BL marker, and with great regularity, is B-cell-derived ALL. This is of great interest in light of the fact that B-ALL probably originates from the same cell type as Burkitt’s lymphoma. B-cell ALL and the majority of the non-African BLs are EBV DNA- and

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EBNA-negative. Nevertheless, they carry the same 8 ; 14 translocation as EBV-positive BL. Taken together with the fact that EBV-transformed LCLs of normal origin do not contain the BL translocation, this excludes the possibility that BV causes the translocation in itself. In mouse plasmacytoma (MPC) the distal region of chromosome 15 was found to be translocated to chromosome 12 in the majority of all examined tumors (Ohno et al., 1979). The distal region of M12 is known to carry the IgH cluster (Me0 et al., 1980). A variant translocation was found in a minority of the kappa light-chain producers. It is generated by a reciprocal exchange between the same distal segment of chromosome 15 that is involved in the typical translocation and the ic gene-carrying chromosome 6 . In BL, the majority of the reported cases were found to carry a reciprocal (8; 14) translocation. Two variant translocations, 2 ; 8 and 8; 22, were identified. They constitute 20% or less of the cases. The same distal segment of H8 is transposed in the typical and the variant translocations (breakpoint at q24). The recipient of the typical translocation, H14, is known to carry the IgH cluster (Croce et al., 1979). Recent in situ hybridization experiments (Leder, personal communication) have shown that IgH is localized in the band region g32. This corresponds to the breakpoint involved in the BLassociated translocation. The variant translocations further reinforce the picture. Chromosome 2 carries the kappa gene that was recently localized to p I 2 or p 1 3 on the short arm of the chromosome (Malcolm et al., 1982). The breakpoint giving rise to the variant BL translocation is in the same region (pl2). Chromosome 22 carries the lambda gene (Wesley-McBride et al., 1982). It is thus clear that the recipient chromosome of the tumor-associated translocation carries immunoglobulin genes in both MPC and BL. But what is known about the remarkably constant donor? In T cell-derived mouse leukemia, the analysis of chromosome 15 trisomy has led to the hypothesis that an oncogene, located in the distal part of chromosome 15, is activated by mutation in a regulator gene or by proviral DNA insertion. This cannot be fully expressed, however, unless the changed chromosome is amplified by nondisjunction (Wiener et al., 1978a,b,c, 1980a, 1981; Spira et ul., 1979, 1980, 1981b). This amplification is necessary to overcome a trans-acting control exerted by the unchanged homologous chromosome (Spira et al., 1981b). In mouse plasmacytoma, the translocation of the distal segment of chromosome 15 to the Ig locus-carrying chromosome was interpreted to suggest a switch-on of what may be the same oncogene, under the influence of a functionally active region in the target cell. It is tempting to speculate that the distal region of human chromosome 8 may carry a corresponding oncogene. It is of interest in this connection that H8 trisomy is the most common of all leukemia-associated trisomies

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(Mitelman and Levan, 1981). The distal region of H8 was also found to be involved in a constitutional 3 ; 8 translocation that was regularly associated with renal carcinoma in every member of the family that carried it, after they have reached middle age (Cohen et af.,1979). In conclusion, one may thus postulate that the BL-associated 2; 8, 8; 14, and 8; 22 translocations act by the same mechanism as the corresponding translocations in MPC: by activation of an oncogene under the influence of a functionally highly active Ig locus. The regularity of the BL-associated translocation is comparable to the association of the Philadelphia chromosome with chronic myeloid leukemia (CML). This suggests that it may play an important and perhaps crucial role in determining the autonomous behavior of the BL clone. The existence of the variant translocations suggests, moreover, that the important genetic determinant is localized in the distal part of chromosome 8, rather than in chromosome 14. Considering the possible mechanisms by which the translocations may favor autonomous growth, two points can be made. The first departs from the fact that the translocation-carrying BL-derived lines show a high agarose clonability and can grow in nude mice, in contrast to EBV-immortalized diploid LCLs. Both types of cells can grow progressively in the brain of the nude mouse, however, suggesting a difference in sensitivity to some controlling host factor that can prevail on the cells in the subcutaneous space of the nude mouse, but not in its brain. This control function is exerted by some immune effector. It is not known whether the same effector or any other immune mechanisms may reflect a control mechanism that prevails in the unmanipulated human host. However this may be, the nude mouse experiment suggests that the BL phenotype differs from the LCL cells in its immunosensitivity. Alternatively, nonimmune feedback controls may be expressed differently in the subcutaneous space and the brain of the nude mouse, respectively. The second point concerns the mechanism by which the translocation may change the behavior of the cell. Before considering this for BL, it may be useful to consider specific translocations associated with murine plasmacytoma (Ohno et af., 1979;Wiener et al., 1980b). Mineral oil-induced murine plasmacytomas were found to carry a 12; 15 translocation, with regular involvement of the same breakpoints. Chromosome 12 is known to carry the genetic loci for the heavy Ig chains in the mouse, localized on chromosome 14 in humans. The kappa but not the lambda light-chain-producer mouse plasmacytomas sometimes contained a reciprocal 6 ; 15 translocation instead. Chromosome 6 is known to carry the kappa-chain locus. The small number of lambda producers so far examined contained the 12; 15 translocation but no additional specific marker.

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The murine plasmacytoma-associated translocations thus resemble the BL-associated translocation, in so far as the recipient chromosome carries an Zg locus expressed in the neoplastic target cells. Because only one of the two homologous Zg genes is active in every Ig-producing cell (allelic exclusion), the question arises whether the active or the inactive homologue serves as the translocation recipient. This is open to direct experimental tests that may be informative with regard to the exact localization of the breakpoints in relation to the Zg loci. Considering the translocated chromosome piece, it is possible that there is a certain analogy between the distal part of human chromosome 8 and murine chromosome 15. The latter was shown to be involved in murine lymphoma development as well (for a review see Klein, 1981). In T-cell lymphomas and, to some extent, in B-cell lymphomas as well, 15-trisomy is the most common nonrandom tumor-associated chromosomal change. Translocation studies have localized the important region to the distal part of the chromosome. The latter includes the segment that translocates to chromosomes 12 and 6, respectively, in murine plasmacytoma. Taken together, these findings were taken to suggest that the region may contain gene(s) that influence the normal growth and/or differentiation of the lymphocyte/plasmacyte series. In humans, the distal part of chromosome 8 may contain gene(s) that have a similar function. In addition to the role of this region in both the typical and the variant BL translocations, it may be noted that trisomy of chromosome 8 is the most common human leukemia-associated trisomy. If human chromosome 8 carries genes that regulate lymphocyte/plasmacyte differentiation, it is conceivable that they are permanently activated by translocation to a highly active chromosomal region. The immunoglobulin region may serve this purpose in the majority of the cases-i.e., in the typical translocations-whereas more infrequently translocation to other chromosomes may have a similar effect as well. Is the Zg locus-carrying translocation recipient chromosome functionally active in the neoplastic cell? Strong indirect evidence suggests that this is probably the case. All rcp(6; 15) translocation-carrying MPCs were found to make kappa chains. Among the variant BLs, all seven 8 ; 22-carrying Ig-producer lines tested made lambda chains (Lenoir et al., 1981). Among five 2; 8 translocation-carrying BL, all three that produced light chains made kappa. This cannot be due to chance. Randomly selected BL biopsies and lines make kappa versus lambda light chains in a ratio of approximately 2: 1 (van Furth et al., 1972; Gunven et al., 1980). Are the Ig-producing chromosome regions preferentially affected by the translocations because they are more vulnerable, due to the DNA rearrangements that take place in the course of normal differentiation? This cannot

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be excluded, but it is unlikely, because lambda-producing cells have rearranged kappa regions (Hieter et al., 1981). The good correlation between the light-chain type and the involvement of the corresponding chromosome in the variant BL translocations makes a functional explanation far more probable. XII. General Discussion

Two main points may be considered with regard to the relationships between EBV-transformed cells and their human host. (1) the status of the EBV-transformed B lymphocyte in the normal and the immunosuppressed human host; and (2) the etiology of Burkitt’s lymphoma. Concerning (l), there can be little doubt that seropositive individuals carry EBV DNApositive B lymphocytes in their peripheral blood and in their tissues. In infectious mononucleosis, these cells have been visualized as a small percentage of the B-cell fraction. In normal seropositives, they have not been visualized. In uitro-explantation lymphocytes from peripheral blood or solid lymphoid tissue of seropositive donors regularly give rise to EBV-carrying permanent lines, however, suggesting that latently infected B cells are present in this case as well. Their number is much lower than in infectious mononucleosis, however; approximately lo6 blood or lymph node cells have to be explanted from a normal donor to give rise to established lines (Bechkt et a/., 1973), whereas lo4 blood mononuclear cells may be already sufficient from cases of infectious mononucleosis (Diehl et al., 1968). It is not known whether the EBV-carrying B cells maintain themselves in uiuo by continuous but controlled division or are steadily generated by de n o w infection from some unknown reservoir. Both alternatives are conceivable. The mitotic potential of the EBV-infected peripheral B lymphocyte has been proved by identifying EBNA-positive mitotic figures in the peripheral blood during acute mononucleosis (Robinson et al., 1980). The number of EBV-carrying B lymphocytes may be controlled in uivo by immune or nonimmune mechanisms. The low agarose clonability of LCL as compared to BL lines suggests that at least some nonimmune mechanisms may be involved. In addition, the role of immune controls is suggested by the fact that EBV-carrying lymphoproliferative disease may occur in immunosuppressed patients, like renal transplant recipients or children with the X-linked lymphoproliferative syndrome (XLP) (Purtilo et al., 1981). Studies on a number of immune effector mechanisms in high EBV antibody-titered, immunologically partially compromised Hodgkins disease and CLL patients in remission, who did not have EBV-carrying lymphoproliferative disease, in comparison with chronic mononucleosis and XLP

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patients who could not properly control their EBV-carrying cells, have given some information about the effector mechanisms that may be involved. In the former group, the degree of immunosuppression was more limited, with regard to both the levels of residual activity and the number of the immune mechanisms affected, compared to the latter groups (Masucci et ul., 1981a). The XLP patients showed particularly serious deficiencies (Masucci el nl., 1981b). Natural killer cells were nearly absent and could not be boosted by interferon treatment; EBV-specific memory T cells were lacking, both according to the leukocyte-migration inhibition (LMI) and the EBV-specific outgrowth inhibition test. They were also deficient with regard to EBVantibody titers. The chronic mononucleosis patients were intermediate in most tests. This suggests, in line with other evidence, that the EBV-carrying B cells are kept latent by multiple effector mechanisms. This is understandable in view of the known fact that the long-standing impact of a ubiquitous, potentially highly oncogenic virus tends to fix all available effector mechanisms that can be brought to bear on the transformed, potentially malignant cells. The lymphoproliferative condition that can be induced by transforming EBV in immunologically (with regard to EBV) naive marmosets also deserves some comment. It has often been suggested that this disease represents a model for BL in hpmans (Miller, 1980). It is more likely, however, that it is akin to fatal IM or the XLP syndrome, than to BL. It is polyclonal, appears after a short latency period, and is due to the proliferation of EBV-carrying diploid cells with differentiation markers that correspond to lymphoblastoid cell lines, rather than to Burkitt’s lymphoma. Because marmosets do not naturally encounter EBV or related herpesviruses, it is not surprising that their behavior should resemble the immunologically compromised human patient . A model for the monoclonal, chromosomally changed Burkitt’s lymphoma has not yet been found in a nonhuman primate. Perhaps it will be necessary to induce a vast multicomponental immunosuppression m.African monkeys that carry EBV-related viruses, or to achieve chronic stimulation of the target tissue, before this can be achieved. The etiology of Burkitt’s lymphoma can be discussed separately for the high-endemic African form and the sporadic nonendemic form. High-endemic African BL is restricted to regions with chronic hyperendemic or holoendemic malaria. Of the studied cases, 97% were EBVcarrying tumors. In contrast, nonendemic Burkitt’s lymphoma is largely EBV-negative. Among the relatively small number investigated, approximately 20% of the nonendemic cases were EBV-carrying, whereas the rest were EBV-negative.

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Because the high-endemic and the nonendemic cases show identical chromosomal translocations, it is likely that the translocation is the ultimate common denominator in the development of BL. Prior to that, the initial stages may be quite different for the EBV-carrying and the EBV-negative forms. For the high-endemic, EBV-positive forms, we have envisaged the following scenario (Klein, 1979b). It is known that African children who later develop Burkitt’s lymphoma have higher anti-EBV antibody titers than those who do not. The difference is statistical; a high EBV-antibody titer per se is neither necessary nor sufficient for the development of BL. Nevertheless, this suggests that children who are at risk to develop BL often have a higher virus load than their brothers and sisters who do not. This would imply that they have a relatively high number of EBV-carrying B cells. As a second step, an environmentally related cofactor must come into operation; Burkitt has postulated that chronic holoendemic malaria is responsible. This is still the most reasonable suggestion, compatible with geographic and epidemiological facts. Heavy malarial infection is also a good candidate, because it causes chronic lymphoproliferation : hyper- and holoendemic malaria often appear in Africa in the form of the “big spleen disease.” EBV-carrying, host-controlled B cells can be expected to be carried along in the chronic proliferation, like normal B cells. Unlike the latter, the vast majority of EBV-carrying B cells are unable to develop into nonproliferative end cells (plasma cells) because the virus has frozen their ability to differentiate. Therefore, they can accumulate a genetic load and are subject to chromosomal aberrations just as are long-propagated in uitro cell lines. If and when the appropriate translocation occurs-and this may happen as a result of chance, or by more specific mechanisms, suggested by Rowley- the monoclonal lymphoma emerges. Alternatively, the occurrence of the chromosomal translocations may lead to a loss of differentiated properties, as also suggested by the appearance of the CALL antigen on the Burkitt’s lymphoma lines, in contrast to lymphoblastoid cell lines (Minowada et al., 1982). Yet another possibility would be the development of BL from a more primitive target cell than those of LCL, localized, e.g., in the bone marrow. For the EBV-negative BL, the antecedents may be quite different, and it is hardly profitable to speculate about them. The final event and the resulting disease appear to be similar or identical. The clinical differences known to exist between the African (largely EBV-positive) and the non-African (largely EBV-negative) cases may be a consequence of the differences in immune response that accompany the growth of an EBV-carrying (and therefore highly antigenic) and an EBV-negative tumor, and/or the immune status of the African and non-African patients in general.

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ACKNOWLEDGMENTS Work by the authors described in this article was supported by the Swedish Cancer Society and PHS grants number 1 R D l , CA 30264-01 and 1 R D 2, CA 28380-01AI.

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TRANSLOCATIONS INVOLVING Ig-LOCUS-CARRYING CHROMOSOMES: A MODEL FOR GENETIC TRANSPOSITION IN CARCINOGENESIS

George Klein Department of Tumor Biology, Karolinska Instilutet, Stockholm, Sweden

Gilben Lenoir International Agencyfor Research on Cancer, Lyon, France

Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Activation of specific oncogenes emerges as the common theme in many different types of carcinogenesis (see Klein, 1981). It can be brought about by a variety of mechanisms. The known oncogenes are unique and highly conserved cellular sequences. They were originally identified because of their incorporation into the genomes of directly transforming and often defective retroviruses (see Bishop, 1981). In this situation the retrovirus provides the necessary apparatus for the integration and constitutive activation of the transforming oncogene. In some cases where the protein product of the oncogene has been identified, e.g., the pp60"' of the Rous system, or the p120 protein codified by the Abelson insert, the same or very similar proteins were also found in some normal tissues, but in much smaller quantities. This finding suggests that transformation may result from the excessive production of a normal protein or from its presence in the wrong place or at the wrong time. A different, although related, mechanism is represented by Hayward's findings with a slow-acting, nontransforming, nondefective avian leukosis virus (Hayward et a/., 1981). In this case, the cellular counterpart (c-om) of a virally transmitted oncogene (v-om), designated myc, was turned on constitutively by the insertion of the retroviral promoter carrying long terminal repeat (LTR) sequence in its immediate neighborhood. It is particularly noteworthy that the same oncogene, myc, can be turned on in different ways and in different types of target cells. When carried by a defective, directly transforming leukemia virus, avian myelocytomatosis virus (MC29), myc induced myeloid and mesenchymal neoplasia (Beard, 1980). The viral promoter insertion model of Hayward was demonstrated in lymphoid leukemia. In DNA 38 1

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transfection experiments, it could induce sarcomatous transformation in fibroblasts (G. van de Woude, personal communication). Other oncogenes were found to transgress tissue barriers in a similar way (Bishop, 1982). Together with their strong evolutionary conservation, this suggests that the c-onc genes may play some basic (“household”) role in the normal cell, perhaps related to the regulation of cell division, rather than a differentiationassociated (“luxury”) function. The mechanisms of oncogene activation in the above-reported systems involve the transposition of DNA. The question therefore arises as to whether the genetic transpositions found in many types of cancer (Cairns, 198l), including those that are expressed in the form of specific chromosomal translocations or deletions, may act by similar mechanisms. Recent findings concerning nonrandom chromosomal changes-mainly reciprocal translocations in two tumor types of B-cell origin, murine plasmacytoma (MPC) and human Burkitt-type lymphoma (even leukemia) (BL)-suggest that this is probably the case. In MPC, the distal region of chromosome 15 was found to be translocated to chromosome 12 in the majority of all examined tumors (Ohno et al., 1979).The distal region of M 12 is known to carry the IgH gene cluster (Me0 et al., 1980). A variant translocation was found in a minority of the IC light-chain producers. It is generated by a reciprocal exchange between the same distal segment of chromosome 15 that is involved in the typical translocation and the K gene carrying chromosome 6 (Ohno et id., 1979). In BL, the majority of the reported cases were found to carry a reciprocal t(8 ;14) translocation. Two variant translocations, t(2;8) and t(8 ;22), were identified (Bernheim et al., 1981). They constitute 20% or less of the cases. The same distal segment of H8 is transposed in the typical and the variant translocations (breakpoint at q24). The recipient of the typical translocation, chromosome H14, is known to carry the ZgH gene cluster (Croce et al., 1979). Recent in situ hybridization experiments (Kirsch et al., 1982) have shown that IgH is localized in the band region q32. This corresponds to the breakpoint involved in the BL-associated translocation. The variant translocations further reinforce the picture. Chromosome 2 carries the K gene that was recently localized between 2p centromere and p13 on the short arm of the chromosome (Malcolm et al., 1982). The breakpoint giving rise to the variant BL translocation is in the same region (pl2). Chromosome 22 carries the 1 gene (Erikson et al., 1982; McBride et al., 1982). It is noteworthy that the translocations represent a characteristic feature of the BL cell and are independent of geographical origin, clinical presentation (with and without leukemia), and Epstein-Barr virus (EBV) association (Bernheim et nl., 1981).

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Thus it is clear that the recipient chromosome of the tumor-associated translocation carries immunoglobulin genes in both MPC and BL. But what is known about the remarkably constant donor? In T-cell-derived mouse leukemia, the analysis of chromosome 15 trisomy has led to the hypothesis that an oncogene, located in the distal part of chromosome 15, is activated by mutation in a regulator gene or by proviral DNA insertion. This cannot be fully expressed, however, unless the changed chromosome is amplified by nondisjunction (Wiener et a/., 1978a,b,c, 1980b, 1981 ; Spira eta/., 1979, 1980, 1981). This amplification is necessary to overcome a trans-acting control exerted by the unchanged homologous chromosome (Spira et a/., 1981). In mouse plasmacytoma, the translocation of the distal segment of chromosome 15 to the Zg-locus-carrying chromosome was interpreted to suggest a switch-on of what may be the same oncogene, under the influence of a functionally active region in the target cell. It is tempting to speculate that the distal region of human chromosome 8 may carry a corresponding oncogene. It is of interest here that 8-trisomy is the most common of all leukemia-associated trisomies (Mitelman and Levan, 1981). The distal region of H8 was also found to be involved in a constitutional 3 ;8 translocation that was regularly associated with renal carcinoma in every member of the family that carried it after they reached middle age (Cohen et al., 1979). In conclusion, one may postulate that the BL-associated t(2;8), t(8;14), and t(8 ;22) translocations act by the same mechanism as the corresponding translocations in MPC: by activation of an oncogene under the influence of a functionally highly active Ig locus. There are a number of ways to check the correctness of this hypothesis. It is already clear that the MPC- and BL-associated translocations arise during the tumorigenic process, and not during the differentiation of the normal Ig-producing cell. No MPC- or BL-type translocations were found in mitogen-stimulated normal B cells, nor have they been seen in EBVtransformed lymphoblastoid lines of normal or mononucleosis origin. Neardiploid BL and MPC contain one translocated and one normal homologous chromosome, whereas near-tetraploid cells (seen only in MPC) have two of each, indicating that the translocation occurs prior to tetraploidization (Ohno et a/., 1979; Wiener et al., 1980a). Is the Zg-locus-carrying translocation-recipient chromosome functionally active in the neoplastic cell? Strong indirect evidence suggests that this is probably the case. All rcp(6;15) translocation-carrying MPC were found to make K chains. Among the variant BLs, all seven 8;22 carrying Ig producer lines tested made A chains and among five 2 ;8 translocation-carrying BL, all three that produced light chains made K (Lenoir et a/., 1981, 1982). This

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GEORGE KLElN A N D GILBERT LENOIR

cannot be due to chance. Randomly selected BL biopsies and lines make K vs I?. light chains in a ratio of approximately 2:l (van Furth et al., 1972; Gunvkn et af., 1980). Are the Ig-producing chromosome regions preferentially affected by the translocations because they are more vulnerable due to the DNA rearrangements that take place in the course of normal differentiation or because they are functionally highly active? This suggestion cannot be excluded, but it is unlikely, since, for example, &producing cells have also rearranged K regions (Hieter et al., 1981). The good correlation between the light-chain type and the involvement of the corresponding chromosome in the variant BL translocations makes a functional explanation far more probable. By what mechanism can the specific translocations contribute to the genesis of B-cell tumors? Burkitt’s lymphomas are particularly interesting in this context. A total of 96% of the African BL tumors are EBV-carrying clones (Klein, 1975). EBV alone is competent to transform normal B lymphocytes into continuous (immortal) cell lines (lymphoblastoid cell lines, LCL). These lines are purely diploid during the first several months of their history (Zech et al., 1976). The LCL cells have only a limited autonomy, however. The proliferation of EBV-transformed lymphocytes in uiuo is controlled by the immune system and there are probably some nonimmunological (feedback) controls as well. EBV-carrying polyclonal lymphoproliferative syndromes are rare but they do occur in certain immunodefective individuals (summarized by Klein and Purtilo, 1981). In contrast to BL-derived lines, LCLs do not grow in the subcutaneous space of the nude mouse (Giovanella et af.,1979). BL cells have a high agarose clonability in contrast to the very limited clonability of LCLs, suggesting a difference in responsiveness to some nonimmunological control (Zech et af.,1976). It would therefore appear that the translocation confers a proliferative advantage on the B lymphocyte in vivo, over and above what is already expressed by EBVinduced in uitro immortalization. This advantage allows the cell clone to escape from growth control mechanism(s) of the organism. A number of hematopoetic neoplasias of diverse origins other than BL and B-ALL were also found to carry 14q+ markers, although much less regularly than BL (Mitelman, 1981 ; Rowley, 1981). These markers are of miscellaneous origin and differ from the BL marker. The segment translocated to chromosome 14 is not derived from chromosome 8 but from a variety of other chromosomes, as a rule. However, the breakpoint on chromosome 14 is often the same as in the BL translocation. It is intriguing to speculate that oncogene activation may occur in these cases as well. This would imply that each translocated fragment carries “its” oncogene and that the same gene region of chromosome 14 is active in the target cells, as in BL.

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Similar questions can now be asked about the oldest and best-known case of a tumor-associated translocation, the Philadelphia chromosome. Does the translocated fragment of chromosome 22 carry an oncogene that becomes activated by translocation to a variety of different chromosomes? The recipient chromosome is 9 in the majority of cases, but at least 18 other chromosomes can serve as recipients in variant translocations (Mitelman and Levan, 1981). The situation is quite different from the variant BL translocations where only chromosomes 2 and 22 have thus far been found to serve as recipients of the translocated chromosome 8 fragment. However, the distribution of the variant Ph translocation recipients is not random, but shows certain regularities. It is not inconceivable that all 18 recipient chromosomes may carry highly active regions that can activate an oncogene carried by the translocated fragment. Alternatively, the mechanism of transformation may be quite different. Since the fragment deleted from the Ph, chromosome cannot always be found in any detectable translocation, it is often suggested that the deletion of chromosome 22 rather than the translocation of the deleted fragment may be responsible for the oncogenic action of the Ph, chromosome. The “disappearance” of the deleted fragment may have spurious (technical) reasons, however. In principle, it is conceivable that pure deletions may switch on oncogenes provided some regulatory gene is lost. The recent DNA transfection experiments of Cooper et al. (1982) and of Weinberg (1982) indicate that the oncogenes of normal cells must be dissociated from flanking regulatory elements, by shearing the DNA to small pieces, before their transforming activity can be demonstrated in transfection experiments. Even when transformation is successful, this event has a low probability of success because of the need to insert the oncogene in the neighborhood of an active chromosomal region. The significance of deletions in oncogenesis is also suggested by the fact that a constitutional 13q - anomaly appears to be responsible for a fraction of the familiar cases of retinoblastoma (Balaban-Malenbaum et al., 1981; Knudson et al., 1976).The same type of 13q- anomaly can also arise during tumorigenesisat the somatic level in patients with no familiar predisposition. A possibly similar situation (involving both familiar and somatic chromosome I 1 deletion) may exist in Wilms tumor (Kaneko et al., 1981). The important question of whether deletion or translocation bears responsibility for the neoplastic behavior of cells that carry both can be subjected to experimental analysis based on the fusion of tumor cells with normal cells, followed by comparative cytogenetic analysis of high- and lowtumorigenic hybrids. This type of analysis has been helpful in clarifying the role of the chromosome 15-associated oncogene in murine T-cell leukemias (Spira et al., 1981).

,

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In conclusion, the analysis of specific tumor-associated chromosomal changes may be helpful in pinpointing the likely localization of oncogenes involved in the genesis of certain tumors and in clarifying the mechanisms involved in their activation. Perhaps the most intriguing question will concern the relationship between the oncogenes postulated from the cytogenetic analysis, and the “cancer genes” deduced from the study of certain familiar tumors (Knudson et id., 1976). Recently developed transfection methods (Shih et al., 1981) may be helpful in characterizing the oncogenes associated with specific translocationcarrying tumors. Transfection of appropriate murine cells with DNA derived from BL cells that carry one of the specific translocations may allow cloning of the relevant oncogene. Purification of the oncogene by serial passage may be followed by its isolation, on the basis of the repetitive (Ah) sequences. Subsequent in situ hybridization or hybridization with FACS-sorted chromosomes may serve to trace its chromosomal localization. As another approach, the murine and human immunoglobulin probes may be helpful in “walking the chromosome” toward the postulated oncogene. If the oncogene attaches directly downstream from the immunoglobulin locus, as postulated, the labor may not be as excessive as it may seem. Thus, the analysis of specific chromosomal translocations in tumors may provide an alternative method for oncogene identification in addition to already available virological and transfection approaches in both human and experimental tumors.

REFERENCES Balaban-Malenbaum, G., Gilbert, F., Nichols, W. W., Hill, R., Shields, J.. and Meadows, A. T. (198 I). Cancer Genet. Cytogenet. 1,243-250. Beard, J . W. (1980). In “Viral Oncology” (G. Klein, ed.), pp. 55-87. Raven, New York. Bernheim, A., Berger, R., and Lenoir, G . (1981). Cancer Genet. Cytoyenet. 3,307-315. Bishop, M. J. (1981). Cell23, 5-6. Bishop, M. J. (1982). Adu. Viral. Oncol., in press. Cairns. J. (1981). Nature (London)289,353-357. Cohen, A. J., Li, F. P., Berg, S., Marchetto, D. J . , Shien Tsai, S. M., Jacobs, S. C., and Brown, R. S. (1979). N . Engl. J . Med. 301,592-595. Cooper, G . M., Lane, M.-A., Krontiris, T. G . , and Goubin, G. (1982). Ado. Viral Oncol., in press. Croce, C. M., Shander. M., Martinis, J., Cicurel, L., and d‘Ancona, G. G. (1979). Proc. Natl. Acad. Sci. U.S.A. 76, 3416-3419. Erikson, J., Martinis, J., and Croce, C. M. (1981). Nature (London) 294, 173-175. Giovanella, B., Nilsson, K., Zech, L., Yim. O., Klein, G., and Stehlin, J. S. (1979). Int. J . Cancer 24, 103-1 13. Gunven, P., Klein, G., Klein, E., Norin, T., and Singh, S. (1980). Int. J . Cancer 25,711-719. Hayward, W. S . , Neel, B. G., and Astrin, S. M. (1981). Nature (London) 290,475-480. Hieter, P. A,, Korsmeyer, S. J., Waldmann, T. A,. and Leder, P. (1981). Nature (London) 290, 368-372.

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Kaneko, Y . ,Egues, M. C., and Rowley, J. D. (1981). Cancer Res. 41,4577-4578. Kirsch, I . R., Morton, C. C., Nakahara, K., and Leder, P. (1982). Science 216, 301-303. Klein, G. (1975). Cold Spring Harbor Symp. Quani. Biol. 39,783-790. Klein, G. (1981). Nature (London) 294,313-318. Klein, G., and Purtilo, D. (1981). Cancer Res. 41,4302-4304. Knudson, A. G., Meadows, A. T., Nichols, W. W., and Hill, R. (1976). N . Engl. J . Med. 295, 1120- 1 123. Lenoir, G., Preud'Homme, J.-L., Bernheim, A., and Berger, R. (1981). C . R . Hebd. Seances Acad. Sci. 293,421-429. Lenoir, G., Preud'Homme, J.-L., Bernheim, A., and Berger, R. (1982). Nature (London), in press. McBride, 0. W., Hieter, P. A., Hollis, G. F., Swan, D., Otey, M. C., and Leder, P. (1982). J . Exp. Med. 155, 1480-1490. Malcolm, S., Barton, P., Murphy, C., Ferguson-Smith, M. A,, Bentley, D., and Rabbits, T. H. (1982). Cytogenei. Cell Genet., in press. Meo, T., Johnson, J., Beechey, C. V., Andrews, S. J., Peters, J., and Searle, A. G. (1980). Proc. Natl. Acad. Sci. U.S.A. 77,550-553. Mitelman, F. (1981). Adu. Cancer Res. 34, 141-167. Mitelman, F., and Levan, A. (1981). Hereditas 95,79-139. Ohno, S., Babonits, M., Wiener, F., Spira, J., Klein, G., and Potter, M. (1979). Cell 18, 10011007. Rowley, J. D. (1981). Cancer Genet. Cytogenei. 1,263-271. Shih, C., Shilo, B.-Z., Goldfarb, M. P., Dannenberg, A,, and Weinberg, R. A. (1981). Proc. Nail. Acad. Sci. U.S.A. 76,5714-5718. Spira, J., Wiener, F., Ohno, S., and Klein, G. (1979). Proc. Natl. Acad. Sci. U.S.A. 76,66196621. Spira, J., Babonits, M., Wiener, F., Ohno, S., Wirschubski, Z., Haran-Ghera, N., and Klein, G. (1980). Cancer Res. 40,2609-2616. Spira, J., Wiener, F., Babonits, M., Gamble, J., Miller, J., and Klein, G. (1981). Ini. J . Cancer 28,185-798. van Furth, R., Gorter, H., Nadkarni, J. S., Nadkarni, J. J., Klein, E., and Clifford, P. (1972). Immunology 22,847-857. Weinberg, R. A. (1982). Adu. Cancer Res. 36, 149-163. Wiener, F., Ohno, S., Spira, J., Haran-Ghera, N., and Klein, G. (1978a). JNCI, J . Nail. Cancer Insi. 61,227-238. Wiener, F., Ohno, S., Spira, J., Haran-Ghera, N., and Klein, G. (1978b). Nature (London) 275,658-660. Wiener, F., Spira, J., Ohno, S., Haran-Ghera, N., and Klein, G. (1978~).Ini. J . Cancer 22, 447-453. Wiener, F., Babonits, M., Spira, J., Klein, G., and Potter, M. (1980a). Som. Cell Genet. 6, 731-738. Wiener, F., Spira, J., Babonits, M., Haran-Ghera, N., and Klein, G. (1980b). Int. J . Cancer 26,661-668. Wiener, F., Babonits, M., Spira, J., Bregula, U., Klein, G., Menvin, R. M., Asofsky, R., Lynes, M., and Haughton, G. (1981). Ini. J . Cancer 27,51-58. Zech, L., Haglund, U., Nilsson, K., and Klein, G. (1976). Int. J . Cancer 17,47-56.

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INDEX

A Abelson virus, tumor antigens of, 79,93-95 Adenovirus, tumor antigens of, 79,86-88 Antigen-presenting cell (APC), definition of, 243 Antipain (AP), anticarcinogenic activity of, 209

B B cells differentiation of, EBV-carrying lymphoid cell lines and, 353 responses, Mhc restriction of, 245-252 Basement membrane proteins, in tumor cells, 138-141 Blood cell neoplasm of Drosophila genes for, 36 mutants, 49-52 B-leukemia/lymphoma cell lines, EBV genome-negative, 349-352 Burkitt’s lymphoma human B-lymphoid cell lines and, 3 19-380 B cell differentiation and, 353-362 EBV-carrying, 338-346

C Calcium, dependence on, of radiation-transformed cultured cells, 173 Cancer, gene role in development of, 33-74 Cancer genes in Drosophila, 33-74 vertebrate gene compared to, 66-69 of retroviruses, 1-32 Carcinogenesis Ig-locus-carrying chromosomes and, 381-387 Cells, matrix interaction and anchorage in, 141-143

Cell culture, radiation oncogenesis in, 159232 Cell membrane, in radiation-transformed cultured cells, 173-175 Chromosomes, of cultured cells affected by radiation oncogenesis, 170- 171 Cocarcinogens, role in radiation oncogenesis, 206-220 Collagen biosynthesis of, I16 in pericellular matrix, 112-113, I14 Contact sensitivity, Mhc restriction of, 264265 Cytolytic responses, Mhc restriction of, 242262

D Delayed-type hypersensitivity (DTH), Mhc restriction of, 262-285 Diploid cell cultures, radiation oncogenesis in, 178- I89 DNA, of cultured cells affected by radiation oncogenesis, 170-171 Drosophila cancer genes in, 33-74 developmental stages of, 34-35 tumor cells, viruses found in, 57-61 tumor mutants of, 35-37

E Embryonic tumors of Drosophila genes for, 36 mutants, 37-39 Entactin in pericellular matrix, 115 in transformation, 12 1- 122 Epithelial cells, radiation oncogenesis in, 163 389

390

INDEX

Epstein-Barr virus (EBV) Burkitt lymphoma cell lines carrying, 338346 genome-negative cell lines, 346-349 role in Burkitt’s lymphoma, 319 role in progression, 362-364 chromosomal changes in, 364-368 transformation by, early events in, 323-325 tumor antigens of, 79, 88-91 b-Estradiol, as radiogenic oncogenesis potentiator. 208-209

F Fibroblasts, radiation oncogenesis in, 163 Fibronectin interactions of, I 1 8 in pericellular matrix, 1 13-1 20 structural model of, I17 Free radical scavengers, as radiogenic ongenesis inhibitors, 215-216

G Glycoproteins, of tumor cell line matrices, 140 Gonial cell neoplasm of Drosophilu genes for, 36 mutants, 52-57

Ir genes, 233-31 7 nonresponsiveness and, 285-309 Is genes, nonresponsiveness and, 294-296

K 32K protein, of SV40 virus tumors, 84-86 56K protein, of SV40 virus tumors, 84-86

L Laminin in pericellular matrix, I I5 properties and role in transformation, 120- 121 Lymphoblastoid cell lines cell surface properties of, 33 1-332 cytogenetic studies of, 336-337 differentiation and, BL cells and, 355-362 as EBV target cells, 321-323 functional properties of, 333-335 growth characteristics of, 335-336 morphology of, 328-33 I phenotypic characteristics of, 326-336 spontaneous establishment of, 325-326 tumorgenicity of, 337-338

M

H Hamster cells, radiation oncogenesis in, 189197 Human cells, radiation oncogenesis in, 197205

I Ig-locus-carrying chromosomes, translocations of, as model for genetic transposition in carcinogenesis, 381-387 Imaginal disc neoplasms of Drosophilu genes for, 36 mutants, 43-49 Immune response, T cell properties involved in, 266-267 Interferon, as radiogenic oncogenesis potentiator, 208-209 Ionizing radiation, oncogenesis by, cell culture studies on, 177-201

Metastasis, pericellular matrix role in, 132138 Methylcholanthrene-induced tumors, antigens of, 79,91-93 Mhc restriction and Mhc genes, 233-317 of cytolytic responses, 242-262 Th cells participating in, 252 discovery of, 238-242 of DTH and CS responses, 262-265 nonresponsiveness and. 289-310 of Ts cells, 257-262

N Neuroblastoma of Drosophila genes for, 36 mutants, 39-43 Nonresponsiveness cause of, 305-309 Mhc restriction and, 289-310

39 1

INDEX

0 Oncogenesis, c-oms role in, 23-26 Oncogenesis, pericellular matrix role in, 111158 Osteonectin, in pericellular matrix, 115

P p53 protein of SV40 virus tumors, 82-84 function of, 103-104 Pericellular matrix attachment proteins in, 119 cellular phenotype and, 123-128 chondronectin in, 122 collagen in, 112-1 13 biosynthesis, 116 properties, 114 components of, 112-123 entactin in, properties, 1 I5 fibronectin in, 113-120 laminin in, properties, 115 in malignant transformation, 11 1-158 altered matrix-cell interaction, 143-146 altered component biosynthesis, 128-132 metastasis, 132-1 38 matrix proteins of, 112 osteonectin in, properties, 1 15 proteins of, from cell cultures, 126 proteoglycans in, properties, 115, 122-123 tropoelastin-elastin in, 1 15 properties, 115 Proteoglycans in pericellular matrix, properties, 115 Proteolytic enzymes, from radiation-transformed cells, 175-177

in vitro, 177-227 ionizing radiation, 177-201 nonionizing radiation, 202-227 inhibitors of, 209-216 thyroid hormone role in, 216-220 Regulatory responses, Mhc restriction of, 245-262 Retinoids, as radiogenic oncogenesis inhibitors, 209-214 Retroviruses cancer genes of, 1-32 activity, 17 as cellular genes, 17-20 characterization, 10-12 discovery, 8-10 expression, 12- 13 function in normal cells, 20-21 origin, 7-8 other species compared to, 62-66 properties, 6-7 proteins encoded by, 13-14 transformation by, 21 -22 genome styles in, 3 transduction by, 19 Rous sarcoma virus (RSV) transformation by, altered cell-matrix interaction in, 143-146 tumor antigens of, 79,96-97

S Selenium, as radiogenic oncogenesis inhibitor, 215 SV40 virus tumor antigens of, 79 properties, 8 1-82 proteins of, 82-86

R

T

Radiation chimeras, in studies of Mhc restriction, 271-285 Radiation oncogenesis in cell culture, 159-232 criteria for, 169-177 initiation, 167-169 systems used in, 161-169 cocarcinogen role in, 206-220 enhancement of, 206-209 in human cells, 197-205

T cells proliferation of, in response to alloantigen stimulation, 255-256 properties of involved in immune response, 266-267 Teratocarcinoma, antigens of, 79,97-100 Thyroid hormone, role in radiation oncogenesis, 216-220 Topography of cells, effects of radiation oncogenesis on, 171-172

392

INDEX

Transfection, vertebrate tumor genes and, 6669 Transformation-associated tumor antigens (TSTA) classification of, 100- 103 as diagnostic tools, 80-81 function of, 75-80 viral and cellular types, 80-81 Transformation of cells, pericellular matrix rolein. 111-158

Tropoelastin-elastin, in pericellular matrix, 115 Tumor cells, basement-membrane proteins of, 138-141 Tumorigenicity, of radiation-transformed cells, 177

v Viruses, in Drosophiln tumor cells, 57-61

CONTENTS OF PREVIOUS VOLUMES

Volume 1 Electronic Configuration and Carcinogenesis 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 Rous No. 1 Sarcoma R. J . C. Harris Applications of Radioisotopes to Studies of Carcinogenesis and Tumor Metabolism Charles Heidelberger The Carcinogenic Aminoazo Dyes James A. Miller and Elizabeth C . Miller The Chemistry o f Cytotoxic Alkylating Agents M . C. J . Ross Nutrition in Relation to Cancer Albert Tannenbaum and Herbert Silverstone Plasma Proteins in Cancer Richard J . Winzler

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 Jeunne C. Bateman Genetic Studies in Experimental Cancer L. W. Law The Role of Viruses in the Production of Cancer C . Oberling and M . Guerin Experimental Cancer Chemotherapy C . Chester Stock

AUTHOR INDEX-SUBJECT INDEX

Volume 2 The Reactions of Carcinogens with Macromolecules Peter Alexander Chemical Constitution and Carcinogenic Activity G. M. Badger

393

AUTHOR INDEX-SUBJECT INDEX

Volume 3 Etiology of Lung Cancer Richard Doll The Experimental Development and Metabolism of Thyroid Gland Tumors Harold t? Morris Electronic Structure and Carcinogenic Activity and Aromatic Molecules: New Developments A . Pullman and B. Pullman Some Aspects of Carcinogenesis t? Rondoni Pulmonary Tumors in Experimental Animals Michael B. Shimkin

394

CONTENTS O F PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues Sidney Weinhouse AUTHOR INDEX-SUBJECT INDEX

Volume 4 Advances in Chemotherapy of Cancer in Man Sidney Farber, Rudolf Toch, Edward Manning Sears, and Donald Pinkel The Use of Myleran and Similar Agents in Chronic Leukemias D. A . G. Galton The Employment of Methods of Inhibition Analysis in the Normal and TumorBearing Mammalian Organism Abraham Godin Some Recent Work on Tumor Immunity P. A. Gorer Inductive Tissue Interaction in Development Clifford Grobstein Lipids in Cancer Frances L. Haven and W. R . Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A . L a c a s s a g n e , N . P. Buu H o i , R . Daudel, and E Zajdela The Hormonal Genesis of Mammary Cancer 0. Miihlbock AUTHOR INDEX-SUBJECT INDEX

Volume 5 Tumor-Host Relations R . W. Begg Primary Carcinoma of the Liver Charles Berman Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal P. N . Campbell The Newer Concept of Cancer Toxin War0 Nakahara and Fumiko Fukuoka Chemically Induced Tumors of Fowls P. R. Peacock

Anemia in Cancer Vincent E. Price and Robert E. Greenfield Specific Tumor Antigens L. A. Zilber Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizabeth K . Weisburger and John H . Weisburger AUTHOR INDEX-SUBJECT INDEX

Volume 6 Blood Enzymes in Cancer and Other Diseases Oscar Bodansky The Plant Tumor Problem Armin C.Braun and Henry N . Wood Cancer Chemotherapy by Perfusion Oscar Creech, Jr. and Edward T. Krementz Viral Etiology of Mouse Leukemia Ludwick Gross Radiation Chimeras P. C. Koller, A . J . S. Davies, and Sheila M . A . Doak Etiology and Pathogenesis of Mouse Leukemia J . E A . P. Miller Antagonists of Purine and Pyrimidine Metabolites and of Folic Acid G. M. Timmis Behavior of Liver Enzymes in Hepatocarcinogenesis George Weber AUTHOR INDEX-SUBJECT INDEX

Volume 7 Avian Virus Growths and Their Etiologic Agents J . W. Beard Mechanisms of Resistance t o Anticancer Agents R . W. Brockman

CONTENTS OF PREVIOUS VOLUMES

Cross Resistance and Collateral Sensitivity Studies in Cancer Chemotherapy Dorris J . Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W. M . Court Brown and Ishbel M . Tough Ethionine Carcinogenesis Emmanuel Farber Atmospheric Factors in Pathogenesis of Lung Cancer Paul Kotin and Hans L . Falk Progress with Some Tumor Viruses of Chickens and Mammals: The Problem of Passenger Viruses G. Negroni AUTHOR INDEX-SUBJECT INDEX

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

Volume 9 Urinary Enzymes and Their Diagnostic Value in Human Cancer Richard Stambaugh and Sidney Weinhouse The Relation of the Immune Reaction to Cancer Louis V. Caso Amino Acid Transport in Tumor Cells R. M . Johnstone and P: G. Scholefield

395

Studies on the Development, Biochemistry, and Biology of Experimental Hepatomas Harold E! Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes, and Bone Marrow Cells I . E Seitz AUTHOR INDEX-SUBJECT INDEX

Volume 10 Carcinogens, Enzyme Induction, and Gene Action H . I! Gelboin In Vitro Studies on Protein Synthesis by Malignant Cells A. Clark Griffin The Enzymatic Pattern of Neoplastic ‘Iissue W. Eugene Knox Carcinogenic Nitroso Compounds P: N. Magee and J . M . Barnes The Sulfiydryl Group and Carcinogenesis J. S. Harrington The Treatment o f Plasma Cell Myeloma Daniel E. Bergsagel, K . M. Griffith, A . Haut, and W. J . Stuckley, Jr. AUTHOR INDEX-SUBJECT INDEX

Volume 11 The Carcinogenic Action and Metabolism of Urethran and N-Hydroxyurethran Sidney S. Mirvish Runting Syndromes, Autoimmunity, and Neoplasia D.Keast Viral-Induced Enzymes and the Problem of Viral Oncogenesis Saul Kit The Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology William Regelsan

396

CONTENTS O F PREVIOUS VOLUMES

Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Joseph C. Arcos and Mary E Argus CUMULATIVE INDEX

Role of Cell Association in Virus Infection and Virus Rescue J . Svoboda and I . Hlotrinek Cancer of the Urinary Tract D. B. Clayson and E. H . Cooper Aspects of the EB Virus M. A. Epsiein AUTHOR INDEX-SUBJECT INDEX

Volume 12 Antigens Induced by the Mouse Leukemia Viruses G. Pasiernak Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses G. I . Deichman Replication of Oncogenic Viruses in VirusInduced Tumor Cells-Their Persistence and Interaction with Other Viruses H. Hunafusa Cellular Immunity against Tumor Antigens Karl Erik Hellsirom and Ingegerd Hellstrom

Perspectives in the Epidemiology of Leukemia Irving L. Kessler and Abraham M. Lilianfeld AUTHOR INDEX-SUBJECT INDEX

Volume 13 The Role of Immunoblasts in Host Resistance and Immunotherapy of Primary Sarcomata l? Alexander und J. G. Hall Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Oswald Jarreii The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Haim Ginsbrtrg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gajanan V. Sherbei The Characteristics of Animal Cells Transformed in Viiro Ian Macpherson

Volume 14 Active Immunotherapy Georges Muihe 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 George Meyer Passive Immunotherapy of Leukemia and Other Cancer Roland Motia Humoral Regulators in the Development and Progression of Leukemia Donald Meicalf Complement and Tumor Immunology Kusuya Nishioka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G. I. Abelev Low Dose Radiation Cancers in Man Alice Stewart AUTHOR INDEX-SUBJECT INDEX

Volume 15 Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J. S . Buiel, S . S. Tevethia, and J . L . Melnick Nasopharyngeal Carcinoma (NPC) J. H. C.Ho Transcriptional Regulation in Eukaryotic Cells A . J. MacGilIivray. J . Paul, and G . Threlfall

CONTENTS OF PREVIOUS VOLUMES

397

Atypical Transfer RNA's and Their Origin in Neoplastic Cells Ernest Borek and Sylvia J . Kerr Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females Philip J . Fialkow Electron Spin Resonance Studies of Carcinogene sis Harold M. Swartz Some Biochemical Aspects of the Relationship between the Tumor and the Host V. S. Shapot Nuclear Proteins and the Cell Cycle Gary Stein and Renato Baserga

Some Aspects of the Epidemiology and Etiology of Esophageal Cancer with Particular Emphasis on t h e T r a n s k e i , South Africa Gerald R Warwick and John S. Harington Genetic Control o f Murine Viral Leukemogenesis Frank Lilly and Theodore Pincus Marek's Disease: A Neoplastic Disease of Chickens Caused by a Herpesvirus K. Nazerian Mutation and Human Cancer Alfred G. Knudson, Jr. Mammary Neoplasia in Mice S. Nandi and Charles M . McGrath

AUTHOR INDEX-SUBJECT INDEX

AUTHOR INDEX-SUBJECT INDEX

Volume 16 Polysaccharides in Cancer Vijai N . Nigam and Antonio Cantero Antitumor Effects of Interferon Ion Gresser Transformation by Polyoma Virus and Simian Virus 40 Joe Sambrook Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing? Sir Alexander Haddow The Expression of Normal Histocompatibility Antigens in Tumor Cells Alena Lengerovrj 1,3-Bis(2-Chloroethyl)-l-Nitrosourea (BCNU) and Other Nitrosoureas in Cancer Treatment: A Review Stephen K. Carter, Frank M . Schabel, Jr., Lawrence E. Broder, and Thomas R Johnston AUTHOR INDEX-SUBJECT INDEX

Volume 18 Immunological Aspects of Chemical Carcinogenesis R. W. Baldwin Isozymes and Cancer Fanny Schapira Physiological and Biochemical Reviews of S e x Differences and Carcinogenesis with Particular Reference to the Liver Yee Chu Toh Immunodeficiency and Cancer John H . Kersey, Beatrice D. Spector, and Robert A . Good Recent Observations Related to the Chemotherapy and Immunology of Gestational Choriocarcinoma K. D. Bagshavr Glycolipids df Tumor Cell Membrane Sen-itiroh Hakomori Chemical Oncogenesis in Culture Charles Heidelberger AUTHOR INDEX-SUBJECT INDEX

Volume 17 Volume 19 Polysaccharides in Cancer: Glycoproteins and Glycolipids Vijai N . Nigam and Antonio Cantero

Comparative Aspects of Mammary Tumors J . M . Hamilton

398

CONTENTS OF PREVIOUS VOLUMES

The Cellular and Molecular Biology of RNA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses, and Their Relatives Howard M. Temin Cancer, Differentiation, and Embryonic Antigens: Some Central Problems J . H . Coggin, Jr. and N . G. Anderson Simian Herpesviruses and Neoplasia Fredrich W. Deinhardt, Lawrence A . Falk, and Lauren G. Wove Cell-Mediated Immunity to Tumor Cells Ronald B . Herberman Herpesviruses and Cancer Fred Rapp Cyclic AMP and the Transformation of Fibroblasts Ira Pastan and George S.Johnson TLlmor Angiogenesis Judah Folkman SUBJECT INDEX

Volume 21 Lung Tumors in Mice: Application to Carcinogenesis Bioassay Michael B . Shimkin and Gary D. Stoner Cell Death in Normal and Malignant Tissues E. H . Cooper, A . J . Bedford, and T. E. Kenny The Histocompatibility-Linked Immune Response Genes Baruj Benacerraf and David H . Katz Horizontally and Vertically Transmitted Oncornaviruses of Cats M . Essex Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms Keef A . Rafferty, Jr. Selection of Biochemically Variant, in Some Cases Mutant, Mammalian Cells in Culture G. B. Clements The Role of DNA Repair and Somatic Mutation in Carcinogenesis James E. Trosko and Ernest H . Y. Chu SUBJECT INDEX

Volume 20 Tumor Cell Surfaces: General Alterations Detected by Agglutinins Annette M. C. Rapin and Max M . Burger Principles of Immunological Tolerance and Immunocyte Receptor Blockade G. J . V. Nossal The Role of Macrophages in Defense against Neoplastic Disease Michael H . Levy and E. Frederick Wheelock Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis P. Sims and I! L. Grover Virion and Tumor Cell Antigens of C-Type RNA TLlmor Viruses Heinz Bauer Addendum to “Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing?” Sir Alexander Haddow SUBJECT INDEX

Volume 22 Renal Carcinogenesis J. M . Hamilton Toxicity of Antineoplastic Agents in Man: Chromosomal Aberrations, Antifertility Effects, Congenital Malformations, and Carcinogenic Potential Susan M . Sieber and Richard H . Adamson

Interrelationships among RNA Tumor Viruses and Host Cells Raymond K Gilden Proteolytic Enzymes, Cell Surface Changes, and Viral Transformation Richard Roblin, lih-Nan Chou, and Paul H . Black Immunodepression and Malignancy Osias Stutman SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 23 The Genetic Aspects of Human Cancer W. E . Heston The Structure and Function of Intercellular Junctions in Cancer Ronald S. Weinstein, Frederick B. Merk, and Joseph Ahoy Genetics of Adenoviruses Harold S.Ginsberg and C. S. H . Young Molecular Biology of the Carcinogen, 4-Nitroquinoline 1-Oxide Minako Nagao and Takashi Sugimura Epstein-Barr Virus and Nonhuman Primates: Natural and Experimental Infection A. Frank, W. A . Andiman, and G. Miller Tumor Progression and Homeostasis Richmond T. Prehn Genetic Transformation of Animal Cells with Viral DNA or RNA Tumor Viruses Miroslav Hill and Jana Hillova SUBJECT INDEX

Volume 24 The Murine Sarcoma Virus-Induced Tumor: Exception or General Model in Tumor Immunology? J . I? Levy and J . C . Leclerc Organization of the Genomes of Polyoma Virus and SV40 Mike Fried and Beverly E. GriBin PI-Microglobulin and the Major Histocompatibility Complex Per A. Peterson, Lars Rask, and Lars Ostberg Chromosomal Abnormalities and Their Specificity in Human Neoplasms: An Assessment of Recent Observations by Banding Techniques Joachim Mark Temperature-Sensitive Mutations in Animal Cells Claudio Basilico

399

Current Concepts of the Biology of Human Cutaneous Malignant Melanoma Wallace H . Clark, Jr., Michael J . Mastrangelo, Ann M . Ainsworth, David Berd, Robert E. Bellet, and Evelina A . Bernardino SUBJECT INDEX

Volume 25 Biological Activity of Tumor Virus DNA E L . Graham Malignancy and Transformation: Expression in Somatic Cell Hybrids and Variants Harvey L. Ozer and Krishna K . Jha Tumor-Bound Immunoglobulins: I n Situ Expressions of Humoral Immunity Isaac f? Witz The A h Locus and the Metabolism of Chemical Carcinogens and Other Foreign Compounds Snorri S. Thorgeirsson and Daniel W. Nebert Formation and Metabolism of Alkylated Nucleosides: Possible Role in Carcinogenesis by Nitroso Compounds and Alkylating Agents Anthony E. Pegg Immunosuppression and the Role of Suppressive Factors in Cancer Isao Kamo and Herman Friedrnan Passive Immunotherapy of Cancer in Animals and Man Steven A. Rosenberg and William D. Terry SUBJECT INDEX

Volume 26 The Epidemiology of Large-Bowel Cancer Pelayo Correa and William Haenszel Interaction between Viral and Genetic Factors in Murine Mammary Cancer J . Hilgers and I? Bentvelzen Inhibitors of Chemical Carcinogenesis Lee W. Wattenberg

400

CONTENTS OF PREVIOUS VOLUMES

Latent Characteristics of Selected Herpesviruses Jack G. Stevens Antitumor Activity of Corynebacterium parvum Luka Milas und Martin T. Scott SUBJECT INDEX

Volume 27 Translational Products of Type-C RNA Tumor Viruses John R . Stephenson, Sushilkumar G . Devare, and Fred H. Reynolds, Jr. Quantitative Theories of Oncogenesis Alice S. Whittemore Gestational Trophoblastic Disease: Origin of Choriocarcinoma, Invasive Mole and Choriocarcinoma Associated with Hydatidiform Mole, and Some Immunologic Aspects J . I . Brewer, E. E. Torok, B. D. Kahan, C . R . Stanhope, and B. Halpern The Choice of Animal Tumors for Experimental Studies of Cancer Therapy Harold B. Hewitt Mass Spectrometry in Cancer Research John Roboz Marrow Transplantation in the Treatment of Acute Leukemia E. Donnull Thomas, C. Dean Buckner, Alexander Fefer, Paul E. Neiman, and Ruiner Storb Susceptibility of Human Population Groups to Colon Cancer Martin Lipkin Natural Cell-Mediated Immunity Ronald B. Herberman and Howurd T. Holden SUBJECT INDEX

Volume 28 Cancer: Somatic-Genetic Considerations E M. Burnet Tumors Arising in Organ Transplant Recipients Isruel Penn

Structure and Morphogenesis of Type-C Retroviruses Ronald C. Montelaro and Dani I? Bolognesi BCG in Tumor Immunotherapy Robert W.Baldwin and Malcolm V. Pimm The Biology of Cancer Invasion and Metastasis Isaiah J. Fidler, Douglas M. Gersten, and Ian R. Hart Bovine Leukemia Virus Involvement in Enzootic Bovine Leukosis A . Burny, E Bex, H. Chantrenne, Y. Cleuter, D. Dekegel, J . Ghysdael, R . Kettmann, M. Leclercq, J . Leunen, M . Mammerickx, and D. Portetelle Molecular Mechanisms of Steroid Hormone Action Stephen J . Higgins and Ulrich Gehring SUBJECT INDEX

Volume 29 Influence of the Major Histocompatibility Complex on T-cell Activation J. E A. P: Miller Suppressor Cells: Permitters and Promoters of Malignancy? David Naor Retrodifferentiation and the Fetal Patterns of Gene Expression in Cancer JosP Uriel The Role of Glutathione and Glutathione STransferases in the Metabolism of Chemical Carcinogens and Other Electrophilic Agents L. E Chasseaud a-Fetoprotein in Cancer and Fetal Development Erkki Ruoslahti and Markku Seppiilu Mammary Tumor Viruses Dan H. Moore, Carole A. Long, Akhil B. Vaidya, Joel B. Sheffield, Arnold S. Dion, and Etienne Y. Lusfurgires Role of Selenium in the Chemoprevention of Cancer A . Clark Griffin SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

401

Volume 30

Volume 32

Acute Phase Reactant Proteins in Cancer E. H . Cooper and Joan Stone Induction of Leukemia in Mice by Irradiation and Radiation Leukemia Virus Variants Nechama Haran-Ghera and Alpha Peled On the Multiform Relationships between the Tumor and the Host V. S. Shapot Role of Hydrazine in Carcinogenesis Joseph Bal6 Experimental Intestinal Cancer Research with Special Reference to Human Pathology Kazymir M . Pozharisski, Alexei J . Likhavchev, Valeri E Klimashevski, and Jacob D. Shaposhnikov The Molecular Biology of Lymphotropic Herpesviruses Bill Sugden, Christopher R . Kintner, and Willie Mark Viral Xenogenization of Intact Tumor Cells Hiroshi Kobayashi Virus Augmentation of the Antigenicity of Tumor Cell Extracts Faye C . Austin and Charles W. Boone

Tumor Promoters and the Mechanism of Tumor Promotion Leila Diamond, Thomas G. O'Brien, and William M . Baird Shedding from the Cell Surface of Normal and Cancer Cells Paul H . Black Tumor Antigens on Neoplasms Induced by Chemical Carcinogens and by DNAand RNA-Containing Viruses: Properties of the Solubilized Antigens Lloyd W.Law, Michael J . Rogers, and Ettore Appella Nutrition and Its Relationship to Cancer Bandaru S. Reddy, Leonard A. Cohen, G . David McCoy, Peter Hill, John H . Weisburger, and Ernst L. Wynder

INDEX

INDEX

Volume 33

The Epidemiology of Leukemia Michael Alderson The Role of the Major Histocompatibility Gene Complex in Murine Cytotoxic T Cell Responses Hermann Wagner, Klaus Pfizenmaier, and Martin Rollinghoff The Sequential Analysis of Cancer Development Emmanuel Farber and Ross Cameron Genetic Control of Natural Cytotoxicity and Hybrid Resistance Edward A. Clark and Richard C . Harmon Development of Human Breast Cancer Sefon R . Wellings

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

INDEX

INDEX

Volume 31

402

CONTENTS OF PREVIOUS VOLUMES

Volume 34 The Transformation of Cell Growth and Transmogrification of DNA Synthesis by Simian Virus 40 Robert G. Martin Immunologic Mechanisms in UV Radiation Carcinogenesis Margaret L. Kripke The Tumor Dormant State E. Federick Wheelock, Kent J . Weinhold, and Judith Levich Marker Chromosome 1 4 9 in Human Cancer and Leukemia Felix Mitelman Structural Diversity among Retroviral Gene Products: A Molecular Approach to the Study of Biological Function through Structural Variability James W. Gautsch, John H . Elder, Fred C. Jensen, and Richard A. Lerner Teratocarcinomas and Other Neoplasms as Developmental Defects in Gene Expression Beatrice Mintz and Roger A . Fleischman Immune Deficiency Predisposing t o Epstein-Barr Virus-Induced Lymphoproliferative Diseases: The X-Linked Lymphoproliferative Syndrome as a Model David T. Purtilo INDEX

Volume 35 Polyoma T Antigens Walter Eckhart Transformation Induced by Herpes Simplex Virus: A Potentially Novel Type of Virus-Cell Interaction Berge Hampar Arachidonic Acid Transformation and Tumor Production Lawrence Levine

The Shope Papilloma-Carcinoma Complex of Rabbits: A- Model System of Neoplastic Progression and Spontaneous Regression John W. Kreider and Gerald L. Bartlett Regulation of SV40 Gene Expression Adolf Graessman, Monika Graessmann, and Christian Mueller Polyamines in Mammalian Tumors, Part I Giuseppe Scalabrino and Maria E. Feriolo Criteria for Analyzing Interactions between Biologically Active Agents Morris C. Berenbaum INDEX

Volume 36 Polyamines in Mammalian Tumors, Part I1 Giuseppe Scalabrino and Maria E. Ferioli Chromosome Abnormalities in Malignant Hematologic Diseases Janet D. Rowley and Joseph R . Testa Oncogenes of Spontaneous and Chemically Induced Tumors Robert A. Weinberg Relationship of DNA Tertiary and Quaternary Structure to Carcinogenic Processes Philip D. Lipetz, Alan ti. Galsky, and Ralph EStephens Human B-Cell Neoplasms in Relation to Normal B-Cell Differentiation and Maturation Processes Tore Godal and Steinar Funderud Evolution in the Treatment Strategy of Hodgkin’s Disease Gianni Bonadonna and Armando Santoro Epstein-Barr Virus Antigens-A Challenge to Modem Biochemistry David A. Thorley-Lawson, Clark M . Edson, and Kathi Geilinger INDEX