Advances in Cancer Research Volume 47

ADVANCESINCANCERRESEARCH VOLUME 47

This Page Intentionally Left Blank

ADVANCES IN CANCERRESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

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

Volume 47-7986

ACADEMIC PRESS, INC. Harcourt Brace Jovanovlch, Publishers

Orlando San Diego New York Austin Boston London Sydney Tokyo Toronto

COPYRIGHT @ 1986 BY ACADEMIC PRESS. INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS. ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY. RECORDING. OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC. Orlando. Florida 32887

United Kingdom Edition published by

ACADEMIC PRESS INC.

(LONDON) 24-28 Oval Road. London NWI 7DX

LTD.

LIBRARY OF CONGRESS CATAL.0G C A R D N U M B E R :

ISBN 0-12-006647-5 PHlNT6.D IN THE UNITED STAT):S OF AMtHtCA

8 6 8 7 ~ 1 ~ 9 8 7 6 5 4 3 2 1

52-1 3360

CONTENTS

Genetic Epidemiology of Familial Aggregation of Cancer NANCYR . SCHNEIDER. W . R . WILLIAMS. AND R . S. K . CHAGANTI

I. I1. 111. IV. V. VI . VII .

1 Introduction .................................................... Early Investigations through the Nineteenth Century . . . . . . . . . . . . . . . . . . 2 5 The Early Twentieth Century: 1900-1930 ........................... 7 The Mid-Twentieth Century: 1930-1970 ............................ Current State.................................................... 14 Recent Developments in Genetic Epidemiology. ..................... 19 Summary ....................................................... 28 References ...................................................... 30

Terminal Transferase in Normal and Leukemic Cells F . J . BOLLUM AND L. M. S . CHANG

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

I1. Biochemistry of Terminal Transferase .............................. 111 Ontogeny ...................................................... IV. Leukemia Marker Studies., ...................................... V . The Nature of TdT+ Cells ........................................ VI . Morphology of TdT+ Cells ....................................... VII . Evolution ...................................................... VIII . Conclusions .................................................... References .....................................................

.

37 38 41 46 49 51 55 58 58

Malignant Metamorphosis: Developmental Genes as Culprits for Oncogenesis in Xiphophorus MANFREDSCHWAB I. I1. 111. IV.

Introduction .................................................... The Teleost Xiphophoms ........................................ Spot Patterns and Melanomas., ................................... Genetic Loci Associated with Susceptibility to Carcinogens ........... V

63 65 65 75

vi

CONTENTS

V. VI . VII . VIII .

Anti-oncogenes ................................................. Molecular Approaches for Identifying Tumor Genes . . . . . . . . . . . . . . . . . . Are Genetic Tumors in Xiphophorus a Peculiarity of Nature? . . . . . . . . . . Summary ...................................................... References .....................................................

82 89 91 93 94

Oncogenes in Retroviruses and Cells: Biochemistry and Molecular Genetics KLAUS BISTERAND HANSW. JANSEN I. I1 I11 IV. V. VI . VII . VIII .

. .

Introduction.................................................... Definition of Oncogenes ......................................... The myc Oncogene ............................................. Themil(rafl0ncogene .......................................... The erbB and erbA Oncogenes .................................... The myb and ets Oncogenes ...................................... Evolution of Retroviral Oncogenes................................. Conclusions and Perspectives ..................................... References ......................................................

99 104 113 131 140 152 160 168 170

Activation of Cellular Oncogenes in Hemopoietic Cells by Chromosome Translocation SUZANNE CORY 189 I . Introduction.................................................... I1 The c-myc Translocation in Burkitt Lymphomas and 190 Murine Plasmacytomas .......................................... 111. Variant Translocations in Burkitt Lymphomas and Murine 212 Plasmacytomas ................................................. IV. Other Translocations Specific to B-Cell Leukemias and Lymphomas . . . . 217 V Translocations Specific to T-cell Leukemias and Lymphomas . . . . . . . . . . 218 VI . The Philadelphia Chromosome in Chronic Myeloid Leukemia . . . . . . . . . 220 VII . Translocations Specific to Acute M yeloid Leukemias . . . . . . . . . . . . . . . . . 224 225 VIII . Concluding Remarks ............................................ 226 References .....................................................

. .

Oncogene Amplification in Tumor Cells KARI ALITALOAND MANFRED SCHWAB I . Introduction.................................................... I1. DMINs and HSRs Contain Amplified Oncogenes ....................

235 241

CONTENTS

vii

.

111 Translocations and Rearrangements May Accompany

Oncogene Amplification.......................................... IV The Mechanisms of Gene Amplification ............................ V. Carcinogen-Induced Gene Amplification and Clonal Selection of Cancer Cells. ................................................ VI . Tumor Specificity of Oncogene Amplification........................ VII Enhanced Expression of Amplified Oncogenes ...................... VIII. Role of c-myc Deregulation in Lymphoid Malignancies . . . . . . . . . . . . . . . IX. Revealing the Normal Functions and Regulation of c-myc . . . . . . . . . . . . . X . Role of Oncogene Amplification in Multistage Carcinogenesis and Tumor Progression .......................................... References .....................................................

.

245 247

.

249 251 261 264 267 270 273

Transcription Activation by Viral and Cellular Oncogenes

.

JOSEPH R NEVINS

I . Introduction ..................................................... I1. Transcription Control by Viral Oncogenes ............................

.

111 Transcription Control by Cellular Oncogenes .........................

IV. Activation of Cellular Transcription by Viral Oncogenes ................ V. Summary and Perspectives ........................................ References ......................................................

283 284 291 291 294 294

Epidemiology and Early Diagnosis of Primary Liver Cancer in China YEH

Fu-SUNAND SHEN KONG-NIEN

I. Introduction .................................................... I1. Distribution .................................................... Environmental Factors ........................................... Family Factors ................................................. Immunosuppression ............................................. Other Factors .................................................. Discussion and Summary ......................................... The Early Diagnosis of Primary Liver Cancer ....................... Summary and Conclusion ........................................ References .....................................................

297 297 299 313 314 314 315 318 326 327

INDEX ...............................................................

331

.

111 IV. V VI VII VIII

. . . .

IX.

This Page Intentionally Left Blank

GENETIC EPIDEMIOLOGY OF FAMILIAL AGGREGATION OF CANCER Nancy R. Schneider,'.' W. R. Williams,t and R. S. K. Chaganti' Laboratory of Cancer Genetics and Cytogenetics and the Department of Pathology. Memorial Sloan-Kettering Cancer Center, New York. New York 10021

t Fox Chase Cancer Center. Philadelphia. Pennsylvania 19111

I. Introduction

Today ample evidence exists that cancer susceptibility in man can be inherited (Lynch, 1976; Bergsma, 1976; Lynch et al., 1977; Mulvihill et al., 1977; Knudson, 1977; Schimke, 1978). Several genetic traits are associated with development of benign or malignant neoplasms. Among them are conditions in which neoplasia is either the sole feature, e.g., Gardner's syndrome (Gardner and Richards, 1953) and Sipple's syndrome (Schimke and Hartmann, 1965); a frequent concomitant, e.g., the primary immunodeficiencies (Spector et al., 1978) and the chromosome breakage syndromes (German, 1972); or an unusual complication, e.g., al-antitrypsin deficiency (Berg and Eriksson, 1972). Predispositions to specific malignancies are also associated with several kinds of abnormal chromosome constitutions: trisomies (Krivit and Good, 1957; Harnden et al., 1971), a monosomy (Simpson and Photopulos, 1976), deletions (Yunis and Ramsay, 1978,1980; Riccardi et al., 1978), and a balanced translocation (Cohen et al., 1979). In addition to the established modes of inheritance of cancer predisposition mentioned above, many kindreds have been described in which a striking familial clustering of malignancy of specific anatomic sites is evident, but with unclear patterns of inheritance (e.g., Lynch et al., 1976b; Blattner et al., 1979b). Indeed, virtually every ki human cancer, even the most common kinds, has been shown clude a small subgroup in which an hereditary component can be recognized as a major factor in its development (Knudson et al., 1973). In this review, studies of the incidence of familial cancer will be discussed from a historical perspective, emphasizing the application Present address: Department of Pathology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235.

I ADVANCES IN CANCER RESEARCH, VOL. 47

Copyright 0 1986 by Academic Press, Inc.

All rights of reproduction in any form reserved.

2

NANCY R. SCHNEIDER ET AL.

of various statistical-epidemiological methods that have evolved. Finally, we will review more recent methodological developments in genetic epidemiology, namely segregation and linkage analysis, and the opportunities they provide to elucidate more fully the heritable nature of cancer susceptibility in man. II. Early Investigations through the Nineteenth Century

The concept that a predisposing or constitutional factor was a necessary “exciting” (i.e,,initiating) cause of disease is as old as Galen (ca. 200 AD). This concept was still widely accepted in the nineteenth century when a constitutional predisposition to cancer specifically was proposed by the pathologists Rokitansky and Virchow in their respective theories of neoplasia (Kardinal and Yarbro, 1979). The earliest known familial aggregate of cancer reported in English (albeit in a footnote) appeared in 1837 in the first book about cancer to be published in the United States (Warren, 1837). The family reported was illustrative of the “scirrho-cancer” type of breast cancer in which “frequently some of the relatives of the affected person are cancerous” (p. 281) and included an affected male among five first-degree relatives with breast cancer. However, familial aggregates of cancer and the theory that cancer is hereditary apparently were known in Great Britain by 1802 when the Society for Investigating the Nature and Cure of Cancer included the query, “Are there any proofs of cancer being an hereditary disease?’ in a questionnaire for English physicians. The Society disbanded a few years later and, unfortunately, nothing is known of the results of its questions (Shimkin, 1957). Nevertheless, data and discussion pertaining to the issue of familial cancer appeared in the publications of some of the most prominent physicians of the middle 1800s; chief among them were Sir James Paget in England and Paul Broca in France. The works of these two influential individuals illustrate both the kind of evidence presented then as “proof’ of cancer heredity, and the earliest recognition of its shortcomings. Paget wrote extensively on the question of cancer heredity (1853, 1957), espoused a “constitutional” theory of neoplasia similar to Virchow’s, and determined the proportion of his patients who gave a family history of “eancerous or other tumors.” He asserted that “It is hence certain that cancerous disease, or a tendency to it, is prone to pass by inheritance from parent to offspring. . . . It may seem unnecessary to bring evidence of a fact so generally believed; but there are some who doubt it” (1857, p. 191). Paget admitted that the basis for

FAMILIAL AGGREGATION OF CANCER

3

the doubt was the high frequency with which so common a disease could occur in family members by “accident,” but he believed (without benefit of statistics) that his own results could not be explained by coincidence. The obvious inadequacies of Paget’s study were criticized a decade later by Broca. However, the average physician of the day accepted Paget’s data, and his work remained a model for studies of cancer heredity for nearly 50 years. Cancer-ridden pedigrees reported by or including famous persons (e.g., Napoleon Bonaparte’s family) made striking impressions and convinced many that hereditary influence in cancer was indisputable (Sokoloff, 1938). Probably the most frequently cited “cancer family” in the literature is that of “Madame Z.,” reported in France in 1866by Broca, In this remarkable family (generally believed to be that of Broca’s wife) many of the female members in three generations died of breast cancer or “liver” cancer. Broca, like Paget, noted that chance alone could account for more than one case of cancer in a family, but he emphasized repeatedly that the question of cancer heredity could not be resolved until “extensive and systematic statistics” of cancer incidence and patterns in the general population would be available, permitting calculation of the probability of coincidence (p. 150), although he admitted that the difficulty of acquiring such data amounted almost to an impossibility. He then analyzed the incidence of cancer in Madame Z.’s pedigree compared to that expected from his estimation of the cancer mortality rate in the French population over 30 years of age, and showed that 15 times more cases had occurred in that family than would be expected in a group of similar size. Broca applied his method of analysis to Paget’s data (vide supra) and concluded that by coincidence alone even more of Paget’s patients should have reported a relative with cancer. He further criticized Paget for failing to note that when tumors of different kinds occur together in a family, one must be even more cautious in attributing an hereditary influence than when they are of a single type. Like Paget, he expressed bewilderment on how a tendency to a late-onset disease such as cancer could be inherited, although neither doubted that that was the case. Paget had remarked that when an hereditary tendency to cancer existed among his cases, it did not lead to an unusually early manifestation of disease, as if this observation were contrary to expectation. Referring to Madame Z.’s kindred, Broca stated that “contrary to the opinion accepted by some authors, these hereditary cancers did not afflict young subjects” (p. 152), but he gives no references to those

4

NANCY R. SCHNEIDER ET AL.

authors or to the source of the opinion. These statements imply that early age of onset had already been observed to be a feature of hereditary or familial disease by the mid-nineteenth century. [Comparison of Madame Z.’s family with recent United States site- and age-specific rates of cancer incidence (Cutler and Young, 1975) reveals that in fact, 7 of the 16 cancer cases died at ages which today would fall into the lowest age decile for diagnosis of those cancers, and that only one of the 16 was above today’s median age of cancer diagnosis at the time of her death,] Many studies like Paget’s were performed in Europe in the late nineteenth century, i.e., studies which found the percentage of cancer cases who had a positive family history of cancer, with regard for neither the size of the families nor the ages of their members. These studies, predictably, were divided almost evenly in their conclusions regarding the heritability of cancer predisposition (Hillier and Tritsch, 1904). Butlin (1887), who made the original observation that cancerous relatives of breast cancer patients were almost invariably in one line of descent, remarked that few people were able to provide adequate information about many of their relatives, and that in general, family histories were difficult to evaluate because “one rarely knows how many members of a family have escaped the disease” (p. 438), a point implicitly recognized by Broca, but unappreciated by other investigators of that era. Perhaps because of difficulty obtaining population data, or possibly because of the esteem accorded Paget at the time, Broca’s approach to evaluation of family cancer data was not used again until the following century, with one exception. In 1878, Cripps compared the incidence of cancer deaths in parents of cancer patients with the incidence among adults in England in general, and concluded that liability to cancer was not transmitted from parent to child. [Four years later, Cripps (1882) first described the familial nature of multiple colon polyps, later recognized as a precancerous autosomal dominant condition. Several of the prototypic “genetic cancers” were described during the late nineteenth century, although their formal genetics had to await definition until some time after the rediscovery of Mendel’s principles in 1900.1 Thus, by the end of the nineteenth century, the first attempts had been made to obtain quantitative data pertaining to familial incidence of cancer. Although reports of individual families with unusual amounts or types of cancer produced striking impressions, surveys of cancer incidence in the relatives of large numbers of cancer patients left the question “Is cancer hereditary?” unresolved.

FAMILIAL AGGREGATION OF CANCER

5

111. The Early Twentieth Century: 1900-1930

Around the turn of the century, three fields contributed novel ways of analyzing data that would, after a latency of several decades, greatly change the approaches, methods, and conclusions of studies of cancer genetics. These were statistics, genetics, and epidemiology.

A. STATISTICS Several fundamental techniques of statistical analysis (such as the chi-square) were developed and introduced around the turn of the century. The first cancer genetics study to be analyzed with statistical methods was that of Hillier and Tritsch in 1904. Their data, which consisted of the family history of cancer taken from the records of 3000 cancer patients and a much smaIler number of non-cancer patients, were analyzed statistically by Karl Pearson (1904).Hillier and Tritsch admitted that most of their patients knew very little about disease in their relatives; further, Pearson lamented the lack of an adequate control group and especially the lack of information about the number of nondiseased relatives of the study patients-serious deficiencies in all previous studies as well. In spite of these difficulties, Pearson undertook the analysis of these data, and detected a small negative correlation between the presence of cancer in a patient and cancer in his relatives. Although not accepted by Pearson himself, these results led initially to ridicule both of Pearson and of biometrical methods in general (see Church et al., 1909). By the following decade, however, others had begun to appreciate the importance of the design deficiencies in past studies that Pearson had noted.

B. EPIDEMIOLOGY During the last decade of the nineteenth century, the British medical community became concerned that cancer mortality rates seemed to be rising rapidly. The now widely used basic epidemiological technique of age adjustment of rates was developed in 1891 by King and Newsholme specifically to permit comparison of cancer mortality rates between populations of different age compositions. They could then show that, because the population was living longer than before, the rising rate of cancer mortality was more apparent than real. Because the most important factor influencing the incidence of cancer is age, the technique of age standardization or adjustment is now considered essential for most epidemiological studies of cancer. Among the first investigations into the genetic epidemiology of can-

6

NANCY R. SCHNEIDER ET AL.

cer were the studies of Cripps (described above) and of Bashford (1908), an outspoken critic of Pearson. Using current cancer mortality rates for the adult British population, Bashford constructed a table showing how many cancer deaths could be expected by chance in families of various sizes, and showed that fully half of all families with more than five adults would have at least one case of cancer by chance alone. Although he expressed the familiar complaint that it was “difficult to impossibility” to obtain complete family histories for even two generations from the great majority of patients, he, like Cripps 30 years before, found that in more than 300 cancer patients, the proportion of individuals with cancer among parents was nearly identical to the proportion of cancerous adults in the general population; Bashford concluded that constitutional predisposition had no part in cancer etiology. In support of his conclusion, he referred to a study made in Germany by Weinberg and Gaspar in 1904 (cited in Church et al., 1909) which was the first cancer genetics study to use spouse controls. Weinberg and Gaspar found the proportion of deaths from cancer to be identical in the parents and siblings of both the cancer patients and their spouses. In contrast, the first study of the genetic epidemiology of cancer to be made in the United States (Little, 1923) found a marked excess of cancer in both the siblings and progeny of cancerous individuals. Little’s study differed from those of Bashford and Cripps not only in its conclusions, but also in the extraordinary (for the time) completeness of the pedigree data analyzed, which were taken from records in the Eugenics Record Office of the Carnegie Institution of Washington, D.C. Little also used newly available American cancer mortality statistics to calculate the number of cancer deaths expectedfor each sex and age group, and compared these with the numbers actually observed. C. GENETICS

The rediscovery of Mendelian genetics in 1900 transformed curious familial aggregates of cancer into collections of data in which meaningful patterns of transmission could sometimes be discerned and predictions of risk made. Unfortunately, however, only a small minority of cancer families showed a clear Mendelian pattern of inheritance, so progress in identifying single genes predisposing to cancer was, and continues to be, slow. In 1912 Levin, in the United States, made the first attempt to analyze the pattern of malignant disease according to the laws of Mendel in hundreds of members of five kin-

FAMILIAL AGGREGATION OF CANCER

7

dreds. He found that although the overall incidence of cancer was not greater than that in the general population, there were certain “fraternities” within the kindreds that showed site-specific susceptibilities to malignancy. He concluded that resistance to cancer is a dominant trait. Levin pointed out, as Pearson had earlier, that each of the two principal methods of study, enumeration of hospital cases with positive family history and description of familial aggregates, contained the same error: failure to compare the number of affected members with the number of unaffected in each generation. Neither Mendelian nor statistical analysis could proceed in the absence of such information. In the following year Warthin (1913) published the results of an 18year study of the family histories of cancer in 1600 patients with histologically documented carcinoma; some of the relatives’ cancers were verified histologically as well. His was the first study of such magnitude based on cases with a tissue diagnosis instead of much less reliable clinical reports or mortality records. Typically, nearly half of the patients could provide no reliable information about their relatives, but among the rest Warthin identified more than 30 with cancer in more than one generation, including four families that showed a striking susceptibility to site-specific malignancies. Warthin noted a marked tendency for the neoplasm to develop at an earlier than usual age in these families, a phenomenon that already had been observed by others for certain types of familial cancer that later would be recognized to be single-gene, Mendelian traits, Warthin concluded that his study, in contrast to Levin’s, showed inherited susceptibility, rather than resistance, to cancer in certain families. [One of Warthin’s families, the “G” family, is still under study today by H. T. Lynch and colleagues (e.g., Lynch et al., 1976b).I Two reviews of the state of knowledge of cancer heredity in man appeared in the early 1920s (Ewing, 1922; Wells, 1923); each concluded that the quality of data in previous studies and the conclusions drawn from them were, in sum, worthless. These two reviews seemed to mark a transition from the haphazard, methodologically unsound work of the past to the more carefully planned and executed studies which were soon to appear. IV. The Mid-Twentieth Century: 1930-1970

By 1930, experiments with mice repeatedly had demonstrated genetic susceptibility to specific types of tumors (e.g., Slye, 1922), and Mendelian patterns of transmission had been identified in several

8

NANCY R. SCHNEIDER ET AL.

familial human disorders that predisposed to certain cancers (Cockayne, 1927).The mouse experiments and consideration of human pedigrees suggested to investigators of this era that when an hereditary predisposition to cancer existed, the predisposition was rarely a general one, but rather almost always limited to a single anatomic site in a given mouse strain or human family. The answer to the previous era’s question “Is cancer hereditary?” seemed to be a qualified negative, and a new, subtler question took its place: “Is there a genetic component in the etiology of some human cancers?” Except for the occasional report of a family affected by neoplasia associated with a known or newly described gene, few cancer families were reported in the literature during this period. Attention was devoted to the carrying out of more sophisticated, rigorous, large-scale studies necessary to demonstrate what was now believed to be a much smaller or less frequent genetic contribution to human cancer. In contrast to the studies of the past, the large, well-planned, statistical surveys of the mid-twentieth century usually did not lump all types of cancer together, but instead investigated only one anatomic site of malignancy at a time for evidence of an hereditary component. The general method of these studies was to select a large group of patients with a specific kind of cancer and a group of non-cancer patients or healthy individuals as controls, matched as closely as possible to the cancer patients for important variables such as age and sex. Family history of cancer was collected with equal care from both groups and analyzed for statistically significant differences. As a second control, some investigators also used cancer experience in the general population. Studies of this type often were called “propositus” studies, referring to the starting point around which data were collected, or, more aptly, “statistical” studies, a term which better differentiates them from studies in which the families of one or a few propositi are subjected to genetic (i.e., Mendelian) analysis. Waaler (1931) in Norway and Wassink (1935) in the Netherlands performed two of the more reliable early surveys. Each compared cancer rates in large numbers of patients’ relatives with cancer mortality rates in the general population; Waaler used spouse controls also. Waaler found more cancer of all sites among the siblings of patients than in either of his control groups. Wassink found more cancer of the same site as the proband’s among the relatives of breast cancer and uterine cancer patients but did not find cancer in general to be more frequent among them. He concluded that heredity determined the site of cancer, not a general predisposition to malignancy. Cancer of the breast was the site most often chosen for study be-

FAMILIAL AGGREGATION OF CANCER

9

cause of its prevalence and its ease of diagnosis compared to visceral malignancies. Lane-Claypon’s classic investigation of breast cancer in England in 1926 was the first modern case control study in cancer research. Family history of cancer was only one of the many variables evaluated; she found a small but consistent excess of cancer among relatives of the breast cancer patients, and that their sisters had twice as many breast cancers as did the controls’ sisters. In the Uriited States, Wainwright (1931) duplicated Lane-Claypon’s study with remarkably similar results. In MOSCOW, Martynova (1937) found 18 times more breast cancer among the relatives of breast cancer patients than among those of a control group of dental clinic patients; no population data were available for comparison. Hers was by far the largest excess reported, but all who since have investigated the familial incidence of breast cancer consistently have found about a twofold increase in its frequency among the female relatives of propositae, e.g., Jacobsen (Denmark), 1946; Smithers (England), 1948; Penrose et al. (England), 1948; and in the United States: Woolf (Utah), 1955; V. E. Anderson et al. (Minnesota), 1958; Macklin (Ohio), 1959; D. Anderson (Texas), 1972. Jacobsen also reported a significant increase of cancer in general in relatives of breast cancer patients, but no subsequent study found this to be the case. It is of interest that Jacobsen also reported that patients who had relatives with any kind of cancer were on average younger than those who had no cancer in the family. Smithers was unable to confirm this observation, but both V. E. Anderson et al. and D. E. Anderson noted such an association. Three often-cited studies were made in Denmark in the late 1940s to assess cancer predisposition in the families of patients with breast cancer (Jacobsen, 1946) (uide supra), leukemia (Videbaek, 1947), or uterine cancer (Brobech, 1949). Each verified all reported cancers in relatives by means of death certificates and medical records, and each used a control group for comparison. However, Busk (1948) reexamined Jacobsen’s and Videbaek’s data and compared them not only to the Danish population’s cancer mortality rates but also to the cancer morbidity (incidence) rates which then were becoming available. Busk found that the control families, compared with families of index cases, provided far less information in general and reported far fewer cases of cancer than expected from population rates, leading to some reinterpretation of the results of both studies. (Jacobsen’s finding of increased breast and other cancer among the relatives of breast cancer patients held up even after reanalysis, but Videbaek’s original conclusion of increased cancer in the families of leukemia victims did not). These studies, summarized by Kemp (1948) before Busk’s critique

10

NANCY R. SCHNEIDER ET AL.

and afterwards by Clemmesen (1949), are good examples of the chief difficulty of using even the most carefully selected control group: underreporting by control subjects. A similar deficiency of information is evident in Martynova’s control group and may explain the magnitude of the difference she found. Fortunately, during this period the first reliable and detailed data concerning cancer incidence in the general population began to become available in Europe and the United States, eventually replacing less reliable mortality data as a standard for comparison and making selection of a separate control group unnecessary. A population-based survey of cancer incidence was first attempted in the United States in Massachusetts in 1927 (Petrakis, 1979). In 1935 the Connecticut Tumor Registry was established, the most complete record of cancer incidence in a population available today (Eisenberg, 1966). In 1937, the National Cancer Institute (NCI) conducted its first National Cancer Survey (Dorn, 1944), repeated in 1948-1949 (Dorn and Cutler, 1958) and again in 1969-1971 (Cutler and Young, 1975). In 1941 a study remarkable at the time for the sophistication of its methods was published by Crabtree in the AmericanJournaZ of Public Health. Crabtree and other National Cancer Institute staff interviewed more than 1000 patients with skin cancer or cancer of the breast, uterine cervix, lip, or lung regarding, among other variables, family history of cancer. (The work was begun at Memorial Hospital in New York City and later extended to hospitals in several other cities.) The chief weakness of this study was that no attempt was made to verify the family members’ cancer reported by the patients. The importance of the study was that the results were analyzed according to now-basic epidemiological techniques that had not been applied previously in investigations of cancer heredity; i.e., the number of person-years at risk was calculated for each relative, and age-specific cancer mortality rates for different periods of calendar time were applied to the various age groups of the study population in order to calculate the expected number of cancer deaths. Crabtree showed that the number of deaths from all causes predicted by this procedure for the parents of his subjects agreed nearly perfectly with the number actually reported, affirming the soundness of the method. This represented an important advance in methodology and permitted much more accurate calculation of the expected numbers of cases by taking into account both the changing cancer mortality rates over time and the actual length of time each person was at risk for developing cancer. (Later, in 1948, Penrose et al. and Karn described and recommended this procedure in the European literature.)

FAMILIAL AGGREGATION OF CANCER

11

Crabtree presented the results for patients grouped according to anatomic site of cancer and, although no reason was given for doing so, according to whether the patient’s age at cancer diagnosis was above or below the median age of onset for the anatomic site. The results showed that when excess cancer mortality in relatives existed, it was confined almost invariably to the parents and sibs of patients younger than the median age. For example, the parents of breast cancer patients under age 45 had twice the expected cancer mortality, whereas the parents of the older group had no more than the expected number of cancer deaths. The tendency for the site affected to be the same as that of the propositus was also more striking in the parents and sibs of the younger group of patients. It is unfortunate that these important and isolated observations of Crabtree did not appear to be known to most of the investigators who conducted the large surveys of the 1940s and 1950s. Although, as mentioned, Jacobsen independently observed an association between age at diagnosis and positive family history that Smithers was unable to confirm, only one other group of investigators (V. E. Anderson et al., 1958, referring specifically to Crabtree) thought to analyze age at onset of the probands in relation to the cancer experience of their relatives. Four well-planned and extensive American studies were published between 1955 and 1960; two of them had begun in the 1940s. Their results and conclusions did not differ from those of earlier studies, but they are notable because each focused detailed attention on methods of data collection and analysis. Each of these investigators was aware of the importance of verifying not only the reported cancers but also other causes of death by means of medical records. Each used more refined epidemiological and statistical methods than earlier studies had, and even though all but one also included control groups, each used cancer experience in the general population as its principal standard of comparison. Woolf (1955), analyzing data with more than a single statistical method, found a significant excess of stomach cancer among first-degree relatives of 200 stomach cancer patients and a significant excess of breast cancer among the mothers and sisters of 200 breast cancer propositae. Weaknesses of his otherwise admirable study are the selection of propositi from death certificates rather than from pathology and hospital records, and verification of relatives’ cancer with death certificates only. However, he confined his study to lifelong residents of the state of Utah, so both his death certificate and genealogical information were exceptionally complete, owing to the record-keeping practices in that state.

12

NANCY R. SCHNEIDER ET AL.

The study of V. E. Anderson et al. (1958) of 621 breast cancer patients concentrated on family history of cancer but included other variables as well. Information was gathered by initial interview, with subsequent mailed questionnaires and letters. These investigators tried to verify all reported cancers by hospital and physician records in addition to death certificates. Like Woolf, they analyzed their data several ways and devoted considerable attention to evaluation of the problems and limitations of the methods used. They found that only breast cancer was in significant excess, and the excess was in the sisters only, not the mothers, of the probands. Referring to Crabtree’s study, they noticed a trend toward increased occurrence of cancer among relatives of younger patients, but the increase did not attain statistical significance. Macklin’s even larger survey of family cancer history of breast cancer patients and two control groups was begun in 1946 but not published until 1959. She sought to verify causes of all deaths among thousands of first- and second-degree female relatives, and found the reliability of the information her patients provided to be much lower than that reported by other investigators. She found a significantly increased rate of breast cancer among the second-degree, as well as the first-degree, relatives of the breast cancer patients compared to both the control groups and the general population. A very large number of striking aggregations of familial leukemia have been reported during the relatively short time since leukemia was first recognized to be a malignant disease, stimulating many statistical surveys of the prevalence of cancer, leukemia, and other variables in the families of large groups of patients. (These are reviewed by Steinberg, 1960, and more recently by Gunz, 1974.) Most early studies lumped together both adult and child probands as well as all types of leukemia. Generally these studies found no increase in malignant disease among relatives, but the methods of many were so inadequate or incompletely described that their results cannot be evaluated. Steinberg’s (1960) smaller survey of familial factors in relation to acute leukemia is noteworthy not only for its high quality but also because of the conflicting opinion and results that continue to characterize the issue of familial leukemia. Steinberg limited his study to families of children with acute leukemia. Parents were interviewed, and second-degree relatives were mailed questionnaires. Attempts were made to obtain medical records or death certificates for all relatives. Steinberg noted the repeated failure of previous investigators to obtain satisfactory control groups; using appropriate epi-

FAMILIAL AGGREGATION OF CANCER

13

demiological and statistical procedures, he compared his results with recent population data for cancer and leukemia only. He found no more malignant disease than expected among the relatives of the probands. The conviction that hereditary factors were (or could be) prominent in leukemia continued to be bolstered by reports of multiple affected family members (e.g., Gunz et al., 1978). Some more recent surveys seeking to document familial predisposition to leukemia have failed to do so (e.g., Marchetto et al., 1978), while Gunz et al. (1975) found two to three times the expected amount of leukemia among even distant relatives of an enormous number of leukemia patients. Two somewhat specialized surveys also showed a genetic influence on leukemia incidence. Kurita et al. (1974) found that parental consanguinity was more than six times more frequent in 20 cases of siblings with leukemia than in 200 sporadic leukemia cases in Japan. More well known is the conservatively analyzed survey of Down’s syndrome cases by Krivit and Good (1957)that established (a year before Lejeune identified the syndrome’s cause as trisomy 21) that the disorder carries a risk of leukemia at least three times higher than the risk for normal individuals. The widespread, large-scale surveys of familial incidence of cancer suggested the conclusion that, although a genetic component often could be demonstrated when anatomic sites were evaluated individually, the hereditary influence (except in rare families) was small and almost certainly dependent upon environmental factors for expression (e.g., V. E. Anderson et al., 1958).The later studies of the period not only were awesome in their proportions and determination to surmount the formidable difficulties of such studies, but also were admirable for their careful rationales, well-balanced evaluations of the data, and explicit discussions of the problems and limitations involved. Clemmensen’s review summarizes this period of research on cancer heredity: [Tlhe statistician, evaluating quantitatively the significance of various factors influencing the incidence of cancer, will probably find genetic factors far less significant in human cancer, than most geneticists will be prepared to admit. . . . Even if the introduction of statistical methods into genetics on human cancer has opened possibilities for critical evaluation of the incidence of malignant disease among relatives of patients, it has at the same time necessitated the restriction of such studies to categories of relatives for which reliable information is available, thereby excluding those more remote relatives that alone can give us indication of the path of inheritance. (Clemmesen, 1965, p. 19)

14

NANCY R. SCHNEIDER ET AL.

V. Current State

The reviews of cancer genetics of the recent past universally have ignored historical studies, usually have mentioned only briefly the statistical surveys of the recent past, and instead have concentrated on summarizing, organ system by organ system, the single-gene premalignant and cancerous disorders and the characteristics of non-Mendelian familial clustering of site-specific malignancy (e.g., D. E. Anderson, 1970). However, some of the later reviews, such as those by Strong (1977) and by Knudson et al. (1973), in addition to assembling catalogs of genes or pedigrees, have organized and presented what is known in a way that suggested a new approach for further investigation or a new hypothesis about the mechanism of gene action in cancer etiology. In two extensive reviews, Knudson and associates (Knudson, 1977; Knudson et aZ., 1973) demonstrated convincingly that for cancer at virtually every anatomic site there is a small minority of cases with a strong, if not primary, genetic basis. The genetic predisposition is not general but has great tissue specificity, even when more than one tissue is specified (e.g., the endocrine adenomatoses), and great specificity for the stage of differentiation at which the malignancy occurs, even when the same tissue is involved in different genetic cancers (e.g., pheochromocytoma in multiple endocrine adenomatosis, type I1 vs familial neuroblastoma). Characteristics of these hereditary and nonhereditary forms were explained plausibly by Knudson’s two-mutation hypothesis of cancer etiology (e.g., Knudson, 1971). Reviews of hereditary cancers of some individual organs and organ systems have appeared in recent years (e.g., Lynch and Frichot, 1978; Dodd, 1977; Kademian and Caldwell, 1976; Horton, 1976; Schimke, 1976; Gunz, 1974). Reports of cancer clusters in individual families have continued to be reported frequently (e.g., Blattner et aZ., 1979a; Li and Fraumeni, 1975; Li et al., 1977; Meisner et al., 1979). There is a high probability that a number of cancers will occur in any given family by chance alone; in the chance occurrence of several cancers in a family, the cancers would be expected to be of the most frequent types, and to occur at the most frequent ages of diagnosis. Familial aggregations of interest are those in which several uncommon cancers occur together, or in which common cancers have occurred at very uncommon ages of onset; these are not easily explainable by chance, and as Macklin pointed out in 1932, their joint probabilities of occurrence are very low indeed. Statistical surveys of aggregation of cancer in families have been

FAMILIAL AGGREGATION OF CANCER

15

reported less frequently in the recent past than formerly. Lynch et al. have published two reports concerning the incidence of familial cancer, one in a large normal population (1976a) and the other in patients attending an oncology clinic (1979). In 1976 they questioned approximately 4500 adults from the general population screened for cancer in two cities about cancer in their first- and second-degree relatives. The distribution of the percentage reporting zero, one, two, or more than two relatives with cancer resembled the Poisson distribution, although calculations of the numbers expected in their sample from such a distribution are not reported by Lynch et al. (1976a). As the number of first-degree relatives with cancer increased, the more likely the proband was to have a history of cancer (approximately 10% of those with one first-degree relative affected vs 22% of those with three or more). The distribution of patients with various numbers of affected relatives in their 1979 survey of 200 patients being treated for cancer was almost identical to that of the normal screened population of the 1976 study, even though the oncology clinic patients were on average 6 years older than the screened subjects. The frequency with which cancers of the same anatomical site as the proband’s occurred in relatives did not seem to be related to either family size or average age of the proband. In a huge population of twin pairs of white male American veterans, Hrubec and Nee1 (1982) found the numbers of twin pairs concordant for cancer before age 60 to be very low and not appreciably different between monozygous and dizygous pairs. In 1977, Albert and Child published the results of a family cancer study in a normal, healthy population that was unusual in several respects. Assays of familial cancer incidence of healthy individuals were rarely made in the past; control probands were all too often noncancerous hospital patients (e.g., Macklin, 1959) because of their accessibility. Even when control probands were healthy, the information obtained tended to be seriously incomplete (e.g., Busk, 1948). Albert and Child chose to study the family history of cancer of the first-degree relatives of the parents of healthy lactating women participating in an unrelated study-an original approach to subject selection. The information they obtained was remarkably complete, and reported cancers were confirmed whenever possible by pathology reports, medical records, or death certificates. Their sophisticated analysis of their data, taking into account family size and ages, revealed more lineages with zero cancers, and fewer lineages with two or more than two cancers, than did the 1976 survey of healthy individuals of Lynch et al. (1976a). Albert and Child calculated expected numbers of cancers in families from the frequency of cancer among individuals

16

NANCY R. SCHNEIDER ET AL.

in their survey and found that two or more cancers in a lineage occurred more often than expected. They found also that significantly more cancers than expected were observed in lineages with several of the more common adenocarcinomas; however, no excess was found in lineages with leukemia, lymphoma, cancer of the cervix, or prostate, i.e., those cancers with a possible viral etiology (Fenoglio, 1982; Gallo and Wong-Staal, 1982; Zeigel, 1979). Other recent investigators have found that the overall two- to threefold increased risk to close relatives of cancer patients could be partitioned and further specified by subclassifying aspects of either the individuals under study or the maligancy itself. Although yet another survey of familial breast cancer might seem to be redundant, D. E. Anderson’s original and detailed analysis of the results of his surveys of familial breast cancer, referred to earlier, has produced some new insights of both scientific and clinical importance. In a series of papers (e.g., D. E. Anderson, 1974, 1977), he rearranged his extensive data into subgroups according to relationships of the women in a kindred, age at diagnosis, or bilaterality of disease, and found that the widely accepted figure of a two- to threefold increased risk of breast cancer for female relatives of propositae was not accurate for any of these subgroups; e.g., the risk for sisters of propositae whose mothers had premenopausal, bilateral disease was nearly 50 times greater than normal, whereas the risk for women whose mothers had unilateral, postmenopausal disease was no higher than normal. Bain et al. (1980) conducted a similar study using an even more complex statistical analysis, and arrived at conclusions essentially the same as Anderson’s. It is logical to assume that the two- to threefold increase in risk reported for relatives of other types of cancer propositae might be similarly modified by subgrouping the population in a meaningful manner analogous to Anderson’s (and as Crabtree had done previously). Studies exploring the possibility that different histological types of cancer of a given anatomical site might have different familial risks have begun. Lehtola (1978)in a large Finnish survey found that relatives of patients with a diffuse, mucin-producing type of gastric cancer had seven times the risk of controls for stomach cancer, but that relatives of gastric cancer patients with a differentiated, glandular histology had no significantly increased risk. The familial tendency to leukemia found by Gunz et al. (1975) was most pronounced for patients with chronic lymphocytic leukemia, less for acute leukemia patients, and virtually absent for chronic myelogenous leukemia. A large study of breast cancer at Memorial Hospital (Rosen et al., 1982) found that a family history of maternal breast cancer was significantly more fre-

FAMILIAL AGGREGATION OF CANCER

17

quent among women with medullary carcinoma than with other histologic types and that breast cancer occurred more frequently in sisters of patients with lobular carcinoma. Another recent type of statistical survey using traditional methods, but beginning with an unusual group of probands, are those surveys whose propositae are children with cancer. Previotlsly, only familial surveys of leukemia have included children as propositi. Surveys of cancer in families of children with various types of malignancies have been made recently, possibly because of evidence for a genetic component in a relatively large proportion of childhood cancers (Knudson, 1976). Li et al. (1977) investigated the cancer experience of relatives in families with cancer in two or more young siblings, compared with that in families with only one child with cancer. They found predisposition to cancer in the former kindreds that extended to parents, other siblings, and other relatives even in the absence of consanguinity or a known inherited disorder. Draper et al. (1977) confined their study to estimation of the frequency with which cancer occurred in siblings by reviewing the records of most of the cases of childhood cancer in Great Britain during a 20-year period. After excluding known genetic tumors such as familial retinoblastoma and neurofibromatosis, they still were able to show that cancer occurred in sibships more frequently than would be expected by chance. They speculated that, although common environmental factors cannot be ruled out, aggregation may be due to “subclinical genetic abnormalities” or heterozygosity for some cancer-predisposing gene. This last speculation, that heterozygosity for a recessive or incompletely penetrant dominant gene that predisposes to cancer could also increase the carrier’s risk of cancer, is the element of originality in several recent surveys, and the hypothesis around which they are organized. Swift and colleagues (1976) were the first to select as probands for a statistical survey of familial cancer individuals who were already known to have a recessively inherited condition with a high risk of cancer (the chromosome breakage syndromes Fanconi’s anemia, ataxia-telangiectasia, and xeroderma pigmentosum). Because heterozygosity for these genes could not be detected (except in parents of an affected person), more sophisticated statistical treatment was necessary, including probability estimates of heterozygosity of each relative in addition to the appropriate epidemiological calculations. The analysis (Swift, 1976) in a small number of Fanconi’s anemia families indicated a significantly increased rate of cancer in the probands’ relatives, but this was not supported as additional families were studied (Caldwell et al., 1979). However, in families of ataxia-

18

NANCY R. SCHNEIDER ET AL.

telangiectasia patients, the analysis showed that risk of dying of cancer was five times the risk for the general population for relatives less than 45 years of age (Swift et al., 1976); this elevated risk is not accounted for by a number of epidemiological factors other than heterozygosity for the ataxia-telangiectasia gene (Daly and Swift, 1978). Swif’t and Chase (1979) also found more skin cancer in relatives of xeroderma pigmentosum patients than in spouse controls, but only four families accounted for the excess, so the significance of this study is uncertain. One small survey (Fedrick and Baldwin, 1978)and one large, extensively analyzed survey (Bonaiti-Pellie and Briard-Guillemot, 1980) of the incidence of cancer of all types in families of children with retinoblastoma have found a significant excess of cancer deaths in relatives, in families of unilateral (presumably nonhereditary) cases as well as of bilateral (presumably hereditary) cases. The interpretation of these results is unclear; however, Bonaite-Pellie and BriardGuillemot’s report illustrates the point that, although results of epidemiological studies are only descriptive, they can stimulate some highly creative interpretations and speculations for further exploration. In 1966, Moertel investigated the family history of cancer of nearly 800 patients with multiple primary neoplasms, a characteristic of known genetic cancers, and found that families of these patients had significantly more malignant disease than families of noncancerous control patients. Moertel’s data indicated that the occurrence of multiple primaries was a sign of increased genetic cancer predisposition in a presumed genetically normal population. Schneider et al. (1983) conducted a statistical study organized to test the specific hypothesis that another characteristic of recognized genetic cancers, unusually early age of onset, can be used to identify individuals in a general cancer patient population who have more pronounced family history of cancer than average. A high incidence of malignant disease among close relatives was selected as the indicator of genetic predisposition to cancer. Family health his tory information was gathered using a short questionnaire from more than 1350 patients. A histological diagnosis was obtained for 90% of the patients. Each patient with a confirmed cancer diagnosis was assigned to one of four study categories after comparison of his age at diagnosis with the distribution of ages at diagnosis for his cancer site compiled by the Third National Cancer Survey (TNCS). Patients whose ages at diagnosis were in the lowest or median deciles of the TNCS distributions were studied further, employing longer questionnaires. Verification of

FAMILIAL AGGREGATION OF CANCER

19

reported deaths or cancer cases was obtained whenever possible from medical records or death certificates. Data were analyzed by computer using standard epidemiological methods to calculate the numbers of cancers expected among the firstdegree relatives in each study category. Half of the comparisons of the numbers of reported vs expected cancers showed significant excesses of reported cancers. These occurred for some relatives of study patients in all age categories, but no study category or type of firstdegree relative had a consistent excess of cancer. Possible reasons for this almost random pattern remained difficult to interpret. The numbers of families reporting cancer in parents or siblings of study patients deviated from randomness, also in the direction of excess cancers. The study demonstrated that a familial tendency to develop cancer exists in the general population of cancer patients, regardless of the age of its onset in the proband. It was also shown that, conversely, early age at diagnosis of cancer may indicate genetic predisposition to malignant disease only in exceptional cases. Investigations of the genetic epidemiology of cancer during the recent past usually have either surveyed the familial cancer experience of individuals who are not cancer patients or have been organized to test a specific hypothesis. For example, the cancer incidence among heterozygotes for some recessively inherited cancer-predisposing genes has been assayed, and the phenotypic attributes of known genetic cancers (e.g., early age at onset, multiplicity of primary tumors) have been used in evaluations of genetic cancer predisposition in the general population. These studies have also been characterized by the increasingly sophisticated use of epidemiological and statistical methods and the computerized storage and analysis of large bodies of data. VI. Recent Developments in Genetic Epidemiology

Since 1970, renewed interest by human population geneticists in the study of disease has led to the development of a scientific discipline known as “genetic epidemiology,” for which the following formal definition was provided by Morton and Chung (1978):“Genetic epidemiology is concerned with etiology, distribution, and control of disease in relatives and with inherited causes of disease in populations.” The relevance of application of methods of genetic epidemiology to the study of familial aggregation of cancer thus is obvious. The methods themselves are primarily in two areas, segregation and linkage analysis.

20

NANCY R. SCHNEIDER ET AL.

A. SEGREGATION ANALYSIS Segregation analysis permits the determinhtion of the mode of transmission of a trait given the distribution of phenotypes in a pedigree or a sample of pedigrees. The word transmission is preferred so as not to imply prejudice for genetic hypotheses. It proceeds by fitting a general model of transmission to the observed segregation pattern of phenotypes. Comparison of likelihoods associated with different parametrizations of the general model leads to tests of hypotheses and identification of the most likely mode of inheritance. The range of inference depends upon the generality of the model as well as entropy of the data. When a proper correction has been applied to account for the manner in which pedigrees were ascertained and extended, the results relate back to the reference population from which pedigrees were originally sampled. In this context, segregation analysis provides a useful method for determining the parameters of a model or models that may serve as the basis for genetic counseling and, when there is evidence for a major gene involvement, the investigation of linkage. From a historical perspective, segregation analysis was applied in its simplest form, namely, mere inspection of pedigrees, by Garrod (1902). Shortly thereafter, Weinberg (1912a,b) introduced the first statistical approach, which consisted of fitting constant segregation parameter (p) for each mating type. More efficient estimates of the segregation frequency were developed by Haldane (1932, 1938) and by Fisher (1934), who introduced maximum likelihood. However, it remained until the 1950s and 1960s and the availability of high-speed computers for the extension of the analysis to more complex models of inheritance (in which recurrence risks are recognized as variable among families) to be attempted (Morton, 1958,1959,1969). The concept of multifactorial inheritance (multiple genetic and environmental factors with small additive effects) to explain familial aggregation of disease was introduced by Falconer (1965, 1967), and by 1970, the geneticist had to choose between this model and the generalized single-locus model. Although strategies to distinguish between the two were presented by Morton et al. (1971) and Smith (1971), it was not until 1974 and the development of the first mixed model (Morton and MacLean, 1974) in which single-locus and multifactorial models could be represented as subhypotheses of the general model that a rigorous test of the two alternatives was available. In its present form (Lalouel and Morton, 1981),the mixed model postulates that a phenotype may result from the effects of a major locus genotype, multifacto-

FAMILIAL AGGREGATION OF CANCER

21

rial contribution, and residual environment, each acting additively and independently. The major locus may be autosomal or sex linked, genotype frequencies and transition matrices incorporate the effects of selection and mutation (Morton and Yasuda, 1979), and multifactorial inheritance has been extended to account for generational differences. While Morton et al. (Morton and MacLean, 1974; Lalouel and Morton, 1981) were developing the mixed model, an alternative model for segregation analysis was being developed by Elston and Stewart (1971). In the latter model, the test for a major gene is provided by determining whether the transmission frequencies, TAAA, T A A,~ Tau A (defined as the probability that an individual with genotype AA, Aa, or aa, respectively, transmits the A gene), deviate from their Mendelian expectations of 1, 0.5, and 0. Although not providing a comparison between major locus and multifactorial inheritance, this model does provide additional non-Mendelian hypotheses that can be tested. Incorporation of variable transmission frequencies into the mixed model has resulted in what has been called a “unified version of the mixed model” (Lalouel et al., 1983). In this context, the variable transmission frequencies provide additional hypotheses that should be rejected prior to accepting a major locus and, as such, provide added protection against inferring a major gene as a result of departures from assumptions of the model. With regard to the unit of analysis, a proper sampling correction has been specified for nuclear families under a variety of ascertainment schemes (see Morton et al., 1983).Although algorithms for extension of segregation analysis to larger pedigree structures have been developed (Elston and Stewart, 1971; Cannings et al., 1978), a proper sampling correction for the manner in which pedigrees have been ascertained and relatives added has been specified only for limiting cases (Cannings and Thompson, 1977; Elston and Sobel, 1979; Boehnke and Greenberg, 1984). A general approximation to permit extension of segregation analysis under the mixed model to larger pedigree structures ascertained and extended in variable manner was suggested by Lalouel and Morton (1981). In this approach, known as the pointer strategy, a pedigree is partitioned into its component nuclear families which are then conditioned upon phenotypes of more distant relatives (pointers) who were responsible for their ascertainment. Development of appropriate ascertainment corrections for segregation analysis of pedigrees is an area where much future effort will undoubtedly be expended. Several recent reviews have been published on segregation analysis (Elston and Rao, 1978; Elston, 1981; Morton, 1982). Be-

22

NANCY R. SCHNEIDER E T AL.

low we review results of segregation analyses that have been performed on families with cancer. The cancer phenotype upon which most segregation analyses have been performed is breast cancer. The largest investigation involved 200 pedigrees that were ascertained through a random series of breast cancer patients who were part of the Danish Tumor Registry, a population-based resource. Family information was collected by Jacobsen (1946) (vide supra) and segregation analysis was performed on these kindreds (Williams and Anderson, 1984; Anderson and Williams, 1985). As reflected by a positive-moderate heritability of 0.3, there was significant aggregation of breast cancer in these families, which could most readily be explained by segregation of an autosomal dominant gene in some of the pedigrees. The frequency of the gene in the general population was estimated to be approximately 0.005, Penetrance of the gene was age dependent and gene carriers have a lifetime risk of approximately 60% of developing breast cancer. The abnormal gene accounts for approximately 10% of breast cancer in women in the general population. An autosomal dominant mode of inheritance for breast cancer has also been reported from segregation analysis of pedigrees that were selected due to a high frequency of breast cancer, such as Kindred 107, the well-known Utah pedigree ascertained by Dr. Eldon Gardner (Gardner and Stephens, 1950; Stephens et al., 1958; Hill et al., 1978; Bishop and Gardner, 1980; Gardner, 1980),as well as from segregation analysis of 18 Midwestern pedigrees that were ascertained by Dr. Henry Lynch (Elston et al., 1981; Go et al., 1983). The only other cancer phenotype for which a large series of families have been subjected to segregation analysis consists of 159 pedigrees that were ascertained through children who had a confirmed histologic diagnosis of soft tissue sarcoma prior to age 16 and had survived at least 3 years. Probands were identified from systematic survey of medical records of The M. D. Anderson Hospital and Tumor Institute for the years 1944 through 1975. The purpose of this investigation was to identify families such as those described by Li and Fraumeni (1969a,b, 1975, 1982) that are characterized by extreme aggregation of a diverse variety of neoplasms, to determine the incidence of such families in the general population, to investigate the mode of inheritance of cancer in these families, and to determine whether high- or low-risk families could be discriminated on the basis of clinical-epidemiological features of the disease. Details of this analysis are given by Williams et al. (1984)and Strong et a2. (1986),results of which are summarized below.

FAMILIAL AGGREGATION OF CANCER

23

In the overall sample of pedigrees the multifactorial heritability was 0.13, which reflects a low but significant degree of familial aggregation of cancer. In comparison with a multifactorial model, an autosomal dominant model provided a more likely explanation of the familial distribution of cancer. The difference in likelihood was explained by a subgroup of 11 pedigrees that presented with a high frequency of cancer. Of clinical significance, 6 of the 11 probands (55%)in these families had either a second benign tumor (2) or a second malignant tumor (4),a much higher frequency than observed overall (25 out of 159 or 16%).Other characteristics of probands that were associated with a positive family history included an early age at onset (less than 5 years of age) and histologic type (specifically embryonal rhabdomyosarcomas). Cancer developed at an early age and a high frequency of second primaries was observed in affected relatives from the 11 families; both features have been associated with a positive family history of cancer of specific sites (e.g., breast cancer, Anderson, 1982). Results of this survey suggest that a much higher frequency of children with soft tissue sarcomas come from families in which cancer appears to be inherited as an autosomal dominant condition than originally thought. This has been corroborated by a recent survey published by Birch et al. (1984), who observed a threefold excess of breast cancer in mothers of children with soft tissue sarcomas. An enriched sample of high-risk families can be obtained by ascertainment through children with soft tissue sarcomas that have developed second malignancies. ANALYSIS B. LINKAGE The demonstration of linear arrangement of genes on chromosomes, the tendency for genes in close proximity (within 50 centimorgans) to segregate nonindependently, and the observation that the frequency of recombination is a reflection of the genetic distance between loci and therefore can be used to derive a genetic map is attributed to T. H, Morgan from his classical work with Drosophila (Morgan, 1911). Within a short period of time, statistical methods had been developed to detect linkage of genes and to estimate recombination in man based upon the joint segregation of two marker loci in pedigrees (Bernstein, 1931; Weiner, 1932; Hogben, 1934; Haldane, 1934).Although statistical improvements were introduced by Fisher (1935) with maximum likelihood which was expanded upon by Finney (1940) and Smith (1953),only in 1955 with the development of “lod scores” (log of the odds ratio) by Morton (1955) was a fully efficient solution available.

24

NANCY R. SCHNEIDER ET AL.

This approach brought together the A statistic of Haldane and Smith (1947) and sequential analysis of Wald (1947) and provided a practical method that could be applied to small numbers of families. In this method, for each pedigree ( i ) one computes a lod score, zi(0), for specified values of the recombination frequency (e), as follows:

Here L ( 8 ) and L (0.5)refer to the likelihood of the pedigree-marker data assuming recombination frequencies of 8 (0 5 6 5 0.5) and 0.5, the latter figure corresponding to no linkage. The test statistic is defined by merely summing lod scores for all pedigrees. When X zi(8 < 0.5) is greater than 3, evidence is considered strong enough to reject the null hypothesis and conclude that there is linkage; when Z zi(8 < 0.5) is less than -2, there is sufficient evidence to accept the null hypothesis of no linkage; and when X zi(8) is between these two bounds, further data are required before acceptance or rejection of the null hypothesis. In this manner, evidence from independent investigations can be accumulated until a decision can be made. In principle, sex-specific values of recombination should be estimated since recombination is not the same in males and females. It is customary to present results in the form of a lod score table in which evidence for linkage between two systems is computed for recombination frequencies of 0.05, 0.1, 0.2, 0.3, and 0.4 (Morton, 1955). A summary of published lod scores has been assembled by Keats et al. (1979) and Keats (1981). In the early days of linkage analysis, tedious computations were performed by hand, which changed as the computer found its way in biomedical research. Although an early computer program for calculating lod scores was introduced by Renwick and Schultze (1961), it remained until 1973 and the development of the computer program LIPED (Ott,1973,1974,1976; Hodge et al., 1979) that computation of lod scores for pedigrees became widely accessible to large numbers of users. Since that time, many other programs have been developed to perform linkage analysis, for example GENPED (Elston and Lange, 1975), PAP (Hasstedt and Cartwright, 1979), LINKAS (Morton and Lalouel, 1981), and LINKAGE (Lathrop and Lalouel, 1984a,b). More extensive reviews of the history and methods in linkage analysis are contained in Conneally and Rivas (1980) and Morton (1982, 1984). Despite the existence of statistical methodology to detect linkage and to estimate recombination, establishment of a map of the human genome proceeded initially at a slow pace, owing to the paucity of

FAMILIAL AGGREGATION OF CANCER

25

known polymorphic markers that were available to test for linkage. Because the human gene map comprises approximately 50,000100,000 genes (McKusick, 1982a) and because they would need to be relatively short distances apart in order to detect linkage, the probability that linkage could be detected for a given disease locus was low. Developments in molecular biology during the last decade, however, have altered this situation by providing the technology to identify a large number of polymorphic loci that conceivably will span the entire genome, and as a result, the likelihood of detecting linkage should approach one. This strategy was first described by Botstein et al. (1980) and takes advantage of two important developments in molecular biology: (1)the availability of libraries of single-copy recombinant DNA fragments or probes whose map location(s) can be determined by in situ molecular hybridization to chromosomes or by somatic cell hybridization and (2) the use of restriction endonucleases which recognize specific sequences in DNA and catalyze cleavages resulting in DNA fragments of defined lengths. Sampling random individuals reveals fragments of different lengths and the resulting variation in molecular weight can be detected by electrophoresis. These DNA polymorphisms have been referred to as restriction fragment length polymorphisms (RFLPs). Lange and Boehnke (1982) and Bishop et al. (1983) have estimated that the number of polymorphic marker loci required to span the autosomes (assuming random distribution of marker loci and a high probability that a randomly selected locus is within 20 centimorgans of a marker polymorphism, a map distance that is associated with a high probability of detecting linkage in a medium-sized family study) is approximately 400. From the 400 markers, a subset of 80 could then be selected that are distributed uniformly throughout the genome as a primary resource for linkage studies. In practice, many fewer than 400 markers will be required, since molecular biologists can sample RFLPs from isolated DNA fragments from specific human chromosomes, rather than defining RFLPs from random single-copy genomic DNA. Skolnick et al. (1984) have recently investigated the extent to which the human gene map is currently covered by RFLPs and classical markers. Their estimates were based upon the probability of detecting linkage based upon a small family study (15 families, each composed of two parents and six children), a medium family study (30 families), and a large family study (60 families). With classical markers alone, 0.08, 0.14, and 0.19% of the genome is covered for a small family study, a medium family study, and a large family study, respectively.

26

NANCY R. SCHNEIDER ET AL.

Using currently mapped RFLPs, these percentages increase to 0.44, 0.62, and 0.71, respectively. Individually, two chromosomes are already completely covered by markers (chromosomes X and 21), and most other chromosomes have between 50 and 75% coverage. Only chromosomes 6 , 7 , 8 , and 11 have less than 50% coverage based upon a medium-sized family study. It should be kept in mind that these figures are quickly becoming obsolete, and that the time is rapidly approaching when any medium-sized family study should have a high probability of detecting linkage. Below we review linkage investigations involving breast cancer, melanoma, and Hodgkin’s disease; all have been the subjects recently of either speculation or controversy, or both. Interest in the application of linkage studies to families with cancer was stimulated in 1980 when King et al. reported a positive lod score of 1.43 between breast cancer and the locus for glutamate-pyruvate transaminase (GPT) which has been provisionally mapped to chromosome 16 (McKusick, 1982b). Reanalysis of the original kindreds and additional pedigrees has resulted in a revised lod score of 1.95 (King et al., 1983).This finding has been contested by a lod score of -5.92 for linkage between GPT and breast cancer that is based upon analysis of 11 Utah pedigrees (McClellan et al., 1983; Cannon et al., 1983). Other linkage studies of breast cancer also fail to confirm the association (Cleton, 1983). Thus, while the present evidence does not support the original finding of King et al. (1980),it is possible that there is genetic heterogeneity in breast cancer, and that more than one major gene that is segregating for the disease may be involved. Many of the pedigrees analyzed by King et al. (1983) included individuals with ovarian cancer, a possibly distinct clinicogenetic entity (Lynch et al., 1978). Analysis of additional pedigrees in which breast and ovarian cancer are segregating will be necessary to resolve this conflict. Results of linkage investigation have also been reported for malignant melanoma. The possibility of linkage between melanoma and the major histocompatibility complex located on chromosome 6 was suggested by reports that melanoma-specific antigens give rise to autochthonous immune response (Roth et al., 1976; Shiku et al., 1976).However, population studies of HLA in melanoma patients have failed to reveal any significant haplotype associations (Lamm et al., 1974; Bergholtz et al., 1976; La1 and Jorgensen, 1976; Pellegris et al., 1980). Nevertheless, Hawkins et al. (1981)reported a lod score of 1.25 (e = 0) between HLA and melanoma in one large pedigree, and linkage was also suggested between HLA and melanoma in two of three families published by Pellegris et aZ. (1982),although no lod scores were com-

FAMILIAL AGGREGATION OF CANCER

27

puted. In contrast, Greene et al. (1983) reported a higher lod score between melanomddysplastic nevus syndrome and the Rh blood group locus which has been mapped to the short arm of chromosome 1 [X zi (0 = 0.3) = 2.01. Again, additional data are necessary to resolve this conflict. [It is interesting to note that cytogenetic analyses of melanoma cells have revealed structural aberrations that most frequently involve chromosomes 1 and 6 (Balaban et al., 1984).1 Hodgkin’s disease, like melanoma, stimulated interest in linkage investigation with the HLA region after population-based studies demonstrated nonrandom distribution of antigens in patients and controls (for summary and reviews, see Ryder et al., 1979; Dausset et al., 1982; Hors and Dausset, 1983). In contrast to breast cancer and melanoma, in which autosomal dominant models of inheritance were used to investigate linkage based on the observed segregation patterns of disease, the aggregation of Hodgkin’s disease in families is not distributed in a discernible Mendelian pattern of transmission. The lack of a known genetic model has ruled out classical linkage analysis; instead, investigation of linkage for Hodgkin’s disease has proceeded by testing whether the extent of haplotype sharing in affected relatives is greater than would be expected by chance. From recent review of published families that have been HLA typed, there is little doubt that there is a relationship between the HLA region and familial Hodgkin’s disease, although the exact nature of this relationship is unclear (Dausset et al., 1982; Hors and Dausset, 1983). The extent of haplotype sharing, although high, is not complete, which suggests the possibility of genetic heterogeneity. The lack of a clear-cut Mendelian pattern of segregation suggests that environmental factors may be involved, thus making this disorder a good example for study of interaction between genotype and environment. Study of familial clusters of Hodgkin’s disease employing more complex forms of analysis will be required to gain greater insight into the genetic etiology of this disease. Linkage investigation of two other cancer phenotypes have been reported. In 1968, Anderson investigated linkage between classical markers and the basal cell nevus syndrome with inconclusive results. Recently, Gatti et al. (1983)investigated linkage between 25 markers and cancer in a large pedigree. The pedigree was ascertained due to a high frequency of cancer of multiple sites. The highest lod score (0.64) was reported for HLA. Application of methods of segregation and linkage analysis will contribute to our understanding of an hereditary basis of cancer in the following ways: (1)determination of the frequency of inherited condi-

28

NANCY R. SCHNEIDER ET AL.

tions that predispose to cancer in the general population, (2) establishment of modes of inheritance (e.g., autosomal dominant, recessive, multifactorial) of “heritable” conditions and determination of parameters that describe their mode of inheritance (e.g., gene frequency, penetrance function, frequency of phenocopies, mutation rate), and (3) resolution of genetic heterogeneity. For instance, in the case of breast cancer, more than 10 different clinicogenetic syndromes have been proposed to explain familial aggregation on the basis of variable clinical-epidemiological features of the disease as well as consideration of associated conditions that also appear to segregate in some pedigrees (see review by Lynch, 1981). Segregation and linkage investigation of such families will clarify which of the syndromes are inherited and whether the same loci or different ones are involved. Finally, results of segregation and linkage can play a role in cancer prevention through genetic counseling. For families in which an inherited form of cancer is segregating, the parameters of the model defined by segregation analysis can be used to predict the level of risk (high or low) for given individuals, and when linked markers are available, they can be used to identify individuals with high probability of having inherited a predisposition to cancer (Chakravarti and Nei, 1982; Chakravarti, 1983; Conneally et al., 1984; Lathrop and Lalouel, 1984a,b). Future advances in the methods in genetic epidemiology can be expected to take advantage of the abundance of DNA polymorphisms in investigating the inherited basis of disease in families. Segregation and linkage analysis are being combined under more complex oligogenic models with allowance for complexities such as linkage disequilibrium that will provide greater clarification of the genetics of diseases that do not follow simple Mendelian modes of inheritance (Morton, 1984; Lalouel, 1984; MacLean et al., 1984; Risch, 1984). Linkage investigations will incorporate information on multiple marker systems that will provide much more information to establish correct gene orders and to estimate correct map distances (Lathrop and Lalouel, 1984a,b; Skolnick et al., 1984). VII. Summary

Literature pertaining to genetic epidemiological studies of familial cancer has been reviewed from a historical perspective. Although interest in the question of heritability of cancer was extant at least as early as the beginning of the nineteenth century, early investigators were unable to produce consistent and meaningful evidence pertain-

FAMILIAL AGGREGATION OF CANCER

29

ing to the issue because of unsystematic methods of data collection and inadequate methods of data analysis. During the early twentieth century, developments in the fields of genetics, statistics, and epidemiology provided concepts and methods that permitted investigators to recognize important deficiencies in past studies, and to design others in which the critical comparisons could be made between patient groups and control groups. Registries of cancer incidence in large populations became available in several countries in the middle twentieth century, providing a standard “control group” for comparison. Large surveys of site-specific cancer experience in families, rigorously designed and analyzed, found for most kinds of cancers a two- to threefold increased risk for close relatives of propositi. These studies also reemphasized the great difficulty in obtaining even minimally complete family health history information, and the importance of verifying all reported cases with medical or vital records. Although clinical and laboratory investigation will be necessary to understand the mechanisms by which human genes may predispose to cancer, epidemiological approaches can estimate the extent to which genetic etiological factors may be present in a population, whether a general population or one defined by other factors under investigation. Population-based studies are already of practical significance to the clinical geneticist in the estimation of risk of eventual cancer development in unaffected family members, and can be expected to continue to identify specific groups and characteristics associated with genetic cancer predisposition. Finally, segregation and linkage analysis and their present applications to family studies of cancer were reviewed. As a result of the increasing number of DNA polymorphisms that are becoming available due to developments in molecular biology, the human gene map can be expected to be well defined in the near future, and investigation of families using segregation and linkage analysis will then be instrumental in defining the role of heredity in the development of cancer in human populations.

ACKNOWLEDGMENTS N.R.S.was the recipient of an Insurance Medical Scientist Scholarship Fund scholarship sponsored by the Equitable Life Assurance Society. This work was supported in part by Grants CA 34097 (W.R.W.)and CA 34775 (R.S.K.C.) from the National Institutes of Health.

30

NANCY R. SCHNEIDER ET AL.

REFERENCES Albert, S., and Child, M. (1977). Cancer (Philadelphia)40, 1674-1679. Anderson, D. E. (1968).Ann. Hum. Genet. 32, 113-123. Anderson, D. E. (1970). Collect. Pap. Annu. Symp. Fundam. Cancer Res. 23,85-104. Anderson, D. E. (1972).J. Natl. Cancer Inst. (U.S.) 48, 1029-1034. Anderson, D. E. (1974). Cancer (Philadelphia)34, 1090-1097. Anderson, D. E. (1977). Cancer (Philadelphia)40, 1855-1860. Anderson, D. E. (1982). In “Cancer Epidemiology and Prevention” (D. Schottenfeld and J. F. Fraumeni, Jr., eds.), pp. 483-493. Saunders, Philadelphia, Pennsylvania. Anderson, D. E., and Williams, W. R. (1984).Am. J . Hum. Genet. 36,24S. Anderson, V. E., Goodman, H. O., and Reed, S. C. (1958).“Variables Related to Human Breast Cancer.” Univ. of Minnesota Press, Minneapolis. Bain, C., Speizer, F. E., Rosner, B. BBlanger, C., and Hennekens, C. H. (1980).Am. J . Epidemlol. 111,301-308. Balaban, G., Herlyn, M., Guerry, D., JV, Bartolo, R., Koprowski, H., Clark, W. H., and Nowell, P. C. (1984). Cancer Genet. Cytogenet. 11,429-439. Bashford, E. F. (1908). Lancet 2, 1508-1512. Berg, N. O., and Eriksson, S. (1972).N. Engl. J . Med. 287, 1264-1267. Bergholtz, B., Klepp, O., Kaakinen, A., and Thorsby, E. (1976).In “HLA and Disease” (J. Dausset and A. Svejgaard, eds.), p. 218. INSERM, Paris. Bergsma, D., ed. (1976). “Cancer and Genetics,” Birth Defects, Orig. Artic. Series, Vol. 12, No. 1. Natl. Found.-March of Dimes, White Plains, New York. Bernstein, F. (1931). Z. Indukt. Abstamm.- Vererblehre. 57, 113-138. Birch, J. M., Hartley, A. L., Marsden, H. B., Harris, H., and Swindell, R. (1984). Br. J . Cancer 49,325-331. Bishop, D. T., and Gardner, E. J. (1980).In “Cancer Incidence in Defined Populations, Banbury Report 4” (J. Cairns, J. L. Lyons, and M. Skolnick, eds.), pp. 389-408. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Bishop, D. T., Cannings, C., Skolnick, M., and Williamson, J. A. (1983). In “Statistical Analysis of DNA Sequence Data” (B. S. Weir, ed.), pp. 181-200. Dekker, New York. Blattner, W. A,, Dean, J. H., and Fraumeni, J. F. (1979a).Ann. Intern. Med. 90,943-944. Blattner, W. A., McCuire, D. B., Mulvihill, J. J., Lampkin, B. C., Hananian, J., and Fraumeni, J. F. (1979b).J . Am. Med. Assoc. 24,259-261. Boehnke, M., and Greenberg, D. (1984).Am. J . Hum. Genet. 36,1298-1308. Bonaiti-Pellie, C., and Briard-Guillemot, M. L. (1980).J . Med. Genet. 17, 95-101. Botstein, D., White, R. L., Skolnick, M. H., and Davis, R. W. (1980).Am.J.Hum, Genet. 32,314-331. Brobech, 0. (1949). “Heredity in Uterine Cancer.” Universitetsforlaget, Aarhus. Broca, P. (1866). “Traite des Tumeurs.” P. Asselin, Paris. Busk, T. (1948).Ann. Eugen. (London) 14,213-229. Butlin, H. T. (1887). Br. Med.1. 1,436-441. Caldwell, R., Chase, C., and Swift, M. (1979).Am. J . Hum. Genet. 31, 132A. Cannings, C., and Thompson, E. A. (1977). Clin. Genet. 12,208-212. Cannings, C., Thompson, E. A., and Skolnick, M. H. (1978).Adv. Appl. Probab. 10,2661. Cannon, L. A,, Bishop, D. T., McLellan, T., and Skolnick, M. H. (1983).Am. J . Hum. Genet. 35,60A. Chakravarti, A. (1983).Am. J. Hum. Genet. 35,592-610.

FAMILIAL AGGREGATION OF CANCER

31

Chakravarti, A,, and Nei, M. (1982). Am. J . Hum. Genet. 34,531-551. Church, W. S., Pearson, K.,Bashford, E. F., Butlin, H. T., Mudge, G. P., inter alia. (1909). Proc. R. SOC. Med. 2, 9-127. Clemmesen, J. (1949). Br. j . Cancer 3, 474-484. Clemmesen, J. (1965). Acta Pathol. Microbiol. Scund., Suppl. 174, 1-543. Cleton, F. J. (1983). In “Proceedings of the Thirteenth International Cancer Congress, 1982. Part C,” pp. 383-389. Alan R. Liss, Inc., New York. Cockayne, E. A. (1927). Cancer Aev. 2,337-347. Cohen, A. J., Li, F. P., Berg, S., Marchetto, D. J., Tsai, S., Jacobs, S. C., and Brown, R. S . (1979). N . Engl. j . Med. 301, 592-595. Conneally, P. M., and Rivas, M. L. (1980). Adu. Hum. Genet. 10, 209-266. Conneally, P. M., Wallace, M. R., Gusella, J. F., and Wexler, N. S. (1984). Genet. Epidemiol. 1,81-88. Crabtree, J. A. (1941). Am. j . Public Health 31,49-56. Cripps, W. H. (1878). St. Barth. Hosp. Rep. 14,287. Cripp~,W. H. (1882). Trans. Patho2. SOC.London 33, 165-168. Cutler, S. J., and Young, J. L. (1975). Natl. Cancer Znst. Monogr. 41. Daly, M. B., and Swift M. (1978).j . Chronic. Dts. 31,625-634. Dausset, J., Colombani, J., and Hors, J. (1982). Cancer Sum. 1, 119-147. Dodd, G. D. (1977). Radiology 123,263-275. Dom, H. F. (1944). Public Health Rep. 59,33-45, 65-77,97-114. Dom, H. F., and Cutler, S. J. (1958). “Morbidity from Cancer in the United States,” Public Health Monogr. No. 56. U.S. Govt. Printing Office, Washington, D.C. Draper, G. J., Heaf, M. M., and Wilson, L. M. K. (1977).J. Med. Genet. 14,81-90. Eisenberg, H. (1966). “Cancer in Connecticut: Incidence and Rates 1935-1962.” Connecticut State Dept. of Health, Hartford. Elston, R. C. (1981). Ado. Hum. Genet. 11, 63-120. Elston, R. C., and Lange, K. (1975). Ann. Hum. Genet. 38,341-350. Elston, R. C., and Rao, D. C. (1978). Annu. Reo. Biophys, Comput. 1, 253-286. Elston, R. C., and Sobel, E. (1979). Am. J . Hum. Genet. 31, 62-69. Elston, R. C., and Stewart, J. (1971). Hum. Hered. 21,523-542. Elston, R. C., Go, R. C. P., King, M. C., and Lynch, H. T. (1981). In “Genetics and Breast Cancer” (H. T. Lynch, ed.), pp. 49-64. Van Nostrand-Reinhold, New York. Ewing, J. (1922).“Neoplastic Diseases,” 2nd ed., pp. 105-108. Saunders, Philadelphia, Pennsylvania. Falconer, D. S. (1965).Ann. Hum. Genet. 29,51-76. Falconer, D. S. (1967). Ann. Hum. Genet. 31, 1-20. Fedrick, J., and Baldwin, J. A. (1978). Br. Med. J . 1, 83-84. Fenoglio, C. M. (1982). Hum. Pathol. 13,785-787. Finney, D. J. (1940). Ann Eugen. (London) 10,171-214. Fisher, R. A. (1934). Ann. Eugen. (London)6, 13-25. Fisher, R. A. (1935). Ann Eugen. (London)6,187-201. Gallo, R. C., and Wong-Staal, F. (1982). Blood 60,545-55Y. Gardner, E. (1980). In “Cancer Incidence in Defined Populations, Banbury Report 4” 0. Cairns, J. L. Lyons, and M. Skolnick, eds.), pp. 365-378. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Gardner, E. J., and Richards, R. C. (1953). Am. j . Hum. Genet. 5, 139-147. Gardner, E. J., and Stephens, F. E. (1950). A m . ] . Hum. Genet. 2,30-40. Garrod, A. E. (1902). Lancet 2,1616-1620.

NANCY R. SCHNEIDER ET AL.

32

Gatti, R. A,, Sparkes, R. S., Field, L. L., Spence M. A., Harris, N. S., and Freidin, M. (1983).Cancer Genet. Cytogenet. 8,9-18. German, J. (1972).Prog. Med. Genet. 8,61-101. Go, R. C. P., King, M. C., Bailey-Wilson, J., Elston, R. C., and Lynch, H. T. (1983).J. Natl. Cancer Znst. (US.) 71,455-461. Greene, M. H., Goldin, L. R., Clark, W. H., Jr., Lovrien, E., Kraemer, K. H., Tucker, M. A., Elder, D. E., Fraser, M. C., and Rowe, S. (1983).Proc. Natl. Acad. Sci. U.S.A.80,

6071-6075.

Gunz, F. W. (1974).Ser. Haematol. 7(2), 164-191. Gunz, F.W., Gunz, J. P., Veale, A. M. O., Chapman, C. I., and Houston, I. B. (1975). Scand. J. Haematol. 15, 117-131. Gunz, F. W., Gunz, J. P., Vincent, P. C., Bergin, M., Johnson, F. L., Bashir, H., and Kirk, R. L. (1978).J . Natl. Cancer Inst. (U.S.) 60, 1243-1250. Haldane, J. B. S. (1932).J . Genet. 25,251-255. Haldane, J. B. S. (1934).Ann. Eugen. (London) 14, 10-31. Haldane, J. B. S . (1938).Ann. Eugen. (London)8,255-262. Haldane, J. B. S., and Smith, C. A. B. (1947).Ann. Eugen. (London) 14, 10-31. Harnden, D.G., Maclean, N., and Lanqlands, A. 0. (1971).J. Med. Genet. 8,460-461. Hasstedt, S., and Cartwright, P. (1979).“PAP-Pedigree Analysis Package,” Dept. of Med. Biophys. and Comput. Tech. Rep. No. 13.University of Utah, Salt Lake City. Hawkins, B. R., Dawkins, R. L., Hockey, A., Houliston, J. B., and Kirk, R. L. (1981). Ttssue Antigens 7,540-541. Hill, J. R., Carmelli, D., Gardner, E. J., and Skolnick, M. (1978).In “Genetic Epidemiology” (N.E. Morton and C. S. Chung, eds.), pp. 304-310. Academic Press, New York. Hillier, W. T., and Tritsch, I. (1904).Arch. Middlesex Hosp. 2,104-126. Hodge, S . E.,Morton, L. A,, Tidemar, S., Kidd, K. K., and Spence, A. (1979).Am.J . Hum. Genet. 31,761-762. Hogben, L. (1934).Proc. R. SOC. London, Ser. B 114,340-363. Hors, J., and Dausset, J. (1983).Immunol. Reu. 70, 167-192. Horton, W. A. (1976).Birth Defects, Orig. Artic. Ser. 12(1),91-97. Hrubec, Z.,and Neel, J. V. (1982).Am. J. Hum. Genet. 34,658-674. Jacobsen, 0.(1946).“Heredity in Breast Cancer.” H. K. Lewis, London. Kademian, M. T., and Caldwell, W. L. (1976).J. Urol. 116,380-381. Kardinal, C. G., and Yarbro, J. W. (1979).Sernin. Oncol. 6,396-408. Kam, M. N. (1948).Ann. Eugen. (London) 14,230-233. Keats, B. J. B. (1981).“Linkage and Chromosome Mapping in Man,” pp. 1-266. Univ. of Hawaii Press, Honolulu. Keats, B. J. B., Morton, N. E., Rao, D. C., and Williams, W. R. (1979).“A Source Book for Linkage in Man,” pp. 1-415. Johns Hopkins Univ. Press, Baltimore, Maryland. Kemp, T. (1948).Br.J. Cancer 2, 144-149. King, G., and Newsholme, A. (1891).Proc. R. SOC. London 54,209-228. King, M. C., Go, R. C. P., Elston, R. C., Lynch, H. T., and Petrakis, N. L. (1980).Science

208,406-408.

King, M.C., Go, R. C. P., Lynch, H. T., Elston, R. C., Terasaki, P. I., Petrakis, N. L., Rodgers, G. C., Lattanzio, D., and Bailey-Wilson, J . (1983).J . Natl. Cancer Inst.

(U.S.) 71,463-467.

Knudson, A. G. (1971).Proc. Natl. Acad. Sci. USA. 68,820-823. Knudson, A. G.(1976).Pediatr. Res. 10,513-517. Knudson, A. G. (1977a).Ado. Hum. Genet. 8,l-66.

FAMILIAL AGGREGATION OF CANCER

33

Knudson, A. G. (197%). In “Cancer Achievements, Challenges and Prospects for the 1980s” (J. H. Burchenal and H. F. Oettgen, eds.), pp. 381-396. Alan R. Liss, Inc., New York. Knudson, A. G., Strong, L. C., and Anderson, D. E. (1973). Prog. Med. Genet. 9, 113158. Krivit, W., and Good, R. A. (1957).AMA J . Dis. Child. 94,289-293. Kurita, S., Kamei, Y., and Ota, K. (1974). Cancer 34, 1098-1101. Lal, V. B., and Jorgensen, G. (1976). In “HLA and Disease” (J. Dausset and A. Svejgaard, eds.), p. 229. INSERM, Paris. Lalouel, J. M. (1984). In “Genetic Epidemiology of Coronary Heart Disease: Past, Present, and Future” (D. C. Rao, R. C. Elston, L. H. Kuller, M.Feinleib, C. Carter, and R. Havlik, eds.), pp. 217-243. Alan R. Liss, Inc., New York. Lalouel, J. M., and Morton, N. E. (1981). Hum. Hered. 31,312-321. Lalouel, J. M., Rao, D. C., Morton, N.E., and Elston, R.C. (1983).Am. J . Hum. Genet. 35,816-826. Lamm, L. U., Kissmeyer-Nielsen, F., Kjerbye, K. E., Mogensen, B., and Petersen, N. C. (1974). Cancer (Philadelphia) 33, 1458-1461. Lane-Claypon, J. E. (1926). “A Further Report on Cancer of the Breast, with Special Reference to Its Associated Antecedent Conditions,” No. 32. Rep. Minist. Health, London. Lange, K.,and Boehnke, M. (1982).Am. J . Hum. Genet. 34,842-845. Lathrop, M., and Lalouel, J. M. (1984a).Am. J. Hum. Genet. 36,460-465. Lathrop, M., and Lalouel, J. M. (198413).In “Genetic Epidemiology of Coronary Heart Disease: Past, Present, and Future” (D. C. Rao, R. C. Elston, L. H. Kuller, M. Feinleib, C. Carter, and R.Havlik, eds.), pp. 267-269. Alan R.Liss, Inc., New York. Lehtola, J. (1978). Scand. J . Gastroenterol. 13, Suppl. 50, 12-54. Levin, I. (1912). 2. Krebsforsch. 11, 547-558. Li, F. P., and Fraumeni, J. F., Jr. (1969a).Ann. Intern. Med. 71, 747-752. Li, F. P., and Fraumeni, J. F., Jr. (1969b).J. Natl. Cancer Inst. (U.S.) 43, 1365-1373. Li, F. P., and Fraumeni, J. F., Jr. (1975).Ann. Intern. Med. 83,833-834. Li, F. P., and Fraumeni, J. F., Jr. (1982).J . Am. Med. Assoc. 247,2692-2694. Li, F. P., McIntosh, S., and Peng-Whang, J. (1977). Cancer (Philadelphia) 39, 26332636. Little, C. C. (1923). Eugenics, Genet. Fam. 1, 186-190. Lynch, H. T., ed. (1976). “Cancer Genetics.” Thomas, Springfield, Illinois. Lynch, H. T. (1981). In “Genetics and Breast Cancer” (H. T. Lynch, ed.), pp. 134-173. Van Nostrand-Reinhold, New York. Lynch, H. T., and Frichot, B. C. (1978). Semin. Oncol. 5,67-84. Lynch, H. T., Brodkey, F. D., Lynch, P., Lynch, J., Maloney, K., Rankin, L., Kraft, C., Swartz, M., Westercamp, T., and Guirgis, H. A. (1976a).J . Am. Med. Assoc. 236, 582-584. Lynch, H. T., Krush, A. J., Thomas, R. J., and Lynch, J. (1976b).In “Cancer Genetics” (H. T. Lynch, ed.), pp. 355-388. Thomas, Springfield, Illinois. Lynch, H. T., Guirgis, H. A., Lynch, P. M., Lynch, J. F., and Harris, R. E. (1977).Cancer (Philadelphia) 39, 1867-1881. Lynch, H. T., Harris, R. E., Guirgis, H. A., Maloney, K., Carmody, L. L., and Lynch, J. F. (1978). Cancer (Philadelphia)41, 1543-1549. Lynch, H. T., Follett, K. L., Lynch, P. M., Albano, W. A., Malliard, J. L., and Pierson, R. L. (1979).J . Am. Med. Assoc. 242, 1268-1272.

34

NANCY R. SCHNEIDER ET AL.

MacLean, C. J., Morton, N. E., and Yee, S. (1984).Comput. Biomed. Res. 17,471-480. McClellan, T.,Cannon, L. A.,Bishop, D. T.,and Skolnick, M. H. (1983).Cytogenet. Cell Genet. 37,536. Macklin, M. T. (1932).Q. Rev. B i d . 7,255-281. Macklin, M. T.(1959).J. Natl. Cancer Inst. (U.S.) 22,927-951. McKusick, V. A. (1982a).“Mendelian Inheritance in Man.” Johns Hopkins Univ. Press, Baltimore, Maryland. McKusick. V. A. (1982b).Clln. Genet. 22,360-391. Marchetto, D. J., Li, F. P., and Meadows, A. 1’.(1978).J . Pediatr. 93,537. Martynova, R. P. (1937).Am. J. Cancer 29,530-540. Meisner, L. F., Gilbert, E., Ris, H. W., and Haverty, G. (1979).Cancer (Philadelphia) 43,

679-689.

Moertel, C. G. (1966).Recent Results Cancer Res. 7 (“Multiple Primary Malignant Neoplasms”). Morgan, T. H. (1911).]. E x p . Zool. 11,365-413. Morton, N. E. (1955).Am.]. Hum. Genet. 7,277-318. Morton, N. E. (1958).Science 127,79-80. Morton, N. E. (1959).Am.J. Hum. Genet. 11, 1-16. Morton, N. E. (1969).In “Computer Applications in Genetics” (N. E. Morton, ed.), pp. 129-139. Univ. of Hawaii Press, Honolulu. Morton, N. E. (1982).Prog. Clin. Biol. Res. 103B, 3-14. Morton, N. E. (1984).In “Genetic Epidemiology of Coronary Heart Disease: Past Present, and Future” (D. C. Rao, R. C. Elston, L. H. Kuller, M. Feinleib, C. Carter, and R. Havlik, eds.), pp. 245-265. Alan R. Liss, Inc., New York. Morton, N. E., and Chung, C. S., eds. (1978).“Genetic Epidemiology.” Academic Press, New York. Morton, N. E., and Lalouel, J. M. (1981).Hum. Hered. 31,3-7. Morton, N. E., and MacLean, C. J. (1974).Am. J . Hum. Genet. 26,489-503. Morton, N. E.,and Yasuda, N. (1979).Am. J . Hum. Genet. 32,202-211. Morton, N. E.,Yee, S., Elston, R.C., and Lew, R.(1971).Clin. Genet. 1,71-94. Morton, N. E.,Rao, D. C., and Lalouel, J. M. (1983).“Methods in Genetic Epidemiology.” Karger Press, New York. Mulvihill, J. J., Miller, R. W., and Fraumeni, F. J., Jr. (1977).Prog. Cancer Res. Ther. 3 (“Genetics of Human Cancer”). Ott, J. (1973).Am. J. Hum. Genet. 24,57A. Ott, J. (1974).Am. J . Hum. Genet. 26,588-597. Ott, J. (1976).Am. J. Hum. Genet. 28,528-529. Paget, J. (1853).“Lectures on Surgical Pathology,” Vol. IT. Longman, Brown, Green, & Longmans, London. Paget, J. (1857).Med. Times Gaz. 15,191-193. Pearson, K. (1904).Arch. Middlesex Hosp. 2, 127-137. Pellegris, G.,Illeni, M. T., Vaglini, M., Rovini, D., Cascinelli, N., and Masserini, C. (1980).Tumori 66,51-58. Pellegris, G., Illeni, M. T., Rovini, D., Vaglini, M., Cascinelli, N., and Chidoni, A. (1982).Znt. J . Cancer 29,621-623. Penrose, L.S.,MacKenzie, H. J., and Karn, M. N. (1948).Ann. Eugen. (London) 14,234-

266.

Petrakis, N. L.(1979).Semin. Oncol. 6,433-444. Renwick, J. H., and Schultze, J. (1961).Znt. Cong. Ser. Excerpta Med. 32, E145. Riccardi, V. M.,Sujansky, E., Smith, A.C., and Francke, U. (1978).Pediatrics 61,604-

610.

FAMILIAL AGGREGATION OF CANCER

35

Risch, N. (1984).Am. J . Hum. Genet. 36,363-386. Rosen, P. P., Lesser, M. L., Senie, R. T., and Kinne, D. W. (1982).Cancer (Philadelphia) 50,171-179. Roth, J. A,, Slocum, H. K., Pellegrino, M. A., Holmes, E. C., and Reisfeld, R. A. (1976). Cancer Res. 365,2360-2364. Ryder, L. P., Anderson, E., and Svejgaard, E. (1979).“HLA and Disease Registry, Third Report,” pp. 1-61. Munksgaard, Copenhagen. Schimke, R. N. (1976).Ado. Intern. Med. 21,249-265. Schimke, R. N. (1978).“Genetics and Cancer in Man.” Churchill-Livingstone, Edinburgh and London. Schimke, R. N., and Hartmann, W. H. (1965).Ann. Intern. Med. 63, 1027-1039. Schneider, N. R., Chaganti, S. R., German, J., and Chaganti, R. S. K. (1983).Am.J . Hum. Genet. 35,454-467. Shiku, H., Takahashi, T., Oettgen, H. F., and Old, L. J. (1976).J.E r p . Med. 144,873881. Shimkin, M . B. (1957).J . Natl. Cancer Znst. (U.S.) 19, 295-315. Simpson, J. L., and Photopulos, G. (1976). Birth Defects, Orig. Artic. Ser. 12, 15-50. Skolnick, M. H., Bishop, D. T., Cannings, C., and Hasstedt, S . J. (1984). In “Genetic Epidemiology of Coronary Heart Disease: Past, Present, and Future” (D. C. Rao, R. C. Elston, L. H. Kuller, M. Feinleib, C. Carter, and R. HavIik, eds.), pp. 271-292. Alan R. Liss, Inc., New York. Slye, M. (1922).J . Cancer Res. 7, 107-147. Smith, C. (1971). C1h. Genet. 2,303-314. Smith, C. A. B. (1953).J . R. Stat. Soc., B 15, 153-192. Smithers, D. W. (1948).Br. J . Cancer 2, 163-167. Sokoloff, B. (1938).Am. J. Surg. 40,673-678. Spector, B. D., Perry, G. S., 111, Good, R. A., and Kersey, J. H. (1978).In “The Immunopathology of Lymphoreticular Neoplasm” (J. J. Twomey and R. A. Good, eds.), pp. 203-222. Plenum, New York. Steinberg, A. G. (1960).Cancer (Philadelphia) 13, 985-999. Stephens, F. E., Gardner, E. J., and Woolf, C. M. (1958).Cancer (Philadelphia)11,967972. Strong, L. C. (1977). Cancer (Philadelphia)40, 438-444. Strong, L. C., Williams, W. R., and Norsted, T. (1986).In preparation. Swift, M. (1976).Ciba Found. Symp. 37,115-134. Swift, M., and Chase, C. (1979).J . Natl. Cancer Inst. (U.S.) 62, 1415-1421. Swift, M., Sholman, L., Perry, M., and Chase, C. (1976).Cancer Res. 36,209-215. Videbaek, A. (1947).“Heredity in Human Leukaemia.” Busek, Copenhagen. Waaler, G. H. M. (1931).Skri. Nor. Vidensk.-Akad.[Kl.] I : Mat.-Naturu. idensk. Kl. No. 2 [English abstract by M. Greenwood, Cancer Rev. 7,464-470 (1932)l. Wainwright, J. M. (1931).Am. J . Cancer 15,2610-2645. Wald, A. (1947).“Sequential Analysis.” Wiley, New York. Warren, J. C. (1837).“Surgical Observations on Tumours, with Cases and Operations.” Crocker & Brewster, Boston, Massachusetts. Warthin, A. S. (1913).Arch. Intern. Med. 12, 546-555. Wassink, W. F. (1935).Genetica (The Hague) 17, 103-144. Weinberg, W. (1912a).Arch. Rass. Ges. B i d . 9, 165-174. Weinberg, W. (1912b).Arch. Rass. Ges. Biol. 9, 694-709. Weiner, A. S . (1932).Genetics 17,335-350. Wells, H. G. (1923).J.Am. Med. Assoc. 81, 1017-1021.

36

NANCY R. SCHNEIDER ET AL.

Williams, W. R., and Anderson, D. E. (1984). Genet. Epidemiol. 1, 7-20. Williams, W. R., Strong, L. C., and Norsted, T. (1984). Am. J . Hum. Genet. 36,39S. Woolf, C. M. (1955). Unfv. Cali$, Berkeley, Publ. Public Health 2,265-349. Yunis, J. J., and Ramsay, N. K. C. (1978). Am. J . Dis. Child. 132, 161-163. Yunis, J. J., and Ramsay, N. K. C. (1980).J . Pedlatr. 96, 1027-1030. Zeigel, R. F. (1979). In “Prostatic Cancer” (C. P. Murphy, ed.), pp. 19-33. PSG Publ., Littleton.

TERMINAL TRANSFERASE IN NORMAL AND LEUKEMIC CELLS F. J. Bollum and L. M. S. Chang Department of Biochemistry. Uniformed Services University for the Health Sciences, Bethesda, Maryland 20814

1. Introduction

Much remains to be known about the DNA polymerase called terminal deoxynucleotidyltransferase (TdT), but recent developments have certainly raised this enzyme out of the realm of biological curiosities. Molecular cloning of terminal transferase cDNA (Peterson et al., 1984; Landau et al., 1984), production of monoclonal antibodies (Bollum et al., 1984), ontogenetic studies (Chang, 1971; Gregoire et al., 1979; Sugimoto and Bollum, 1979; Sasaki et al., 1980; Bodger et al., 1983; Deibel et al., 1983), and correlative investigations of TdT function (Desiderio et al., 1984) should soon illuminate its true function. It is now clear that TdT is indeed a creative DNA polymerase, quite separate in its role from the replicative DNA polymerases, and that its function is in producing diversification in the immune system. These findings are long-awaited confirmations of early speculations about participation of this enzyme in thymus biology (&to et al., 1967) and diversification in the immune system (Chang, 1971; Baltimore, 1974). In this review we present some of the biological investigations that have been of primary importance for understanding the position of TdT in the normal hematopoietic system and for using TdT determinations in leukemia research. Earlier reviews have described the biochemical properties of TdT (Bollum, 1974) and some of the preliminary biological investigations (Bollum, 1978). A symposium volume, Terminal Transferase in Zmmunobiology and Leukemia, published in 1982 reviewed many aspects of terminal transferase research (Bertazzoni and Bollum, 1982). Articles covering methodology used in TdT research (Cibull et al., 1982)and use of TdT determinations in clinical diagnosis are also available (Bollum, 1979).Prior to 1975 there are less than 100 original articles on TdT in the scientific literature. Since then publications have been accelerating at a rate of about 25 publications per year, many of them related to use of TdT determinations in leukemia diagnosis. This review is intended as an aid in interpreting the biological position of TdT+ cells and to assist in resolving the status of TdT in leukemia research. 37 Copyright 0 1986 by Academic Press. Inc. ADVANCES IN CANCER RESEARCH, VOL. 47

All rights of reproduction in any forni reserved.

38

F. J. BOLLUM AND L. M. S. CHANG

II. Biochemistry of Terminal Transferase

A. ENZYMATIC PROPERTIES The reactions catalyzed by TdT are simple linear condensation polymerizations requiring activated deoxynucleotides in the form of dNTPs, divalent ions, and a suitable initiator molecule. The initiator molecule must have a chain length of at least three nucleotides and a free 3’-OH (Bollum, 1963; Kato et al., 1967). The enzyme catalyzes a distributed synthesis, producing an average chain length in the product simply related to the molar ratio of monomer to initiator, and proceeds to completion (Chang and Bollum, 1971a). Reversal of synthesis has not been demonstrated. The polymerization reaction has been useful for synthesis of oligodeoxynucleotides and polydeoxynucleotides, and for “tailing” DNA fragments for use in recombinant DNA technology. Readers should refer to other publications for specific details in this area (Michelson and Orkin, 1982). For the present discussion it is useful to know that measurement of enzymatic activity was the original method for specifically demonstrating the presence of this protein in various biological samples. It remains useful for that purpose. Terminal transferase is quite specific in its preference for deoxynucleotide substrates and initiators. Ribonucleotides, arabinonucleotides, dideoxynucleotides, and 3’-deoxynucleotides are also used as substrates but often at considerably lower rates. In most cases analog nucleotides behave as chain terminators in TdT-catalyzed polymerizations. The homogeneous enzyme has a specific enzyme activity that varies somewhat with the nucleotide being polymerized and divalent ion. Maximum rates for purine nucleotide polymerization are achieved with Mg2+,and the dATP polymerization rate of 100,000 nmoles per hour per milligram protein compares with a dGTP rate of 400,000. Pyrimidine nucleotides polymerize best in the presence of Co2+and both dCTP and dTTP show maximum rates of around 300,000 nmoles per hour per milligram of protein. dATP and dTTP polymerizations proceed well and long linear molecules can be produced in greater that 95% yield. dCTP polymerizations are somewhat limited due to formation of double-stranded interactions in the product. dGTP polymerizations cease after about 25 nucleotides are added due to aggregation of the product. Thus, analysis of enzyme activity in crude biological extracts is best carried out by polymerization of dGTP onto a short polydeoxyadenylate (n = 50) initiator, allowing sensitive detec-

TERMINAL TRANSFERASE IN CELLS

39

tion of low levels of enzyme with minimal effects from degradative activities in the extract (Chang, 1971). Analysis of purified fractions is preferably done with dATP to avoid kinetic inhibition (Bollum, 1974). B. PROTEINSTRUCTURE Homogeneous enzyme was first obtained from calf thymus gland in 1971 (Chang and Bollum, 1971b). An unusual structure was found, with native enzyme having a molecular weight of 32 kDa and consisting of two peptides of 24 and 8 kDa. The complete amino acid sequence of human TdT has now been deduced from the cloned cDNA sequence (Peterson et aZ., 1984; Bollum et d.,1985). The sequence and amino acid composition are listed in Table I. The protein contains 508 amino acids, giving a molecular weight of 58,308, in good agreement with the 58,000-Da estimate from in viva labeled human TdT on SDS gels (Bollum and Brown, 1979). Analysis of the distribution of cysteines and methionines in the 58kDa protein indicates that the 32-kDa TdT protein originates from carboxyl terminal residues 159-508. The p peptide is contained within residues 159-402 and the a peptide is within residues 403508. Phosphorylation sites (Chang and Bollum, 1982; Elias et al., 1982) and a nuclear localization sequence (Kalderon et al., 1985) are probably present in the first 17 amino-terminal residues. The catalytic activity measured by free polymerization of dNTPs onto low molecular weight initiators resides in the carboxy-terminal 70% of the protein. Since the structure of the true biological substrate for this enzyme is unknown it is not easy to imagine what the function of the 159 amino-terminal residues might be. We believe that this part of the molecule may be responsible for specific binding to regions of DNA. Potential nucleic acid binding sites (nuclear localization sites) reside in the hydrophilic tail. The 58-kDa enzyme also shows tighter binding to high molecular weight initiator molecules (Augl et aZ., 1983).

C. POLYCLONAL ANTIBODIES The availability of pure 32-kDa protein in useful quantity eventually permitted the development of rabbit antibodies to TdT (Bollum, 1975). Antibodies were needed for cytochemical studies on tissue localization of TdT since the early biochemical studies had indicated an extremely limited occurrence of the enzyme activity (Chang, 1971; Coleman et al., 1974). The polyclonal antibodies produced from ho-

40

F. J. BOLLUM AND L. M. S. CHANC

TABLE I

AMINOACIDSEQUENCEAND COMPOSITION OF HUMAN TdT Translated human TdT amino acid sequence 5

10

15

20

25

30

1

M D P P R A S H L S P R K K R P R Q T G A L M A S S P Q D I

31

K F Q D L V V F I L E K K M G T T R R A F L M E L A R R K G F R V E N E L S D S V T H I V A E N N S G S D V L E W L Q A

61 91 121 151 181 211 241 271 301 331 361

Q K V Q V S S Q P E L L D V S W L I E C I G A G K P V E M T C K H Q L V V R R D Y S D S T N P G P P K T P P I A V Q K I

S F T L F S T L

391

S

421

W G R

451 481

Q R E N R C G Q R Q S I

Y E G D T V G K K E R F

A N I E L T F V V G F L

C E P R S R R M D K E K

Q D C Y K A R N A T R A

R S L Q V E G L L W D E

R C G S R A K W D K L S

T V S F S E K E H A R E

T T K K D A M K F I R E

L F V L K V G K Q R Y E

N M K F S S H G K V A T

N R G T L V D L C D T F

C A I S K L V L F L H A

N A I V F V D L

Q S E F T K F Y

L.1

V L E R H L

I F T D A F D I L A E N C E V L K S L P F T I I S M K D E I I E D G E S S E V K A V G V G L K T S E K W F R M G R M Q K A G F L Y Y E D L V E A V W A F L P D A F V T M L I T S P G S T E D E E Q L Y D L V E S T F E K L R L P F K L P R Q R V D S D Q S S C P Y E R R A F A L L G W T K M I L D N H A L Y D K T K G L D Y I E P W E R N A

Amino acid composition (508residues;M, = 58,308)

-L

31Ala-A

20 Gln - Q

50Leu

37 Arg

41 Glu

-E 26 Gly - G 8 His - H

39 Lys - K

27 Thr - T

-M

9Try-W Y 35 Val - V

-R 13 Asn - N

30Asp-D

9cys-c

23 Ile

-I

13 Met

28 Phe -

F u)Pro - P

38 Ser - S

11 Tyr -

mogeneous antigen were purified on antigen columns and have been the primary cytochemical reagent for ontogenetic studies on cell populations and leukemia marker studies to be described later. The use of the polyclonal antibodies immediately produced some interesting biochemical findings about TdT. First of all, the antibodies produced against calf TdT exhibited a broad cross-reactivity in inhibition tests against TdT from various other species (Bollum, 1975), indicating the possibility of highly conserved protein sequence. TdT from species as distant as humans and birds were all inhibited by the polyclonal antibody developed against the bovine antigen. Second, when the polyclonal antibody was used to immuno-

TERMINAL TRANSFERASE IN CELLS

41

precipitate crude extracts from radiolabeled TdT+ human leukemia cell lines only a 58-kDa peptide was detected (Bollum and Brown, 1979). In later studies antibody applied to immunoblots of crude preparations from a variety of species, including calf thymus, demonstrated the presence of a 58- to 60-kDa peptide species in all samples studied (Bollum and Chang, 1981).Thus the principal form of TdT present in all TdT+ biological specimens is indeed the 58-kDa species, and the low molecular weight protein originally isolated in homogeneous form from calf thymus gland is a severely degraded but enzymatically active form of the enzyme (Chang et al., 1982). Degradation reactions also occur in other tissues (Bollum and Chang, 1981; Deibel et al., 1981), but it is now fairly certain that most (but not necessarily all) degradation is an artifact of the tissue homogenization and purification procedures. The tissue degradation reactions can be mimicked by the action of trypsin (Chang et al., 1982). These findings need not detract from any of the studies conducted using polyclonal antibody, since the low molecular weight form used as antigen used must contain a subset of the epitopes present in the complete enzyme. The polyclonal antibody has allowed extensive study of the presence and absence of TdT in various tissues and the localization of the stable and transient populations of TdT-containing cells (Gregoire et al., 1979; Sasaki et al., 1980), and was used in the molecular cloning of TdT (Peterson et al., 1984; Landau et al., 1984) to be described later. D. MONOCLONALANTIBODIES We prepared mouse monoclonal antibodies against calf thymus TdT using the 32-kDa enzyme prepared by conventional protein purification as antigen. The monoclonal antibodies now permit rapid isolation of pure 32-kDa and 58-kDa proteins from calf thymus gland with ease and complete recovery of activity of the 32-kDa form on monoclonal immunoadsorbent columns. The monoclonal anti-calf TdT columns have fair cross-reactivity with human TdT, allowing the isolation of antigen in suitable amounts and purity for production of mouse antihuman TdT monoclonal antibodies (Bollum et al., 1984). These materials should provide resolution for continuing studies on TdT biology in humans and for leukemia diagnosis. 111. Ontogeny

The original study on the distribution of terminal transferase enzyme activity was carried out in calf fetuses (Chang, 1971). Here it was

42

F. J. BOLLUM AND L. M. S. CHANC

demonstrated that terminal transferase activity was present only in the thymus gland, appearing in later stages of fetal growth, and increasing in amount during the early postnatal period. Measurement of enzyme activity suffers from lack of sensitivity in tissues that contain only a minor population of TdT+ cells. This early work concluded that the enzyme was only present in thymus, later shown to be present in 6070% of cells and restricted to the cortex (Goldschneider et al., 1977). Tissues such as spleen, liver, lymph nodes, lung, muscle, and brain did not contain detectable levels of activity. Improved sensitivity eventually showed the enzyme to be present in human and rodent bone marrow (Coleman et al., 1974; Vines et al., 1980) where the TdT+ cell population is only around 1-3% of nucleated cells. Subsequent work has relied on cytochemical methods using specific immunochemical reagents that are capable of detecting minor and transient populations of TdT+ cells. Since the original biochemical studies in cow fetuses (Chang, 1971), the development of cell populations containing TdT has been studied rather intensively in rodents, birds, and man. The course of appearance differs rather widely in each biological system studied so each must be considered separately.

A. INRODENTS The data obtained on TdT+ populations from extensive studies on rodent embryos and neonates do not fit the expected pattern for hematological development (Gregoire et al., 1979; Sasaki et al., 1980). These investigations discovered the existence of transient TdT+ populations in secondary lymphoid organs and followed the development of the stable populations in primary lymphoid organs. The transient and stable populations in rodents are shown in Fig. 1. The earliest population is found in the thymus, developing in late fetal life, and maturing to the adult level in the early neonate. The bone marrow population is not found in the fetus and reaches its fully developed level about 10 days after birth, remaining in adults as a fairly stable minor lymphocyte population. Transient populations are found in liver, spleen, lung, and blood in the first several weeks after birth, decreasing to practically undetectable levels within the first 4 weeks of life. The expected course of hematopoietic development moving from liver to bone marrow to thymus seen in other hemopoietic systems seems to be almost reversed in this species. Attempts to characterize origins of some of the transient populations in rodents have been carried out in mutant mouse strains (Hutton and Bollum, 1977; Sasaki et al., 1980) since the general course of develop-

TERMINAL TRANSFERASE IN CELLS

43

TIME (WEEKS)

ment is similar in rats and mice. Homozygous nude mice have all transient populations except that normally found in peripheral blood. Since nude mice also lack the stable thymus population of TdT+ cells it seems obvious that the transient population in blood must arise from

44

F. J. BOLLUM AND L. M. S. CHANC

the thymus. Further analysis using monoclonal antibodies to establish surface phenotype in normal rats (Goldschneider, 1982) confirms the predominant origin from thymus, but also indicates a minor component that could have arisen from bone marrow. These results should be contrasted to the analysis of circulating TdT+ cells in humans, where the predominant source is bone marrow (Bradstock et al., 1985). Of course, it may be that the immunological analyses performed are not completely appropriate. Most of the marker antibodies available for this kind of study are derived from surface antigens present on the circulating lymphocytes of adult animals. Rare phenotypic markers produced from early precursor cells might be more appropriate for the final analysis of the derivation of the transient TdT+ population in the circulation. The immunological phenotyping also suggested that there may be several waves of immigration into the thymus, perhaps with eventual differentiation into several separate functional classes. B. INHUMANS The presence of TdT+ cells in normal adults is almost exclusively restricted to thymus cortex and bone marrow lymphocytes. Rare TdT+ cells are found in circulating lymphocytes (Froehlich et al., 1981; Bradstock et al., 1985) and spleen. Values in these tissues are usually less than 0.02%, making detailed phenotypic analysis rather difficult. Remarkable changes in location and cell number may occur in lymphoid leukemias and lymphomas (Bollum, 1979). The developmental pattern follows that expected for hematopoietic cells in man (Bodger et al., 1983). TdT+ cells are first detected in 12to 13-week embryos in the lymphoid cells of the liver. At this time no TdT+ cells are present in the fetal thymus or bone marrow. The thymus and bone marrow populations develop at 19-21 weeks and 15-16 weeks, respectively. Fetal thymus has low level of TdT+ cells constituting 5-10% of total thymocytes. The level of TdT+ cells in the thymus increases during development and after birth, reaching 6 0 4 5 % positivity between 10 and 40 months of age (Janossy et al., 1980c; Deibel et al., 1983). During childhood (5-12 years), about 65% of thymic cortical lymphocytes are TdT+ and HTA-1+and only 1-3% are TdT+ and HTA-1-. Only 3% of lymphoid cells in the medulla expressed TdT and most medullary thymocytes are TdT- and HTA-1-. The TdT+ cells in the thymus decrease with age as in the normal thymic involution (Deibel et al., 1983). The TdT+populations in fetal bone marrow and liver have recently

TERMINAL TRANSFERASE IN CELLS

45

been shown to have similar phenotypes (Bofill et al., 1985). Membrane markers of early B lineage, but no T-lineage markers, are found on TdT+ cells in these tissues. Other human embryo tissues have not been extensively studied due to virtual absence of TdT+ cells. In infant and regenerating bone marrow 0.5-11% TdT+ cells are found (Campana et al., 1985)and these cells show a predominant B-lineage phenotype (BA-l+, AL-I+, and to a lesser extend Bl+). Young adult bone marrow contains 0.2-2% TdT+ cells with presence of similar immature B-lineage markers and absence of more mature B-cell markers. There is some indication that neonatal blood lymphocytes may contain detectable levels of TdT+ cells, decreasing to very low levels during the first few years of life. Characterization of the rare TdT+ cells (<0.02% of Ficoll-Hypaque-separated lymphocytes) in the circulation of children and young adults (Bradstock et al., 1985) shows that the majority are TdT+, HLA-A,B+, and Ia+. Minor populations expressing surface markers characteristic of B lineage (CALLA and BA-1) are seen but no T-characteristic markers (UCHT-1, RFT-2, NAI34, or Lyt-3) were demonstrated. This population seems to be derived from B-lineage bone marrow cells, quite in contrast to results obtained in rodents. The ontogeny of TdT+ cells in humans is in keeping with the idea that hematopoietic cells migrate from yolk sac to fetal liver, then move to the primary lymphoid organs in maturing embryos where they become the sources of continued renewal for the secondary lymphoid tissues. C. INBIRDS In chick embryo the TdT+ population begins to develop in thymus on day 10-12 of incubation and is fully developed to the adult level at hatching on day 21 (Penit and Chapeville, 1977; Sugimoto and Bollum, 1979). In marked contrast to the mammals studied, no other stable population of TdT+ cells has been found in birds. In fact, no other measurable stable or transient populations of TdT+ cells have been found in birds even though many embryonic tissues were tested (Sugimoto and Bollum, 1979). Rare TdT+ cells were seen in the bursa of Fabricius at levels too low for careful enumeration. Studies on the immigration of host embryo cells of different ages into uncolonized embryonic thymus rudiment show that development of the TdT+ population in rudiments is related to explant age and not to the age of the host. The immigration of lymphoid stem cells into thymus rudiments is cyclical. The first wave enters the thymus at 6.5-

46

F. J. BOLLUM AND L. M. S . CHANC

8 days and does not differentiate to TdT+ cells. The second wave occurs between 12 and 14 days and 95% of the TdT+ cells develop from this influx. Thymic rudiments colonized in uitro by bone marrow cells before explantation in embryo hosts show even greater percentage of TdT+ cells in their progeny (Penit et al., 1985). These studies suggest that precursors of TdT+ cells reside in the bone marrow in birds. It seems that time in the thymic microenvironment is less important than the source of immigrating stem cells in determining development potential. This might also explain the different developmental potential of TdT+cells in successive immigrating stem cells (Gregoire et al., 1979) in rodent thymus. IV. Leukemia Marker Studies

Evaluation of terminal transferase as a leukemia marker began with the finding that large amounts of enzyme were present in circulating blast cells from a patient with childhood T-cell acute lymphocytic leukemia (T-ALL) (McCaffrey et al., 1973). Since TdT was specifically associated with thymus at that time it was thought that it might be a marker for leukemia derived from thymic cells. Continued examination of patients with various forms of lymphoid malignancy extended terminal transferase positivity to include most forms of acute leukemia, excepting mature forms classified as B-ALL and mature T-ALL (Stass et al., 1979), and in some forms of lymphoblastic lymphoma (Cossman et al., 1983; Murphy and Jaffe, 1984). Analysis of blood and bone marrow lymphocytes and lymph node biopsies for the presence of TdT+ cells now provides a rather useful diagnostic test for lymphoblastic proliferation, and is widely used in conjunction with various membrane markers to aid in the detailed classification of cell populations in leukemic patients (Janossy et al., 1980a; Hutton et al., 1982; San Miguel et al., 1985). The usefulness of TdT analysis in leukemia diagnosis stems from several observations. In the normal situation TdT+ cells are restricted to thymus and bone marrow. In acute lymphocytic leukemia TdT+ cells are found in large numbers in the circulation (Bollum, 1979), and in certain cases in spleen (Braziel et al., 1984), lymph nodes (Cossman et al., 1983), cerebrospinal fluid (Bradstock et al., 1980), testes (Thomas et al., 1982), and possibly other extramedullary locations where these cells do not occur normally. In the circulation and bone marrow a major fraction (50-90%) of the blasts present in ALL patients may express TdT.

TERMINAL TRANSFERASE IN CELLS

47

Elevated populations of TdT+ cells have not been observed regularly in chronic leukemias of granulocytic, lymphocytic, erythrocytic, or other differentiation oriented disease types, but occasional instances are reported in the literature. In the blastic or accelerated phase of chronic granulocytic leukemia the proliferating population is usually very immature and not easily differentiated as myeloblastic or lymphoblastic. In these cases TdT positivity in a high percentage of cells (greater than 20%of nucleated cells) is used to classify the proliferation as lymphoblastic (Sarin and Gallo, 1974; McCaffrey et al., 1982; San Miguel et al., 1985),whereas TdT-negative cases are called myeloblastic, allowing confident diagnosis of acute-phase proliferation and suggesting therapeutic protocol (Marks et al., 1978). Generally, the majority of acute myeloid leukemia (AML) cases do not contain elevated levels of TdT+ cells (Jani et al., 1983). But there are exceptions to this rule that still require analysis and explanation. In early work certain AML patients were found to show the presence of the enzyme (Srivastava et al., 1978). Retrospective study of a large series of patient samples using TdT immunofluorescence (Jani et al., 1983) demonstrated that as many as 10%of AML patients may exhibit elevation of the TdT+ population, as had been suggested by earlier limited case studies (Stass et al., 1979; Hutton et al., 1982; Bradstock et al., 1983).For the most part the population of cells is not as large in AML patients as in ALL patients, but nevertheless greater than 10% and up to 50% of the blasts may be TdT+. As more sensitive TdT immunoperoxidase or TdT alkaline phosphatase detection systems have been developed, increased numbers of AML patient samples are judged to be positive (Lanham et al., 1985)! Monoclonal antibody staining has not detected TdT+ cells in a limited series of AML patients (Lanham et d., 1986). What might be the explanation of these seeming inconsistencies? One must first consider the differentiation pathways of the lymphoid and myeloid cells. Since these cell lines are thought to come from common progenitors that eventually become committed to either myeloid or lymphoid differentiation, it is possible that a common immature precursor may contain TdT and retain this expression for some period of time after commitment is made. This argument implies that early myeloid committed blasts may contain vestigal TdT. Sensitive immunoenzymatic cytochemistry would detect this vestigal protein, and since microscopy is primarily subjective, an increased number of positive cells would be scored. The availability of specific early myeloid markers and cytofluorometry of TdT fluorescence might aid in clarification of this point.

48

F. J. BOLLUM AND L. M. S. CHANC

An interesting alternative explanation concerns the possibility that malignant blasts may exhibit “aberrant expression,” expressing markers not expected during normal differentiation. This phenomenon has also been called lineage infidelity or biphenotypy, terms that are semantic refinements of what older hematologists call mixed leukemias. The original finding was that malignant blast cells may show dual expression of lymphoid and myeloid antigens on the same cell (Smith et al., 1983, Lanham et al., 1984). Dual expression is not limited to TdT and myeloid markers and may in rare instances show promyelocytic (Paietta et al., 1985), monocytic (Cuttner et al., 1984), and probably other recognizable differentiation lineages. At the present time this is often attributed to loss of control of gene expression resulting from oncogene insertions (Cline et al., 1984). Readers are referred to more detailed discussion of these ideas (Olsson, 1983), since the explanation for aberrant expression of TdT in AML and other forms of acute leukemia is not clearly evident at the present time. In this list of several types of TdT-positive leukemia it is important to note that most are acute leukemias. The majority of the totaZ leukemic population, and of blood dyscrasias generally, is TdT negative (Stass et al., 1979). Current consensus interprets TdT positivity as a marker of immature lymphoid populations, but just how immature? Chronic leukemias of mature B- and T-cell phenotypes are routinely negative for TdT. This includes the cutaneous leukemias (Sbzary syndrome and hairy cell leukemia), mature B- and mature T-cell leukemias, and the great majority of lymphomas with mature phenotype. On the other hand, those rare cases in the literature passing editorial judgment as “stem cell leukemias” (J. Kurtzberg and M. Hershfield, personal communication) are usually TdT negative. Thus it would seem that the TdT+ cell is not the stem cell, and might be best classified as a prelymphocyte-lineage undefined. The participation of viruses and oncogenes in the etiology of leukemic processes is beyond the scope of this review. We should mention that the HTLV-I-positive leukemias and lymphomas of limited geographic distribution are mature cell leukemias and are TdT negative (R. Gallo, personal communication). We should be reminded that the strength of the arguments about the virus association with this and other malignant diseases is due to the availability of recombinant DNA technology and cytogenetic confirmation of chromosome abnormalities (Croce et aZ., 1985). Generalizing from this, it might be worthwhile to consider that ultimate understanding of the leukemic process will require knowledge of malignant aberrations at the DNA level. Thus, overexpression or malexpression in all forms of leukemia may

TERMINAL TRANSFERASE IN CELLS

49

be due to loss of control of genetic regulation of differentiation, induced by viral control elements. We need to know which genes to study and the method for the study in order to extend our knowledge of these processes. Recombinant DNA technology appears to be the promise for the future in this arena. V. The Nature of TdT+ Cells

At certain stages of the development of ideas about a natural phenomenon the reasons for the phenomenon are obvious. This is a double aphorism since its truth depends, like beauty, on the eye of the beholder as well as that beheld. When terminal transferase was first demonstrated, ideas about the participation of thymus in cellular immunity were in their infancy and the detailed structure of immunoglobulin molecules and immunoglobulin genes was unknown. The molecular bases of genetic diversification were not as obvious as they are in 1985. Noting the presence of TdT in thymus plus the rising evidence for the role of the thymus in cellular immunity it seemed reasonable to conjecture that TdT might be related to the specific function of this organ (Kato et al., 1967). Viewing thymocytes with the plethora of monoclonal markers available from the technology of the 1980s provides impressions that were not visible in the 1970s when the primary classification for thymocytes was small, medium, or large! Thus when the unique tissue location of terminal transferase was first appreciated, along with its nonreplicative mechanism for DNA synthesis, speculation that this enzyme might participate in antibody DNA diversification in the immune system (Chang, 1971) did not seem outlandish. It would be ridiculous to claim then that we knew all along that TdT was responsible for diversification, as it would be premature to claim now. It does seem to be a good idea. While we can now talk about germ line diversity, recombinational diversity, somatic diversity, and perhaps mutational diversity with considerable Blan, it remains to be determined how important each of these mechanisms is for functional diversity. Current research is exploring these ideas in detail (Jeske et al., 1984). To classify mature T- and B-cell phenotypes is a simple matter at the present time. This is because most monoclonal antibodies currently available were developed against antigens present on major mature cell populations. Classification of immature cell populations is not such a simple matter, because immature cells generally do not express surface antigens characteristic of mature functional cells in

50

F. J. BOLLUM AND L. M. S. CHANG

the immune system, One can readily isolate subsets of mature populations using monoclonal antibody labels in the cell sorter, and measure the functionality of the separated cells. Within the general population of T cells one finds subclasses that are killer cells, helper cells, and suppressor cells. But one must induce differentiation to maturation of surface antigen or functionality in order to adduce the subset lineage of a population of immature T cells. Some progress has been made in this direction with leukemia cells and cell lines, following the technology that was first used for differentiation induction in erythroid precursors. The compounds used for differentiation in lymphoblasts are not as wide ranging as those used effectively for other cell lineages, but phorbol esters have been found to be active, probably effecting activation of a Ca2+-activated CAMP phosphodiesterase (Neidel et al., 1983; Kraft and Anderson, 1983). Studies of this kind now indicate that phorbol ester-induced differentiation of certain preT TdT+ cell lines leads to the expression of markers characteristic of the helperhppressor subclass of T lymphocytes (Sacchi and Bollum, 1985; Sacchi et al., 1984). Unfortunately, this does not immediately clarify the molecular activities of either helperhppressor cells or terminal transferase! But it does narrow the scope of interest. Terminal transferase is also present in bone marrow lymphocytes and pre-B leukemic cell lines. The bone marrow TdT+ cells are most likely also associated with the B-cell lineage. Phorbol ester-induced differentiation of pre-B TdT+ cell lines to demonstrable immunoglobulin synthesis or to further rearrangement of immunoglobulin genes (Korsmeyer et al., 1981) has been attempted but so far not achieved (Sacchi et al., 1984). Phorbol ester-treated TdT+ leukemic blasts with p and K rearranged have been induced to synthesize cytoplasmic and surface IgM (Cossman et al., 1982). Whether TdT plays a role in immunoglobulin diversification has not been clearly shown in this kind of system. Since immunoglobulin diversification proceeds as cells mature to IgG producers not expressing TdT, it is clear that this enzyme is not instrumental in all diversification, Most specifically, it does not appear to have the sort of recombinational activity that would serve to generate junctions. Diversity at junctions could of course be amplified if the enzyme were to add deoxynucleotides to genes undergoing recombinational repair. In addition to the recombinational diversity occurring at VD and DJ joints, non-germ-line sequences are also found in the DNA coding for the third hypervariable region of immunoglobulin heavy chains. These have been called N regions by Alt and Baltimore (1982) and non-germ-line elements by others (Sakano et al., 1981). They consist

TERMINAL "SFERASE

IN CELLS

51

of 5-12 nucleotides not present in germ line sequence and have some bias toward being rich in guanine nucleotides. N-region diversity aIso occurs in T-cell receptor variable regions (Clark et al., 1984). Mouse immunoglobulin heavy chain sequences assembled in TdT+ cell lines develop N regions at recombination junctions during rearrangement whereas TdT- lines do not (Desiderio et al., 1984).Thus, circumstantial evidence for the participation of terminal transferase in N-region production and associated diversification has finally been produced. It should be noted that all of the N regions detected in these experiments would produce VH coding sequence out-of-frame with the J H sequence and would not produce recognizable immunoglobulin. It will be of considerable interest to have more information about this phenomenon in T-cell receptor and immunoglobulin diversification. VI. Morphology of TdT+ Cells

Most of the cytochemical studies related to TdT+ cell populations have been done using indirect immunofluorescence and phase microscopy. This is a useful technique for subjective enumeration of positive and negative cells, especially for clinical specimens, but it does not provide much structural detail. Nevertheless, these studies did suggest some gross structural differences. Generally TdT is found only in the nucleus of positive cells. There are exceptions to this localization in mitotic cells, in which chromatin condensation and nuclear membrane dissolution result in redistribution of TdT into the cytoplasm. Daughter cells appear to be redistributing the enzyme to the nucleus but the origin of this material from premitotic protein or postmitotic synthesis has not been determined. Also, in certain cultured leukemic cell lines it appears that some immunoreactive material is present in the cytoplasm. The simplest interpretation of this is that cells having polyribosomes actively engaged in synthesis might have a detectable level of cytoplasmic antigen, although it is equally possible that some form of degradation is taking place. In rare patient samples, particularly blast crisis chronic myelogenous leukemia, cytoplasmic as well as nuclear localization (Cibull et al., 1981) has been reported. Finally, when one views the fluorescence in thymocyte preparations, rather than seeing a large brilliant TdT+ population and smaller numbers of negative cells, a disappointing gradation of fluorescence intensity makes positive and negative difficult to discriminate. So what should be the best test material turns out to be the most difficult for microscopic assessment, at least using fluorescence. Morphologic evaluation of terminal transferase localization and

52

F. J. BOLLUM AND L. M. S. CHANG

FIG.2. Immunoperoxidase staining ofTdT+ cells. (A) and (B) Bone marrow smears of two ALL patients; (a) indicates TdT+ cells and (b) TdT- cells. (C) A mixture of human

lymphoblastoid KM-3cells with mouse myeloma SP-2 cells. (D) A cytospin preparation of rat thymocytes. (A) ~ 1 6 0(B) ; x160; (C) ~ 6 3 0 (D) ; x630.

transport requires better microscopic resolution. The development of immunoperoxidase procedures for staining TdT+ cells (Stass et al., 1982; Racklin et al., 1983),allowing bright-field microscopy and counterstaining, has improved resolution somewhat. Figure 2 shows TdT immunoperoxidase-stained and hematoxylin-counterstained cell smears from several sources, Staining of human lymphoblastoid KM-3 cells mixed with mouse SP-2 myeloma cells (Fig. 2C)reveals cells in mitosis with the redistribution into the cytoplasm of TdT+ material. The heterogeneity of staining by rat thymocytes (Fig. 2D) shows a

TERMINAL TRANSFERASE IN CELLS

53

remarkable gradation of immunoperoxidase deposit in the nucleus of a large fraction of the small to medium-sized thymocytes, as expected from the immunofluorescence. The most interesting class of cells are the large thymocytes showing both nuclear and cytoplasmic stain for TdT. These are thought by some authors to be recent immigrants to the thymus (Janossy et al., 1980c; Gregoire et al., 1979), and possibly precursors of the medium to small cells. It seems that cells in all stages of differentiation with respect to expression of TdT are present. Results of this kind have not yet been detected in other tissues, but this does not mean that it does not occur there. TdT immunoperoxidase staining of bone marrow smears from two ALL patients is presented in Fig. 2A and B. Electron microscopic examination of the intracellular localization of TdT confirms the results obtained in bright-field microscopy. The electron micrographs of a KM-3 cell (Fig. 3B) and a KM-3 cell in mitosis (Fig. 3A) show that when the nuclear membrane disappears during mitosis, TdT is distributed into the cytoplasm while nonmitotic cells show TdT staining only in the nucleus. It is now possible to consider the morphological results in terms of molecular structure. Immunochemical studies on TdT have shown that the thymus exhibits an unusual pattern of protein degradation (Chang et al., 1982), making it almost impossible to isolate the 58-kDa form of the protein from this tissue (see Section 11,B). Most tissues will show varying amounts of 58-kDA and 56-kDa forms, but thymus also shows a 42- to 44-kDa doublet and sometimes 32- to 34-kDa forms, as well as the “limit” form having the a and p peptides of the original 32-kDa protein (Chang and Bollum, 1971). As mentioned earlier the degradation is currently ascribed to artifactual degradation during purification but there may be biological degradation accompanying differentiation as well. It is now well established that domains within protein structure provide signaling for intracellular transport and localization (Blobel, 1980; Watson, 1984). Thus signal sequences at the amino terminus may induce extracellular transport and other domains may dictate membrane localization. Certain protein codes must also direct nuclear localization and transport since large proteins cannot penetrate nuclear pores by simple diffusion (Paine and Feldherr, 1982). Residues 11-17 of the TdT sequence (Table I) have the sequence -Pro-Arg-LysLys-Arg-Pro-Arg-, five highly basic amino acids in a flexible region near the amino-terminal end of the protein. A sequence shown to control nuclear localization for SV40 T antigen has been found to be -Pro-Lys-Lys-Lys-Arg-Lys-Am-, starting at Lys-128 in this protein

FIG.3. Electron micrographs of immunoperoxidase-stained KM-3 cells. (A) The TdT staining of a cell in mitosis. (B) The nuclear localization of a nondividing TdT' cell. The bar at the lower right comer of each panel represents 1 pm.

TERMINAL TRANSFERASE IN CELLS

55

(Kalderon et al., 1985). The similarity in charge density for these two sequences is remarkable, suggesting that TdT residues 11-17 might direct the nuclear localization of this protein. Residues 5-9 of the TdT sequence contain the sequence -Pro-ArgAla-Ser-His-Leu-, which resembles typical serine phosphorylation site sequences in many proteins (Walsh and Krebs, 1973). It has been demonstrated in uitro that the 58-kDa form of calf thymus TdT is phosphorylated but the 56-kDa form is not (Chang and Bollum, 1982). Analysis of phosphorylation sites of human TdT phosphorylated in uiuo showed 85% phosphoserine and 15% phosphothreonine (Elias et al., 1982). The amino-terminal residues 1-17 are highly hydrophilic, probably forming a tail on the more hydrophobic core. One then might speculate that hormone-induced phosphorylation may activate proteolysis at Met-23 to remove the nuclear localization sequence (1123) and force the marked protein to pass to the cytoplasm for further degradation. The availability of the complete sequence, possibilities for experimental mutation of the sequence, monoclonal antibodies, and DNA probes should soon provide an understanding of the synthesis, transport, localization, and degradation of TdT. The possibilities for studying the ability of mutated proteins to carry out the biological function of TdT is most intriguing. It is indeed a sign of the power of modern molecular biology when cell morphology and protein sequence can be discussed in the same paragraph! VII. Evolution

The uniqueness of TdT in biological occurrence causes some wonder about the origins of this kind of DNA polymerase. Earlier studies using immunological reagents showed cross-reactivity of TdT in species ranging from chicken to human (Bollum, 1975).In addition to the conservation of immunological determinants, the size of the TdT molecules from these diverse species was also found to be similar (Bollum and Chang, 1981). With DNA probes questions concerning the origin of this unique DNA polymerase can be addressed directly. Figure 4 illustrates dot blots of DNA samples from a set of organisms spanning considerable evolutionary time. The close sequence relationship expected for the human and bovine gene is seen, and there is even good hybridization with bony fish DNA (Saluelinus fontinalis). The latter relationship is not completely unexpected since even cartilaginous fish have a primitive immune system. The weak hybridization with crabs (Cancer productus), barnacles (Pisaster ochraceus), and clams

56

F. J. BOLLUM AND L. M. S. CHANG

FIG.4. DNA homology studies by dot DNA hybridization. Serial twofold dilutions of various DNA samples starting with 5 pg in lane 1 were fixed on nitrocellulose filters, hybridized with nick-translated pT17, and visualized by autoradiography. Positive controls of the hybridization were serial twofold dilution of pT17 starting with 5 ng in lane 1. The negative control is pBR322 DNA.

(Chlumys rindsii) is rather unusual and may be indicative of some interesting evolutionary history for TdT in invertebrates. No reactivity with Tetruhymena or Pneumococcus DNA is found. These studies must now be pursued with a variety of DNA samples and by Southern blot hybridizations. Some preliminary work on evolutionary structure of the TdT gene is shown in Fig. 5. In this figure total DNA from man, mouse, and chicken has been fragmented with the restriction endonuclease Hind111 and transferred to nitrocellulose

TERMINAL TRANSFERASE IN CELLS

57

FIG.5. DNA hybridization of human (A), mouse (B), and chicken (C) genomic DNAs with TdT cDNA. DNAs (10 pg) were digested with the restriction endonuclease HindIII, separated on a 0.6%agarose gel, transferred onto a nitrocellulose filter, and hybridized with nick-translated pUC-8 containing the total TdT cDNA sequence.

from agarose electrophoresis gels. The figure illustrates fragments that hybridize to a mixture of DNA probes spanning the total TdT cDNA sequence. Since Hind111 does not cut within the TdT-cDNA sequence the hybridizing fragments are due to breakage within introns. Therefore, although differences in intron structure may be deduced from the fragments differing in molecular weight, a fragment at about 3.8 kb is conserved between these diverse species. It will be of future interest to find the part of the TdT molecule that is produced by this conserved gene sequence.

58

F. J. BOLLUM AND L. M.

S. CHANG

VIII. Conclusions

The next few years should produce clarification of the role of terminal transferase in lymphocyte biology. Vague ideas expressed in the past about DNA diversification can be approached directly now that experimental vehicles are possible to construct. This could not be done in the past since the DNA segments postulated to be subject to diversification reactions were not precisely known, and if known or suspected, were impossible to isolate and manipulate. Because the only known enzyme activity for TdT is dNTP polymerization it is fairly certain what the final result may be. The basic biological findings should provide some deeper understanding of the participation of TdT+ cells in the malignant process. It is of considerable utility to have the use of TdT as a marker for differential diagnosis of acute leukemias, but whether the presence of expanded populations of this kind contributes to the disease process, or will provide leverage for therapy, remains to be determined. The fact that cells may express antigens in an aberrant manner is of descriptive interest, but whether aberration has deleterious effects on the cells still is not known. Resolution of these questions should lead us deeper into the mechanisms of differentiation in cellular and humoral immunity. At the present time TdT is the only protein that leads to this junction. Tracing this system through its evolutionary origins might lead us to the true roots of the immune systems of the higher vertebrates. ACKNOWLEDGMENTS The opinions or assertions contained in this paper are those of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The unpublished work presented in this review was supported by United States Public Health Service Grants CA23262 and GM31393. We acknowledge the contributions of our co-workers R. C. Peterson and Ling C. Cheung for molecular cloning and DNA sequencing, Roberto DePrimio for electron microscopy, and R. J. Mattaliano for protein sequencing. Expert technical assistance has been provided by Christel Augl, Elizabeth Rafter, and Stephen T. White.

REFERENCES Alt, F., and Baltimore, D. (1982). Proc. Natl. Acad. Sci. U S A . 79,4118-4122. Augl, C., Lee, S., Brevario, D., Chang, L. M. S., and Bollum, F. J. (1983).Fed. Proc., Fed. Am. SOC. E x p . Blol. 42, 2147. Baltimore, D. (1974). Nature (London)248,409-411. Bertazzoni, U., and Bollum, F. J., eds. (1982).“Terminal Transferase in Immunobiology and Leukemia.” Plenum, New York. Blobel, G. (1980).Proc. Natl. Acad. Sci. U.S.A. 77, 1496-1500.

TERMINAL “RANSFERASE IN CELLS

59

Bodger, M. P., Janossy, G., Bollum, F. J., Burford, G. D., and Hoarand, A. V. (1983). Blood 61,1125-1131. Bofill, M., Janossy, G., Janossa, M., Burford, G. D., Seymour, G. J., Wernet, P., and Kelemen, E. (1985).J. Immunol. 134, 1531-1538. Bollum, F. J. (1963).Prog. Nucleic Acid Res. 1, 1-26. Bollum, F. J. (1974).In “The Enzymes” (P. D. Boyer, ed.), Vol. 10, pp. 145-171. Academic Press, New York. Bollum, F. J. (1975).Proc. Natl. Acad. Sci. U S A . 72,4119-4122. Bollum, F. J. (1978).Ado. Enzymol. 47,347-374. Bollum, F. J. (1979).Blood 54, 1203-1215. Bollurn, F. J., and Brown, M. (1979).Nature (London)278, 191-192. Bollum, F. J., and Chang, L. M. S. (1981).J. Biol. Chem. 256,8767-8770. Bollum, F. J., Augl, C., and Chang, L. M. S. (1984).J. Biol. Chem. 259,5848-5850. Bradstock, K.F., Papageorgiou, E. S., Janossy, G., HoMbrand, V. A,, Willoughby, M. L., Roberts, P. D., and Bollum, F. J. (1980).Lancet 1, 1144. Bradstock, K. F., Hewson, J., Kerr, A., Kabral, A., Lee, C. H., and Hughes, W. G. (1983). Am. J . Clin. Pathol. 80,800-805. Bradstock, K. F., Kerr, A., and Bollum, F. J. (1985).Cell. Immunol. 90,590-598. Braziel, R. M.,Keneklis, T., Donlon, J. A., Hsu, S. M., Cossman, J., Bollum, F. J., and Jaffe, E. S. (1984).Am. J. Clin. Pathol. 80,655-659. Campana, D., Janossy, G., Bofill, M., Trejdosiewicz, L. K., Ma, D., H o a r a n d , A. V., Mason, D. Y., Le Bacq, A.-M., and Forster, H. K. (1985).J . Immunol. 134, 1524-

1530.

Chang, L. M. S. (1971).Biochem. Biophys. Res. Commun. 44,124-131. Chang, L. M. S., and Bollum, F. J. (1971a).Biochemistry 10,536-542. Chang, L. M. S., and Bollum, F. J. (1971b).J. Biol. Chem. 246,909-916. Chang, L. M. S., and Bollum, F. J. (1982).J. Biol. Chem. 257,9588-9592. Chang, L. M. S., Plevani, P., and Bollum, F. J. (1982).J . Biol. Chem. 257, 5700-5706. Cibull, M. L.,Coleman, M. S., Hutton, J. J.. Bollum, F. J., and Jackson, D. V. (1981).Am. J . Clin. Pathol. 75,363-366. Cibull, M. L., Coleman, M. S., Nelson, O., Hutton, J. J., Gordon, D. and Bollum, F. J. (1982).Am. J . Clin. Pathol. 77,420-423. Clark, S. P., Yoshikai, Y., Taylor, S., Sui, G., Hood, L., and Mak, T. W. (1984).Nature (London) 311,387-389. Cline, M. J., Slamon, D. J., and Lipsick, J. S. (1984).Ann. Intern. Med. 101,223-233. Coleman, M. S., Hutton, J. J., DeSimone, P., and Bollum, F. J. (1974).Proc. Natl. Acad. Sci. U.S.A.71,4404-4408. Cossman, J,, Neckers, L. M., Arnold, A., and Korsmeyer, S. J. (1982).N . Engl. J . Med.

307,1251-1254.

Cossman, J., Chused, T. M., Fisher, R. I., Magrath, I., Bollum, F., and Jaffe, E. (1983). Cancer Res. 43,4486-4490. Croce, C. M., Isobe, M., Palumbo, A. P., Puck, J., Erikson, J., Davis, M., and Rovera, G. (1985).Science 227, 1044-1047. Cuttner, J., Seremetis, S., Nafield, V., Dimitriu-Bona, V., and Winchester, R. A. (1984). Blood 64,237-243. Deibel, M. R., Jr., Coleman, M. S., Acree, K., and Hutton, J. J. (1981).J . Clin. Invest. 67,

725-734.

Deibel, M. R., Jr., Riley, L. K., Coleman, M. S., Cibull, M. L., Fuller, S. A., and Todd, E. (1983).J . Immunol. 131,195-200.

60

F. J. BOLLUM AND L. M. S. CHANG

Desiderio, S. V., Yancopoulos, G. D., Paskind, M., Thomas, E., Boss, M. A,, Landau, N., Alt, F. W., and Baltimore, D. (1984).Nature (London) 311,752-755. Elias, L., Longmire, J., Wood, A,, and Ratliff, R. (1982).Biochem. Biophys. Res. Commun. 106,458-465. Froehlich, T. W., Buchanan, G. R., Comet, J. A., Sartain, P. A., and Smith, R. G. (1981). Blood 58,214-220. Goldschneider, I. (1982).Ado. E x p . Med. Biol. 145, 115-132. Goldschneider, I., Gregoire, K. E., Barton, R. W., and Bollum, F. J. (1977).Proc. Natl. Acad. Sci. U.S.A. 74,734-738. Gregoire, K., Goldschneider, I., Barton, R. A., and Bollum, F. J. (1979).J. Immunol. 123, 1347-135 1. Hutton, J. J., and Bollum, F. J. (1977).Nucleic Acids Res. 4,457-460. Hutton, J. J., Coleman, M. S., Moffitt, S., Greenwood, M. F., Holland, P., Lampkin, B., Kisker, T., Krill, C., Kastelic, J. E., Valdez, L., and Bollum, F. J. (1982).Blood 60,

1267-1276.

Jani, P., Werbi, B., Greaves, M. F., Bevan, D., and Bollum, F. J. (1983).Leuk. Res. 7,17-

29.

Janossy, G., Bollum, F. J., Bradstock, K. F., and Ashley, J. (1980a).Blood 56,430-441. Janossy, G., Hoffbrand, A. V., Greaves, M. F., Ganeshaguru, K., Pain, C., Bradstock, K. F., Prentice, H. G., and Kay, H. E. M. (1980b).J . Haematol. 44,221-234. Janossy, G., Thomas, J. A., Bollum, F. J., Granger, S., Pizzolo, G., Bradstock, K. F., Wong, L., McMichael, A., Ganeshaguru, K., and H o a r a n d , A. V. (1980~). J . Immunol. 125,202-212. Jeske, D. J., Jarvis, J., Milstein, C., and Capra, J. D. (1984).J. Immunol. 133,1090-1092. Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1985).Cell 39,499-

509.

Kato, K., Goncalves, J. M., Houts, G. E., and Bollum, F. J. (1967).J . Biol. Chem. 242,

2780-2789.

Korsmeyer, S . J., Hieter, P. A., Ravetch, J. W., Poplack, D. G., Waldmann, T. A., and Leder, P. (1981).Proc. Natl. Acad. Sci. U.S.A. 78, 7096-7100. Kraft, A. S., and Anderson, W. B. (1983).Nature (London) 301,621-623. Landau, N. R., St. John, T. P., Weissman, I. L., Wolf, S . C., Silverstone, A. E., and Baltimore, D. (1984).Proc. Natl. Acad. Sci. U S A . 81,5836-5840. Lanham, G. R., Bollum, F. J., Williams, D. L., and Stass, S. A. (1984).Blood 64,318-320. Lanham, G. R., Melvin, S . L., and Stass, S . A. (1985).Am. J . Clin. Pathol. 83,366-370. Lanham, G. R., Stass, S. A,, and Bollum, F. J. (1986).Am. J. Clin. Pathol., in press. McCaffrey, R. A., Bell, R., Lillquist, A., Wright, G., and Baril, E. (1982).Ado. E x p . Med. B i d . 145,221-239. McCaErey, R. A., Smoler, D., and Baltimore, D. (1973).Proc. Natl. Acad. Sci. USA. 70,

521-525.

Marks, S. M., Baltimore, D., and McCaffrey, R. P. (1978).N . Engl.J.Med. 298,812-814. Michelson, A. M.,and Orkin, S. H. (1982).1. B i d . Chem. 257, 14773-14782. Murphy, S., and JaEe, E. (1984).N . Engl.1. Med. 311,1373-1374. Neidel, J. E.,Kuhn, L. J., andvanderbark, G. R. (1983).Proc. Natl. Acad. Sci. U S A . 80,

36-40.

Olsson, L. (1983).Cancer Metastasis Reo. 2, 153-163. Paietta, E.,Dutcher, J. P., and Wiernik, P. H. (1985).Blood 65, 107-114. Paine, P. L., and Feldherr, C. M. (1972).Exp. Cell Res. 74,81-98. Penit, C., and Chapeville, F. (1977).Btochem. Biophys. Res. Commun. 74,1096-1101. Penit, C., Jotereau, F., and Gelabert, M. J. (1985).J. Immunol. 134,2149-2154.

TERMINAL TRANSFERASE IN CELLS

61

Peterson, R. C., Cheung, L. C., Mattaliano, R. J., Chang, L. M. S., and Bollum, F. J. (1984). Proc. Natl. Acad. Sci. U S A . 81,4363-4367. Racklin, B., Bearman, R., Sheibani, K., Winberg, C., and Rappaport, H. (1983). Led. Res. 7,431-437. Sacchi, N., and Bollum, F. J. (1985).Serono Symp. 17,187-199. Sacchi, N., LeBien, T. V., Trost, S., Breviario, D., and Bollum, F. J. (1984).Cell. Immunol. 84,65-73. Sakano, H., Kurosawa, Y.,Weigert, M., and Tonegawa, S. (1981).Nature (London)290, 562-565. San Miguel, J. F., DeCastro, J. T., Matutes, E., Rodriguez, B., Polli, N., Zola, H., McMichael, A. J., Bollum, F. J., Thompson, D. S., Goldman, J. M., and Catovsky, D. (1985).J. Haematol. 59,297-309. Sarin, P., and Gallo, R. C. (1974).J. B i d . Chem. 249,8051-8053. Sasaki, R.,Bollum, F. J., and Goldschneider, I. (1980).J. Immunol. 125,2501-2503. Smith, L. J., Curtis, J. E., Messner, H. A., Senn, J. S., Furthmayr, H., and McCulloch, E. A. (1983).Blood 61,1138-1145. Srivastava, B. I. S., Khan, S. A., Minowada, J., and Freeman, A. (1978).Int. J. Cancer22, 4-9. Stass, S. A., Schumacher, H. R.,Keneklis, T. P., and Bollum, F. J. (1979).Am J . Clin. Pathol. 72,898-903. Stass, S . A., Dean, L., Peiper, S. C., and Bollum, F. J. (1982).Am. J. Clin. Pathol. 77, 174-176. Sugimoto, M., and Bollum, F. J. (1979).J . Immunol. 122,392-397. Thomas, J. A., Janossy, G., Eden, 0.B., and Bollum, F. J. (1982).Br. J . Cancer 45,709717. Vines, R. L., Coleman, M. S., and Hutton, J. J. (1980).Blood 56, 501-509. Walsh, D. A., and Krebs, E. G. (1973).In “The Enzymes” (P. D. Boyer, ed.), Vol. P. Academic Press, New York. Watson, M. E. E. (1984). Nucleic Acids Res. 12, 5145-5164.

This Page Intentionally Left Blank

MALIGNANT METAMORPHOSIS: DEVELOPMENTAL GENES AS CULPRITS FOR ONCOGENESIS IN Xip hopho rus Manfred Schwab The George Williams Hooper Foundation, School of Medicine, University of California. San Francisco. California 94143

I. Introduction

The term oncogenes refers to a class of genes that contribute as etiological factors to the development of tumors. It is firmly established now that certain viruses, most notably retroviruses, do contain oncogenes whose expression within the cells of a suitable host results in tumor development (for reviews, see Bishop, 1985; Varmus, 1984). All vertebrates, and at least many invertebrates as well, carry in their genome genes that are homologous to the oncogenes of retroviruses. These cellular homologs are referred to as proto-oncogenes (for reviews, see Varmus, 1984; Bishop, 1985). Experimental approaches have revealed a series of additional proto-oncogenes with partial similarity (see Schwab, 1985) or not homologous to retrovirus transforming genes (see Cooper, 1982; Weinberg, 1982). The development of cancer in animals and humans is often associated with alterations of the structure and the expression of proto-oncogenes. This process is generally referred to as “activation” and represents the metamorphosis of the proto-oncogene into the virulent cellular oncogene. It is a major tenet in contemporary cancer research that this activation is causally related to tumorigenesis. The generation of direct evidence for such a relation has proved to be a difficult task, however. Despite recent excitement about “cancer genes,” the idea that cellular genes might be etiological factors in tumorigenesis is rather old. As early as 1914 Boveri formulated his farsighted ideas concerning chromosomal control of cancer, and, perhaps more significant, not much later K. H. Bauer (1928) implicated somatic mutations in human cancer. During the early years of this century, the fish fanciers Loesslein (1912), H a h e r (1913), and particularly Gramsch (1913) 63 ADVANCES IN CANCER RESEARCH, VOL. 47

Copyright 0 1986 bv Academic Press. Inr All rights of reproduction in any form reserved.

64

MANFRED SCHWAB

called attention to a “cancer-like” growth in certain fish hybrids derived from species of Xiphophorus, which normally carry specific pigment cell spots. The fish breeders, apparently aware of genetic factors involved in this neoplastic growth in the hybrid fish, also noted that the nonspotted hybrid siblings were free of cancer. These observations were taken up and extended later by Myron Gordon (1927) in New York, Curt Kosswig (1927) in Muenster, and Georg Haeussler (1928) in Heidelberg. During the subsequent 30 years until the early 1960s Gordon (mainly) worked out both the genetic and cytological basis of the development of the pigment cell neoplasia (“melanoma”) in the hybrid fish, enabling us to explain the results of genetic experiments on the melanomas within the framework of Mendelian inheritance (Gordon, 1957,1959; for review, see Anders, 1967). Occurrence of genetic melanoma in Xiphophorus provides direct evidence that cellular genes normally having a function during the developmental differentiation program can be activated to direct formation of cancer. The genetic analyses have also revealed activity of pleiotropic loci with anti-oncogenic effects. Presence of these “anti-oncogenes” results either in a gene-dosage-dependent manner in reduced malignancy of the melanomas or even in complete abolishment of melanoma development. Although hypothetical schemes attempting to explain the basis for genetic tumors in Xiphophorus have been brought up (Comings, 1973; Ahuja and Anders, 1976; Anders et al., 1985),the genetic mechanisms leading to oncogenic activation of the cellular genes involved in melanoma remain completely enigmatic. The molecular identification of the genes that are in Xiphophorus normally directing development of pigment cell spots and whose abnormal expression appears to be the culprit for pigment cell cancer presents an exciting challenge for future experimentation. Hybrids between Xiphophorus species not only are characterized by their ability to develop pigment cell neoplasia but also show increased sensitivity, compared to the parental species, for carcinogenic induction of a broad spectrum of different tumor types (Schwab and Anders, 1981; Schwab et al., 1982). This indicates that the regulation of normal development and cellular differentiation of a number of cell types of different embryonic lineages is less stable in the hybrids than in the parental species. This discussion will critically review the current state of information available on tumorigenesis in Xiphophorus and review the strategies to which we owe the identification of genetic loci in malignancy: Mendelian segregation analysis and carcinogenesis studies.

ONCOGENES IN Xiphophorus

65

II. The Teleost Xiphophorus

In the light of a comprehensive review covering the biology of Xiphophorus (Kallman, 1975), only a short introduction shall be given here. The genus Xiphophorus combines 16 species according to the last revision by Rosen (1979).Rosen also combined the former genera PZatypoeciZus (platyfish) and Xiphophorus (swordtail) into the genus Xiphophorus. All species of the genus live in Central America, and the intraspecific populations are defined by both specific spot patterns and the river system from which they are derived (for overview, see Kallman, 1975). Under laboratory conditions all species can be mated to each other to yield, with few exceptions, fertile hybrids. Hybridization in nature has not been observed yet, and even in the laboratory efficient hybridization is usually achieved by artificial insemination. The various species exhibit distinct dermal spot patterns consisting of guanophores, pterinophores, or melanophores. These are due to the expression of genetic loci linked either to sex chromosomes (X, Y,W, Z; normally XX = female, XY = male, WZ = female, ZZ = male, WY female) or to autosomes. All species tested so far have 48 (2n) small chromosomes (Friedman and Gordon, 1934). The complexity of the genome of the species Xiphophorus maculatus, X . variatus, and X . helleri is low, with a size of approximately 4 x lo8 base pairs, a value that corresponds to a mean DNA content of approximately 1.20 pg (Schwab, 1982). This size represents approximately 20% of the mammalian genome and is thought to be the basic genome size of vertebrate evolution (Ohno and Atkin, 1966). Ill. Spot Patterns and Melanomas

As described by Gordon (1931),fishes of the various species exhibit basically two types of dermal melanophores (Fig. 1). One type is distributed more of less evenly over the body of the fish and does not exceed 100 pm in diameter (“micromelanophores”); the other is arranged in colonies to form specific spot patterns, which may be up to several millimeters in diameter (“macromelanophores”). Fish of all wild-type populations carry micromelanophores , and most of them also display macromelanophores. Although a large number of melanophore patterns have been described (Gordon, 1931; Kallman, 1975), most of them are not associated with neoplasia, and the major features of the system can be described employing few wild-type marker loci. The following discussion shall be restricted to seven selected loci, most of them present in the platyfish ( X . maculatus and X . variatus; see Table I).

66

MANFRED SCHWAB

FIG. 1. Dermal melanophores of X. maculatus carrying the X chromosome marked by the macromelanophore spot pattern locus “spotted” ( S p ) . Small arrowhead: micromelanophores; large arrowhead: macromelanophores. Scale bar is -500 pm.

TABLE 1 SELECTED PROTOTYPE MACROMELANOPHORE SPOTPATTERNSUSED FOR THE STUDY OF SPONTANEOUS AND CARCINOGEN-INDUCED MELANOMAIN XiphophorusiJ Macromelanophore locus Sd (spotted dorsal) S p (spotted) N (nigra)

Li (lineatus) Sr (stripe sided) Pu (punctatus) Db (dabbed)

a

Phenotype Fig. 2a Fig. 2a Patches on body side Fig. 7a Fig. 8b Spots on back Spots on body side

Melanoma in backcross hybrids

Sex chromosome

Species

Spontaneous Spontaneous Spontaneous

X X 2

X . maculatus X . maculatus X . maculatus

Inducibleb Inducible Difficult even to induce Not implicated in melanoma

X Y Y

X . uariatus X . maculatus X. variatus

?

X . helleri

For details see Cordon (1927) and Kallman (1975). B y N-methyl-N-nitrosourea (MNU) or by X rays.

ONCOGENES IN

A. SPOTPATTERN LOCIIN

THE

XiphOphOrUs

67

PLATYFISH

Members of the platyfish group exhibit the greatest variety of spot patterns, some of which are listed in Table I. Gordon (1957) recognized five dominant loci in natural populations of the platyfish (X. maculatus). Fish of the Rio Jamapa population of the platyfish, for instance, exhibit two macromelanophore patterns: one confined to the body side (“spotted”), the other to the dorsal fin (“spotted dorsal”). Both patterns are determined by X-chromosomal loci (Sp and Sd, respectively) that are expressed codominantly. Individual females may carry either Sp or Sd, or Sp plus Sd (Fig. 2a). The same principle applies for the other loci listed in Table I. In crossings the pattern phenotypes segregate according to Mendelian rules, showing that the Sd and Sp are located on the homologous X chromosomes. However, it is unclear whether these two loci are alleles, although lack of genetic recombination, which averages 1% between the pattern locus and the sex determining region for the XY and the WZ chromosome pair (Gordon, 1951a), would indicate an allelic relationship (for crossing-over rates in X. maculatus, see also Kallman, 1975). It is also possible that the number of fish looked at so far has not been sufficient to detect crossover between pattern loci. Linked to Sd on the X chromosome is a spot pattern locus, “dorsal red” (Dr) which directs development of a pterinophore pattern in the dorsal fin. This pattern is not present on fish carrying the Xsp chromosome instead of the XSd chromosome. A strain of X. maculatus has been bred that stably lacks the spotted dorsal phenotype (Anders et al., 1973a; Schwab and Scholl, 1981), but does exhibit the dorsal red pattern. In earlier publications the locus has been referred to as Sddel; in this discussion the term Dr’ shall be used throughout. The altered locus has been interpreted as resulting from deletion of Sd (Anders et al., 1973a), partly on the basis of cytogenetic analyses suggesting a lack of part of the X chromosome (Ahuja et al., 1979). Interestingly enough, platyfish carrying the Dr’ locus display a consistent polymorphism at a DNA sequence related to the c-src proto-oncogene as compared to their wild-type Sd counterparts (Vielkind and Dippel, 1985).The matter is somewhat complicated by the finding that both macromelanophores and melanoma can be induced by the carcinogen N-methyl-N-nitrosourea (MNU) and by X rays in backcross fish carrying the locus Dr‘ (Schwab and Scholl, 1981), which would also allow the interpretation that Sd is silent rather than deleted. Altogether, the basis for the lack of dermal macromelanophores in the Dr’ strain of X.

FIG.2. (a) Xiphophonrs mculatus from Rio Jamapa, Mexico. XX female carrying on one X chromosome the macromelanophore pattern locus “spotted dorsal” (Sd), which directs development of macromelanophores in the dorsal fin, and carrying on the other X chromosome the macromelanophore locus “spotted (Sp), which is responsible for spots on the body side. (b) X i p h o p h o m s helleri from Rio Lancetilla, Mexico. Male of a strain lacking the macromelanophore spot pattern “dabbed” (Db),which directs development of spots on the body side.

ONCOGENES IN

Xiphophorus

69

rnuculatus should be addressed in future molecular and cytogenetic analyses. B. SPOTPATTERN LOCIIN

THE

SWORDTAIL

Fish of the Rio Lancetilla population of the swordtail (X. helleri) exhibit in addition to micromelanophores and macromelanophore spot pattern “dabbed” (locus Db). This locus has not been implicated in melanoma. Because of the polygenic sex determining mechanism, with apparent lack of dominating sex factors, it is not known whether Db is linked to a sex chromosome. The population is polymorphic with respect to expression of Db, and a strain has been bred that stably lacks the macromelanophore pattern (Fig. 2b). This strain is usually employed as the genetic background for testing expressivity of macromelanophore loci derived from the platyfish (see Table I), and it has been also used as a recipient strain for gene transfer experiments (see later). It is not known, however, whether the Db present in the fish of the natural population is missing in the laboratory strain or is silent due to rigid control of expression. C. ONCOGENIC ACTIVATIONOF CELLULAR GENES THROUGH SELECTIVE CROSSINGS Animals of the F1 generation between the platyfish and the swordtail exhibit a highly increased number of macromelanophores, either on the body side or in the area of the dorsal fin, depending upon whether they carry, for instance, the S p or Sd of the platyfish. Hybrids carrying the XSd chromosome also show increased pterinophore coloration. Reciprocal crosses yield identical results. In the backcross (BC) F1 X swordtail, in accordance with the segregation of the macromelanophore locus, 50% of the individuals develop melanosis (Fig. 3) and 50%are free of cells of the macromelanophore lineage and resemble the swordtail phenotypically. Among the segregants with melanosis there are often, but not always, individuals that develop malignant overgrowth of pigment cells regarded as malignant melanoma (Fig. 4a displays a fish with an exceptionally large melanoma). The melanomas show invasiveness into the surrounding tissue (Fig. 5 ) and distant metastases have been found (Vielkind and Vielkind, 1982). Detailed histological examination has confirmed that these malignant melanomas of Xiphophorus are very similar to melanomas found in mouse or man (Grand et al., 1941; Grand and Cameron, 1948; Weissenfels et al., 1970; Vielkind et al., 1971).

70

MANFRED SCHWAB

X. macdafus Sd/Sd

F1 Sd/-

1-

BC

X helleri -/-

X. helleri -/-

vB1

FIG.3. Illustration of the breeding strategy for obtaining melanomas in backcross hybrids (BC) derived from X. maculatus and X. helleri. The locus Sd is used as an example; corresponding results are obtained with S p .

Melanomas in backcross hybrids homozygous for the autosomal recessive locus “albino” (a) are unpigmented, showing that the process of melanoma formation is independent from synthesis of melanin (Breider, 1938; Gordon, 1948; Fig. 4b). Homozygosity for a is also correlated with increased malignancy (Greenberg et a1., 1956): pigment cells of the amelanotic melanoma show a lower degree of differentiation and a higher rate of division than cells of the melanotic melanoma (Vielkind, 1976). In line with this, carcinogen-induced neuroblastoma (Schwab et aZ., 1979) is considerably more malignant in albinotic than in wild-type animals (G. Kollinger, personal communication). The mechanism for albinism is unclear, but aa animals are tyrosinase positive (Vielkind, 1976; Schwab, 1982). When the X chromosome carrying the Dr’ locus is introduced into X. helleri melanosis does not develop (Fig. 6a,b), yet pterinophore coloration is still increased. The formation of melanomas is reversible. When backcross hybrids carrying malignant melanomas are back-hybridized to the platyfish,

ONCOGENES IN

Xiphophorus

71

FIG.4. Backcross hybrids X. maculatus x helleri x helleri showing malignant melanomas associated with Sd. (a) Wild type; (b) homozygous for albino (aa).

the offspring resembles in its phenotype animals of the F1. Further back-hybridization to the platyfish eventually yields fish of normal phenotype. The primary area of the body susceptible for melanomas is determined genetically by the macromelanophore locus: Sd evokes spots as well as melanomas predominantly in the dorsal fin, and only secondary melanoma growth may invade the body side, whereas S p elicits spots and melanomas on the body side and does not affect the dorsal fin. The locus “nigra” (N) of X . maculatus (Table I) is another macromelanophore locus that directs melanoma formation when introduced into the swordtail. The X-chromosomal “lineatus” (Li)of X . variatus allows melanoma development in the background of the swordtail only when present in double dosage (Fig. 7). Single dosage evokes an increase of the number of macromelanophores, although in these animals the malignant phenotype has been induced by the tumor promoter 12-0-tetradecanoylphorboll3-acetate (TPA; Schwab, 1982).

FIG.5. (a, b) Sections through a melanoma corresponding to that shown in Fig. 4a, which resulted from abnormal expression of the macromelanophore locus Sd. Dark masses consist of melanoma cells, mostly melanocytes that invade the underlying muscular tissues.

ONCOGENES IN

Xiphophorus

73

FIG.6. Phenotype of fish carrying the locus Dr’. (a)X. rnaculatus (compare with wild belled x helleri (compare with wild type in Fig. 4a). type in Fig. Pa). (b) BC hybrid X. rnaculatus x

Certain intraspecific hybrids develop melanomas as well. For instance, malignant melanomas develop when X. maculatus from the Belize River is backcrossed to X. maculatus from Rio Jamapa (Kallman, 1975). As a general rule, both penetrance and expressivity, even of a specific macromelanophore locus, vary considerably in the various intra- and interspecific hybrid combinations, and there seems to be no basic difference in the reaction between the intraspecific hybrids of members of two geographically distant populations and interspecific hybrids (Gordon, 1951a). The remaining macromelanophore loci in Table I, Sr (Fig. 8) and Pu, do not give rise to spontaneous hybrid melanomas, but as discussed later, they may be triggered by mutagens to yield melanomas. Altogether it appears interesting, although not explainable at the present, that all spot patterns implicated in spontaneous hybrid melanomas are located on sex chromosomes. The genetic mechanism evoking development of malignant melanoma is not understood and it is unknown whether the genes in the

74

MANFRED SCHWAB

FIG.7. Effect of dosage of the macromelanophore locus “lineatus” ( L i ) on development of melanomas in BC hybrids. Note that in hybrids an increase of pigmentation is associated with a single dosage of Li,and melanoma is associated with double dosage. (a) X. uariatus, female homozygous for Li. (b) Single dosage: BC hybrid X. uariatus x helleri x helleri. (c) Double dosage: Fa hybrid.

melanoma cells are in the same configuration as in the germ line or have undergone somatic rearrangement or other alterations. Somatic alteration in development of hybrid melanoma has recently been implicated also by Wakamatsu (1980; see also Ozato and Wakamatsu, 1983). An important step toward analyzing gross rearrangements of the genome has been made by Morizot and Siciliano (1979, 1982) through establishing several linkage groups identifiable by isozyme polymorphisms. The establishment of linkage maps should be ex-

ONCOGENES IN

Xiphophorus

75

FIG.8. Macromelanophore pattern not associated with spontaneous hybrid melanoma. (a) X. maculatus male showing spot pattern “stripe sided” directed by the Ylinked locus Sr; the X chromosome carries in this individual the locus Dr’. (b) BC hybrid X. helleri x maculatus x helleri. The number of macromelanophores is increased as compared to the platyfish; however, this condition is not considered melanosis.

tended and refined through the anaiysis of restriction length polymorphisms on the level of DNA. Two conclusions may be drawn from the basic experiments discussed here. (1)Certain species of Xiphophorus carry genes that normally direct development of macromelanophores, but which when introduced into a suitable heterologous genetic environment allow formation of melanosis or malignant melanoma. (2) The genes directing development of macromelanophores or of melanoma remain intact in the germ line during the crossings. In other words, it appears that the same locus, e.g., Sp or Sd, directs either development of a spot, a benign melanosis, or a full-fledged malignant melanoma. IV. Genetic Loci Associated with Susceptibility to Carcinogens

It is a puzzling feature that as the result of interspecific crossings in Xiphophows only the normal development of macromelanophores appears to be disrupted, but not that of other cell types. There is evidence now that normal differentiation of cells derived from various

76

MANFRED SCHWAB

embryonal lineages is less stable in backcross hybrids than in the fish parental strains. The detection of such a relationship stems from experiments designed to determine whether development of tumors other than melanoma may be under direction of cellular genes. Because hybridization of species failed to yield nonmelanotic tumors, attempts have been made to induce tumors in specific genotypes with carcinogens. It was astonishing to discover that fish of wild-type strains resist developing tumors when exposed to the nitrosamide MNU or to X rays (Schwab and Anders, 1981). Experiments in which other chemical carcinogens such as benzo[a]pyrene, 3,4,9,10-dibenzpyrene, 3-methylcholanthrene7and benzanthracene were applied to platyfish also failed to yield melanomas or other tumors (Shelton et al., 1982). The general picture that has emerged suggests that (1)certain backcross hybrids are hypersensitive to MNU (Schwab et al., 1982) and X rays (Schwab and Anders, 1981) and respond upon treatment with development of a broad spectrum of tumors, and (2) at least X rays induce changes in the germ line that lead to spontaneous melanomas in hybrid constructions, the normal counterparts of which do not develop melanomas (Anders et al., 1973a).

A. SUSCEPTIBILITY OF HYBRID FISHTO CARCINOGENS In these studies an experimental strategy was employed that combines selective breeding with mutagen/carcinogen treatment. Each experimental protocol was constructed according to the same principle (Fig. 9). A dominantly expressed locus determining development of a dermal pigment cell pattern in species A, either a macromelanophore locus (in Fig. 9 the locus Li was chosen as example) or another melanophore, pterinophore, or guanophore locus, was introduced by crossing and backcrossing into the genetic background of another species (B). The pool of the backcross segregants of such a cross differs genotypically with respect to the marker locus and phenotypically in their spot pattern. The idea behind this strategy was that if both parental strains and backcross segregants are treated with mutagens, differences in susceptibility to mutagenesis and possible contributions of the marker locus or its chromosomally linked loci to carcinogenesis should emerge. Both MNU and X rays have been employed as the mutagenic/carcinogenic agent. The experiments have generally shown that nonhybrids were resistant, but that backcross hybrids responded to treatment by developing a broad spectrum of tumors (for a summarizing trend see Table 11).

ONCOGENES IN

x. variatus Li/Li

Fl

Xiphophorus

77

X hellm' -/-

X. helleri

1

BC

I

J

-/-

FIG.9. Illustration of the experimental strategy for detecting hybrid hypersensitivity and spot pattern marker associated with sensitivity for carcinogens. Genotype constructions resulting from crossing X.variatus with X. helleri are used as example.

These tumors included melanoma (Schwab and Scholl, 1981), a certain neuroblastic type of tumor, presumably neuroblastoma (Schwab et al., 1979), various types of carcinoma, fibrosarcoma, and rhabdomyosarcoma (Schwab et al., 1978; Schwab and Anders, 1981; Figs. TABLE I1 SENSITIVITY OF FISH OF WILD-TYPE STRAINS AND OF INTERSPECIES HYBRIDS TO CARCINOGENS' Fish

Number treatedb

Individuals with tumors

Wild types Fi Backcross

3500 1200 3300

0 0.6 7.1

N-Methyl-N-nitrosourea (MNU) or X rays were employed; the results were combined to show the general trend. For genotypes of fish and experimental details see Schwab et al. (1979). * Approximate number.

MANFRED SCHWAB

FIG.10. Retroocular tumor, presumably neuroblastoma, induced by N-methyl-N-nitrosourea (MNU) in BC hybrids. (a) Phenotype of BC hybrid X. oariatus x helleri x helleri showing the tumor. (b) Section through the tumor. (c) Histology of the tumor. Note typical rosette-like arrangement of tumor cells (arrow). (d) Electron microscopic appearance of tumor cells. Nuclei (N) are large and frequently bipartite with nuclear pockets (P). Giant mitochondria (M) and numerous vesicles (V) are seen. (Inset) Cilia showing a 9 + 0 pattern of microtubules are a conspicuous feature of the tumor cells.

10-12 show sections of MNU-induced neuroblastoma, fibrosarcoma, and epidermal carcinoma, respectively). Spontaneous tumors were not observed in these hybrids, except for melanomas in those backcross hybrids carrying macromelanophore loci, such as Sp or Sd. Even among the hybrids, susceptibility does not appear to be distributed at random. Hypersensitivity, i.e., high sensitivity for malignant tumors and for multiple tumors, was particularly observed in individuals derived from backcrossing X. variatus x helled hybrids with X. helleri (See Fig. 9). Animals of these hybrids showed high incidence for melanoma and neuroblastoma, especially

ONCOGENES IN X i p h o p h o r u s

79

FIG.11. Fibrosarcoma induced by MNU.(a) Phenotype of BC hybrid X. uariatus x hellen' x helleri (segregantwithout Li)showing fibrosarcoma. (b) Section through fibrosarcoma illustrating invasion of tumor cells into muscular tissue.

when carrying the X-chromosomal locus Li of X . uariatus. As discussed earlier, homozygosity for Li in hybrids involving the swordtail causes spontaneous melanoma (Fig. 7c). It would be interesting to learn (1)what genetic mechanism confers hypersensitivity, and (2) whether hypersensitivity for the two neuroectodermal tumors, melanoma and neuroblastoma, is coincidental or due to enhanced susceptibility of specific neuroectodermal stem cells for transformation. Differential sensitivity for carcinogens, particularly MNU, has also been described for other vertebrates. In Sprague-Dawley rats, for instance, a series of intravenous injections of MNU elicits myelogenous leukemia in high yield, whereas erythroleukemia is not evoked; conversely, 7,8,12-trirnethylbenz[a]anthracene elicits erythroleukemia, whereas myelogenous leukemia is not produced (Huggins et a,?., 1982). BuUN rats appear to be particularly sensitive to induction of mammary carcinomas by MNU (Gullino et al., 1975), and boxer dogs, which show high spontaneous tumor incidence (Cohen et al., 1974), are highly sensitive to MNU (Denlinger et al., 1978). The general picture emerging is that the process of carcinogenesis

80

MANFRED SCHWAB

FIG.12. Epidermal carcinoma induced by MNU. (a) Phenotype of BC hybrid X. helleri x helleri showing epidermal carcinoma. (b) Section through the

oadatus x

tumor showing densely packed, round to ovoid tumor cells.

in Xiphophorus can be experimentally dissected into two steps. The first step (achieved by hybridization of species) generates a sensitive genotype. The second step (treatment with a mutagen or carcinogen) seems to trigger tumor development. Hybrid sensitivity in Xiphophorus is further testimony to the multistep nature of tumorigenesis. Yet, the molecular mechanisms that might be involved are

ONCOGENES IN

Xiphophorus

81

obscure. What appears to be safe to say is that hybridization of Xiphophorus species obviously disposes certain cells of the progeny to undergo neoplastic transformation upon mutagenkarcinogen treatment. It appears particularly interesting, although not explainable at present, that the model tumor melanoma can be caused both by selective hybridizations and by carcinogen treatment.

B. HEREDITARY CHANGES CAUSING DEVELOPMENT OF MELANOMA Subsequent to X-irradiation, germ line alterations affecting expression of macromelanophore loci were obtained that evoke melanoma in hybrid combinations, the normal counterparts of which either are free of melanoma or have a different melanoma phenotype (for detailed discussion, see the overview by Anders et al., 1973a,b). When the Sr locus, which normally does not direct spontaneous hybrid melanoma, of the X-irradiated X . maculatus was introduced into X . helleri, the offspring unexpectedly developed highly malignant melanomas (Anders et al., 1973a,b). This altered Sr is generally referred to as Sr’. Genetic analyses showed that the alteration is inherited as a Mendelian trait. The locus Sd directs development of spots in the platyfish and melanoma in backcross hybrids in the dorsal fin. Following X-irradiation an altered Sd was obtained that allows development of spots and melanoma, both in the dorsal fin area and on the body side. The genetic nature of these alterations is not clear, but it appears possible that either the macromelanophore locus itself or regulatory sequences in cis position have been mutated. However, more complex changes such as chromosomal rearrangements that have altered the control of expression of the macromelanophore loci cannot be ruled out. The most provocative new combination of macromelanophore loci concerns an alteration that has created a chromosomal linkage of Dr’ locus of X. maculatus and the Li of X . uariatus (Anders et al., 1973a). In its new linkage group, the Li elicits in single dosage highly malignant melanomas when introduced into the test strain X . helleri. Expression of Li in the nonhybrid strain is balanced as in the case of the altered Sr. Other new combinations that do not result in melanoma include translocation of the Y-linked Sr next to the Dr‘ (a number of these nonproductive rearrangements occurred spontaneously; see Anders et al., 1973a; Kallman, 1975).

82

MANFRED SCHWAB

V. Anti-oncogenes

The cells of the melanoma are, like all vertebrate pigment cells including melanophores, pterinophores, and guanophores, derived from the neural crest (Humm and Young, 1956). Little data beyond this point have been presented to warrant much speculation about the differentiation and developmental relationships of the pigment cells in Xiphophorus. There exist, however, detailed studies in other vertebrates, including a number of teleosts (see Moore, 1974, and references therein), the lamprey (Newth, 1951), the chick (Dorris, 1938), and mice (Rawles, 1947). Referring to dermal chromatophores, a recent theory is that the three types derive from a common precursor, the unpigmented chromatoblast, capable of producing any of the three (Taylor and Bagnara, 1972; Bagnara et al., 1979). We assume that this is also true for Xiphophorus. Concerning the two types of melanophores, macromelanophore and micromelanophore, circumstantial evidence has been generated that differentiation of both types may be determined at the level of the chromatoblast. Macromelanophore differentiation, assumed to be determined as the result of a macromelanophore locus, appears to proceed via the stages of melanoblast and melanocyte. It was Gordon (1957,1959) who recognized that the development of the melanoma is a consequence of incomplete differentiation of the macromelanophores, a view that was recently confirmed (Vielkind, 1976; Vielkind and Vielkind, 1982; Esaka et al., 1981):the spots in the platyfish consist of macromelanophores, while the melanoma in the backcross hybrids is predominantly composed of incompletely differentiated cells of the macromelanophore lineage, melanocytes and melanoblasts (Fig. 13a and b). Macromelanophores are the terminally differentiated pigment effector cells. Eventually they become senile and degenerate, and the cellular debris is apparently removed by true macrophages (Gordon and Lansing, 1943). The phenotype of both the spontaneous and the carcinogen-induced melanoma may vary considerably among different inter- and intraspecific hybrids. At least two pleiotropic loci have so far been identified to control the phenotype of the melanoma: “differentiation”

FIG. 13. Ultrastructure of melanoma cells. (a) Terminally differentiated macromelanophore possessing large masses of fully melanized melanosomes and a pleomorphic nucleus (n). (b) Incompletely differentiated pigment cell (late melanoblast to early melanocyte) characterized by numerous incompletely melanized melanosomes (arrowheads), many vesicles, and a round to ovoid nucleus (n).

ONCOGENES I N

Xiphophorus

83

a4

MANFRED SCHWAB

(Ddffl and “golden” (g).Presence of Diffis associated with less malignant phenotype, homozygosity for g completely abolishes melanoma development. In other words, either of the two loci has anti-oncogenic activity and, therefore, may be referred to as “anti-oncogenes.” A. DIFFERENTIATION In backcross animals X. maculatus x X . helleri, FI, x X. helleri carrying Sd or Sp two melanoma phenotypes are detectable. Melanoma is benign in most of the animals, and malignant in a minor number of individuals (Fig. 14). In some crossing experiments animals of the two phenotypes occurred in nearly equal number, and it has been proposed that the appearance of the two phenotypes is the result of segregation of a “differentiation gene” (Diff, transmitted from the platyfish and involved in determining the melanoma phenotype (Vielkind, 1976).According to this idea, homozygosity for Dqjas, for instance, present in the nonhybrid strain carrying Sd or Sp is associated with the development of terminally differentiated macromelanophores. A single dosage of Dvf as present in the F1 X. macuZatus x helleri and in 50% of the Sd or Sp backcross hybrids F1 x helleri is associated with failure of terminal differentiation, leading to an increase of the ratio of incompletely differentiated versus terminally differentiated pigment cells. Lack of Dqfin the remaining 50% of the Sd or Sp backcross hybrids is associated with the majority of the pigment cells remaining in an incompletely differentiated state, predominantly as melanocytes or melanoblasts, a condition that eventually leads to massive increase in the number of pigment cells and to the formation of a melanoma. Circumstantial support for this view comes from biochemical studies. An isozyme locus for esterase-1 (Est-1) encoding a presumably polymorphic, electrophoretically identifiable product (Est-l) has been found to cosegregate often with the benign phenotype (Siciliano and Wright, 1976; Ahuja et al., 1980) and is thought to be loosely linked to D v f . The Est-l band is detectable in electrophoretic separations of tissues from animals carrying spots or benign melanoma, and appears to be absent in most analyses of animals showing malignant melanoma. It seems that similar mechanisms operate in the MNU-induced melanoma in the Dr’ fish, which normally do not develop macromelanophores (Section 111,A). A significant number of backcross segregants in which benign melanoma has been induced (Fig. 15) showed Est-1, while most of the segregants in which malignant melanoma was induced (Fig. 16) were found to lack Est-1 (Fig. 17; Schwab and

FIG.14. Association of the alleged locus “differentiation” (Dqfl in BC hybrids with the phenotype of the meIanoma directed by the locus Sd (a,b) and Sp (c, d). (a, c) Diff/-. (b, d) -I-. A marker locus, esteruse-1 (Est-I),cosegregateswith DqJ Consequently, animals in (a) and (c) are Est-1 positive; those in (b) and (d) are negative (see also Fig. 15).

86

MANFRED SCHWAB

FIG.15. Benign variant of melanoma induced by MNU in Dr' BC segregants X. maculatus x helleri x helleri. (a)Phenotype of BC segregants showing melanoma cells.

(b) The benign variant of MNU-induced melanoma consists of terminally differentiated highly dendritic macromelanophores (arrows). Note size difference to micromelanophores in the surrounding. (c) Section showing that melanoma cells, mostly terminally differentiated macromelanophores (arrows), do not invade the muscular tissue

(MI.

Scholl, 1981).For the Sr, Li, and Pu loci a putative accompanying DVf locus has not been identified yet. Crossing procedures that lead to melanoma in case of the Sd and Sp generally fail to produce melanoma in case of Sr, Li,and Pu. Altogether, the role of the Difflocus, if there is any, is not clear at present and should be addressed in future experiments. One should bear in mind, however, that in the absence of any objective biochemical markers it is difficult, if not impossible, to assess the degree of

ONCOGENES IN

XiphOphOrUS

87

FIG.16. Malignant variant of melanoma induced by MNU in Dr' segregants. (a) Phenotype of fish showing melanoma. (b) Section through melanoma showing that the tumor cells infiltrate the muscular tissue. (c) Histology of melanoma. Incompletely differentiated pigment cells are abundant. The turnover rate of cells in this type of tumor is high. Cellular debris, to a large extent melanin, is phagocytized and released through the epidermis (arrow).

malignancy that melanosis in a given individual has. The development of such biochemical markers, perhaps in the form of monoclonal antibodies, should have priority in the future, because it is an essential step for further classifying the various types of melanomas observed and for further analyses of genetic factors possibly involved in modifying the degree of malignancy that melanoma assumes in a given individual.

88

MANFRED SCHWAB

FIG. 17. Association of Est-I marker locus with the degree of malignancy of the melanoma induced by MNU in Dr’ BC hybrids. Esterase patterns are resolved by polyacrylamide gel electrophoresis and visualized by substrate staining. Arrows point to the position of the Est-I band. -, Chromosome ofX. maculatus; - - -,chromosome of X. helleri.

B. GOLDEN The effect of the autosomal recessive locus g can be best studied in

X.maculatus. Platyfish homozygous for g usually lack dermal melanophores, except for rare individuals (Fig. 18; compare with wild-type platyfish in Fig. 2a), but in contrast to the albino phenotype, possess retinal pigment cells. A feasible hypothesis for the lack of dermal melanophores is that differentiation of their precursor cells is blocked at an early stage. Guanophores and pterinophores are produced at an apparently normal level. Consequently, gg backcross hybrids carrying either the Sp or Sd fail to yield melanoma (Fig. 18b). Their wild-type counterparts do develop melanosis or melanoma (compare Fig. 18b with Fig. 4a). Recent studies indicate that the apparent differentiation block can to some extent be overcome by treatment of the animals with tumor promoters, such as TPA (Schwab, 1981). Macromelanophores determined by the various macromelanophore loci differ: while the gg condition usually abolishes the differentiation of macromelanophores determined by Sd or Sp, macromelanophores deter-

89

FIG. 18. Effect of the autosomal recessive locus “golden” ( g ) on the macromelanophore phenotype. (a)X. maculatus SdlSd; g/g. Note that dermal melanophoresare missing (compare with Fig. 2a). (b) BC-segregant X. maculatus x helleri x helleri carrying Sd (Sdl-; glg). Note lack of melanoma (compare with wild type in Fig. 4a). Further crossing with wild-type X. helleri yields melanoma phenotype.

mined by other loci such as Li differentiate to a normal degree, although micromelanophores are still missing. VI. Molecular Approaches for Identifying Tumor Genes

Two molecular approaches for identification of tumor genes that may presently be thought of are (1)DNA-mediated gene transfer and (2) the study of proto-oncogenes discovered in other vertebrates. A. DNA-MEDIATED GENETRANSFER DNA-mediated gene transfer aiming at identifying dominant transforming genes can be pursued at two levels, using as the recipient for the donor DNA either the intact organism including its embryonal

90

MANFRED SCHWAB

developmental stages or tissue culture cells. However, because several earlier reports dealing with gene transfer, in which morphological characters were claimed to be transferred to intact organisms such as ducks, plants, and others (for discussion, see Schwab et aZ., 1976), were never reproducible, the modern definition of successful gene transfer should include (1)the molecular demonstration of the gene transfer and (2) undisputable proof by objective means that the new phenotype is due to the transfer of a specific gene. Rather promising results in this direction have been recently reported (Vielkind et al., 1982; Vielkind and Vielkind, 1982).Total DNA from fish that carry several macromelanophore loci was microinjected according to a previously reported procedure (Schwab et al., 1976) into the neural crest region of fish embryos of a strain of X. helleri that normally does not exhibit macromelanophores (Fig. 2b). Subsequently the development of abnormal pigment cells resembling macromelanophores was observed. This approach could turn out to be a powerful tool for the isolation and characterization of cellular genes involved in development of macromelanophores and possibly in that of melanoma. The recent construction of genomic libraries (M. Schwab and J. Vielkind, unpublished) of fish carrying macromelanophore loci should help to answer this question and should make the gene transfer an even more defined task. DNA transfection experiments utilizing cultured mouse NIH/3T3 cells as indicators for tumor genes (Weinberg, 1982; Cooper, 1982) have failed so far to generate conclusive results that would indicate the presence of dominant tranforming genes (M. Schwab, unpublished). The establishment of continuous cell lines from a hybrid melanoma (Wakamatsu, 1981; Wakamatsu et d . , 1984) will provide a source for larger amounts of DNA for use in transfection experiments and should be a basis to address this issue again.

B. STUDY OF PHOTO-ONCOGENES DISCOVERED IN OTHER VERTEBRATES Studies during the past decade in various vertebrates have led to molecular identification of evolutionary conserved cellular genes that are thought to contribute to tumorigenesis (for reviews, see Bishop, 1985; Varmus, 1984). These genes are generally referred to as protooncogenes. A cellular gene is being classified as a proto-oncogene (1) if it is the cellular homolog of a retrovirus transforming gene, or (2) if its activated version is capable of inducing tumorigenic conversion or contributes,together with another factor to tumorigenic conversion of

ONCOGENES IN

Xiphophorus

91

suitable cultured target cells (for further discussion, see Land et al., 1983). Although the etiological role of proto-oncogenes in tumorigenesis is not yet definitively established, there is strong circumstantial evidence for involvement of at least a few such genes in specific forms of cancer. For instance, translocations with chromosomal breakpoints in the vicinity of the c-myc gene typify a human B-cell neoplasm, Burkitt’s lymphoma (for review, see Klein, 1983); and abnormally high expression consequent to amplification of the gene N-myc is frequent in human neuroblastoma cells (for overview, see Schwab et aZ., 1984; Schwab, 1985). To date, we know that at least most of the cellular homolog of retroviral transforming genes with the possible exception of c-mos are, as expected, detectable in the genome of the various strains of Xiphophorus (Vielkind and Dippel, 1985; also unpublished data of M. Schwab and J. Vielkind). These include c-src, c-yes, c-erb, c-myb, cmyc, c-Harvey-rus, c-Kirsten-ras, and c-abl. Representative patterns for cellular sequences in X-helleri homologous to the viral oncogenes v-src and v-myc are shown in Fig. 19. A multitude of cellular sequences is detectable with these two probes, and further molecular studies employing clones isolated from a genomic library of X. helleri indicate that at least three c-src and four to five c-myc loci are present (M. Schwab, unpublished). Their further characterization is in progress. At least c-src seems to be expressed at significant level in embryos of different developmental stages and in melanoma (Barnekow et al., 1982; Schartl and Barnekow, 1982), and other homologs are expressed in different organs and tissues of Xiphophorus as well (M. Schwab, unpublished). Yet no indication has emerged so far that any one of these genes tested might be causally related to tumorigenesis in Xiphophorus. Nevertheless, the study of cellular homologs to retrovirus transforming genes in Xiphophorus might provide interesting clues concerning evolution of this class of genes. VII. Are Genetic Tumors in Xiphophorus a Peculiarity of Nature?

Do cellular genes also play a role in neoplasia in other multicellular organisms? A large body of information has been generated by Mendelian strategies both through experimentation in plant and animal settings (a selection is presented in Table 111) and through pedigree studies in humans (for an excellent account of genetics of human cancer, see Mulvihill et al., 1977). The picture emerging makes it difficult to avoid the conclusion that cellular genes, whose normal

92

MANFRED SCHWAB

a

b

FIG.19. Cellular hoinologs in X . helleri to retroviral transforming genes v-src and v-myc. Total genomic DNA was digested with the restriction endonuclease EcoRI and analyzed by DNA blotting procedures.

function in the cell remains to be determined, play a causative role in the etiology of neoplasia in a wide variety of plant and animal species as well as in man. Aside from Xiphophorus two experimental systems appear particularly amenable for studying genomic contributions to neoplasia, the fruit fly Drosophila (Gateff, 1978, 1982) and the tobacco plant Nicotiana (Braun, 1969; Smith, 1972), because turnorigenesis occurs in a highly reproducible pattern. Among all the systems, macromelanophore genes in Xiphophorus that can trigger development of melanoma should be a particularly suitable tool for analyzing the genetic basis of tumorigenesis. First, these genes display a simple highly reproducible Mendelian inheritance mostly linked to sex chromosomes, and second, their presence can easily be detected due to their dominant expression behavior resulting in development of clearly visible dermal macromelanophore spot patterns.

ONCOGENES IN

93

Xiphophorus

TABLE 111 SELECTION OF ANIMALSAND PLANTS SHOWING TUMORS OF PRESUMABLY GENETICORIGIN Organism

References

Animals Within species Sinclair swine Mouse

Millikan et al. (1974) Many reports, for review see Heston (1982) For review see Heston (1982) Sonstegard (1977) Crew and Koller (1936) Bridges (1916); Gateff (1978)

Rat carp Duck Drosophila In species hybrids Xiphophorus

This review

Plants Within species Melilotus alba (sweet clover) Pteridium aquilinum (Bracken fern prothalli) Picea glauca (white spruce) Pharbitis nil (Japanese morning glory) Thea sinesis (tea) Sorghum bicolor (sorghum) Pisum satioum (pea) In species hybrids Brassica Datura Prunus Bryophyllurn Lilium Lycopersicon Nicotiana

Littau and Black (1952) Steeves et al. (1955) White and Millington (1954) Takenaka and Yoneda (1963) Shaw and Burnett (1968) Lin and Ross (1969) Nuttal and Lyall (1964) Caspary (1873); Kehr (1965) Rappoport et 01. (1950) Sludskaya (1953) Resende (1957) Emsweller et al. (1962) Martin (1966) Kostoff (1930); Naef (1958); Ahuja (1962)

VIII. Summary

Neoplastic growth is a widespread developmental aberration among multicellular organisms ranging from primitive avertebrates such as coelenterates (Brien, 1961) and annelids (Cooper, 1969) to man. A major goal of the studies concerning neoplasia has been to obtain insight into its cellular and molecular basis, and it has been suggested as early as the beginning of this century that cellular genes are paramount in the etiology of neoplasia. Early support for this idea has been gained by Mendelian strategies applied to a number of experimental systems, such as Xiphophorus.

94

MANFRED SCHWAB

During the last years molecular biology has provided some insight into the genetic mechanisms that might be involved in neoplasia in higher vertebrates, and it has been possible to identify proto-oncogenes as candidates for the agents directing cellular transformation and/or maintainance of the neoplastic state of the cell. The high degree of evolutionary conservation of the proto-oncogenes points to basic functions that these genes normally might have for the cell and at the same time indicates that crucial steps associated with tumorigenesis might take similar pathways in different classes of vertebrates. There are now four main lines of molecular evidence that relate cellular genes to neoplasia: (1)insertional mutagenesis or chromosomal rearrangement that juxtaposes an exogenous or endogenous genetic element which augments gene expression next to a cellular gene, resulting in elevated expression (for review, see Varmus, 1982; Klein, 1983); (2) gene amplification that results in an increase of the copy number and an elevated expression of a particular gene and, to date, has been found in tumors of humans and mice (for overviews, see Schwab et al., 1984; Schwab, 1985; Alitalo and Schwab, 1986); (3) structural alteration of a cellular gene itself which results in the synthesis of an altered protein (Weinberg, 1982; Cooper, 1982); and (4) generation of fusion genes as a result of gene translocation with possibly altered biological activities (for review, see Adams, 1985). It remains to be addressed in future experiments which genetic mechanisms are operative in development of tumors of genetic origin in Xiphophorus.

ACKNOWLEDGMENT Parts of the original work of the author referred to in this article were supported by the Deutsche Forschungsgemeinschafk through a Heisenberg Fellowship.

REFERENCES Adams, J. M. (1985). Nature (London)315, 542-543. Ahuja, M. R. (1962). Mol. Gen. Genet. 103, 176-184. Ahuja, M. R., and Anders, F. (1976) Prog. E x p . Tumor Res. 20,380-397. Ahuja, M. R., Lepper, K., and Anders, F. (1979). Experientia 35,28-29. Ahuja, M. R., Schwab, M., and Anders, F. (1980).J . Hered. 71,403-407. Alitalo, K.,and Schwab, M. (1986).Ado. Cancer Res. (this volume). Anders, A., Anders, F., and Klinke, K. (1973a). In “Genetics and Mutagenesis of Fish” (J. H. Schroeder, ed.). pp. 33-52. Springer-Verlag, Berlin and New York. Anders, A,, Anders, F., and Klinke, K. (1973b).In “Genetics and Mutagenesis of Fish” (J. H. Schroeder, ed.), pp. 53-63. Springer-Verlag, Berlin and New York. Anders, F. (1967).Erperientla 23, 1-10.

ONCOGENES IN

XiphOphO?US

95

Anders, F., Schartl, M., Bamekow, A., and Anders, A. (1984).Ado. Cancer Res. 42,191275. Bagnara, J. T., Matsumoto, J., Ferris, W., Frost, S., Turner, W., Tchen, T., and Taylor, J. (1979). Science 203,410-415. Barnekow, A., Schartl, M., Anders, F., and Bauer, H. (1982).Cancer Res. 42,2429-2433. Bauer, K. H. (1928). “Mutationstheorie der Geschwu1stentstehung.- Uebergang von Koerperzellen in Geschwulstzellen.” Springer-Verlag, Berlin and New York. Bishop, M. (1985). Trends Genet. 1,245-249. Bouck, N., and diMayorca, G. (1976). Nature (London) 264,722-727. Boveri, T. (1914). “Zur Frage der Entstehung maligner Tumoren.” Fischer, Jena. Braun, A. C. (1969).“Understanding the Cancer Problem.” Columbia Univ. Press, New York. Breider, H. (1938).Z. Wiss. Zool. 152, 784-828. Bridges, C. B. (1916). Genetics 1, 1-52. Brien, P. (1961). BulE. Biol. Fr. Belg. 95, 301-363. Cohen, D., Reif, J. S., Brodey, R. S., and Keiser, H. (1974).Cancer Res. 34,2859-2862. Comings, D. E. (1973). Proc. Natl. Acad. Sci. U S A . 70,3324-3328. Cooper, E. L. (1969). Natl. Cancer lnst. Monogr. 31,665-669. Cooper, G. M. (1982). Science 218,801-817. Crew, F. A. E., and Koller, P. (1936). Proc. R. Soc. Edinburgh 56,210-220. Denlinger, R., Koestner, A., and Swenberg, J. A. (1978). Cancer Res. 38, 1711-1717. Dorris, F. (1938). Wilhelm Roux’ Arch. Entwicklungsmech. Org. 138, 323-334. Esaka, T., Asada, M., Wakamatsu, Y., and Ozato, K. (1981).J. Exp. Zool. 215, 133-142. Friedman, B., and Gordon, M. (1934).Am. Nut. 58,446-455. Gateff, E. (1978). Science 200, 1448-1459. Gateff, E. (1982).Adu. Cancer Res. 37,33-69. Gordon, M. (1927). Genetics 12,253-283. Gordon, M. (1931).Am. J . Cancer 15, 732-787. Gordon, M. (1948).Ann. N. Y. Acad. Sci. 4,216-268. Gordon, M. (1951a).Growth 10,153-219. Gordon, M. (1951b). Cancer Res. 11,676-686. Gordon, M. (1957).Ann. N. Y. Acad. Sci. 71, 1213-1222. Gordon, M. (1959). In “Pigment Cell Biology” (M. Gordon, ed.), pp. 215-239. Academic Press, New York. Gordon, M., and Lansing, W. (1943).J . Morphol. 73(2), 231-245. Gramsch, E. (1913). Wochenschr. Aquar. Terrarienkd. 10,97. Grand, C. G., and Cameron, G. (1948). Spec. Publ. N. Y. Acad. Sci. 4, 171-176. Grand, C. G., Gordon, M., and Cameron, G. (1941). Cancer Res. 1,660-666. Greenberg, S . S., Kopac, M. J., and Cordon, M. (1956).Ann. N. Y. Acad. Sci. 67(4), 55122. Gullino, P. M., Pettigrew, H. M., and Grantham, F. H. (1975).J . Natl. Cancer. lnst. (U.S.) 54,401-409. Haeussler, G. (1928). Klin. Wochenschr. 7,1561-1562. Haffner, K. (1913). BZ. Aquar. Temurienkd. 24,533-535. Heston, W. E. (1982).In “Cancer-A Comprehensive Treatise” (F. F. Becker, ed.), Vol. 1, pp. 47-71. Plenum, New York. Huggins, C. B., Grand, L., and Keda, N. (1982).Proc. Natl. Acad. Sci. U S A . 79,54115414. Humm, D. C., and Young, R. S. (1956). Zoologica (N.Y.)41, 1-10,

96

MANFRED SCHWAB

Kallman, K. D. (1975). Handb. Genet. 4, 81-132. Klein, G. (1983). Cell 32, 311-315. Kosswig, C. (1927). 2.Indukt. Abstamm.-Vererbungsl. 44,253. Kostoff, D. (1930).Zentralbl. Bakteriol., Parasitenkd., Infektionskr. Hyg., Abt. 2, Naturwiss.: Allg., Landwirtsch. Tech. Mikrobiol. 81,244-260. Land, H., Parada, L., and Weinberg, R. A. (1983). Science 222, 771-778. Lin, P. S., and Ross, J. G. (1969).]. Hered. 60, 183-185. Littau, V. C., and Black, L. M. (1952).A m . ] . Bot. 39,191-194. Loesslein, F. (1912). BZ. Aquar.-Terran’enkd. (London) [N. S.] 23,346-347. Martin, F. W. (1966).Ann Bot. 30,701-709. Millikan, L. E., Boylon, J. L., Hock, R.R.,and Manning, P. J. (1974).]. Inuest. Dermatol. 62,20-30. Moore, W. S. (1974).]. Hered. 65, 326-330. Morizot, D. C., and Siciliano, M. J. (1979). Genetics 93,947-956. Morizot, D. C., and Siciliano, M. J. (1982). Biochem, Genet. 20, 505-518. Mulvihill, J. J., Miller, R. W., and Fraumeni, J. F. eds. (1977). “Genetics of Human Cancer.” Raven Press, New York. Naef, U. (1958). Growth 22, 167-180. Newth, D. R. (1951).J . E x p . Biol. 28,248-260. Nuttal, V. W., and Lyall, L. H. (1964).J . Hered. 55, 184-186. Ohno, S., and Atkin, N. B. (1966). Chromosoma 18,455-466. Ozato, K., and Wakamatsu, Y. (1983). Differentiation 24, 181-190. Rawles, M. E. (1947). Physiol. 2001.20, 248-266. Rosen, D. (1979). Bull. Am. Mus. Nut. Hist. 162, 267-376. Schartl, M., and Barnekow, A. (1982). Differentiation 23, 109-114. Schwab, M. (1981).In “Carcinogenesis and Biological Effects of Tumor Promoters” (E. Hecker, ed.), pp. 417-426. Raven Press, New York. Schwab, M. (1982). Mol. Gen. Genet. 188,410-417. Schwab, M. (1985). Trends Genet. 1,271-275. Schwab, M., and Anders, A. (1981). In “Neoplasma: Comparative Pathology of Growth in Animals, Plants and Man” (H. E. Kaiser, ed.), pp. 451-459. Williams & Wilkins, Baltimore, Maryland. Schwab, M., and Scholl, E. (1981). Diflerentiation 19, 77-83. Schwab, M., Vielkind, J., and Anders, F. (1976).Mol. Gen. Genet. 34, 151-158. Schwab, M., Abdo, S., Ahuja, M. R., Kollinger, Anders, A., Anders, F., and Frese, K. (1978).2. Krebsforsch. 91,301-315. Schwab, M., Kollinger, G., Haas, J., Ahuja, M. R.,Abdo, S., Anders, A., and Anders, F. (1979). Cancer Res. 39,519-526. Schwab, M., Abdo, S.,and Kollinger, G. (1982).In “Symposium: Carcinogenic Polynuclear Aromatic Hydrocarbons in the Marine Environment” (N. L. Richardson and B. L. Jackson, eds.), pp. 212-232. Environ. Res. Lab., U.S. Environ. Prot. Agency, Gulf Breeze, Florida. Schwab, M., Alitalo, K., Varmus, H., and Bishop, J. (1984). In “Cancer Cells 2: Oncogenes and Viral Genes” (G. F. vandewoude, A. J. Levine, W. C. Topp, and J. D. Watson, eds.), pp. 215-291. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Shelton, L. S., Bellamy, M. L., and Humm, D. G. (1982).In “Symposium: Carcinogenic Polynuclear Aromatic Hydrocarbons in the Marine Environment” (N. L. Richard-

ONCOGENES IN

XiphOphOWS

97

son and B. L. Jackson, eds.), pp. 233-243. Environ. Res. Lab., U.S. Environ. Prot. Agency, Gulf Breeze, Florida. Siciliano, M. J., and Wright, A. (1976). Prog. Exp. Tumor Res. 20,398-411. Sludskaya, L. A. (1953). Tr. Prikl, Bot., Genet. Sel. 30,59-64. Smith, H. H. (1972). Prog. Exp. Tumor Res. 15,138-164. Sonstegard, R. (1977).Ann. N. Y. Acad. Sci. 298,261-269. Steeves, T. A., Sussex, I. M., and Partenen, C. R. (1955).Am. J . Bot. 42,232-245. Taylor, J. D., and Bagnara, J. T. (1972).Am. Zool. 12,43-62. Varmus, H. E. (1982). Science 216,812-820. Varmus, H. E. (1984).Annu. Reu. Genet. 18,553-612. Vielkind, U. (1976).J. Exp. Zool. 196, 197-204. Vielkind, J. and Dippel, E. (1985). Can. J . Genet. Cytol. 26, 607-614. Vielkind, J., and Vielkind, U. (1982).Can. J . Genet. Cytol. 24, 133-149. Vielkind, J., Haas-Andela, H., Vielkind, U., and Anders, F. (1982).Mol. Gen. Genet. 185, 379-389. Vielkind, J., Vielkind, U.,and Anders, F. (1971).Cancer Res. 31,868-875. Wakamatiu, Y. (1980). Deu. Growth Differ. 22, 731-740. Wakamatsu, Y. (1981).Cancer Res. 41.679-680. Wakamatsu, Y., Oikawa, A,, Obika, M., Hirobe, T., and Ozato, K. (1984).Dev. Growth Differ. 26,503-513. Weinberg, R. A. (198.2). Adu. Cancer Res. 36, 149-164. Weissenfels, N., Schafer, D., and Bretthauer, R. (1970).Virchows Arch. B 5, 144-158.

This Page Intentionally Left Blank

ONCOGENES IN RETROVIRUSES AND CELLS: BIOCHEMISTRY AND MOLECULAR GENETICS Klaus Bister and Hans W. Jansen Otto-Wa~urg-Laboratorium.Max-Planck-lnstitut fur molekulare Genetik, 0-loo0 Berlin 33 (Dahlem). Federal Republic of Germany

1. Introduction

A.

SCOPE

Oncogenes were first identified as transforming (or oncogenic) principles of retroviruses that induce rapid tumor formation in infected animals and morphological transformation of animal cells in tissue culture. Cellular oncogenes were then discovered by the demonstration of close nucleotide sequence homology between retroviral oncogenes and their apparent progenitors, normal cellular genes of vertebrates. Some of these and additional cellular oncogenes have also been identified by structural and functional investigations of mutated or rearranged genes in animal and human tumor cells. Cellular oncogenes have been highly conserved in metazoan evolution, and their normal nonmutated alleles appear to fulfill essential physiological functions in cell growth and development. Hence, biochemical and genetic analyses of oncogenes will possibly not only provide an understanding of some underlying principles of the malignant state, but also contribute to our knowledge about structure, function, and regulation of essential eukaryotic genes. In this chapter we will focus specifically on the biochemistry and the molecular genetics of a particular set of oncogenes originally identified as transforming genes of avian acute leukemia viruses. This group of retroviruses played a key role in establishing the concept that normal cells contain multiple genes with the potential to become dominant oncogenic determinants, like upon transduction in retroviral genomes. Six different cell-derived oncogenes were identified in this virus group alone, among them the myc oncogene, by now one of the most intensively studied genes implicated in viral and nonviral carcinogenesis. Furthermore, several basic features and some unique 99 ADVANCES IN CANCER RESEARCH,VOL.47

Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

100

KLAUS BISTER AND HANS W. JANSEN

variations of oncogene structure and expression were first diagnosed in this virus group, such as the peculiar structure of hybrid oncogenes composed of cell-derived and viral coding elements, or the intriguing assembly of two different cell-derived oncogenes on a single retroviral genome. Also, a crucial progress in the search for physiological functions of cellular alleles of oncogenes was achieved by the discovery of close sequence homology between an oncogene of an avian acute leukemia virus and a human gene encoding a growth factor receptor, Hence, the oncogenes of avian acute leukemia viruses will serve here as a model case to review the current knowledge about structure and function of eukaryotic genes involved in malignant cell transformation. OF B. SYNOPSIS

THE

AVIANLEUKEMIA VIRUSSYSTEM

Avian leukemia viruses are members of a taxonomic subfamily termed Oncovirinae (RNA tumor viruses) within the family of Retroviridae (retroviruses) (Fenner, 1976; Vogt, 1977). Specifically, they belong to the avian leukosis-sarcoma group of type C RNA tumor viruses which frequently cause sarcomas, leukemias, and a broad spectrum of visceral tumors in infected fowl (Beard, 1963, 1980; Purchase and Burmester, 1972; Hanafusa, 1977). The leukemia viruses were first distinguished from the sarcoma viruses, in particular from Rous sarcoma virus (RSV), solely on the basis of their unique oncogenic properties. These include the induction of slow lymphoid leukoses by the lymphatic leukemia viruses and of rapid erythroid and myeloid leukemias by acute leukemia viruses such as avian myeloblastosis virus (AMV), avian erythroblastosis virus (AEV), or avian myelocytomatosis virus (MC29). The oncogenic spectra of some of these acute-transforming viruses are rather broad, including the induction of carcinomas, endotheliomas, and also sarcomas, in addition to the characteristic leukemic disorders (Beard, 1963, 1980; Purchase and Burmester, 1972; Graf and Beug, 1978). The original classification of the avian leukemia viruses was exclusively based on their biological and pathogenic properties, They were divided into two main classes: the weakly oncogenic lymphatic leukemia (or leukosis) viruses and the highly oncogenic acute leukemia viruses (Hanafusa, 1977; Graf and Beug, 1978). The molecular basis for the profound differences in pathogenicity between these two classes has now emerged from the biochemical analyses of viral genomes, and the explanation is, at hindsight, remarkably simple: highly oncogenic viruses contain oncogenes, and weakly oncogenic ones do

ONCOGENES IN RETROVIRUSES AND CELLS

101

not (Bister and Duesberg, 1980,1982; Bishop et al., 1980; Bishop and Varmus, 1982; Graf and Stehelin, 1982; Bishop, 1983; Bister, 1984, 1986).

I . Lymphatic Leukemia Viruses (Leukosis Viruses) Viruses of this class are in general weakly oncogenic and induce neoplastic disease only after a long latent period of several months or more. The most common neoplasm induced is B-cell lymphoma originating in the bursa and metastasizing to other visceral organs such as liver, kidney, and spleen. Erythroblastosis, osteopetrosis, nephroblastomas, and occasional fibrosarcomas have also been observed in birds infected with certain strains of lymphatic leukemia viruses (Beard, 1963,1980; Purchase and Burmester, 1972).Transformation of cells in tissue culture has not been reported for any of these viruses. A very characteristic genetic property of lymphatic leukemia viruses is their replication competence. Their genomic RNAs carry full complements of the essential virion genes, gag, pol, and envybut lack any oncogene sequences (see also Section 11,A).Due to their ability to replicate and to complement defective viruses in replicative functions, lymphatic leukemia viruses are also found in infectious stocks of replicationdefective highly oncogenic retroviruses. They are then commonly called associated or helper viruses. Although oncogenes have not been identified in the genomes of lymphatic leukemia viruses, they still appear to be involved in carcinogenesis induced by these viruses. The activation of cellular oncogenes by insertion of proviral DNA in the vicinity of chromosomal oncogene loci has been observed in numerous cases of tumors induced by lymphatic leukemia viruses (see Section 11,B). 2. Acute Leukemia Viruses Viruses belonging to this class induce acute and often fatal neoplastic disease in the infected animal, usually within a few weeks, even within days. Based on the predominant response of the hemopoietic system of the infected host, basically three subgroups of avian acute leukemia viruses could be distinguished: agents inducing predominantly erythroblastosis, myeloblastosis, or myelocytomatosis, respectively (Beard, 1963,1980; Purchase and Bunnester, 1972; Beard et al., 1973). These apparent preferences in the interaction with different hemopoietic compartments is reflected in the names of the prototype strains AEV, AMV, and MC29 (Table I). A similar classification was established on the basis of extensive characterizations of hemopoietic target cells in bone marrow cultures transformed in vitro by these

102

KLAUS BISTER AND HANS W.JANSEN TABLE I LEUICEMIA VIRUSES

AVIAN ACUTE

Subgroup and virus strain"

Predominant neoplasm(s) induced in vivo

Viral oncogene(s)*

MC29 subgroup Strain MC29 Strain CMII Strain OK10 Strain MH2

v-myc v-myc v-myc v-myc, v-mil

AEV subgroup Strain AEV-R Strain AEV-H

v-erbB, v-erbA v-erbB

AMV subgroup Strain AMV Strain E26

v-myb v-myb, v-ets

I

M yelocytomatosis, endothelioma, carcinoma

1

Erythroblastosis, fibrosarcoma

M yeloblastosis Erythroblastosis, myeloblastosis

Cell type(s) transformed in vitro

I

M yeloid, epithelioid, fibroblastic

1

Erythroid, fibroblastic Myeloid Erythroid, myeloid

a Abbreviations: MC29, myelocytomatosis virus 29; MH2, Mill Hill virus 2; AEV, avian erythroblastosis virus; AMV, avian myeloblastosis virus; E26, erythroblastosis virus 26. b For a general definition of oncogenes, see Section 11. For the molecular properties of the genes listed here, see Sections 111-VI.

viruses (Grafand Beug, 1978; Beug et al., 1979; Moscovici and Gazzolo, 1982). However, the broad spectra of tumors induced by these virus strains in addition to the predominant neoplasms (Table I) show considerable overlap, and several uncertainties remained about the identity of the preferred target cells in uivo and in uitro.An unambiguous biochemical definition of the MC29, AEV, and AMV subgroups of the avian leukosis-sarcoma group is now based on the identification of homologous oncogenes in viruses belonging to the same subgroup (Table I; see Sections 111-VI). Strain MC29, isolated in Bulgaria (Ivanov et al., 1964), strain CMII, discovered in Germany (Loliger, 1964),strain OK10, found in Finland (Oker-Blom et al., 1978), and strain MH2, an isolate from England (Begg, 1927), all induce broad overlapping spectra of malignant growths, including myelocytomatosis, endotheliomas, liver and kidney carcinomas, and occasionally sarcomas and erythroblastosis in chickens and other fowl (Beard, 1963,1980; Purchase and Burmester, 1972).A remarkably high incidence of liver carcinomas was observed in birds infected with MH2 (Alexander et al., 1979), and this virulent strain appears to be particularly oncogenic (Linial, 1982). In tissue culture, viruses of the MC29 subgroup transform fibroblasts of avian

ONCOGENES I N RETROVIRUSES AND CELLS

103

or mammalian origin (Langlois et al., 1967; Bister et al., 1977; Graf et al., 1977,1979; Hu et al., 1978; Quade, 1979)and also cells of epithelioid morphology (Graf and Beug, 1978; Zeller et al., 1980; Bister, 1984).Bone marrow cells transformed in vitro by these viruses closely resemble the immature myeloid cells observed in in viuo tumorigenesis (Langlois et al., 1969; Graf and Beug, 1978; Beug et al., 1979).The immediate in vitro target cells appear to include fully differentiated macrophages (Durban and Boettiger, 1981a,b). The apparently identical strains AEV-R and AEV-ES4, isolated in Denmark (Engelbreth-Holm and Rothe-Meyer, 1935), and strain AEV-H, a recent isolate from Japan (Hihara et al., 1983), have similar pathogenic properties. The typical disease induced in chickens is acute erythroid leukemia, characterized by the appearance of large numbers of primitive red blood cells, mainly erythroblasts, in the peripheral blood (Beard, 1963, 1980; Purchase and Burmester, 1972; Hihara et al., 1983). Sarcomas are also frequently induced, especially when the virus is injected intramuscularly. In vitro transformation of chicken bone marrow cells yields proliferating erythroblasts, indistinguishable from the leukemic cells observed in vivo (Graf and Beug, 1978; Beug et al., 1979). AEV strains also transform cultured fibroblasts, even of mammalian origin (Ishizaki and Shimizu, 1970; Graf et al., 1976; Quade, 1979; Hihara et al., 1983). Strain AMV, isolated in the United States (Hall et al., 1941),induces a profound myeloblastosis in infected chickens, with exceedingly high numbers of immature myeloid cells, mainly myeloblasts, in the peripheral blood (Beard, 1963, 1980; Purchase and Burmester, 1972; Beard et al., 1973; Moscovici, 1975). No other disease is commonly observed, indicating a rather narrow oncogenic spectrum for AMV. The hemopoietic cells transformed in vitro resemble immature myeloid cells, similar to the leukemic cells observed in vivo (Baluda dn Goetz, 1961; Moscovici, 1975; Graf and Beug, 1978; Beug et al., 1979; Moscovici and Gazzolo, 1982).Interestingly, fully differentiated macrophages were shown to be susceptible to in uitro transformation by AMV (Durban and Boettiger, 1981a,b). Strain E26, isolated in Bulgaria (Ivanov et al., 1962), induces a typical erythroblastosis in infected fowl (Sotirov, 1981; Moscovici et al., 1981),but low numbers of leukemic cells of the myeloid lineage are also observed (Radke et al., 1982). Under selective culture conditions, E26 induces in uitro transformation of either erythroid or myeloid cells in bone marrow cultures (Radke et al., 1982). Fibroblasts are generally not transformed by either AMV or E26; only fibroblastic cells derived from quails have been reported to be susceptible to in uitro transformation by E26

104

KLAUS BISTER AND HANS W. JANSEN

(Graf et al., 1979). Despite their pronounced differences in pathogenicity, AMV and E26 contain related oncogenes and are grouped together solely on this basis (Table I). Another strain of avian acute leukemia viruses, S13, has been described by Stubbs and Furth (1935). Interestingly, initial molecular analyses have recently revealed that S 13 presumably contains a novel oncogene and hence does not belong to any of the three subgroups defined in Table I (Benedict et al., 1985; Hayman et al., 1985). A distinctive genetic feature of all avian acute leukemia viruses is their replication defectiveness. In order to replicate, they are dependent on the presence of replication-competent helper viruses. Replication defectiveness of the transforming component of acute leukemia virus stocks was demonstrated by the isolation of in vitro transformed cell clones that did not release any infectious virus (Ishizaki et al., 1971; Bister et al., 1977; Graf et al., 1977, 1979; Hu et al., 1978; Hayman et al., 1979a,b; Duesberg et al., 1980). Such cells, harboring only the replication-defective transforming virus, are called nonproducer cells. By using such cells in genetic complementation experiments it was first demonstrated for MC29 and MH2 that they are defective in all three replicative genes, gag, pol, and env (Bister and Vogt, 1978; Hu and Vogt, 1979). Biochemical analyses of the genome structures of acute leukemia viruses then revealed that the defectiveness is due to the loss of complete or partial complements of replicative genes. In place of the deleted replicative genes, oncogenes have been inserted into the genomes of these highly oncogenic viruses (Table I; see Sections II-VI).

II. Definition of Oncogenes

The concept that initiation, maintenance, and progession of cancerous growth is based on genetic alterations in the neoplastic cell (Temin, 1974, 1984; Cairns, 1975, 1981; Klein, 1981; Varmus, 1984) received dramatic support by the discovery of genes, first in retroviruses and then in animal and human cells, that have the potential to act as dominant oncogenic determinants. A summary of the general experimental strategies for the identification and molecular definition of such genes, commonly called oncogenes, and a conceptional definition of oncogenes emerging from a compilation of their basic properties will serve as an appropriate background for the detailed description of the oncogenes discovered in avian acute leukemia viruses (Sections III-VI).

105

ONCOGENES I N RETROVIRUSES AND CELLS

5’ LTR

3’ LTR

--- WEAKLY ONCOGENIC RETROVIRUS

H I G H L Y ONCOGENIC RETROVIRUS

CELLULAR LOCUS

c-onc

FIG.1. Retroviral oncogenes (v-onc) are transduced mutant alleles of cellular oncogenes (c-onc). In a typical example, the diagram shows the structural relationship of proviral DNA from a highly oncogenic defective retrovirus to both proviral DNA from a weakly oncogenic nondefective retrovirus and to DNA from a cellular c-onc locus. Exons of the c-onc gene are shown as large boxes; domains transduced are indicated by hatching. gag, pol, eno: Essential virion genes; A: partial gene complement; LTR: long terminal repeat of proviral DNA; R, U3, U5: sequences corresponding to terminal repeat sequences, to unique 3’ sequences, and to unique 5‘ sequences, respectively, of viral RNA. Broken lines indicate cellular DNA sequences flanking integrated proviral DNAs or the c-onc locus.

A. RETROVIRAL (v-onc) AND CELLULAR (c-onc) ALLELESOF ONCOGENES Nondefective weakly oncogenic retroviruses carry on their genomes three genes essential for their replication: gag, encoding the virion core proteins: pol, directing synthesis of the RNA-dependent DNA polymerase (reverse transcriptase); and e m , specifying virion envelope glycoproteins (Fig. 1).The capped 5’ and the polyadenylated 3’ termini of viral genomic RNA contain repeated (R) and unique ( U 5 and U3, respectively) regulatory sequences. After reverse transcription of viral RNA and integration of viral DNA into the host chromosome, the structure U3-R-U5 is found repeated at both ends of proviral DNA. This long terminal repeat (LTR; F&*.l) contains nucleotide sequences typical for eukaryotic transcriptional control elements. The primary protein product of the gag gene is synthesized by translation of genome-sized mRNA, physically indistinguishable from genomic RNA. The precursor protein of the reverse transcriptase encoded by gag and po2 is synthesized by translation of a mRNA species in which the gag termination codon is presumably bypassed by ribosomal frameshifting (for out-of-frame gag and pol

106

KLAUS BISTER AND HANS W. JANSEN

genes) or in-frame amber suppression (for in-frame gag and pol genes). The primary enu gene product is generated by translation of a spliced subgenomic enu mRNA which contains a leader sequence from the 5' end of viral RNA (Coffin, 1982; Varmus and Swanstrom, 1982; Schwartz et al., 1983; Yoshinaka et al., 1985; Jacks and Varmus, 1985). The first evidence that highly oncogenic retroviruses contain genetic information other than the genes and control elements essential for retroviral replication was obtained in the course of pioneering genetic and biochemical analyses of avian Rous sarcoma virus (RSV) (Vogt, 1977; Hanafusa, 1977; Duesberg, 1980). By classical genetic and biochemical approaches, a gene, termed src, was unequivocally defined as the transforming principle of RSV. The src gene, the very first oncogene to be recognized, is inserted between the enu gene and the 3' terminus of RSV RNA. It was shown to be essential and sufficient for the initiation and maintenance of cell transformation, but to be dispensable for retroviral replication. RSV is the only known highly oncogenic retrovirus carrying all genes essential for replication and, in addition, an oncogene. All other highly oncogenic retroviruses are defective for replication and were found to contain transformationspecific sequences inserted into their genomes at the expense of replication-specific genes (Fig. 1). The search for the origin of transformation-specific sequences in retroviral genomes led to the striking discovery that normal cells contain genes that are closely related in their nucleotide sequence to retroviral transforming genes (Stehelin et aZ., 1976; Bishop, 1981). Based on available evidence, it appears almost certain that highly oncogenic retroviruses arose by recombination between replicationcompetent, weakly oncogenic retroviruses and the apparent cellular progenitors of retroviral oncogenes (Fig. 1). Hence, retroviral oncogenes, termed v-onc genes, represent transduced mutant alleles of cellular oncogenes, termed c-onc genes. Cellular c-onc genes represent typical eukaryotic split genes with coding sequences interrupted by multiple intervening sequences (Fig. 1). In all cases, retroviral transduction of cellular oncogenes leads to incorporation of only partial complements of the c-onc gene into the genome of the transducing retrovirus. In particular, the terminal transcriptional control regions are always removed and replaced by retroviral control elements. Hence, the v-onc alleles in retroviral genomes represent truncated forms of the c-onc alleles in cellular chromosomes (Fig. l),although the integrity of the protein coding region is not always affected by the transduction. Further structural distinctions between viral and cellular onc alleles include the removal of intervening sequences from c-

ONCOGENES I N RETROVIRUSES AND CELLS

107

onc genes upon transduction by retroviral genomes, and the appearance of scattered point mutations in the mutant v-onc alleles (compare Sections III-VII). A catalog of all cell-derived genes identified so far in highly oncogenic retroviral isolates from various species is shown in Table 11. Complete nucleotide sequences for nearly all of them have been determined. Based on predicted amino acid sequences of gene products, families of related genes, such as the src family and the rus family, have been recognized. Some of these genes have been found in several independent retroviral isolates of the same or of different species. The existence of these multiple independent transductions of specific cellular genes in highly oncogenic retroviruses is one of the strongest indications for their oncogenic potential. However, for most of the transduced genes a classical genetic definition of oncogenic function is not yet available. In most cases, justification for their classification as dominant oncogenic determinants rests exclusively on the biochemical definition of a protein-coding nucleotide sequence in the genomes of highly oncogenic retroviruses that is not related to virion genes but closely related to a cellular gene of the host (Fig. 1).The benefits and drawbacks of such a definition will be discussed in a later part of this section.

B. ACTIVATIONOF CELLULAR ONCOGENES BY MUTATIONAND REARRANGEMENT Cellular oncogenes have been recognized independently of retroviral transduction by direct experimental search for genetic alterations in tumor cells. The genetic alterations of known or presumed cellular oncogenes implicated in tumorigenesis include (1)functionally relevant mutations within the coding sequences, (2) transcriptional activation by insertion mutagenesis, (3)chromosomal translocation, and (4) gene amplification. Many genes found to be affected by somatic mutations or rearrangements in tumor cell DNA proved to be c-onc alleles of retroviral transforming genes. The overlaps between the groups of oncogenes identified by retroviral transduction or by somatic mutational events support the belief that the number of cellular genes with oncogenic potential is fairly limited. This opinion is also based on the observation of multiple transduction of the same oncogene in different retroviral isolates and on the recognition of oncogene families (Table 11; Weinberg, 1982a). Cellular oncogenes activated by mutations in their nucleotide sequence were identified in the course of gene transfer experiments using transfection of tumor cell DNA into cultured cells of the NIH/

108

KLAUS BISTER AND HANS W. JANSEN

TABLE I1 GENESTRANSDUCED IN HIGHLY ONCOCENIC RETROVIRUSES

Gene"

Species of originb

Isolation of dominant oncogenic allelec

+ +

Multiple transductiond

mos abl

Chicken Chickenlcat Chicken Cat Chicken Chicken Chickenlmouse Cat Cat Mouse Mouse, cat

Ha-raslbas Ki-ras

Ratlmouse Rat

+

myc mvb

Chicken Chicken

+ +

+ +

fos re1 sish ski erbA

Mouse Turkey Monkey, cat Chicken Chicken Chicken Mouse Cat

+ + + +

+ +

src fpslfes yes*

fsr

ros erbBf millraf fmsg

kit

ets

fox actin

+ +

+ + +

+ + + + +

+ + + + + + +

0 Names of cognate genes transduced from different species are separated by a slash. Genes for which close (ras family), close or intermediate (src Family, src through uhl), distant (myc and myb), or no (fos through actin) relationship between predicted amino acid sequences of gene products has been reported are grouped together. Selected references to nucleotide sequences of transduced or chromosomal alleles: src (Schwartz et al., 1983; Takeya and Hanafusa, 1983),fpslfes (Shibuya and Hanafusa, 1982; Hampe et al., 1982; Huang et al., 1985; Roebroek et al., 1985), yes (Kitamura et al., 1982),fgr (Naharro et al., 1984; Nishizawa et al., 1986),ros (Neckameyer and Wang, 1985),erbB (Yamamotoet al., 1983b; Privalsky et al., 1984),millraf (Sutrave et al., 1984a; Kan et ul., 1984; Galibert et al., 1984; Mark and Rapp, 1984; Jansen and Bister, 1985; Bonner et al., 1985, 1986),fms (Hampe et al., 1984; Coussens et al., 1?86), kit (Besmer et ul., 1986), mos (Van Beveren et al., 1981a,b;Watson et ul., 1982),ah1 (Reddy et ul., 1983b; Wang et al., 1984a),Ha-raslbas (Dhar et al., 1982; Capon et al., 1983a; Reddy et ul., 1985),Ki-rus (Tsuchida et ul., 1982; Shimizu et al., 1983a; McGrath et al., 1983; Capon et al., 1983b), myc (Alitalo et al., 1983a;Watson et al., 1983a,b; Reddy et al., 1983a; Battey et ul., 1983; Colby et al., 1983; Watt et al., 1983a,b; Bernard et al., 1983; Shih et al., 1984), myb (Rushlow et al., 1982; Klempnauer et a!., 1982; Nunn et ul., 1984),fos (Van Beveren et al., 1983, 1984; van Straaten et al., 1983), re1 (Stephens et ul., 1983; Wilhelmsen et ul., 1984). sis (Devare et al., 1983; Chiu et al., 1984;Josephs et al., 1984),ski (Van Beveren

ONCOGENES IN RETROVIRUSES AND CELLS

109

3T3 mouse fibroblast line (Weinberg, 198213; Cooper, 1982). Morphological transformation of the recipient cells is taken as an indication for the transfer of a dominant mutant allele of a cellular oncogene present in the DNA of the donor cell. The majority of oncogenes identified in this manner in the DNA of a variety of human and animal primary tumors and established cell lines proved to be mutant alleles of the ras gene family, including c-Ha-rusl and c-Ki-ras2, which are cellular alleles of retroviral transforming genes (Table 11), and the related N-ras gene (Parada et al., 1982; Der et al., 1982; Santos et al., 1982; Pulciani et al., 1982; McCoy et al., 1983; Shimizu et al., 198313; Taparowsky et al., 1983). Structural comparisons between the mutant ras alleles and their normal counterparts revealed that the active M S oncogenes (including the transduced viral alleles) contain nucleotide substitutions in at least one of three defined codons, leading to concomitant amino acid changes in the ras gene protein products (Tabin et al., 1982; Reddy et al., 1982; Taparowsky et al., 1982, 1983; Santos et al., 1983; Capon et al., 1983a,b; Shimizu et al., 1983a). Other genes identified by transfection of tumor DNAs include the B-lym and T-

et al., 1985),erbA (Debuire et al., 1984),ets (Nunn et al., 1983),fox (Van Beveren et al., 1984), actin (Naharro et al., 1984). b Species of origin are separated by a slash when different names have been assigned to cognate genes; when the same name is used for cognate genes, species are separated by a comma. c In most cases, potential to act as dominant oncogenic determinant has been demonstrated only for the transduced mutant alleles of these genes based on genetic and biochemical analyses of viral genomes. In somes cases, oncogenic potential has been demonstrated for mutated or rearranged cellulfr alleles isolated from tumor cells or obtained by in oitro manipulation of cloned oncogene DNA (see text). d A + indicates that the gene has independently been transduced at least in two different retroviral isolates from the same or from different species. For a complete list of virus strains, compare Coffin et nl. (1981), Weiss et al. (1982), and Varmus (1984). Although the chicken yes and felinefgr genes are closely related, they probably are not cognate genes, since human yes and fgr genes represent related but distinct loci (Tronick et ol., 1985; Nishizawa et al., 1986). f The human homolog of erbB was identified as the gene encoding the epidermal growth factor (EGF) receptor (Downward et al., 1984; Ullrich et al., 1984). In addition, the gene products of all members of the src gene family show noticeable sequence relationship with the human insulin receptor (Ullrich et al., 1985)and with mammalian CAMP-dependent protein kinase (Barker and Dayhoff, 1982). 1 The fms protooncogene product is closely related (or identical) to the receptor for the mononuclear phagocyte growth factor, CSF-1 (Sherr et al., 1985). h The human homolog of sis was identified as the gene encoding the platelet-derived growth factor (PDGF) (Doolittle et al., 1983; Waterfield et al., 1983).

110

KLAUS BISTER AND HANS W. JANSEN

lym genes obtained from B-cell and T-cell lymphomas, respectively (Cooper and Neiman, 1981; Diamond et al., 1983; Goubin et al., 1983; Lane et al., 1984), the erbB-related neu oncogene obtained from rat neuro/glioblastomas (Schechter et al., 1984; Bargmann et al., 1986; Hung et al., 1986), and the met oncogene which was isolated from chemical carcinogen-treated human osteosarcoma cells and which maps to a chromosomal locus that is linked with the human hereditary disease cystic fibrosis (Cooper et al., 1984; Dean et al., 1985; White et al., 1985). It is noted that the authenticity and the significance of the B-lym genes have recently been doubted (Rogers, 1986; Cooper et al., 1986). Transcriptional activation of cellular oncogenes was originally proposed as a mechanism by which lymphatic leukemia viruses that lack v-onc genes (Fig. 1)induce B-cell lymphomas in chickens (Hayward et al., 1981; Payne et al., 1982). In many of these clonally derived tumors proviral insertions were found in the vicinity of the c-myc oncogene and it is assumed that the strong promoter or enhancer elements present in the viral LTR sequences are involved in the observed transcriptional up-regulation of the c-myc gene. Many murine T-cell lymphomas also contain proviral insertions in the vicinity of the c-myc locus (Corcoran et al., 1984; Steffen, 1984; Selten et al., 1984; Li et al., 1984). Other known c-onc genes were found to be activated by proviral insertion mutagenesis: the c-erbB gene in cases of chicken erythroblastosis (Fung et al., 1983), the c-myb gene in plasmacytoid lymphosarcomas of mice (Mushinski et al., 1983), the c-mos gene in mouse plasmacytomas (Rechavi et al., 1982; Cohen et al., 1983), and the c-ra. gene in transformed mouse fibroblasts (Muller and Muller, 1984). Other genes possibly involved in tumorigenesis are the int-1 and int-2 loci identified in mammary carcinomas as common targets for insertion mutagenesis by mouse mammary tumor viruses (Nusse and Varmus, 1982; Peters et al., 1983; Fung et al., 1985; van Ooyen et al., 1985), or the pim-1 locus found as a preferred proviral integration site in murine leukemia virus-induced T-cell lymphomas (Cuypers et al., 1984; Selten et al., 1985). Specific chromosomal translocations are frequently and consistently observed as karyotypic abnormalities in many tumors, especially in particular types of leukemia and lymphoma (Klein, 1981; Rowley, 1983, 1985; Yunis, 1983). An intriguing link between chromosomal abnormalities and possible oncogene activation emerged from the discovery that known c-onc genes are involved in specific translocations. In mouse plasmacytomas and human Burkitt’s lymphoma the c-myc oncogene is translocated into one of the immunoglobulin loci apparently leading to transcriptional activation or deregulation of the onco-

ONCOGENES IN RETROVIRUSES AND CELLS

111

gene (Klein, 1983; Perry, 1983; Leder et al., 1983; Croce and Klein, 1985; Vogt et al., 1985). Another example is the specific reciprocal translocation between chromosomes 9 and 22 generating the characteristic Philadelphia (Phl) chromosome commonly observed in chronic myelogenous leukemia in man (Rowley, 1983, 1985). In the course of this translocation the c-abl oncogene is moved to the Phl chromosome, resulting in augmented transcription and possibly also local rearrangement and amplification (see below) of the oncogene (de Klein et al., 1982; Heisterkamp et al., 1983; Collins et al., 1984). Amplification of cellular oncogenes and concomitant augmented expression has been observed in a variety of tumors. Amplified oncogenes have been identified on homogeneously staining regions or double minute chromosomes, the karyotypic markers of gene amplification (Alitalo et al., 198313; Schwab et al., 1983a). The human c-myc oncogene was found to be amplified in primary tumor cells or cell lines derived from promyelocytic leukemias, colon or lung carcinomas, and glioblastomas (Collins and Groudine, 1982; Dalla Favera et al., 1982a; Nowell et al., 1983; Alitalo et al., 1983b; Little et al., 1983; Yokota et al., 1986; Trent et al., 1986). The N-myc gene, a putative oncogene with limited homology to c-myc, was identified as an amplified gene in human neuroblastomas, retinoblastomas, and small cell lung cancer (Schwab et al., 1983b, 1984a, 1985; Kohl et al., 1983, 1986; Lee et al., 1984; Yancopoulos et al., 1985; Nau et al., 1986). Correlation between advanced stage of disease and amplification of N-myc suggests that amplification of activated oncogenes may contribute to tumor progression (Brodeur et al., 1984; Schwab et al., 1984b). Other oncogenes found amplified in specific human leukemia- and carcinoma-derived cells include c-abl (Collins and Groudine, 1983), c-myb (Alitalo et al., 1984; Pelicci et al., 1984), c-erbB (Lin et al., 1984; Merlin0 et al., 1984; Ullrich et al., 1984), the erbB- (and new) related c-erbB-2 gene (Semba et al., 1985; King et ul., 1985;Yamamoto et al., 1986), c-Ki-ras2 (McCoy et al., 1983), and the c-myc-related L-myc gene (Nau et al., 1985). C. BASICPROPERTIES OF ONCOGENES The basic definition of an oncogene (Table 111; Vogt et al., 1985) emerges from a compilation of the common properties of experimentally studied genes implicated in carcinogenesis. They are eukaryotic protein-encoding genes that are well preserved in evolution and that appear to fulfill essential functions in the normal cell. To be further qualified as an oncogene, however, requires the unequivocal demonstration of the most outstanding functional property of this class of

112

KLAUS BISTER AND HANS W. JANSEN

TABLE 111 BASICDEFINITION OF AN

ONCOGENE"

An oncogene (1) Is a eukaryotic gene (2) Codes for a protein (3) Is preserved in evolution and presumably fulfills an essential physiological function in the normal cell (4) Has the potential to become a dominant oncogenic determinant a

From Vogt et al. (1985).

genes: the potential to act as a dominant oncogenic determinant. Any gene for which the occurrence as a dominant oncogenic mutant allele has been documented would be called an oncogene by the definition given in Table 111. The normal nononcogenic alleles of such genes are often designated as protooncogenes, and the mutant alleles as active oncogenes (Bishop, 1981; Bister and Duesberg, 1982; Duesberg, 1983; Bister, 1984, 1986; Temin, 1984; Varmus, 1984). Since protooncogenes are expressed in normal cells and hence are active genes, the term active oncogene refers to activation of oncogenic function. The transition from a normal allele with physiological function to a mutant allele with oncogenic function can be achieved in the course of totally different events: retroviral transduction or somatic mutation and rearrangement (see above), or in vitro manipulation of the cloned normal allele (Blair et al., 1981; DeFeo et al., 1981; Chang et al., 1982; Miller et al., 1984). Oncogenic function of the mutant allele is generally demonstrated by the reintroduction of the active oncogene into normal cells, resulting in their transformation. Experimental reintroduction is done by retroviral infection or DNA transfection (see above). Most of the genes which have been transduced in highly oncogenic retroviruses are oncogenes according to the given definition (Tables I1 and 111). Although classical genetic definition using conditional and nonconditional mutants for oncogenic function is available for only a few of these genes, notably for the STC gene of RSV, there is strong supportive genetic evidence, such as multiple independent transductions of the same gene, for their oncogenicity (Table 11). Nevertheless, a rigorous definition of the nucleotide sequences essential and sufficient for cell transformation has not been provided for many of these genes, especially not for those which are expressed as hybrid genes including viral coding sequences (compare Sections III-VI). For some of the biochemically defined transduced genes, the autonomous function as a dominant oncogenic allele has not been documented at all. These are the genes that were only found in retroviral genomes which already harbor a known autonomous oncogene (Bister, 1986)

ONCOGENES I N RETROVIRUSES AND CELLS

113

(Table 11): erbA is only found together with erbB (Section V), ets together with myb (Section VI),fox together withfos in the genome of FBR murine osteosarcoma virus (Van Beveren et ul., 1984), and actin only together with f g r in the genome of Gardner-Rasheed feline sarcoma virus (Naharro et ul., 1984). It is apparent that the mere biochemical definition of a cell-derived nucleotide sequence in a retroviral genome is not sufficient justification to call the gene of origin an oncogene. For erbA and ets, there is at least circumstantial evidence that they may modulate or enhance oncogenicity of the companion oncogene, erbB or myb, respectively (Section V and VI). Hence, they will be called oncogenes in this review, keeping in mind that this is a tentative assignment. Among the genes which have been defined by the recognition of mutations and rearrangements in tumor cell DNA, the ones with documented transforming activity upon introduction into cultured cells clearly represent oncogenes. In particular for the genes of the rus family, this definition is independently based on the identification of mutant alleles in highly oncogenic retroviruses and on the discovery of active rus oncogenes in tumor cell DNA. In contrast, the genes for which implication in tumorigenesis is based on the description of their structural or regulatory modifications in tumor cells, such as transcriptional activation, translocation, or amplification, are not ips0 fucto oncogenes, unless their function as dominant oncogenic determinants has been demonstrated. For instance, direct demonstration of autonomous oncogenic activity is missing for the amplified, transcriptionally activated, or translocated c-myc, N-myc, int-I, int-2,or pim-1 genes (see above). Of course, for the myc oncogene dominant oncogenic activity was demonstrated for the transduced allele, and the involvement of the cellular allele in genetic alterations in tumor cells is an intriguing observation. 111. The rnyc Oncogene

Specific protein products (Bister et al., 1977) and nucleic acid sequences (Duesberg et al., 1977) of the myc oncogene were originally discovered for the transduced allele in the genome of acute leukemia virus MC29. The discovery of myc alleles in the genomes of three other independently isolated leukemia viruses with pathogenic properties similar to those of MC29 (see Section I; Table I) lend strong support to the assumption that myc represented a new specific oncogene that was unrelated to src, the only avian oncogene then known. The search for the origin of v-myc led to the identification of the cellular c-myc gene which proved to be highly conserved among ver-

114

KLAUS BISTER AND HANS W. JANSEN

tebrate species and which is now suspected to be possibly implicated in viral or nonviral animal and human malignancies (see Section I1 and below). A. THEv-myc ALLELESIN MC29, CMII, OK10, AND MH2

1 . Structure and Expression The genetic contexts in which the four v-myc alleles were found in the genomes of the four leukemia viral isolates MC29, CMII, OK10, and MH2 are shown in Fig. 2, and the genetic origin of the nucleotide sequences of these v-myc alleles is depicted in Fig. 3. For an easier understanding of v-myc structure and expression, the structure of the chicken c-myc gene, which will be discussed in more detail in a later part of this section, is briefly described here. The c-myc gene is composed of three exons separated by two intervening sequences (Fig. 3). Since the first exon ( E l ) is untranslatable, translation of c-myc mRNA apparently initiates at the AUG codon near the 5’ border of the second exon (E2), continues through an open reading frame encoding 416 amino acids, and terminates at the UAG codon in the third exon (E3) (Fig. 3; see below). The most intriguing structural feature of the v-myc alleles is that in all four cases the entire protein coding domain of the cellular gene is transduced, but that sequences from the noncoding exon are always excluded. Sequences derived from the coding domains of E2 and E3 are precisely fused in the transduced alleles (Fig. 3), implying that transduction includes removal of intervening sequences between coding exons by RNA splicing (see Section VII). At their 3’ ends the v-myc alleles contain various complements of the untranslated region of E3 downstream from the common translational stop codon, but they all lack the polyadenylation signal of the cellular allele. At the 5’ ends three of the v-myc alleles contain sequences derived from the intron between E l and E2 (Fig. 3; see below). The genetic structure of the MC29 genome, in particular the map location and complexity of contiguous MC29-specific (i.e.,v-myc) nucleotide sequences (Duesberg et al., 1977; Sheiness et al., 1978), was originally determined by mapping of RNase TI-resistant oligonucleotides (TI-oligonucleotides) of viral RNA and by heteroduplex analysis of RNA-cDNA hybrids (Mellon et al., 1978; Duesberg et al., 1979; Hu et al., 1979; Bister and Duesberg, 1980). The structure was then confirmed and refined by molecular cloning and nucleotide sequence analysis of proviral DNA (Lautenberger et al., 1981; Vennstrom et al., 1981; Alitalo et al., 1983a; Reddy et al., 1983a). The

115

ONCOGENES M RETROVIRUSES AND CELLS

AUG SO

IUAGI' 909

I

Apol

MH2

U

1

4-H p59/61 FIG.2. Structure and expression of v-myc alleles. Genome-sized and subgenomic mRNAs of viruses from the MC29 subgroup (compare Table I), aligned with respect to homologous v-myc sequences, and their v-myc protein products are shown. Translation is symbolized by the open arrows; RNA splicing by the bent arrows. Nucleotide sequences (v-myc) transduced from the chicken c-myc gene are indicated by hatching, with large boxes representing sequences derived from c-myc exons and small boxes representing sequences derived from a c-myc intron (see also Fig. 3). All possibly relevant translational initiation and termination codons and splice donor (SD) and acceptor (SA) signals on all RNA species are indicated. Codons or signals on a given RNA species not used for translational initiation or termination, or for RNA splicing, respectively, are shown in parentheses. The termination codon marked by an asterisk is bypassed for the translation of v-myc from genome-sized OK10 RNA, but utilized in the synthesis of the gag protein product (not shown). Translation of the v-myc sequences is apparently always initiated from the gag AUG codon, although initiation from the v-myc AUG (in broken parentheses) on subgenomic mRNAs has not been rigorously ruled out. The designations ofprotein products refer to their apparent molecular weight; i.e., p l l o indicates that the MC29 gag-myc hybrid protein has an apparent molecular weight of 110,OOO. Protein domains encoded by v-myc sequences corresponding to c-myc coding domains are indicated by heavy lining. Complexities of RNA molecules are approximately to scale, with the bar representing 1 kilobase (kb). 1 kb

116

KLAUS BISTER AND HANS W. JANSEN ATG

TAG chick en c - m y c gene

5'

3' c-myc

)

1

(-

--

](---I

v - m y c (MC29)

-1

v - m y c (CMII)

( - - - ) r - I c -

v - m y c (OK101

)---(]-))---(

,

0.5- k-b ,( ) ) -(- -

mRNA

-1

v - m y c (MH 2 )

FIG.3. Structural relationship between c-myc and v-myc alleles. The three exons (E) of the chicken c-myc gene are shown as large boxes with black regions indicating the protein coding domain defined by the single large open reading frame. The nucleotide sequences found in mature c-myc mRNA and in transduced v-myc alleles are schematically aligned below the corresponding sequences of the c-myc gene. Sequences fused by RNA splicing are connected by thin angled lines. Broken lines in parentheses indicate flanking sequences on viral genomes (compare Fig. 2). Within the shared coding domains, v-myc alleles differ from c-myc by single nucleotide substitutions and by a small deletion (MH2).

genome contains no full complement of any of the three replicative genes, which is the evident explanation for the extensive defectiveness of MC29 diagnosed by biochemical analysis of viral protein synthesis and genetic complementation studies (Bister et al., 1977; Bister and Vogt, 1978). The v-myc allele replaces all of pol and 3' and 5' segments of gag and enu, respectively. The expression of the MC29 v-myc allele is mediated in a unique way which is now recognized as a widely used strategy for the expression of many v-onc genes. Using immunoprecipitation with antisera against gag proteins, a single virus-specific protein with an apparent molecular weight of 110,000 (PllOgng-m~c) was discovered in MC29transformed nonproducer cells, which represents a hybrid translational product of both the partial gag sequences and the v-myc allele of the MC29 genome (Fig. 2) (Bister et al., 1977; Mellon et at., 1978). The mRNA utilized for the synthesis of pllOgn~-myc appears to be indistinguishable from MC29 genomic RNA (Mellon et al., 1978; Sheiness et al., 1981).The open reading frame on this mRNA encompasses 450

ONCOGENES IN RETROVIRUSES AND CELLS

117

gag codons including the AUG start codon, 4 codons derived from cmyc intron sequences apparently fused to the sequences derived from

the second c-myc exon by a splicing process, 5 codons derived from the 5‘ end of the second exon and preceding the c-myc AUG start codon, and 416 codons derived from the protein coding domain of cmyc (Figs. 2 and 3) (Alitalo et al., 1983a; Reddy et al., 1983a; Watson et al., 1983a; Shih et al., 1984). The 875-amino acid protein product has a calculated molecular weight of -94,100. The nucleotide sequences of MC29 v-myc and chicken c-myc are very closely related with single nucleotide substitutions leading to only five (Alitalo et al., 1983a) or seven (Watson et al., 1983a) amino acid exchanges in the predicted sequences of v-myc and c-myc gene products. Confirmation of the predicted protein sequences and of the genetic origin of p l 10gag-m~cwas achieved by specific immunoprecipitation using antisera raised against synthetic peptides whose amino acid sequences were deduced from the nucleotide sequence of v-myc (Hann et al., 1983; Patschinsky et al., 1984), or using sera raised against proteins specified by v-myc DNA expressed in bacteria (Alitalo et al., 1983~). The genetic structure of the CMII genome (Fig, 2) was determined by mapping of TI-oligonucleotides of viral RNA (Bister et al., 1979) and confirmed by molecular cloning and nucleotide sequence analysis of proviral DNA (Walther et al., 1985, 1986). The CMII genome contains partial complements of all three structural genes: a 5’ segment of gag, a central segment of pol, and a 3‘ segment of enu. The CMII v-myc allele is closely related to MC29 v-myc (Bister et al., 1979; Roussel et al., 1979; Bister and Duesberg, 1980; Jansen et al., 1983a), and is mainly distinguished by the absence of 5’ intron-derived sequences and by a shorter stretch of 3’ untranslated sequences (Figs. 2 and 3) (Walther et al., 1985, 1986). The CMII v-myc allele is expressed via genome-sized mRNA as a hybrid protein with an apparent molecular weight of 90,000 (p90gaemgc)which, like the MC29 pllOgag-mgcprotein, is initiated from the gag AUG codon (Fig. 2) (Bister et al., 1979; Hayman et al., 1979b). Assuming that the unsequenced part of the CMII gag complement is isogenic to that of the RSV gag gene, the open reading frame on CMII genome-sized mRNA encompasses 278 gag codons including the start codon, the 5 codons derived from the 5’ end of E2, and the 416 codons corresponding to the entire coding domain of c-myc (Fig. 3) (Walther et al., 1985,1986). The 699-amino acid protein product has a calculated molecular weight of -75,400. The nucleotide sequence of the CMII v-myc coding domain is distinguished from that of chicken c-myc by a single

118

KLAUS BISTER AND H A N S W. JANSEN

nucleotide substitution leading to one amino acid exchange (Walther et al., 1985, 1986). The structural similarities between the MC29 and CMII encoded proteins were directly revealed by tryptic peptide analysis (Kitchener and Hayman, 1980) and by the demonstration of common antigenic determinants not only in the gag-encoded regions but also in the myc-specific domains (Hann et al., 1983; Patschinsky et al., 1984). The genetic structure of the OKlO genome (Fig. 2) was determined by TI-oligonucleotide mapping of viral RNA (Bister et al., 1980a) and confirmed and refined by molecular cloning and nucleotide sequence analysis of proviral DNA (Pfeifer et al., 1983; Hayflick et al., 1985). The OKlO genome contains a complete and functional gag gene and 5’ or 3’ segments of the pol and enu genes, respectively. The gag gene directs synthesis of the virion core proteins which are used for the assembly of noninfectious virus particles released by OK10-transformed nonproducer cells (Bister et al., 1980a; Ramsay and Hayman, 1980).The OKlO v-myc allele is closely related to those of MC29 and CMII (Roussel et al., 1979; Bister et al., 1980a; Jansen et al., 1983a; Pfeifer et al., 1983; Hayflick et al., 1985), and is distinguished mainly by the presence of a 5’ segment derived from c-myc intron sequences immediately preceding the second exon and including the splice acceptor signal at the 5’ border of E2 (Figs. 2 and 3). Two entirely different strategies are used for the expression of the OKlO v-myc allele. The first is principally similar to v-myc expression in MC29 and CMII: translation of genome-sized mRNA initiates at the gag AUG start codon and extends through the gag and pol sequences and the v-myc sequences to the conserved myc UAG stop codon (Fig. 2). The UAG stop codon at the 3‘ terminus of the gag gene is presumably bypassed by the same mechanism that is utilized in the synthesis of the Pr180gag-po2precursor protein for the reverse transcriptase of nondefective retroviruses. It has been proposed for the avian leukosis-sarcoma viruses that the fusion of the overlapping out-of-frame gag and pol reading frames (Schwartz et al., 1983) is achieved by ribosomal frame shifting (Varmus, 1985; Jacks and Varmus, 1985).The OKlO v-myc gene product synthesized by translational initiation at the gag AUG codon was identified as a protein with an apparent molecular weight of 200,000 (p200Bag’B-p01-m~c) in OK10-transformed cells (Ramsay and Hayman, 1980). The open reading frame encoding this protein encompasses the fused coding domains of gag and pol genes except the 3’ 115 pol codons, 20 codons derived from c-myc intron sequences, 5 codons from the noncoding 5’ end of the second c-myc exon, and the 416 codons corresponding to the entire c-myc coding

ONCOGENES IN RETROVIRUSES AND CELLS

119

domain (Figs. 2 and 3) (Hayflick et al., 1985). Assuming that the frameshift occurs at the last gag codon (Jacks and Varmus, 1985) and that the unsequenced part of the gag-pol complement of OKlO is isogenic to the corresponding region of the RSV genome, the protein product contains a total of 1929 amino acid residues (including 701 encoded by gag and 787 encoded by p o l ) and has a calculated molecular weight of -212,000. The nucleotide sequences of OKlO v-myc and chicken c-myc coding regions are distinguished only by single nucleotide substitutions with the resultant exchange of just two amino acids in the predicted protein sequences (Hayflick et al., 1985). The second mode of OKlO v-myc gene expression involves RNA splicing and translation of a subgenomic mRNA species (Fig. 2) which is found in OK10-transformed cells in addition to genome-sized mRNA (Chiswell et al., 1981; Saule et al., 1982). The generation of this subgenomic mRNA is based on the availability of the c-mycderived splice acceptor signal which is present in the OKlO v-myc allele (and in that of MH2; see below), but not in those of MC29 and CMII (Figs. 2 and 3).The splice donor presumably utilized is located just downstream of the g a g start codon and is analogous to the signal that is used in the generation of enu mRNA of nondefective avian retroviruses and src mRNA of RSV (Hackett et al., 1982; Schwartz et al., 1983). The spliced OKlO mRNA manufactured in that way then contains the first six g a g codons fused in-frame to the v-myc sequences at the position corresponding to the 5' end of c-myc exon E2 (Figs. 2 and 3). The translational product of this mRNA was identified in OK10-transformed cells as a protein with an apparent molecular which, like the p200g4g-po2-myc weight of approximately 60,000 (p60V-m~c) protein, can be precipitated by antisera against myc-encoded protein domains (Hann et al., 1983; Alitalo et al., 1983c; Patschinsky et al., 1984).It has not been directly resolved yet whether translation of the subgenomic mRNA initiates at the gag or at the myc AUG start codon; these codons are separated by only 10 codons on that RNA species (Fig. 2). It may appear more plausible that initiation occurs at the first, i.e., the g a g AUG codon, although the scanning model for eukaryotic translational initiation is not directly applicable to avian retroviral mRNAs (Kozak, 1980, 1981; Schwartz et al., 1983). Assuming that contains 427 amino acid translation initiates at the g a g AUG, p60v-mYC residues (6 encoded by g a g , 5 encoded by sequences derived from the noncoding 5' terminus of the second c-myc exon, and 416 encoded by sequences corresponding to the c-myc coding region) and has a calculated molecular weight of -47,000. The presence in MH2 genomic RNA of sequences related to the

120

KLAUS BISTER AND HANS W. JANSEN

v-myc allele of MC29 was demonstrated by TI-oligonucleotide analysis and nucleic acid hybridization (Duesberg and Vogt, 1979; Roussel et al., 1979; Sheiness et al., 1980a).However, the genetic structure of the MH2 genome (Fig. 2) could only be resolved by molecular cloning and structural analysis of proviral DNA (Jansen et aZ., 1983a,b; Coll et al., 1983a; Kan et al., 1983). The MH2 genome contains a 5' segment of the gag gene and no pol or enu sequences at all. The v-myc allele of MH2 is located near the 3' terminus of the genome and separated from the partial gag gene by v-mil, the second oncogene of MH2 (see Section IV). The MH2 v-myc allele contains a 5' segment derived from intron sequences directly adjacent to the second c-myc exon including the splice acceptor signal, and a short untranslated sequence of only 39 nucleotides 3' of the conserved UAG stop codon (Figs. 2 and 3) (Jansen et al., 1983a; Sutrave et al., 1984a,b; Kan et al., 1984). The noncoding region between v-myc and the 3' LTR of MH2 proviral DNA (Fig. 2) contains a surprising arrangement of sequences related to avian sarcoma viral genomes, including sequences homologous to the 3' end of the v-src coding region (Sutrave et ul., 1984b; Dutta et al., 1985). Expression of the MH2 v-myc allele by translational initiation at the gag AUG start codon of genome-sized mRNA is prohibited by the presence of the in-frame mil UAG stop codon (Fig. 2; Section IV). Instead, expression is mediated by translation of a subgenomic mRNA species (Fig. 2) which is found in MH2-transformed cells in addition to genome-sized mRNA (Pachl et al., 1983a; Saule et al., 1983). The splicing process is apparently analogous to the one involved in the genesis of OKlO v-myc mRNA. Direct evidence for the presumed splice junction was obtained by nucleotide sequence analysis of proviral DNA of a transmissible and transforming retrovirus (MH2E21) that was apparently generated by encapsidation and reverse transcription of MH2 v-myc mRNA (Patschinsky et al., 1986a). The analysis revealed a precise fusion of the first six gag codons to the v-myc sequences corresponding to the 5' terminus of E2 in c-myc. The translational product of MH2 v-myc mRNA was identified by immunoprecipitation using antisera against myc-specific protein domains as a protein appearing in gel electrophoresis as a characteristic doublet corresponding to apparent molecular weights of 59,000 and 61,000 (p59/6lV-*VC) (Hann et al., 1983; Patschinsky et al., 1984). Like for the OKlO v-myc mRNA, it has not been directly demonstrated yet by sequence analysis of the protein product whether translation of the MH2 v-myc mRNA initiates at the gag or at the myc AUG codon, or at both (Fig. 2). However, oligonucleotide-directed mutagenesis of the

ONCOGENES IN RETROVIRUSES AND CELLS

121

gag translational initiation codon on the MH2E21 genome generated a

transmissible and transforming retrovirus (MH2E21ml) specifying a smaller v-myc protein product, p57/59"-'"yCc, which is presumably initiated at the myc AUG codon (Patschinsky et al., 1986a). This strongly suggests that on standard MH2 v-myc mRNA the gag and not the myc initiation codon is utilized. The nucleotide sequence of MH2 v-myc differs from that of chicken c-myc in the shared coding domains by single nucleotide substitutions leading to 27 (Kan et al., 1984) or 25 (Patschinsky et al., 1986a) amino acid substitutions in the predicted protein sequences and by the deletion of four consecutive codons. Y~ Assuming that translation initiates at the gag AUG, ~ 5 9 / 6 1 " - ~contains 423 amino acid residues (6 encoded by gag, 5 encoded by sequences derived from the noncoding 5' terminus of the second c-myc exon, and 412 encoded by sequences corresponding to the c-myc coding region) and has a calculated molecular weight of -46,100. 2. Function Conditional and nonconditional mutants in src function have played a key role in the definition of the RSV transforming gene (Hanafusa, 1977; Vogt, 1977). Only a few mutants in transforming function have so far been isolated from acute leukemia viruses, and in general the lesions only partially affect the transforming ability of the virus. The phenotype of most of these mutants points to the fundamental genetic difference between replication-competent RSV and the replication-defective acute leukemia viruses: mutants of the latter with a complete onc- phenotype would be nonreplicating, nontransforming entities, impossible to select for by standard biological assays. For the v-myc-containing viruses of the MC29 subgroup, only a few nonconditional mutants have been isolated. The permanent quail cell line QlO, originating from a cell clone transformed by unmutagenized MC29 virus (Bister et al., 1977), spontaneously generated nonconditional mutants of MC29 which were selected on the basis of altered vmyc gene products (Ramsay et al.,1980). These mutants have retained the capacity to transform cultured fibroblasts, but have a strongly reduced potential to transform hemopoietic cells in vitro (Ramsay et al., 1980) or to induce tumors in animals (Enrietto et al., 1983a). Interestingly, a similar phenotype at the nonpermissive temperature has recently been reported for a conditional mutant of MH2 with a presumed lesion in the v-myc allele (Palmieri, 1986). Molecular analyses of the lesions of the nonconditional mutants of MC29 revealed that their v-myc alleles had suffered overlapping in-

122

KLAUS BISTER AND HANS W. JANSEN

frame deletions in their central domains (Bister et al., 1982a; Ramsay and Hayman, 1982; Enrietto and Hayman, 1982). Hence, wild-type and mutant gag-myc hybrid proteins differ by the lack of internal amino acid sequences in the mutant v-myc domains, but share identical carboxyl termini (Patschinsky et al., 1984). Nucleotide sequence analyses of these mutant v-myc alleles revealed that the deletions encompass 56 or 120 consecutive codons corresponding to sequences from the 3' end of E2 and the 5' end of E3 in c-myc (compare Fig. 3) (H. W. Jansen, K. Bister, and C. Trachmann, unpublished data). From one of these mutants with the larger deletion, a back-mutant was isolated for which recovery of the deleted sequences by presumed recombination with c-myc correlated with the restoration of full in uitro transforming activity and in v i m tumorigenicity, albeit with an altered tumor spectrum (Ramsay et aZ., 1982a; Bister et al., 1983; Enrietto et al., 1983b; Smith et aZ., 1985). Based on molecular structure and biological properties of these mutants it is concluded that the central segment of v-myc appears to be essential for hemopoietic cell transformation but dispensable for fibroblast transformation. Whether this reflects the presence of different functional domains on the wild-type v-myc gene product or quantitative changes in the activity of the mutant proteins is unknown. Although analysis of these mutants as well as the observation of independent transductions of the myc oncogene provide strong genetic evidence for the direct involvement of myc in oncogenesis, a rigorous definition of nucleotide sequences essential and sufficient for cell transformation has not been provided yet. The recent isolations of a spontaneous deletion mutant and of a unique variant of MH2 may aid in such a definition. The deletion mutant, MH2D12, has lost almost all v-mil sequences but has retained the v-myc gene (Jansen et al., 1985a). The variant, MH2E21, has a genetic structure precisely as the one expected for the subgenomic v-myc mRNA of MH2 (Fig. 2), and it was apparently generated by encapsidation and reverse transcription of this mRNA species (Patschinsky et al., 1986a). Both viruses transform cultured fibroblasts, indicating that expression of v-myc via subgenomic mRNA is sufficient, and that the major structural protein sequences in v-myc hybrid proteins (Fig. 2) or the concomitant expression of a second oncogene are not essential for fibroblast transformation. Furthermore, analyses of the genetic structure and the transforming properties of the in uitro mutagenized MH2E21ml virus (see above) directly demonstrated that v-myc protein synthesis initiated at the authentic myc initiation codon is sufficient for fibroblast transformation (Patschinsky et al., 1986a). Also, a nontransmissible variant

ONCOGENES I N RETROVIRUSES AND CET.LS

123

MC29 provirus has been isolated that has lost almost all of the gag complement and yet has transforming activity in transfection experiments (Shaw et al., 1985). The biochemical function of v-myc protein products responsible for cell transformation is unknown. However, some of the properties of these proteins may provide a hint to their unknown function. All vmyc gene products (Fig. 2) were reported to be phosphoproteins containing phosphoserine and phosphothreonine residues in their mycspecific domains (Bister et al., 1980b; Ramsay et al., 1982b; Hann et al., 1983).An intriguing observation was that the proteins encoded by the mutant v-myc alleles of the MC29 deletion mutants (see above) have lost specific phosphorylation sites at threonine and possibly also serine residues within the myc-specific domain, and that the protein encoded by the back-mutant (see above) has regained those acceptor sites (Ramsay et al., 1982b). Employing subcellular fractionation procedures and immunofluorescence techniques, it has been demonstrated that the bulk of v-myc gene products is found in the nucleus of transformed cells (Abrams et al., 1982; Donner et al., 1982; Hann et al., 1983; Alitalo et al., 1983~). It was also reported that v-myc protein products have in vitro DNA-binding activity, albeit without any apparent sequence specificity, and are associated with chromatin preparations from transformed cells (Donner et al., 1982; Bunte et al., 1982). However, recent results indicate that only a minor fraction of v-myc protein products is associated with chromatin, that the major fraction is tightly associated with the nuclear matrix of transformed cells, and that these proteins are dispersed throughout the cytoplasm during mitosis (Winqvist et al., 1984; Eisenman et al., 1985). A more recent report showed that myc protein products can be extracted from nuclei by mild salt concentrations and that they become insoluble and apparently associated with matrix structures only after exposure to physiological temperatures during the in vitro manipulations of nuclear preparations (Evan and Hancock, 1985). Attempts to correlate the in uitro DNA affinity of v-myc protein products in a quantitative manner with their oncogenic function have yielded inconsistent results. It was reported that the proteins specified by the mutant v-myc alleles of the MC29 deletion mutants (see above) have a reduced in vitro DNAbinding activity, leading the authors to propose that efficient DNA binding by v-myc protein products is necessary for tansformation of hemopoietic cells but not for fibroblast transformation (Donner et al., 1983). However, using nondenaturing salt extracts of transformed cells other investigators observed equal in vitro DNA affinity for the proteins encoded by one of these partially transformation-defective

124

KLAUS BISTER AND HANS W. JANSEN

deletion mutants or by wild-type MC29, respectively (Eisenman et al., 1985). B. THEc-myc GENE

1, Structure and Expression The presence in normal chicken DNA of sequences related to the vmyc oncogene of MC29 was originally detected by nucleic acid hybridization (Sheiness and Bishop, 1979; Roussel et al., 1979). It was further shown that the v-myc-related sequences in the chicken genome have homologous counterparts in other vertebrate DNA, that they appear to represent a single constant locus in the chicken genome, and that they are expressed as a polyadenylated RNA species in normal cells (Sheiness and Bishop, 1979; Sheiness et al., 1980b). The structure of the chicken c-myc gene (Fig. 3) was then extensively characterized by molecular cloning of genomic DNA (Robins et al., 1982; Vennstrom et al., 1982; Nee1 et al., 1982), by nucleotide sequence analysis (Watson et al., 1983a; Shih et al., 1984; Papas et al., 1984), and by transcriptional mapping (Linial and Groudine, 1985). The c-myc locus contains three exons with complexities of -340 ( E l ) , 703 (E2), and -980 (E3) base pairs, respectively, divided by two intervening sequences of -710 and 971 base pairs, respectively (Fig. 3) (Watson et al., 1983a; Linial and Groudine, 1985).The first exon contains no translational initiation codon but several termination codons, and hence is untranslatable (Shih et al., 1984; Linial and Groudine, 1985). The first translational initiation codon is located at the sixth codon position in exon E2, and a large open reading frame, encoding a protein of 416 amino acids with a calculated molecular weight of about 46,000, extends to the stop codon in exon E3 (Fig. 3). The corresponding polyadenylated c-myc mRNA with a complexity of -2.4 kilobases is translated to yield a protein with an apparent molecular weight of -60,000, detected by in vitro translation of hybridselected c-mvc mRNA or by immunoprecipitation of cellular proteins using antisera against myc-specific protein domains (Pachl et al., 1983b; Hann et al., 1983; Alitalo et al., 1983~). The structure of mammalian c-myc genes is very similar to the one described above for the chicken c-myc locus. Transcriptional mapping and nucleotide sequence analyses of cloned c-myc genomic DNA and cDNA of human (Dalla Favera et al., 198213; Hamlyn and Rabbits, 1983; Colby et al., 1983; Watt et al., 1983a,b; Watson et al., 1983b; Battey et al., 1983) and murine (Adams et al., 1983, Stanton et al., 1983, 1984; Bernard et al., 1983) origin established that these genes

ONCOGENES IN RETROVIRUSES AND CELLS

125

contain a 5’ noncoding exon and two coding exons. The first exon of both the human and the mouse c-myc gene has a complexity of about 560 base pairs, contains two active promoters located about 150 nucleotides from another, and is untranslatable due to the absence of a translational initiation codon and the presence of multiple termination codons in each reading frame (Battey et al., 1983; Bernard et al., 1983; Watt et al., 1983b; Stanton et al., 1984). The second exon in both mammalian genes is separated from the first by an intron of about 1600 base pairs, has a complexity of 772 base pairs, and contains the translational start codon at the same sixth codon position as in the chicken cmyc exon E2 (Fig. 3). The third exon in mouse and human c-myc is separated from the second by an intron of approximately 1100 or 1370 base pairs, respectively, has a complexity of about 900 base pairs, and contains the translational stop codon terminating the open reading frame that starts at the ATG codon near the 5’ end of the second exon and encodes a protein of 439 amino acid residues. The homology between human and mouse c-myc genes is about 90% for the nucleotide sequences, and about 92-94% for the predicted amino acid sequences. A comparison between the coding regions of the chicken and the two mammalian c-myc genes revealed that sequence conservation is highest in the third exon. There are no insertions or deletions between the avian and mammalian predicted polypeptide sequences, and about 75% of the 187 amino acid residues are identical. For the second exon, which encodes 252 amino acid residues in the mammalian genes but 229 in the chicken gene, several insertions and deletions in the predicted polypeptide sequences are required for optimal alignment, with a resultant homology of about 70% (Bernard et al., 1983; Watson et aZ., 1983b). For all three genes, the sequence analyses revealed conservation of sequences at the 3’ boundary of exon 2 and the 5’ boundary of exon 3, ensuring reading frame continuity (Stanton et al., 1983; Bernard et al., 1983). The noncoding first exons of human and murine c-myc genes show a nucleotide sequence homology of about 70%, a surprisingly high conservation of an untranslatable sequence (Battey et al., 1983; Bernard et al., 1983). In contrast, the first exon of the avian c-myc gene appears to be unrelated in nucleotide sequence to the corresponding mammalian exons (Shih et al., 1984; Linial and Groudine, 1985). The mRNAs derived from mammalian c-myc genes (Erikson et al., 1983) contain large untranslated leader sequences like the chicken c-myc mRNA (Fig. 3) and have complexities of 2.25 and 2.40 kilobases. The two species differ at their 5‘ ends, indicating that apparently both promoter sites in the first exon are used in transcription (Battey et al., 1983; Bernard et al., 1983).

126

KLAUS BISTER AND HANS W. JANSEN

The translational products of mouse and human c-myc genes have been identified using in vitro translation of hybrid-selected c-myc mRNA or employing immunoprecipitation of cellular proteins with myc-specific antisera as a major and a minor protein corresponding to apparent molecular weights of 64,000 and 67,000, respectively (Hann and Eisenman, 1984; Ramsay et al., 1984; Persson et al., 1984; Evan et al., 1985). Human c-myc protein products have been synthesized from c-myc alleles cloned in expression vectors in bacterial, yeast, and insect cells (Miyamoto et al., 1985a,b). Like for the avian v-myc and cmgc encoded proteins, there is a large discrepancy between calculated molecular weights of mammalian c-myc-encoded proteins (about 48,800) and their apparent molecular weights deduced from electrophoretic mobilities (see above). This may be due to unusual structural features of these proteins inherent in their primary structure, such as high proline content, and clustering of basic residues in their carboxy-terminal domain.

2. Function The physiological role of the c-myc gene in normal cells and the relevant biochemical function of its protein product are unknown. The c-myc gene is highly conserved among vertebrate species (see above) and expressed in a variety of animal and human cells and tissues (Sheiness et al., 1980b; Gonda et al., 1982; Eva et al., 1982; Westin et al., 1982b; Coll et al., 1983b). Like the v-myc gene products, avian and mammalian c-myc-encoded proteins were shown to be nuclear phosphoproteins that are found to be associated with the nuclear matrix under certain conditions of in uitro preparation (see above) and that have nonspecific in vitro DNA affinity (Hann et al., 1983; Alitalo et al., 1983c; Hann and Eisenman, 1984; Ramsay et al., 1984; Persson and Leder, 1984; Eisenman et al., 1985; Watt et al., 1985; Evan and Hancock, 1985). A major approach in the elucidation of c-myc function is the analysis of differential expression of this gene in normal or malignant cells. Such analyses have revealed differential expression of c-myc in several experimental systems designed to unveil parameters of cell growth, differentiation, and development (Reitsma et al., 1983; Lachmann and Skoultchi, 1984; Pfeifer-Ohlsson et al., 1984; Slamon and Cline, 1984; Stewart et al., 1984a; Gonda and Metcalf, 1984; McCormack et al., 1984; Yaswen et al., 1985; Knight et al., 1985; Einat et al., 1985; Dean et al., 1986; Zimmerman et al., 1986). Elevated c-myc transcripts have been observed during regeneration of rat liver (Goyette et al., 1984; Makino et al., 1984) and c-myc RNA and protein levels rapidly increase after delivery of proliferation signals to quies-

ONCOGENES IN RETROVIRUSES AND CELLS

127

cent cells (Kelly et al., 1983; Campisi et al., 1984;Armelin et al., 1984; Persson et al., 1984). It has been inferred from these data that expression of c-myc is regulated in a cell cycle-specific manner and that activation of this gene is necessary and specific for the transition from the Go through the G1 period of the cell cycle (Kelly et al., 1984; Calabretta et al., 1986).However, experiments using counterflow centrifugation methods to enrich for populations of cells at different stages of the cell cycle clearly demonstrated that the levels of c-myc mRNA and protein are invariant throughout the cell cycle and that the increase in c-myc expression on stimulation of resting cells is only transient, with the elevated levels of c-myc mRNA and protein falling back to constant levels throughout subsequent cell cycles (Thompson et al., 1985; Hann et al., 1985). Furthermore, elevated c-myc expression can be stimulated by a variety of agents, including phorbol ester tumor promoters and hormones activating protein kinase C, and does not directly account for the mitogenic effects of growth factors (Coughlin et al., 1985; Rabin et al., 1986). Hence, the typical mode of c-myc expression is continuous synthesis and rapid turnover throughout the cell cycle of normal and transformed cells (Dani et al., 1984; Hann et al., 1985; Thompson et al., 1985; Blanchard et al., 1985; Rabbitts et al., 1985a; Persson et al., 1985; Lacy et al., 1986). Another approach toward an understanding of the physiological role of vertebrate oncogenes is the structural and functional analysis of homologs of these genes in genetically well-defined and easily manipulated eukaryotic organisms. Structural and presumably functional homologs of ras oncogenes have been found in yeast (DeFeo-Jones et al., 1983; Gallwitz et al., 1983; Powers et al., 1984),and genes related to ras, src, abl, and myb have been found in Drosophila (Hoffmann et al., 1983; Simon et al., 1983; Neuman-Silberberg et al., 1984; Katzen et al., 1985). Nucleic acid hybridization of Drosophila genomic DNA with v-myc probes had suggested that c-myc-related genes may also exist in this organism (Shilo and Weinberg, 1981). However, recent analyses indicated that the Drosophila nucleic acid sequences detected by hybridization do not represent structural homologs of the vertebrate c-myc gene (Madhavan et al., 1985). Oncogenic activation of nontransduced myc alleles has not been rigorously established yet, nor are the molecular mechanisms underlying such presumed activations or the subsequent molecular events causing cell transformation known in any definitive way. In particular, all attempts to demonstrate autonomous function of c-myc DNA as a dominant oncogenic determinant in cell transformation assays have failed so far. Furthermore, since expression of c-myc is observed in normal cells (see above) as well as in tumor cells of essentially all

128

KLAUS BISTER AND HANS W. JANSEN

tested human malignancies (Slamon et al., 1984), a straightforward correlation between c-myc expression and specific tumor induction does not exist. Nevertheless, there is multiple circumstantial evidence that activated c-myc alleles may be involved in carcinogenesis. In the majority of bursal (B-cell) lymphomas in chickens induced by avian leukosis virus infection, proviral DNA was found integrated within or in the vicinity of the c-myc locus and augmented transcription of this gene was observed (Hayward et al., 1981; Payne et al., 1982). In many cases proviral integration occurred downstream of the first exon (compare Fig. 3) within the first intron in the same transcriptional orientation as the cellular gene, and c-myc transcripts were found to contain viral LTR sequences and sequences from the second and third, but not from the first, c-myc exon (Hayward et al., 1981; Shih et al., 1984). Hence, a promoter insertion mechanism was postulated which implied transcriptional initiation of c-myc transcripts from the inserted viral promoter rather than from the c-myc promoter in the first exon, resulting in elevated levels of c-myc RNA (Hayward et al., 1981). However, other relative orientations of integrated proviral DNA and different transcriptional patterns have been observed to be compatible with augmented c-myc transcription in primary bursal tumors and in lymphoma cell lines. Proviruses were found integrated 3’ of c-myc, or 5’, but in the opposite transcriptional orientation, hence prohibiting initiation of c-myc transcripts from viral promoters (Payne et al., 1982). Furthermore, elevated c-myc transcripts in some bursal lymphoma cell lines were found to lack viral LTR sequences but to contain all sequences from the first exon, and hence they were apparently initiated at the authentic c-myc promoter, even in cases where integration of the proviral DNA was in the same transcriptional orientation (Pachl et al., 1983b; Linial and Groudine, 1985). Based on these observations it was proposed that inserted viral LTR sequences can function solely as an enhancer of the c-myc locus (Linial and Groudine, 1985). The idea that insertion mutagenesis may involve other mechanisms than provision of a transcriptional promoter (Payne et al., 1982) was also supported by the report that leukosis virus integration in the vicinity of the c-myc locus alters the chromatin structure in this region (Schubach and Groudine, 1984). Viral gene products are not necessary for the transcriptional activation of c-myc. In most cases of insertion mutagenesis of this locus, the proviral DNA is extensively deleted, commonly at its 5’ terminus, precluding viral gene expression (Nee1 et al., 1981; Payne et al., 1981; Fung et al., 1981; Westaway et al., 1984). It has been argued that the apparent selection for proviral defectiveness reflects the selection

ONCOGENES IN RETROVIRUSES AND CELLS

129

for advanced tumor development, either because tumor cells not expressing viral antigens escape immune surveillance, or because transcription from the 3’ LTR (in the promoter insertion model) is more efficient when the 5’ LTR is deleted. At least for the second proposal, experimental support has been provided (Cullen et al., 1984). Despite the strong correlation of c-myc activation with lymphoid tumorigenesis in chickens, there is no definitive proof that it is necessary for tumor induction, and it appears almost certain that it is not sufficient (Cooper and Neiman, 1981) (compare Section VI1,C). In a substantial number of virus-induced T-cell lymphomas of mice and rats, proviral DNA was found integrated in the vicinity of the cmyc locus. In most cases analyzed in detail, the provirus was integrated 5’ of the first c-myc exon in the opposite transcriptional orientation, thereby implying that the observed increased levels of c-myc RNA may be due to enhancing effects of the LTR sequences (Corcoran et al., 1984; Steffen, 1984; Selten et al., 1984; Li et al., 1984). A surprising arrangement of proviral DNA and c-myc sequences was detected in several feline leukemia virus-positive spontaneous T-cell lymphomas of cats. Although the majority of such tumors showed no rearrangements in their c-myc loci, in some cases c-myc sequences were found to be incorporated within proviral DNA sequences (Neil et al., 1984; Mullins et al., 1984; Levy et al., 1984). A few of the resultant recombinant proviruses were shown to be transmissible, although without demonstrated transforming activity. An experimental extension of the proviral insertion mutagenesis theme was achieved when constructed LTR-c-myc hybrid genes were injected into early murine embryos. Some of the transgenic mice, both founder and progeny animals, developed spontaneous mammary carcinomas with high expression of the hybrid gene (Stewart et al., 1984b). Similarly, transgenic mice bearing a c-myc gene coupled to an immunoglobulin enhancer frequently developed fatal B-cell lymphoma (Adams et al., 1985). Increased or inappropriate transcription of c-myc genes has also been observed in chemically induced tumors in quails, rats, and mice (Saule et al., 1984; Hayashi et al., 1984; Chinsky et al., 1985).Amplification of the c-myc gene and concomitant increased levels of c-myc RNA and protein were observed in cell lines or primary tumor cells derived from cases of human myeloid leukemia, lung and colon carcinoma, gastric adenocarcinoma, or glioblastomas (Collins and Groudine, 1982; Dalla Favera et al., 1982a; Little et al., 1983; Alitalo et al., 1983b; Nowell et al., 1983; Hann and Eisenman, 1984; Shibuya et al., 1985; Yokota et al., 1986; Trent et al., 1986). The discovery that the c-myc gene is involved in and presumably

130

KLAUS BISTER AND HANS W. JANSEN

activated by specific chromosomal translocations in many B-cell tumor lines of human and murine origin supported the original suggestion by Klein (1981)predicting oncogene activation by genetic transpositions, and initiated extensive investigations into the mechanisms of translocation and activation at the molecular level. The basic observation was that specific translocations in murine plasmacytoma cell lines and human Burkitt’s lymphoma cell lines join immunoglobulin genes to c-myc (Dalla Favera et al., 1 9 8 2 ~Taub ; et al., 1982; ShenOng et al., 1982; Crews et al., 1982; Adams et al., 1983; Marcu et al., 1983; Hamlyn and Rabbits, 1983).An overwhelmingly extensive and complex collection of data has since been provided on this topic, and many details have been comprehensively reviewed and referenced (Klein, 1983; Perry, 1983; Leder et al., 1983; Varmus, 1984; Croce and Hein, 1985; Vogt et al., 1985; Rabbits, 1985). Some of the common features that have emerged from the analyses of translocations examined so far will be briefly described. In the most common translocation in Burkitt’s lymphoma cell lines, t(8;14), c-myc is transferred from its normal position on chromosome 8 to the immunoglobulin heavy chain constant region locus on chromosome 14. In most murine plasmacytoma cell lines, an analogous translocation, t(12;15), is observed joining the c-myc locus from chromosome 15 to the heavy chain locus on chromosome 12. In both the human and the murine translocations, the heavy chain gene and the cmyc gene are joined in opposite transcriptional orientations. The less frequent variant translocations found in some of these lymphoid tumors involve exchange between the chromosomes carrying the c-myc gene and chromosomes carrying light chain genes: chromosome 2 (in man) or 6 (in mice) bearing the K locus, and chromosome 22 (in man) bearing the A locus. In the Burkitt variant translocations, t(2;8) and t(8;22), the scissions occur 3’ of c-myc and the gene remains on chromosome 8. The translocated light chain regions are joined on the 3’ side of c-myc in the same transcriptional orientation. In the common Burkitt t(8;14) translocations, the breakpoints in the c-myc locus were found on either side of the first noncoding exon, leading to transfer of a complete or a truncated gene to chromosome 14. However, breakpoints 3‘ of the first exon are always located in the first intron and do not affect the coding region of c-myc (compare Fig. 3). Most of the heavy chain breakpoints in t(8;14) translocations are within the Sp switch region. In the predominant plasmacytoma t( 12;15) translocations, most breakpoints in the c-myc locus occur in a region encompassing the first exon and the first intron, thus separating the c-myc coding region from the authentic c-myc promoter region. Breakpoints

ONCOGENES IN RETROVIRUSES AND CELLS

131

within the heavy chain locus are generally found in the Sa switch region. A major consequence of these translocations appears to be that the affected c-myc allele is constitutively expressed whereas the other allele remains silent. Hence, inappropriate constitutive expression of c-myc is suspected to be involved in the genesis of these tumors. The molecular mechanisms of the c-myc deregulation are not yet understood, in particular because the position of breakpoints varies and often occurs at a large distance of 10,000or more base pairs from the cmyc locus. Also, it is still a matter of controversy whether only the loss of transcriptional controls or also the disturbance of posttranscriptional regulation, like that of mRNA turnover or translation, is of any relevance (Saito et al., 1983; Darveau et al., 1985; Butnick et al., 1985; Piechaczyk et al., 1985; Eick et al., 1985; Rabbitts et al., 1985b; Feo et al., 1986), or whether somatic mutations observed in the coding regions of some translocated c-myc alleles are of any functional significance (Rabbitts et al., 1983, 1984; Battey et al., 1983; Stanton et al., 1984; Showe et al., 1985). Finally, circumstantial evidence for the oncogenic potential of the c-myc gene was obtained from in vitro transformation assays. It was shown that augmented expression of c-myc genes placed under control of strong promoters or enhancers is sufficient for the cotransformation of rat embryo cells together with a mutant M S gene (Land et al., 1983; Lee et al., 1985) or for the induction in mouse NIH/3T3 fibroblasts of many properties of transformed cells, however without a greatly altered morphology (Kelekar and Cole, 1986). IV. The mil(raf) Oncogene

Specific protein products (Hu et al., 1978; Rapp et al., 1983a) and nucleic acid sequences (Jansen et al., 1983a,b; Coll et al., 1983a; Kan et al., 1983; Rapp et al., 198313) of the mil(raf) oncogene were independently discovered for the transduced alleles in the genomes of avian acute leukemia virus MH2 and murine sarcoma virus 3611 (3611-MSV).The avian oncogene, termed v-mil, and the murine onaogene, termed v - r a . were then recognized to be closely related and to represent transduced alleles of cognate cellular genes, the c-mil(raf) genes of chicken and mice. Curiously, the protein product of the MH2 v-mil allele (Hu et al., 1978) was long mistaken for the product of the previously identified MH2 v-myc allele mainly because the mode of gene expression is similar for both MC29 v-myc (compare Section 111) and MH2 v-mil (see below). Notably, c-mil(raf) and c-myc are unlinked in the cell.

132

KLAUS BISTER AND HANS W. JANSEN

1

UbG

AUG SO

v-myc

It

,

l k b ,

1

UAG

SO AUG

MH2

Apol

I

env

a

13611 MSV

P75

FIG.4. Structure and expression of v-mil(raf) alleles. Genome-sized mRNAs of avian retrovirus MH2 and murine retrovirus 3611-MSV aligned with respect to homologous vrniZ(raf3 sequences and their v-mil(rafl protein products are shown. Nucieotide sequences transduced from either the chicken or the mouse c-mil(raf) gene are indicated by hatching. For the explanation of further symbols, see legend to Fig. 2. The splice donor sites on both RNA species are utilized (for v-myc and eno expression, respectively), but they are not involved in v-mil(raf) expression.

A. THEv-mil ALLELEIN MH2 AND THE v-raf

ALLELE IN

3611-MSV

1. Structure and Expression

The structures and the modes of gene expression of the v-mil and vrufalleles are shown in Fig. 4, and the genetic origin of the nucleotide sequences of v-mil and the region of homology to v-ruf are depicted in Fig. 5. Determination of the genetic structure of the MH2 genome (Figs. 2 and 4) by molecular cloning of proviral DNA (Jansen et al., 1983a,b; Coll et al., 1983a; Kan et al., 1983) and nucleotide sequence analysis (Sutrave et al., 1984a; Kan et al., 1984; Galibert et al., 1984) revealed that the v-mil allele is inserted between partial gag sequences at its 5’ end and intron-derived v-myc sequences at its 3’ end (compare Sections I11 and VII). Comparison of v-mil with the chicken c-mil gene (Fig. 5; see below) showed that in the course of the transduction, coding domains derived from 11 exons of the cellular gene were precisely fused (Jansen and Bister, 1985).The 5’ terminus of vmil is derived from sequences within the coding domain of an exon, and the 3’ terminus corresponds to a position located in the untranslated sequence 12 base pairs downstream of the translational stop codon shared between v-mil and c-mil (Jansen and Bister, 1985). In the MH2 genome, the partial gag and the v-mil sequences are fused in one large reading frame. Assuming that the unsequenced part of the MH2 gag complement is isogenic to the corresponding region of the RSV gag gene, this reading frame encompasses 515 gag codons in-

133

ONCOGENES IN RETROVIRUSES AND CELLS TAG

'--'

v-mil

c

I homology t o v-raf

k

(MH2)

& FIG.5. Structural relationship between c-mil and v-mil alleles. The exons (E) of the chicken c-mil gene sharing sequence homology with v-mil are shown as large boxes, with black regions indicating the protein coding domain defined by an open reading frame for which only the translational termination codon but not the initiation codon has been identified. The c-mil gene contains more exons (coding and possibly noncoding) 5' of the region defined by homology to v-mil, and more 3' untranslated sequences. The nucleotide sequences of c-mil mRNA and of the transduced v-mil allele are schematically aligned below the corresponding sequences of the region of the c-mil gene shown here. Within the shared coding domains, v-mil and c-mil differ by single nucleotide substitutions. The region in v-mil homologous to the murine v-raf allele is indicated. For further symbols and explanations, see legend to Fig. 3.

cluding the AUG start codon and 379 v-mil codons predicting a 894amino acid protein with a calculated molecular weight of approximately 97,000 (Sutrave et al., 1984a; Kan et ul., 1984; Galibert et al., 1984). The corresponding protein product of genome-sized mRNA, a hybrid protein with an apparent molecular weight of 100,000 (plWag-mi'),had been identified in MH2-transformed nonproducer cells as the only virus-specific protein product, based on immunoprecipitation experiments with antisera against viral structural proteins (Hu et al., 1978). The c-mil and v-mil nucleotide sequences differ at only 7 out of 1153 nucleotide positions, and the predicted sequences of v-mil and c-mil (see below) protein products differ by one chemically conservative and four nonconservative substitutions among the 379 amino acid residues of the shared domains (Jansen and Bister, 1985). Confirmation of the predicted protein sequences and of the genetic origin of plOOBag-miz was achieved by specific immunoprecipitation, using antisera raised against synthetic peptides whose amino acid sequences were deduced from the nucleotide sequence of v-mil or v-ruf (Patschinsky et al., 1986b). The mammalian retrovirus 3611-MSV was isolated from mice inoculated with a stock of murine leukemia virus obtained by iododeoxyuridine induction of methylcholanthrene-transformed C3H/lOT1/2 mouse cells (Rapp et al., 1983a). The 3611-MSV isolate transforms fibroblasts and epithelial cells in culture and induces fibrosarcomas in uiuo. The genetic structure of the 3611-MSV genome (Fig. 4) was

134

KLAUS BISTER AND HANS W. JANSEN

determined by molecular cloning and nucleotide sequence analysis of proviral DNA (Rapp et al., 1983b; Mark and Rapp, 1984).The genome contains a 5' segment of gag, a 3' segment of pol, and a complete and functional en0 gene. These structural sequences are homologous with the corresponding regions of the Moloney murine leukemia virus (Mo-MLV) genome. The v-rafallele is inserted between the partial complements of gag and pol with a resultant in-frame fusion of g a g and v-raf reading frames. Assuming that the unsequenced part of the 3611-MSV gag complement is isogenic to the corresponding region of the Mo-MLV g a g gene, the gag-ruf reading frame encompasses 384 gag codons including the start codon and 326 v-rufcodons, and specifies a 710-amino acid protein with a calculated molecular weight of -79,500 (Mark and Rapp, 1984; Bonner et al., 1985). The corresponding translational product of genome-sized mRNA detected in 3611-MSV-transformed cells has an apparent molecular weight of 75,000 (Rapp et al., 1983a). A minor protein of 90,000 molecular weight, p90Bagraf,is also observed in addition to p75gagmrafin 3611-MSV-transformed cells (Rapp et aZ., 1983a). Since ~ 7 5 @ g - ~phosphorylated ~fis (see below) and myristylated, and is glycosylated (Schultz et al., 1985), the two protein species apparently correspond in translational initiation and posttranslational modification to the P&5g0g and gPr8oBw proteins, respectively, of nondefective murine leukemia viruses (Dickson et al., 1982; Schultz and Oroszlan, 1983). Both ~ 7 5 g ~ g ~ ~p9@wraf ~ f a n d can be immunoprecipitated using antisera raised against g a g proteins (Rapp et al., 1983a) or against v-raf-specific synthetic peptides (Rapp et al., 1985; Schultz et al., 1985). The close homology between the nucleotide sequences of the avian v-mil and the murine v-raf alleles was originally discovered by nucleic acid blot hybridization and heteroduplex analyses (Jansen et al., 1984a; Bister et al., 1984),and was confirmed by nucleotide sequence analyses (Sutrave et al., 1984a; Kan et al., 1984). The two alleles share homologous coding domains including a conserved translational termination codon (Figs. 4 and 5). The 3' untranslated sequences, only 12 base pairs in v-mil and about 180 base pairs in v-mJ show no homology. The v-mil coding domain contains 379 codons, and the vraf coding domain encompasses 326 codons. The additional 54 v-mil codons are all located toward the 5' terminus of the avian allele (Figs. 4 and 5), and v-rafcontains in its 3' domain one codon that is deleted in the aligned v-mil allele, For the shared coding domains, the homology was found to be 80% for the nucleotide sequences, and 94% for the predicted amino acid sequences, confirming the conclusion that v-

ONCOGENES IN RETROVIRUSES AND CELLS

135

mil and v-raf were derived from cognate cellular genes of avian and mammalian species (Jansen et al., 1984a). 2. Function Mutants in oncogenic function have not been described yet for 3611-MSV7and genetic definition of v-mil function is considerably complicated by the fact that the MH2 genome harbors another cellderived gene for which autonomous oncogenic function is already known (compare Section 111). In fact, the homology between v-mil and v-ra., the single oncogene of 3611-MSV7is the strongest circumstantial evidence that v-mil presumably contributes to the oncogenic specificities of MH2. A spontaneously generated variant of MH2, MH2YS3, was reported to be as oncogenic as wild-type MH2 although it did not direct synthesis of plOOgag-miz in transformed cells. However, subcloning experiments revealed that MH2Y S3 stocks contained a mixture of viruses, some encoding plOO~'~-miz, others not, and hence the correlation between phenotype and genotype and the nature of the possible lesion in MH2YS3 are not clear (Pachl et al., 1983a). Two recently isolated spontaneous variants of MH2, well defined by molecular cloning of their proviral DNAs, and several mutants constructed by in uitro mutagenesis of cloned MH2 proviral DNA were used to define possible contributions of v-mil to the oncogenic potential of MH2. One of the spontaneous variants, MH2D12, has lost nearly all sequences of v-mil by apparent recombination between short homologous segments of gag and mil sequences in MH2, but has retained a functional v-myc gene (Jansen et al., 1985a), and the other variant, MH2E21, was apparently generated by encapsidation and reverse transcription of subgenomic v-myc mRNA (Patschinsky et al., 1986a). Both mutants are capable of transforming cultured fibroblasts, although the morphology and proliferation rate of such cells appear to differ from those observed for wild-type MH2-transformed fibroblasts (Jansen et al., 1985a; Patschinsky et al., 1986a). Hence, v-mil expression is not necessary for transformation of fibroblasts by MH2, but transformation-associated parameters may be differentially affected in cells transformed by viruses containing the v-myc or both the v-myc and the v-mil oncogene (see also Section VI1,C). Essentially the same results in fibroblast transformation were obtained with mutants generated in uitro by construction of out-of-frame deletions in the v-mil domain of cloned MH2 proviral DNA (Jansen et al., 1985b; Bechade et al., 1985; Zhou et al., 1985).The spontaneous mutant MH2D12 and the constructed v-mil deletion mutants are also capable of inducing

136

KLAUS BISTER AND HANS W. JANSEN

transformation of macrophage-like cells in chicken bone marrow transformation assays. However, mutant-transformed macrophage cultures are growth factor dependent, like cultures transformed by other viruses containing the v-myc allele only, whereas macrophages transformed by MH2, containing v-mil and v-myc, are growth factor independent, Furthermore, the mutant viruses appear to be remarkably less oncogenic than MH2 when injected into chickens (Graf et al., 1986; see also Section VI1,C). Surprisingly, spontaneous or constructed v-myc deletion mutants of MH2 retaining a complete v-mil allele were shown to lack the ability to induce morphological transformation of avian fibroblasts (Bechade et al., 1985; Zhou et al., 1985; T. Patschinsky, H. W. Jansen, and K. Bister, unpublished data). This is in marked contrast to the transforming ability of v - r a . which apparently is sufficient for the transformation of mammalian fibroblasts (Rapp et al., 1983a; see above). Whether this difference in oncogenic potential is due to the more extensive truncation of the transduced murine allele (see Figs. 4 and 5), leading to more efficient oncogenic activation, or due to the different environments in the avian or mammalian cells is not known. The biochemical functions of v-mil and v-raf protein products responsible for or involved in cell transformation are unknown. The first hint at a possible function came from the observation that the predicted amino acid sequences of v-mil(ruf3 gene products show partial homologies with the predicted sequences of proteins specified by the oncogenes belonging to the src family (compare Table 11) (Sutrave et d.,1984a; Mark and Rapp, 1984; Galibert et al., 1984). The sequence homology is highest (ranging from 25 to 35%) with the protein domains presumably bearing the tyrosine-specific kinase activity associated with the protein products of most members of the src gene family (Hunter, 1984). Based on these structural homologies, the mil(raf) oncogene is grouped into the src family (Table 11), although tyrosinespecific kinase activity could not be demonstrated for the v-mil(raf) gene products (Sutrave et al., 1984a; see below). Both plOOgap-miz and ~75g~R-'~f are located predominantly in the cytoplasm of transformed cells (Hann et al., 1983; Blasi et al., 1985; Cleveland et al., 1986) and are phosphoproteins containing predominantly phosphoserine residues and very low amounts of phosphothreonine residues (Ramsay et al., 1982b; Rapp et al., 1983a).Also, both proteins become phosphorylated when immunoprecipitates are incubated in uitro with radioisotopically labeled ATP; however, phosphate is now predominantly transferred to threonine residues and less frequently to serine residues (Sutrave et al., 1984a). The protein kinase activity associated with preparations of these proteins was also shown to act on exoge-

ONCOGENES IN RETROVIRUSES AND CELLS

137

nous substrates added to the in uitro phosphorylation assay and to copurify in immunoaffinity purifications with a lipid kinase activity of apparently cellular origin (Molling et al., 1984). Whether the in uitro protein phosphorylation is due to an intrinsic enzymatic activity of the v-mil(raf3 protein products reflecting their relevant in viuo activity is unknown. Notably, in uitro serine- or threonine-specific kinase activity has also been reported to be associated with the protein products of v-mos alleles (Kloetzer et al., 1983, 1984; Maxwell and Arlinghaus, 1985) or of v-ras alleles (Shih et al., 1980).

B. THEc-miZ(raf3 GENE 1 . Structure and Expression The presence in genomic DNA of avian and mammalian species of sequences closely related to v-mil(raf3 sequences was originally detected by nucleic acid hybridization (Jansen et al., 1983b; Rapp et al., 1983b; Coll et al., 1983a). The chicken c-mil gene was then extensively characterized by molecular cloning, heteroduplex analysis, and nucleotide sequencing (Jansen et al., 198313, 1984b; Jansen and Bister, 1985). The v-mil nucleotide sequences are derived from 11exons of the cellular gene, ranging in size from 28 to 177 base pairs (Fig. 5). All exon-intron boundaries of c-mil, except the 5' boundary of exon 1 and the 3' boundary of exon 11, were unambiguously defined by the identification of consensus splice donor and acceptor sites precisely at positions where homology to v-mi2 ceases or resumes. The homology to v-mil starts within the coding sequence of exon 1 and ends within the 3' untranslated region of exon 11 (see Fig. 5, and above). The nucleotide sequences of v-mil and c-mil are very closely related (see above), and the open reading frames of both the transduced and the cellular allele terminate at a common stop codon. Hence, the carboxylterminal domains of plOWag-m'z(Fig. 4) and of the c-mil protein are nearly identical. However, the cellular gene product contains aminoterminal protein sequences encoded by c-mil 5' coding domains which were not transduced into the v-mil allele. The chicken c-mi2 mRNA has a complexity of -4.0 kilobases (Coll et al., 1983a; Flordellis et al., 1985) and encodes a protein with an apparent molecular weight of 71,000/73,000, p71/73'-"", detected in extracts of chicken cells by immunoprecipitation using antisera raised against v-mil(raf3specific protein domains and which was shown to be structurally related to the v-mil protein product (Patschinsky et al., 1986b). The vmil-homologous region of the chicken c-mil gene has a coding capacity of 379 amino acid residues (see above), corresponding to a polypeptide with a calculated molecular weight of -42,000. Hence,

138

KLAUS BISTER AND HANS W. JANSEN

the c-mil gene apparently contains additional 5‘coding sequences of about 850 base pairs and additional 3‘and presumably 5’untranslated sequences. This is in good agreement with the structural analyses of chicken c-mil cDNA clones (C. Trachmann, M. Koenen, and K. Bister, unpublished data) and of human c-mil(raf3 genomic and cDNA clones (see below). On the chicken genome, c-mil is located distant from cmyc (Jansen et ul., 1983b,1984b),and in mice and men the two loci are asyntenic (see below and Section 111). The progenitor of v-ruf, the mouse c-rufgene, is located on chromosome 6 (Kozak et ul., 1984).Although the structure of mouse c-rufhas not been reported in detail, the structural homologies between v-mil and v-ruf(Figs. 4 and 5)and between v-rufand human c-mil(ruf) (see below) indicate that v-rufis derived from the nine 3’most exons of mouse c-ruf. In man, the active c-mil(ru. gene is located on chromosome 3, and an apparent pseudogene is located on chromosome 4 (Bonner et ul., 1984). The structural organization of the human cmil(ruf) gene is very similar to that of the chicken gene (Bonner et ul., 1985).The coding domain homologous to v-mil (v-rufl is organized in 11 (9)exons exactly as in the chicken gene (Fig. 5). The predicted amino acid sequence differs at 21 out of 379 positions from that of vmil (17 of these changes and one additional one are also found in chicken c-mil) and, like in the v-rufpredicted sequence (see above), contains an additional amino acid residue near the carboxyl terminus. At only 7 out of 326 positions, differences are found between the predicted amino acid sequences of the shared coding domains of human c-mil(ruf) and v-ruf. The very 3’exon of the human gene, corresponding to Ell in the diagram of the chicken gene (Fig. 5),contains the translational termination codon conserved in all transduced and cellular mil(ru. alleles and an untranslated sequence of about 900 base pairs defined by the identification of a polyadenylation signal in the genomic clone and by comparison with the structure of a human cmil(ru.cDNA clone (Bonner et ul., 1985,1986).The sequence analysis of the cDNA and comparative analyses of the genomic locus (Bonner et d., 1986) indicated that additional coding sequences are organized in five more exons, located 5‘ of the 11 exons defined by homology to v-mil or v-ruf, and also revealed the presence of a very 5’ untranslated exon. Hence, the human c-mil(ruf) gene (and presumably also the chicken and the mouse homologs) contains 17 exons, 16 of which are coding. The sequence analysis of the cDNA predicts that the human cmil(raf3 protein product contains 648 amino acid residues and that it has a calculated molecular weight of -73,000 (Bonner et ul., 1986).

ONCOGENES IN RETROVIRUSES AND CELLS

139

This is in good agreement with the apparent molecular weight of 71,000/73,000 observed for the human, rat, and mouse c-mil proteins (Patschinsky et al., 1986b) and for the homologous chicken protein (see above; Patschinsky et al., 1986b). The sizes reported for human cmil(ra.. mRNA (-3.6 kilobases; Coll et al., 1983a) and for mouse c-ru.. mRNA (-3.5 kilobases; Molders et al., 1985) are compatible with the complexity of the human cDNA clone (-3.0 kilobases; Bonner et al., 1986). 2. Function

The physiological role of the c-miZ(raf3 gene in normal cells and the biochemical function of its protein product are unknown. It is well conserved among avian and mammalian species and expressed in various tissues (Coll et al., 1983a; Patschinsky et al., 198613). The cmil(raf) protein products are cytoplasmic phosphoproteins (Patschinsky et al., 1986b). It has been reported that the predicted amino acid sequence of the v-raf protein, and hence that of the c-mil(ra.) protein in the shared domain, is partially (24%) homologous to the primary structure of a cell division control gene (CDC28) product of yeast (Lorincz and Reed, 1984). Whether this sequence homology extends to any functional properties is unknown. Oncogenic activation (other than by transduction) of c-mil(ra. has been inferred from experiments in which augmented expression of partial complements of the gene has been mediated by linkage to retroviral control elements. It was shown that human c-mil(raf) DNA can substitute for the homologous 3’ domain of v-raf in 3611-MSV proviral DNA (lacking the 3‘ terminus) with a resultant transforming activity in DNA transfection assays, albeit with much lower efficiency than that obtained with authentic 3611-MSV DNA (Bonner et al., 1985). In a variation of the insertion mutagenesis theme, it was reported that transfection of LTR DNA into NIH/3T3 cells in one case led to the isolation of a transformed cell clone in which the LTR sequences had integrated in the same transcriptional orientation into the fifth intron of the mouse c-raf gene (Muller and Miiller, 1984; Molders et al., 1985). The transformed cells contained amplified levels of truncated c-raf transcripts initiated at the retroviral promoter and high levels of truncated c-raf protein products corresponding in size approximately to the protein domain encoded by v-mil. These results are consistent with the observation that the internal LTR integration site was located 5’ of the region in mouse c-raf that is homologous to v-mil (Molders et al., 1985). Recently, the human c-mil(raf3 gene has been identified in transforming DNA sequences isolated

140

KLAUS BISTER AND HANS W. JANSEN

from human cancer cells derived from cases of stomach cancer and glioblastoma (Shimizu et al., 1985; Fukui et al., 1985). V. The erbB and erbA Oncogenes

Specific nucleic acid sequences (Bister and Duesberg, 1979; Lai et al., 1979; Roussel et al., 1979) and protein products (Hayman et al,, 1979a; Privalsky and Bishop, 1982) of the erbA and erbB oncogenes were originally discovered for the transduced alleles in the genomes of the apparently identical strains R and ES4 of acute leukemia virus AEV. Since v-erbA and v-erbB form a nearly contiguous stretch of cell-derived sequences in the AEV-R(ES4) genome (see below), they were first believed to represent a single transformation-specific region (v-erb). Recognition of independent expression of the 5‘ end 3’ “domains” of v-erb and of their origin from unlinked chicken loci (c-erbA and c-erbB) led to the designations erbA and erbB (Coffin et al., 1981). Hence, the potentially misleading similarity between these gene designations has only historical reasons and does not reflect any structural or functional relationship. A. THEv-erbB ALLELESIN AEV-R AND AEV-H AND THE v-erbA ALLELE IN AEV-R

I . Structure and Expression The structures and modes of gene expression of the v-erbB alleles in the genomes of AEV-R and AEV-H are shown in Fig. 6. The structural features shown here for AEV-R were elucidated using both the R or the ES4 strain of AEV. Since these strains are identical in structure (Hayman et al., 1979a; Nishida et al., 1984) and biological properties (see Section I), only the term AEV-R will be used throughout this section for structural distinction from AEV-H. The genetic structure of AEV-R was originally determined by mapping of TI-oligonucleotides of viral RNA and heteroduplex analysis (Bister and Duesberg, 1979, 1980; Lai et al., 1979), and then confirmed and refined by molecular cloning and nucleotide sequence analyses of proviral DNA (Vennstrom et al., 1980; Nishida et al., 1984; Debuire et al., 1984; Henry et al., 1985). The AEV-R genome contains a 5’ segment of gag, a 3’ segment of env, and a very short region of env-related sequences interspersed between the v-erbA and v-erbB alleles (Fig. 6). At its 5’ terminus the v-erbB allele contains a segment apparently derived from c-erbB intron sequences directly adjacent to the 5’ terminus of a c-erbB exon including the splice acceptor site (Fig. 6) (Henry et al.,

ONCOGENES IN RETROVIRUSES AND CELLS

UAG SA (AUGI

+5

(UGA)

Aenv

Aenv

p75

AEV- R AUG< >AUGi

UGA

Aenv

P62

-

UAA

Aenv

AEV-H

AUG, jAUG)

, 1kb ,

+

I!

Aenv

i

P67

FIG.6. Structure and expression of v-erbB and v-erbA alleles. Genome-sized and subgenomic mRNAs of viruses from the AEV subgroup (compare Table I), aligned with respect to homologous v-erbB sequences, and their v-erbB and v-erbA protein products are shown. Nucleotide sequences transduced from the chicken c-erbB or the c-erbA gene are indicated by hatching or stippling, respectively. For the explanation of further symbols, see legend to Fig. 2.

1985). At its 3' terminus, the junction between v-erbB and enu sequences apparently corresponds to sequences within a coding exon of c-erbB (Nishida et al., 1984). Partial or preliminary nucleotide sequence analyses of the remainder of the AEV-R v-erbB allele indicate that it is closely related to the completely sequenced v-erbB allele of AEV-H (see below), but that it is distinguished from the homolog in AEV-H by an internal deletion of 21 codons near the 3' terminus and by the lack of 39 codons at the 3' terminus (Nishida et al., 1984; Privalsky et al., 1984; Schatzman et al., 1986; M. Hayman, personal communication). Expression of the v-erbB allele of AEV-R by translational initiation at the gag AUG start codon is prohibited by the presence of an in-frame stop codon within the 5' intron-derived v-erbB sequences (Fig. 6; see below). Instead, the cell-derived splice acceptor site is utilized for the generation of a subgenomic mRNA species with a complexity of -3.5 kilobases (Fig. 6), which was observed

142

KLAUS BISTER AND HANS W. JANSEN

in AEV-R-transformed cells in addition to genome-sized RNA (Anderson et al., 1980; Sheiness et al., 1981; Saule et al., 1981).Predicted from the nucleotide sequence analysis of the 5' domain of v-erbB (Debuire et al., 1984; Henry et al., 1985), the six gag codons upstream of the presumed splice donor site (see above) would be fused in-frame to the v-erbB coding sequences (Fig. 6). An AUG codon is present at the sixth codon position of the v-erbB coding domain. This AUG apparently is not the translational initiation codon utilized in the synthesis of the cellular c-erbB protein product (see below). It has not been directly determined yet whether translation of the v-erbB mRNA initiates at the gag or at the v-erbB AUG codon. The open reading frame on this mRNA encompasses the six gag codons and 545 v-erbB codons (M. Hayman, personal communication), and terminates at a UGA stop codon which is located immediately 3' of the v-erbBlenu junction and which is out-of-frame with respect to the enu coding sequence (Nishida et al., 1984). Assuming initiation at the gag AUG codon, the predicted translational product of this mRNA is a 551-amino acid protein with a calculated molecular weight of -61,700. The corresponding protein was identified by in uitro translation of apparently virionencapsidated mRNA, and by immunoprecipitation from extracts of AEV-R-transformed cells, using either antisera from tumor-bearing rats which had been inoculated with rat cells transformed in uitro by AEV-R or sera raised against proteins specified by v-erbB DNA expressed in bacteria (Privalsky and Bishop, 1982; Hayman et al., 1983; Privalsky et al., 1983). The primary translational product with an ap~ 6), is subsequently parent molecular weight of 62,000, ~ 6 2 ' " ' ~(Fig. modified by phosphorylation and glycosylation (see below). The genetic structure of AEV-H (Fig. 6) was determined by molecular cloning and nucleotide sequence analysis of proviral DNA (Yamamot0 et al., 1983a,b; Nishida et al., 1984). The genome contains a complete gag gene, a 5' segment of the poZ gene, and a 3' segment of the env gene. The v-erbB allele is inserted between the partial pol and enu genes. The gag gene directs synthesis of the standard Pr76gag precursor protein, and hence AEV-H-transformed nonproducer cells release noninfectious virus particles (Yamamoto et al., 1983~). Surprisingly, these particles contain functional reverse transcriptase, although the pol gene is deleted at its 3' end with a resultant loss of 36 codons and of the natural pol termination codon. The reading frame on the AEV-H mRNA encoding the polymerase precursor starts at the gag AUG, bypasses the gag stop codon (see Sections I1 and 111),and continues through the pol sequences and then for six codons into intron-derived v-erbB sequences, before it terminates at a UAA stop

ONCOGENES IN RETROVIRUSES AND CELLS

143

codon (Fig. 6). Apparently, the resultant amino-terminal modification of the gag-pol precursor protein does not affect either the processing of this protein or the function of the mature reverse transcriptase (Yamarnoto et al., 1983b,c). A t its 5' terminus, the structure of the AEV-H v-erbB allele is very similar to that of v-erbB in AEV-R. The only distinction is that the allele in AEV-H contains a smaller and internally deleted complement of intron-derived sequences. However, both v-erbB alleles contain the same splice acceptor site at the 5' border of the shared coding domains (Fig. 6) (Yamamoto et al., 1983b; Henry et al., 1985). In the AEV-H genome, there is another splice acceptor site located just upstream of the pollv-erbB junction (Fig. 6) which is utilized for the generation ot subgenomic e m mRNA in nondefective viruses (Schwartz et al., 1983). It has been postulated that this splicing signal is used for the generation of subgenomic v-erbB mRNA which is observed in AEV-H-transformed cells (Yamamoto et al., 1983a,b,c). However, utilization of the cell-derived splice acceptor appears equally plausible and was in fact directly demonstrated by transcriptional mapping in the case of the v-erbB mRNA of AEV-R (Henry et aZ., 1985). The putative structure of the AEV-H subgenomic mRNA generated by usage of the downstream splice acceptor is shown in Fig. 6. The open reading frame on this mRNA encompasses the first six gag codons including the AUG, 605 v-erbB codons, and 4 enu codons (Yamamoto et al., 198313).The env codons and the UAG stop codon (Fig. 6) are out-of-frame with respect to the env coding sequence. As in the case of AEV-R, translation of this mRNA species could either initiate at the gag AUG or at the v-erbB AUG at the sixth codon position downstream of the cell-derived splice acceptor site. If the upstream splice acceptor is actually used for the generation of the mRNA, then frame shifting and stop codons in the intron-derived verbB sequences would not allow translation of the coding v-erbB sequences by initiation at the gag AUG, but would predict usage of the v-erbB AUG (Yamamoto et al., 1983b). Assuming utilization of the downstream splice acceptor and initiation at the gag AUG, the predicted translational product of the subgenomic v-erbB mRNA of AEVH is a 615-amino acid protein with a calculated molecular weight of -69,000. In transformed cells, a protein with an apparent molecular weight of 67,000, ~67'"'&~,was identified using the specific antisera described above in immunoprecipitation experiments (Nishida et al., 1984). The protein is further modified like p62v-erbB of AEV-R (see below). The 5' border of the v-erbA allele in AEV-R is well defined by its

144

KLAUS BISTER AND HANS W. JANSEN

fusion with recognizable gag sequences (Debuire et al., 1984). Between its 3’ end and the 5’ end of v-erbB, unambiguously defined by comparison with the nucleotide sequence of chicken c-erbB, is a stretch of 30 nucleotides with a sequence homology of 82% with a segment of the RSV enu gene (Henry et al., 1985). Clearly, the localization of the exact 3’ border of v-erbA and a more definitive proof that viral (Aenu) sequences are interspersed between v-erbA and v-erbB (Fig. 6) will depend on nucleotide sequence analysis of the corresponding region of the chicken c-erbA locus. However, the structure as depicted in Fig. 6 is supported by the recent observation that sequence homology between v-erbA and the human c-erbA allele only extends to a position in v-erbA 5’ of the presumed Aenv sequences (D. Stehelin, personal communication). Expression of the v-erbA allele of AEV-R is mediated by translation of genome-sized mRNA initiating at the gag AUG start codon. Assuming that the unsequenced portion of the AEV-R gag complement is isogenic to the corresponding region of the RSV gag gene, the open reading frame encompasses 255 gag codons, 383 v-erbA codons, and 15 codons provided by the enu-related sequences (which are out-of-frame with respect to the RSV env gene), and by intron-derived v-erbB sequences which also provide the UAG stop codon (Fig. 6) (Henry et al., 1985). The calculated molecular weight of the predicted 653-amino acid protein product is 72,000. The corresponding translational product of genome-sized mRNA has an apparent molecular weight of 75,000 (p75@’K-er’’A; Fig. 6), and was detected by immunoprecipitation from extracts of AEV-R-transformed cells using antisera against gug proteins (Hayman et al., 1979a; Rettenmier et ul., 1979; Kitchener and Hayman, 1980). 2. Function There is clear evidence from the analyses of conditional and nonconditional mutants of AEV strains that v-erbB is the dominant oncogenic determinant of these viruses. In fact, the genetic structure of the AEV-H strain (Fig. 6) which induces both erythroblastosis and sarcoma (compare Section I) (Hihara et al., 1983) is the most obvious illustration that v-erbA is not necessary, and that v-erbB is necessary and possibly sufficient, for the induction and maintenance of erythroid and fibroblastic cell transformation. A nonconditional mutant of AEV-H, td (transformation-defective)-130,was isolated that has retained the ability to transform cultured avian fibroblasts and to induce sarcomas in chickens, but has lost the capacity to induce erythroblastosis (Yamamoto et al., 1983a). The lesion was identified as an out-offrame deletion of 169 base pairs from the 3’ domain of v-erbB, predict-

ONCOGENES IN RETROVIRUSES AND CELLS

145

ing a mutant protein product which lacks 192 authentic amino acid residues at the carboxyl terminus (Yamamoto et al., 1983b). As in the case of the partially transformation-defective deletion mutants of MC29 (see Section 111), it is not clear whether the differential loss of oncogenic potential in td-130 is due to the inactivation of one of several functional domains encoded by v-erbB, or due to a quantitative change in activity of a functionally unaltered mutant protein. A nonconditional mutant with a very similar phenotype, called td-359, was described for AEV-R (Royer-Pokora et al., 1979). Although the lesion in td-359 was originally believed to reside only in v-erbA (Beug et al., 1980), it is now clear that v-erbB of td-359 is also affected (T. Graf, personal communication). The biological properties of mutants constructed in vitro by the introduction of large deletion or frame shift mutations in the v-erbA or v-erbB allele of AEV-R proviral DNA are consistent with the notion that the main transforming potential is encoded by v-erbB (Frykberg et al., 1983; Sealy et al., 1983a,b). Mutations in the v-erbB allele, except when they are very close to the 3' terminus, abolish all celltransforming capacities of AEV-R. In contrast, mutants that have suffered large deletions in the Agag-v-erbA domain are still capable of transforming fibroblasts and erythroid cells in uitro, and some of them induce erythroid leukemia or sarcomas in chickens. However, the efficiency of cell transformation and the differentiation phenotype of the leukemic cells are different from those observed with wild-type AEV-R. This may suggest an enhancing effect of the v-erbA gene product in AEV-R-induced leukemogenicity (see also Section VII). Conditional mutants of AEV-R have also been isolated (Graf et al., 1978; Palmieri et al., 1982). These temperature-sensitive (ts) mutants transform erythroid cells at 37°C but not at 42"C, and have a reduced leukemogenic potential presumably due to the elevated body temperature of chickens (41.5"C). Erythroid cells transformed by tsAEV-R mutants at 37°C terminally differentiate into mature erythrocytes when they are shifted to the nonpermissive temperature, provided that the culture medium contains an erythropoietin-like component needed for the promotion of erythroid differentiation (Graf et al., 1978; Beug et al., 1982a). The terminal differentiation is accompanied by changes in globin gene chromatin structure, increased transcription of the gene, and accumulation of hemoglobin (Weintraub et al., 1982). In contrast, erythroblasts transformed by wild-type AEV-R cannot be induced to differentiate at either temperature (Graf et al., 1978). These observations indicate that the transforming principle of AEV-R, apparently the v-erbB protein product (see above), is neces-

146

KLAUS BISTER AND HANS W. JANSEN

sary for the induction and maintenance of a state of erythroid progenitor cells that allows them to proliferate but not to differentiate terminally. The tsAEV-R mutants are also temperature-sensitive for the transformation of fibroblasts, albeit not in all parameters of cell transformation (Beug and Graf, 1980; Palmieri et al., 1982). Therefore, disregarding the unlikely possibility that all these mutants contain multiple lesions, their phenotype confirms that the basic capacity of AEV-R for both fibroblastic and erythoid cell transformation is encoded by one gene, i.e., the v-erbB allele (see above). The biochemical functions of v-erbB protein products and the mechanisms leading to cell transformation have not yet been identified unequivocally. However, the discoveries of structural homology of v-erbB with oncogenes of the src family (Yamamoto et al., 1983b; Privalsky et al., 1984) and of its strikingly close relationship with a segment of the human gene encoding the epidermal growth factor (EGF) receptor (see below) (Downward et al., 1984; Ullrich et al., 1984) provided a strong basis for assessing the functional significance of some of the biochemical properties of v-erbB protein products. The primary translational product of the v-erbB allele of AEV-R, p62v-erbB (Fig. 6), is modified by glycosylation and phosphorylation through intracellular membrane-located precursors of apparent molecular weights of 66,000 and 68,000, gp66V-erbB and gp68v-erbB, respectively, to a mature integral plasma membrane glycoprotein of 74,000 molecular weight ( g ~ 7 4 " - ~ 'which ~ ~ ) , is expressed at the cell surface (Hayman et al., 1983; Privalsky et al., 1983; Hayman and Beug, 1984; Privalsky and Bishop, 1984). A possible further processing product of 82,000 molecular weight has recently been detected using antisera raised against the human EGF receptor protein for immunoprecipitation of proteins from a specific line of AEV-R-transformed fibroblasts (Decker, 1985). In tsAEV-R-transformed cells, processing of the verbB protein product is arrested at the immature gp68v-erbB form at the nonpermissive temperature and surface expression is abolished, implying that it is the mature gp74v*rbBform which may be critically involved in cell transformation (Beug and Hayman, 1984). However, this view had to be refined in the light of the surprising observation that incorrectly glycosylated v-erbB protein products synthesized by AEV-R-transformed cells incubated with glycoprotein processing inhibitors are apparently routed normally to the plasma membrane and have full transforming activity. Hence, correct glycosylation is apparently unimportant for subcellular routing and biological activity, and the lesions in the v-erbB protein products of the t s mutants presumably impair primarily the insertion into the plasma membrane with a

ONCOGENES IN RETROVIRUSES AND CELLS

147

secondary effect on glycosylation (Schmidt et al., 1985). In AEV-Htransformed cells, ~ 6 7 ' ~(Fig. ' ~ ~6) is processed into the glycosylated form gp72v-mbB, presumably corresponding to gp68v-e'bBspecified by AEV-R (Nishida et al., 1984). Further processing of gp72 v-erbB has not been described yet. The predicted amino acid sequences fiom the central domains of verbB protein products show considerable homology (34-38% on average, and up to 50% in local regions) with the protein domains presumably bearing the tyrosine-specific kinase activity associated with the protein products of most members of the src gene family (Yamamoto et al., 1983b; Privalsky et al., 1984). Indeed, tyrosine-specific protein phosphorylation has recently been implicated in v-erbB-induced cell transformation. The 68,000 and 74,000 molecular weight species of AEV-R v-erbB protein products and the corresponding proteins of AEV-H were found to become phosphorylated in vitro at tyrosine residues when immunoprecipitates were incubated with [y-32P]ATP (Kris et al., 1985; Decker, 1985). Either polyclonal antisera against the human EGF receptor or a serum against a synthetic peptide from the cytoplasmic domain of this receptor protein were used in these experiments. Furthermore, it was reported that the v-erbB protein products and cellular proteins became phosphorylated in vitro at tyrosine residues when membrane preparations from AEV-R-transformed cells were incubated with [y-32P]ATP,and that enhanced in vivo tyrosine phosphorylation of cellular proteins was observed in these cells (Gilmore et al., 1985). The v-erbB-encoded protein products, however, are phosphorylated in vlvo predominantly at serine residues and to a very low extent at threonine and tyrosine residues (Gilmore et al., 1985; Decker, 1985).Analyses of conditional and unconditional mutants of AEV-R failed to reveal any apparent correlation between associated kinase activity and transforming function of v-erbB protein products (Hayman et al., 1986). The biochemical function of the protein product of the v-erbA allele of AEV-R is unknown. The p75@"'-erbAprotein is a cytoplasmicphosphoprotein containing predominantly phosphoserine residues (Bister et al., 1980b; Anderson and Hanafusa, 1982; Abrams et al., 1982; Hayman et al., 1983). It has been reported that the predicted amino acid sequence of the v-erbA protein product shows statistically significant homologies to the sequences of carbonic anhydrases (Debuire et al., 1984) or of human steroid receptors (Weinberger et al., 1985; Green et al., 1986; Greene et al., 1986). Whether these structural homologies extend to any functional properties is unknown.

148 B. THEc-erbB

KLAUS BISTER AND HANS W. JANSEN AND

c-erbA GENES 1. Structure and Expression

The presence in normal chicken and other vertebrate DNA of sequences related to the combined v-erbA and v-erbB sequences was originally detected by nucleic acid hybridization using cDNA probes containing all cell-derived sequences of the AEV-R genome (Roussel et aZ., 1979; Saule et al., 1981; Wong et al., 1981). The c-erbA and cerbB genes represent distant loci and are even located on different chromosomes in some, but not all species (see below). The chicken c-erbB locus located on a large chromosome (Symonds et al., 1984a) was molecularly cloned and analyzed by restriction enzyme cleavage site mapping and heteroduplex formation with v-erbB DNA or RNA (Vennstriim and Bishop, 1982; Sergeant et al., 1982). Distributed over approximately 20 kilobase pairs of the chicken genome, 12 regions of homology to v-erbB were defined by the heteroduplex analyses. Complete nucleotide sequence analysis of the chicken c-erbB gene is not yet available. Based on the homology to the human EGF receptor gene (see below) and on the complexity of transcriptional and translational products, it is clear that the chicken cerbB gene extends 5' and 3' from the region defined by homology to verbB (Nilsen et al., 1985). Two v-erbB-related transcripts with complexities of approximately 9.0 and 12.0 kilobases, respectively, were observed in chicken cells (Vennstrom and Bishop, 1982). The presumed translational product of chicken c-erbB mRNA, a protein with an apparent molecular weight of 170,000, was identified by immunoprecipitation from solubilized chicken liver cells using antibodies against a synthetic peptide from the human EGF receptor and subsequent in vitro phosphorylation (Kris et al., 1985). The human c-erbB gene was found to be located on chromosome 7 (Spurr et al., 1984; Zabel et al., 1984a) in the same region that had previously been identified as the chromosomal localization of the EGF receptor gene (Shimizu et al., 1980). Human genomic DNA clones presumably containing the c-erbB locus have been isolated but were not further characterized (Jansson et al., 1983). The recognition of the apparent identity (or close homology) of the human c-erbB locus with the gene encoding the EGF receptor (or a closely linked gene) greatly facilitated the structural analysis of c-erbB. The close homology between v-erbB and the human EGF receptor gene was first discovered by comparison of the predicted amino acid sequence of the AEV-H v-erbB protein product (see above) with the sequences of

ONCOGENES IN RETROVIRUSES AND CELLS

ATG

, 0.5 kb,

149

TGA

L

homology t o v-erbB

J

FIG.7. Structure of a cDNA clone of the human epidermal growth factor receptor (HER) gene. The complexity of the protein coding domain is indicated by the position of translational initiation and termination codons. Regions encoding the extracellular, the transmembrane, or the cytoplasmic domains of the receptor protein are shown as cross-hatched, black, or hatched boxes, respectively. The stippled box represents sequences encoding a signal peptide. The borders of homology to the avian v-erbB allele are indicated.

several peptides derived from the human receptor protein (Downward et uZ., 1984). The EGF receptor was purified by monoclonal immunoaffinity chromatography from human placental tissue or from the human epidermoid carcinoma cell line A431 containing dramatically increased EGF receptor levels. The partial amino acid sequence of an A431 EGF receptor cyanogen bromide peptide was used to design a long synthetic DNA hybridization probe for screening of placental and A431 cDNA libraries (Ullrich et al., 1984). Nucleotide sequence analysis of overlapping cDNA clones revealed the complete structure of human EGF receptor cDNA shown in Fig. 7. The open reading frame encodes a 1210-amino acid E G F receptor precursor polypeptide containing a 24-amino acid signal peptide sequence and a 1186-amino acid sequence of the mature receptor. This agrees well with the apparent molecular weight of 138,000 observed for the polypeptide backbone of the 175,000-molecular weight glycosylated receptor protein (Mayes and Waterfield, 1984; Downward et al., 1984). As was observed for c-erbB in chicken cells (see above), two EGF receptor cDNA-related transcripts were detected in human cells, with complexities of 5.8 and 10.5 kilobases, respectively (Ullrich et al., 1984). The complexity of the smaller mRNA species is consistent with the size of the cDNA clone (Fig. 7). It is not clear whether the two transcripts result from transcription of two closely related genes, or whether they represent differentially terminated or processed transcripts of the same gene. Preliminary analyses indicated that the gene corresponding to the 5.8-kilobase transcript is greater than 50 kilobase pairs in size (Ullrich et al., 1984). The region of homology between the EGF receptor cDNA and v-erbB deduced from the predicted amino acid sequences of the v-erbB protein prod-

150

KLAUS BISTER AND HANS W. JANSEN

uct and of the EGF receptor protein is indicated in Fig. 7. Alignment of the 605-amino acid sequence of the AEV-H v-erbB protein product (see above) with the carboxyl-terminal domain of the EGF receptor sequence revealed a 95% homology for a central 376-amino acid sequence. Sequence conservation is apparent, but reduced in sequences flanking the 376-amino acid core region (Ullrich et al., 1984). The chicken c-erbA locus is located on a microchromosome (Symonds et al., 1984a) and contains four separate regions of homology to v-erbA as determined by heteroduplex analysis of cloned DNAs (Vennstrom and Bishop, 1982). Based on the nucleotide sequence analysis of v-erbA (see above) and on the complexity of transcriptional products, it is clear that the chicken c-erbA gene extends 5' and 3' from the region defined by homology to v-erbA. Two v-erbA-related transcripts with complexities of -3.0 and 4.5 kilobases were detected in chicken cells (Vennstrom and Bishop, 1982). The translational products of these presumed c-erbA mRNAs have not been identified yet. A human gene closely related to v-erbA and a more distantly related locus were molecularly cloned (Jansson et al., 1983). The closely related c-erbA gene is located on chromosome 17 (Spurr et al., 1984; Zabel et al., 1984a), and its presumed transcript was identified as a 5.0-kilobase RNA species (Jansson et al., 1983). Translational products have not been identified. Interestingly, c-erbA and c-erbB loci are syntenic in mice (Zabel et al., 1984a; Silver et al., 1985). However, although physically linked, their distance on mouse chromosome 11 appears to be significant (Zabel et al., 1984a).

2. Function The most plausible interpretation of the observed close homology between v-erbB protein sequences and the human EGF receptor sequence appears to be that the chicken and human c-erbB loci are the genes encoding the avian and the human EGF receptor, respectively. EGF is a mitogenic polypeptide hormone that initiates cellular responses by binding with high affinity to specific receptor molecules on the surface of target cells (Carpenter and Cohen, 1979; Guroff, 1983).Binding of EGF to its receptor triggers a cascade of intracellular events including stimulation of DNA replication and cell proliferation. The temporal order and the causative links of events following EGF binding are unknown. The human receptor molecule has been well characterized (see above). It contains an extracellular ligand binding domain of 621 amino acids, a hydrophobic transmembrane region of 23 amino acids, and a cytoplasmic domain of 542 amino acids

ONCOGENES IN RETROVIRUSES AND CELLS

151

(Fig. 7). Characteristic features of the extracellular domain are the presence of multiple glycosylation sites and the striking clustering of cysteine residues in two separate regions of possibly common evolutionary origin (Ullrich et al., 1984). The intracellular domain shares considerable sequence homology with the putative tyrosine-specific kinase domains of proteins encoded by members of the src gene family (Ullrich et d., 1984), in agreement with the observed homology between v-erbB and the src gene family (Yamamoto et al., 1983b; Privalsky et al., 1984). It has been reported that the human EGF receptor has intrinsic tyrosinespecific kinase activity, measured by in vitro autophosphorylation, which can be stimulated by the addition of EGF to cell membrane preparations or to purified receptor molecules (Cohen et al., 1980; Ushiro and Cohen, 1980). Similar activities have been described for the putative chicken EGF receptor (see above) (Kris et al., 1985). Recently, a Drosophila EGF receptor gene homolog has been isolated which shows a 41% homology of predicted extracellular amino acid sequences with the homologous domain of the human receptor, and shows a 55% homology in the cytoplasmic domain (Livneh et al., 1985). Associated tyrosine-specific kinase activity has also been reported for the human platelet-derived growth factor (PDGF) receptor (Ek et al., 1982), for the insulin receptor (Kasuga et al., 1982), and for the insulin-like growth factor I receptor (Jacobs et al., 1983). For the insulin receptor, structural homologies to the src oncogene family have been documented (Ullrich et al., 1985). Hence, tyrosine-specific protein phosphorylation is believed to be critically involved in mitogenic signal transmission (Hunter, 1984; Heldin and Westermark, 1984; Feramisco et al., 1985). Oncogenic function of nontransduced erbB alleles has not been directly demonstrated, but is inferred from circumstantial evidence. Insertion mutagenesis of the chicken c-erbB locus has been reported as a possible cause of avian leukosis virus-induced long-latency erythroblastosis in a particular line of chickens (151) (Fung et al., 1983; Raines et al., 1985). Except for the remarkable difference in latency (about 70 days for leukosis virus-induced leukemia versus 10 days for AEV-induced leukemia), AEV- or leukosis virus-induced erythroblastoses are diagnostically indistinguishable. In all leukosis virus-induced erythroblastoses a high level of c-erbB expression was observed, and in all leukemic birds tested the c-erbB locus was structurally altered. In most cases, integration of proviral DNA had occurred in the same transcriptional orientation just upstream of the c-

152

KLAUS BISTER AND HANS W. JANSEN

erbB region that is homologous to v-erbB, implying promoter insertion as a possible mechanism for transcriptional activation of a truncated c-erbB gene (Nilsen et al., 1985; Lax et al., 1985). It has recently been reported that leukosis virus-induced erythroblastosis in 151 chickens is associated with a surprisingly high frequency of c-erbB transduction (Miles and Robinson, 1985; Beug et al., 1986). The new viral isolates induce erythroblastosis, but no fibrosarcomas and no transformation of cultured cells. Their genome structures and the modes of expression of the transduced erbB sequences have not been described yet. In the A431 human carcinoma cell line (see above), the EGF receptor gene is amplified, rearranged, and transcribed at normal levels into authentic receptor mRNAs (see above) and at high levels into aberrant mRNA species encoding only the extracellular domain of the receptor. How this relates to the increased EGF receptor levels in A431 cells or to the neoplastic character of this cell line is unknown (Ullrich et al., 1984; Merlin0 et al., 1985). The physiological role of c-erbA in normal cells and the biochemical function of its undefined protein product are unknown. Partial nucleotide sequence analysis revealed that human c-erbA predicted protein sequences are distantly related to those of carbonic anhydrases, as was shown for the v-erbA sequences (Debuire et al., 1984). Recently, possibly more significant sequence homologies have been reported between the predicted protein sequences of erbA products and of human steroid receptors (Weinberger et al., 1985; Green et al., 1986; Greene et al., 1986).The functional significance of these homologies is still obscure. There is no evidence for oncogenic activation of c-erbA, although it has been inferred from the observation that a chromosome translocation breakpoint in acute promyelocytic leukemia occurs proximal to the human c-erbA locus on chromosome 17 (Dayton et al., 1984). VI. The myb and ets Oncogenes

Specific nucleic acid sequences (Roussel et al., 1979) and protein products (Bister et al., 1982b; Klempnauer et al., 1983; Boyle et al., 1983) of the myb oncogene were originally discovered for the transduced alleles in the genomes of acute leukemia viruses AMV and E26. Specific protein products (Bister et al., 1982b) and nucleic acid sequences (Leprince et al., 1983a; Nunn et al., 1983)of the ets oncogene were first discovered for the transduced allele in the genome of E26. Although intimately linked in E26, v-myb and v-ets alleles are derived from unlinked and unrelated cellular genes, c-myb and c-ets.

153

ONCOGENES IN RETROVIRUSES AND CELLS

AUG SD

I

(UAGI' gag

1

Apol UAG AUG

AMV

UAG

+

henv

nP45 AUG, (SO)

\I

I

,

Agag v-myb

............................... ... $$$ v - e t s :$:: ..........................

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

Aenv

1

E26

l k b ,

FIG.8. Structure and expression of v-myb and v-ets alleles. Genome-sized and subgenomic mRNAs of viruses from the AMV subgroup (compare Table I), aligned with respect to homologous v-myb sequences, and their v-myb and v-ets protein products are shown. Nucleotide sequences transduced from the chicken c-myb or the c-ets gene are indicated by hatching or stippling, respectively. For the explanation of further symbols, see legend to Fig. 2.

A. THEv-myb ALLELESIN AMV AND E26 AND THE v-ets ALLELEIN E26 1 . Structure and Expression

The structure and mode of gene expression of the v-myb alleles in AMV and E26 is depicted in Fig. 8, and their genetic origin is shown in Fig. 9. Comparison of their structures with that of the chicken cmyb gene (see below) revealed that in the course of the transductions coding sequences from seven (AMV v-myb) or six (E26 v-myb) exons of the cellular gene were precisely fused. The 5' terminus of AMV vmyb corresponds to intron sequences within c-myb, and the 3' terminus of AMV v-myb and both termini of E26 v-myb correspond to different locations within the c-myb coding region (Fig. 9). The genetic structure of the AMV genome (Fig. 8) was determined by TIoligonucleotide mapping of viral RNA (Duesberg et al., 1980) and by molecular cloning and nucleotide sequence analysis of proviral DNA (Souza et al., 1980; Klempnauer et al., 1982; Rushlow et al., 1982). The AMV genome contains a complete and functional gag gene and 5' or 3' segments of the poZ and the e m gene, respectively. The v-myb allele is inserted between the partial complements of poZ and enu.

KLAUS BISTER AND H A N S W. JANSEN

154

___

-

I

m ---

E6

El

El

E2

E3

E4 E5

c-myb mRNA

**-

( - - I

I---)

- -I

-l(i--l

chicken c-myb g e n e

3'-

5' --.

,

!---

rn

v-myb (AMV)

v-myb lE261

l k b ,

FIG.9.Structural relationship between c-myb and v-myb alleles. The exons (E) of the chicken c-myb gene sharing sequence homology with v-myb alleles are shown as large boxes, with black regions indicating the protein coding domain defined by an open reading frame for which neither translational initiation nor termination codons have been identified in this region. The c-myb gene contains more coding sequences 5' and 3' of the region defined by homology to v-myb. The nucleotide sequences of c-myb mRNA and of the transduced v-myb alleles are schematically aligned below the corresponding sequences of the region of the c-myb gene shown here. Within the shared coding domains, v-myb and c-myb alleles differ by single nucleotide substitutions. For further symbols and explanations, see legend to Fig. 3.

The gag gene directs synthesis of the precursor for the virion core proteins which are used for the assembly of noninfectious virus particles released by AMV-transformed nonproducer myeloblasts (Duesberg et aZ., 1980). The pol gene is truncated at its 3' end immediately downstream of the splice acceptor site used for the generation of subgenomic enu mRNA of nondefective viruses (Fig. 8). AMV directs synthesis of a p180B'g-P01 protein which is, however, not processed into a functional reverse transcriptase (Duesberg et al., 1980).This protein is encoded by the fused gag-poZ reading frames lacking 36 authentic pol codons at the 3' terminus, and by 27 codons of intron-derived vmyb sequences which also provide the UAG stop codon (Fig. 8). Interestingly, truncation of the poZ gene in AEV-H (see above) occurred at a position extremely close to that described for the AMV poZ gene, and yet AEV-H encodes a functional polymerase whereas AMV does not. The in-frame stop codon in the intron-derived v-myb sequences prevents expression of the v-myb allele by translational initiation at the g a g AUG start codon on genome-sized mRNA. Instead, expression is mediated by translation of a subgenomic v-myb mRNA of -2.3 kilobases (Fig, 8) which was identified in AMV-transformed cells in

ONCOGENES IN RETROVIRUSES AND CELLS

155

addition to genome-sized RNA (Gonda et al., 1981; Chen et al., 1981). This mRNA species is generated by utilization of both the splice donor site near the 5’ terminus of the gag gene and the authentic cellderived splice acceptor site within the v-myb sequences (Figs. 8 and 9) (Klempnauer et al., 1982; Klempnauer and Bishop, 1983).The open reading frame on this v-myb mRNA encompasses the first six gag codons including the AUG codon, 371 v-myb codons, and 11 enu codons, before it terminates at the authentic enu UAG codon (Fig. 8). The predicted 388-amino acid protein product has a calculated molecular weight of -43,700. The corresponding translational product of this mRNA was identified in AMV-transformed cells as a protein with by immunoprecipian apparent molecular weight of 45,000, tation using antisera raised against proteins specified by v-myb DNA expressed in bacteria (Klempnauer et al., 1983; Evan et al., 1984), or against synthetic peptides whose sequences were deduced from the v-myb sequence (Boyle et al., 1983). The predicted amino acid sequences of v-myb and c-myb (see below) protein products differ at l l positions within the shared domains (Klempnauer et al., 1982). The genetic structure of the E26 genome (Fig. 8) was originally deduced from structural analyses of viral RNA and its protein product (Bister et aZ., 1982b), and then confirmed and refined by molecular cloning and nucleotide sequence analyses of proviral DNA or cDNA (Leprince et al., 1983a; Nunn et al., 1983, 1984). The E26 genome contains a 5’ complement of gag and a 3’ complement of enu, and no pol sequences at all. The v-myb allele and the v-ets allele, unique for E26, are inserted next to each other between the partial gag and enu sequences. Nucleotide sequence analysis (Nunn et al., 1984) confirmed the observations that E26 and AMV share closely related vmyb alleles (Roussel et al., 1979; Duesberg et al., 1980; Bister et al., 1982b). The v-myb allele of E26 mainly differs from that of AMV by the lack of intron-derived sequences and 9 codons at the 5’ terminus, and the lack of 79 codons at the 3’ terminus (Figs. 8 and 9). The nucleotide sequence analysis of the v-ets allele (Nunn et al., 1983) suggests that the 3‘ terminus corresponds to 3‘ untranslated sequences of the c-ets gene, which is not extensively characterized yet (see below), and that the 5’ terminus may be derived from within or upstream of the c-ets coding domain. This is deduced from the presence of an open reading frame in v-ets extending from the 5’ terminus to the UGA stop codon just upstream of the 3’ terminus. The v-myb and v-ets alleles of E26 are expressed in one translational unit generated by the fusion of gag, myb, and ets reading frames. Assuming that the unsequenced part of the E26 gag comple-

156

KLAUS BISTER AND HANS W. JANSEN

ment is isogenic to the corresponding region of the RSV gag gene, the open reading frame on genome-sized mRNA encompasses 272 gag codons including the AUG start codon, 283 v-myb codons, and 491 vets codons, before it terminates at the UGA within v-ets (Fig. 8). The predicted 1046-amino acid protein product has a calculated molecular weight of -1 15,600. The corresponding translational product of this mRNA was identified in E26-transformed cells as a protein of apparent molecular weight 135,000, p135g'g-mvb-ets, by immunoprecipitation using antisera against gag proteins (Bister et al., 198213).The genetic origin of the protein was confirmed by tryptic peptide analysis and by immunoprecipitation using antisera specific for myb protein domains (see above) (Boyle et al., 1983; Klempnauer and Bishop, 1984; Evan et al., 1984). The predicted amino acid sequence of the v-myb domain in p135g'8-m~b-ets differs at only one position from that of the c-myb protein (see below), and at the same and nine other positions from that of p45v-mvb in the shared domains (see above) (Nunn et al., 1984). 2. Function Nonconditional mutants have not been described either for AMV or E26. A ts mutant has been isolated from mutagen-treated AMV that was described as nonleukemogenic and temperature-sensitive for the in vitro transformation of myeloid cells and for the production of vmyb mRNA and protein (Moscovici and Moscovici, 1983; Moscovici et al., 1985). Some of the tsAMV-transformed myeloblasts differentiated into macrophages at the nonpermissive temperature. Similar ts mutants have also been described for E26 (Beug et aZ., 1984). Myeloid precursor cells transformed by these mutants differentiated into macrophage-like cells at the nonpermissive temperature. However, the mutant viruses retained their leukemogenicity and their ability to transform erythroid cells at the nonpermissive temperature. It is not clear whether the phenotype of these mutants reflects the presence of independent functional domains on the single p135gag-mYb-ets protein product of E26. Interestingly, it has been reported that myeloblasts transformed by E26 are hormone-dependent for growth and for the expression of p135gug-mvb-efs (Beug et aZ., 1982b). The biochemical functions of v-myb and v-ets protein products are unknown. Both p135g'g-myb-ets and p45v-myb are phosphoproteins (Bister et al., 1982b; Klempnauer et al., 1983), and both were found to be located predominantly in the nucleus of transformed cells, using subcellular fractionation procedures or immunofluorescence techniques (Klempnauer et al., 1984; Boyle et al., 1984). In subnuclear fractionation procedures, v-myb protein products were found associated with

ONCOGENES IN RETROVIRUSES AND CELLS

157

both chromatin and nuclear matrix structures, and they were also shown to exhibit in vitro DNA binding activity (Boyle et al., 1985; Klempnauer and Sippel, 1986). When AMV-transformed myeloblasts were induced to differentiate into macrophages by the addition of lipopolysaccharide and tumor promoter 12-0-tetradecanoylphorbol lSacetate, p45v-mvb continued to be synthesized at similar levels (Symonds et al., 1984b), but was now distributed mainly to perinuclear compartments of the cytoplasm (Klempnauer et al., 1984). In fibroblasts infected but not transformed by AMV (compare Section I), p45v-myb was found mainly in the nucleus, indicating that nuclear localization of this protein is not sufficient for transformation of these cells (Klempnauer et al., 1984).A distant relationship of yet-unknown functional significance between v-myb, v-m yc, and adenovirus E l a predicted protein sequences has been reported (Ralston and Bishop, 1983), although the validity of the proposed homologies has been questioned (McLachlan and Boswell, 1985). B. THEc-myb AND c-ets GENES

1 . Structure and Expression

The presence in normal chicken DNA and in other vertebrate DNA of sequences closely related to the v-myb oncogene of AMV and E26 was originally detected by nucleic acid hybridization (Roussel et al., 1979; Souza et al., 1980; Bergmann et al., 1981; Gonda et al., 1981). The structure ofthe chicken c-myb locus (Fig. 9) was then extensively characterized by molecular cloning of genomic DNA (Klempnauer et al., 1982; Perbal et al., 1983), by nucleotide sequence analysis (Klempnauer et al., 1982),and by transcriptional mapping (Gonda and Bishop, 1983; Klempnauer and Bishop, 1983). Seven exons of the gene were defined by homology to v-myb and by transcriptional mapping (Klempnauer et al., 1982; Klempnauer and Bishop, 1983). The nucleotide sequence analysis revealed that the open reading frame in c-myb shared with v-myb (see above) extends throughout the seven exons since there is no in-frame termination codon within this region. Furthermore, analyses of transcriptional and translational products of c-myb indicated that this gene extends both 5' and 3' of the region defined by homology to v-myb. The c-myb mRNA in chicken cells was identified as a 4.0-kilobase species that contains in addition to the vmyb-related domain sequences derived from 5' and 3' domains of cmyb that are not present in v-myb (Gonda et al., 1982; Gonda and Bishop, 1983).The translational product of c-myb mRNA was identi-

158

KLAUS BISTER AND HANS W. JANSEN

fied in chicken cells as a 75,000-molecular weight protein, p75"-"vb, by immunoprecipitation using the specific antibodies described above (Klempnauer et al., 1983). A candidate c-myb protein product of 110,000 molecular weight has also been described (Boyle et al., 1983), but its relationship to v-myb proteins has been doubted (Evan et al., 1984; Boyle et al., 1984). The utilization of monoclonal antibodies recognizing epitopes on both p4SV-"vband p7SC-"vb(and on p135gag'8-mvbbets) strongly supports the conclusion that p75c-mvbis the authentic chicken c-myb protein product (Evan et al., 1984). Furthermore, a recent analysis of chicken c-myb cDNA clones revealed that this gene encodes a 699-amino acid protein with a calculated molecular weight of -77,000 which contains sequences at both the amino and the carboxyl terminus that are not present in v-myb protein products (Rosson and Reddy, 1986). The structure of the human c-myb locus which is located on chromosome 6 (Dalla Favera et al., 1982d; Harper et al., 1983; Zabel et al., 1984b) was analyzed by molecular cloning, restriction enzyme cleavage site mapping, and heteroduplex analysis (Leprince et al., 198313; Franchini et al., 1983). The overall organization of the gene and the complexity of the mature transcript are very similar to those observed for the chicken gene. In mice, c-myb is located on chromosome 10 (Sakaguchi et al., 1984), and analysis of cDNA clones has revealed its close relationship to the chicken homolog (Gonda et al., 1985).Recently, the c-myb gene of Drosophila has been isolated and characterized (Katzen et al., 1985). Within a limited domain, the amino acid sequence homology between Drosophila and chicken c-myb protein products is greater than 70%. The presence of v-ets-related sequences in avian and human DNA was detected by nucleic acid hybridization (Leprince et al., 1983a). Sequences related to v-ets are distributed over 35-50 kilobase pairs of the chicken genome and are located distant from the c-myb gene (Leprince et al., 1983a; Nunn et al., 1983, 1984). The major c-ets transcript in chicken cells was identified as a 7.5-kilobase RNA species (Leprince et al., 1983a) which is presumably translated into a 56,000 molecular weight protein (Chen, 1985; Ghysdael et al., 1986). The human ets homolog is apparently discontiguous and located on chromosomes 11(c-ets-1) and 21 (c-ets-2) (de Taisne et al., 1984; Watson et al., 1985).Similarly, two different c-ets loci have been found in mice and cats (Watson et al., 1986). 2. Function The physiological role of the c-myb gene in normal cells and the biochemical function of its protein product are unknown. Like v-myb

ONCOGENES IN RETROVIRUSES AND CELLS

159

protein products, p75emvb is located predominantly in the nucleus, where it is associated with both chromatin and the nuclear matrix (Klempnauer et al., 1984; Klempnauer and Sippel, 1986). Analyses of a possible differential expression of c-myb revealed that this gene is expressed primarily in immature hemopoietic cells (Gonda et al., 1982; Westin et al., 1982a; Coll et al., 1983b; Sheiness and Gardiner, 1984). In particular, it was reported that immature thymocytes and macrophage progenitor cells contain exceptionally high levels of cmyb mRNA, suggesting a critical role of this gene in the normal differentiation of these cells (Duprey and Boettiger, 1985; Thompson et al., 1986).On the other hand, studies on c-myb expression during induced differentiation of a murine myeloid leukemia cell line revealed that decrease in transcription occurred only very late in monocytic differentiation, suggesting a role for c-myb in the proliferation, rather than the differentiation, of myeloid cells (Gonda and Metcalf, 1984). Concordantly, c-myb expression in several cell types was found to vary as a function of cellular proliferation and to be presumably regulated by post-transcriptional mechanisms (Thompson et al., 1986). Oncogenic function of nontransduced myb alleles has not been directly demonstrated, but is inferred based on several lines of circumstantial evidence. In murine plasmacytoid lymphosarcomas the c-myb locus was found to be rearranged presumably due to retroviral insertion mutagenesis, with a resultant high level of aberrant c-myb transcripts (Mushinski et al., 1983; Shen-Ong et al., 1984, 1986). Amplification of c-myb and high levels of normal c-myb transcripts were observed in human cell lines derived from a colon carcinoma and from an acute myelogenous leukemia (Alitalo et at., 1984; Pelicci et al., 1984). Also, increased c-myb transcription was observed in human small-cell lung cancer cell lines (Griffin and Baylin, 1985) and in the lymphoid organs of mice with autoimmune lymphoproliferative syndrome (Mountz et al., 1984). The physiological role of the c-ets gene and the biochemical function of its protein product are unknown. The chicken c-ets gene was found to be expressed predominantly in lymphoid cells (Chen, 1985; Ghysdael et al., 1986). A significant, albeit distant and locally confined, homology between the predicted amino acid sequences of ets (Nunn et al., 1983) and yeast cell cycle gene products has been reported (Peterson et al., 1984). It is unknown whether this reflects any functional homologies. There is no direct evidence for oncogenic activation of c-ets, although the human c-ets loci (see above) have recently been implicated in chromosomal translocations in human acute leukemias (Diaz et al., 1986; Sacchi et aE., 1986).

160

KLAUS BISTER AND HANS W. JANSEN

VII. Evolution of Retroviral Oncogenes

Most retroviral alleles of oncogenes have the capacity to function as dominant oncogenic determinants in many or specific target cells (Table 11). Hence, retroviral transduction is an efficient and well-established way of activating a cellular oncogene. The transition from a normal cellular allele to an oncogenic transduced allele is accompanied by various structural and regulatory changes. The functionally relevant changes underlying these transitions are not well understood, but the intensive structural comparisons between v-onc and cone alleles have unveiled some distinctive features.

A. MECHANISMSOF TRANSDUCTION There are as yet no reports on reproducible experimental transductions of cellular genes by retroviral vectors allowing a direct investigation into the mechanisms of recombination between internal proviral DNA or viral RNA sequences and cellular nucleic acids. Hence, all models for transduction of cellular oncogenes are based on the structural features of the final products of such transductions, i.e., the natural isolates of highly oncogenic retroviruses (compare Fig. 1). Nevertheless, some common structural features of all transduced alleles of oncogenes are clearly established: (1)v-onc genes are integral parts of retroviral genomes, hence recombination with viral sequences has to occur at 5' and 3' locations of the cellular gene, (2)the 5' recombination point is located either within intronic or within exonic sequences of the cellular gene, (3)the 3' recombination point is always within an exon and upstream of the polyadenylation or 3' processing signals of the cellular gene, (4) introns between transduced exons are always removed, and ( 5 ) v-onc and c-onc sequences are nonpermuted (compare Figs. 3, 5, and 9). Based on this structural consensus, a favored model for transduction has been formulated (Varmus, 1982, 1984; Temin, 1984). The initial event is believed to be proviral integration in the same transcriptional orientation 5' of the gene to be transduced. A presumed deletion then removes a 3' portion of proviral DNA and adjacent cellular DNA. The deletion extends either into an intron or an exon of the cellular gene. As a result of the deletion, proviral and cellular sequences would be fused in one transcriptional unit, with the primary transcript initiating at the 5' LTR and terminating at the 3' end of the cellular gene. This transcript would then be processed by splicing removing all introns between exons, and would be packaged into virions, presumably along with authentic viral RNA to form a heterozygotic dimer. On

ONCOGENES IN RETROVIRUSES AND CELLS

161

subsequent infections, recombination between the 3' domains of the viral and the hybrid RNA species, presumably during reverse transcription, would complete the genesis of a transmissible, highly oncogenic retrovirus. As an alternative to the DNA deletion mechanism (see above), hybrid transcripts could be generated by occasional overriding of the transcriptional termination signal in the 3' LTR of the provirus integrated upstream of the cellular gene, and internal viral sequences and cellular sequences would subsequently be fused by a splicing or deletion process (see below). The model described above is compatible with the structural features of all transduced alleles of oncogenes described to date. Nevertheless, there is no clear evidence as yet for a common molecular mechanism by which the actual recombination of viral and cellular nucleic acids is mediated. Such a mechanism would have to facilitate apparently illegitimate recombinations between unrelated nucleic acid sequences, i.e., sequences of retroviral structural genes and of cellular oncogenes (Bister and Duesberg, 1982; Duesberg, 1983). Nucleotide sequence analyses of the junctions between transduced alleles of oncogenes and retroviral structural genes (or a second transduced oncogene; see below) provided some clues about the nature of recombination in certain cases of transduction, but failed to do so in others (Fig. 10). The nucleotide sequence analysis of CMII proviral DNA (compare Fig. 2 for the genetic structure) at the Agaglv-myc and the v-myclApoZ junctions (Fig. 10A) revealed significant homologies between avian retroviral structural genes and the cellular oncogene c-myc precisely at the positions corresponding to the gene junctions in CMII (Walther et al., 1985). Particularly striking (including an 11 out of 13 match of identical nucleotides) is the homology between the pol gene and the chicken c-myc gene right at the positions corresponding to the v-mycl Apol junction in CMII (Fig. 10A). Similar homologies between short gene segments have been observed in other cases: at the 3' border of the MC29 v-myc allele, a 5-nucleotide homology between the corresponding c-myc and enu sequences has been reported (Reddy et al., 1983a); homologies from 5 to 11nucleotides at both the 5' and the 3' junctions of cellular and viral sequences may have facilitated the transductions of the c-fos (Van Beveren et al., 1983) and the c-raf (Bonner et al., 1985) genes; and a 10-nucleotide homology between enu and c-src has been implicated in the recombination between partial src deletion mutants of RSV and the chicken gene c-src (Wang et aZ., 1984b). Interestingly, not only transduction of oncogenes into, but also their excision from, retroviral genomes may be mediated by recombination between short homologous sequence elements of other-

KLAUS BISTER AND HANS W. JANSEN

162 A RSV

gag

CHI1 A g a g / v - m y c Ck

c-nyr

ck c - m y c

CMII v - m y c / A p o l RSV pol

B RSV gag

MH2 A g a g / v - m i l ck

c -mil

ACAATGCCAGTAGACAGC ACAATGCCAGTAGACAGC

ck c - m i l M H 2 v-mmil/v-myc ck r - m y r

FIG.10. Sites of recombination involved in transductions of c-myc and c-mil. The nucleotide sequences at the vectodoncogene junctions on proviral DNA of CMII (A) or at the vector/oncogene and oncogene/oncogene junctions on proviral D N A of MH2 (B) are compared with the corresponding nucleotide sequences of avian retroviral structural genes (RSV proviral sequences are used assuming that they are closely related to those of the original transducing viruses) and of the chicken c-myc and c-mil genes. Homology between nucleotide sequences is indicated by boxes. The arrow points to a position corresponding to the authentic splice acceptor site at the 5‘ border of exon 2 in c-myc (compare Fig. 3) and to a possible cryptic splice donor site in the RSV gag gene.

wise unrelated genes: the Agag and v-mil sequences of MH2 share a 9-nucleotide homology precisely at the positions corresponding to the AgaglAv-mil junction in the mil deletion mutant MH2D12 (Jansen et al., 1985a). As an alternative to recombination between homologous gene segments, RNA splicing may have been involved in joining cellular and viral sequences at the 5’ junction of Agaglv-myc sequences in CMII (Fig. 1OA). The junction point corresponds to the authentic splice acceptor site in the c-myc gene, and, in a striking coincidence, to a possible cryptic splice donor signal within the RSV gag sequence (Walther et al., 1985). A similar structure has been reported for the 5’ vectorlv-re2 junction in the genome of avian reticuloendotheliosis virus (Wilhelmsen et al., 1984). Whether this implies that in these cases of transduction the initial event was recombination at the RNA level is not clear. It is probably more plausible that, again, nearby proviral

ONCOGENES IN RETROVIRUSES AND CELLS

163

integration was the first event (see above), and that a larger viralcellular hybrid transcript was a substrate for the presumed splicing process. I n several cases of retroviral transduction of cellular oncogenes, no clues to the recombination mechanism were obtained from nucleotide sequence analysis. As an example, the junctions of the MH2 v-mil allele with flanking gag and v-myc sequences are shown in Fig. 10B. At both junctions, no significant sequence homologies between the corresponding gene segments participating in the recombination were observed, and no consensus splicing signals were found in these locations (Jansen and Bister, 1985). Similarly, no extensive homologies were found between viral and cellular sequences corresponding to the 5' and 3' junctions of v-myb in AMV (Klempnauer et al., 1982), to the 5'junction of v-myc in MC29 (Reddy et al., 1983a), or to the 3' junction of v-myc in MH2 (Sutrave et al., 1984b). A possible explanation for these findings is that the sequence elements of the original transducing viruses involved in these particular recombinational events were not isogenic to the RSV proviral sequences which are commonly used for such nucleotide sequence comparisons, and hence possible limited homologies would remain undetected. Furthermore, genetic changes in vector and oncogene sequences after the transduction may obscure clues to the mechanism of recombination. In any case, the vector-oncogene recombinations appear to involve rare processes and may be the limiting steps in the evolution of highly oncogenic retroviruses.

B. STRUCTURALAND FUNCTIONAL DIFFERENCES BETWEEN c-onc AND v-onc GENES A central issue in the analyses of oncogenes is the quest for the mechanisms by which the physiological (i.e., obviously nononcogenic) forms of cellular oncogenes may be rendered oncogenic. Basically two relevant mechanisms are considered, referred to as the quantitative and the qualitative model, respectively, of oncogene activation (Bishop, 1981; Bister and Duesberg, 1982; Duesberg, 1983; Bister, 1984; Varmus, 1984).According to the quantitative model, disturbance of the normal transcriptional (and/or translational) controls of the oncogene with a resultant production of large amounts of a functionally unaltered protein product would lead to cell transformation. This model, also called the dosage hypothesis, would principally also apply to the assumption that expression of an oncogene at comparably normal levels, but in an inappropriate environment (i.e., in a cell type

164

KLAUS BISTER AND HANS W. JANSEN

in which it is normally silent), would lead to neoplastic cell transformation. The qualitative model holds that activation of cellular oncogenes is based on mutational changes in the coding domain with a resultant normal-level production of functionally altered protein products. Transduction of cellular oncogenes, as far as it has been analyzed in detail, is always accompanied by structural changes of the transduced gene, and usually leads to high-level expression of the transduced mutant allele when reintroduced into cells by proviral integration. There is as yet very little knowledge about which of the qualitative changes in structure or which of the quantitative changes in expression are of any functional significance in the sense of the qualitative or the quantitative model of oncogene activation. This is mainly due to the fact that there is no example of an oncogene for which the biochemical function and the cellular targets of both the physiological and the oncogenic form of the protein product have been unequivocally identified. Many oncogenes, such as erbB, myb, or miZ(ruf) (see above), were more or less severely truncated in their coding domains in the course of retroviral transduction. In these cases, qualitative changes in oncogene function appear most plausible but have yet to be demonstrated in a rigorous manner. In contrast, all v-myc alleles contain the entire protein coding domain of the c-myc gene (compare Fig. 3). The degree of mutation within the shared coding domains varies between the different v-myc alleles: CMII v-myc, at the one extreme, differs at a single nucleotide position from chicken c-myc (leading to one amino acid substitution in the predicted protein sequence), and MH2 v-myc, at the other extreme, differs at 31 nucleotide positions from the cellular counterpart (leading to 27 amino acid substitutions) and has completely lost four internal codons (see above). Notably, no specific mutation common to all four v-myc alleles has been identified (Walther et al., 1986). Whether this implies that oncogenic activation of c-myc by transduction is mainly a quantitative phenomenon is uncertain. All v-myc protein products, presumably even those expressed via subgenomic mRNAs, are synthesized as hybrid proteins containing large (several hundred amino acid residues) or small (six amino acid residues) complements of structural viral protein sequences at their amino termini (see above). Hence, v-myc protein products differ from the normal cmyc-encoded protein by one or more, albeit no common, amino acid substitutions and by additional' amino-terminal amino acid residues. However, recent analyses of spontaneous or constructed derivatives of MC29 and MH2 have revealed that neither major structural changes, such as in-frame fusion with virion genes or internal deletions (Jansen

ONCOGENES I N RETROVIRUSES AND CELLS

165

et al., 1985a; Shaw et al., 1985; Patschinsky et al., 1986a), nor specific, if any, missense mutations of the c-myc coding region (Patschinsky et al., 1986a; Walther et al., 1986) are necessary for the activation of the basic oncogenic function of the c-myc gene upon transduction. Nevertheless, mutations in the c-myc coding region may enhance oncogenic function and may play an important role in the evolution of fully tumorigenic v-myc alleles. The possible relevance of mutations in the coding domains of nontransduced c-myc alleles for their oncogenic activation, like upon chromosomal translocation or insertion mutagenesis, is not clear yet (Rabbitts et al., 1983,1984; Battey et al., 1983; Stanton et al., 1984; Westaway et al., 1984; Showe et al., 1985).Interestingly, it was recently reported that augmented expression of c-myc genes with unaltered coding sequence is sufficient for cotransformation of rat embryo cells with a mutant ras gene (Lee et al., 1985) or for the induction in mouse NIH/3T3 cells of many properties of transformed cells except greatly altered morphology (Kelekar and Cole, 1986). Examples of structural changes in coding regions that appear to be necessary or at least beneficial for full oncogenic activation were described for transduced v-ras and v-src alleles (Varmus, 1984; Hanafusa et al., 1984). However, it is not clear whether these mutations are also sufficient for full activation of the oncogenic potential of the gene or whether positioning in the retroviral genome and transcriptional control by the retroviral promoter are also essential elements of activation. For ras genes, the latter supposition is supported by the observation that mutations in the coding region are not required for basic activation of the transforming potential (DeFeo et al., 1981; Chang et al., 1982; Cichutek and Duesberg, 1986).Furthermore, as pointed out above, the qualitative or quantitative effects of the apparently necessary changes in gene structure and expression on the biologically relevant functions of the protein products are not known. In summary, available circumstantial evidence suggests that full activation of oncogenes by retroviral transduction (and possibly also activation other than by transduction; see Section 11)may require multiple changes in both structure and expression of those genes. C. RETROVIRUSES WITH Two ONCOGENES AND MULTISTEP CARCINOGENESIS Based on the findings of classical and molecular oncology, it is generally believed that cancer is the final product of a multistep process presumably involving multiple genes (Foulds, 1958; Knudson, 1973, 1981; Klein and Klein, 1984, 1985; Temin, 1984). The initiation and

166

KLAUS BISTER AND HANS W. JANSEN

progression of avian B-cell lymphomas has been considered as a model case for the involvement of at least two different genes in carcinogenesis. In such tumors the c-myc gene is transcriptionally activated (see Section 111),but a different gene (B-lym) was identified as the presumed transforming gene in lymphoma DNA when assayed by transfection on NIH/3T3 cells (Cooper and Neiman, 1981; Goubin et al., 1983).It has recently been suggested that activated myc oncogenes may be involved in the formation of transformed follicles, the presumed preneoplastic lesions within the bursa (Baba and Humphries, 1985; Neiman et al., 1985). Activation of the B-lym gene is then hypothesized to lead to progression of preneoplastic lymphocytes to clonal neoplastic growths. In a similar fashion, human c-myc and B-lym genes are believed to be involved in different stages of development of Burkitt’s lymphoma (Diamond et al., 1983; Klein and Klein, 1985).While the progressive development of these lymphomas may well involve several genes, the authenticity and significance of the B-lym genes have recently been doubted (Rogers, 1986; Cooper et al., 1986). The multistep nature of carcinogenesis was also deduced from the observation that activated oncogenes, such as c-Ha-ras genes from tumor DNA, were capable of transforming cells of the established preneoplastic NIH/3T3 mouse cell line, but were dependent on the presence of a second activated oncogene, such as v-myc, for the transformation of primary rodent fibroblasts (Land et al., 1983a,b; Ruley, 1983).However, the proposed cooperation between two oncogenes is apparently not essential for the transformation of normal embryo cells. Both v-myc and activated c-Ha-ras genes have been shown to be individually capable of transforming rat and mouse embryo cells (Quade, 1979; Quade et al., 1983; Spandidos and Wilkie, 1984; Vennstrom et al., 1984; Pozzatti et al., 1986). Retroviral oncogenes are capable of transforming cells in a single step, and hence their action would not be compatible with the concept of the multistep nature of carcinogenesis. However, it has been argued that the multiple changes in the evolution of retroviral oncogenes from their cellular progenitors (see above) are analogous to the multiple stages in nonviral carcinogenesis (Temin, 1984). Retroviruses with two oncogenes may represent the products of a natural selection for enhanced oncogenicity caused by additive, complementary, or even directly cooperative functions of two oncogenes whose simultaneous and augmented expression is guaranteed by their presence on a single retroviral genome. The v-erbA gene appears to enhance the oncogenic effects of the v-erbB gene in AEV-R (Frykberg et al., 1983; Sealy et al., 1983a,b; Graf and Beug, 1983), and the v-etsencoded domain of the single gag-myb-ets protein product of the E26 genome (compare Fig. 8) may either modulate the function of the

ONCOGENES IN RETROVIRUSES AND CELLS

167

v-myb-encoded domain or add new functional properties to the protein, leading to broadening of the oncogenic spectrum of E26 compared to that of AMV (compare Table I) (Sotirov, 1981; Moscovici et al., 1981; Radke et al., 1982). MH2 is the only known retrovirus that carries in its genome alleles of two oncogenes, v-myc and v-mil, which have been found in modified form and in different genetic context (i.e., in the genomes of MC29 or 3611-MSV7respectively) as autonomous oncogenic mutant alleles. The autonomous oncogenic function of the MH2 v-myc allele was directly shown by the isolation of transforming variants of MH2 that have lost the v-mil gene, but have retained the v-myc gene (compare Sections I11 and IV). MH2 was reported to be significantly more oncogenic than other viruses carrying alleles of the v-myc oncogene (Alexander et al,, 1979; Linial, 1982), suggesting that v-mil may contribute to the oncogenic specificities of MH2. However, it is also possible that the multiple mutations in the MH2 v-myc allele (see above) reflect the progressive evolution of an activated myc oncogene selected for high oncogenicity. In this context it should be noted that the CMII v-myc allele which most closely resembles the c-myc allele (see above) was reported to be inefficient in affecting certain parameters of cell transformation in comparison to the activities of the other v-myc alleles (Palmieri et al., 1983). Additive or complementary function of the v-myc and v-mil alleles has been inferred from several lines of circumstantial evidence. Although v-mil deletion mutants of MH2 are capable of transforming cultured fibroblasts, some transformation-associated parameters may be affected differently in cells transformed by mutant or wild-type MH2 (Jansen et al., 1985a; Patschinsky et al., 1986a). Natural or constructed v-mil deletion mutants of MH2 (Jansen et al., 1985a,b) were shown to be less oncogenic in uiuo than wild-type MH2 (Graf et al., 1986).Furthermore, these v-mil deletion mutants are capable of transforming macrophages in a bone marrow transformation assay, but these cells are growth factor dependent, whereas macrophages transformed by wild-type MH2 are growth factor independent (Graf et al., 1986).The abrogation of growth factor requirement of myeloid cells transformed by viruses containing v-myb or v-myc can be achieved by superinfection of these cultures by MH2 (and other viruses containing oncogenes of the src family), confirming that v-mil may be responsible for this effect (Adkins et al., 1984). Cooperation or complementation between v-myc and v-mil of MH2 has also been suggested as a critical event in the transformation of chicken neuroretina cells (Bechade et aZ., 1985) and in the alterations of the differentiated phenotype and of the proliferative capacity of definitive chondroblasts infected with MH2 (Alema et al., 1985). Constructed murine retroviruses containing both v-mil(ra. and v-myc genes (Jansen et al., 1985b)were shown to induce the rapid growth of hemopoietic neoplasms and carcinomas in

168

KLAUS BISTER AND HANS W. JANSEN

newborn mice or to induce growth factor independent growth and immortalization of hemopoietic cells in uitro (Rapp et al., 1985; Blasi et al., 1985; Cleveland et al., 1986). In summary, it appears that enhanced oncogenicity of retroviruses with two oncogenes may be due to cooperation between these genes. However, the significance and the nature of such a presumed cooperation can only be assessed when the functions of the oncogene protein products relevant for the induction and maintenance of the transformed state become known. The v-onc alleles of retroviruses with two oncogenes were shown to be derived from cellular genes which are unIinked in their normal chromosomal locations (compare Sections IV-VI). It is entirely unknown how the common transduction of two unlinked cellular genes in one retroviral genome was accomplished. It is possible that these genes were first joined in the course of a chromosomal translocation in the tumor from which the virus was isolated, and that they were then transduced in a manner similar to the mechanisms believed to be involved in the transduction of single oncogenes (see above). Alternatively, the transduction of two oncogenes by one retrovirus could have occurred via subsequent and independent recombinational events. The latter mechanism would have to be favored if viral sequences are found interspersed between the two transduced oncogenes, as was reported for v-erbA and v-erbB in the AEV-R genome (compare Fig. 6) (Henry et al., 1985). In the case of MH2 v-myc and v-mil, however, sequences of the two oncogenes appear to be directly adjacent on the viral genome (compare Fig, 10) (Jansen and Bister, 1985), and in the E26 genome, v-myb and v-ets coding sequences are even cotranslated since they are fused in one reading frame (compare Fig. 8) (Bister et al., 1982b; Nunn et al., 1984). VIII. Conclusions and Perspectives

Based on extensive biochemical definitions of oncogene structure, commonly at the nucleotide sequence level, active oncogenes including the transduced and reintegrated retroviral oncogenes can be viewed as mutant alleles of protooncogenes, the normal nonmutated alleles of cellular oncogenes. Evolution of active oncogenes, in particular of the autonomous and dominant v-onc alleles, involves multiple changes of the original protooncogene in structure and expression. The exhaustive knowledge about oncogene structure leads to several questions of obvious relevance. (1)Which genetic alterations in active oncogenes are of functional significance? (2) What is the biochemical function of the protein products of active oncogenes in transformed

ONCOGENES IN RETROVIRUSES AND CELLS

169

cells? (3)What is the role of proto-oncogenes in function, growth, and development of normal cells? (4)Are nontransduced mutant alleles of cellular oncogenes relevant to cancer, including human malignancies? With respect to the first question it would be important to know whether the well-characterized structural changes, for instance in the transition from c-onc to v-onc alleles, lead to qualitative or to quantitative changes in relevant gene function. Assessing the extent of functional differences between v-onc and c-onc protein products would probably directly bear on answers to the fourth question (see below). Obviously, definitive answers to the second and third question are needed first. Biochemical functions for several v-onc protein products have been proposed based on in vitro assays, but there is as yet no direct and often not even circumstantial evidence that these in vitro activities reflect functions crucially involved in neoplastic cell transformation. Knowing more about v-onc functions will probably advance our general knowledge about the induction and maintenance of the neoplastic state, irrespective of the unresolved issue whether nontransduced mutant alleles of cellular oncogenes are relevant to cancer (see below). Determining the function of a mutant allele will be inherently linked to assessing the function of the normal allele. For at least two c-onc genes, the physiological function as a growth factor or as a growth factor receptor gene appears to be established. Significantly, this was achieved by the unequivocal demonstration of structural homology (if not identity) of c-onc genes with genes of presumably known function, rather than by a direct search for the unknown function of the c-onc protein product. The fourth issue, which addresses the relevance of oncogenes to nonviral cancers and cancers induced by weakly oncogenic retroviruses, is of vital interest. There is multiple circumstantial evidence that activation of cellular oncogenes other than by transduction may be involved in animal and human carcinogenesis. However, there is as yet no direct proof that the mutated, rearranged, translocated, or transcriptionally activated genes can cause neoplastic transformation of normal cells as autonomous oncogenes or, more importantly, that they are the cause of the tumor from which they were isolated (Temin, 1983; Rubin, 1983, 1984; Duesberg, 1983, 1985).On the other hand, the provocatively close relationship of conserved vertebrate genes to genes with demonstrated autonomous oncogenic function, i.e., the v-onc alleles, provides a compelling motive to explore the possible role of nontransduced mutant alleles of cellular oncogenes in carcinogenesis. Once the functional changes in the tran-

170

KLAUS BISTER AND HANS W. JANSEN

sition from c-onc to v-onc alleles become known, it will be possible to assess whether similar changes of c-onc alleles can be achieved without the incorporation in retroviral vectors.

ACKNOWLEDGMENTS We thank Christiane Trachmann for invaluable help with the preparation of the manuscript, and Hannelore Markert for typing. Work of the authors has been supported by the Max-Planck-Gesellschaft, by the Deutsche Forschungsgemeinscha, and by the Fonds der Chemischen Industrie.

REFERENCES Abrams, H. D., Rohrschneider, L. R., and Eisenman, R. N. (1982). Cell 29,427-439. Adams, J. M., Gerondakis, S., Webb, E., Corcoran, L. M., and Cory, S. (1983).Proc. Natl. A d . Sct. U . S A . 80, 1982-1986. Adams, J. M., Hams, A. W., Pinkert, C. A., Corcoran, L. M., Alexander, W. S., Cory, S., Palmiter, R. D., and Brinster, R. L. (1985).Nature (London)318, 533-538. Adkins, B., Leutz, A., and Graf, T. (1984). Cell 39,439-445. Alema, S., Tato, F., and Boettiger, D. (1985). Mol. Cell. Biol. 5, 538-544. Alexander, R.W., Moscovici, C., and Vogt, P. K. (1979).J . Natl. Cancer Inst. (U.S.)62, 359-366. Alitalo, K., Bishop, J. M., Smith, D. H., Chen, E. Y., Colby, W. W., and Levinson, A. D. (1983a).Proc. NatLAcad. Sci. U S A . 80, 100-104. Alitalo, K., Schwab, M., Lin, C. C., Varmus, H. E., and Bishop, J. M. (198313).Proc. Natl. A d . S C ~U . . S A . 80, 1707-1711. Alitalo, K., Ramsay, G., Bishop, J. M., Ohlsson-Pfeifer, S., Colby, W. W., and Levinson, A. D. (1983~). Nature (London)306,274-277. Alitalo, K., Winqvist, R.,Lin, C. C., de la Chapelle, A., Schwab, M., and Bishop, J. M. (1984).Proc. Natl. Acad. Sci. U.S.A. 81,4534-4538. Anderson, S. M., and Hanafusa, H. (1982). Virology 121, 32-50. Anderson, S. M., Hayward, W. S., Neel, B. G., and Hanafusa, H. (1980)J. Virol. 36,676683. Armelin, H. A., Armelin, M. C. S., Kelly, K., Stewart, T., Leder, P., Cochran, B. H., and Stiles, C. D. (1984). Nature (London) 310,655-660. Baba, T. W., and Humphries, E. H. (1985). Proc. Natl. Acad. Sci. U . S A . 82,213-216. Baluda, M.A,, and Goetz, I. E. (1961). Virologv 15, 185-199. Bargmann, C. I., Hung, Ma-C.,and Weinberg, R. A. (1986).Nature (London) 319,226230. Barker, W. C., and Dayhoff, M. 0. (1982).Proc. Natl. Acad. Sci. U S A . 79,2836-2839. Battey, J., Moulding, C., Taub, R., Murphy, W., Stewart, T., Potter, H., Lenoir, G., and Leder, P. (1983). Cell 34, 779-787. Beard, J. W. (1963).Ado. Cancer Res. 7, 1-127. Beard, J. W. (1980). In “Viral Oncology” (G. Klein, ed.), pp. 55-87. Raven Press, New York. Beard, J. W., Langlois, A. J., and Beard, D. (1973).In “Unifying Concepts of Leukemia” (R. M. Dutcher and L. Chieco-Bianchi, eds.), pp. 31-44. Karger, Basel. Bechade, C., Calothy, G., Pessac, B., Martin, P., Coll, J., Denhez, F., Saule, S., Ghysdael, J., and Stehelin, D. (1985). Nature (London)316,559-562.

ONCOGENES IN RETROVIRUSES AND CELLS

171

Begg, A. M. (1927).Lancet 1,912-915. Benedict, S . H., Maki, Y., and Vogt, P. K. (1985).Virology 145, 154-164. Bergmann, D. G., Souza, L. M., and Baluda, M. A. (1981).J . Virol. 40,450-455. Bernard, O., Cory, S., Gerondakis, S., Webb, E., and Adams, J. M. (1983).E M B O J . 2, 2375-2383. Besmer, P., Murphy, J. E., George, P. C., Qiu, F., Bergold, P. J., Lederman, L., Snyder, H. W., Brodeur, D., Zuckerman, E. E., and Hardy, W. D. (1986).Nature (London) 320,415-421. Beug, H., and Graf, T. (1980).Virology 100,348-356. Beug, H., and Hayman, M. J. (1984).Cell 36,963-972. Beug, H . , von Kirchbach, A., Doderlein, G., Conscience, J.-F., and Graf, T. (1979).Cell 18,375-390. Beug, H., Kitchener, G., Doderlein, G., Graf, T., and Hayman, M. J. (1980).Proc. Natl. Acad. Sci. U S A . 77, 6683-6686. Beug, H., Palmieri, S., Freudenstein, C., Zentgraf, H., and Graf, T. (1982a). Cell 28, 907-919. Beug, H., Hayman, M. J., and Graf, T. (1982b).EMBOJ. 1, 1069-1073. Beug, H., Leutz, A., Kahn, P., and Graf, T. (1984).Cell 39,579-588. Beug, H., Hayman, M. J., Raines, M. B., Kung, H. J., and Vennstrom, B. (1986).J.Virol. 57, 1127-1138. Bishop, J. M. (1981).Cell 23,5-6. Bishop, J. M. (1983).Annu. Aeo. Biochem. 52,301-354. Bishop, J. M., and Varmus, H. (1982).In “RNA Tumor Viruses” (R. Weiss, N. Teich, H. Varmus, and J. Coffin, eds.), pp. 999-1108. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Bishop, J. M., Courtneidge, S. A., Levinson, A. D., Oppermann, H., Quintrell, N., Sheiness, D. K., Weiss, S . R., and Varmus, H. E. (1980).Cold Spring Harbor Symp. Quant. Biol. 44, 919-930. Bister, K. (1984).In “Mechanisms of Viral Leukaemogenesis” (J. M. Goldman and J. 0. Jarrett, eds.), pp. 38-63. Churchill-Livingstone, Edinburgh and London. Bister, K. (1986).Adu. Viral Oncol. 6 (in press). Bister, K., and Duesberg, P. H. (1979).Proc. Natl. Acad. Sci. U.S.A. 76, 5023-5027. Bister, K., and Duesberg, P. H. (1980).Cold Spring Harbor Symp. Quant. Biol. 44,801822. Bister, K., and Duesberg, P. H. (1982).Adu. Viral Oncol. 1,3-42. Bister, K., and Vogt, P. K. (1978).Virology 88, 213-221. Bister, K., Hayman, M. J., and Vogt, P. K. (1977).Virology 82,431-448. Bister, K., Loliger, H.-C., and Duesberg, P. H. (1979).J . Virol. 32,208-219. Bister, K., Ramsay, G., Hayman, M. J., and Duesberg, P. H. (1980a).Proc. Natl. Acad. Sci. U S A . 77,7142-7146. Bister, K., Lee, W.-H., and Duesberg, P. H. (1980b).J. Virol. 36, 617-621. Bister, K., Ramsay, G. M., and Hayman, M. J. (1982a).J . Virol. 41, 754-766. Bister, K., Nunn, M., Moscovici, C., Perbal, B., Baluda, M. A,, and Duesberg, P. H. (1982b).Proc. Natl. Acad. Sci. U S A . 79, 3677-3681. Bister, K., Jansen, H. W., Graf, T., Enrietto, P., and Hayman, M. J. (1983).J.Virol. 46, 337-346. Bister, K., Lurz, R., Jansen, H. W., Sutrave, P., Bonner, T. I., and Rapp, U. R. (1984).In “Genes and Cancer” (J. M. Bishop, J. D. Rowley, and M. Greaves, eds.), pp. 315328. Alan R. Liss, Inc., New York.

172

KLAUS BISTER AND HANS W. JANSEN

Blair, D. G., Oskarsson, M., Wood, T. G., McClements, W. L., Fischinger, P. J., and Vande Woude, G. F. (1981). Science 212,941-943. Blanchard, J.-M., Piechaczyk, M., Dani, C., Chambard, J.-C., Franchi, A,, Pouyssegur, J., and Jeanteur, P. (1985).Nature (London)317,443-445. Blasi, E., Mathieson, B. J., Varesio, L., Cleveland, J. L., Borchert, P. A., and Rapp, U. R. (1985).Nature (London)318,667-670. Bonner, T.I., O’Brien, S. J,, Nash, W. G., Rapp, U. R., Morton, C. C., and Leder, P. (1984). Science 223,71-74. Bonner, T. I., Kerby, S. B., Sutrave, P., Gunnell, M. A., Mark, G., and Rapp, U. R. (1985). Mol. Cell. Biol. 5, 1400-1407. Bonner, T. I., Oppermann, H., Seeburg, P., Kerby, S. B., Gunnell, M. A., Young, A. C., and Rapp, U. R. (1986).Nucleic Acids Res. 14, 1009-1015. Boyle, W. J., Lipsick, J. S., Reddy, E. P., and Baluda, M. A. (1983).Proc. Natl. Acad. Sci. U S A . 80,2834-2838. Boyle, W. J., Lampert, M. A., Lipsick, J. S.,and Baluda, M. A. (1984).Proc. Nutl. Acad. Sct. U . S A . 81,4265-4269. Boyle, W. J., Lampert, M. A,, Li, A. C., and Baluda, M. A. (1985).Mol. Cell. Biol. 5, 3017-3023. Brodeur, G . M., Seeger, R. C., Schwab, M., Varmus, H. E., and Bishop, J. M. (1984). Science 224, 1121-1124. Bunte, T., Greiser-Wilke, I., Donner, P., and Molling, K. (1982).EMBOJ. 1, 919-927. Butnick, N. Z., Miyamoto, C., Chizzonite, R., Cullen, B. R., Ju, G., and Skalka, A. M. (1985).Mol. Cell. Biol. 5,3009-3016. Cairns, J. (1975).Nature (London)255, 197-200. Cairns, J. (1981).Nature (London)289,353-357. Calabretta, B., Venturelli, D., Kaczmarek, L., Narni, F., Talpaz, M., Anderson, B., Beran, M., and Baserga, R. (1986).Proc. Natl. Acad. Sci. U.S.A.83, 1495-1498. Campisi, J., Gray, H. E., Pardee, A. B., Dean, M., and Sonenshein, G. E. (1984).Cell 36, 241-247. Capon, D. J., Chen, E. Y., Levinson, A. D., Seeburg, P. H., and Goeddel, D. V. (1983a). Nature (London) 302,33-37. Capon, D. J., Seeburg, P. H., McGrath, J. P., Hayflick, J. S., Edman, U., Levinson, A. D., and Goeddel, D. V. (1983b).Nature (London)304,507-513. Carpenter, G., and Cohen, S. (1979).Annu. Reo. Biochem. 48,193-216. Chang, E. H., Furth, M. E., Scolnick, E. M., and Lowy, D. R. (1982).Nature (London) 297,479-483. Chen, J. H. (1985).Mol. Cell. Biol. 5,2993-3000. Chen, J. H., Hayward, W. S.,and Moscovici, C. (1981).Virology 110, 128-136. Chinsky, J., Lilly, F., and Childs, G. (1985).Proc. Natl. Acad. Sci. U.S.A.82,565-569. Chiswell, D. J., Ramsay, G., and Hayman, M. J. (1981).J. Virol. 40, 301-304. Chiu, LM., Reddy, E. P., Givol, D., Robbins, K. C., Tronick, S. R., and Aaronson, S . A. (1984).Cell 37, 123-129. Cichutek, K.,and Duesberg, P. H. (1986).Proc. Nutl. Acad. Sci. U S A . 83, 2340-2344. Cleveland, J. L., Jansen, H. W., Bister, K.,Fredrickson, T. N., Morse, H. C., 111, Ihle, J. N., and Rapp, U. R. (1986).J . Cell. Biochem. 30, 195-218. Coffin, J. M. (1982). In “RNA Tumor Viruses” (R. Weiss, N. Teich, H. Varmus, and J. Coffin, eds.), pp. 261-368. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Coffin, J. M., Varmus, H. E., Bishop, J. M., Essex, M., Hardy, W. D., Martin, G. S.,

ONCOGENES IN RETROVIRUSES AND CELLS

173

Rosenberg, N. E., Scolnick, E. M., Weinberg, R. A., and Vogt, P. K. (1981).J.Virol. 40,953-957. Cohen, J. B., Unger, T.,Rechavi, G., Canaani, E., and Givol, D. (1983).Nature (London) 306,797-799. Cohen, S., Carpenter, G., and King, L. (1980).J. Biol. Chem. 255,4834-4842. Colby, W. W., Chen, E. Y., Smith, D. H., and Levinson, A. D. (1983).Nature (London) 301,722-725. Coll, J., Righi, M., d e Taisne, C., Dissous, C., Gegonne, A., and Stehelin, D. (1983a). EMBO J . 2,2189-2194. Coll, J., Saule, S., Martin, P., Raes, M. B., Lagrou, C., Graf, T., Beug, H., Simon, I. E., and Stehelin, D. (1983b).Ezp. Cell Res. 149, 151-162. Collins, S. J., and Groudine, M. T. (1982).Nature (London) 298,679-681. Collins, S. J., and Groudine, M. T. (1983).Proc. Natl. Acad. Sci. U . S A . 80,4813-4817. Collins, S. J., Kubonishi, I., Miyoshi, I., and Groudine, M. T. (1984).Science 225,72-74. Cooper, G. M. (1982). Science 218,801-806. Cooper, G. M., and Neiman, P. E. (1981).Nature (London)292,857-858. Cooper, C. S., Park, M., Blair, D. G., Tainsky, M. A., Huebner, K., Croce, C. M., and Vande Woude, G. F. (1984).Nature (London) 311,29-33. Cooper, G. M., Goubin, G., Diamond, A., and Neiman, P. (1986).Nature (London) 320, 579-580. Corcoran, L. M., Adams, J. M., Dunn, A. R., and Cory, S. (1984).Cell 37, 113-122. Coughlin, S. R., Lee, W. M. F., Williams, P. W., Giels, G. M., and Williams, L.T. (1985). Cell 43,243-251. Coussens, L., Van Beveren, C., Smith, D., Chen, E., Mitchell, R. L., Isacke, C. M., Verma, I. M., and Ullrich, A. (1986). Nature (London) 320,277-280. Crews, S., Barth, R., Hood, L., Prehn, J.,andCalame, K. (1982).Science2l8,1319-1321. Croce, C. M., and Klein, G. (1985). Sci.Am. 252(3),44-50. Cullen, B. R., Lomedico, P. T., and Ju, G. (1984).Nature (London) 307,241-245. Cuypers, H. T., Selten, G., Quint, W., Zijlstra, M., Maandag, E. R., Boelens, W., van Wezenbeek, P., Melief, C., and Berns, A. (1984).Cell 37,141-150. Dalla Favera, R., Wong-Staal, F., and Gallo, R. C. (1982a).Nature (London)299,61-63. Dalla Favera, R., Gelmann, E. P., Martinotti, S., Franchini, G., Papas, T. S., Gallo, R. C., and Wong-Staal, F. (1982b).Proc. Natl. Acad. Sci. U S A . 79,6497-6501. Dalla Favera, R., Bregni, M., Erikson, J., Patterson, D., Gallo, R. C., and Croce, C. M. (1982~). Proc. Natl. Acad. Sci. U.S.A. 79, 7824-7827. Dalla Favera, R., Franchini, G., Martinotti, S., Wong-Staal, F., Gallo, R. C., and Croce, C. M. (1982d).Proc. Natl. Acad. Sci. U.S.A. 79,4714-4717. Dani, C., Blanchard, J. M., Piechaczyk, M., El Sabouty, S., Marty, L., and Jeanteur, P. (1984).Proc. Natl. Acad. Sci. U S A . 81, 7046-7050. Darveau, A., Pelletier, J., and Sonenberg, N. (1985).Proc. Natl. Acad. Sci. USA. 82, 2315-23 19. Dayton, A. I., Selden, J. R., Laws, G., Dorney, D. J., Finan, J., Tripputi, P., Emanuel, B. S., Rovera, G., Nowell, P. C., and Croce, C. M. (1984).Proc. Nutl. Acad. Sci. U.S.A. 81,4495-4499. Dean, M., Park, M., Le Beau, M. M., Robins, T. S., Diaz, M. O., Rowley, J. D., Blair, D. G., and Vande Woude, G. F. (1985).Nature (London) 318,385-388. Dean, M., Levine, R. A., and Campisi, J. (1986).Mol. Cell. Biol. 6, 518-524. Debuire, B., Henry, C., Benaissa, M., Biserte, G., Claverie, J. M., Saule, S., Martin, P., and Stehelin, D. (1984). Science 224, 1456-1459. Decker, S. J. (1985).J . Biol. Chem. 260,2003-2006.

174

KLAUS BISTER AND HANS W. JANSEN

DeFeo, D., Gonda, M. A., Young, H. A., Chang, E. H., Lowy, D. R., Scolnick, E. M., and Ellis, R. W. (1981).Proc. Natl. Acad. Sci. U S A . 78, 3328-3332. DeFeo-Jones, D., Scolnick, E. M., Koller, R., and Dhar, R. (1983).Nature (London)306, 707-709. de Klein, A., van Kessel, A. G., Grosveld, G., Bartram, C. R., Hagemeijer, A., Bootsma, D., Spurr, N. K., Heisterkamp, N., Groffen, J., and Stephenson, J. R. (1982).Nature (London)300,765-767. Der, C. J., Krontiris, T. G., and Cooper, G. M. (1982).Proc. Natl. Acad. Sci. USA. 79, 3637-3640. de Taisne, C., Gegonne, A., Stehelin, D., Bernheim, A., and Berger, R. (1984).Nature (London)310,581-583. Devare, S. G., Reddy, E. P., Law, J. D., Robbins, K. C., and Aaronson, S. A. (1983).Proc. Natl. Acad. Sci. U S A . 80, 731-735. Dhar, R., Ellis, R. W., Shih, T. Y., Oroszlan, S., Shapiro, B., Maizel, J., Lowy, D., and Scolnick, E. (1982).Science 217,934-937. Diamond, A., Cooper, G. M., Rik, J., and Lane, M.-A. (1983).Nature (London) 305, 112-116. Diaz, M. O., Le Beau, M. M., Pitha, P., and Rowley, J. D. (1986).Science 231,265-267. Dickson, C., Eisenman, R., Fan, H., Hunter, E., and Teich, N. (1982).In “RNA Tumor Viruses” (R. Weiss, N. Teich, H. Varmus, and J. Coffin, eds), pp. 513-648. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Donner, P., Greiser-Wilke, I., and Molling, K. (1982).Nature (London)296,262-266. Donner, P., Bunte, T., Greiser-Wilke, I., and Molling, K. (1983).Proc. Natl. Acad. Sci. U.S.A.80,2861-2865. Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K. C., Aaronson, S. A., and Antoniades, H. N. (1983). Science 221,275-277. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A., Schlessinger, J., and Waterfield, M. D. (1984).Nature (London)307,521-527. Duesberg, P. H. (1980).Cold Spring Harbor Symp. Quant. Biol. 44, 13-29. Duesberg, P. H. (1983).Nature (London)304,219-225. Duesberg, P. H. (1985).Science 228, 669-677. Duesberg, P. H., and Vogt, P. K. (1979).Proc. Natl. Acad. Sci. U . S A . 76, 1633-1637. Duesberg, P. H., Bister, K., and Vogt, P. K. (1977).Proc. Natl. Acad. Sci. U S A . 74, 4320-4324. Duesberg, P. H., Bister, K., and Moscovici, C. (1979).Virology 99, 121-134. Duesberg, P. H., Bister, K., and Moscovici, C. (1980).Proc. Natl. Acad. Sci. USA. 77, 5120-5124. Duprey, S. P., and Boettiger, D. (1985).Proc. Natl. Acad. Sci. U.S.A.82,6937-6941. Durban, E. M., and Boettiger, D. (1981a).J. Virol. 37,488-492. Durban, E. M., and Boettiger, D. (1981b).Proc. Natl. Acad. Sci. U S A . 78,3600-3604. Dutta, A., Wang, L.-H., Hanafusa, T., and Hanafusa, H. (1985).]. Virol. 55, 728-735. Eick, D., Piechaczyk, M., Henglein, B., Blanchard, J.-M., Traub, B., Kofler, E., Wiest, S., Lenoir, G. M., and Bornkamm, G. W. (1985).EMBOJ. 4,3717-3725. Einat, M.,Resnikky, D., and Kimchi, A. (1985).Nature (London)313,597-600. Eisenman, R. N., Tachibana, C. Y., Abrams, H. D., and Hann, S. R. (1985).Mol. Cell. B i d . 5, 114-126. Ek, B., Westermark, B., Wasteson, A., and Heldin, C.-H. (1982).Nature (London)295, 419-420. Engelbreth-Holm, J., and Rothe Meyer, A. (1935).Acto Pathol. Microbiol. Scand. 12, 352-365.

ONCOGENES IN RETROVIRUSES AND CELLS

175

Enrietto, P. J., and Hayman, M. J. (1982).J . Virol. 44,711-715. Enrietto, P. J., Hayman, M. J., Ramsay, G. M., Wyke, J. A., and Payne, L. N. (1983a). Virology 124, 164-172. Enrietto, P. J., Payne, L. N., and Hayman, M. J. (1983b).Cell 35,369-379. Erikson, J.,ar-Rushdi, A., Drwinga, H. L., Nowell, P. C., and Croce, C. M. (1983).Proc. Natl. Acad. Sci. U.SA. 80,820-824. Eva, A,, Robbins, K. C., Andersen, P. R., Srinivasan, A., Tronick, S. R., Reddy, E. P., Ellmore, N. W., Galen, A. T., Lautenberger, J. A,, Papas, T. S., Westin, E. H., WongStaal, F., Gallo, R. C., and Aaronson, S. A. (1982).Nature (London)295, 116-119. Evan, G. I., and Hancock, D. C. (1985).Cell 43,253-261. Evan, G. I., Lewis, G. K., and Bishop, J. M. (1984).Mol. Cell. Biol. 4,2843-2850. Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985).Mol. Cell. Biol. 5,3610-

3616.

Fenner, F. (1976).lnteruirology 7, 1-115. Feo, S., Harvey, R., Showe, L., and Croce, C. M. (1986).Proc. Natl. Acad. Sci. USA. 83,

706-709.

Feramisco, J., Ozanne, B., and Stiles, C., eds. (1985).“Cancer Cells 3:Growth Factors and Transformation.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Flordellis, C. S., Kan, N. C., Lautenberger, J. A., Samuel, K. P., Garon, C. F., and Papas, T. S. (1985).Virology 141,267-274. Foulds, L. (1958).]. Chronic Dis. 8,2-37. Franchini, G., Wong-Staal, F., Baluda, M. A,, Lengel, C., and Tronick, S. R. (1983).Proc. Natl. A d . Sci. U.SA. 80,7385-7389. Frykberg, L., Palmieri, S., Beug, H., Graf, T., Hayman, M. J., and Vennstrom, B. (1983). Cell 32,227-238. Fukui, M.,Yamamoto, T., Kawai, S., Maruo, K.,and Toyoshirna, K. (1985)Proc. Natl. A d . Sci. U . S A . 82,5954-5958. Fung, Y.-K. T., Fadly, A. M., Crittenden, L. B., and Kung, H.-J. (1981).Proc. Natl. Acad. Sci. U S A . 78,3418-3422. Fung, Y.-K. T., Lewis, W. G., Crittenden, L. B., and Kung, H.-J. (1983).Cell 33, 357-

368.

Fung, Y.-K.T.,Shackleford, G. M., Brown, A. M. C., Sanders, G. S., and Varmus, H. E. (1985).Mol. Cell. Biol. 5,3337-3344. Galibert, F., Dupont de Dinechin, S., Righi, M., and Stehelin, D. (1984).EMBO J . 3,

1333-1338.

Gallwitz, D., Donath, C., and Sander, C. (1983).Nature (London)306,704-707. Ghysdael, J., Gegonne, A., Pognonec, P., Dernis, D., Leprince, D., and Stehelin, D. (1986).Proc. Natl. Acad. Sci. USA. 83, 1714-1718. Gilmore, T., DeClue, J. E., and Martin, G. S. (1985).Cell 40,609-618. Gonda, T. J., and Bishop, J. M. (1983).J. Virol. 46,212-220. Gonda, T.J., and Metcalf, D. (1984).Nature (London)310,249-251. Gonda, T. J., Sheiness, D. K., Fanshier, L., Bishop, J. M., Moscovici, C,, and Moscovici, M. G. (1981).Cell 23,279-290. Gonda, T. J., Sheiness, D. K., and Bishop, J. M. (1982).Mol. Cell. Biol. 2,617-624. Gonda, T.J., Gough, N. M.,Dunn, A. R., and de Blaquiere, J. (1985).EMBOJ. 4,20032008.

Goubin, G., Goldman, D. S., Luce, J., Neiman, P. E., and Cooper, G. M. (1983).Nature (London)302,114-119. Goyette, M.,Petropoulos, C. J., Shank, P. R., and Fausto, N. (1984).Mol. Cell. B i d . 4,

1493-1498.

176

KLAUS BISTER AND HANS W. JANSEN

Graf, T., and Beug, H. (1978).Biochim. Biophys. Acta 516,269-299. Graf, T., and Beug, H. (1983).Cell 34,7-9. Graf, T., and Stehelin, D. (1982).Biochim. Biophys. Acta 651, 245-271. Graf, T., Royer-Pokora, B., Schubert, G. E., and Beug, H. (1976).Virology 71,423-433. Graf, T., Royer-Pokora, B., Meyer-Glauner, W., Claviez, M., Gotz, E., and Beug, H. (1977).Virology 83,96-109. Graf, T., Ade, N., and Beug, H. (1978).Nature (London)275,496-501. Graf, T., Oker-Blom, N., Todorov, T. G., and Beug, H. (1979).Virology 99,431-436. Graf, T., v. Weizsaecker, F., Grieser, S., Coll, J., Stehelin, D., Patschinsky, T., Bister, K., Bechade, C., Calothy, G., and Leutz, A. (1986). Cell (in press). Green, S., Walter, P., Kumar, V., Krust, A,, Bornert, J.-M., Argos, P., and Chambon, P. (1986).Nature (London) 320, 134-139. Greene, G. L., Gilna, P., Waterfield, M., Baker, A., Hart, Y.,and Shine, J. (1986).Science 231,1150-1154. Griffin, C. A., and Baylin, S. B. (1985).Cancer Res. 45,272-275. Guroff, G., ed. (1983). “Growth and Maturation Factors,” Vol. 1. Wiley, New York. Hackett, P. B., Swanstrom, R., Varmus, H. E., and Bishop, J. M. (1982).]. Virol. 41,527534. Hall, W. J., Bean, C. W., and Pollard, M. (1941).Am. J. Vet. Res. 2, 272-279. Hamlyn, P. H., and Rabbits, T. H. (1983).Nature (London)304, 135-139. Hampe, A., Laprevotte, I., and Galibert, F. (1982).Cell 30, 775-785. Hampe,A., Gobet, M., Sherr, C. J., and Galibert, F. (1984).Proc. Natl. Acad. Sci. U . S A . 81,85-89. Hanafusa, H. (1977). In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 10, pp. 401-483. Plenum, New York. Hanafusa, H., Iba, H., Takeya, T., and Cross, F. R. (1984).In “Cancer Cells 2: Oncogenes and Viral Genes” (G. F. Vande Woude, A. J. Levine, W. C. Topp, and J. D. Watson, eds.), pp. 1-7. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Hann, S. R., and Eisenman, R. N. (1984). Mol. Cell. Biol. 4,2486-2497. Hann, S. R.,Abrams, H. D., Rohrschneider, L. R., and Eisenman, R. N. (1983).Cell 34, 789-798. Hann, S. R., Thompson, C. B., and Eisenman, R. N. (1985).Nature (London)314,366369. Harper, M. E., Franchini, G., Love, J,, Simon, M. l., Gallo, R. C., and Wong-Staal, F. (1983).Nature (London)304,169-171. Hayashi, K., Makino, R., and Sugimura, T. (1984).Gann 75,475-478. Hayflick, J., Seeburg, P. H., Ohlsson, R., Pfeifer-Ohlsson, S., Watson, D., Papas, T., and Duesberg, P. H. (1985).Proc. Natl. Acad. Sci. U.S.A. 82,2718-2722. Hayman, M. J., and Beug, H. (1984).Nature (London)309,460-462. Hayman, M. J., Royer-Pokora, B., and Graf, T. (1979a).Virology 92,31-45. Hayman, M. J., Kitchener, G., and Graf, T. (1979b).Virology 98, 191-199. Hayman, M. J., Ramsay, G. M., Savin, K., and Kitchener, G . (1983). Cell 32,579-588. Hayman, M. J,, Kitchener, G., Vogt, P. K., and Beug, H. (1985).Proc. Natl. Acad. Sci. U S A . 82,8237-8241. Hayman, M. J., Kitchener, G., Knight, J., McMahon, J., Watson, R., and Beug, H. (1986). Virology 150,270-275. Hayward, W. S., Neel, B. G., and Astrin, S. M. (1981).Nature (London)290,475-479. Heisterkamp, N., Stephenson, J. R., Groffen, J., Hansen, P. F., de Klein, A., Bartram, C. R., and Grosveld, G. (1983).Nature (London)306,239-242. Heldin, C.-H., and Westermark, B. (1984).Cell 37,9-20.

ONCOGENES IN RETROVIRUSES AND CELLS

177

Henry, C., Coquillaud, M., Saule, S., Stehelin, D., and Debuire, B. (1985).Virology 140,

179-182.

Hihara, H., Yamamoto, H., Shimohira, H., Arai, K., and Shimizu, T. (1983).J . Natl. Cancer Inst. (U.S.) 70,891-897. Hoffmann, F. M., Fresco, L. D., Hoflinan-Falk, H., and Shilo, B.-Z. (1983).Cell 35,393-

401.

Hu, S. S. F., and Vogt, P. K. (1979).Virology 92,278-284. Hu, S. S. F., Moscovici, C., and Vogt, P. K. (1978).Virology 89, 162-178. Hu, S.S.F., Lai, M. M. C., and Vogt, P. K. (1979).Proc. Natl. Acad. Sci. U S A . 76,1265-

1268.

Huang, C.-C., Hammond, C., and Bishop, J. M. (1985).J. Mol. Biol. 181, 175-186. Hung, M.-C., Schechter, A. L., Chevray, P.-Y. M., Stern, D. F., and Weinberg, R. A. (1986).Proc. Natl. Acad. Sci. U S A . 83,261-264. Hunter, T. (1984).Sci. Am. 251(2),60-69. Ishizaki, R., and Shimizu, T. (1970).Cancer Res. 30,2827-2831. Ishizaki, R., Langlois, A. J., Chabot, J., and Beard, J. W. (1971).J. Virol. 8,821-827. Ivanov, X . , Mladenov, Z., Nedyalkov, S., and Todorov, T. G. (1962).Bull. Inst. Pathol. Comp. Anim. 9,5-36. Ivanov, X.,Mladenov, Z., Nedyalkov, S., Todorov, T. G., and Yakimov, M. (1964).Bull. Inst. Pathol. Comp. Anim. 10, 5-38. Jacks, T.,and Varmus, H. E. (1985).Science 230, 1237-1242. Jacobs, S., Kull, F. C., Earp, H. S., Svoboda, M. E., Van Wyk, J. J., and Cuatrecasas, P. (1983).J . Biol. Chem. 258,9581-9584. Jansen, H. W., and Bister, K. (1985).Virology 143,359-367. Jansen, H. W., Patschinsky, T., and Bister, K. (1983a).J . Virol. 48,61-73. Jansen, H. W., Riickert, B., L u n , R., and Bister, K. (198313).EMBO J. 2,1969-1975. Jansen, H. W., Lurz, R., Bister, K., Bonner, T. I., Mark, G. E., and Rapp, U. R. (1984a). Nature (London)307,281-284. Jansen, H. W., Trachmann, C., and Bister, K. (1984b).Virology 137,217-224. Jansen, H. W., Patschinsky, T., Walther, N.,Lurz, R., and Bister, K. (1985a).Virology

142,248-262.

Jansen, H. W., Trachmann, C., Patschinsky, T., and Bister, K. (1985b).Mod. Trends Hum. Leuk. 6,280-283. Jansson, M., Philipson, L., and Vennstriim, B. (1983).EMBO J. 2,561-565. Josephs, S. F., Guo, C., Ratner, L., and Wong-Staal, F. (1984).Science 223,487-491. Kan, N. C., Flordellis, C. S., Garon, C. F., Duesberg, P. H., and Papas, T. S. (1983).Proc. Natl. Acad. Sci. U S A . 80,6566-6570. Kan, N. C., Flordellis, C. S., Mark, G. E., Duesberg, P. H., and Papas, T. S. (1984).Proc. Natl. Acad. Sci. U S A . 81,3000-3004. Kasuga, M., Zick, Y., Blithe, D. L., Crettaz, M., and Kahn, C. R. (1982).Nature (London)

298,667-669.

Kaken, A. L., Kornberg, T. B.,and Bishop, J. M. (1985).Cell 41,449-456. Kelekar, A,, and Cole, M. D. (1986).Mol. Cell. Biol. 6,7-14. Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. (1983).Cell 35,603-610. Kelly, K., Cochran, B., Stiles, C., and Leder, P. (1984).Curr. Top. Microbiol. Immunol.

113,117-126.

King, C. R.,Kraus, M. H., and Aaronson, S. A. (1985).Science 229,974-976. Kitamura, N., Kitamura, A., Toyoshima, K., Hirayama, Y.,and Yoshida, M. (1982).Nature (London) 297,205-208. Kitchener, G., and Hayman, M. J. (1980).Proc. Natl. Acad. Sci. U S A . 77,1637-1641.

178

KLAUS BISTER AND HANS W. JANSEN

Klein, G. (1981).Nature (London)294,313-318. Klein, G . (1983).Cell 32,311-315. Klein, G., and Klein, E. (1984).Carctnogenesis 5,429-435. Klein, G., and Klein, E. (1985).Nature (London)315, 190-195. Klempnauer, K.-H., and Bishop, J. M. (1983).J. Virol. 48,565-572. Klempnauer, K.-H., and Bishop, J. M. (1984).J. Virol. 50,280-283. Klempnauer, K.-H., and Sippel, A. E. (1986).Mol. Cell. Biol. 6,62-69. Klempnauer, K.-H., Gonda, T. J., and Bishop, J. M. (1982).Cell 31,453-463. Klempnauer, K.-H., Ramsay, G., Bishop, J. M., Moscovici, M. G., Moscovici, C., McGrath, J. P., and Levinson, A. D. (1983).Cell 33, 345-355. Klempnauer, K.-H., Symonds, G., Evan, G. I., and Bishop, J. M. (1984).Cell 37,537-

547.

Kloetzer, W. S., Maxwell, S. A., and Arlinghaus, R. B. (1983).Proc. Natl. Acad. Sci. U.SA. 80,412-416. Kloetzer, W. S., Maxwell, S. A., and Arlinghaus, R. B. (1984).Virology 138, 143-155. Knight, E., Anton, E. D., Fahey, D., Friedland, B. K., and Jonak, G. J. (1985).Proc. Natl. Acad. Sci. U.S.A.82, 1151-1154. Knudson, A. G. (1973).Ado. Cancer Res. 17,317-352. Knudson, A.G. (1981).In “Cancer” (J.H. Burchenal and H.F. Oettgen, eds.), Vol. 1, pp. 381-396. Grune & Stratton, New York. Kohl, N. E., Kanda, N., Schreck, R.R., Bruns, G., Latt, S. A., Gilbert, F., and Alt, F. W. (1983).Cell 35,359-367. Kohl, N. E.,Legouy, E., DePinho, R. A., Nisen, P. D., Smith, R.K., Gee, C. E., and Alt, F.W.(1986).Nature (London)319,73-77. Kozak, M. (1980).Cell 22,7-8. Kozak, M. (1981).Nucleic Acidr Res. 9,5233-5252. Kozak, C.,Gunnel], M. A,, and Rapp, U. R. (1984).J . Virol. 49,297-299. Kris, R. M.,Lax, I., Gullick, W., Waterfield, M. D., Ullrich, A., Fridkin, M., and Schlessinger, J. (1985).Cell 40,619-625. Lachman, H. M., and Skoultchi, A. I. (1984).Nature (London)310,592-594. Lacy, J., Sarkar, S. N., and Summers, W. C. (1986).Proc. Natl. Acad. Sci. U S A . 83,

1458-1462.

Lai, M. M. C., Hu, S. S. F., and Vogt, P. K. (1979).Virology 97,366-377. Land, H., Parada, L. F., and Weinberg, R. A. (1983a).Nature (London)304, 596-602. Land, H., Parada, L. F., and Weinberg, R. A. (1983b).Science 222, 771-778. Lane, M.-A., Sainten, A., Doherty, K. M., and Cooper, G. M. (1984).Proc. Natl. Acad. S C ~U . S A . 81,2227-2231. Langlois, A. J., Sankaran, S., Hsiung, P.-H. L., and Beard, J. W. (1967).].Virol. 1,1082-

1084.

Langlois, A. J., Fritz, R. B., Heine, U., Beard, D., Bolognesi, D. P., and Beard, J. W. (1969).Cancer Res. 29,2056-2074. Lautenberger, J. A., Schulz, R.A., Garon, C. F., Tsichlis, P. N., and Papas, T. S. (1981). Proc. Natl. Acad. Sci. U S A . 78, 1518-1522. Lax, I., Kris, R., Sasson, I., Ullrich, A., Hayman, M. J., Beug, H., and Schlessinger, J. (1985).EMBO J. 4,3179-3182. Leder, P., Battey, J., Lenoir, G., Moulding, C., Murphy, W., Potter, H., Stewart, T., and Taub, R. (1983).Science 222,765-771. Lee, W.-H., Murphree, A. L.,and Benedict, W. F. (1984).Nature (London)309,458-

460.

ONCOGENES IN RETROVIRUSES AND CELLS

179

Lee, W. M. F., Schwab, M., Westaway, D., and Varmus, H. E. (1985). Mol. Cell. B i d . 5, 3345-3356.

Leprince, D., Gegonne, A., Coll, J., de Taisne, C., Schneeberger, A., Lagrou, C., and Stehelin, D. (1983a). Nature (London) 306, 395-397. Leprince, D., Saule, S., de Taisne, C., Gegonne, A., Begue, A., Righi, M., and Stehelin, D. (1983b). E M B O J . 2,1073-1078. Levy, L. S., Gardner, M. B., and Casey, J. W. (1984).Nature (London) 308, 853-856. Li, Y., Holland, C. A., Hartley, J. W., and Hopkins, N. (1984). Proc. Natl. Acad. Sci. U S A . 81,6808-6811. Lin, C. R., Chen, W. S., Kruiger, W., Stolarsky, L. S., Weber, W., Evans, R. M., Verma, I. M., Gill, G. N., and Rosenfeld, M. G. (1984). Science 224,843-848. Linial, M. (1982). Virology 119,382-391. Linial, M., and Groudine, M. T. (1985). Proc. Natl. Acad. Sci. U.S.A. 82,53-57. Little, C. D., Nau, M. M., Carney, D. N., Gazdar, A. F., and Minna, J. D. (1983).Nature

(London)306,194-196.

Livneh, E.,Glazer, L., Segal, D., Schlessinger, J., and Shilo, B.-Z. (1985).Cell 40,599607.

Loliger, H.-C. (1964). Dtsch. Tierdrztl. Wochenschr. 71,207-212. Lorincz, A. T., and Reed, S. I. (1984).Nature (London) 307, 183-185. McCormack, J. E., Pepe, V. H., Kent, R. B., Dean, M., Marshak-Rothstein, A., and Sonenshein, G. E. (1984). Proc. Natl. Acad. Sci. U S A . 81,5546-5550. McCoy, M. S., Toole, J. J., Cunningham, J. M., Chang, E. H., Lowy, D. R., and Weinberg, R. A. (1983). Nature (London) 302,79-81. McGrath, J. P., Capon, D. J., Smith, D. H., Chen, E. Y., Seeburg, P. H., Goeddel, D. V., and Levinson, A. D. (1983).Nature (London) 304,501-506. McLachlan, A. D., and Boswell, D. R. (1985).J. Mol. Biol. 185,39-49. Madhavan, K., Bilodeau-Wentworth, D., and Wadsworth, S. C. (1985). Mol. Cell. Biol. 5, 7-16.

Makino, R., Hayashi, K., and Sugimura, T. (1984).Nature (London) 310,697-698. Marcu, K. B., Harris, L. J., Stanton, L. W., Erikson, J., Watt, R., and Croce, C. M. (1983). Proc. Natl. Acad. Sci. U S A . 80,519-523. Mark, G. E., and Rapp, U. R. (1984). Science 224,285-289. Maxwell, S. A., and Arlinghaus, R. B. (1985). Virology 143,321-333. Mayes, E. L. V., and Waterfield, M. D. (1984). E M B O J . 3,531-537. Mellon, P., Pawson, A., Bister, K., Martin, G. S., and Duesberg, P. H. (1978). Proc. Natl. Acad. Sci. U S A . 75,5874-5878. Merlino, G. T., Xu, Y.-H., Ishii, S., Clark, A. J. L., Semba, K., Toyoshima, K., Yamamoto, T., and Pastan, I. (1984). Science 224,417-419. Merlino, G. T., Ishii, S., Whang-Peng, J., Knutsen, T., Xu, Y.-H., Clark, A. J. L., Stratton, R. H., Wilson, R. K., Ma, D. P., Roe, B. A,, Hunts, J. H., Shimizu, N., and Pastan, I. (1985). Mol. Cell. Biol. 5, 1722-1734. Miles, B. D., and Robinson, H. L. (1985). J . Virol. 54,295-303. Miller, A. D., Curran, T., and Verma, I. M. (1984). Cell 36,51-60. Miyamoto, C., Chizzonite, R., Crowl, R., Rupprecht, K., Kramer, R., Schaber, M., Kumar, G., Poonian, M., and Ju, G. (1985a). Proc. Natl. Acad. Sci. U . S A . 82, 72327236.

Miyamoto, C., Smith, G. E., Farrell-Towt, J., Chizzonite, R., Summers, M. D., and Ju, G. (198513). Mol. Cell. Biol. 5,2860-2865. Molders, H., Defesche, J., Muller, D., Bonner, T. I., Rapp, U. R., and Miiller, R. (1985). E M B O I. 4,693-698.

180

KLAUS BISTER AND HANS W. JANSEN

Miilling, K., Heimann, B., Beimling, P., Rapp, U. R., and Sander, T. (1984).Nature (London) 312,558-561. Moscovici, C. (1975).Curr. Top. Microbiol. Immunol. 71,79-101. Moscovici, C.,and Gazzolo, L. (1982).Adu. Vtral Oncol. 1,83-106. Moscovici, C., Samarut, J., Gazzolo, L., and Moscovici, M. G. (1981).Virology 113,765-

768.

Moscovici, M. G., and Moscovici, C. (1983).Proc. Natl. Acad. Sci. U.S.A. 80,1421-1425. Moscovici, M. G., Klempnauer, K.-H., Symonds, G., Bishop, J. M., and Moscovici, C. (1985).Mol. Cell. B i d . 5,3301-3303. Mounk, J. D., Steinberg, 4.D., Klinman, D. M., Smith, H. R., and Mushinski, J. F. (1984).Science 226, 1087-1089. Muller, R., and Muller, D. (1984).EMBO J . 3, 1121-1127. Mullins, J. I., Brody, D. S., Binari, R. C., and Cotter, S. M. (1984).Nature (London)308,

856-858.

Mushinski, J. F., Potter, M., Bauer, S. R., and Reddy, E. P. (1983).Science 220,795-798. Naharro, G.,Robbins, K. C., and Reddy, E. P. (1984).Science 223,63-66. Nau, M. M., Brooks, B. J., Battey, J., Sausville, E., Gazdar, A. F., Kirsch, I. R., McBride, 0.W., Bertness, V., Hollis, G. F., and Minna, J. D. (1985).Nature (London)318,69-

73.

Nau, M. M., Brooks, B. J., Carney, D. N., Gazdar, A. F., Battey, J. F., Sausville, E. A., and Minna, J. D. (1986).Proc. Natl. Acad. Sci. U.S.A. 83, 1092-1096. Neckameyer, W. S., and Wang, L.-H. (1985).J . Vdrol. 53,879-884. Neel, B. G., Hayward, W. S., Robinson, H. L., Fang, J., and Astrin, S. M. (1981).Cell 23,

323-334.

Neel, B. G., Gasic, G. P., Rogler, C. E., Skalka, A. M., Ju, G., Hishinuma, F., Papas, T., Astrin, S. M., and Hayward, W. S. (1982).J . Virol. 44, 158-166. Neil, J. C., Hughes, D., McFarlane, R.,Wilkie, N. M., Onions, D. E., Lees, G., and Jarrett, 0.(1984).Nature (London) 308,814-820. Neiman, P., Wolf, C., Enrietto, P. J., and Cooper, G. M. (1985).Proc. Natl. Acad. Scf. U.S.A. 82,222-226. Neuman-Silberberg, F. S., Schejter, E., H o h a n n , F. M., and Shilo, B.-2. (1984).Cell

37,1027-1033.

Nilsen, T.W., Maroney, P. A., Goodwin, R. G., Rottman, F. M., Crittenden, L. B., Raines, M. A,, and Kung, H.-J. (1985).Cell 41,719-726. Nishida, T.,Sakamoto, S.-I., Yamamoto, T., Hayman, M., Kawai, S., and Toyoshima, K. (1984).Gann 75,325-333. Nishizawa, M., Semba, K., Yoshida, M. C., Yamamoto, T., Sasaki, M., and Toyoshima, K. (1986).Mol. Cell. B i d . 6,511-517. Nowell, P., Finan, J., Dalla Favera, R., Gallo, R. C., ar-Rushdi, A., Romanczuk, H., Selden, J. R., Emanuel, B. S., Rovera, G., and Croce, C. M. (1983).Nature (London)

306,494-497.

Nunn, M. F., Seeburg, P. H., Moscovici, C., and Duesberg, P. H. (1983).Nature (London) 306,391-395. Nunn, M. F., Weiher, H., Bullock, P., and Duesberg, P. (1984).Virology 139,330-339. Nusse, R., and Varmus, H. E. (1982).Cell 31,99-109. Oker-Blom, N.,Hortling, L., Kallio, A., Nurmiaho, E.-L., and Westermarck, H. (1978).J . Gen. Virol. 40,623-633. Pachl, C.,Biegalke, B., and Linial, M. (1983a).J . Virol. 45, 133-139. Pachl, C., Schubach, W., Eisenman, R., and Linial, M. (19838).Cell 33,335-344. Palmieri, S. (1986).J . Virol. 58, 134-141.

ONCOGENES IN RETROVIRUSES AND CELLS

181

Palmieri, S., Beug, H., and Graf, T. (1982).Virology 123,296-311. Palmieri, S., Kahn, P., and Graf, T. (1983).E M B O J . 2,2385-2389. Papas, T. S., Kan, N. K., Watson, D. K., Flordellis, C. S., Psallidopoulos, M. C., Lautenberger, J., Samuel, K. P., and Duesberg, P. H. (1984).In “Cancer Cells 2: Oncogenes and Viral Genes” (G. F. Vande Woude, A. J. Levine, W. C. Topp, and J. D. Watson, eds.), pp. 153-163. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Parada, L. F., Tabin, C. J., Shih, C., and Weinberg, R. A. (1982).Nature (London)297,

474-478.

Patschinsky, T., Walter, G., and Bister, K. (1984).Virology 136,348-358. Patschinsky, T., Jansen, H. W., Blocker, H., Frank, R., and Bister, K. (1986a).J. Virol. (in press). Patschinsky, T., Schroeer, B., and Bister, K. (1986b).Mol. Cell. B i d . 6,739-744. Payne, G. S., Courtneidge, S. A., Crittenden, L. B., Fadly, A. M., Bishop, J. M., and Varmus, H. E. (1981).Cell 23,311-322. Payne, G. S., Bishop, J. M., and Varmus, H. E. (1982).Nature (London)295,209-214. Pelicci, P.-G., Lanfrancone, L., Brathwaite, M. D., Wolman, S . R., and Dalla Favera, R.

(1984).Science 224, 1117-1121.

Perbal, B., Cline, J. M., Hillyard, R. L., and Baluda, M. A. (1983).J. Virol. 45,925-940. Perry, R. P. (1983).Cell 33,647-649. Persson, H., and Leder, P. (1984).Science 225, 718-721. Persson, H., Hennighausen, L., Taub, R., DeGrado, W., and Leder, P. (1984).Science

225,687-693.

Persson, H., Gray, H. E., and Godeau, F. (1985).Mol. Cell. Biol. 5,2903-2912. Peters, G., Brookes, S., Smith, R., and Dickson, C. (1983).Cell 33,369-377. Peterson, T. A., Yochem, J., Byers, B., Nunn, M. F., Duesberg, P. H., Doolittle, R. F., and Reed, S. I. (1984).Nature (London)309,556-558. Pfeifer, S., Zabielski, J., Ohlsson, R., Frykberg, L., Knowles, J., Pettersson, R., OkerBlom, N., Philipson, L., Vaheri, A., and Vennstrom, B. (1983)J Virol. 46,347-354. Pfeifer-Ohlsson, S., Goustin, A. S., Rydnert, J., Wahlstrom, T., Bjersing, L., Stehelin, D., and Ohlsson, R. (1984).Cell 38,585-596. Piechaczyk, M., Yang, J.-Q., Blanchard, J.-M., Jeanteur, P., and Marcu, K. B. (1985).Cell

42,589-597.

Powers, S., Kataoka, T., Fasano, O., Goldfarb, M., Strathern, J., Broach, J., and Wigler, M. (1984).Cell 36,607-612. Pozzatti, R., Muschel, R., Williams, J., Padmanabhan, R., Howard, B., Liotta, L., and Khoury, G. (1986).Science 232,223-227. Privalsky, M. L., and Bishop, J. M. (1982).Proc. Natl. Acad. Sci. U S A . 79,3958-3962. Privalsky, M. L., and Bishop, J. M. (1984).Virology 135,356-368. Privalsky, M. L., Sealy, L., Bishop, J. M., McGrath, J. P., and Levinson, A. D. (1983).

Cell 32,1257-1267.

Privalsky, M. L., Ralston, R., and Bishop, J. M. (1984).Proc. Natl. Acad. Sci. U S A . 81,

704-707.

Pulciani, S., Santos, E., Lauver, A. V., Long, L. K., Aaronson, S. A., and Barbacid, M. (1982).Nature (London)300,539-542. Purchase, H. G., and Burmester, B. R. (1972).In “Diseases of the Poultry” (M. S. Hofstad, B. W. Calnek, C. F. Helmboldt, W. M. Reid, and H. W. Yoder, eds.), pp. 502-568. Iowa State Univ. Press, Ames. Quade, K. (1979).Virology 98,461-465.

182

KLAUS BISTER AND HANS W. JANSEN

Quade, K., Saule, S., Stehelin, D., Kitchener, G., and Hayman, M. J. (1983).J . Gen. Virol. 64,83-94. Rabbitts, T . H. (1985).Trends Genet. 1,327-331. Rabbitts, T. H., Hamlyn, P. H., and Baer, R. (1983). Nature (London)306, 760-765. Rabbitts, T. H., Forster, A., Hamlyn, P., and Baer, R. (1984).Nature (London)309,592597. Rabbitts, P. H., Watson, J. V., Lamond, A., Forster, A., Stinson, M. A., Evan, G., Fischer, W., Atherton, E., Sheppard, R.,and Rabbitts, T. H.(1985a).E M B O J . 4,2009-2015. Rabbitts, P. H., Forster, A., Stinson, M. A,, and Rabbitts, T. H. (1985b). E M B O J . 4, 3727-3733. Rabin, M. S., Doherty, P. J., and Gottesman, M. M. (1986).Proc. Natl. Acad. Sci. U.S.A. 83,357-360. Radke, K., Beug, H., Kornfeld, S., and Graf, T. (1982). Cell 31,643-653. Raines, M. A., Lewis, W. G., Crittenden, L. B., and Kung, H.-J. (1985).Proc. Natl. Acad. sci. U.S.A.8a72287-229i. Ralston, R., and Bishop, J. M. (1983). Nature (London)306,803-806. Ramsay, G . M., and Hayman, M. J. (1980). Virology 106,71-81. Ramsay, G. M., and Hayman, M. J. (1982).J . Virol. 41, 745-753. Ramsay, G. M., Graf, T., and Hayman, M. J. (1980).Nature (London)288, 170-172. Ramsay, G. M., Enrietto, P. J., Graf, T., and Hayman, M. J. (1982a).Proc. Natl. Acad. Sci. U.S.A.79,6885-6889. Ramsay, G. M., Hayman, M. J., and Bister, K. (1982b). E M B O J . 1,1111-1116. Ramsay, G . M., Evan, G. I., and Bishop, J. M. (1984). Proc. Natl. Acad. Sci. U.S.A.81, 7742-7746. Rapp, U. R.,Reynolds, F. H., and Stephenson, J. R. (1983a).J. Virol. 45,914-924. Rapp, U. R.,Goldsborough, M. D., Mark, G. E., Bonner, T. I., Groffen, J., Reynolds, F. H., and Stephenson, J. R. (1983b). Proc. Natl. Acad. Sci. U.S.A. 80,4218-4222. Rapp, U. R., Cleveland, J. L., Fredrickson, T. N., Holmes, K. L., Morse, H. C., 111, Jansen, H. W., Patschinsky, T., and Bister, K. (1985).J . Virol. 55,23-33. Rechavi, G., Givol, D., and Canaani, E. (1982). Nature (London)300,607-611. Reddy, E. P., Reynolds, R. K., Santos, E.,and Barbacid, M. (1982).Nature (London)300, 149-152. Reddy, E. P., Reynolds, R. K., Watson, D. K., Schultz, R.A., Lautenberger, J., and Papas, T. S. (1983a). Proc. Natl. Acad. Sci. U S A . 80,2500-2504. Reddy, E.P., Smith, M. J., and Srinivasan, A. (198313). Proc. Natl. Acad. Sci. U.S.A.80, 3623-3627. Reddy, E. P., Lipman, D., Andersen, P. R.,Tronick, S. R.,and Aaronson, S.A. (1985).J. Virol. 53,984-987. Reitsma, P. H., Rothberg, P. G., Astrin, S. M., Trial, J., Bar-Shavit, Z., Hall, A., Teitelbaum, S. L., and Kahn, A. J. (1983). Nature (London)306,492-494. Rettenmier, C. W., Anderson, S. M., Riemen, M. W., and Hanafusa, H. (1979).J . Virol. 32,749-761. Robins, T., Bister, K.,Garon, C., Papas, T., and Duesberg, P. (1982).J . Virol. 41,635-

642.

Roebroek, A. J. M., Schalken, J. A., Verbeck, J. S., Van den Ouweland, A. M. W., Onnekink, C., Bloemers, H. P. J., and Van de Ven, W. J. M. (1985). E M B O J . 4, 2897-2903. Rogers, J. (1986).Nature (London) 320,579. Rosson, D., and Reddy, E. P. (1986).Nature (London)319,604-606.

ONCOGENES IN RETROVIRUSES AND CELLS

183

Roussel, M., Saule, S., Lagrou, C., Rommens, C., Beug, H., Graf, T., and Stehelin, D. (1979).Nature (London)281,452-455. Rowley, J. D. (1983).Nature (London)301,290-291. Rowley, J. D. (1985). I n “Leukemia” (I. L. Weissman, ed.), pp. 179-202. SpringerVerlag, Berlin and New York. Royer-Pokora, B., Grieser, S., Beug, H., and Graf, T. (1979).Nature (London)282,750752. Rubin, H. (1983). Science 219, 1170-1172. Rubin, H. (1984).Nature (London)309, 518. Ruley, H. E. (1983).Nature (London) 304,602-606. Rushlow, K. E., Lautenberger, J. A., Papas, T. S., Baluda, M. A., Perbal, B., Chirihian, J. G., and Reddy, E. P. (1982).Science 216,1421-1423. Sacchi, N.,Watson, D. K., Guerts van Kessel, A. H. M., Hagemeijer, A., Kersey, J., Drabkin, H. D., Patterson, D., and Papas, T. S. (1986). Science 231,379-382. Saito, H., Hayday, A. C., Wiman, K., Hayward, W. S., and Tonegawa, S. (1983). Proc. Natl. Acad. Sci. U S A . 80, 7476-7480. Sakaguchi, A. Y., Lalley, P. A., Zabel, B. U., Ellis, R. W., Scolnick, E. M., and Naylor, S. L. (1984).Proc. Natl. Acad. Sci. USA. 81, 525-529. Santos, E., Tronick, S. R., Aaronson, S. A., Pulciani, S., and Barbacid, M. (1982).Nature (London) 298,343-347. Santos, E., Reddy, E. P., Pulciani, S., Feldmann, R. J., and Barbacid, M. (1983).Proc. Natl. Acad. Sci. U.SA. 80, 4679-4683. Saule, S., Roussel, M., Lagrou, C., and Stehelin, D. (1981).J . Virol. 38,409-419. Saule, S., Sergeant, A., Torpier, G., Raes, M. B., Pfeifer, S., and Stehelin, D. (1982).J. Virol. 42, 71-82. Saule, S., Coll, J., Righi, M., Lagrou, C., Raes, M. B., and Stehelin, D. (1983).E M B O J . 2, 805-809. Saule, S.,Martin, P., Gegonne, A., Begue, A., Lagrou, C.,and Stehelin, D. (1984).E r p . Cell Res. 155,496-506. Schatzman, R. C., Evan, G. I., Privalsky, M. L., and Bishop, J. M. (1986).MoZ. Cell. Btol. 6,1329-1333. Schechter, A. L., Stem, D. F., Vaidyanathan, L., Decker, S.J., Drebin, J. A., Greene, M. I., and Weinberg, R. A. (1984).Nature (London)312,513-516. Schmidt, J. A., Beug, H., and Hayman, M. J. (1985).E M B O J . 4, 105-112. Schubach, W., and Groudine, M. (1984).Nature (London) 307,702-708. Schultz, A. M., and Oroszlan, S. (1983).J . Virol. 46, 355-361. Schultz, A. M., Copeland, T. D., Mark, G. E., Rapp, U. R., and Oroszlan, S. (1985). Virology 146,78-89. Schwab, M., Alitalo, K.,Varmus, H. E., Bishop, J. M., and George, D. (1983a).Nature (London) 303,497-501. Schwab, M., Alitalo, K.,Klempnauer, K.-H., Varmus, H. E., Bishop, J. M., Gilbert, F., Brodeur, G., Goldstein, M., and Trent, J. (198313).Nature (London) 305,245-248. Schwab, M., Varmus, H. E., Bishop, J. M.,Grzeschik, K.-H., Naylor, S. L., Sakaguchi, A. Y., Brodeur, G., and Trent, J. (1984a).Nature (London) 308,288-291. Schwab, M., Ellison, J., Busch, M., Rosenau, W., Varmus, H. E., and Bishop, J. M. (1984b).Proc. Natl. Acad. Sci. U S A . 81,4940-4944. Schwab, M., Varmus, H. E., and Bishop, J. M. (1985).Nature (London)316, 160-162. Schwartz, D. E., Tizard, R., and Gilbert, W. (1983).Cell 32,853-869. Sealy, L., Privalsky, M. L., Moscovici, G., Moscovici, C., and Bishop, J. M. (1983a). Virology 130, 155-178.

184

KLAUS BISTER AND HANS W. JANSEN

Sealy, L., Moscovici, G., Moscovici, C., and Bishop, J. M. (1983b).Virology 130, 179-

194.

Selten, G., Cuypers, H. T., Zijlstra, M., Melief, C., and Berns, A. (1984).EMBO J . 3,

3215-3222.

Selten, G.,Cuypers, H. T., and Berns, A. (1985).E M B O J . 4,1793-1798. Semba, K., Kamata, N., Toyoshima, K., and Yamamoto, T. (1985).Proc. Natl. Acad. Sci. U.S.A. 82,6497-6501. Sergeant, A., Saule, S., Leprince, D., Begue, A,, Rommens, C., and Stehelin, D. (1982). E M B O I . 1,237-242. Shaw, J., Hayman, M. J., and Enrietto, P. J. (1985).J . Virol. 56,943-950. Sheiness, D., and Bishop, J. M. (1979).J . Virol. 31,514-521. Sheiness, D., and Gardinier, M. (1984).Mol. Cell. Blol. 4, 1206-1212. Sheiness, D.,Fanshier, L., and Bishop, J. M. (1978).J . Virol. 28,600-610. Sheiness, D.,Bister, K., Moscovici, C., Fanshier, L., Gonda, T., and Bishop, J. M. (1980a).J . Virol. 33,962-968. Sheiness, D. K., Hughes, S. H., Varmus, H. E., Stubblefield, E., and Bishop, J. M. (1980b).Virology 105,415-424. Sheiness, D., Vennstrom, B., and Bishop, J, M. (1981).Cell 23,291-300. Shen-Ong, G. L. C., Keath, E. J., Piccoli, S. P., and Cole, M. D. (1982).Cell 31,443-452. Shen-Ong, G. L. C., Potter, M., Mushinski, J. F., Lavu, S., and Reddy, E. P. (1984). Science 226,1077-1080. Shen-Ong, G. L. C., Morse, H. C., 111, Potter, M., and Mushinski, J. F. (1986).M o l . Cell. Biol. 6,380-392. Sherr, C. J., Rettenmier, C. W., Sacca, R.,Roussel, M. F., Look, A. T., and Stanley, E. R. (1985).Cell 41,665-676. Shibuya, M., and Hanafusa, H. (1982).Cell 30, 787-795. Shibuya, M., Yokota, J., and Ueyama, Y. (1985).Mol. Cell. Biol. 5,414-418. Shih, C.-K., Linial, M., Goodenow, M. M., and Hayward, W. S.(1984).Proc. Natl. Acad. Sci. U.SA. 81,4697-4701. Shih, T. Y.,Papageorge, A. G., Stokes, P. E., Weeks, M. O., and Scolnick, E. M. (1980). Nature (London)287,686-691. Shilo, B.-Z., and Weinberg, R. A. (1981).Proc. Natl. Acad. Sci. U S A . 78,6789-6792. Shimizu, K., Birnbaum, D., Ruley, M. A., Fasano, O., Suard, Y., Edlund, L., Taparowsky, E., Goldfarb, M., and Wigler, M. (1983a).Nature (London)304,497-500. Shimizu, K.,Goldfarb, M., Suard, Y., Perucho, M., Li, Y., Kamata, T., Feramisco, J., Stavnezer, E., Fogh, J.,and Wigler, M. H. (1983b).Proc. Natl. Acad. Sci. U . S A .80,

2112-2116.

Shimizu, K., Nakatsu, Y., Sekiguchi, M., Hokamura, K., Tanaka, K., Terada, M., and Sugimura, T. (1985).Proc. Natl. Acad. Sci. U S A . 82,5641-5645. Shimizu, N., Behzadian, M. A., and Shimizu, Y. (1980).Proc. Natl. Acad. Sci. U.S.A.77,

3600-3604.

Showe, L. C., Ballantine, M., Nishikura, K., Erikson, J., Kaji, H., and Croce, C. M. (1985).Mol. Cell. Biol. 5,501-509. Silver, J., Whitney 111, J. B., Kozak, C., Hollis, G., and Kirsch, I. (1985).Mol. Cell. Biol.

5,1784-1786.

Simon, M.A., Kornberg, T. B., and Bishop, J. M. (1983).Nature (London)302,837-839. Slamon, D. J., and Cline, M. J. (1984).Proc. Natl. Acad. Sci. U.S.A.81,7141-7145. Slamon, D.J., de Kernion, J. B.,Verma, I. M., and Cline, M. J. (1984).Science 224,256-

262.

185

ONCOGENES IN RETROVIRUSES AND CELLS

Smith, D. R., Vennstrom, B., Hayman, M. J., and Enrietto, P. J. (1985).J.Virol. 56,969-

977.

Sotirov, N. (1981).J. Natl. Cancer Znst. (U.S.) 66, 1143-1148. Souza, L. M., Strommer, J. N., Hillyard, R. L., Komaromy, M. C., and Baluda, M. A. (1980).Proc. Natl. Acad. Sci. U S A . 77,5177-5181. Spandidos, D. A., and Wilkie, N. M. (1984).Nature (London)310,469-475. Spurr, N. K., Solomon, E., Jansson, M., Sheer, D., Goodfellow, P. N., Bodmer, W. F., and Veenstrom, B. (1984).EMBOJ. 3, 159-163. Stanton, L. W., Watt, R., and Marcu, K. B. (1983).Nature (London)303,401-406. Stanton, L. W.,Fahrlander, P. D., Tesser, P. M., and Marcu, K. B. (1984).Nature (London) 310,423-425. Steffen, D. (1984).Proc. Natl. Acad. Sci. U S A . 81,2097-2101. Stehelin, D., Varmus, H. E., Bishop, J. M., and Vogt, P. K. (1976).Nature (London)260,

170-173.

Stephens, R. M., Rice, N. R., Hiebsch, R. R., Bose, H. R., and Gilden, R. V. (1983).Proc. Natl. Acad. Sci. U.S.A.80,6229-6233. Stewart, T. A., BellvB, A. R., and Leder, P. (1984a).Science 226,707-710. Stewart, T. A., Pattengale, P. K., and Leder, P. (1984b).Cell 38,627-637. Stubbs, E. L., and Furth, J. (1935).J . Exp. Med. 61,593-615. Sutrave, P., Bonner, T. I., Rapp, U. R.,Jansen, H. W., Patschinsky, T., and Bister, K. (1984a).Nature (London)309,85-88. Sutrave, P., Jansen, H. W., Bister, K., and Rapp, U. R. (1984b).J . V4rol. 52,703-705. Symonds, G., Stubblefield, E., Guyaux, M., and Bishop, J. M. (1984a).Mol. Cell. Biol. 4,

1627-1630.

Symonds, G., Klempnauer, K.-H., Evan, G. I., and Bishop, J. M. (1984b).Mol. Cell. B i d .

4,2587-2593.

Tabin, C . J., Bradley, S . M., Bargmann, C. I., Weinberg, R. A., Papageorge, A. G., Scolnick, E. M., Dhar, R., Lowy, D. R.,and Chang, E. H. (1982).Nature (London)

300,143-149.

Takeya, T., and Hanahsa, H. (1983).Cell 32,881-890. Taparowsky, E.,Suard, Y.,Fasano, O., Shimizu, K., Goldfarb, M., and Wigler, M. (1982). Nature (London) 300,762-765. Taparowsky, E., Shimizu, K., Goldfarb, M., and Wigler, M. (1983).Cell 34,581-586. Taub, R., Kirsch, I., Morton, C., Lenoir, G., Swan, D., Tronick, S., Aaronson, S., and Leder, P. (1982).Proc. Natl. Acad. Sci. U S A . 79,7837-7841. Temin, H. M. (1974).Cancer Res. 34,2835-2841. Temin, H. M. (1983).Nature (London)302,656. Temin, H. M. (1984).J . Cell. Physiol., Suppl. 3, 1-11. Thompson, C.B., Challoner, P. B., Neiman, P. E., and Groudine, M. (1985).Nature (London)314,363-366. Thompson, C. B., Challoner, P. B., Neiman, P. E., and Groudine, M. (1986).Nature (London)319,374-380. Trent, J., Meltzer, P., Rosenblum, M., Harsh, G., Kinzler, K., Mashal, R., Feinberg, A., and Vogelstein, B. (1986).Proc. Natl. Acad. Sci. U.S.A. 83,470-473. Tronick, S . R.,Popescu, N. C., Cheah, M. S . C., Swan, D. C., Amsbaugh, S.C., Lengel, C. R., DiPaolo, J. A., and Robbins, K. C. (1985).Proc. Natl. Acad. Sci. U S A . 82,

6595-6599.

Tsuchida, N., Ryder, T., and Ohtsubo, E. (1982).Science 217,937-939. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A. Lee, J., Yarden, Y.,Libermann, T. A., Schlessinger, J., Downward, J., Mayes, E. L. V.,

w.,

186

KLAUS BISTER AND HANS W. JANSEN

Whittle, N., Waterfield, M. D., and Seeburg, P. H. (1984).Nature (London) 309,

418-425.

Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, 0. M., and Ramachandran, J. (1985).Nature (London) 313,756-761. Ushiro, H., and Cohen, S. (1980).J . B i d . Chem. 255, 8363-8365. Van Beveren, C., Galleshaw, J. A., Jonas, V., Berns, A. J. M., Doolittle, R. F., Donoghue, D. J., and Verma, I. M. (1981a).Nature (London) 289,258-262. Van Beveren, C., van Straaten, F., Galleshaw, J. A., and Verma, I. M. (1981b).Cell 27,

97-108.

Van Beveren, C., van Straaten, F., Curran, T., Muller, R., and Verma, I. M. (1983).Cell

32, 1241-1255.

Van Beveren, C., Enami, S., Curran, T., and Verma, I. M. (1984).Virology 135,229-243. Van Beveren, C., Coffin, J., and Hughes, S.(1985).In “RNA Tumor Viruses. 2.Supplements and Appendixes” (R. Weiss, N. Teich, H. Varmus, and J. Coffin, eds.), pp. 744-747. Cold Spring Harbor Lab., Cold Spring Harbor, New York. van Ooyen, A., Kwee, V., and Nusse, R. (1985).E M B O J. 4,2905-2909. van Straaten, F., Muller, R., Curran, T., Van Beveren, C., and Verma, I. M. (1983).Proc. Natl. Acad. Sci. USA. 80,3183-3187. Varmus, H. E. (1982).Science 216,812-820. Varmus, H. E. (1984).Annu. Reu. Genet. 18,553-612. Vamrus, H. E. (1985).Nature (London) 314,583-584. Varmus, H. E.,and Swanstrom, R. (1982).In “RNA Tumor Viruses” (R. Weiss, N. Teich, H. Varmus, and J. Coffin, eds.), pp. 369-512.Cold Spring Harbor Lab., Cold Spring Harbor, New York. Vennsbom, B., and Bishop, J. M. (1982).Cell 28, 135-143. Vennstram, B., Fanshier, L., Moscovici, C., and Bishop, J. M. (1980).J . Virol. 36,575-

585.

Vennstrom, B., Moscovici, C., Goodman, H. M., and Bishop, J, M. (1981).J. Virol. 39,

625-631.

Vennstrom, B., Sheiness, D., Zabielski, J., and Bishop, J. M. (1982).J. ViroZ. 42,773-

779.

Vennstrom, B., Kahn, P., Adkins, B., Enrietto, P., Hayman, M. J., Graf, T., and Luciw, P. (1984).E M B O J. 3,3223-3229. Vogt, P. K. (1977).In “Comprehensive Virology” (H.Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 9,pp. 341-455. Plenum, New York. Vogt, P. K., Bister, K., Burny, A. L., Croce, C. M., Haseltine, W. A., Hayman, M. J., Hayward, W. S., Klein, G., Moiling, K., Neth, R. D., Pragnell, I. B., and Rowley, J. D. (1985).In “Leukemia” (I. L. Weissman, ed.), pp. 275-292. Springer-Verlag, Berlin and New York. Walther, N., Lun, R., Patschinsky, T., Jansen, H. W., and Bister, K. (1985).J . Virol. 54,

576-585.

Walther, N., Jansen, H. W., Trachmann, C., and Bister, K. (1986).Virology (in press). Wang, J. Y.J., Ledley, F., Goff, S., Lee, R., Groner, Y., and Baltimore, D. (1984a).Cell

36,349-356.

Wang, L.-H., Beckson, M., Anderson, S. M., and Hanahsa, H. (1984b).J. Vlrol. 49,881-

891.

Waterfield, M. D., Scrace, G. T., Whittle, N.,Stroobant, P., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C.-H., Huang, J. S., and Deuel, T. F. (1983).Nature (London) 304,35-39.

ONCOGENES IN RETROVIRUSES AND CELLS

187

Watson, D. K., Reddy, E. P., Duesberg, P. H., and Papas, T. S. (1983a).Proc. Natl. Acad. S d . U . S A . 80,2146-2150. Watson, D. K., Psallidopoulos, M. C., Samuel, K. P., Dalla Favera, R., and Papas, T. S. (1983b). Proc. Natl. Acad. Sci. U S A . 80,3642-3645. Watson, D. K., McWilliams-Smith, M. J., Nunn, M. F., Duesberg, P. H., O’Brien, S. J., and Papas, T. S. (1985).Proc. Natl. Acad. Sci. U S A . 82, 7294-7298. Watson, D. K., McWilliams-Smith,M. J., Kozak, C., Reeves, R., Gearhart, J., Nunn, M. F., Nash, W., Fowle, J. R., Duesberg, P. H., Papas, T. S., and O’Brien, S.J. (1986). Proc. Natl. Acad. Sci. U S A . 83, 1792-1796. Watson, R., Oskarsson, M., and Vande Woude, G. F. (1982).Proc. Natl. Acad. Sci. U.S.A. 79,4078-4082. Watt, R. A., Stanton, L. W., Marcu, K. B., Gallo, R. C., Croce, C. M., and Rovera, G. (1983a). Nature (London)303,725-728. Watt, R. A., Nishikura, K., Sorrentino, J., ar-Rushdi, A., Croce, C. M., and Rovera, G. (198313). Proc. NatE. Acad. Sci. U S A . 80, 6307-6311. Watt, R. A., Shatzman, A. R., and Rosenberg, M. (1985).Mol. Cell. Biol. 5,448-456. Weinberg, R. A. (1982a). Cell 30, 3-4. Weinberg, R. A. (1982b). Ado. Cancer Res. 36, 149-163. Weinberger, C., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. (1985). Nature (London) 318,670-672. Weintraub, H., Beug, H., Groudine, M., and Graf, T. (1982). Cell 28,931-940. Weiss, R., Teich, N., Varmus, H., and Coffin, J., eds. (1982). “RNA Tumor Viruses.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Westaway, D., Payne, G., and Varmus, H. E. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 843-847. Westin, E. H., Gallo, R. C., Arya, S. K., Eva, A., Souza, L. M., Baluda, M. A., Aaronson, S. A., and Wong-Staal, F. (1982a). Proc. Natl. Acad. Sci. U S A . 79,2194-2198. Westin, E. H., Wong-Staal, F., Gelmann, E. P., Dalla Favera, R., Papas, T. S., Lautenberger, J. A., Eva, A., Reddy, E. P., Tronick, S. R., Aaronson, S.A., And Gallo, R. C. (1982b). Proc. Natl. Acad. Sci. U S A . 79,2490-2494. White, R., Woodward, S., Leppert, M., O’Connell, P., Hoff, M., Herbst, J., Lalouel, J.M., Dean, M., and Vande Woude, G. (1985).Nature (London)318,382-384. Wilhelmsen, K. C., Eggleton, K., and Temin, H. M. (1984).J . Virol. 32, 172-182. Winqvist, R., Saksela, K., and Alitalo, K. (1984). EMBO]. 3, 2947-2950. Wong, T. C., Tereba, A., Vogt, P. K., and Lai, M. M. C. (1981).Virology 111,418-426. Yamamoto, T., Hihara, H., Nishida, T., Kawai, S., and Toyoshima, K. (1983a). Cell 34, 225-232. Yamamoto, T., Nishida, T., Miyajima, N., Kawai, S., Ooi, T., and Toyoshima, K. (1983b). Cell 35, 71-78. Yamamoto, T., Kawai, S., Koyama, T., Hihara, H., Shimizu, T., and Toyoshima, K. (1983~).Virology 129,31-39. Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Nomura, N., Miyajima, N., Saito, T., and Toyoshima, K. (1986).Nature (London) 319,230-234. Yancopoulos, G. D., Nisen, P. D., Tesfaye, A., Kohl, N. E., Goldfarb, M. P., and Alt, F. W. (1985). Proc. Natl. Acad. Sci. USA. 82, 5455-5459. Yaswen, P., Goyette, M., Shank, P. R., and Fausto, N. (1985).Mol. Cell. Biol. 5,780-786. Yokota, J., Tsunetsugu-Yokota, Y., Battifora, H., Le Fevre, C., and Cline, M. J. (1986). Science 231,261-265. Yoshinaka, Y., Katoh, I., Copeland, T. D., and Oroszlan, S. (1985).Proc. Natl. Acad. Sci. USA. 82,1618-1622.

188

KLAUS BISTER AND HANS W. JANSEN

Yunis, J. J. (1983). Science 221,227-236. Zabel, B. U., Fournier, R. E. K., Lalley, P. A., Naylor, S.L., and Sakaguchi, A. Y. (1984a). Proc. Natl. Acad. Sci. U S A . 81,4874-4878. Zabel, B. U., Naylor, S.L., Gneschik, K.-H., and Sakaguchi, A. Y. (1984b). Somat. Cell Genet. 10,105-108. Zeller, N. K., Gazzolo, L., and Moscovici, C. (1980). Virology 104,239-242. Zhou, R.-P., Kan, N., Papas, T., and Duesberg, P. (1985). Proc. Natl. Acad. Sci. U S A . 82,6389-6393. Zimmerman, K. A., Yancopoulos, G. D., Collum, R. G., Smith, R. K., Kohl, N. E., Denis, K. A., Nau, M. M., Witte, 0. N., Toran-Allerand, D., Gee, C. E., Minna, J. D., and Alt, F. W. (1986). Nature (London) 319, 780-783.

ACTIVATION OF CELLULAR ONCOGENES IN HEMOPOIETIC CELLS BY CHROMOSOME TRANSLOCATION Suzanne Cory The Walter and Ellza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia

I. Introduction

The last few years have witnessed a revolution in oncology. The identification of a specific set of cancer-promoting genes has brought the genetic basis of malignancy under molecular scrutiny. About 30 cellular oncogenes (c-onc) have now been identified (for reviews, see Varmus, 1984;Bishop, 1983),largely through the auspices of rapidly transforming retroviruses. Each viral transforming gene (v-onc) was originally acquired by recombination with a particular cellular gene. While the cellular genes do not themselves cause cancer, and are therefore properly called proto-oncogenes, their function is presumed to be crucial to the normal cell because many are strongly conserved between species as diverse as yeast, Drosophila, and man. Some proto-oncogenes are known to encode growth factor receptors and one a growth factor, but the normal function of most remains to be defined. Conversion of a proto-oncogene to a frank oncogene may occur in a number of ways, and involve structural and/or regulatory changes. Karyotypic alterations are a long-recognized feature of neoplasia, particularly of hematopoietic cancers (Yunis, 1983;Mitelman, 1984; Rowley, 1984).With the development of sophisticated chromosome banding techniques, an ever-increasing fraction of tumors prove to have distinctive alterations which include monosomy, trisomy, deletions, and amplified chromosomal regions. Among the most striking karyotypic changes are the specific chromosome translocations. While these had long been suspected to play a crucial role in the induction or progression of malignancy, it was difficult to rule out the possibility that they were merely adventitious by-products. Molecular characterization of the typical translocations in human Burkitt lymphomas and murine plasmacytomas provided the first compelling evidence for a causative role. In both of these B lymphoid tumors, translocation activates constitutive expression of the c-myc oncogene by fusion to the immunoglobulin heavy chain locus. The paradigm of the c-myc trans189 ADVANCES IN CANCER RESEARCH, VOL.47

Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

190

SUZANNE CORY

location raised the expectation that other chromosome breakpoints would prove to be associated with oncogenes. This view has been strongly buttressed by a second example: the Philadelphia chromosome associated with chronic myeloid leukemia has been shown to result from linkage of the c-abl oncogene to a gene of unknown function, bcr, resulting in synthesis of a hybrid bcr-abl protein. The chromosomal locations of many oncogenes are now known relatively precisely, largely due to the development of high-resolution in situ hybridization. Several oncogenes map close to other known translocation breakpoints (Yunis and Soreng, 1984) and may prove to be directly involved. Conversely, those translocations which do not map near known oncogenes may ultimately lead to the discovery and isolation of new oncogenes. This review will focus particularly on the myclIgH translocation, about which most is known, and then consider the nature of the variant forms of the plasmacytoma and Burkitt translocations. The bcr-abl recombination which creates the Philadelphia chromosome will also be described, and its role in the development of chronic myeloid leukemia considered. Finally, several other chromosomal abnormalities specific to particular lymphoid and myeloid neoplasms will be assessed for possible involvement of oncogenes. II. The c-myc Translocation in Burkitt Lymphomas and Murine Plasmacytomas

The predominant translocation associated with Burkitt lymphomas is the t(8;14), and its murine counterpart is the t(15;12)' in plasmacytomas (Klein, 1981).Molecular analysis (reviewed by Klein, 1983, 1986; Perry, 1983; Leder et al., 1983) revealed that both of these translocations result from a reciprocal recombination between the c-myc locus (at band q24 on human chromosome 8, D2/3 on murine chromosome 15) and the immunoglobulin heavy chain (IgH) locus (at band q32 on human chromosome 14, F2 on murine chromosome 12), as depicted in Fig. 1. Many acute lymphocytic leukemias of the B-cell type (B-cell ALL) also bear a t(8;14)(q24;q32)(Berger et al., 1979; Mitelman et al., 1979; Williams et al., 1984), and one has been shown to result, as expected, from a myclIgH recombination (Peschle et al., 1984). The t(7;6) in rat immunocytomas is likely to be equivalent to the human t(8;14) and murine t(15;12) (Sumegi et al., 1983). The strong association of the myc translocation with B lymphoid 1 To facilitate comparison between species, the myc-bearing chromosome is listed first, rather than the lowest numbered chromosome.

ONCOGENES IN HEMOPOIETIC CELLS t(8;14) W i t Lymphoma 14

191

t(15;12) MurinePlasmacytoma 12 15 n

FIG.1. The major translocations in Burkitt lymphomas and murine plasmacytomas: schematic representation, showing the position of the chromosomal breaks and the resulting recombinant chromosomes. The transcriptional orientation of each locus is indicated by an arrow on the blowup of the translocation sites. Cleavage near c-myc occurs either 5‘ to the gene or within the first exon or intron, while the IgH locus is usually cleaved within a switch site in the CHregion (see text). It is now clear that the orientation of the murine loci is the same as the human (Wirschubsky et al., 1985; Erikson et al., 1985).

neoplasia argued persuasively for a causal role in tumorigenesis. Direct proof that this is indeed the case has now been provided by studies with transgenic mice (Adams et al., 1985). Mice bearing the c-myc transgene coupled to the immunoglobulin heavy chain enhancer almost all developed fatal B lymphomas, some as early as 3 weeks after birth, whereas mice bearing a “normal” c-myc transgene were unaffected. These transgenic mice thus dramatically illustrate the tumorigenic potential of the c-myclIgH translocation. Before discussing the translocations in detail, the nature of the normal c-myc gene will be considered.

A. THEc-myc GENE The c-myc gene is strongly conserved between species and was originally identified by its homology to v-myc, the transforming gene carried by the avian myelocytomatosis retrovirus MC29. Figure 2 summarizes the structure of the human, murine, and chicken c-myc genes (Colby et al., 1983;Watt et al., 1983; Bernard et al., 1983; Battey et al., 1983; Watson et al., 1983; Gazin et al., 1984; Shih et al., 1984). Each comprises three exons, only the second and third of which are homologous to v-myc. Significantly, it is these exons which encode the myc polypeptide, starting at nucleotide 16 of exon 2. The first exon corresponds to an unusually long untranslated sequence.

I

1

2 3 4 5 6 'IIQ FIG.2. The c-myc locus. Exons are indicated as boxes, with coding regions solid and untranslated regions hatched. The second murine intron has not been sequenced, so its size is from restriction analysis. The precise boundaries of chicken exon 1 have not yet been delineated; the broken box indicates the restriction fragment known to hybridize to chicken myc mRNA, while the solid box indicates the region considered to be the best candidate for exon 1 (Shih et al., 1984). Arrows labeled P1 and P2 indicate alternative initiation sites for transcription. Roman numerals below the human and chicken loci indicate the location of DNase-hypersensitivity sites (see text), while broken lines indicate the eight regions of the murine 5' flanking DNA which exhibit maximal homology with the human (Corcoran et al., 1985; Fahrlander et al., 1985b).Horizontal arrows under exons 1 and 2 indicate inverted repeats which might form a hairpin loop in the mRNA (see text). 0

1

ONCOGENES IN HEMOPOIETIC CELLS

193

Conservation of the coding sequences is marked, particularly for exon 3, where amino acid homology is 94% between the human and murine proteins and 76% between the avian and mammalian. Variability is somewhat greater in the N-terminal portion encoded by exon 2, particularly between the avian and mammalian sequences (Bernard et al., 1983). The exon boundaries may correspond to functional domains in the polypeptide: the C-terminal half encoded by exon 3 is very basic, which presumably accounts for the DNA binding properties of the protein (see below), while the N-terminal region, encoded by exon 2, is acidic (Alitalo et al., 1983a; Bernard et al., 1983). Transcription initiates at two sites in exon 1(P1 and P2 in Fig. 2), resulting in two mRNAs which differ by 160 nucleotides at the 5’ end (Battey et al., 1983; Bernard et al., 1983; Hamlyn and Rabbitts, 1983; Saito et al., 1983). Homology between the human and murine exon 1 sequences is surprisingly high for a noncoding region (-70%; Bernard et al., 1983), but the first chicken exon lacks any detectable homology to its mammalian analogs (Shih et al., 1984). Since homology between the mouse and human genes extends at least 2 kb 5’ from exon 1 (Battey et al., 1983; Corcoran et al., 1985; Dunnick et al., 1985; Fahrlander et al., 1985b), it seems likely that this region contains sequences controlling myc expression. Conservation is particularly marked (>80%) in eight regions shown as broken lines beneath the murine locus in Fig. 2 (Corcoran et al., 1985; Fahrlander et al., 1985b). These regions represent candidate control regions, as do the DNase-hypersensitivity sites which are indicated by roman numerals in Fig. 2 for the human and chicken genes (Schubach and Groudine, 1984; Siebenlist et al., 1984; Dyson and Rabbitts, 1985; Dyson et al., 1985). The conserved region closest to the gene is in the same location as a DNase-hypersensitivity site, and almost certainly corresponds to a promoter region. The murine locus exhibited hypersensitivity sites equivalent to those observed for human c-myc and, in addition, even in fibroblasts, another site within the first intron, near the cryptic promoter sequences (see below) (Fahrlander et al., 1985b). Opinions differ as to whether the distal site I is involved in repression (Siebenlist et al., 1984; Dyson et al., 1985) or activation (Fahrlander et al., 1985b) of c-myc expression. Like its v-myc-encoded counterpart, the c-myc protein is a nuclear phosphoprotein that binds to double-stranded DNA in vitro (Donner et al., 1982; Alitalo et aZ., 1983b; Persson et al., 1984; Persson and Leder, 1984; Ramsay et al., 1984; Hann and Eisenman, 1984; Beimling et al., 1985). Many anti-myc antisera precipitate two polypeptides

-

194

SUZANNE CORY

of similar size (-60, 66 kDa), the size difference almost certainly reflecting a conformational difference (Persson et al., 1984). These values are substantially larger than the 49 kDa predicted from the cDNA sequence. Again, the discrepancy can be ascribed to conformation rather than modification because a 64-kDa c-myc protein is also synthesized in a rabbit reticulocyte lysate (Persson et al., 1984, 1985) and in bacteria (Watt et al., 1985), where modification would not be expected. The clustering of proline residues and long stretches of basic and acidic residues may well confer an unusual tertiary structure. Nuclear sequestration of c-myc seems to be governed primarily by ionic interactions because the protein can readily be extracted from nuclei by moderate salt concentrations at neutral pH (Evans and Hancock, 1985). These results were reconciled with an earlier report of strong binding to the nuclear matrix (Eisenman et al., 1985), when it was found that heat shock (>35”)of “low salt nuclei” induced irreversible binding of myc and a discrete subset of other nuclear proteins to the nuclear matrix (Evans and Hancock, 1985). During mitosis, c-myc protein is primarily cytoplasmic rather than chromosome bound (Alitalo et al., 198313; Winquist et al., 1984). The c-myc protein is very labile, with a half-life of about 30 min (Hann and Eisenman, 1984; Rabbitts et al., 1985),although the larger murine protein is substantially longer lived (Persson et al., 1985).The mRNA is also unusually unstable (t1,2,15minutes) (McCormack et al., 1984; Dani et al., 1984; Rabbitts et al., 1985a), and degradation may be controlled via the 5’ untranslated region specified by exon 1, since RNA lacking this sequence has enhanced stability (Rabbitts et al., 1985; Piechaczyk et al., 1985). A potential hairpin loop structure involving the arrowed sequences in exons 1 and 2 in Fig. 1 (Saito et al., 1983; Darveau et al., 1985) might serve as a recognition site for degradative enzymes. Rapid turnover would facilitate rapid adjustment of cmyc protein levels in response to changes in the cellular environment. c-myc appears to be associated with growth control. c-myc mRNA is detected in a wide range of cell types and tissues (Gonda et al., 1982) and, in general, its level correlates closely with the rate of cell division (Pfeifer-Ohlsson et al., 1984; Stewart et al., 1984). In early embryos, however, myc expression is low compared to that in corresponding extra-embryonic tissues, and later in embryonic development only certain cell types display abundant transcripts (Pfeifer-Ohlsson et al., 1985).These results suggest that gene(s) other than c-myc govern proliferation during early embryogenesis. Rapid (but transient) increases in myc RNA levels have been ob-

ONCOGENES IN HEMOPOIETIC CELLS

195

served in several cell types in response to proliferative signals, preceding the onset of cellular RNA and DNA synthesis by several hours. When quiescent, serum-deprived fibroblasts are treated with PDGF, or lymphocytes are stimulated with mitogen, a 10- to 40-fold increase in myc RNA ensues within 3 hours (Kelly et al., 1983).An early wave of myc RNA expression also occurs in regenerating liver (Makino et al., 1984; Goyette et al., 1984), in B cells stimulated with anti-p antibody (Smeland et al., 1985) and in normal human peripheral blood mononuclear cells treated with phytohemagglutinin, phorbol ester, or calcium ionophore (Reed et al., 1985). These results suggested that cmyc expression might be specific to the GI phase of the cell cycle (Kelly et al., 1983; Campisi et al., 1984), a hypothesis which fitted well with the extreme instability of c-myc mRNA. It was subsequently shown, however, that both c-myc RNA and protein synthesis are expressed at a constant level throughout the normal cell cycle (Thompson et al., 1985; Hann et al., 1985; Rabbitts et al., 198513; Lachman et al., 1985),and that the rate of c-myc protein turnover at various phases of the cycle is similar (Rabbitts et al., 1985a). Moreover, induction seems to be independent of the growth state of the cells, since c-myc is induced in exponentially growing A431 cells by treatment with epidermal growth factor (Bravo et al., 1985).Thus myc expression may be necessary to render resting cells competent to enter the cell cycle, but its expression is maintained in the presence of appropriate growth stimuli, irrespective of the cell cycle stage (Thompson et al., 1985). Since the c-myc gene is transcribed at a high rate in Go-arrested fibroblasts (Blanchard et al., 1985), and transcription is only modestly increased in response to PDGF (Ziff and Greenberg, 1984), it seems likely that the increased mRNA levels result from a post-transcriptional change, such as enhanced mRNA stability (Blanchard et al., 1985). Indeed, decreased levels of nuclease could account for the super-induction of myc mRNA by cycloheximide, which inhibits protein synthesis (Kelly et d.,1983). Induction of c-myc expression after interaction of growth factor with its cognate receptor may be mediated via the kinase C pathway, since many agents that activate kinase C, either directly or indirectly, also trigger rapid increases in c-myc expression (Bravo et al., 1985; Coughlin et al., 1985). Enforced c-myc expression cannot, however, bypass the need for growth factors for a mitogenic response. Thus, enhanced expression of c-myc, either via transfection of an appropriately engineered exogenous c-myc gene (Armelin et al., 1984), or by direct injection of c-myc protein (Kaczmarek et al., 1985),reduced but did not replace the need for platelet derived growth factor for stimula-

196

SUZANNE CORY

tion of quiescent fibroblasts. Similarly, c-myc expression driven by a viral LTR did not render immortalized myeloid cells factor-independent even though growth in agar in very low concentrations of growth factor was enhanced (S. Cory, 0. Bernard, D. Bowtell, and J. Schrader, in preparation). [Factor-independent myeloid and T cells have been obtained using an LTR-driven v-myc gene (Rapp et al., 1985),but the low frequency of such clones suggests that a second genetic alteration may be involved.] Decreased c-myc expression may be required to allow a proliferating cell to escape from the cell cycle. In support of this notion, c-myc RNA levels have been shown to decrease in Daudi Burkitt lymphoma cells when proliferation was inhibited by interferon-a or -/3 (Jonak and Knight, 1984; Einat et al., 1985). Reduced c-myc transcription preceded accumulation of the cells in GdG1 phase (Einat et al., 1985), while the level of most mRNAs, including that for actin, was unchanged (Jonak and Knight, 1984). Differentiation in several cell types is associated with down-regulation of myc (Gonda and Metcalf, 1984; Reitsma et al., 1983; Campisi et al., 1984; McCormack et al., 1984; Lachman and Skovitchi, 1984; Filmus and Buick, 1985). Induction of mouse erythroleukemia (MEL) cells to differentiate leads to a very rapid decline in myc RNA followed by a wave of expression and then another decline (Lachman et al., 1985). Intriguingly, myc expression takes place primarily in GI phase in the differentiating MEL cells, instead of being relatively constant throughout the cell cycle as is the case prior to induction (Lachman et aZ., 1985). These data suggest that the balance between self-renewal and differentiation within a cell population may be set by a switch controlling the level of myc expression at a specific stage in the cell cycle. Although the function of myc remains unknown, an intriguing clue has been provided by the observation that, like the E1A protein of adenovirus, activated myc can complement the TUS oncogene in transforming primary fibroblasts (Land et al., 1983; Lee et al., 1985) and kidney cells (Ruley, 1983). Slight homology between c-myc and E1A (and c-myb) has been noted (Ralstan and Bishop, 1983). Since E1A controls transcription of other adenovirus early genes, an attractive hypothesis is that myc, too, is a transcriptional regulator, a function consistent with its nuclear location and ability to bind DNA. Transfection studies showing that myc can stimulate expression from the heatshock protein promoter (Kingston et al., 1984) seem to lend support to this hypothesis. Genes involved in sustaining cells in cycle would appear to be good candidates for genes subject to myc control.

ONCOGENES IN HEMOPOIETIC CELLS

197

FIG.3. Formation of immunoglobulin heavy chain genes by somatic recombination. The organization of the locus in germline DNA is shown on the top line, the transcriptional orientation being right to left (to enable easier comparison with subsequent IgHl c-myc recombination figures). V, D, J, and C genes are indicated as boxes or vertical lines and switch (S)regions within introns as smaller boxes. E is the IgH enhancer. Distances are given in kilobase pairs (kb). There are probably -160 VHgenes, and their relative orientation and distance from the D gene cluster is still not established. Assembly of a complete V gene from V, D, and J elements requires at least two recombination events, with concomitant deletion of intervening DNA. The second line shows the resulting organization of the functional allele in a p-producing B lymphocyte. Switching expression from p to, in this case, a,requires recombination between p and a S regions, resulting in a hybrid (S,S,) switch region and deletion of the intervening DNA. The resultant rearranged locus in the a-producing cell is shown on the third line. Bold vertical arrows (a to h) indicate sites which have undergone recombination with the c-myc locus in various Burkitt 8;14 or plasmacytoma 15;12 translocations (see Table I).

B.

RECOMBINATION BETWEEN THE

c-myc AND IgH LOCI

To understand the interchromosomal recombination in B lymphoid tumors, it is valuable to first consider the intrachromosomal recombination which enables normal B cells to undertake Ig synthesis (reviewed by Adams and Cory, 1983). As shown in Fig. 3, two different types of somatic recombination take place at the IgH locus in B cells. First, a complete variable region sequence is assembled by site-specific recombination between V, D, and J elements, allowing synthesis of p and 6 heavy chains. Expression of other CH sequences usually requires a second recombination event, this time involving the reiterated sequences termed switch (S) regions, which are located within

198

SUZANNE CORY

the intron 5‘ to each C H gene except Cg. Recombination between S, and S,, as in the example shown, deletes the intervening DNA and places the VDJ gene proximal to C,, enabling synthesis of an a heavy chain. As originally suggested by Ohno et al. (1979), the Burkitt and plasmacytoma translocations are in a sense aberrant Ig gene rearrangements, and they were initially detected as such at the molecular level by several groups (Adams et al., 1982, 1983; Cory et al., 1983a,b; Harris et al., 1982; Marcu et al., 1983; Calame et al., 1982; Taub et al., 1982). Others recognized them by alterations in c-myc structure or expression (Shen-Ong et al., 1982; Mushinski et al., 1983; Dalla Favera et al., 1983).The salient features of the c-myclIgH translocations are summarized in Fig. 4.The two loci recombine in opposite transcriptional orientation (“head to head”) (Shen-Ong et al., 1982; Crews et al., 1982; Adams et al., 1983; Marcu et al., 1983; Dalla Favera et al., 1983) and both reciprocal products are usually retained in the tumor (Cory et al., 1983b; Bernard et al., 1983), even after years in culture. The precise nature of the recombination varies between tumors as discussed below. 1 . The c-myc Breakpoints As indicated in Fig. 4,the c-myclIgH translocations can be usefully classified by the location of the breakpoint with respect to c-myc: within the gene itself (class I), immediately 5’ to it (class 11),or distant (class 111).Figure 5 depicts the location of the c-myc breakpoints in different human and murine tumors. Significantly, no translocation breakpoint violates the coding region. Instead, a dramatic clustering of breakpoints within exon 1 and intron 1 is evident, accounting for 70% of the murine sites (23 of 33) and nearly half of the human ones (11or 12 of 26). Since translocation in these class I tumors has “decapitated” the c-myc gene, its normal promoters have been relegated to the reciprocal chromosome product (Fig. 4). In class I1 tumors, the breakpoints map within a 9-kb region 5‘ to the c-myc promoters and most fall within the conserved region 2-kb 5’ to P l ywhere the DNasehypersensitivity sites have been mapped. It is noteworthy that many murine T lymphomas arising after retroviral infection bear proviruses inserted within the same region (Corcoran et al., 1984), as indicated by the broken line in Fig. 5. While these translocations (and insertions) leave the c-myc transcriptional unit intact, they may well have disrupted regulatory sequences and, in particular, they may have interfered with feedback control of c-myc expression (see below). The

FIG.4. Three classes of exchange between omyc and the IgH locus, defined by location of the c-myc breakpoints. The c-myc and IgH genes are in opposite transcriptional orientation, as indicated by placing them either above or below the bar. The IgH gene may be either switched or not, as indicated by the slashed S region. Recombination within the IgH locus is shown (a) within switch (SH)regions or (b) between the enhancer (E)and the (V)DJgene, resulting in the alternative products shown to the right. Class I1 translocations have been somewhat arbitrarily defined as falling within -9 kb upstream from exon 1, the position of the EcoRI site 5‘ to human c-myc. At least some class I11translocations (shown on a reduced scale) may involve recombination within the V, locus, but that is not shown here because none have been characterized at the molecular level.

FIG.5. Translocationbreakpoints near c-myc. The positions of sequenced breakpoints are shown by vertical solid arrows, while bars indicate locations determined by Southern blot analysis. P1 and P2 indicate alternative transcriptional start points, and roman numerals show DNase-hypersensitivity sites (see text). The broken line below the murine locus indicates the region bearing proviral inserts in certain T lymphomas (Corcoranet al., 1984).References defining the plasmacytoma breakpoints are J558 (Adams et aZ., 1982; Stanton et al., 1983;Gerondakis et al., 1984); P3 (Dunnicket al., 1983; Neuberger and Calabi, 1983; Gerondakis et al., 1984); MPC 11(Harris et al., 1983; Stanton et al., 1984); HOPC 1, WEHI 267 (Cerondakis et al., 1984); MOPC 315, MOPC 104E (Piccoli et al., 1984); MOPC 167, MOPC 603 (Cahme et al., 1982); ABPC 45, ABPC 33, TEPC 1194, TEPC 1165, TEPC 1033, ABPC 47, ABPC 52, ABPC 89, ABPC 60, ABPC 48 (Yang et al., 1984; Fahrlander et al., 1985);SAMM 368, Y5606,TEPC 609, WEHI 279, TEPC 1017, Baltnlm 17A, S194, BFPC 61, MOPC 173, S107, EPC 109, S117, Baltelm 1131 (Cory et al., 1983b).ABPC 65 and C6T TEPC 1156 also bear a t(15;12)involving a break distant to c-myc (Yang et al., 1985). References to the Burkitt breakpoints are ST486 (Gelmann et al., 1983);BL31 (Siebenlist et al., 1984); Raji (Dyson and Rabbitts, 1985);BL22 (Mouldinget al., 1985);Ramos, Manca (Wiman et al., 1984);CA46 (Showe et al., 1985);Lou, Joy, W1, BM, LS (Bernard et al., 1983);ALL (Peschle et al., 1984);LY65, BLA2, Seraphina (Taub et al., 1984);BL-3, BL-1, BL-4 (Rothberget al., 1984); Daudi, JD38IV (ar-Rushidiet al., 1983; Erikson et al., 1982);P3HR-1 (ar-Rushidiet al., 1983; Magrath et al., 1983; Erikson et al., 1983a), MC116, AK778, EW36 (Dalla Favera et al., 1983);380 (Pegoraroet al., 1984). For ABPC 17, the arrow indicates the site of insertion of the IgH enhancer region (Corcoran et al., 1985). If they are 5' to c-myc (see text), the distant breakpoints are >18 kb from P1, except for Seraphina, where the site is between 12.5 and 18 kb (Taub et al., 1984).The break in the human locus indicates 4 kb deleted horn this fiq-' 2 to save space.

ONCOGENES IN HEMOPOIETIC CELLS

201

TABLE I TRANSLOCATION BREAKPOINTS IN THE IgH Locus“ IgH target

Burkitt lymphoma

(a) Germline S ,

,ST486 (p), Ramos (p), BL22 (PI JOY, JD38 akaji ( p ) @LS CA46 ( p ) OALL

(b) Switched S , (c) Germline S , (d) Switched S ,

(e) Germline (9 Near JH (g) 5’ to JH (h) VH locus

S,ep

Plasmacytoma (BALB/c) eT1194

,HOPCl (y2a), M104E ( p ) ,T1165 aM173 (y2a), S368 (y2b,a), T609 (y2b,a), B17A nJ.558 (a),W267 (a),M315 (a), M167 (a), M603 (a) aB1131, Y5606 (y3), S107 (a),S117 (a),S194 (a), El09 (a),B61 (a), A (a), M41 "Mil (y2b), W1033

“Manca (p), BL31 w 1 , Lou ,P3HR-l, 380 PDaudi ( p )

a The breakpoints are classified according to their location in the IgH locus, (a) through (h)referring to arrows on Fig. 3. “Germline” refers to a switch region which has not undergone recombination with another switch region and would therefore be still in its germline configuration had it not undergone interchromosomal translocation. Superscript a indicates breakpoints established by sequencing; /3, those deduced by Southern blot analysis. The heavy chain synthesized in the tumor is indicated in parentheses, where published. References to the various lines are given in the legend to Fig. 5. The ABPC 33 breakpoint apparently lies within a “switched” S% region (Yang et al., 1985).

breakpoints for class I11 tumors have yet to be located. Indeed, only for the Burkitt lymphomas Daudi (Erikson et al., 1982; Davis et al., 1984) and Seraphina (Taub et al., 1984b) is there clear evidence that the breakpoints are 5’ to c-myc. 2. Translocation Targets in the IgH Locus Several sites in the IgH locus can provide breakpoints for translocation, as indicated by vertical arrows (a-h) in Fig. 3 and summarized for specific tumors in Table I. Switch regions are the most frequent targets for both class I and class I1 tumors [recombination (a) in Fig. 41, suggesting that the cleavage mechanism for normal IgH switch recombination renders the chromosome temporarily vulnerable to “invasion” by other DNA. Most Burkitt lymphomas are relatively

202

SUZANNECORY

early B cells that express surface IgM (Lenoir et al., 1982). S, is a frequent translocation site in these tumors, although unswitched or germline” S, is occasionally involved (Table I). Taken together, these observations suggest that the precursor B cell becomes susceptible to translocation at the onset of competence for switch recombination, and that transformation arrests further maturation. In strong contrast, Table I shows that S, sites predominate as translocation targets for the BALB/c plamacytomas, which are tumors of mature antibodysecreting cells that have generally switched expression from IgM to another Ig class. This remarkable preference for S, targets correlates with the predominance of IgA expressors among BALB/c tumors, and may reflect a propensity for S, breaks in lymphocytes associated with the granuloma which forms after injection of mineral oil during tumor induction (Cancro and Potter, 1976; Potter et al., 1984). In several Burkitt lymphomas, the IgH breakpoint is clearly not within a switch region (Table I). In some cases (Manca, BL31, W1, Lou), recombination has occurred close to the JHregion (Wiman et al., 1984; Siebenlist et al., 1984; Cory et al., 1983b), while in Daudi the site may be among the VH genes (Erikson et al., 1982). Although it is tempting to conclude that these breaks were catalyzed by the enzyme(s) involved in VDJ joining, the sites in Manca and BL31 are actually a few hundred base pairs away from the sequence used for joining. As shown in Fig. 4, the disposition of the known IgH enhancer (E) with respect to c-myc varies with the site of cleavage within the IgH locus. Recombination within an SH region [(a) in Fig. 41 places the enhancer and the myc coding exons on different chromosomes, while recombination 5’ to the enhancer, such as (b) in Fig. 4, links the two. About 56% of Burkitt lymphomas have the enhancer and myc on the same chromosome (Table I), but no plasmacytomas with a conventional t(15;12) do. Translocation involves only one allele. For the CH locus this is usually the silent or “excluded” allele, as shown for several Burkitt lymphomas by analysis of segregating somatic cell hybrids (Croce et al., 1983; Erikson et al., 1982, 1983a,b). Translocation could itself inactivate an IgH allele by severing a functional VDJ gene from its associated CH gene (see Fig. 4). It should be pointed out, however, that translocation might sometimes involve the functional IgH allele, since there is no obvious reason why translocation should favor the nonexpressed allele. In a cell expressing a C, gene, for example, translocation could well occur at the downstream S, region (Gerondakis et al., 1984). 68

ONCOGENES IN HEMOPOIETIC CELLS

203

3. Clues to the Translocation Mechanism Homologous recombination is clearly not responsible for the c-mycl IgH translocations. No extensive sequence homology exists between the c-myc breakpoint regions and IgH “targets.” Nor is there any evidence for crossover within an homologous oligonucleotide. The preference for SH breaks on the IgH-bearing chromosome raises the question whether the c-myc locus is a pseudo-switch region that becomes accidentally caught up in Ig class switch recombination. A computer-aided search revealed no greater occurrence of switch region sequence motifs in the c-myc region than in any random sequence (Bernard et al., 1983). Nevertheless, certain c-myc breakpoints such as the “hot-spot” used in M104, W267, and M603 (see Fig. 5) are tantalizingly like some switch recombination sites (Gerondakis et al., 1984; Dunnick et al., 1983, 1984), raising the possibility that switch recombination enzymes make at least some c-myc scissions. While several other features of translocation sites have been noted, none appears to be invariant. Piccoli et al. (1984), for example, noted that the tetranucleotide GAGG occurred near the c-myc breakpoint in each of the five SJc-myc plasmacytoma junctions published at that time, but HOPC 1subsequently proved to be an exception (Gerondakis et al., 1984). Comparison of the sequences of both chromosomal junctions from several plasmacytomas (Gerondakis et al., 1984; Neuberger and Calabi, 1983; Dunnick et ul., 1983; Stanton et ul., 1983) unexpectedly revealed that the exchange is not a precise crossover: nearly all junctions displayed deletions and/or small insertions, and one demonstrated a duplication. Deletions of SH sequences ranged from 0.3 to 1.6 kb. myc deletions were usually small, ranging from 7 to 11 bp, but about 1 kb was deleted in two or three tumors (Gerondakis et al., 1984; Cory et al., 1983b). Similarly, Burkitt lymphoma BL22 has a 16bp deletion from c-myc and a 2-kb deletion from S, (Moulding et al., 1985). More surprisingly than the deletions was the presence of the same 106-bp c-myc sequence on both exchange products in plasmacytoma HOPC 1(Gerondakis et al., 1984). [Deletions and a duplication have also been noted for translocations involving the CAlocus and sites 3‘ to c-myc (Hollis et al., 1984; Denny et al., 1985; see below).] The fact that the deletions and duplications are located precisely at the interchromosomal junctions argues strongly that they occur during the recombination. I t has therefore been proposed that translocation can be initiated with staggered single-strand breaks on each chromosome (Gerondakis et al., 1984). If the single-stranded tails are “filled

204

SUZANNE CORY

in” by polymerization prior to ligation of the flush ends to the other chromosome, then the sequence between the staggered breaks will be retained by both translocation products, as in HOPC 1. Nucleolytic degradation of the single strands, on the other hand, would generate a deletion. The frequency of deletions argues that nucleolytic action is more common. The frequent insertion at the junctions of a few (1-5) residues, usually pyrimidines, presumably reflects a polymerizing activity of the enzyme(s) carrying out the recombination. In summary, while c-myclIgH translocations may be catalyzed frequently (but not always) by switch recombination enzymes, the clustering of the c-myc breakpoints around exon 1 cannot be satisfactorily explained by the marginal homology to S H or other IgH regions. Presumably, therefore, the location of these translocation sites reflects strong selection for the resulting modulation of c-myc expression.

C. CONSEQUENCES OF TRANSLOCATION FOR c-myc EXPRESSION What fundamental changes are wrought in c-myc expression by the translocation? The answer has been sought at the level of both transcription and translation. 1 . The c-myc Protein Is Usually Unchanged Sequence analysis of c-myc cDNAs and genes has established that the c-myc polypeptide expressed in Burkitt lymphomas and plasmacytomas is often identical in amino acid sequence to that in normal cells (Battey et al., 1983; Stanton et al., 1984). Thus, in contrast to the situation for ras gene products, a mutated myc amino acid sequence is not required for the progression to malignancy. However, in certain Burkitt lymphomas, notably Raji (Rabbitts et al., 1983) and CA46 (Showe et al., 1985), mutations have been observed within the Nterminal region encoded by exon 2. The tendency for mutations around amino acid 58 is particularly striking and a mutation has also been detected in this region in a chicken bursa1 lymphoma (Westaway et al., 1984).While such mutations might confer a selective advantage in tumor progression (Westaway et al., 1984),they might instead indicate a region of the protein that is relatively unimportant for transformation (Showe et al., 1985).The ratio of the two major c-myc proteins differs for tumors and normal cells (Hann and Eisenman, 1984; Eisenman and Hann, 1985), but the significance of this observation is unclear.

205

ONCOGENES IN HEMOPOIETIC CELLS 1

NORMAL

2

P1 P2

ALTERED _ _ _ ~

’

‘H

3

-A.

7% 19-*’

FIG.6.Alternative modes of c-myc transcription. In normal cells or in tumors where translocation occurs 5‘ to exon 1 (class I1 and 111), two mRNAs of 2.4 and 2.25 kb are spliced from transcripts initiating at two alternative sites P1 and P2. In tumors where translocation has severed the transcriptionalunit (class I), multiple mRNAs, ranging in size from about 1.9 to 2.7 kb, result from alternative starts within intron 1. The intron 1derived sequences provide alternative 5’ untranslated leader sequences, most of which are unspliced (see text).

2. Translocation Can Alter the Mode of c-myc Transcription Both c-myc promoters function during normal c-myc transcription, generating two mRNAs of -2.4 and 2.25 kb (Fig. 6). In normal human and murine lymphoid cells (Stewart et al., 1984; Yang et al., 1984) and in EBV-transformed lymphoblastoid lines (Taub et al., 1984b; Nishikura et at., 1985), P2 is the preferred promoter and the smaller mRNA species predominates. This also holds for normal nonlymphoid cells, where P2 preference can be even more marked than in Iymphoid cells (Stewart et al., 1984). The mode of c-myc transcription in tumors bearing c-myclIgH translocations varies, depending on where the c-myc breakpoint occurred. Tumors with breakpoints 5’ to the first promoter (class I1 and I11 in Fig. 4) bear the conventional transcripts initiated at P1 and P2. However, P1 promoter usage is substantially increased, possibly as a result of regulatory influences from the Ig locus. Many human lines have comparable amounts of the 2.4 and 2.25 kb RNAs (Hamlyn and Rabbitts, 1983; Taub et al., 1984b; Nishikura et al., 1985),while in others the larger RNA species predominates (Bernard et at., 1983), as it does in many plasmacytomas (Yang et at., 1984; 1985). The available data for class I11 tumors are somewhat conflicting: the Burkitt lymphomas Daudi and EW36 both seem to use P1 and P2 equally (Hamlyn and Rabbitts, 1983; Nishikura et al., 1985),while P2 is preferred in several murine lines (Yang et al., 1984, 1985). A very different mode of transcription is found in tumors where translocation has disrupted the normal transcriptional unit by scission

206

SUZANNE CORY

within exon 1 or intron 1 (class I in Fig. 4), Instead of the canonical 2.4- and 2.25-kb RNAs, a number of myc RNAs are generated, ranging in size from about 1.9 to 2.7 kb. The relative concentration of the different mRNAs varies in different tumors, but they all bear sequences from within the 3’ two-thirds of the first c-myc intron (Adams et al., 1983; Stanton et al., 1983)and initiate from multiple sites within the intron (Keath et al., 1984a; Prehn et al., 1984; Calabi and Neuberger, 1985). The most frequently used sites lie within the 170 bp immediately 5’ to exon 2 (Stanton et al., 1983; Calabi and Neuberger, 1985) but some are up to -900 bp further upstream (Keath et al., 1984a; Prehn et al., 1984). There is no evidence that these cryptic promoters” ever function in normal cells. New splices may be involved for some transcripts (Prehn et al., 1984), but not for most (Calabi and Neuberger, 1985).Hence, since no AUG within intron 1is in phase with exon 2, the intron sequences represent new 5’ untranslated sequences, replacing exon 1. The heterogeneity of initiation sites within intron 1almost certainly reflects the lack of a conventional promoter in this region (Calabi and Neuberger, 1985; Prehn et al., 1984), although several TATA-like sequences are present, including one in a region strongly conserved between mouse and man (Bernard et al., 1983). The heterogeneous initiation is reminiscent of that in the SV40 early region when its promoter is deleted by mutation (Benoist and Chambon, 1981). Somewhat surprisingly, RNA transcripts of the opposite (noncoding) strand of the c-myc intron also occur in most tumor lines (Keath et al., 1984a; Calabi and Neuberger, 1985).These RNAs are also transcribed from multiple initiation sites, most of which map 5‘ to those utilized for coding strand transcripts (Keath et al., 1984a; Calabi and Neuberger, 1985). These results suggest that intron 1 bears a bipolar regulatory element (Calabi and Neuberger, 1985). This element could be an enhancer which would normally favor the P2 promoter. While the transcriptional activity of a “decapitated” myc gene is no higher than that achievable with the normal promoters of an intact cmyc gene (Piechaczyk et al., 1985), myc mRNA lacking the exon 1 sequence is considerably more stable than normal c-mgc mRNA (Rabbitts et al., 1985; Piechaczyk et al., 1985). However, no dramatic increase in the overall concentration of c-myc mRNA ensues. While some workers (Croce et al., 1983; Nishikura et al., 1983) have emphasized the relatively high levels of c-myc transcripts in Burkitt lymphomas, the level is not uniformly higher than in lymphoblastoid lines immortalized by Epstein-Barr virus (Bernard et al., 1983; Hamlyn and Rabbitts, 1983; Taub et al., 1984b), which have a normal karyo6‘

ONCOGENES IN HEMOPOIETIC CELLS

207

type. Similarly, although plasmacytomas exhibit a considerable range, the average level of c-myc transcripts is only a fewfold higher than the average in other lymphoid tumors lacking c-myc rearrangement (Adams et d., 1982, 1983). The most appropriate comparison to make is with normal B cells. Significantly, proliferating (mitogen-stimulated) B lymphocytes have comparable levels to the average plasmacytoma (Keath et al., 1984a), while the concentration of myc RNA in normal quiescent B cells is some 10- to 30-fold lower (Bernard et at., 1983; Keath et al., 1984a). While the changes in c-myc mRNA levels may not be dramatic, altered translational efficiency could result in elevated levels of myc protein. In vitro tests of the effect of removal of exon 1sequences on translational efficiency have yielded conflicting results; Persson et al. (1984) found no difference, whereas Darveau et al. (1985) observed increased efficiency. I n general, the amount of myc protein in vivo appears to be directly proportional to the level of mRNA (see, for example, Hann and Eisenman, 1984; Persson et al., 1984). To summarize, the concentration of neither myc mRNA nor myc protein is markedly elevated by the 8;14 translocation in Burkitt lymphomas or the t(15;12) in murine plasmacytomas, in contrast to the considerable enhancement reported to occur in chicken bursa1 lymphomas as a result of proviral insertion near c-myc (Hayward et al., 1981). The level of c-myc expression in Burkitt lymphomas and murine plasmacytomas is, however, at least as high as in normal dividing B cells.

3. Deregulation of c-myc Expression The most dramatic change induced by translocation lies in the regulation of c-myc expression. This crucial point was recognized when it was found that the untranslocated allele is silent (or nearly so) in plasmacytomas and Burkitt lymphomas. If transcription occurred from both alleles, normal size mRNAs from the untranslocated allele would be detectable with an exon 1probe in tumors where the translocated gene has been “decapitated” (class I). Virtually no such mRNA was detectable, however, in 4 of 5 such Burkitt lines or in 12 of 13 such plasmacytomas (Adams et al., 1983; Bernard et al., 1983) and the lesion is at the transcriptional level (Fahrlander et al., 1985b). Nishikura et al. (1983) independently concluded that the normal allele is inactive after analysis of somatic cell hybrids between Burkitt lymphomas and a murine plasmacytoma: human myc transcripts could be detected in segregants bearing the IgHlc-myc gene on the 14q+chromosome, but not in those bearing only a normal chromosome 8. These

208

SUZANNE CORY

results have been confirmed for at least eight other Burkitt lymphomas (ar-Rushdi et al., 1983; Taub et al., 1984; Wiman et al., 1984; Rabbitts et al., 1984; Feo et al., 1985; Showe et al., 1985),representing all three classes of 8;14 translocation. Consistent with their difference in transcriptional activity, the translocated c-myc gene is strikingly more sensitive to DNase than the silent allele (Kakkis et al., 1986) and the latter is extensively methylated (Dunnick et al., 1985). The silence of the untranslocated allele in B cell tumors has been interpreted in two ways. One view is that a mature B lymphocyte or plasma cell is essentially nonpermissive for expression of a normal cmyc gene and that inactivation can either be prevented or overcome by translocation to the IgH locus (e.g., Bernard et al., 1983; Nishikura et al., 1985). On this hypothesis, myc expression in Ig-secreting lymphoblastoid lines is presumably also abnormal and may be the basis for their immortalization. The other view explains the repression of the untranslocated allele in terms of a feedback control loop: c-myc expression activates a repressor, either directly or indirectly, which can prevent expression of a conventional c-myc allele but not of a translocated allele (Leder et al., 1983).In a more specific model, the cmyc protein is its own repressor (Rabbitts et al., 1984).In either view, constitutive c-myc expression is seen as the key event predisposing to B cell malignancy. If the negative regulation model is correct, forced expression of an exogenous myc gene should turn off the resident myc gene. Striking confirmation of this prediction was obtained with transgenic mice bearing a myc gene controlled by the immunoglobulin heavy chain enhancer: expression of the endogenous c-myc gene was undetectable in cell lines derived from pre-B and B cell tumors arising in these mice (Adams et al., 1985).Moreover, copious expression of an avian vmyc gene under the control of a murine retroviral LTR apparently suppressed expression of the endogenous c-myc gene in both fibroblasts and hemopoietic cells (Rapp et al., 1985).In contrast, fibroblasts carrying an LTR-driven c-myc gene still expressed the endogenous gene when grown in tissue culture although not after being grown as a tumor in nude mice (Keath et al., 1984b).The basis for this difference is unclear, but may relate to the level of expression of the exogenous gene and/or sequence difference between v- and c-myc. The frequent occurrence of exon 1 mutations in class I1 and I11 Burkitt lymphomas (Taub et al., 1984; Rabbitts et al., 1983, 1984) suggests that they confer a selective advantage for myc deregulation. Such mutations may sometimes be generated by the somatic mutational mechanism that operates on the VDJ region for a brief period

ONCOGENES IN HEMOPOIETIC CELLS

209

during normal B cell differentiation (Crews et al., 1981), particularly in cases such as Raji, where the disposition of c-myc exon 1 with respect to CHapproximates that of VDJ in an heavy chain gene. However, since there are tumors with apparently no mutations in exon l (Corcoran et al., 1985; Fahrlander et al., 1985a; Yang et al., 1985), or very few (Wiman et al., 1984),mutation of exon 1 does not seem to be an essential prerequisite for c-myc deregulation. It may instead play an augmenting role by influencing transcription or mRNA stability. How does translocation to the IgH locus deregulate c-myc expression? Do the various types of recombination events represent different ways of achieving the same objective, or is there a common underlying mechanism? Is proximity to the Ig locus crucial, or is c-myc activation solely due to the damage inflicted on the c-myc locus by translocation, the Ig locus simply providing a convenient (frequently broken) recipient chromosome? For at least some translocations, it seemed reasonable to postulate that physical removal of control sequences to another chromosome accounted for deregulation. Class I translocations (Fig. 4) remove not only the conventional promoters but also any 5' regulatory sequences. Class I1 translocations leave the transcriptional unit intact, but remove much of the immediate 5' flanking sequence. While class I11 translocations might also remove regulatory sequences, these would have to be located a very great distance from the gene. Studies with transgenic mice (Adams et al., 1985) have now established, however, that removal of regulatory sequences is insufficient and that, as suspected, the Ig locus plays an active role in the deregulation. Mice bearing a c-myc transgene subjugated to the immunoglobulin heavy chain enhancer (E,) invariably developed fatal lymphosarcomas, as did others bearing c-myc linked to the kappa enhancer (E,J. In marked contrast, mice bearing a c-myc transgene from which exon 1 and the 5' flanking sequence had been removed were unaffected, as were those carrying an intact c-myc transgene. The nature of the regulatory influence of the IgH locus on the expression of the translocated c-myc gene remains a puzzle. The transgenic mouse study (Adams et al., 1985) vividly illustrated the potency of the heavy chain enhancer, but in many myc translocations, including almost all those in murine plasmacytomas, this enhancer is not coupled to myc but lies on the reciprocal translocation product. While it is conceivable that the IgH locus harbors other enhancers or enhancer-like elements, searches for such sequences have so far been negative (Mercola et al., 1984). This dilemma can be resolved if it is postulated that activation of c-myc simply requires conjunction with

210

SUZANNE CORY

an active CH locus and that E, is needed for the establishment of “Igcompetent” chromatin but not for subsequent maintenance of this state. The surprising observation that IgH genes remain active in vivo even when deletions remove E, (Wabl and Burrows, 1984; Klein et al., 1984) provides support for this notion. Judging from the class I11 tumors, the IgH locus must be able to exert its deregulating influence on c-myc from a considerable distance. Precedent exists for this notion from studies of the activation of VH genes. In early B cells, VH gene families are apparently sequentially activated to undergo VDJ joining, starting with those closest to JH and C p (Yancopoulos et al., 1984; Perlmutter et aZ., 1985): even the closest VHgenes must be more than 80 kb from the JHlocus (Wood and Tonegawa, 1983). Moreover, activation of transcription by the heavy chain enhancer is not restricted to the VH gene joined to JH but can also extend to a VHgene located -13 kb upstream (Wang and Calame, 1985). Studies with somatic cell hybrids strongly suggest that expression of a translocated myc gene is governed by lymphoid-specific factors. When Burkitt lymphoma cells were fused to murine fibroblasts, the translocated myc gene was turned off, as was the functional p gene (Nishikura et al., 1984), presumably because expression of both genes requires lymphoid-specific factors not present (or active) in the hybrids. In contrast, the translocated c-myc gene remained active in hybrids made with a murine plasmacytoma (Nishikura et al., 1983; Croce et al., 1984).The results from fusions of various Burkitt lymphomas to a lymphoblastoid line are more puzzling. While, as expected, the translocated c-myc allele from Daudi was expressed in such hybrids (Croce et aZ., 1985a), those from three other tumors were not, even though immunoglobulin genes from both parental lines continued to be expressed (Croce et al., 1984; Nishikura et al., 1985). The basis for the difference is unclear but it is worth noting that each of the translocated c-myc genes inactivated by the lymphoblastoid cell environment has recombined within an IgH switch region (S, or Sa),while that in Daudi has apparently recombined within the VH locus. BETWEEN MURINE D. Two ATYPICALRECOMBINATIONS CHROMOSOMES 15 AND 12

Plasmacytoma ABPC 17 was originally described as bearing a t(15;6) (Ohno et al., 1984), but subsequent analysis suggests that chromosome 6 was not involved (F. Wiener, personal communication). The rearranged c-myc gene in this tumor proved to be most unusual: a

ONCOGENES IN HEMOPOIETIC CELLS

211

2.3-kb region bearing the lymphoid-specific IgH locus enhancer from chromosome 12 has been inserted within the conserved region immediately 5’ to c-myc exon 1 (Corcoran et al., 1985). The insertion represents a true physical transposition because an IgH gene that has suffered the equivalent 2.3-kb deletion was also cloned from this line. The transcriptional orientation of the insert is the same as that of crnyc, in contrast to the organization of IgH and myc sequences in a t(15;12). This class of interaction would previously have escaped attention, being cytogenetically invisible. Oncogene activation by transposition of a tissue-specific enhancer may therefore be more widespread than might be supposed. Another plasmacytoma (ABPC 45) which has neither a t(15;12) nor a t(15;6) has a band deletion near the c-myc locus on chromosome 15 (Ohno et al., 1984). Sequencing the myc locus suggests that multiple exchanges have occurred with the IgH locus (Fahrlander et al., 1985). The first probably was a conventional t( 15;12), recombination having occurred between sequences 5’ to c-myc and an S, region. A second recombination event then introduced the IgH enhancer but in the opposite orientation to S , (Fahrlander et aZ., 1985). Strikingly, the translocation in ABPC 45 occurred at precisely the same nucleotide as the insertion in ABPC 17. In addition, the translocation in Burkitt lymphoma Ramos (Wiman et al., 1984) occurred only three nucleotides away, if the human and murine sequences are aligned to maximize homology (Corcoran et al., 1985). These observations suggest that alterations at this site are particularly effective for rnyc deregulation.

E. ARE OTHERGENESREQUIRED TO COMPLEMENT c-myc ACTIVATION? While oncologists have long emphasized multistep scenarios for tumor development (Klein and Klein, 1985a), the nearly invariant leukemogenesis in transgenic mice bearing the E,-myc gene (Adams et aZ., 1985) might be taken as evidence that an activated myc gene is sufficient for lymphoid neoplasia. Several observations suggested, however, that other events (presumably genetic) influenced tumor development (Adams et al., 1985). First, the onset time for tumors varied greatly (from 3 weeks to 6 months), even though E,-driven myc expression presumably commenced very early in B cell ontogeny (before birth). Second, the tumors were almost all clonal. Third, and most important, even though the bone marrow of young mice was replete with atypical blast cells, it failed to elicit tumors in syngeneic

212

SUZANNE CORY

recipients. Thus, while an activated c-myc gene very significantly predisposes toward tumor development, by itself it is insufficient (Adams et d.,1985). A similar conclusion has been reached from a study of the development of mammary adenocarcinomas in transgenic mice carrying a c-myc gene fused to the LTR of murine mammary tumor virus (Stewart et al., 1984). Translocation of the c-myc gene to the IgH locus therefore probably represents only one of at least two genetic events required before a B lymphocyte clone becomes fully tumorigenic. Different genes probably complement the activated myc gene in different types of tumor. Gene(s) encoded by Epstein-Barr virus seem likely to be involved in the many Burkitt lymphomas that are EBV positive, because EBV itself readily immortalizes B cells. At least one Burkitt lymphoma (Ramos) bears an activated N-ras gene (Murray et al., 1983) and, significantly, activated myc and ras genes are known to complement each other in transforming primary fibroblasts (Land et al., 1983). Another candidate complementing oncogene, Blym-1,has been implicated in several Burkitt lymphomas (Diamond et al., 1983). Plasmacytomas can be induced by mineral oil injection only in BALB/c and NZB mice, so it is likely that alleles specific to these strains play a role in plasmacytoma development (Potter and Wax, 1981; Potter et al., 1984). Most plasmacytomas arising in mice infected with Abelson murine leukemia virus (the ABPC series) bear the typical 15;12 or 15;6 translocation, but the onset of tumor development is greatly accelerated (Ohno et al., 1984), presumably because v-abl provides a complementing function. In a few plasmacytomas, activation of the c-mos gene by insertion of an intracisternal A particle element may contribute to the progression of malignancy (Rechavi et al., 1982), while in the plasmacytoma which bears a t(6;lO) as well as a conventional t(15;12), a gene on chromosome 6 may be involved (Perlmutter et al., 1984). 111. Variant Translocations in Burkitt Lymphomas and Murine Plasmacytomas

While the major Burkitt lymphoma and plasmacytoma translocations have been studied extensively, much less is understood about the variant translocations carried by some 15% of such tumors. As Fig. 7 shows, variant translocations also involve the myc-bearing chromosome band, but exchange has occurred with a chromosome bearing an immunoglobulin light chain locus: in mice, chromosome 6, which bears the K chain locus and, in man, chromosome 2 and 22, which bear

2 13

ONCOGENES I N HEMOPOIETIC CELLS

J7 8q+

22q-

as'

J7

0 2P-

15q*

6q-

FIG.7. The variant translocations in Burkitt lymphomas and murine plasmacytomas, Conventions are as described for Fig. 1. For the variant Burkitt lymphomas, cleavage near c-myc occurs 3' to myc, usually at a considerable (unknown) distance (depicted stippled), while cleavage within the IgH locus can be 5' to the J region or apparently within the V locus. For the variant plasmacytomas, >94 kb cleavage is 3' to c-myc and within a few kilobases 5' to C, (see text).

the K and A loci, respectively (reviewed by Klein, 1983).In situ hybridization had previously established that the human K- and A-bearing bands, 2p12 and 22ql1, respectively, were precisely those involved in translocation (Erikson et al., 1981; McBride et al., 1982; Malcolm et al., 1982). It thus seemed highly likely that the variant translocations resulted from recombination of c-myc and an immunoglobulin light chain locus. Only the second part of this hypothesis proved to be correct. In molecular terms, nearly all the variant breakpoints map far from myc and may even involve another gene. A. VARIANTTRANSLOCATION SITESARE NOT CLOSETO c-myc Cytogenetic data could not initially distinguish between the breakpoints on the myc-bearing chromosome for the major and variant translocations (Klein, 1983; Ohno et al., 1984). Nevertheless, for all ten variant Burkitt translocations subsequently analyzed by in situ hybridization, it became clear that breakage was 3' to c-myc rather than 5'.Thus c-myc remains associated with the 8q+,while C, translocates from 2 to 8 in the t(8;2) (Erikson et aZ., 1983; Davis et aZ., 1984; Taub et al., 1984; Rappold et al., 1984), and CI translocates from 22 to 8 in the t(8;22) (Croce et aZ., 1983; Hollis et aZ., 1984; de la Chapelle et al., 1983). In situ hybridization analysis of a variant plasmacytoma has recently revealed that the t( 15;6) also involves breakage 3' to c-myc (Banejee et al., 1985).

214

SUZANNE CORY

Molecular analysis showed that in two Burkitt lymphomas, recombination to the CAlocus has taken place only 400 bp (Hollis et al., 1984) and about 5 kb downstream from the c-myc poly(A) addition signal (Denny et al., 1985). However, no rearrangement corresponding to a variant translocation has been detected within 8 kb 3’ to c-rnyc in 4 other Burkitt lymphomas bearing a t(8;22) (Bernard et al., 1983; McGrath et al., 1983) or in any bearing a t(8;2) (Taub et al., 1982; 1984; Bernard et al., 1983; Rappold et al., 1984). For several plasmacytomas bearing a 15;6 translocation, cloning data have established that the breakpoints are more than 94 kb 3’ to c-myc (see below). Recent cytogenetic analysis has unexpectedly revealed that the chromosome 8 breakpoint in all four 8;22 translocations studied differs from that in the standard t(8;14) (Manolov et al., 1986).This raises the possibility that the chromosome 8 breakpoints lie thousands of kilobases from myc in certain 8;22 translocations and therefore may well involve a different oncogene. The 8;2 translocations do not involve this site because the chromosome 8 breakpoints in five tumors could not be distinguished from those in the t(8;14).

B. VARIANT TRANSLOCATIONS UTILIZEIg LIGHTCHAINLOCI The hypothesis that the variant translocations involve Ig light chain loci proved to be correct and several interchromosomal junctions have been cloned (Hollis et aZ., 1984; Taub et al., 1984; Webb et al., 1984; Denny et al., 1985). In all cases analyzed thus far, recombination occurs 5’ to the CL gene. Is this predilection related to V-J joining? Cloning has established that the breakpoint in one Burkitt lymphoma bearing a t(8;2) is several kb 5’ to the J, region (Taub et al., 1984). For another t(8;2), the site appears to lie within the V, locus, as judged by hybridization analysis of somatic cell hybrids (Erikson et al., 1983; Emanuel et al., 1984). I n situ hybridization studies suggested that another t(8;2) involved a site between J, and C, while two others involved breakage either within the distal portion of the V, locus or between the V, and J, loci (Rappold et aZ., 1984). In tumors bearing a t(8;22), recombination has been observed at a site -5 kb upstream from the J region for CAI (Hollis et al., 1984), immediately 5‘ to a joined VA-JA gene (Denny et al., 1985), within the Ch cluster (de la Chapelle et al., 1983), or within the Vh locus (Emanuel et al., 1985). The chromosome 6 breakpoints in six of nine plasmacytoma 15;6 translocations have been mapped near C, (Van Ness et al., 19831 Webb et al., 1984; Cory et al., 1985); three fall within the JK-C, intron, two near JK, and one 5’ to JK (Fig. 8, bottom line). Since many of the

ONCOGENES IN HEMOPOIETIC CELLS

K

Locus

hl

I

215

@cK

6

E

X&L,

!%2

FIG.8. Plasmacytoma variant (15;6)translocations. The put-1 locus on chromosome 15 and the K locus in chromosome 6 are shown, together with the reciprocal recombination products in ABPC4 (Webb et al., 1984).Also indicated by bars are the sites at both loci utilized for 15;6 translocation in TEPC 1198, Baltnlm 17A, ABPC 20, ABPC 103 (Cory et al., 1985) and the complex 15;12,6 translocation in the NZB plasmacytoma PC 7183 (Van Ness et al., 1983). E indicates the approximate location of the K enhancer. The cmyc locus lies at least 94 kb to the left of the ABPC 4 breakpoint on chromosome 15 (see text). The break in the put-1 locus indicates 2 kb deleted from this diagram to save space.

Burkitt and plasmacytoma K breakpoints thus lie some distance away from the signal sequences used for V-J joining, which are immediately adjacent to each J gene, it seems unlikely that the variant translocations are catalyzed by the V-J joining enzyme(s). The JK-CKregion seems prone to aberrant recombination events. In addition to the t(15;6), an unusual plasmacytoma 10;6 translocation has been mapped within the intron (Perlmutter et al., 1984). In another plasmacytoma, there has been an insertion of an intracisternal A particle element (Hawley et al., 1982),while two others bear deletions starting within the J,-C, intron (Durdik et a,?.,1984).The tendency for recombination within the JK-CKregion may be related to its involvement in V gene assembly, to the constitutive C, expression in B lymphocytes (Van Ness et al., 198l), and/or the nuclease hypersensitivity (Weischet et al., 1982; Parslow and Granner, 1982)associated with the K enhancer.

C. put-1: THE MAJORMURINELocus FOR VARIANTTRANSLOCATION@ The major chromosome 15 locus for plasmacytoma variant translocations has now been identified and denoted put-1. A region of 108 kb has been cloned (Webb et al., 1984; Cory et al., 1985; Graham et al., 1985). As shown in Fig. 8, the recombination sites in five of eight

2 16

SUZANNE CORY

tumors with a t(15;6) lie within a 4.5-kb region of put-1 (Cory et al., 1985),and the site of a complex 15;12;6 rearrangement originally characterized by Van Ness et al. (1983)lies only about 11kb distant. Thus all 15;6 exchanges so far characterized at the molecular level involve the put-1 locus. Comparison of the pot-1 and c-myc loci revealed no overlap and established that the minimal distance of the put-1 breakpoints from the c-myc promoters is 72 kb (Cory et al., 1985). Since the breakpoint is now known to be 3' to c-myc (Banejee et al., 1985), it is clear that the separation is in fact more than 94 kb. The levels of c-myc RNA in plasmacytomas with variant translocations are comparable to those in tumors with a conventional t(15;12), and in at least one tumor (Baltnlm 17A), it is clear that the c-myc allele linked to the 15;6 translocation is being expressed (Cory et al., 1985). Thus the put-C, exchange may activate myc expression, either directly or indirectly. Could the effect of translocation be conveyed in cis from put to c-myc over more than 94 kb? While models of longrange chromatin folding can be invoked (see above), these become less tenable the greater the separation between the loci. Could put-1 bear a gene that encodes a trans-acting regulator of myc expression? By stimulating (or depressing) activity of a putative put-1 gene, the translocation might then stimulate myc expression indirectly. Of course such trans-acting factors would be expected to activate both c-myc alleles. The observation that hypersensitivity site I (see Fig. 2) could only be detected on one c-myc allele in each of two variant plasmacytomas may indicate that only one allele is active (Fahrlander et al., 198513). Moreover, a study of somatic cell hybrids made between variant Burkitt lymphomas and a mouse plasmacytoma bearing a conventional t(15;12) has been interpreted as evidence that only the translocated human c-myc gene is expressed. Thus, in hybrid lines derived from J1, which bears a t(8;2) (Erikson et al., 1983b) or BL2, which bears a t(8;22) (Croce et al., 1983), human c-myc RNA can be detected in hybrid lines bearing c-myc on an 8q+ but not in those containing only a normal chromosome 8. This result would also be expected, however, if the gene encoding the trans-acting factor is located on the 8q+,between c-myc and C,, and is therefore not available in the latter lines for activation of c-myc on the normal chromosome 8. Could put-1 encode an oncogene? In this view, myc expression would be a secondary rather than a primary consequence of the 15;6 translocation. The notion gains in credibility by evidence that put-1 is the site of proviral integration in about 10% of AKR T lymphomas (Graham et al., 1985) and corresponds to mis-1, a site favored for

ONCOGENES I N HEMOPOIETIC CELLS

217

integration of Moloney leukemia virus in rat T lymphomas (Villeneuve et al., 1986).The parallel which can be drawn with activation of the c-myc by proviral insertion in both chicken bursa1 lymphomas (Hayward et al., 1981) and retrovirally induced T cell tumors (Corcoran et al., 1984) is obvious. It is unclear why put-1 translocation always involves the C, locus rather than, for example, the IgH locus. Perhaps put-1 only becomes accessible (or active) relatively late in B-cell development, after heavy chain rearrangement has ceased. The propensity for recombination with the C, locus presumably reflects a selective advantage. One of several possible activation mechanisms is linkage to the K enhancer (E in Fig. 8),which is located -0.6 kb 5‘ to C,(Queen and Staf!ford, 1984; Pickard and Schaffner, 1984). Indeed, all the characterized variant translocation breaks lie 5‘ to this enhancer, suggesting that the putative gene might lie to the left of the put-1 breakpoint cluster in Fig. 8. IV. Other Translocations Specific to B-Cell Leukemias and Lymphomas

Other translocations involving the IgH-bearing band 14q32 have been observed in human B lymphoid tumors. The precedent of the myclIgH translocations suggested that these might also involve the IgH locus and that the breakpoints could be sought by cloning “aberrant” Ig rearrangements. Using this approach, two candidate B-cell oncogenes, designated bcl-1 and bcl-2 (bcl is B-cell leukemidlymphoma), have recently been identified (Erikson et al., 1984; Pegoraro et al., 1984). The t(11;14) (q13;q32) translocation occurs in some B-cell chronic lymphocytic leukemias (CLL), diffuse small-cell lymphocytic leukemia and diffuse large-cell lymphoma (Yunis, 1983), and multiple myeloma (van den Berghe et al., 1984). The chromosome 11 breakpoints in two CLL patients have been shown to occur only seven nucleotides away from each other, while that in a diffuse B-cell lymphoma is -0.9 kb distant (Tsujimoto et al., 1984a, 198513) and the region has been denoted bcl-1. In the case of the two CLL lines, the recombination site on chromosome 14 was within a JH gene and, significantly, a sequence homologous to those thought to be essential for VDJ joining was found near the bcl-1 breakpoint. Thus the t(11;14) may be sequence specific and catalyzed by enzymes normally involved in VDJ joining (Tsujimoto et al., 198513). bcl-2 is strongly implicated in another B-cell neoplasm, follicular cell lymphoma. This lymphoma is almost invariably (26 of 32 cases) associated with a t( 14;18) (q32;q21) (Yunis, 1983). Interchromosomal

218

SUZANNE CORY

junctions have been cloned from an acute pre-B-lymphocytic leukemia (ALL) cell line (Pegoraro et al., 1984) and from several follicular lymphomas (Tsujimoto et al., 1985c; Cleary and Sklar, 1985). The recombining region of chromosome 18, bcl-2, is rearranged in DNA of some 60% of follicular lymphomas (Tsujimoto et al., 1984b, 1985a; Cleary and Sklar, 1985). Most breakpoints cluster within a 2.1-kb region, and a transcript of 6 kb has been detected in various cell types. The recombination sites on chromosome 14 lie close to the 5’ end of the JH elements. Since extraneous nucleotides (N regions) were found at the recombination junction and specific signal-like sequences were detected on chromosome 18 close to the breakpoints, the t(14;18) is believed to result from a mistake in the VDJ joining process at the preB-cell stage of differentiation (Tsujimoto et al., 1985c; Cleary and Sklar, 1985). If so, the translocation does not “freeze” further differentiation, as most follicular lymphomas are of a more mature B cell stage. Indeed, in two of four tumors, bcl-2 was found to be linked to C, rather than C,, presumably because switch recombination had occurred on that “excluded” allele. A new non-random translocation, t( 1;19) (q23;p13.3), has recently been described for pre-B ALL (7 of 23 cases; Williams et al., 1984). While no oncogenes have yet been mapped to either breakpoint region, it is intriguing that the insulin receptor gene has recently been localized to band p13.2-p13.3 on chromosome 19 (Yang-Feng et al., 1985). The insulin receptor is a member of the tyrosine kinase family. This group of proteins includes two oncogenes known to encode growth factor receptors: erbB which is a truncated form of EGF receptor (Downward et al., 1984) andfms which corresponds to the receptor for macrophage colony stimulating factor, CSF-1 (Sherr et al., 1985). Clearly, it will be most exciting to learn whether or not the t(1;19) directly involves the insulin receptor gene. Certain pre-B ALL are characterized by abnormalities of chromosome 12 with a common breakpoint of p12 (Williams et al., 1984). This site may be close to the location of the Ki-rase oncogene (Jhanwar et al., 1983). V. Translocations Specific to T-cell Leukemias and Lymphomas

The T-cell antigen receptor is a disulfide-linked heterodimer comprising an a chain and a p chain. Like immunoglobulin polypeptides, each chain is encoded by variable, joining, and constant region genes and a functional T-cell receptor gene must be “created” by somatic recombination during T-cell differentiation (for a review, see Hood et

ONCOGENES IN HEMOWETIC CELLS

219

al., 1985).As in B cells, this somatic recombination process is prone to mistakes and certain T-cell lymphomas and leukemias may prove to have been triggered by accidental recombination of oncogenes with T-cell receptor loci. The a subunit of the T-cell antigen receptor has recently been mapped to human chromosome 14 (Collins et al., 1985) at band q l l q12 (Croce et al., 1985). Significantly, “breaks in 14qll-13 probably constitute the most common nonrandom abnormality in T cells” (Ueshima et al., 1984) and can take the form of translocations or inversion. Four of 16 cases of T-cell ALL were found to carry a t(11;14) (p13;q13) (Williams et al., 1984).The breakpoint on chromosome 14 is likely to be q l l rather than 913, because segregation analysis of somatic cell hybrids has recently shown that the breakpoints for two patients occur between the variable and constant region genes for the a chain of the T-cell receptor (Lewis et al., 1985; Erikson et al., 1985b). It is of great interest that the region of chromosome 11 that participates in the translocation, p13, which has been termed tcl-2 by Erikson et al. (1985b), is the site of the interstitial deletion characteristic of patients with Wilms’ tumor and aniridia (Riccardi et al., 1980; Kaneko et al., 1981). The chromosome 14 breakpoint involved in a t(8;14) (q24;qll) carried by a cell line established from a patient with T cell CLL also seems to lie within the a variable region sequences (Shima et al., 1986).Significantly, a rearranged c-myc allele is present in these cells. If this rearrangement proves to be the result of the translocation, the data suggest that recombination occurred about 9 kb 3‘ to c-myc, the cmyc and T-cell receptor C, genes then lying in tandem (head to tail) orientation, separated from each other by V, genes. The T-cell antigen receptor a locus at 1 4 q l l may also undergo exchanges with 14q32. Recombination can occur between homologs, creating a t(14;14), or within the same chromosome, by inversion. The inv(l4) (Hecht et al., 1984) has been detected in seven of nine patients with T-cell CLL (Zech et al., 1984; Hecht et al., 1984; Ueshima et al., 1984)and in another with childhood lymphoblastic lymphoma (Hecht et al., 1984) and the frequency of this marker may previously have been underestimated (Ueshima et al., 1984). The “target” oncogene in 14q32 was tentatively dubbed tcl-1 (Croce et al., 1985),but molecular cloning of the inv(l4) in one cell line (SVP-T1) has recently revealed the lesion to have been mediated by a site-specific recombination event between an immunoglobulin variable region gene and a T-cell receptor a joining segment (Baer et al., 1985; Denny et al.,

220

SUZANNE CORY

t (9:22)Chronic Mveloid Leukemia

9

22

9q+

22q-

FIG.9. The 9;22 translocation in chronic myeloid leukemia. Conventions are as described in Fig. 1.

1986). While the hybrid gene is transcribed (Denny et al., 1986), the hybrid receptor has not yet been demonstrated on the cell surface and its role in T-cell malignancy remains to be established. The p subunit of the T cell receptor is located on human chromosome 7 (M. K. L. Collins et al., 1984), probably at q35 or q36 (Le Beau et aZ., 1985b), although an earlier assignment placed it at 7p13-p21 (Caccia et al., 1984). To date no strong correlations have been noted between T-cell neoplasias and specific chromosomal abnormalities involving this site, but clonal and nonclonal rearrangements occur at high frequency at this site in circulating T cells of patients with ataxia telangiectasia (Aurias, 1981). VI. The Philadelphia Chromosome in Chronic Myeloid Leukemia

The hallmark of chronic myeloid leukemia is the presence of a shortened chromosome 22, the well-known Philadelphia (Ph') chromosome (Nowell and Hungerford, 1960). In 96%of Ph'-positive CML, this chromosomal abnormality results from a reciprocal translocation (Rowley, 1973) between band q l l of chromosome 22 and band q34 of chromosome 9 (Figure 9). The molecular consequence of the t(9;22) has recently been shown to be fusion of the abl oncogene to another gene, bcr, resulting in production of a novel tyrosine kinase. The conclusion that the altered protein is crucial to the development of CML seems inescapable and opens exciting new avenues for investigating the induction of CML. A. THESEARCH FOR

THE

9;22 JUNCTION

Among the known oncogenes, the most likely candidates for involvement in the 9;22 translocation were c-abl, on chromosome 9

ONCOGENES IN HEMOPOIETIC CELLS

221

(Heisterkamp et al., 1982), and c-sis, on chromosome 22 (Dalla Favera et al., 1982). In situ hybridization and analysis of somatic cell hybrids established that c-sis was translocated to chromosome 9 (Groffen et al., 1983) in CML, while c-abl was located on the relatively small fragment of chromosome 9 that translocated to chromosome 22 (de Klein et al., 1982). The failure to detect c-sis transcripts in CML (Gale and Canaani, 1984) decreased the likelihood that this proto-oncogene was involved in the disease, but c-abl became an attractive candidate when it was discovered that the breakpoint in one CML lay either immediately 5’ of, or within, the c-abl gene (Heisterkamp et al., 1983a). Molecular cloning of the 9;22 junction provided access to the chromosome 22 sequences involved in the translocation, and this region was designated bcr, for “breakpoint cluster region,” when it was found that the breakpoints in DNA from 17 of 17 Ph’-positive CML patients mapped within 5.8 kb of each other (Groffen et al., 1984).The clustering of bcr breakpoints on chromosome 22 contrasted strikingly with the dispersed pattern for chromosome 9, where the breakpoints are spread over a region of up to 100 kb (Heisterkamp et al., 1983b, 1985). Nevertheless, essentially all CML samples and Ph’-positive cell lines examined were found to contain a novel 8-kb abl RNA, larger than the normal species of 6 and 7 kb (Gale and Canaani, 1984; Canaani et al., 1984; S. Collins et al., 1984). Moreover, a new 210-kDa abl protein ( ~ 2 1 0larger ) ~ than the normal human 145-kDa c-abl protein, was identified in the K562 line which bears the Ph’ chromosome (Konopka et al., 1984; Kloetzer et al., 1985) and subsequently in leukemic cells from most CML patients examined (Konopka et al., 1985). The molecular basis for the new abl product has recently been elucidated by partial sequence analysis of transcripts of the fused abl and bcr genes (Shtivelman et al., 1985). Fusion occurred at an identical position for the RNAs in two lines and joined the abl and bcr coding regions in phase, bcr being N terminal. Rearranged bcr and abl sequences have also been identified in a Ph’-negative CML patient (Bartram et al., 1985).

B. THEHYBRIDbcrhbl ONCOGENE Although many details remain to be established about the t(9;22), the emerging picture is summarized in Figs. 9 and 10. The orientation of the bcr and c-abl genes is centromere-5’-3’-telomere and it is noteworthy that each gene is very large, bcr spanning a minimum of 45 kb (Heisterkamp et al., 1985) and c-abl at least 60 kb (Heisterkamp

222

SUZANNE CORY

FIG.10. The 9;22 translocation creates a hybrid bcrlabl gene. Boxes and lines indicate the approximate position of large and small exons, respectively. In the abl locus, the hatched exon is one of several alternative 5’ exons (Ben-Meriah et al., 1986).Most of the abl breakpoints have not been located but seem to be a considerable distance 5‘ to the hatched exon. For bcr, not all exons have been defined but all breakpoints fall within the bracketed region. In the bcr-abl locus, a nuclear precursor RNA (thin line) is presumed to be spliced in such a way that sequences of the most 5’ abl exon (hatched) are excised during generation of the 8.7-kb hybrid bcr-abl mRNA, which encodes the 210K polypeptide that contains the abl tyrosine kinase domain. The molecular counterpart of the 9q+ chromosome is not shown. Reprinted by permission from Nature, 315, 542. Copyright 0 1985 Macmillan Journals Limited.

et al., 1983b; Shtivelman et al., 1985). The sequence of chromosome junctions in two CMLs suggests that, as in plasmacytomas, translocation occurs by reciprocal recombination and a few nucleotides can be inserted during the exchange (Heisterkamp et al., 1985). The data argue against homologous recombination as the mechanism and suggest instead a random event selected for its biological consequence. The chromosome 22 breakpoints in different CML cluster within two introns of the bcr gene (Heisterkamp et al., 1985), while most of those on chromosome 9 all lie 5’ to the hatched exon in Fig. 10, which represents one of several alternative 5‘ c-abl exons (Ben-Neriah et al., 1986). The Philadelphia chromosome thus bears the 5’ portion of the bcr gene fused head to tail with most of the c-abl gene. Splicing produces a hybrid mRNA of -8 kb even in CMLs where the chromosome 9 breakpoints differ greatly, so presumably the splice of bcr sequences to the first exon common to all abl mRNAs takes precedence over the alternative abl splices. The chimeric mRNA accounts for the p210 abl protein associated with CML. The bcrlabl polypeptides in different CML may differ by the presence or absence of the 24 amino acids encoded by the third

ONCOGENES IN HEMOPOIETIC CELLS

223

mini bcr exon (see Fig. 10).In all cases, at least 25 residues from the N terminus of the normal abl polypeptide have been replaced by some 600-700 residues from the N terminus of bcr. Intriguingly, the v-abl polypeptide encoded by Abelson virus is also an N-terminal substitution of c-abl: 236 amino acids of the viral gag polypeptide replace 100 or so residues of c-abl protein. Hence N-terminal substitution may be critical for conversion of the c-abl proto-oncogene to a bonafide oncogene. Significantly, tyrosine phosphorylation of the p210 bcrlabl fusion protein is considerably greater than that of the normal c-abl protein (Konopka et d., 1984; Davis et d., 1985). The new N-terminal sequence of the protein may alter the conformation of the tyrosine kinase domain and account for this increase. The bcr-abl protein is expressed in hybrids made between mouse fibroblasts and CML cells (Kozbor et al., 1986), so expression of bcrl abl is not specific to myeloid cells. However, the hybrids were phenotypically indistinguishable from the parental fibroblasts, in contrast to v-abl-bearing fibroblasts, which round up and are clearly transformed. Thus the consequences of bcrlabl expression may differ for myeloid cells and fibroblasts. The function of the bcrlabl protein has yet to be determined. It will be very important to ascertain the nature of the bcr protein and its pattern of expression in hemopoietic cells, especially stem cells. If the pattern of bcr expression is very different from that of abl, a crucial function of the translocation may be to put abl under the control of bcr regulatory influences. Since the tyrosine kinase domain is essential for transformation of lymphoid cells (and fibroblasts) by v-abl (Prywes et al., 1985), it seems likely that the tyrosine kinase activity of the bcrabl protein will play a key role in transformation of myeloid cells. The acquisition of increased autophosphorylation activity is an important clue, because it strongly suggests that replacement of the N-terminal amino acids of c-abl by bcr sequences has changed the activity of the kinase. The receptors for several growth factors, including those for epidermal growth factor (Downward et aZ., 1984), insulin (Ullrich et al., 1985), and macrophage colony stimulating factor (Sherr et al., 1985), are transmembrane proteins with tyrosine kinase activity. The c-ab2 protein could be a component of a growth factor receptor. Fusion to bcr may interfere with normal factor-receptor interactions and allow the hybrid protein to continually deliver a proliferation signal to the cell, even in the absence of factor. An intriguing alternative, should bcr itself prove to be (part of) a receptor, is that bcr-abl represents a hybrid receptor.

224

SUZANNE CORY

VII. Translocations Specific to Acute Myeloid Leukemias

Several nonrandom chromosome abnormalities are associated with acute myeloid leukemia. Eighteen percent of all patients with M2 subtype of acute myeloblastic leukemia bear an 8;21 translocation (Drabkin et al., 1985). The chromosome 21 breakpoint at q22.3 is within the region implicated in Down’s syndrome, which is associated with an increased risk of leukemia (Evans and Steward, 1972), while that for chromosome 8 (q22.1) corresponds to the region bearing the c-mos oncogene (Nee1 et al., 1982). The role, if any, of c-mos in this translocation remains to be established. However, it is clear that no rearrangement occurs within a region of 12.4 kb around c-mos and that this oncogene is not within the region of chromosome 8 which is translocated to chromosome 21 (Drabkin et al., 1985); Diaz et al., 1985). A 15;17 translocation is strongly associated with acute promyelocytic leukemia (42 of 42 cases; Le Beau et al., 1985c), the breakpoint on chromosome 15 mapping at band 922, and that on chromosome 17 at band q21 (Sheer et al., 1983).The gene(s) affected by this translocation have not yet been identified. The oncogene c-fes is probably ruled out, because it maps too far away, at 15q24-q25 (Dalla Favera et al., 1982), but the Pz-microglobulin gene maps near or at the chromosome 15 breakpoint (Sheer et al., 1983). The chromosome 17 breakpoint is bracketed by two putative oncogenes: the cellular homolog of the erbA gene of avian erythroblastosis virus is proximal and thus remains on the 17q- chromosome (Dayton et al., 1984; Le Beau et al., 1985c),while the p53 gene is distal and is translocated to chromosome 15 (Le Beau et al., 1985~).While the 15q+ chromosome may bear the critical junction (see Le Beau et al., 1985c), any role for p53 in the pathogenesis of APL remains to be established, particularly since no rearrangement has been detected. It should also be noted that the neu oncogene, which is homologous to erbB, has also recently been mapped to the relevant region of chromosome 17 (Schechter et al., 1985). The frequent association of abnormalities involving band llq23 in acute leukemias with monocytic differentiation (Berger et al., 1982) suggests they involve alteration of a gene important in myelomonocytic differentiation. Intriguingly, the cellular homolog of ets, a gene associated with the mvb oncogene in the acutely transforming avian retrovirus E26, is located at llq23-q24 (de Taisne et al., 1984). About 10% of patients with acute monocytic leukemia bear the translocation t(q; 11)(p22;q23). Recent cytogenetic analysis has revealed that c-ets-l

ONCOGENES IN HEMOPOIETIC CELLS

225

is translocated in this translocation, while the breakpoint on chromosome 9 splits the interferon gene cluster (Diaz et al., 1986). While no DNA rearrangement has yet been defined, the results raise the possibility that juxtaposition of c-ets-1 and the interferon genes may be involved in the pathogenesis of this monocytic leukemia. Recent localization of the metallothionein gene cluster to band q22 of chromosome 16 raises intriguing questions regarding chromosome abnormalities associated with acute myelomonocytic leukemia (AMML) (Le Beau et al., 1985a). An inversion of chromosome 16, [inv (16)(p13q22)],has been identified in 25% of AMML patients and is associated with specific morphological changes in bone marrow eosinophils (Le Beau et al., 1983). Certain deletions and translocations which involve 16q22 have also been noted (see Le Beau et al., 1985b). Both inv(l6) and t(16;16) have been shown to result from cleavage within the metallothionein gene cluster, raising the possibility that leukemogenesis involved in either abnormal metallothionein gene expression or activation of another cellular gene by association with methallothionein gene control elements (Le Beau et al., 1985a).

VIII. Concluding Remarks

The marriage of cytogenetics and molecular oncology has already proved both exciting and fruitful. Two cellular oncogenes, c-myc and c-abl have been shown to be directly involved in specific chromosome translocations and the role of other known oncogenes is being vigorously pursued. Three putative oncogenes (pvt-1, bcl-1, bcE-2) have been identified at translocation junctions in lymphoid tumors by molecular cloning of “aberrant” immunoglobulin gene rearrangements. It is already clear that different translocations can exemplify very different ways of activating a proto-oncogene. The myc translocation does not alter the myc protein but brings its synthesis under constitutive control. The Philadelphia translocation, on the other hand, drastically changes the nature of the abl protein by fusing it to part of the bcr protein. The conclusion that these changes influence the development of malignancy seems inescapable, but the fundamental mechanisms involved remain enigmatic. The challenge for the future is not only to delineate the molecular basis for other translocations but also to identify the normal function of the genes involved. Only by learning where the cellular oncogenes fit into the normal pathway of growth and differentiation will we start to comprehend how their modulation by translocation, insertion, or

226

SUZANNE CORY

mutation contributes to the transformation of a normal cell to a malignant clone.

ACKNOWLEDGMENT I warmly thank all my colleagues, especially Dr. Jerry M. Adams, for their helpful comments and continuing support and stimulation.

REFERENCES Adams, J. M., and Cory, S. (1983).In “Eukaryotic Genes: Their Structure, Activity and Regulation” (M. Maclean, S. Gregory, and R. Flavell, eds.), pp. 343-358. Butterworth, London. Adams, J. M., Gerondakis, S., Webb, E., Mitchell, J., Bernard, O., and Cory, S. (1982). Proc. Natl. Acad. Scl. U S A . 79,6966-6970. Adams, J. M., Gerondakis, S., Webb, E., Corcoran, L. M., and Cory, S. (1983).Proc. Natl. A d . Scf. U . S A . 80, 1982-1986. Adams, J. M., Harris, A. W., Pinkert, C. A., Corcoran, L. M., Alexander, W. S., Cory, S., Palmiter, R. D., and Brinster, R. L. (1985).Noture (London) 318,533-538. Alitalo, K., Bishop, J. M., Smith, D. H., Chen, E. Y.,Colby, W. W., and Levinson, A. D. (1983a).Proc. Natl. Acad. Sci. U S A . 80, 100-104. Alitalo, K., Ramsay, G., Bishop, J. M., Ohlsson-Pfeifer, S., Colby, W., and Levinson, A. D.(198313).Nature (London)306,274-277. Armelin, H. A., Armelin, M. C.S.,Kelly, K., Stewart, T., Leder, P., Cochran, B. H., and Stiles, C. D. (1984).Nature (London)310,655-660. ar-Rushdi, A., Nishikura, K., Erikson, J., Watt, R., Rovera, G., and Croce, C. M. (1983). Science 222,390-393. Aurias, J. (1981).J . Genet. Hum. 29,235-247. Baer, R., Chen, K.-C., Smith, S. D., and Rabbitts, T. H. (1985).Cell 43,705-713. Banejee, M., Wiener, F., Spira, J., Babonits, M., Nilsson, M.-G., Sumegi, J., and Klein, G. (1985).EMBO J. 4,3183-3188. Bartram, C. R., Kleihauer, E., de Klein, A., Grosveld, G., Teyssier, J. R., Heisterkamp, N., and Groffen, J. (1985).EMBO J . 4,683-686. Battey, J., Moulding, C., Taub, R., Murphy, W., Stewart, T., Potter, H., Lenoir, G., and Leder, P. (1983).Cell 34,779-787. Beimling, P., Benter, T.,Sander, T., and Moelling, K. (1985).Biochemistry 24,6349-

6355.

Ben-Neriah, Y.,Bernards, A., Paskind, M., Daley, G. Q., and Baltimore, D. (1986).Cell (in press). Benoist, C., and Chambon, P. (1981).Nature (London)290,304-310. Berger, R., Bernheim, A., Brouet, J. C., Daniel, M. T., and Flandrin, G. (1979).Br. J. Haematol. 43,87. Berger, R. (1982).k u k . Res. 6, 17. Bernard, O.,Cory, S., Gerondakis, S.,Webb, E., and Adams, J. M. (1983).E M B O J . 2,

2375-2383.

Bishop, J. M. (1983).Annu. Reo. Biochem. 52,301-354. Blanchard, J.-M., Piechaczyk, M., Dani, C., Chambard, J.-C., Franchi, A., Pouyssegur, J., and Jeanteur, P. (1985).Nature (London)317,443-445. Bravo, R., Burckhardt, J., Curran, T., and Miiller, R. (1985).EMBOJ. 4, 1193-1197. Caccia, N., Kronenberg, M., Saxe, D., Haars, R., Bruns, G. A. P., Boverman, J., Malissen,

ONCOGENES IN HEMOPOIETIC CELLS

227

M., Willard, H., Yoshikai, Y., Simon, M., Hood, L., and Mak, T. W. (1984).Cell 37, 1091-1099. Calabi, F., and Neuberger, M. S. (1985).E M B O ] . 4,667-674. Calame, K., Kim, S., Lalley, P., Hill, R., Davis, M., and Hood, L. (1982). Proc. Natl. Acad. Sci. U.SA. 79,6994-6998. Carnpisi, J., Gray, H. E., Pardee, A. B., Dean, M., and Sonenshein, G. E. (1984).Cell 36, 241-247. Canaani, E., Steiner-Saltz, D., Aghai, E., Gale, R. P., Berrebi, A., and Januszewicz, E. (1984). Lancet 1,593-595. Cancro, M., and Potter, M. (1976).J. E z p . Med. 144, 1554. Cleary, M. L., and Sklar, J. (1985).Proc. Natl. Acad. Sci. U S A . 82, 7439-7443. Colby, W. W., Chen, E. Y., Smith, D. H., and Levinson, A. D. (1983).Nature (London) 301,722-725. Collins, M. K.L., Goodfellow, P. N., Dunn, M. J., Spurr, N. K., Solomon, E., and Owen, M. J. (1984). E M B O J . 3,2347-2349. Collins, M. K. L., Goodfellow, P. N., Spurr, N. K., Solomon, E., Tanigawa, G., Tonegawa S., and Owen, M. J. (1985). Nature (London) 314,273-274. Collins, S., Kabonishi, I., Miyoshi, I., and Groudine, M. (1984). Science 225,72-74. Corcoran, L. M., Adams, J. M., Dunn, A. R., and Cory, S. (1984). Cell 37, 113-122. Corcoran, L. M., Cory, S., and Adams, J. M. (1985).Cell 40,71-79. Cory, S., Adams, J. M., Gerondakis, S. D., Miller, J. F. A. P., Gamble, J., Wiener, F., Spira, J., and Francke, U. (1983a).E M B O J . 2,213-216. Cory, S., Gerondakis, S., and Adams, J. M. (1983b). E M B O J . 2, 697-703. Cory, S., Graham, M., Webb, E., Corcoran, L., and Adams, J. M. (1985).E M B O J . 4,675681. Coughlin, S. R., Lee, W. M. F., Williams, P. W., Giels, G. M., and Williams, L. T. (1985). Cell 43, 243-251. Crews, S., Griffen, J., Huang, H., Calame, K., and Hood, L. (1981). Cell 25, 59-66. Crews, S., Barth, R., Hood, L., Prehn, J., andCalame,K. (1982).Science218,1319-1321. Croce, C. M., Thierfelder, W., Erikson, J., Nishikura, K., Finan, J., Lenoir, G. M., and Howell, P. C. (1983).Proc. NQtl. Acad. Sci. U.S.A. 80, 6922-6926. Croce, C. M., Erikson, J., ar-Rushidi, A,, Aden, D., and Nishikura, K. (1984).Proc. Natl. Acad. Sci. U . S A . 81,3170-3174. Croce, C. M., Erikson, J., Huebner, K., and Nishikura, K. (1985a). Science 227, 12351238. Croce, C. M., Isobe, M., Palumbo, A., Puck, J., Ming, J., Tweardy, D., Erikson, J., Davis, M., and Rovera, G. (198513).Science 227, 1044-1047. Dalla Favera, R., Gallo, R. C., Giallongo, A., and Croce, C. M. (1982).Science 218,686688.

Dalla Favera, R., Martinotti, S.,Gallo, R. C., Erikson, J., and Croce, C. M. (1983). Science 219,963-967. Dani, C., Blanchard, J. M., Piechaczyk, M., El Sabouty, S., Marty, L., and Jeanteur, P. (1984). Proc. Natl. Acad. Sci. USA. 81, 7046-7050. Darveau, A., Pelletier, J., and Sonenberg, N. (1985). Proc. Natl. Acad. Sci. U S A . 82, 2315-2319. Davis, M., Malcolm, S., and Rabbitts, T. H. (1984).Nature (London) 308,286-288. Davis, R. L., Konopka, J. B., and Witte, 0. N. (1985).MoZ. Cell. Biol. 5,204-213. Dayton, A. I., Seldon, J. R., Laws, G., Dorney, D. J., Finan, J., Tripputi, P., Emanuel, B. S., Rovera, G., Nowell, P. C., and Croce, C. M. (1984).Proc. Natl. Acad. Sci. U.SA. 81,4495-4499.

228

SUZANNE CORY

De Klein, A., van Kessel, A. G., Grosveld, G., Bartram, C. R., Hagemeijer, A., Bootsma, D., Spurr, N. K., Heisterkamp, N., Groffen, J,, and Stephenson, J. R. (1982).Nature (London)300,765-767. d e la Chapelle, A,, Lenoir, G., BovB, J., Bov6, A., Gallano, P., Huerre, C., Szajnert, M.F., Jeanpierre, M., Lalouel, J.-M., and Kaplan, J.-C. (1983).Nucleic Acids Res. 11, 1133-1 142.

Denny, C. T., Hollis, G. F., Magrath, I. T., and Kirsch, I. R. (1985). Mol. Cell Biol. 5, 3199-3207.

Denny, C. T., Yoshikai, Y., Mak, T. W., Smith, S. D., Hollis, G. F., and Kirsch, I. R. (1986).Nature (London) 320,549-551. de Taisne, C., Gegonne, A,, Stehelin, D., Bemheim, A,, and Berger, R. (1984).Nature (London)310,581-583. Diamond, A., Cooper, G. M., Ritz, J., and Lane, M. A. (1983).Nature (London)305,112116.

Diaz, M. D., Le Beau, M. M., Pitha, P., and Rowley, J. D. (1986). Science 231,265-267. Diaz, M.O., Le Beau, M. M., Rowley, J. D., Drabkin, H. A., and Patterson, D. (1985). Science 229,767-769. Donner, P., Greiser-Wilke, I., and Moelling, K. (1982).Nature (London)296,262-266. Downward, J., Yarden, Y.,Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A,, Schlessinger, J., and Waterfield, M. D. (1984).Nature (London)307,521-527. Drabkin, H. A., Diaz, M., Bradley, C. M., Le Beau, M. M., Rowley, J. D., and Patterson, D. (1985). Proc. Natl. Acad. Sci. U S A . 82,464-468. Dunnick, W., Shell, B. E., and Dery, C. (1983).Proc. Natl. Acad. Sci. U S A . 80,72697273.

Dunnick, W., Baumgartner, J., Fradkin, L., and Schultz, C. (1984). Curr. Top. Microbiol. Immunol. 113, 154-160. Dunnick, W., Baumgartner, J., Fradkin, L., Schultz, C., and Szurek, P. (1985).Gene 39, 287-292.

Durdik, J., Moore, M., and Selsing, E. (1984). Nature (London)307, 749-752. Dyson, P. J., Littlewood, T. D., Forster, A., and Rabbitts, T. H. (1985).E M B O ] . 4,28852891.

Dyson, P. J., and Rabbitts, T. H. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 1984-1988. Einat, M., Resnitzky, D., and Kimchi, A. (1985).Nature (London)313,597-600. Eisenman, R. N., and Hann, S. R. (1985). Proc. R. SOC. B226,73-78. Eisenman, R. N., Tachibana, C. Y.,Abrams, H. D., and Hann, S. R. (1985). Mol. Cell. BbZ. 5, 114-126. Emanuel, B. S., Cannizzaro, L. A., Magrath, I., Tsujimoto, Y., Nowell, P. C., and Croce, C. M. (1985). Nucleic Acids Res. 13,381-387. Emanuel, B. S., Selden, J. R., Chaganti, R. S. K., Jhanwar, S.,Nowell, P. C., and Croce, C. M. (1984). Proc. Natl. Acad. Sci. U S A . 81,2444-2446. Erikson, J., Martinis, J., and Croce, C. M. (1981).Nature (London)294, 173-175. Erikson, J., Finan, J., Nowell, P. C., and Croce, C. M. (1982). Proc. Natl. Acad. Sci. USA. 79,5611-5615. Erikson, J,, ar-Rushdi, A., Dnvinga, H., Nowell, P. C., and Croce, C. M. (1983a). Proc. Natl. Acad. Sci. U S A . 80,820-824. Erikson, J., Nishikura, K., ar-Rushidi, A., Finan, J,, Emanuel, B., Lenoir, G., Nowell, P. C., and Croce, C. M. (1983b).Proc. Natl. Acad. Sci. U S A . 80,7581-7585. Erikson, J., Finan, J., Tsujimoto, Y., Nowell, P. C., and Croce, C. M. (1984).Proc. Natl. Acad. S C ~U.S.A. . 81,4144-4148.

ONCOGENES IN HEMOPOIETIC CELLS

229

Erikson, J., Miller, D. A., Miller, 0. J., Abcarian, P. W., Skurla, R. M., Mushinski, J. F., and Croce, C. M. (1985a).Proc. Natl. Acad. Sci. U.S.A.82,4212-4216. Erikson, J., Williams, D. L., Finan, J., Nowell, P. C., and Croce, C. M. (1985b).Science am, 784-786. Evans, D. J. K., and Steward, J. K. (1972). Lancet 2, 1322. Evan, G. I., and Hancock, D. C. (1985). Cell 43,253-261. Fahrlander, P. D., Piechaczyk, M., and Marcu, K. B. (1985b).E M B O J . 4,3195-3202. Fahrlander, P. D., Sumegi, J,, Yang, J.-Q., Wiener, F., Marcu, K. B., and Klein, G. (1985a).Proc. Natl. Acad. Sci. U S A . 82,3746-3750. Feo, S., ar-Rushidi, A,, Huebner, K., Finan, J., Nowell, P. C., Clarkson, B.,and Croce, C. M. (1985).Nature (London) 313,493-495. Filmus, J. and Buick, R. N. (1985).Cancer Res. 45,822-825. Gale, R. P., and Canaani, E. (1984). Proc. Natl. Acad. Sci. U S A . 81,5648-5652. Gazin, C., Dupont de Dinechin, S., Hampe, A,, Masson, J.-M., Martin, P., Stehelin, D., and Galibert, F. (1984).E M B O J. 3,383-388. Gelmann, E. P., Psallidopoulos, M. C., Papas, T. S., and Dalla Favera, R. (1983).Nature (London)306,799-803. Gerondakis, S., Cory, S., and Adams, J. M. (1984).Cell 36, 973-982. Gonda, T. J., and Metcalf, D. (1984).Nature (London) 310,249-251. Gonda, T., Sheiness, D. K., and Bishop, J. M. (1982).M o l . Cell. Biol. 2,617-624. Goyette, M., Petropoulos, C. J., Shank, R. R., and Fausto, N. (1984).Mol. Cell. Biol. 4, 1493-1498. Graham, M., Adams, J. M., and Cory, S. (1985).Nature (London)314,740-743. Greenberg, M. E., and Ziff, E. B. (1984).Nature (London) 311,433-438. Groffen, J., Heisterkamp, N., Stephenson, J. R., Van Kessel, A. G., De Klein, A,, Grosveld, G., and Bootsma, D. (1983).J . Erp. Med. 158, 9-15. Groffen, J., Stephenson, J. I., Heisterkamp, N., de Klein, A., Bartram, C. R., and Grosveld, G. (1984).Cell 36,93-99. Hamlyn, P., and Rabbitts, T. H. (1983). Nature (London)304, 135-139. Hann, S. R., and Eisenman, R. (1984).Mol. Cell. Biol. 4, 2486-2497. Hann, S. R., Thompson, C. B., and Eisenman, R. N. (1985).Nature (London)314,366369. Harris, L. J., Lang, R. B., and Marcu, K. B. (1982).Proc. Natl. Acad. Sci. U S A . 79,41754179. Harris, L. J., Remmers, E. F., Brodeur, P., Riblet, R., D’Eustachio, P., and Marcu, K. B. (1983).Nucleic Acids Res. 11, 8303-8315. Hawley, R., Shulman, M., Murialdo, H., Gibson, D., and Hozumi, N. (1982).Proc. Natl. Acad. Sci. U S A . 79, 7425-7429. Hayday, A. C., Gillies, S. D., Saito, H., Wood, C., Wiman, K., Hayward, W. S., and Tonegawa, S. (1984).Nature(London)307,334-340. Hayward, W., Neel, B. G., and Astrin, S. (1981).Nature (London) 290,475-480. Hecht, F., Morgan, R., Hecht, B. K.-M., and Smith, S. (1984).Science 226, 1445-1447. Heisterkamp, N., Groffen, J., Stephenson, J. R., Spun; N. K., Goodfellow, P. N., Solomon, E., Carritt, B., and Bodmer, W. F. (1982).Nature (London)299,747-750. Heisterkamp, N., Stephenson, J. R., Groffen, J., Hansen, P. F., de Klein, A,, Bartram, C. R., and Grosveld, G. (1983a).Nature (London)306,239-242. Heisterkamp, N., Groffen, J., and Stephenson, J. R. (1983b).J.Mol. Appl. Genet. 2,5768. Heisterkamp, N., Stam, K., Groffen, J., de Klein, A., and Grosveld, G. (1985). Nature (London)315,758-761.

230

SUZANNE CORY

Hollis, G. F., Mitchell, K. F., Battey, J., Potter, H., Taub, R., Lenoir, G. M., and Leder, P (1984). Nature (London)307,752-755. Hood, L., Kronenberg, M., and Hunkapiller, T. (1985). Cell 40, 225-229. Jhanwar, S. C., Neel, B. G., Hayward, W. S., and Chaganti, R. S . (1983). Proc. Natl. Acad. Sci. U.SA. 80,4794-4797. Jonak, G. J., and Knight, E. (1984). Proc. Natl. Acad. Sci. U S A . 81, 1747-1750. Kaczmarek, L., Hyland, J. K., Watt, R., Rosenberg, M., and Baserga, R. (1985). Science 228, 1313-1314.

Kakkis, E., Prehn, J., and Calame, K. (1986). Mol. Cell. B i d . 6, 1357-1361. Kaneko, Y.,Egues, M. C., and Rowley, J. D. (1981). Cancer Res. 41,4577-4578. Keath, E. J., Kelekar, A., and Cole, M. D. (1984a). Cell 37, 521-528. Keath, E. J,, Caimi, P. G. and Cole, M. D. (1984b). Cell 39,339-348. Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. (1983).Cell 35,603-610. Kingston, R. E., Baldwin, A. S., and Sharp, P. A. (1984). Nature (London)312,280-282. Klein, G. (1981). Nature (London)294,313-318. Klein, G. (1983). Cell 32,311-315. Klein, G. (1985). I n “Genetic Rearrangements in Leukaemia and Lymphoma” (J. M. Goldman and D. E. Harnden, eds.), pp. 117-135. Churchill-Livingstone, London. Klein, G. and Klein, E. (1985).Nature (London)315, 190-195. Klein, S., Sablitzky, F., and Radbruch, A. (1984). E M B O ] . 3,2473-2476. Kloetzer, W., Kurzvock, R., Smith, A., Talpaz, M., Spiller, M., Gutterman, J., and Arlinghaus, A. (1985). Virology 140,230-238. Knight, E., Anton, E. D., Fahey, D., Friedland, B. K., and Jonak, G. J. (1985). Proc. Natl. Acad. Scf. U.S.A.82, 1151-1154. Konopka, J., Watanabe, S., Singer, J., Collins, S . , and Witte, 0. (1985). Proc. Natl. Acad. Sci. U.SA. 82,1810-1814. Konopka, J. B., Watanabe, S . M., and Witte, 0. N.(1984). Cell 37,1035-1042. Kozbor, D., Giallongo, A., Sierzega, M. E., Konopka, J. B., Witte, 0. N., Showe, L. C., and Croce, C. M. (1986). Nature (London)319,331-333. Lachman, H. M., Hatton, K. S., Skoultchi, A. I., and Schildkrant, C. L. (1985). Proc. Natl. Acad. Sci. U S A . 82,5323-5327. Lachman, H.M., and Skoultchi, A. J. (1984). Nature (London)310,592-594. Land, H., Parada, L., and Weinberg, R. (1983). Nature (London)304,596-602. Le Beau, M. M., Larson, R. A., Bitter, M. A., Vardiman, J. W., Golombe, H. M., and Rowley, J. D. (1983).N. Engl. I. Med. 309,630-636. Le Beau, M.M., Diaz, M. O., Karin, M., and Rowley, J. D. (1985a).Nature (London)313, 709-711.

Le Beau, M. M., Diaz, M. O., Rowley, J. D., and Mak, T. W. (198%). Cell 41,335. Le Beau, M. M., Westbrook, C. A., Diaz, M. O., Rowley, J. D., and Oren, M. (1985~). Nature (London)316,826-828. Leder, P., Battey, J., Lenoir, G., Moulding, C., Murphy, W., Potter, H., Stewart, T., and Taub, R. (1983). Science 222,765-771. Lee, W.M.F., Schwab, M., Westaway, D., and Varmus, H. E. (1985). Mol. Cell. Biol. 5, 3345-3356.

Lenoir, G., Preud’homme, J. L., Bernheim, A., and Berger, R. (1982). Nature (London) 298,473-476.

Lewis, W. H., Michalopoulos, E. E., Williams, D. L., Minden, M. D., and Mak, T. W. (1985). Nature (London)317, 544-546. Magrath, I., Erikson, J., Whang-Peng, J., Sieverts, H., Armstrong, G., Benjamin, D., Triche, T., Alabaster, O., and Croce, C. M. (1983). Science 222, 1094-1098. Makino, B., Hayashi, K., and Sugimura, T. (1984). Nature (London) 310, 697-698.

ONCOGENES IN HEMOPOIETIC CELLS

231

Malcolm, S., Barton, P., Murphy, C., Ferguson-Smith, M. A., Bently, D. L., and Rabbitts, T. H. (1982).Proc. Natl. Acad. Sci. U.S.A.79,4957-4961. Manolov, G., Manolova, Y., Klein, G., Lenoir, G., and Levan, A. (1986).Cancer Genet. Cytogenet. 20,95-99. Marcu, K. B., Harris, L. J., Stanton, L. W., Erikson, J., Watt, R., and Croce, C. M. (1983). Proc. Natl. Acad. Sci. U S A . 80,519-523. McBride, D. W., Heiter, P. A., Hollis, G. F., Swan, D., Otey, M. C., and Leder, P. (1982). j . Erp. Med. 155, 1480-1490. McCormack, J. E., Pepe, V. H., Kent, R. B., Dean, M., Marshak-Rothstein, A., and Sonenshein, G. (1984).Proc. Natl. Acad. Sci. U S A . 81,5546-5550. Mercola, M., Kakkis, E.,Prehn, J., Wang, X., and Calame, K. (1984).In “Regulation of the Immune System, UCLA Symposium on Molecular and Cellular Biology” (Cantor, H., Chess, L., and Sercarz, E., eds.), 18th ed. Alan R. Liss, New York. Mitelman, F. (1984).Nature (London)310,325-327. Mitelman, F., Anderson-Anvret, M., Brandt, L., Catovsky, D., Klein, G., Manolov, G., 1nt.J.Cancer 24,27-38. Manolova, Y., Mark-Vendel, E., and Nilsson, P. G. (1979). Moulding, C., Rapopoxt, A., Goldman, P., Battey, J., Lenoir, G. M., and Leder, P. (1985). Nucleic Acids Res. 13,2141-2152. Murray, M. J., Cunningham, J. M., Pavada, L. F., Dantry, F., Lebowitz, P., and Weinberg, R. A. (1983).Cell 33,749-756. Mushinski, F., Bauer, S. R., Potter, M., and Reddy, E. P. (1983).Proc. Natl. Acad. Sci. U.SA. 80,1073-1077. Neel, B. G., Jhanwar, S.C., Chaganti, R. S., and Hayward, W. S. (1982).Proc. Natl. Acad. Sci. U . S A . 79,2971-2975. Neuberger, M. S., and Calabi, F. (1983).Nature (London) 305,240-243. Nishikura, K., ar-Rushdi, A., Erikson, J,, Watt, R., Rovera, G., and Croce, C. M. (1983). Proc. Natl. Acad. Sci. U S A . 80,4822-4826. Nishikura, K.,ar-Rushdi, A., Erikson, J., DeJesus, E., Dugan, D., and Croce, C. M. (1984).Science 224,399-402. Nishikura, K.,Erikson, J., ar-Rushdi, A., Huebner, K., and Croce, C. M. (1985).Proc. Natl. Acad. Sci. USA. 82,2900-2904. Nowell, P. C., and Hungerford, D. A. (1960).Science 132, 1197. Ohno, S., Babonits, M., Wiener, F., Spira, J., Klein, G., and Potter, M. (1979).Cell 18,

1001-1007.

Ohno, S.,Migita, S.,Wiener, F., Babonits, M., Klein, G., Mushinski, J. F., and Potter, M. (1984).j . E r p . Med. 159, 1762-1777. Parslow, T., and Granner, D. (1982).Nature (London)299,449-451. Pegoraro, L., Palumbo, A., Erikson, J., Falda, M., Giovanazzo, B., Emanuel, B. S., rovera, G., Nowell, P. C., and Croce, C. M. (1984).Proc. Natl. Acad. Sci. U.S.A. 81,

7166-7170.

Perlmutter, R., Klotz, J., Pravtcheva, D., Ruddle, F., and Hood, L. (1984).Nature (Lon-

don) 307,473-476.

Perlmutter, R., Kearney, J. F., Chang, S. P., and Hood, L. (1985).Science 227, 15971601. Perry, R. P. (1983).Cell 33, 647-649, Person, H., Gray, H.E., and Godeau, F. (1985).Mol. Cell. Biol. 5,2903-2912. Person, H., and Leder, P. (1984).Science 225,718-721. Person, H., Lenninghausen, L., Taub, R., DeGrado, W., and Leder, P. (1984).Science

225,687-693.

Peschle, C., Mavilio, F., Sposi, N. M., Giampaolo, A., C a d , A., Bottero, L., Bruno, M.,

232

SUZANNE CORY

Mastroberardino, G., Gastaldi, R., Testa, M. G., Alimena, G., Amadori, S., and Mandelli, F. (1984).Proc. Natl. Acad. Sci. U S A . 81,5514-5518. Pfeifer-Ohlsson, S . , Goustin, A. S., Rydnert, J,, Wahlstrom, T., Bjersing, L., Stehelin, D., and Ohlsson, R. (1984).Cell 38, 585-596. Pfeifer-Ohlsson, S., Rydnert, J., Goustin, A. S., Larsson, E., Belsholtz, C. and Ohlsson, R. (1985).Proc. Natl. Acad. Sci. U S A . 82, 5050-5054. Picard, D., and Schafher, W. (1984).Nature (London) 307,80-82. Piccoli, S . P., Caimi, P. G., and Cole, M. D. (1984).Nature (London) 310,327-330. Piechaczyk, M., Yang, J.-Q., Blanchard, J.-M., Jeanteur, P., and Marcu, K. B. (1985).Cell 42,589-597. Potter, M., and Wax, J. S. (198l).J.Immunol. 127, 1591-1595. Potter, M., Wiener, F., and Mushinski, F. (1984).Ado. Viral Oncol. 4, 139-162. Prehn, J., Mercola, M., and Calame, K. (1984).Nucleic Acids Res. 12,8987-9007. Prywes, R., Foulkes, J. G., and Baltimore, D. (1985).J. Virol. 54, 114-122. Queen, C., and Stafford, J. (1984). MoZ. Cell. Biol. 4, 1042-1049. Rabbitts, T. H., Hamlyn, P. H., and Baer, R. (1983).Nature (London) 306,760-765. Rabbitts, T. H., Forster, A., Hamlyn, P., and Baer, R. (1984).Nature (London)309,592597. Rabbitts, P. H., Forster, A., Stinson, M. A., and Rabbitts, T. H. (1985a).E M B O J . 4, 2009-2015. Rabbitts, P. H., Watson, J. V., Lamond, A., Forster, A., Stinson, M. A., Evan,'G., Fischer, W.,Atherton, E., Sheppard, R., and Rabbitts, T. H. (1985b).E M B O J . 4,2009-2015. Ralston, R.,and Bishop, J. M. (1983).Nature (London) 306, 803-806. Ramsay, G., Evan, G. I., and Bishop, J. M. (1984).Proc. Natl. Acad. Sci.U.S.A.81,77427746. Rapp, U. R., Cleveland, J. L., Brightman, K., Scott, A., and Ihle, J. N. (1985).Nature (London) 317,434-438. Rappold, G . A., Hameister, H., Cremer, T., Adolph, S., Henglein, B., Freese, U.-K., Lenoir, G . M., and Bornkamm, G. W. (1984).Cell 3,2951-2955. Rechavi, G., Givol, D., and Canaani, E. (1982).Nature (London) 300,607-611. Reed, J. C., Nowell, P. C., and Hoover, R. G. (1985).Proc. Natl. Acad. Sci. U.S.A. 82, 4221-4224. Reitsma, P. H., Rothberg, P. G., Astrin, S . M., Trial, J., Bar-Shavit, Z., Hall, A., Teitelbaum, S. L., and Kahn, A. J. (1983).Nature (London) 306,492-493. Riccardi, V. M., Hittner, H. M., Francke, U., Yunis, J., Ledbetter, D., and Borges, W. (1980). Cancer Genet. Cytogenet. 2, 131-137. Rothberg, P. G., Erisman, M. D., Diehl, R. E., Rovigatti, U. G., and Astrin, S. (1984). M o l . Cell Biol. 4, 1096-1103. Rowley, J. D. (1973).Nature (London) 243, 190-293. Rowley, J. D. (1984). Cancer Res. 44,3159-3168. Rowley, J. D., Golomb, H. M., Vardiman, J., Fukuhara, S . , Dougherty, C., and Potter, D. (1977). Znt. J . Cancer 20,869. Ruley, H. E. (1983).Nature (London) 304,503-607. Saito, H.,Hayday, A. C., Wiman, K., Hayward, W. S., and Tonegawa, S. (1983). Proc. Natl. Acad. Sci. U,SA. 80, 7476-7480. Schechter, A. L., Hung, M.-C., Vaidyanathan, L., Weinberg, R., Yang-Feng, T. L., Francke, U., Ulrich, A. and Coussens, L. (1985).Science 229,976-978. Schubach, W., and Groudine, M. (1984).Nature (London) 307,702-708. Sheer, D., Hiorns, L. R., Stanley, K. F., Goodfellow, P. N., Swallow, D. M., Povey, S.,

ONCOGENES IN HEMOPOIETIC CELLS

233

Heisterkamp, N., Groffen, J., Stephenson, J. R., and Solomon, E. (1983).Proc. Natl. Acad. Sci. U.S.A. 80,5007-5011. Shen-Ong, G. L. C., Keath, E. J., Piccoli, S. P., and Cole, M. D. (1982).Cell 31,443-452. Sherr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F., Look, A. T., and Stanley, E. R. (1985).Cell 41,665-676. Shih, C.-K., Linial, M., Goodenow, M. M., and Hayward, W. S . (1984).Proc. Natl. Acad. Sci. U.S.A. 81,4697-4701. Shima, E. A., Le Beau, M. M., McKeithan, T. W., Minowada, J., Showe, L. W., Mak, T. W., Rowley, J. D., and Diaz, M. 0. (1986).Proc. Natl. Acad. Sci. U.S.A.(in press). Showe, L. C., Ballantine, M., Nishikura, K., Erikson, J., Kaji, H., and Croce, C. M. (1985).Mol. Cell. Biol. 5, 501-509. Shtivelman, E., Lifshitz, B., Gale, R. B., and Canaani, E. (1985).Nature (London)315,

550-554.

Siebenlist, U., Hennighausen, L., Battey, J., and Leder, P. (1984).Cell 37,381-391. Smeland, E., Godal, T., Ruud, E., Beiske, K., Funderud, S., Clark, E. A., Pfeifer-Ohlsson, S., and Ohlsson, R. (1985).Proc. Natl. Acad. Sci. U S A . 82,6255-6259. Stanton, L. W., Watt, R., and Marcu, K. B. (1983).Nature (London)303,401-406. Stanton, L. W., Fahrlander, P. D., Tesser, P. M., and Marcu, K. B. (1984).Nature (London) 310,423-425. Stanton, L. W., Yang, J.-Q., Eckhardt, L. A., Harris, L. J., Birshtein, B. K., and Marcu, K. B. (1984).Proc. Natl. Acad. Sci. U.S.A. 81,829-833. Stewart, T. A., BellvB, A. R., and Leder, P. (1984).Science 226, 707-710. Stewart, T.A., Pattengale, P. K., and Leder, P. (1984).Cell 38,627-637. Sumegi, J., Spira, J., Bazin, H., Szpirer, J., Levan, G., and Klein, G. (1983).Nature (London)306,497-498. Taub, R., Kirsch, I., Morton, C., Lenoir, G., Swan, D., Tronick, S., Aaronson, S., and Leder, P. (1982).Proc. Natl. Acad. Sci. U S A . 79, 7837-7841. Taub, R., Kelly, K., Battey, J., Latt, S., Lenoir, G. M., Tantravahi, U., Tu, Z., and Leder, P. (1984a).Cell 37,511-520. Taub, R.,Moulding, C., Battey, J., Murphy, W., Vasicek, T., Lenoir, G. M., and Leder, P. (1984b).Cell 36,339-348. Thompson, C. B., Challoner, P. B., Neiman, P. E., and Groudine, M. (1985).Nature (London) 314,363-366. Tsujimoto, Y., Yunis, J., Onorato-Showe, L., Erikson, J., Nowell, P. C., and Croce, C. M. (1984a).Science 224, 1403-1406. Tsujimoto, Y., Finger, L. R., Yunis, J., Nowell, P. C., and Croce, C. M. (198413).Science

226,1097-1099.

Tsujimoto, Y., Cossman, J., Jaffe, E., and Croce, C. M. (1985a).Science 228,1440-1443. Tsujimoto, Y., Jaffe, E., Cossman, J., Gorham, J., Nowell, P. C., and Croce, C. M. (1985b).Nature (London) 315,340-343. Tsujimoto, Y., Gorham, J., Cossman, J., Jaffe, E., and Croce, C. M. (1985~). Science 229,

1390.

Ueshima, Y., Rowley, J. D., Variakojis, D., Winter, J., and Gordon, L. (1984).Blood 63,

1028-1038.

Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A,, Seeburg, P. H., Grunfeld, C., Rosen, 0. M., and Ramachandran, J. (1985).Nature (London)313,756-761. Van den Berghe, H. (1984).Cancer Genet. Cytogenet. 11,381-387. Van Ness, B., Weigert, M., Coleclough, C., Mather, E., Kelley, D. E., and Perry, R. P. (1981).Cell 27,593-602.

234

SUZANNE CORY

Van Ness, B. G., Shapiro, M., Kelley, D. E., Perry, R. P., Weigert, M., D’Eustachio, P., and Ruddle, F. (1983). Nature (London)301,425-427. Varmus, H. E. (1984).Annu. Rev. Genet. 18,553-612. Villeneuve, L., Rassart, E., Jolicoeur, P., Graham, M., and Adams, J. M. (1986). Mol. Cell. Btol. (in press). Wabl, M. R., and Burrows, P. D. (1984). Proc. Natl. Acad. Sci. U S A . 81,2452-2455. Wang, X.-F., and Calame, K. (1985). Cell 43,659-665. Watson, D. K., Reddy, E. P., Duesberg, P. H., and Papas, T. S. (1983).Proc. Natl. Acad. Sci. U.SA. 80, 2146-2150. Watt, R., Stanton, L. W., Marcu, K. B., Gallo, R. C., Croce, C. M., and Rovera, G. (1983). Nature (London)303,725-728. Watt, R. A,, Shatzman, A. R., and Rosenberg, M. (1985). Mol. Cell. Biol. 5, 448-456. Webb, E., Adams, J. M., and Cory, S. (1984). Nature (London)312,777-779. Weischet, W., Glotov, B., Schnell, H., and Zachau, H. (1982). Nucleic Acids Res. 10, 3627-3645.

Westaway, D., Payne, G., and Varmus, H. (1984). Proc. Natl. Acad. Sci. U S A . 81,843847.

Williams, D. L., Look, A. T., Melvin, S. L., Roberson, P. K., Dahl, G., Flake, T., and Stass, S. (1984). Cell 36, 101-109. Wiman, K. G., Clarkson, B., Hayday, A. C., Saito, H., Tonegawa, S., and Hayward, W. S. (1984). Proc. Natl. Acad. Sci. U S A . 81, 6798-6802. Winquist, R., Saksela, K., and Alitalo, K. (1984). EMBO J , 3,2947-2950. Wirschubsky, Z., Ingvarsson, S., Carstensen, A., Wiener, F., Klein, G. and Sumegi, J. (1985). Proc. Natl. Acad. Sct. U S A . 82, 6975-6979. Wood, C., and Tonegawa, S. (1983).Proc. Natl. Acad. Sci. U.S.A. 80, 3030-3034. Yancopoulos, G. D., Desiderio, S. V., Paskind, M., Kearney, J. F., Baltimore, D., and Alt, F. W. (1984). Nature (London) 311,727-733. Yang, J.-Q,, Bauer, S . R., Mushinski, J. F., and Marcu, K. B. (1985). EMBOJ. 4, 14411447.

Yang, J. Q., Mushinski, J. F., Stanton, L. W., Fahrlander, P. D., Tesser, P. C., and Marcu, K. B. (1984). Curt. Top. Microbiol. Immunol. 113, 146-153. Yang-Feng, T. L., Francke, U., and Ullrich, A. (1985). Science 228, 728-731. Yunis, J. J. (1983). Science 221,227-236. Yunis, J. J,, and A. L. Soreng, (1984). Science 226, 1199-1204. Zech, L., Gahrton, G., Hammarstrom, L., Juliusson, G., Mellstedt, H., Robert, K. H., and Smith, C. I. E. (1984). Nature (London) 308,858-860.

ONCOGENE AMPLIFICATION IN TUMOR CELLS Kari Alitalo' and Manfred Schwabt

* Department of Virology, University of Helsinki. 00290 Helsinki 29, Finland t The Qeorge Williams Hooper Foundation, University of California, San Francisco, California 94143

1. Introduction

Regulatory or structural alterations of cellular oncogenes have been implicated in the causation of various cancers. Oncogene alteration by point mutations can result in a protein product with a strongly enhanced oncogenic potential. Aberrant expression of cellular oncogenes may be due to tumor-specific chromosomal translocations that deregulate the normal expression of proto-oncogenes. This review summarizes data on the third mechanism of oncogene activation: oncogene amplification. It is not the purpose of this review to deal with all forms of DNA damage that have been found to activate cellular oncogenes (see Bishop, 1985; Klein and Klein, 1985). For the purpose of integrating the review into a coherent picture, however, the reader is given a list of known cellular oncogenes in Table I, and Fig. 1 illustrates the various ways by which the oncogenic potential of different proto-oncogenes can be activated. Because of the involvement of myc oncogenes in amplifications in a variety of tumors, other lesions that also activate the cellular oncogene c-myc and aspects of the normal regulation of this oncogene will be described in Section VIII. Since its discovery in drug-resistant eukaryotic cells, somatic amplification of specific genes has been implicated in an increasing variety of adaptive responses of cells to environmental stresses (Schimke, 1982; Stark and Wahl, 1984; Pohjanpelto et al., 1985). Cytogenetic abnormalities, double minute chromosomes (DMINs) associated with DNA amplification, were found in tumor cells followed by the discovery of DMINs and homogeneously staining chromosomal regions (HSRs) in cells selected for drug resistance (Biedler and Spengler, 235 ADVANCES IN CANCER RESEARCH, VOL.47

Copyright 0 1986 by Academic Press, Inc.

All rights of reproduction in any form reserved.

TABLE I SOMECURRENTLY KNOWNONCOGENES Gene product Oncogene

Cellular location

Function of protein

Class

Oncogenesfound in retroviruses

Retrovirus (example) RSV w Q,

STC

Plasma membrane

Y73V GR-FeSV

Yes fqr

Ab-MuLV FuSV, ST-and GA-FeSV

abl fpslfes

Plasma membrane Plasma membrane/ cytoplasm Plasma membrane Cytoplasm (plasma membrane?) Plasma membrane

uR2v

ros

HZ4FeSV

kit

AEV

erbB

SM-FeSV

fms

S13ASV

sea

Plasma membrane and cytoplasmic membranes Plasma membrane and cytoplasmic membranes Plasma membrane and cytoplasmic membranes

Tyrosine-specific protein kinases

Class l a (Tyrosine protein kinases)

(fgr contains sequences

homologous to actin)

EGF receptor’s transmembrane and cytoplasmic domain M-CSF growth factor receptor Growth factor receptor

Class Ib (Class la-related proteins)

- - - - - - - - - - _ _ _ - -

MH-2V, 3911-MSV Mo-MSV

mos

millraf

Cytoplasm Cytoplasm

Serine/threonine kinase Serinelthreonine kinase

ssv

sis

Secreted

Ha-MSV Ki-MSV

Ha-ras Ki-rus

Plasma membrane Plasma membrane

Signal transducing G proteins

Class 3 (Cytoplasmic GTPases)

FBJ-MuSV

fos

Nucleus

?

Class 4 (Nuclear phosphoproteins)

OK-1OV AMV

mYc mYb

Nucleus Nucleus

Nuclear “matrix” protein ?

SKV 770

ski

Nucleus?

?

REV

re1

? ? ?

~

8 4

erbA

E26V

ets

PDGF-likegrowth factor

?

Class 2 (Growth factors)

Unclassified AEV Steroid receptor?

?

Oncogenes found in tumor cells but not in retroviruses Tumor cell Neuroblastoma Neuroblastomas Small-cell lung cancer Friend virus-induced erythroleukemia Neuro/glioblastomas Osteosarcoma Colon carcinoma T-cell lymphoma

N-ras N-myc L-myc P53

Plasmamembrane ? ? Nucleus

GTP-binding ? ? Nuclear phosphoprotein

Class3 Class 4 Class 4 Class 4

neu (c-erbB2) met trk pim-1

Plasma membrane

Growth factor receptor

Class l b

Tyrosine kinase Tyrosine kinase Tyrosine kinase

Class la Class lb Classl a

238

KARI ALITALO AND MANFRED SCHWAB

FIG.1. Activation of cellular oncogenes. The haploid complement of a proto-oncogene, schematically depicted in (A), is composed of three exons (black boxes) in a segment of DNA. The different activated forms are schematically outlined in (B-G). The abbreviation c-onc stands for cellular oncogene, and v-onc for viral oncogene; DNA sequences with associated promoterlenhancer functions are striated, and an actively transcribed gene is marked with radiations. (B) Acute transforming retroviruses have the capacity to transduce cellular oncogenes (c-onc) into their genome, modify them, and reinsert the activated viral oncogenes (v-onc) into the genome of host animal cells as a part of the provirus. The activity of the v-onc gene is greatly enhanced due to the associated promoter of the proviral long terminal repeat (LTR). Both increased dosage of the oncogene and its structural mutations may contribute to tumorigenesis. (C) Slow transforming retroviruses without oncogenes replicate and reinsert their proviral copies into the host cell DNA during a latency period from infection to tumorigenesis. Tumor initiation through hyperplastic growth may begin when the provirus integrates sufficiently close to a proto-oncogene to activate it through promoter or enchancer functions of the proviral LTR element. It should be noted, however, that mutations have also been found in the oncogenes thus activated and that mutational damage to other oncogenes has been described in the resulting tumors. (D) In some mouse plasmacytomas, a retrovirus-like DNA element (directing the synthesis of the so-called intracisternal Atype particles, IAPs) has been found in association with a transcriptionally activated oncogene c-mos. The IAP insertion also disrupts the 5' part of c-mos (Rechavi et al., 1982). (E) In humans, as well as in animals, chromosome translocations may place proto-oncogenes into transcriptionally active regions of chromatin or create fused transcripts and proteins from two genes. The details of these mechanisms have not been worked out. Translocations activate c-myc and c-abl genes in Burkitt lymphomas and Philadelphia chromosome-positive leukemias, respectively (Heisterkamp et al., 1983; Klein, 1983). (F) Increased amounts of oncogene-specific RNA and protein can also result from an excess of DNA template for transcription acquired through oncogene amplification. The present review concentrates primarily on this mechanism. (G) Mutationally activated oncogenes have been found in nearly one-fifth of human malignant

ONCOGENE AMPLIFICATION IN TUMOR CELLS

239

FIG.2. DMINs in metaphases of cells from a case of acute myeloid leukemia. The DNA from the leukemic cells was found to contain about 30-fold amplification of the cmyc oncogene (Alitalo et al., 1985).

1976; Cox et al., 1965; Levan et al., 1968, 1977; Mark, 1967, 1971; Spriggs and Boddington, 1962).In metaphase spreads, DMINs appear as small, spherical, usually paired chromosomelike structures that lack a centromere and may contain circular DNA in chromatin form (Hamkalo et al., 1985; Fig. 2). HSRs stain with intermediate intensity throughout their length rather than with the normal pattern of alternating dark and light bands in both trypsin-Giemsa- and quinacrine dihydrochloride-stained preparations (Fig. 3). Both kinds of abnormalities contain amplified DNA and are occasionally found in metaphases of freshly isolated cancer cells but not of normal cells (Barker, 1982). DMINs and HSRs are apparently rare in tumor cells in uiuo, although exact data are difficult to obtain since the abnormalities are easily missed in routine cytogenetic analysis (Barker, 1982; Gebhart et al., 1984; Kovacs, 1979; Li, 1983). DMINs and HSRs have been described in most types of in uitro cultured malignant tumor cells, tumors. Oncogene loci activated by somatic structural mutations are revealed by transfection experiments, where they are introduced into supposedly normal genetic background of cultured immortalized cells. Several such transforming loci have been cloned and many of them belong to the c-ras oncogene family. It should be pointed out that both structural mutations and either increased expression or activation of a complementing oncogene may be required to achieve a fully tumorigenic phenotype (Land et al., 1983a).

240

KARI ALJTALO AND MANFRED SCHWAB

FIG.3. (Left) The homogeneously staining regions (HSR) in the G-banded HSRmarker chromosome (GTG) comprise a major portion of both its long and short arms. The HSR-marker chromosome has evolved from an X chromosome (Alitalo et al., 1983b). (Right) The about 30-fold amplified copies of the c-myc oncogene in COLO 320 cells were found to be located to HSRs by in situ hybridization (Alitaloet al., 1983b; Lin et ol., 1985).

with a notable frequency in neuroblastoma cell lines (Biedler et al., 1983). Initial growth in cell culture apparently selects for tumor cells that contain either DMINs or HSRs. Moreover, in culture, DMINs are frequently lost, concomitant with the appearance of clonal populations of cells that have developed an HSR, suggesting that the two cytogenetic abnormalities are alternative forms of gene amplification (Biedler et al., 1983). It has been assumed that HSRs can break down to form DMINs and that DMINs can integrate into chromosomes to generate HSRs (Biedler et al., 1983, Cowell, 1982). Amplified genes may also occupy abnormally banding regions (ABRs) and C-bandless chromosomes (CMs) (Levan et al., 1977, 1981; Lewis et al., 1982; Nowell et al., 1983; Schwab et al., 1985). Experimental work on drug-resistant cells has shown that in the absence of a selection pressure (drug), DMINs and the amplified genes that they contain are lost, whereas amplified DNA in the form of HSRs is retained in the cells (Schimke et al., 1981).This is explained by the fact that DMINs are segregated unevenly in mitosis and are

ONCOGENE AMPLIFICATION IN TUMOR CELLS

24 1

frequently lost from the nucleus due to their lack of centromeres (Levan et al., 1981). HSR chromosomes carry centromeres and are therefore divided equally between daughter cells at mitosis. If DMINs and HSRs contain amplified genes that encode drug-resistant or growth-stimulating protein products, it would follow that the more stable chromosomal form, the HSR, confers a greater selective growth advantage for cells (see Wigley and Cowell, 1984). Although DMINs and HSRs have been described predominantly in tumor cells selected for resistance to cytotoxic drugs, it is also clear that DMINs and HSRs may be present in cancer cells before the start of therapy (Barker, 1982). It was in this setting that we and others first chose to explore the possible amplification of cellular oncogenes. II. DMlNs and HSRs Contain Amplified Oncogenes

Table I1 summarizes some of the somatic amplifications of cellular oncogenes so far reported in tumor cells. The finding of known cellular oncogenes among amplified DNA represented by DMINs and HSRs of cancer cells is provocative. Amplification has been found to affect at least 6 out of 20 or so known cellular oncogenes, and the degree of gene amplification varies up to many hundredfold over the single haploid copies found in normal cells. Although small heritable variations in the copy numbers of some oncogenes may exist between animal species and strains, they are apparently rare. One report has described a relatively recent, 10-fold amplification of the c-Hams oncogene in the germ cell line of the Mus pahari mice and approximately a six-fold amplification of the c-Ki-ras oncogene in Chinese hamsters as compared with Syrian hamsters (Chattopadhyay et al., 1982). However, these amplifications, in contrast to somatic oncogene amplifications, are not expressed at the protein level. The first report of a somatic amplification of a cellular oncogene involved the c-myc oncogene (see Table 11)in a promyelocytic leukemia cell line HL-60 (Collins and Groudine, 1982; Dalla Favera et al., 1982). The degree of c-myc amplification was between 8- and 32-fold both in the HL-60 cell line and in primary leukemic cells from the patient. Original clonal lines of HL-60 were later found to contain some DMINs in culture but their number was insufficient to establish any clear correlation with amplified c-myc. Such a correlation, however, was discovered for c-myc amplification in a neuroendocrine cell line from a colon carcinoma, COLO 320 (Alitalo et al., 1983~). In these cells, the approximately 30-fold amplified c-myc copies were mapped

TABLE I1 S w m r c AMPLIFICATIONS OF CELLULAR ONCOGENES"

Tumor cells

H M (acute promyelocytic leukemia, M3) COLO 320 (colon carcinoma)

Y1 (adrenocortical tumor) COLO 20U!205 (colon carcinoma) K562 [chronic myelogenous leukemia (CML)]

Oncogene

Fold

Chromosomal location of amplifiedgene

c-myc

20x

8q(ABR)

c-myc

30x

DMIN, HSR

c-Ki-ras c-myb

5OX

lox

DMIN, HSR marl, mar2

c-abl

lox

mar(ABR)

A431 (epidermoid carcinoma)

c-erbB

15-2ox

n.d.

ML1-3 (acute myeloid leukemia, M2)

c-myb

&lox

n.d.

SK BR-3 (breast carcinoma)

c-myc N-ras

10x 5-lox

n.d. n.d.

MCF-7 (breast carcinoma)

Remarks and references Amplification present in primary leukemic cells Part of the amplified c-myc sequences rearranged Levels of ~ 2 1 " " -protein ~ elevated Patient treated with Sfluorouracil prior to culturing of the tumor cells CA coamplified in the marker that may be derived from chromosome 22, c-abl protein-associated tyrosine kinase activated Amplificationlinked to and sequence rearrangements. Amount of protein product, the EGF receptor, elevated Abnormalities of chromosome 6q22-24, where c-myb is normally located (Pelicci et al., 1984) Kozborand Croce (1984) Cells contain activated oncogenes mcf-2 and

mf4

SEWA (polyoma virus-induced mouse tumor) SC-2, NS-3, Shiraishi (gastric adenocarcinomas) Lu-65 (giant-cell lung carcinoma) Primary leukemic cells &om an acute myeloid leukemia (M2) patient

~

c-myc

30 x

n.d.

c-myc

15-3Ox

n.d.

8x 10 x 33 x

n.d. n.d. n.d.

At least some copies of cKi-ras mutated

Increased level of amplification during growth of the cells in nude mice (Modjtahedi et al., 1985) King et al. (1985)

c-myc c-Ki-ras c-myc

Cells have DMINs depending on culture conditions; c-myc amplification correlates with growth as a tumor Shibuya et al. (1985)

SW 613-S

mYC

5-9Ox

DMN, chromosomes

MAC 117 (mammary carcinoma) UY (salivary adenocarcinoma) Plasma cell leukemias (2J3) Small-cell lymphocytic lymphoma (1) A-MuLV-transformed fibroblasts (3/3) Glioblastoma

neu (c-erbB2) neu c-myc c-ets-1

%lox

n.d.

30X 8-32 X 10x

n.d. n.d.

c-myc

8-20 x

n.d.

Nepveu et al. (1985)

c-myc

25 x

n.d.

Trent et al. (1986)

1lq23

Semba et al. (1985) S ~ m e get i al. (1985) Rovigatti et al. (1986)

a n.d., Not determined; mar, marker chromosome; M2 and M 3 refer to the French-American-British classification of acute myeloid leukemias. References appear in the text unless indicated.

244

KARI ALITALO AND MANFRED SCHWAB

FIG.4. Localization of amplified c-myb in COLO 201/205 cells by in situ hybridization (Alitalo et al., 1984b). Shown is a characteristic, large marker chromosome (marl) with C-banding (CTC) and c-myb autoradiographicgrains (In situ). Note the absence of HSRs. marl has probably evolved from chromosome number 6, the resident site of the c-myb oncogene in normal cells (Harper et al., 1983; Winqvist et al., 1984; Zabel et al., 1984).

either to HSRs of a marker chromosome (Alitalo et al., 1983c; Fig. 3) or to DMINs (Alitalo et al., 1983c; Lin et al., 1985), depending on the particular subline studied. Since DMINs were present already in the primary tumor cells from this colon carcinoma (Quinn et al., 1979), it is very likely that also c-myc had been amplified during in vivo growth of the tumor. Similarly, amplified copies of the c-Ki-ras oncogene were mapped to DMINs and HSRs of a mouse adrenocortical tumor Y 1 (Schwab et al., 1983a). An extensive search for changes in other oncogenes and tumor cells has since revealed amplifications that do not show up in cytogenetic analysis. Thus, for example, the c-myb oncogene is amplified in a characteristic marker chromosome of a colon carcinoma without evidence of HSRs or DMINs (see Alitalo et al., 1984b; Fig. 4)and in other tumors, the amplified c-abl and c-myc oncogene loci map to abnormally banding regions (ABRs) in translocated or resident chromosomal segments (Nowell et al., 1983; Selden et al., 1983). The finding of moderately amplified oncogenes also in chromosomal sites lacking HSRs suggests that (onco)gene amplification may be more common than the structural alterations revealed by chromosome banding and microscopy (Alitalo et al., 1984a,b; Winqvist et al., 1985). The minimum size for persistence of DMINs in the nucleus may be of the order of lo3 kilobase pairs (Hamkalo et aZ., 1985). Apparently then, the limits of resolution of chromatin DNA with the light microscope, 1-5 x lo6 base pairs (Yunis, 1981) may be insufficient to reveal the smallest of both the chromosomal and extrachromosomal DNA amplifications.

ONCOGENE AMPLIFICATION IN TUMOR CELLS

245

111. Translocations and Rearrangements May Accompany Oncogene Amplification

The evolution and progression of the karyotype of tumor cells are complex processes (see Rowley and Ultmann, 1983). Concomitant with amplification, DNA sequences acquire an increased mobility in the genome with extrachromosomal intermediates visualized as DMINs, transpositions, and translocations to other chromosomal segments, etc. (see Biedler et al., 1983; Nowell, 1976).There may not be preferred chromosomal sites for the apparent reintegration of DMINs as HSRs (Schwab et al., 1983b). In at least one case, however, an oncogene may have been caught amplifying in situ in its resident chromosomal site (Nowell et al., 1983). In at least five cases reported amplification has been accompanied with a DNA rearrangement of the oncogene (Alitalo et al., 1983c; Collins and Groudine, 1982; Liebermann et al., 1985; Nau et al., 1984; Ullrich et al., 1984). In the colon carcinoma COLO 320, both damaged and normal versions of the c-myc gene are amplified (Alitalo et al., 1983~). Although individual cell clones have not yet been examined, our unpublished experiments suggest that the same DMIN-containing cells harbor and express both normal and rearranged forms of cmyc. However, the normal version of the amplified gene predominates in COLO 320 cells containing HSRs; the rearranged version is present only in what appears to be a single copy (Fig. 5A). In the chronic myeloid leukemia (CML:erythroleukemia) cell line K562, an amplified, translocated DNA segment consists of portions of both the c-abl oncogene and the immunoglobulin C A locus (Selden et al., 1983). In both cases abnormal transcripts are produced from the rearranged amplified oncogenes (Fig 5B; Collins et al., 1984). In K562 cells, the abnormal c-ab2 oncogene product has also been activated as a tyrosine kinase (Konopka et al., 1984). It seems likely that a chromosomal translocation of c-abl to the Philadelphia chromosome occurred before DNA amplification in the K562 cells, since all amplified DNA copies are also rearranged (Collins and Groudine, 1983). It could also be speculated that the fusion of the c-abl and bcr genes in K562 cells upon Philadelphia translocation [t(9;22)] (Klein et al., 1983) activated the c-abl protein tyrosine kinase typical of CML cells (Groffen et al., 1984; Konopka et al., 1985), but that subsequent growth as a tumor required increased dosage of the oncogene. Amplification of the bcr-cabl DNA has been reported to occur during a blast crisis of a CML patient (Bartram et al., 1986). However, it is not known whether structural alterations of the genes preceded amplification or whether they

246

KARI ALITALO AND MANFRED SCHWAB

FIG. 5. (A) Amplification and rearrangement of c-myc in COLO 320 cells. Cellular DNA (10pg) was digested with SstI, electrophoresed, blotted, and probed with a v-myc PstI fragment (see Alitalo et al., 1983c, left panel). Fragments of 2.7 and 1.4 kbp are seen in both normal and amplified c-myc DNA. The 3.3-kbp fragment is derived from a DNA segment of unknown origin translocated to the 5' region of c-myc with a concomitant deletion of its first exon. HSF, Human skin fibroblasts; DM, COLO 320 DM cells; HSR, COLO 320 HSR cells. Different amounts of DNA from COLO 320 DM cells as indicated were mixed with calf thymus DNA to give 24 pg of total DNA, cleaved with SstI, electrophoresed, blotted, and probed with a fragment of 3' human c-myc sequences. The intensities of the 2.7-kbp c-myc fragment in different samples were compared to assess its copy number, estimated to be about 30 (Alitalo et al., 1983c, 1984a). (B) Comparison of the electrophoretic mobilities of c-myc mRNAs from COLO 320 DM and HSR cells. The size of the normal c-myc mRNA is 2.3 kb. The c-myc locus in DM cells seems to be predominantly expressed, giving rise to a shortened RNA (A).

ONCOGENE AMPLIFICATION IN TUMOR CELLS

247

were acquired during the process of gene amplification. Although they have not been sequenced, most reported cases of amplified oncogenes are apparently normal on the basis of mapping with restriction endonucleases (see Tables I1 and 111).Therefore we cannot presently view mutation as a necessary companion of oncogene amplification. IV. The Mechanisms of Gene Amplification

The mechanism of gene amplification and the structure of the amplified DNA have been worked out mainly in experimental settings involving selection for drug resistance in cell culture. Although the mechanisms are still incompletely known and may vary in different cases, some general features have emerged. There seems to exist a spontaneous degree of illegitimate DNA replication in normal cells, so that various segments of DNA are replicated more than once during a single cell cycle (Johnston et al., 1983). In unselective conditions this DNA is probably lost, e.g., through formation of micronuclei, because the newly synthesized extra copies of DNA are not covalently linked to chromosomal DNA of mitotic cells (Roberts et at., 1983; Schimke et al., 1981). However, if there is a selective pressure to retain an increased gene dosage, a progressive multiplication of gene copy number is obtained. Thus, the generation of DMINs in some human tumors and during in uivo growth of some experimentally induced tumor cells has been thought to reflect changes in the copy number of genes involved in malignancy (Levan et at., 1981; Pall, 1981). The incidence of cells bearing amplified genes under conditions of cytotoxic selection can vary by two orders of magnitude and is greatly increased by the presence of mitogenic substances (hormones or tumor promoters) during selection (Barsoum and Varshavsky, 1983; Varshavsky, 198la,b) or certain carcinogenic or cytotoxic agents before selection (Brown et at., 1983; Mariani and Schimke, 1984; Stark and Wahl, 1984; Tlsty et al., 1984; Varshavsky, 1981b). An interesting hypothesis suggested by Varshavsky (1981a,b) supposes that origins of DNA replication “fire” (initiate replication) illegitimately several times during a single cell cycle, and that this kind of “replicon misfiring” may be increased by substances such as tumor promoters and mitogenic hormones. Mitogenic hormones probably increase disproportionate DNA replication but also enhance the colony forming efficiency of drug-resistant cells in selective conditions (Barsoum and Varshavsky, 1983). Mariani and Schimke (1984) pointed out that most of the cytotoxic agents which increase the incidence of gene amplifi-

248

KARI ALITALO AND MANFRED SCHWAB

cation are inhibitors of DNA synthesis. Aberrant replication is known to take place after transient inhibition of DNA synthesis and this response can lead to gene amplification (Laughlin and Taylor, 1979; Lavi, 1981; Mariani and Schimke, 1984; Woodcock and Cooper, 1981). Tumor promoters could in principle have major effects on the expression and amplification of oncogenes or even possible recessive cancer genes that have suffered carcinogenic insults. On the other hand, many carcinogens seem to induce specific DNA amplification in experimental conditions (Lavi, 1981; Heilbronn et al., 1985). For example, the herpes simplex virus (HSV) is an effective inducer of selective DNA amplification of SV40 sequences in Chinese hamster embryo cells (Schlehofer et al., 1984). There is also a preliminary report of oncogenes amplified in advanced stages of cervical cancer to which HSV and human papillomaviruses have been linked (Riou et al., 1984). According to the studies of Axel and collaborators (Roberts et al., 1983),the multiple cycles of unscheduled DNA replication at a single locus during a single cell cycle result in a structure schematically outlined in Fig. 6. The hydrogen-bonded amplified copies of DNA depicted in Fig. 6 must resolve into a tandem linear array prior to the next mitosis. It is suggested that this occurs by homologous recombination between any of several repeated sequences within the amplified domain (Roberts et al., 1983).Part of the recombinations would lead to extrachromosomal circles possessing an origin for replication (Bullock and Botchan, 1982; Pellegrini et al., 1984); these could be the precursors of DMINs. Due to the unequal recombinations, the resolved linear structures would consist of tandemly repeated, but heterogeneous units. According to the model of Axel, a gradient of amplification is formed so that centrally located sequences are amplified more than sequences distal to the origin of replication (Roberts et al., 1983). This in fact has been found to be the case also for the large, complex DNA domain containing the N-myc oncogene amplified in 1983a; neuroblastoma cells in vivo (Kanda et al., 1983; Schwab et d., Shiloh et al., 1985; see also below). The chromosomal site of integration of transfected genes significantly affects the frequency and cytogenetic result of their experimentally induced amplification (Wahl et aE., 1984). The amplification frequency in some tranfectants has been found to be 100-fold that of the others (Wahl et al., 1984). This suggests that there are preferred chromosomal positions for amplification of cellular genes and that chromosomal rearrangements, such as translocations, may facilitate gene amplification by positioning chromosomal sequences in a favorable array.

ONCOGENE AMPLIFICATION IN TUMOR CELLS

249

FIG.6. Model of DNA amplification in tumor cells. In the top part of the figure, chromosomal DNA is replicated bidirectionally from a fixed site of origin during the S phase of the cell cycle. With a certain probability, unscheduled replications of the already replicated DNA may occur during a single cell cycle. This leads to a structure shown as superimposed replication bubbles. The multiple copies of DNA must resolve into a linear array before the next mitosis. This can occur by homologous or illegitimate recombinations between different amplified segments. The process can lead to a linear, tandem, but heterogenous multicopy unit, shown on the left, which may in some cases evolve to an HSR. If suitable recombinations occur, extrachromosomal circular DNA elements containing origins for DNA replication are formed; these may be the precursors of DMINs. The m denotes a hypothetical (onco)gene within the amplified domain. Note that in tumors such as the colon carcinoma COLO 320, DMINs and HSRs can apparently interchange. The model is drawn according to Roberts et al. (1983).

V. Carcinogen-Induced Gene Amplification and Clonal Selection of Cancer Cells

Although cell sorting experiments have demonstrated a basal spontaneous rate of gene amplification (up to to eventdcell generation) in eukaryotic cells (Johnston et al., 1983), this can be in-

250

KARI ALITALO AND MANFRED SCHWAB

creased severalfold by metabolic inhibitors or cytotoxic agents (Brown et al., 1983; Johnston et al., 1983; Schimke, 1982; Varshavsky, 1981a,b).In many respects the latter response is reminiscent of the socalled SOS response elicited in bacteria by noxious stimuli (see Echols, 1981; Rossman and Klein, 1985; Sarassin, 1985). Teleologically thinking, the rapid induction of gene amplification which apparently occurs frequently through extrachromosomal intermediates may provide tumor cells with genetic material for subsequent selective pressures operating in harmful conditions (Pall, 1981). In cancer cells, the mechanism may enhance the emergence of clonal populations of cells with increasingly malignant properties (Nowell, 1976). Such genetic instability of cancer cells is clearly enhanced, leading to the rapid evolution of increasingly malignant tumor cell populations (Cifone and Fidler, 1981; Nowell, 1976). A serious question of practical importance is whether drug resistance in treated patients also selects cells that have an enhanced ability to amplify (onco)genes important for growth and progression of the tumor (Barsoum and Varshavsky, 1983; Varshavsky, 1981a,b).An important component of the malignant progression could be a relaxation or loss of the control of numbertiming of initiations of DNA replication (Shimke et al., 1986). The question whether the basal spontaneous rate of DNA amplification in untreated cancer cells is increased has not been generally answered (Sager et al., 1985). Amplified DNA in DMINs must contain an origin for DNA replication (Pellegrini et al., 1984) and must be selected for in daughter cell populations, where it is unevenly segregated (Schimke et al., 1981). Cells carrying amplified DNA either as an HSR or as DMINs often grow more slowly unless the dosage of the amplified genes provides a growth advantage. In the absence of such selective pressures DMINs are lost (Schimke et al., 1981). The persistence of DMINs in some tumors therefore suggests that there are selective pressures for their retention (Barker, 1982; Barker et al., 1980; Levan et al., 1981). The loss of DMINs occurs reversibly in a polyomdvirus-induced mouse tumor when explanted into culture conditions (Levan et al., 1981). Interestingly, c-myc has been found ampIified in this tumor (Schwab et al., 1985). In another study, subclones of a mouse cell line containing HSRs were found to be more tumorigenic than subclones containing DMINs, and when reisolated from the tumors, the latter contained more DMINs than the cells used to initiate them (Wigley and Cowell, 1984). In at least two studies the length of an HSR has been found to increase during a selection of malignant cells for enhanced tumorigenicity (Gilbert et al., 1983; Shtromas et al., 1985).

ONCOGENE AMPLIFICATION IN TUMOR CELLS

25 1

The amplified c-erbB gene in A431 cells codes for epidermal growth factor (EGF) receptor (Downward et al., 1984).The abundant amounts of receptor protein on A431 cell surface may, however, provide the cells with an abnormal growth response, because unlike normal cells, these cells tend to die upon EGF treatment (Gill and Lazar, 1981). However, the A431 cells are an exception; a naive supposition is that the amplified sequences in DMINs and possibly in HSRs of tumors contain growth-promoting genes (see Section VI,C and Heldin and Westermark, 1984). This hypothesis fits seemingly well with recent findings concerning amplified oncogenes, though in many cases the search for an amplified oncogene is still continuing. Conceivably, overproduction of some proteins might be also deleterious, thus counterselecting against amplifications that include the corresponding genes. We are also inherently biased toward searching for amplifications of oncogenes already known, and even positive findings do not mandate a role for amplified cellular oncogenes, because the domain of amplified DNA is inevitably much larger than a single genetic locus-sometimes even hundreds to thousands of kilobase pairs (e.g., Kanda et al., 1983). VI. Tumor Specificity of Oncogene Amplification

A. N-myc

IN

NEWROBLASTOMAS

Both DMINs and HSRs are found with a remarkable frequency in human neuroblastomas (Biedler et al., 1983). The finding of sporadic amplifications of c-myc in colon carcinoma cells containing DMINs and HSRs therefore prompted an intense search for amplified oncogenes in neuroblastoma cells. In these experiments an amplified DNA fragment was found that hybridized with a labeled fragment from the second exon of the c-myc oncogene (Schwab et al., 198313).The cloning of the cross-hybridizing neuroblastoma DNA has revealed a related gene, called N-myc, that is very similar in its structural organization to that of c-myc (Fig. 7). In addition, amino acid sequences of c-myc and N-myc proteins show disperse segments of homology (overall about 32%) and hydropathy blots indicate that the physical properties implied by their primary sequences are very similar (Fig. 7; Battey et al., 1983; Michitsch et al., 1984; Kohl et al., 1986; Stanton et al., 1986). Further evidence for the related functions of c-myc and N-myc comes from cotransfection studies with activated c-ras oncogenes, where N-myc shows transformation complementing activity similar to c-myc, when linked to strong transcriptional promoters

252

KARI ALITALO AND MANFRED SCHWAB

A c-rns 5

'

3'

F

1

0 1-1

I-

EXON 2

1-

EXON3

kbp

50 aa

-I

FIG.7.Structural features of c-myc and N-myc genes. (A) Exon structures and coding portions of c-myc and N-myc drawn to the same scale. Hatched box indicates noncoding portions of exons; black boxes show the coding portions. The N-myc promoters are only tentatively assigned at present. The c-myc gene possesses two promoters, indicated as P1 and P2.It cannot be excluded, however, that c-myc includes an alternative, small exon buried in the first intron (S.Hann, personal communication). The figure is drawn according to Battey et al. (1983)and Stanton et al. (1986).(B)Comparison of the proteins encoded by N-myc and c-myc. The predicted amino acid sequence of human N-myc (Kohl et al., 1986;Stanton et al., 1986)is schematically presented to indicate positions that are similar to human c-myc protein (Battey et al., 1983;Watt et al., 1983;Colby et al., 1983).The two sequences were optimally aligned and regions of at least two amino acids with more than 70% identity are illustrated as black areas.

(Schwab et al., 1985; Yancopoulos et al., 1985). The homology with c-myc, amplification of N-myc, loss of N-myc expression upon neuroblastoma differentiation (Jacobovitz et al., 1986; Thiele et al., 1985) and biological activity of the N-myc clone classify N-myc as a cellular oncogene. Even further c-myc-related sequences are present in the human genome (Dalla Favera et al., 1982) and may be amplified as well, as shown by Nau et aZ. (1984)for the as yet incompletely characterized L-myc DNA in some small-cell lung cancers. The N-myc gene is consistently amplified in most neuroblastoma cell lines and in neuroblastoma tumors (Table 111; Schwab et al., 1983b; Brodeur et al., 1984). In addition, at least one retinoblastoma cell line and some small-cell lung cancers contain multiple copies of N-myc (Lee et al., 1984; Nau et al., 1984). Retinoblastomas and Wilms' tumors, which do not show N-myc amplification, often still express abundant amounts of the N-myc RNA (Lee et al., 1984; F. Alt, personal communication). Levels of N-myc expression are compara-

TABLE I11 TUMOR-SPECIFIC ONCOGENE AMPLIFICATIONS Tumor Small-cell lung carcinomas

Neuroblastomas Glioblastomas and some carcinomas

a

References appear in the text.

Oncogene

Fold

Chromosomal location

c-myc N-myc Gmyc N-myc

Up to 80x

n.d.

u p to 250x

DMIN, HSR

c-erbB c-erbB2 be4

u p to l O O X

n.d.

Remarks' Most c-mycamplifications in the variant phenotype of SCLC N-myc amplified in 50% of primary tumors of grade 111-IV About 30% of glioblastoma multiforme tumors show cerbB amplification; rearrangements of the gene also may be common

254

KARI ALITALO AND MANFAED SCHWAB

ble in human fetal retinas of 6- to 12-week gestational age and in retinoblastomas (A. Goddard, personal communication). The unamplified diploid copies of N-myc in normal cells are located at chromosomal bands 2p23-p24, but the amplified DNA containing N-myc may be translocated to any of the several other chromosomal sites bearing HSRs in neuroblastoma cells (Schwab et al., 1984~). Another chromosomal anomaly seen in about 70% of neuroblastoma cells and tumors is deletion of portions of the short arm of chromosome 1 (lp-) (Brodeur et al., 1981). The implications of the deletion are not known, but there may be a hereditary background in some of the neuroblastoma cases. The L-myc gene maps to lp32 (Nau et al., 1985) and one of its two alleles may thus be deleted in some neuroblastomas. Also, it is interesting to note that chromosomal deletions have been implicated in the genesis of retinoblastomas (Benedict et al., 1983; Cavenee et al., 1983), Wilms’ tumors, and small-cell lung cancer (Whang-Peng et al., 1982a,b), which also shows myc-oncogene amplifications. The short arm of chromosome 2 contains three genes related to cell growth or transformation, namely the genes for N-myc, TGF-a (located in 2pll-pl3, Brissenden et al., 1985), and ornithine decarboxylase [(ODC) Winqvist et al., 19861. Sublocalization of the ODC gene on chromosome 2 mapped it to 2p23-pter7which was relatively close to N-myc (Winqvist et al., 1986). Because of the frequent involvement of segments of the short arm of chromosome 2 in DNA amplification in neuroblastomas (Schwab et al., 1984c; Shiloh et al., 1985) and smallcell lung cancers (J. Minna and K. Saksela, personal communications), we estimated the copy number of ODC and TGF-a sequences in the well-characterized neuroblastoma cell line IMR-32 (Kohl et al., 1983), and in fresh tumor samples from neuroblastomas and lung carcinomas, which all contain amplified N-myc. Only “single-copy” hybridization signals for ODC and TGF-a genes were obtained. Also, N-myc is not included in the amplicon containing the ODC gene in tumor cells grown in the presence of 2-difluoromethylornithine (DFMO), an inhibitor of ODC (Alhonen-Hongisto et al., 1986). The high frequency of N-myc amplifications suggests that neuroblastoma tumor cells with preexisting amplifications may adapt particularly well to growth in vitro. Although the great majority of neuroblastoma cell lines have amplified N-myc, at least one, the SH-SYSY, does not (Schwab et al., 1983a). Transfection of genomic DNA from SH-SY5Y cells to NIH/3T3 cells has indentified a mutationally activated dominant oncogene, N-ras, in this tumor cell line (Shimizu et al., 1983; Taparowsky et al., 1983). However, the activation of the

ONCOGENE AMPLIFICATION IN TUMOR CELLS

255

N-ras gene is not specific to neuroblastomas but occurs in other tumors as well, notably in leukemias (Mariano Barbacid, personal communication). N-ras is found amplified in MCF-7 breast carcinoma cells (Fasano et al., 1984). An active N-ras oncogene has also been found to coexist with amplified c-myc in the HL-60 promyelocytic leukemia cells and with a translocated c-myc in an American Burkitt’s lymphoma cell line (Murray et aZ., 1983). In both instances, an abundantly expressed c-myc gene was found together with a mutationally activated oncogene in the same cells, conditions sufficient to malignantly transform primary rat embryo fibroblasts in transfection experiments (Land et al., 1983b). However, mutated N-ras and amplified Nmyc have not yet been found in the same tumor cells, nor have neuroblastomas been described where the N-rus gene, located in the proximal part of the short arm of chromosome 1 (Hall et al., 1983; de Martinville et al., 1983), would have been affected by the frequent deletion of the distal part of the short arm of this chromosome. The extent of N-myc amplification in neuroblastomas may be bimodal from 3- to 10- or 100- to 300-fold in different tumors (Brodeur et al., 1984) and is strongly correlated with advanced stages (111-IV) of neuroblastoma tumors; about 50% of these show evidence of amplified Nmyc. In contrast, no amplifications were found in stage I and I1 tumors (Brodeur et al., 1984). The prognosis for patients at stages I11 and IV is usually very poor; 2-year survival is 10-30%, compared with 75-90% for stages I and I1 (Reynolds and Smith, 1982). It should be noted, however, that clinical (anatomical) staging of neuroblastomas does not follow biological properties of the tumor cells. Stage I indicates a tumor that is confined to the organ or structure of origin. Stage I1 tumors extend in continuity beyond the organ or structure of origin but do not cross the midline; regional ipsilateral lymph nodes may be involved. Stage I11 tumors extend beyond the midline and may involve regional lymph nodes bilaterally, while stage IV tumors have distant metastases involving distant lymph nodes, or hematogeneous dissemination to organs, tissues, bone, or bone marrow. If one assumes that stages represent a form of malignant progression in a homogeneously behaving group of tumors, it would follow that amplification of N-myc is associated with tumor progression. In fact Reynolds and Smith (1982) can identify two “biologic classes” of neuroblastoma that may well correspond to tumors with and without N-myc amplification. But it cannot be excluded that there initially are two or more types of tumors, of which the ones with amplified N-myc grow more rapidly and therefore often present with an advanced stage upon clinical diagnosis. Results of in situ hybridization suggest that even within

256

KARI ALITALO AND MANFRED SCHWAB

the same tumor individual cells vary in their degree of expression and therefore presumably also amplification of N-myc with high-level expression occurring predominantly in undifferentiated neuroblasts (Schwab et al., 1984a).

1. Structure of Ampl$ed DNA in Neuroblastoma Cells Although it is well established that the amplification of mammalian genes often involves a much larger DNA segment than is occupied by the target gene for selection (Schimke, 1984; Stark and Wahl, 1984; Hamlin et al., 1984; Montgomery et al., 1983), the nature of the coamplified sequences is poorly understood. Some of the few defined examples of co-amplification are the ribosomal genes in cells exposed to N-(phosphonacety1)-L-aspartatewith the genes of the trifunctional enzyme complex CAT (consisting of the enzymes carbamoyl phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase) required for pyrimidine synthesis (Stark and Wahl, 1984). More recent examples are the expression of several amplified genes in a multidrugresistant Chinese hamster ovary cell line (Van der Bliek et al., 1986) and in Chinese hamster fibroblasts overproducing adenylate deaminase (Debatisse et al., 1984). The possibility that a sequence other than N-myc is the functional and structural core of the amplicons in neuroblastomas and small-cell lung cancers cannot be ruled out. Only circumstantial evidence points to a role of N-myc in the progression of neuroblastoma, retinoblastoma, and smaII-cell lung cancer. N-myc belongs to DNA sequences that are most amplified in IMR-32 DNA (Shiloh et al., 1985) and increased expression of N-myc accompanies its amplification. In neuroblastomas, N-myc expression is most abundant in undifferentiated neuroblasts (Schwab et al., 1984~). The HSR on the abnormal chromosome 1 of IMR-32 cells consists of particularly large repeat units of approximately 3000 kilobase pairs, containing sequence information from three separate domains of the short arm of chromosome 2 (Shiloh et al., 1985).The distance between any two of these domains is several thousands of kilobase pairs. The data suggest the existence of a novel splicing and amplification event, which relocates distant sequences, which are then amplified, and, at times, rearranged in neuroblastomas (Shiloh et al., 1985). 2. N-myc Copy Number and Prognosis of Neuroblastoma Patients Recently, Seeger et al. (1985) studied 89 patients with untreated prima.ry neuroblastomas to determine the number of copies of the N myc oncogene and survival without disease progression, Genomic

ONCOGENE AMPLIFICATION I N TUMOR CELLS

257

amplification (up to 300 copies) of N-myc was detected in 2 of 16 tumors in stage 11, 13 of 20 in stage 111, and 19 of 40 in stage IV. In contrast, 8 stage I and 5 stage IV-S tumors all had one haploid copy of the gene. The analysis indicated that amplification of N-myc was associated with poor prognosis. The estimated progression-free survival at 18 months was 70%,30%, and 5%for patients whose neuroblastomas had 1,3-10, or more than 10 N-myc copies, respectively. The statistics also suggested that genomic amplification in advanced tumors is independent of the patient’s age at diagnosis. The aggressiveness of neuroblastomas is thus related to genomic amplification of N-myc. Stage IV tumors with amplification progressed most rapidly: 9 months after diagnosis the estimated progression-free survival rates were 61%, 47%,and 0%in patients whose tumors had 1, 3-10, or more than 10 copies of N-myc, respectively (Seeger et al., 1985). Comparison of N-myc copy number and other factors of importance for the prognosis of neuroblastoma patients has not yet been performed. Both histopathological characteristics of the primary tumor, the amount of tumor-cell DNA and neuron-specific enolase and ferritin in serum at diagnosis, differentiate good from poor prognoses among patients with different disease categories (Hann et d.,1985). However, the number of N-myc copies in primary untreated neuroblastomas is a new and clinically important prognostic indicator that is independent of tumor stage. An even better prognostic indicator might be the concentration of N-myc protein in the tumors, because even a single copy of N-myc might be activated by faulty regulation or mutation. For example, elevated expression of N-myc might be activated by faulty regulation or mutation. For example, elevated expression of N-myc without genomic amplification is a prevalent feature of retinoblastomas and Wilms’ tumors (Lee et al., 1984; F. Alt, personal communication). B. myc ONCOGENES IN SMALL-CELL LUNGCANCER Lung cancers are classified into four diagnostic categories on basis

of their light microscopic features. Non-small-cell lung cancers consist of adenocarcinomas, large-cell carcinomas, and epidermoid carcinomas. Small-cell lung cancer (SCLC) comprises about 25% of all lung cancers. This latter group can also be divided into pure SCLC and its morphological and biochemical variants (SCLC-V) (Minna et aZ., 1982; Radice et al., 1982).The variant forms are highly malignant and respond poorly to chemo- and radiotherapy (Gazdar et al., 1981: Radice et d.,1982). These features are also reflected in the phenotypes

258

KARI ALITALO AND MANFRED SCHWAB

of SCLC-V cells in uitro: they have a faster growth rate, less clustered morphology, higher cloning efficiency in soft agar, and a decreased expression of SCLC neuroendocrine markers such as L-dopa decarboxylase, neurosecretory granules, and peptide hormones related to bombesin (Carney et al., 1985; Gazdar et al., 1985; Little et al., 1983). SCLC-V cell lines frequently also show DMINs and HSRs (WhangPeng et al., 1982a,b). Furthermore, there is evidence of progression of the SCLC phenotype from “classical” to the variant form: the SCLC-V is seen histologically in approximately 6-15% of diagnostic biopsies taken before therapy, but at autopsy approximately 30-40% of patients diagnosed to have “classical” SCLC will have SCLC-V (Hirsch, 1983; Nau et al., 1984). These cytogenetic and clinical features prompted a study of cellular oncogenes in SCLC cells. The c-myc or related oncogene sequences were found to be amplified in 13 of 25 SCLC tumors and in 21 of 31 cell lines derived from SCLC (Little et al., 1983; Nau et al., 1984, 1986). The degree of c-myc amplification in SCLC cell lines varies from 20- to 80-fold and the amplifications are regularly associated with the SCLC-V phenotype. One lung adenocarcinoma cell line was found to contain about 20-fold amplified c-myc (Little et al., 1983)and one lung adenocarcinoma tumor about 30-fold amplified N-myc (Saksela et al., 1986). Different metastatic deposits of one patient had one or two DNA fragments hybridizing to N-myc, suggesting that the development of metastasis may be associated with an ongoing progression of amplification or rearrangement of myc-related sequences (Nau et al., 1984). J. D. Minna and co-workers have cloned amplified DNA fragments that show hybridization to c-myc and N-myc probes in SCLC cell lines. This strategy, which originally led to the discovery of the N-myc gene, has revealed a third putative oncogene of the myc family, named L-myc (Nau et al., 1985). A portion of the biochemical features of SCLC-V phenotype may be caused by elevated c-myc expression, since electroporation-transfection of additional c-myc copies into cells representing the classical SCLC phenotype causes some variant features in their phenotype (J. Minna, personal communication). In contrast to c-myc amplifications, most N-myc and L-myc amplifications were found in cell lines representing the classic form of SCLC (A. Gazdar, personal communication). These in vitro findings were also reflected in the survivals of the corresponding patients: the N-myc and L-myc amplifications do not correlate with a poor prognosis, whereas c-myc copy number does. In addition, c-myc has been found amplified in some large-cell carcinoma cell lines.

ONCOGENE AMPLIFICATION IN TUMOR CELLS

259

Several lines of evidence and recent experiments by Cuttitta et al., (1985) suggest that bombesin-like peptides (BLPs) can function as autocrine growth factors in human small-cell lung cancer. Bombesinlike peptides, which are homologs of the carboxyl-terminal half of human gastrin-releasing peptide are potent stimulators of DNA synthesis and cell division in several cell lines (Rozengurt and SinnettSmith, 1983). Human SCLC cell lines produce and secrete bombesinlike peptides and can express a single class of high-affinity receptors for BLPs (Cuttitta et al., 1985; Zachary and Rozengurt, 1985). However, many SCLCs with a variant phenotype secrete smaller amounts of BLPs than classic SCLC lines. An attractive hypothesis is that cmyc amplification in some variant cell lines could partly substitute for the competence- and progression-associated cell-cycle effects of autocrine BLPs. It has been claimed that small-cell lung cancers have a consistent deletion in the short arm of chromosome 3 (3p14-3pter; Whang-Peng et al., 1982a). The c-ra.1 oncogene has been mapped in 3p25, proximal to the common deletions (Bonner et al., 1984). Although this could result in a hemizygous state for the c-raf-1 gene and although craf is active in many SCLC tumors (Ulf Rapp, personal communication), no molecular lesions of c-raf have been reported in the tumor cells. Also, no comprehensive studies are available on the frequency of activating point mutations in the c-Ki-ras oncogene in SCLC, although such have been reported in other types of lung cancer (Nakano et al., 1984; Santos et al., 1984).

C. c-erbB ONCOGENES IN GLIOBLASTOMAS AND CARCINOMAS Epidermal growth factor is a prototype hormonal mitogen that induces a pleiotropic response in its target cells. The 6000 molecular weight EGF peptide is produced from a large, integral membrane protein precursor, from which mature EGF is cleaved off proteolytically (Gray et al., 1983; Scott et al., 1983). EGF is found in cerebrospinal fluid, pancreatic secretions, milk, and urine, but prepro-EGF mRNA is mainly found in the submaxillarly gland and kidney in the mouse (Rall et al., 1985).While the exact physiological functions of EGF remain unknown in uiuo, it seems to accelerate several developmental processes related to fetal and postpartal growth and maturation. In in uitro culture, various cells express EGF receptors and respond to EGF stimulation by an intricate sequence of events including tyrosine phosphorylation of the internal part of the transmem-

260

KARI ALITALO AND MANFRED SCHWAB

brane receptor protein and culminating in mitogenesis in permissive conditions (Carpenter and Cohen, 1984). Comparison of the amino acid sequence of purified human EGF receptor (EGF-R) with several deduced amino acid sequences in a data bank by Downward et al. (1984) showed that EGF-R is the homolog of the v-erbB oncogene of avian erythroblastosis virus. The virus causes erythroblastosis and sarcomas in chicks (Graf and Beug, 1983).This immediately suggested that molecular lesions of the EGFR gene c-erbB may be involved in human malignancy as well. Indeed, about a 15-to 20-fold amplification and sequence rearrangements of cerbB were found in A431 vulva1 carcinoma cells, which express exceptionally high amounts of EGF receptors on their surface and which have therefore been used as a model system to study the rapid effects of EGF stimulation (Ullrich et al., 1984; Lin et al., 1984; Xu et al., 1984a). Besides squamous cell carcinomas (Cowley et al., 1984), brain tumors such as glioblastoma multiforme (GM) and occasional meningiomas were found by Libennann et al. (1984) to contain elevated concentrations of EGF receptor kinase. The receptor gene was therefore examined in 21 brain tumors, of which four showed an amplification of 6- to 60-fold and in two tumors rearranged versions of the c-erbB gene were detected (Libermann et al., 1985). Because the v-erbB oncogene transforms cells through overexpression of a truncated EGF receptor (Graf and Beug, 1983; Downward et al., 1984), a similar role can also be envisioned for amplification and rearrangement of c-erbB gene in human gliomas. It is also of interest that c-erbB is highly expressed in many glioblastomas which do not have amplification of the gene (Josef Schlessinger, personal communication), and a related transforming gene (neu, c-erbB2) is activated in rat neuro/glioblastomas induced by ethylnitrosourea (Schechter et al., 1984). The latter gene is rearranged or amplified in many adenocarcinomas (Yokota et al., 1986).The c-erbB gene is also highly expressed and amplified in some squamous cell carcinomas (B. Ozanne, personal communication). However, Xu et al. (1984b) failed to find further c-erbB amplifications among nine unrelated nonglioblastoma tumor cell lines expressing abundant EGF-R. Glioblastoma multiforme is a highly anaplastic tumor of relatively undifferentiated neuroglial cells. Overall GM accounts for about 2530% of all intracranial tumors and for more than 50% of all primary gliomas. GM may also arise by the development of progressive anaplasia in a preexisting astrocytoma, which represents a more differentiated tumor composed of astrocyte-like cells. In most cases, some

ONCOGENE AMPLIFICATION IN TUMOR CELLS

26 1

areas of GM tumors reveal typical astrocyte-like cells, but in contrast to astrocytoma the prognosis with GM is very poor; about 10% of patients survive more than 2 years after diagnosis, with little help from radio- or chemotherapy. The human c-erbB oncogene has been assigned to chromosome 7 and is located between 7q22 and 7qter (Spurr et al., 1984). Several human GM tumors have increased copies of chromosome 7 (Shapiro et al., 1981), and occasional gliomas have contained DMINs (Mark and Granberg, 1970). The A431 cells, which have 15-to 20-fold amplified c-erbB contain two copies of intact chromosome 7 and two chromosome 7-derived markers (Shimizu et al., 1984). The expression of high EGF binding capacity in A431-mouse cell hybrids correlates with the presence of marker containing the region 7q22-qter (Shimizu et al., 1984).

VII. Enhanced Expression of Amplified Oncogenes

In all cases where they have been studied, the amplified oncogenes have been found abundantly expressed at the RNA level, roughly in proportion to the amount of DNA amplification (see Table 11). Described cases of elevated RNA expression include examples of abnormal and ectopic high-level transcription (Alitalo et al., 1983c, 1984b; Collins et al., 1984). In at least four cases this enhancement is not limited to synthesis of RNA (Hann and Eisenman, 1984; Konopka et al., 1984; Schwab et al., 1983a; Ullrich et al., 1984). High amounts of the c-myc-encoded protein are found in COLO 320 cells that have amplified the gene, but disproportionately less can be immunoprecipitated from HL-60 cells, which also have amplified c-myc, for reasons unknown to us (unpublished experiments). The Y 1 cells that have amplified c-Ki-ras also contain exeptionally large amounts of its protein product (Fig. 8) located on their plasma membranes (Schwab et al., 1983a). Most retinoblastomas and Wilms’ tumors sustain elevated expression of N-myc even in the absence of its amplification (Lee et al., 1984). Also, relatively small hereditary and nonhereditary retinoblastomas and embryonic retinae and kidneys were expressing Nmyc, suggesting that high-level expression of the gene reflects a tissue-specific transcription pattern (F. Alt and A. Goddard, personal communications; Lee et al., 1984). In contrast, the c-myc oncogene is not detectably expressed in many neuroblastomas having amplified N-myc (our unpublished observations). It would appear that N-myc in contrast to c-myc is a very tightly

KARI ALITALO AND MANFRED SCHWAB

F1~8..Elevatedlevelsofthe p21"n-m*protein in Y 1 cells. The Y 1 DM and HSR cells and control cells which harbor a 50-fold amplified c-Ki-ras oncogene (Schwab et al., 1983a) were labeled with [WSImethionine and the p21"G-m" protein was immunoprecipitated, with normal rat serum (NRS)or rat monoclonal anti-pel serum. The proteins were electrophoresed in a 15% polyacrylamide gel in the presence of SDS. In addition to a major p21 band, a labeled band at about 16 kDa was present in the immunoprecipitates. The amount of radioactivity in p21 was about 50-fold that in normal rat kidney cells.

ONCOGENE AMPLIFICATION IN TUMOR CELLS

263

controlled gene, since its expression has only been detected in neuroblastomas, retinoblastomas, and Wilms’ tumors. The amount of DNA template is not the only factor regulating levels of N-myc RNA in tumors. For example, the IMR-32 and Kelly neuroblastomas both have about 60- to 80-fold elevated levels of N-myc RNA as compared to the SK-N-SH neuroblastoma line which has single-copy N-myc DNA, yet amplification of N-myc DNA in IMR-32 cells is only 15- to 20-fold compared with 100- to 120-fold in Kelly cells (Schwab et al., 1984b). Retinoic acid, an analog of vitamin A which has cytosolic receptors in neuroblastoma cells, can be used to differentiate many human neuroblastoma cell lines morphologically and biochemically (Haussler et al., 1983).Decreased expression of amplified N-myc precedes retinoic acid-induced differentiation in SMS-KCNR neuroblastoma cells (Thiele et al., 1985).Thus, even an amplified oncogene can respond to differentiative stimuli. In fact, very similar results have been observed for the c-myc oncogene in differentiating HL-60 cells (see Section IX).N-myc is also active in some teratocarcinoma cell lines (Jacobowitz et al., 1986; James Siimegi, personal communication). This provides a model for the studies of N-myc activity during differentiation. It is interesting to note that oncogene expression can be selective among other transcriptional units in the amplified DNA. The K562 erythroleukemia cells which have amplified both the c-abl and C A sequences to a similar extent transcribe only the former gene in significant amounts (Collins et al., 1984). As already mentioned, ectopic activation of the c-myb oncogene may have preceded its amplification in COLO 201/205 cells. Transcriptional activity of normal c-myb in chromosome 6q22-24 (Harper et al., 1983) is known to be associated with immature, proliferating hematopoietic cells and to cease upon their differentiation (Westin et al., 1982). Both the transcriptional and apparently also transforming activities of c-myb are enhanced by retrovirus-associated DNA rearrangements in murine plasmacytoid lymphosarcomas (Mushinski et al., 1983) and possibly by structural alteration of chromosome 6 in the COLO 201/205 tumor cells (Winqvist et al., 1985).Subsequent clonal selection for cells producing more c-myb protein during tumor growth may have resulted in c-myb amplification in the latter. These positive findings are fortunate for the teleological model of oncogene amplification induced by selection pressures during tumor growth, because lack of expression of amplified oncogenes would exclude their involvement in the growth advantage some tumor cells enjoy. An interesting link between c-erbB and c-myc activity comes from the studies of Bravo et aZ. (1985): EGF and other protein kinase C

264

KARI ALITALO AND MANFRED SCHWAB

activators induce increased expression of c-fos and c-myc oncogenes in A431 cells, but at the same time, the growth of different A431 sublines may be stimulated, inhibited, or not affected at all. Regardless, one must consider the possibility that in GM cells the high-level expression of c-erbB could elevate the expression of c-myc or even crus functions (Kamata and Feramisco, 1984); the effect would then resemble that of c-myc and C-rusamplifications. There may also occur an autocrine growth stimulation of glioma cells and their c-myc expression by transforming growth factor a! or by platelet-derived growth factor, the protein product of the c-sis oncogene, which is active in many gliomas (Heldin and Westermark, 1984). Elevated expression of c-myc has been shown to partially replace platelet-derived growth factor in induction of competence for DNA replication (Arme1984). Thus, autocrine stimulation might no longer be essenlin et d., tial when c-myc is amplified. VIII. Role of c-myc Deregulation in Lymphoid Malignancies

In order to understand amplification of myc oncogenes it may be helpful to look at other types of myc activation (deregulation) in tumor cells. Recent work on a variety of animal models and human tumors suggests that besides its role in amplification, an activated c-myc oncogene is involved in the genesis of several types of B- and T-cellderived tumors. Rearrangement of the c-myc locus may be caused by integration of retroviral proviruses into host chromosomal DNA or by chromosomal translocations. The damaged versions of c-myc found in lymphoid tumors are often aberrantly expressed, either in excess or autonomously, in a fashion not controlled by regulatory mechanisms during lymphocyte differentiation. The pathogenetic spectrum of avian leukemia viruses carrying the v-myc oncogene is very broad (Beard, 1980). These viruses, commonly called the myelocytomatosis viruses, induce carcinomas, endotheliomas, and sarcomas in addition to the characteristic leukemic disorder called myelocytomatosis. In tissue culture, the myelocytomatosis viruses are able to transform fibroblasts and macrophages into a malignant phenotype (Bishop, 1983; Beug et al., 1982; Bister, 1984). In uiuo, the constitutive expression of v-myc in myelocytomatosis virus-infected cells may be sufficient to prevent cellular differentiation and thus enhance the self-renewal of infected cells, leading to tumor formation. Slow transforming retroviruses lack oncogenes and are able to induce tumors in vim after a long period of latency. The bursa of Fabri-

ONCOGENE AMPLIFICATION IN TUMOR CELLS

265

cius is the target site for tumors induced in chickens susceptible to avian lymphoid leukosis virus (LLV)-induced B-cell lymphomas. It is generally held that in the vast majority of LLV-induced lymphomas, oncogenesis is initiated from the occasional integration of an LLV provirus adjacent to the cellular oncogene c-myc (Hayward et al., 1981; Payne et al., 1982).Most insertions of LLV proviruses in bursal lymphomas reside in the first intron of c-myc and result in the transcriptional activation of the c-myc gene. In many tumors, the integrated provirus is damaged and frequently deletions have occurred which may have eliminated an LTR element. In these cases transcription promoted by the LTR may be directed into adjacent oncogene sequences, thus unleashing c-myc from its normal regulatory controls (Hayward et al., 1981; Payne et al., 1982; Cullen et al., 1984). But cases have also been described where the viral long terminal repeat element (LTR) can activate transcription of c-myc without involvement of the viral promoter. In these tumor cells, the viral LTR apparently functions as an enhancer (Linial and Groudine, 1985). Enhanced levels of c-myc RNA are indeed found in the tumors, though it is difficult to know the level of c-myc expression in the nonhal cell that gave rise to the tumor. Furthermore, activation of cmyc may not be sufficient alone for lymphoma development (Baba and Humphries, 1985; Neiman et al., 1985). It may be that the activation of c-myc only results in an intermediate stage in the development of bursal lymphoma, the so-called transformed follicles, that may also regress, perhaps in the absence of additional mutagenic events (Baba and Humphries, 1985). In fact, the clonal development of these LLVinduced B-cell tumors is believed to be a multistep process. Thus, mutations have been described in the coding sequences of activated c-myc (Westaway et al., 1984) and at least some c-myc loci activated by the chicken syncytial virus are also amplified in the tumors (NooriDaloii et al., 1981). It should also be mentioned that c-myc alone is insufficient to transform primary fibroblasts in culture, even when linked to a strong transcriptional promoter such as an LTR. Cotransfection with a complementing oncogene (such as c-ras ) appears necessary for myc to effect its oncogenic function in cultured fibroblastic cells (Land et al., 1983b). However, even cotransfection has not been successful in inducing transformation with the insertionally mutagenized c-myc alleles from bursal lymphomas (Varmus, 1984). An augmented expression of normal c-myc is sufficient for cotransformation of rat embryo cells with a mutant ras gene (Lee et al., 1985).A transformed phenotype can be induced in v-myc, rearranged c-myc, or C-MS oncogene

266

KARI ALITALO AND MANFRED SCHWAB

transfected rat embryo fibroblasts by treatment with a tumor promoter (TPA, Connan et al., 1985; Dotto et al., 1985) that also induces gene amplification (Varshavsky, 1981a,b). Murine T-cell lymphomas induced by chronic retroviral infection, X rays, or chemicals are frequently trisomic for chromosome 15, where the mouse c-myc gene resides (Klein, 1981, 1983). In a number of retrovirus-induced T-lymphomas, the MuLV-provirus has been found integrated adjacent to c-myc, in an orientation precluding the use of the LTR promoter for transcriptional activation of c-myc (Steffen, 1984; Corcoran et al., 1984; Li et al., 1984). In cats, the majority of spontaneous lymphomas are associated with feline leukemia virus (FeLV) infection. FeLV does not carry an oncogene, and the mechanism of the T-cell leukemogenesis it causes is still poorly known. However, Jarrett and others have detected c-myc-derived sequences in a number of FeLV proviruses found in tumor DNA (Neil et al., 1984; Levy et al., 1984; Mullins et al., 1984).In only a few cases has cmyc been affected by proviral insertion; in others proviruses seem to carry spliced versions of the feline c-myc in their genomes. Tumor-specific chromosomal translocations may be found in most hematopoietic neoplasms (Rowley, 1983). Breakpoints of consistent reciprocal translocations frequently affect the loci for immunoglobulin heavy, A, and K chains in B-cell tumors such as Burkitt’s lymphoma (BL), mouse plasmacytoma (PC), and rat immunocytoma. This observation led George Klein to propose in 1981 that the immunoglobulin gene region may provide a translocated cellular proto-oncogene with promoters active in B cells, thus activating it (Klein, 1981). Evidence has indeed accumulated that fits into this hypothesis. It has been shown by several laboratories that the human c-myc gene, normally found on chromosome 8, is involved in the translocations in BL and that analogous translocations occur in PC (for a review, see the excellent review by Cory et al., in this volume; Croce and Nowell, 1985; Klein, 1983). Studies of somatic cell hybrids between BL cells and either fibroblasts, PC cells, or lymphoblastoid cells by Croce and coworkers (reviewed in Croce and Nowell, 1985) have shown that the expression of translocated c-myc is deregulated so that it loses its ability to respond to normal regulatory signals during B-cell differentiation. While the expression of the normal c-myc gene on chromosome 8 is shut off in plasma cells, translocated c-myc is transcribed constitutively at high levels. On the other hand, cell hybrids between BL cells and lymphoblastoid cells, which represent a less mature stage of B-cell differentiation, express only c-myc from the normal chromosome 8 and not the translocated c-myc gene. Also, the expression of the translocated c-myc is greatly reduced in hybrids between

ONCOGENE AMPLIFICATION IN TUMOR CELLS

267

BL cells and fibroblasts, suggesting that high-level transcription of the translocated c-myc requires a defined stage of B-cell differentiation (Croce et al., 1985). Exactly how c-myc is deregulated in the translocations remains an enigma, although various hypotheses have been suggested including loss of feedback inhibition of c-myc expression (Siebenlist et al., 1984), altered transcription, altered stability of RNA from the rearranged c-myc (rc-myc) (Dani et al., 1984; Piechaczyk et al., 1985), increased translational efficiency of rc-myc mRNA (Saito et al., 1983), and mutations in the coding sequences of rc-myc (Rabbitts et al., 1984). It is obvious that the failure of rc-myc to be regulated by B-cell differentiation (Croce et al., 1985) is a fundamental change that may account for a large part of the tumorigenicity of BL cells, but a mechanistic explanation requires the definition of both the control elements of c-myc expression and the normal functions of c-myc protein. An interesting observation by Stephen Hann (personal communication; Hann and Eisenman, 1984) is that damage of the c-myc in bursa1 and Burkitt’s lymphomas correlates with alterations of the normal pattern of c-myc polypeptides. IX. Revealing the Normal Functions and Regulation of c-myc

Lesions of c-myc obviously became under scrutiny because of our desire to understand retrovirus-induced tumorigenesis. But so far we have learned very little about the normal functions of the myc genes to explain the contribution of c-myc in the loss of growth control in mechanistic terms. However, properties reIevant to the subject of cmyc amplifications have emerged. The c-myc RNA is expressed from two promoters which are differentially used in different tissues and in Burkitt’s lymphomas (Battey et al., 1983; Stewart et al., 1984b). No expression was found in mitotically and meiotically active germ cells by Stewart et al. (198413). Several DNase I-hypersensitive sites can be identified within 2 kbp of DNA 5‘ of the first c-myc exon (Siebenlist et al., 1984). Differential DNase I sensitivity of these sites in lymphoblastoid cells and in translocated and nontranslocated cmyc alleles in BL has led to a model of c-myc control by feedback inhibition of expression through nuclear protein-binding sites (Siebenlist et al., 1984; Dyson et al., 1985). This model would also explain the frequent suppression of expression of the normal c-myc allele in Burkitt’s tumor cell lines. However, results from analysis of cmyc expression in hybrid cell lines argue against the model (Croce and Nowell, 1985). The c-myc mRNA is extremely unstable, having a half-life of 10-20

268

KARI ALITALO AND MANFRED SCHWAB

min in different cells (Dani et al., 1984). Inhibition of protein synthesis results in a marked stablization of the c-myc messenger, suggesting that its degradation could be mediated by a protein of rapid turnover (Dani et al., 1984). Interestingly, a messenger RNA devoid of sequences from the first exon in COLO 320 DM colon carcinoma and PC cells has a considerably longer half-life than the normal-sized messenger in the same cells (Piechaczyk et al., 1985; Terry Rabbitts, personal communication). Deletion of the first exon also removes a sequence of internal complementarity with a sequence in the second exon; this may have an effect on mRNA stability or translational efficiency or both (Saito et al., 1983). However, results of immunoprecipitation of the c-myc protein from lymphoblastoid and BL cell lines argue against such possibility (Stephen Hann and Robert Eisenman, personal communication). The amino acid sequences encoded by the. myc genes contain a large number of uncharged polar amino acids (Alitalo et al., 1983a; Colby et al., 1983). Also, the amino acid sequences derived from the third exon contain several basic residues (Alitalo et al., 1983a; Colby et at?.,1983). These features may be relevant to the DNA-binding properties of the myc proteins (Abrams et at?., 1982, Donner et al., 1982). Antibodies to synthetic myc peptides (Hann et al., 1983; Evan et al., 1985; Persson et al., 1984; Hann and Eisenman, 1984; Ramsay et al., 1984) or myc proteins expressed in bacteria (Alitalo et al., 1983a; Persson et al., 1984; Watt et al., 1985) have been used to characterize the c-myc protein. In general, two major polypeptides are precipitated by such antisera from lysates of cells active in the synthesis of c-myc protein. The polypeptides have apparent molecular weights of 58,000 and 62,000 (Alitalo et al., 1983a; Hann et al., 1983) in chicken cells and 64,000 and 67,000 in human cells (Persson et al., 1984; Hann and Eisenman, 1984). Despite the difference in molecular weights the polypeptides show virtually identical peptide maps and both are phosphorylated (Hann and Eisenman, 1984). The half-life of the cmyc protein, like its mRNA, is very short, of the order of 20-30 min (Ramsay et al., 1984). Interestingly, however, the protein is greatly stabilized by a heat shock or UV-irradiation of the cells which produce it (unpublished data of Gerard Evan). The c-myc protein is located in the nuclei both by immunofluorescence and by subcellular fractionation (Alitalo et al., 1983a; Hann et al., 1983; Eisenman et al., 1985; Persson and Leder, 1984).A majority of the immunofluorescence for c-myc protein is extranucleolar and most of the protein is found in the nuclear matrix subfraction, as operationally determined by standard experimental manipulations (Eisenman et al., 1985). By varying the extraction protocols, however, a

ONCOGENE AMPLIFICATION IN TUMOR CELLS

269

majority of the c-myc protein can be recovered from the nuclei in a soluble form (Evan and Hancock, 1985). In mitotic cells, the myc proteins are distributed in the cytoplasm and granular nuclear fluorescence is again discerned upon reformation of the nuclear envelope in postmitotic cells (Winqvist et al., 1984). The c-myc RNA increases 10-to 20-fold after treatment of quiescent cells with various mitogenic stimuli such as platelet-derived growth factor (PDGF) (Kelly et al., 1983).The slow c-myc induction by transforming growth factor beta (TGF-P), apparently occurs through autocrine stimulation by PDGF (Leof et al., 1986).Concanavalin A treatment of T cells and lipopolysaccaride stimulation of B cells lead to an approximately 10- to 20-fold increase in c-myc mRNA about 2 hours after stimulation; the mRNA concentrations decline slowly thereafter (Kelly et al., 1983).Part of the regulation of c-myc RNA levels appears to occur at the level of mRNA degradation (Blanchard et al., 1985). Interestingly, induction of interferon production might function as a feedback inhibitor of c-myc response in growth factor-treated cells (Zullo et al., 1985).Microinjection of high amounts of c-myc protein into cultured cells induces DNA synthesis as a competence factor (Kaczmarek et al., 1985).However, expression of c-myc is not specific to the GI phase of the cell cycle. Rather, the levels of c-myc mRNA and protein remain invariant throughout the cell cycle in cultured cells (Thompson et al., 1985;Hann et al., 1985).Transformed cells in general fail to become quiescent and also have a more constitutive, elevated expression of c-myc RNA (Campisi et al., 1984). Even similar mitogenic or differentiative stimuli can lead to very different responses depending on the state of differentiation of treated cells. It has been shown that activation of protein kinase C by tumor promoters results in an increase in c-myc mRNA levels (Bravo et al., 1985;Coughlin et al., 1985).In contrast, induction of macrophage-like differentiation of HL-60 human promyelocytic leukemia cells by retinoic acid (Westin et al., 1982), 12-0-tetradecanoy1phorbo1-13-acetate (TPA)or dihydroxyvitamin DS(Reitsma et al., 1983)is associated with a loss of amplified c-myc mRNA expression within 2-8 hr of treatment, well before any morphological changes become apparent (Muller et al., 1984, 1985).A similar decrease of c-myc RNA accompanies the granulocytic differentiation of HL-60 cells induced by dimethyl sulfoxide (Westin et al., 1982; Grosso and Pitot, 1985; Filmus and Buick, 1985).As already mentioned, the translocated c-myc allele, in contrast to the normal one, is not shut off by signals of B-cell differentiation into plasma cells (Croce et aE., 1985).It thus appears possible that the failure to shut off myc functions in myelocytomatosis virusinfected cells, bursa1 lymphocytes, or Burkitt’s lymphoma cells may

270

KARI ALITALO AND MANFRED SCHWAB

lead to continuous proliferation of the affected cells in the presence of additional mitogens in an undifferentiated state, a change required for the development of the myelomonocytic leukemia or the characteristic lymphomas. X. Role of Oncogene Amplification in Multistage Carcinogenesis and Tumor Progression

It is obvious from data summarized in the preceding sections that amplification of certain oncogenes is a common correlate of the progression of some tumors and also occurs as a rare sporadic event affecting various oncogenes in different types of cancer. Amplified copies of oncogenes may or may not be associated with chromosomal abnormalities signifying DNA amplification: double minute chromosomes and homogeneously staining chromosomal regions. Amplified oncogenes, whether sporadic or tumor type specific, are also expressed at elevated levels, in some cases in cells where their diploid forms are normally silent. Increased dosage of an amplified oncogene may therefore contribute to the multistep progression of at least some cancers. In a few cases, specific genes are amplified in normal cells during developmental processes; there are examples of prokaryotes, yeast, Drosophila, and vertebrates (see reviews by Kefatos et al., 1985; Stark and Wahl, 1984).Amplification can be transient during growth or permanent in terminally differentiated cells. It cannot yet be excluded that amplification of, for example, N-myc occurs in rare stem cells of potential future neuroblastomas or transiently during some phases of differentiation of neural cells. However, if this were the case, the finding of amplified oncogenes in cancer cells could just reflect their stage of differentiation in a developmental cell lineage. If they exist, normal cells with amplified oncogenes should be rare, since they have not revealed themselves in analysis of normal cell populations or tissues. Also, one would have to postulate specific mechanisms for ensuring differential and synchronized gene amplification in specific normal cells whenever expression must be faster than can be achieved by transcription from a single copy of the gene. In cancer cells, less specific mechanisms will suffice, because tumors appear to contain ongoing clonal expansions that occur at the cost of phenotypically inferior sibling cell lines. One possibility is that preexisting mutations of oncogenes could ignite the process of amplification in some tumors by first converting a proto-oncogene into an active oncogene. Subsequently, a selective pressure for increased amounts of the mutated transforming protein in

ONCOGENE AMPLIFICATION IN TUMOR CELLS

271

the actively dividing initiated cells could lead to amplification of the corresponding gene. Examples include translocation of c-abl in K562 cells (Selden et al., 1983) and transcriptional activation of c-myb in COLO 201/205 cells (Alitalo et al., 1984b). Taya et al. (1984) have recently described a human lung giant cell carcinoma grown in nude mice, where both c-Ki-ras and c-myc oncogenes were amplified about 10-fold. Sequencing studies indicated that at least some of the amplified c-Ki-ras copies were also mutationally activated in their twelfth codons. Rearrangement and amplification had also occurred in the DNA of the c-Ha-ras oncogene from a biopsy of bladder cancer (Hayashi et aE., 1983). These results fit to the multistage theory of cancer development and progression (Nowell, 1976; Klein and Klein, 1985). Apparently, cooperating lesions in cellular oncogenes accumulate during tumor growth and clonal selection and increase the malignant potential of the tumor cells. It may be that activated oncogenes have specific roles in the accelerated genomic evolution of tumor cells. For example, several “immortalizing” oncogenes induce sister chromatid exchanges in cultured cells (Cerni et al., 1986). Loss of a suppressor gene or activation by conversion to hemizygosity of a recessive cancer gene may occur in neuroblastomas and smallcell lung cancer showing consistent amplifications. According to the models of Knudson (1985),the first lesion is inherited; amplification could be a subsequent early step in retinoblastomas or a later step in progression of neuroblastomas and small-cell lung cancer to more malignant phenotypes. Exceptional cases of disseminated neuroblastomas classified as stage IV-S tumors can also regress spontaneously (D’Angio et al., 1971). It has been proposed that these tumors only represent a nonautonomous proliferation of neural crest cells possessing an inherited mutation that interferes with their differentiation (Knudson and Meadows, 1980). They would therefore lack any subsequent dominant somatic lesions such as amplifications that activate cellular oncogenes. Indeed, stage IV-S tumors lack the N-myc amplification so commonly seen in usual stage IV neuroblastomas (Seeger et al., 1985). Although somatic amplifications of cellular oncogenes may be very rare in most common tumors, two recent case reports illustrate their role in leukemias. The first, a patient with Phl-positive chronic granulocytic leukemia had three separate episodes of blast crisis with promyelocytic and cytogenetic changes. Cells from the second blast crisis, but not from the first or third episodes of promyelocytic expansion, showed an 8- to 16-fold amplification of the c-myc oncogene (McCarthy et al., 1984). It was concluded that progression of chronic myeloid leukemia from chronic phase to episodes of blast transforma-

272

KARI ALITALO AND MANFRED SCHWAB

tion can reflect successive expansions of distinct clones of progressively more malignant cells and cells more resistant to chemotherapy. Amplification of c-myc in HL-60 promyelocytic leukemia cells also occurred in vivo and has persisted in culture conditions (Collins and Groudine, 1982; Dalla Favera et al., 1982). In a patient that we studied, DMIN chromosomes were already found in bone marrow cells during a preleukemic period of 8 months. Analysis of DNA from peripheral blood cells during subsequent acute myeloid leukemia (M2) showed an approximately 30-fold amplification of c-myc, a figure which corresponds to approximately two copies of c-myc per DMIN chromosome in the leukemic cells (Alitalo et al., 1985). In this case the DMINs and presumably also c-myc amplification persisted throughout the disease. It seems that the molecular diagnosis of oncogene amplification might serve to denote additional genetic lesions associated with a more aggressive disease in small-cell lung cancer and maybe also in neuroblastomas, squamous cell carcinomas, and glioblastomas, and an alternative lesion associated with tumor progression in sporadic cases. However, most leukemias, for example, do not show myc amplifications (Rothberg et al., 1984) and therefore multiple other somatic lesions such as c-myc translocation or point mutations in the c-TUSgenes could suffice for the clonal expansions of these tumor cells. An interesting question or paradox concerns the cell line specificity of amplifications and other lesions of c-myc. It could be speculated that translocation of c-myc is an irreversible event necessary early in the development of Burkitt’s lymphoma and that the lesion leads to permanent failure of the cells to respond to differentiative stimuli. Thus the specificity of the lesion would result from cell-specific regulation of transcription and existence of mechanisms for their damage in B cells at some stages of differentiation. In contrast, the role of cmyc amplification would be to provide increased dosage of a protein that is advantageous for growth during tumor progression, especially in cells which would not otherwise divide easily. The development of solitary mammary carcinomas in transgenic pregnant mice that abundantly express c-myc in response to corticosteroids in sensitive tissues is also compatible with the view that, besides an elevated dosage of c-myc, some other factors are necessary for the emergence of malignant neoplasms (Stewart et al., 198413). Multiple lesions would have accumulated in tumor DNA at the time when diagnostic material is obtained (Balmain, 1985). Although most of these lesions would concern different and complementing oncogenes, the experiments of Spandidos and Wilkie (1984) suggest that overexpression even of a single mutated M S oncogene is sufficient to

ONCOGENE AMPLIFICATION I N TUMOR CELLS

273

malignantly transform primary rodent cells. There appears to be selection for overexpression of mutant c-ras alleles during tumorigenesis (Winter et aZ., 1985) and the dosage of mutant c-ras genes also has a significant effect on transformed properties of in vitro transfected cell lines (Sistonen et al., 1986), pointing to the role of c-ras dosage in tumor progression. It may be that elevated expression of specific cmyc functions is necessary for the growth transformation-immortalization aspect of the phenotype of cancer cells that may also contribute to tumor progression (Armelin et aZ., 1984; Keath et al., 1984; Heldin and Westermark, 1984). Some oncogene amplifications might sensitize cells to growth factors (Leof et aZ., 1986; Kelekar and Cole, 1986; Mougneau et al., 1984). However, there may be no mandatory sequence for activation of oncogenes in the genesis of any particular tumor. Amplification of an oncogene could play its part in malignant progression of already initiated cells whenever it happened to occur. Generally, enhanced expression of an oncogene could be a necessary prerequisite for acquisition of a growth advantage by cells having extra copies of the gene. This effect could also be the principal contribution of amplification to tumorigenesis. ACKNOWLEDGMENTS We are grateful to Dr. Robert Winqvist for help in preparing a part of the review during his thesis work at the University of Helsinki. We thank our colleagues J. Michael Bishop, Harold Varmus, Kalle Saksela, Tomi Makelti, Jorma Keski-Oja, C. C. Lin, Arthur Levinson, Wendy Colby, and Donna George for collaboration, Stephen Hann for critical comments and for communicating results before publication, and Ms. Mervi Laukkanen for expert secretarial assistance. The studies in the authors’ laboratories were supported by the Finnish Cancer Research Fund, by the Academy of Finland, and by Deutsche Forschungsgemeinschafi. Part of this work was carried out under a contract with the Finnish Life Insurance Companies.

REFERENCES Abrams, H. D., Rohrschneider, L. R., and Eisenman, R. N. (1982). Cell 29,427-239. Alhonen-Hongisto, L., Leinonen, P., Sinervirta, R., Laine, R., Winqvist, R., Alitalo, K., Jhnne, O., and J h n e , J. (1986). Submitted for publication. Alitalo, K. (1984). Med. Biol. 62,304-317. Alitalo, K., Bishop, J. M., Smith, D. H., Chen, E. Y.,Colby, W. W., and Levinson, A. D. (1983a). Proc. Natl. Acad. Sci. U S A . 80, 100-104. Alitalo, K., Ramsay, G., Bishop, J. M., Ohisson-Pfeifer, S., Colby, W. W., and Levinson, A. D. (1983b). Nature (London)306,274-277. Proc. Nutl. Alitalo, K., Schwab, M., Lin, C. C., Varmus, H. E., and Bishop, J. M. (1983~). Acad. Sci. U.S.A. 80, 1707-1711. Alitalo, K.,Saksela, Winqvist, R., Schwab, M., and Bishop, J. M. (1984a).I n “Genes and

274

KARI ALITALO AND MANFRED SCHWAB

Cancer” (J. M. Bishop, J. Rowley, and M. Greaves, eds.), pp. 383-397.Alan R. Liss, Inc., New York. Alitalo, K., Winqvist, R., Lin, C. C., de la Chapelle, A., Schwab, M., and Bishop, J. M. (1984b).Proc. Natl. Acad. Sci. U.S.A. 81,4534-4538. Alitalo, K., Saksela, K., Winqvist, R., Alitalo, R., Laiho, M., Keski-Oja, J., Ilvonen, M., Knuutila, S., and de la Chapelle, A. (1985).Lancet 2, 1035-1039. Armelin, H. A.,Armelin, M. C. S., Kelly, K., Stewart, T., Leder, P., Cochran, B. H., and Stiles, C. D. (1984).Nature (London) 310,655-660. Baba, T. W., and Humphries, E. H. (1985).Proc. Natl. Acad. Sci. U S A . 82,213-216. Balmain, A. (1985).Br. J . Cancer 51, 1-7. Barker, P. E. (1982).Cancer Genet. Cytogenet. 5,81-94. Barker, P. E., Drwinga, H. L., Mittelman, W. N., and Maddox, A. M. (1980).Exp. Cell Res. 130,353-360. Barsoum, J., and Varshavsky, A. (1983).Proc. Natl. Acad. Sci. USA. 80,5330-5334. Bartram, C. R., de Klein, A., Hagemeijer, A., Carbonell, F., Kleihauer, E., and Grosveld, G. (1986).Leuk. Res. (in press). Battey, J., Moulding, C., Taub, R.,Murphy, W., Stewart, T., Potter, H., Lenoir, G., and Leder, P. (1983).Cell 34,779-787. Beard, J. W. (1980).In “Viral Oncology” (G. Klein, ed.), pp. 55-87. Raven Press, New York. Benedict, W. F., Banejee, A., Mark, C., and Murphree, A. L. (1983).Cancer Genet. Cytogenet. 10,311-333. Beug, H., Hayman, M.J., and Graf, T. (1982).Cancer Sum. 1,205-230. Biedler, J. L., and Spengler, B. A. (1976).Science 191,185-187. Biedler, J. L., Meyers, M. B., and Spengler, B. A. (1983).Adv. Cell. Neurobiol. 4,268-

301.

Bishop, J. M. (1983).Annu. Rev. Biochern. 52,301-354. Bishop, J. M. (1985).Cell 42,s-38. Bister, K. (1984).In “Leukaemia and Lymphoma Research” (J. M. Goldman and 0. Jarrett, eds.), Vol. 1, pp. 38-63. Churchill-Livingstone, Edinburgh and London. Blanchard, J.-M., Piechaczyk, M., Dani, C., Chambard, J.-C., Franchi, A., Pouyssegur, J., and Jeanteur, P. (1985).Nature (London) 317,443-445. Bonner, T.I., Kerby, S., Sutrave, P., Gunnell, M., Mark, G., and Rapp, U. R. (1984). Science 223,71-74. Bravo, R., Burckhardt, J., Curran, T., and Muller, R. (1985).EMBOJ. 4, 1193-1197. Brodeur, G. M., Green, A. A., Hayes, F. A., Williams, K. J., Williams, D. L., and Tsiatis, A. A. (1981).Cancer Res. 41,4678-4686. Brodeur, G. M., Seeger, R. C., Schwab, M., Varmus, H. E., and Bishop, J. M. (1984). Science 224,1121-1124. Brown, P. C., Tlsty, T. D., and Schimke, R. T. (1983).Mol. Cell. B i d . 3, 1097-1107. Bullock, P., and Botchan, M. (1982).In “Gene Amplification” (R. T. Schimke, ed.), pp. 215-230. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Campisi, J., Gray, H. E., Pardee, A. B., Dean, M., and Sonenshein, G. E. (1984).Cell 36,

241-247.

Carney, D. N., Gazdar, A. F., Bepler, G., Cuccion, J. G., Marangos, P. J., Moody, T. W., Zweig, M. H., and Minna, J. D. (1985).Cancer Res. 45,2913-2923. Carpenter, G., and Cohen, S . (1984).Trends Biochern. Sci. 99,169-171. Cavenee, W. K., Dryja, T. P., Phillips, R. A., Benedict, W. R., Codbout, R., Gallie, B. L., Murphree, A. L., Strong, L. C.,-and White, R. L. (1983).Nature (London)305,779-

784.

ONCOGENE AMPLIFICATION IN TUMOR CELLS

275

Cerni, C., Mougneau, E., and Cuzin, F. (1986). Submitted for publication. Chattopadhyay, S. K., Chang, E. H., Lander, M. R., Ellis, R. W., Scolnick, E. M., and Lowy, D. R. (1982). Nature (London) 296,361-363. Cifone, M. S., and Fidler, I. J. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 6949-6952. Colby, W. W., Chen, E. Y., Smith, D. H., and Levinson, A. D. (1983). Nature (London) 301,722-725.

Collins, S. J.. and Groudine, M. T. (1982).Nature (London)298,679-681. Collins, S. J., and Groudine, M. T. (1983).Proc. Natl. Acad. Sci. U S A . 80,4813-4817. Collins, S. J., Kubonishi, I., Miyoshi, I., and Groudine, M. T. (1984). Science 225,72-74. Connan, G., Rassoulzadegan, M., and Cuzin, F. (1985). Nature (London)314,277-279. Corcoran, L. M., Adams, J. M., Dunn, A. R., and Cory, S. (1984). Cell 37, 113-122. Coughlin, S. R., Lee, W. M. F., Williams, P. W., Giels, G. M., and Williams, L. T.(1985). Cell 43,243-251. Cowell, J. K. (1982). Annu. Reu. Genet. 16,21-52. Cowley, G., Smith, J. A., Gusterson, B., Hendler, F., and Ozanne, B. (1984). In “The Cancer Cells” (G. F. Vande Woude, A. J. Levine, W. C. Topp, J. D. Watson, eds.), Vol. 1, pp. 5-10. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Cox, D., Yuncken, C., and Spriggs, A. I. (1965). Lancet 1,55-58. Croce, C. M., and Nowell, P. C. (1985). Blood 65, 1-7. Croce, C. M., Erikson, J., Huebner, K., and Nishikura, K. (1985). Science 227, 12351238.

Cullen, B. R., Lomedico, P. T., and Ju, G. (1984). Nature (London) 307,241. Cuttitta, F., Carney, D. N., Mulshine, J., Moody, T. W., Fedorko, J., Fischler, A., and Minna, J. D. (1985). Nature (London) 316, 823-826. Dalla Favera, R. D., Wong-Staal, F., and Gallo, R. C. (1982). Nature (London)299,6163.

D’Angio, G. J., Evans, A. E., and Koop, C. E. (1971).Lancet 1,1046-1049. Dani, C. H., Blanchard, J. M., Piechaczyk, M., El Sabouty, S.,Marty, L., and Jeanteur, P. H. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 7046-7050. Dani, C. H., Mechti, N., Piechaczyk, M., Lebleu, B., Jeanteur, P. H., and Blanchard, J. M. (1985). Proc. Natl. Acad. Sci. U S A . 82,4896-4899. de Martinville, B., Cunningham, M., Murray, J., and Francke, U.(1983). Nucleic Acids Res. 11,5267-5271. Donner, P., Greiser-Wilke, I., Moelling, K. (1982).Nature (London) 296,262-266. Dotto, G. P., Parada, L. F., Weinberg, R. A. (1985).Nature (London) 318,472-475. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A,, Schlessinger, J., and Waterfield, M. D. (1984). Nature (London) 307, 521-527. Echols, H.(1981). Cell 25, 1-2. Eisenman, R. N., Tachibana, C. Y., Abrams, H. D., and Hann, S. R. (1985). Mol. Cell. B i d . 5, 114-126. Evan, G. I., and Hancock, D. C. (1985). Cell 43,253-261. Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985). MoZ. Cell. Bid. 5,36103616.

Fasano, O., Birnbaum, D., Edlund, L., Fogh, J., and Wigler, M. (1984).Mol. Cell. Biol. 4,1695-1705.

Filmus, J., and Buick, R. N. (1985). Cancer Res. 45,822-825. Gazdar, A. F., Carney, D. N., Guccion, J. G.,and Baylin, S . B. (1981). In “Small Cell Lung Cancer” (F. A. Greco, R. K. Oldham, and P. A. Bunn, Jr., eds.), pp. 145-175. Grune & Stratton, New York.

276

KAFU ALITALO AND MANFRED SCHWAB

Gazdar, A. F., Carney, D. N., Nau, M. M., and Minna, J. D. (1985).Cancer Res. 49,

2924-2930.

Gebhart, E., Bruderlein, S., Tulusan, A. H., Maillot, K., and Birkman, J. (1984).Int. J . Cancer 34,369-373. Gilbert, F., Balaban, G., Brangman, D., Henmann, N., and Lister, A. (1983).Int. J . Cancer 31,765-768. Gill, G. N.,and Lazar, C. S . (1981).Nature (London)293,305-307. Graf, T., and Beug, H. (1983).Cell 34, 7-9. Gray, A,, Dull, T. J., and Ullrich, A. (1983).Nature (London)303,722-725. Groffen, J., Stephenson, J. R., Heisterkamp, N., de Klein, A., Bartram, C. R., and Grosveld, G. (1984).Cell 36,93-99. Grosso, L. E., and Pitot, H. C. (1985).Cancer Res. 45,847-850. Hall, A., Marshall, C. J., Spurr, N. K., and Weiss, R. A. (1983).Nature (London)303,

396-400.

Hamkalo, B. A., Farnham, P. J., Johnston, R., and Schimke, R. T. (1985).Proc. Natl. Acad. Sci. U . S A . 82, 1126-1130. Hann, S. R., and Eisenman, R. N. (1984).Mol. Cell. Biol. 4,2486-2497. Hann, S. R., Abrams, H. D., Rohrschneider, L. R., and Eisenman, R. N. (1983).Cell 34,

789-798.

Hann, S. R., Thompson, C. B., and Eisenman, R. N. (1985).Nature (London)314,366-

369.

Harper, M . E., Franchini, G., Love, J., Simon, M. I., Gallo, R. C., and Wong-Staal, F. (1983).Nature (London)304,169-171. Haussler, M., Sidell, M., Kelly, M., Donaldson, C., Altman, A., and Mangelsdorf, D. (1983).Proc. Natl. Acad. Sci. U S A . 80,5525-5529. Hayashi, K., Kakizoe, T., and Sugimura, T. (1983).Gann 74,798-801. Hayward, W., Neel, B. G., and Astrin, S . (1981).Nature (London)290,475-480. Heilbronn, R., Schlehofer, J. R., Yalkinoglu, A. 6.,and zur Hausen, H. (1985).Int. J . Cancer 36,85-91. Heisterkamp, N., Stephenson, J. R., Groffen, J., Hansen P. F., de Klein, A., Bartman, C. R., and Grosveld, G. (1983).Nature (London)306,239-242. Heldin, C.-H., and Westermark, B. (1984).Cell 37,9-20. Hirsch, F. R. (1983).Cancer 52,2144-2150. Jacobovits, A,, Schwab, M., Bishop, J. M., and Martin, G. R. (1985).Nature (London)

318, 188-191.

Johnston, R. N., Beverley, S. M., and Schimke, R. T. (1983).Proc. Natl. Acad. Sci. U.S.A.

80,3711-3715.

Kaczmarek, L., Hyland, J. K., Watt, R., Rosenberg, M., and Baserga, R. (1985).Science

228, 1313-1315.

Kafatos, F.C., Orr, W., and Delidakis, C. (1985).Trends Genet. Noo., 301-305. Kamata, T., and Feramisco, J. R. (1984).Nature (London)310, 147-150. Kanda, N.,Schreck, R., Alt, F., Bruns, G., Baltimore, D., and Latt, S . (1983).Proc. Natl. Acad. Sci. U.S.A. 80,4069-4073. Keath, E. J., Caimi, P. G., and Cole, M. D. (1984).CelZ 39, 339-348. Kelekar, A., and Cole, M. D. (1986).M o l . Cell. Biol. 6,7-14. Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. (1983).Cell 35,603-610. King, C. R., Kraus, M. H., and Aaronson, S. A. (1985).Science 229,974-978. Klein, G. (1981).Nature (London)294,313-318. Klein, G. (1983).Cell 32,311-315. Klein, G., and Klein, E. (1985).Nature (London)315, 190-195.

ONCOGENE AMPLIFICATION IN TUMOR CELLS

277

Klein, A., Bartram, C. R., and Grosveld, G. (1983).Nature (London) 306,239-242. Knudson, A. G. (1985).Cancer Res. 45, 1437-1443. Knudson, A. G., and Meadows, A. T. (1980).N . Engl. J . Med. 302, 1254-1256. Kohl, N. E., Kanda, N., Schreck, R. R.,Bruns, G., Latt, S. A., Gilbert, F., and Alt, F. W. (1983).Cell 35,359-367. Kohl, N. E., Legouy, E., DePinho, R.A., Nisen, P. D., Smith, R. K., Gee, C. E., and Alt, F. W. (1986).Nature (London)319, 73-77. Konopka, J. B., Watanabe, S. M., Witte, 0. N. (1984).Cell 37,1035-1042. Konopka, J. B., Watanabe, S. M., Singer, J. W., Collins, S. J., and Witte, 0. N. (1985). Proc. Natl. Acad. Sci. U S A . 82, 1810-1814. Kovacs, G. (1979).Int.]. Cancer 23,299-301. Kozbor, D., and Croce, C. M. (1984).Cancer Res. 44,438-441. Land, H., Parada, L. F., and Weinberg, R. A. (1983a).Nature (London)304,596-602. Land, H., Parada, L. F., and Weinberg, R. A. (1983b).Science 222, 771-778. Laughlin, T.J., and Taylor, J. H. (1979).Chrornosorna 75, 19-35. Lavi, S. (1981).Proc. Natl. Acad. Sci. U.S.A.78, 6144-6148. Lee, W.-H., Murphree, A. L., and Benedict, W. F. (1984).Nature (London) 309,458-

460.

Lee, W. M. F., Schwab, M., Westaway, D., and Varmus, H. E. (1985).Mol. Cell. Biol. 5,

3345-3356.

Leof,E. B., Proper, J. A., Hash, C. A., Branum, E. L., and Moses, H. L. (1986).Submitted for publication. Leof, E.B., Proper, J. A., Goustin, A. S., Shipley, C . D., DiCorleto, P., and Moses, H. L. (1986).Proc. Natl. Acad. Sci. U.S.A. 83,2453-2457. Levan, A., Manolov, G., and Clifford, P. (1968).J. Natl. Cancer Inst. (U.S.) 41, 1377-

1387.

Levan, A., Levan, G., and Mitelman, F. (1977).Hereditas 86, 15-29. Levan, A,, Levan, G., and Mandahl, N. (1981).In “Genes Chromosomes and Neoplasia” (Frances, Arrighi, Potu,Rao, Stubblefield, eds.), pp. 223-250. Raven Press, New York. Lewis, J. A., Biedler, J. L., and Melera, P. W. (1982).J. Cell Biol. 94,418-424. Levy, L. S., Gardner, M. B., and Casey, J. W. (1984).Nature (London) 308,853-856. Li, Y.-S., Holland, C. A., Hartley, J. W., and Hopkins, N. (1984).Proc. Natl. Acad. Sci. U . S A . 81,6808-6811. Li, Y . 4 . (1983).Int. J . Cancer 32,455-459. Libermann, T.A., Razon, N., Bartal, A. D., Yarden, Y., Schlessinger, J., and Soreq, H. (1984).Cancer Res. 44,753-760. Libermann, T.A., Nusbaum, H. R., Razon, N., Kris, R., Lax, I., Soreq, H., Whittle, N., Waterfield, M. D., Ullrich, A., and Schlessinger, J. (1985).Nature (London) 313,

144-147.

Lin, C. C.,Alitalo, K., Schwab, M., George, D., Varmus, H. E., and Bishop, J. M. (1985). Chrornosorna 92, 11-15. Lin, C. R.,Chen W. S., Kuiger, W., Stolarsky, L. S., Weber, W., Evans, R. M., Verma, I. M., Gill, G. N., and Rosenfield, M. G. (1984).Science 22,843-848. Linial, M . , and Groudine, M. (1985).Proc. Natl. Acad. Sci. U S A . 82,53-57. Little, C. D., Nau, M. M., Carney, D. N., Gazdar, A. F., and Minna, J. D. (1983).Nature (London)306, 194-196. McCarthy, D., Rassool, F. V., Goldman, J. M., Graham, S. V., and Birnie, G. D. (1984). Lancet 1, 1362-1365. Mariani, B. D., and Schimke, R. T. (1984).J. Biol. Chem. 259, 1901-1910.

278

KARI ALITALO AND MANFRED SCHWAB

Mark, J. (1967).Hereditas 57, 1-22. Mark, J. (1971).Hereditas 68,61-100. Mark, J., and Granberg, J. (1970).Acta Neuropathol. 16, 194-209. Michitsch, R. W.,Montgomery, K. T., and Melera, P. W. (1984).Mol. Cell. Biol. 4,2370-

2380.

Minna, J. D., Higgins, G. A., and Glatstein, E. J. (1982).In “Principles and Practice of Oncology” (DeVita, Hellman, and Rosenberg, eds.), pp. 396-474. Lippincott, Philadelphia, Pennsylvania. Modjtahedi, N., Lavialle, C., Poupon, M.-F., Landin, R.-M., Cassingena, R., Monier, R., and Brison, 0. (1985).Cancer Res. 45,4372-4379. Montgomery, K. T., Biedler, J. L., Spengler, B. A,, and Melera, P. W. (1983).Proc. Natl. Acad. Sci. U.S.A. 80, 5724-5728. Mougneau, E., Lemieux, L., Rassoulzadegan, M., and Guzin, F. (1984).Proc. Natl. Acad. Sci. U S A . 81, 5758-5762. Muller, R., Muller, D., and Guilbert, L. (1984).EMBOJ. 3, 1887-1890. Muller, R.,Curran, T., Muller, D., and Guilbert, L. (1985).Nature (London) 314,546548.

Mullins, J. T., Brody, D. S., Binari, R. C., and Cotter, S. M. (1984).Nature (London) 308,

856858.

Murray, M. J., Cunningham, J. M., Parada, L. F., Dautry, F., Lebowitz, P., and Weinberg, R. A. (1983).Cell 33, 749-757. Mushinski, J. F., Potter, M., Bauer, S.R., and Reddy, E. P. (1983).Science 220,795-798. Nakano, H., Yamamoto, F. Neville, C., Evans, D., Mizuno, T., and Perucho, M. (1984). Proc. Natl. Acad. Scl. U S A . 81,71-75. Nau, M. M., Carney, D. N., Battey, J., Johnson, B.,Little, C., Gazdar, A., and Minna, J. D. (1984).Cum. Top. Microbiol. Immunol. 113,172-177. Nau, M. M., Brooks, B. J., Battey, J., Sausville, E., Gazdar, A. F., Kirsch, I. R., McBride, 0.W., Bertness, V., Hollis, G. F., and Minna, J. D. (1985).Nature (London) 318,69-

73.

Neil, J. C., Hughes, D., McFarlane, R.,Wilkie, N. M., Onions, D. E., Lees, G., and Jarrett, 0.(1984).Nature (London) 308,814-820. Neiman, P., Wolf, C., Enrietto, P. J., and Cooper, G. M. (1985).Proc. Natl. Acad. Sci. U.S.A. 82,222-226. Nepveu, A., Fahrlander, P. D., Yang, J.-Q., and Marcu, K. B. (1985).Nature (London)

317,440-443.

Noori-Daloii, M. R., Swift, R. A., Kung, H.-J., Crittenden, L. B., and Witter, R.L. (1981). Nature (London) 294,574-576. Nowell, R. C.(1976).Science 194,23-28. Nowell, R. C., Finan, J,, Favera, R. D., Gallo, R. C., ar-Rushdi, A., Romanczuk, G., Selden, J. R., Emanuel, B. S.,Rovera, G., and Croce, C. M. (1983).Nature (London)

306,494-497.

Pall, M. L. (1981).Proc. Natl. Acad. Sci. U.S.A.78,2465-2468. Payne, G. S., Bishop, J. M., and Varmus, H. E. (1982).Nature (London) 295,209-217. Pelicci, P.-G., Lanfrancone, L., Brathwaite, M. D., WoIman, S. R., and Dalla Favera, R.

(1984).Science 224, 1117-1121.

Pellegrini, S., Dailey, L., and Basilico, C. (1984).Cell 36,943-949. Persson, H., and Leder, P. (1984).Science 225, 718-720. Persson, H., Hennighousen, L., Taub, R., DeGrado, W., and Leder, P. (1984).Science

225,687-693.

ONCOGENE AMPLIFICATION IN TUMOR CELLS

279

Piechaczyk, M., Yang, J.-Q., Blanchard, J.-M., Jeanteur, P., and Marcu, K. B. (1985).Cell 42,589-597. Pohjanpelto, P., Alitalo, K., Holttli, E., JBnne, 0. A., and Knuutila, S. (1985).J . Biol. Chem. 260,8532-8537. Quinn, L. A., Moore, G. E., Morgan, R. T., and Woods, L. K. (1979). Cancer Res. 39, 4914-4924. Rabbitts, T. H., Forster, A., Hamlyn, P., and Baer, R. (1984).Nature (London)309,592597. Radice, P. A., Matthews, M. J., Ihde, D. C., Gazdar, A. F., Carney, D. N., Bunn, P. A., Cohen, M. H., Fossieck, B. E., Makuch, R. W., and Minna, J. D. (1982).Cancer 50, 2894-2902. Rall, L. B., Scott, J., Graeme, I. B., Crawford, R. J., Penschow, J. D., Niall, H. D., and Coghlan, J. P. (1985).Nature (London)313,228-231. Ramsay, G., Evan, G. I., and Bishop, J. M. (1984).Proc. NatLAcad. Sci. U S A . 81,77427746. Rechavi, G., Givol, D., and Canaani, E. (1982).Nature (London)300,607-611. Reitsma, P. H., Rothberg, P. G., Astrin, S. M., Trial, J., Bar-Shavit, Z., Hall, A., Teitelbaum, S. L., and Kahn, A. J. (1983).Nature (London) 306,492-494. Reynolds, G. P., and Smith, R. G. (1982).In “Neuroblastoma” (C. Pochedly, ed.), pp. 313-167. Elsevier Biomedical Press, New York. Riou, G., Barrois, M., Tordjman, 0. S., Dutronquay, V., and Orth, G. (1984). C. R. Seances Acad. Sci. 14,575-580. Roberts, J. M., Buck, L. B., and Axel, R. (1983).Cell 33,53-63. Rossman, T. G., and Klein, C. B. (1985).Cancer Inuest. 3, 175-187. Rothberg, P. G., Erisman, M. D., Diehl, R. E., Rovigatti, U. Go,and Astrin, S. M. (1984). Mol. Cell. B i d . 4, 1096-1103. Rovigatti, U., Watson, D. K., and Yunis, J. J. (1986).Science 232,398-400. Rowley, J. D. (1983).Nature (London) 301,290-291. Rowley, J. D., and Ultmann, J. E. (1983).“Chromosomes and Cancer.” Academic Press, New York. Rozengurt, E., and Sinnett-Smith, J. (1983).Proc. Natl. Acad. Sci. U S A . 80,2936-2940. Sager, R., Gadi, I. K., Stephens, L., and Grabowy, C. T. (1985).Proc. Natl. Acad. Sci. U.S.A.82,7015-7019. Saito, H., Hayday, A. C., Wiman, K., Hayward, W.S., and Tonegawa, S . (1983). Proc. Natl. Acad. Sci. U.S.A.80, 7476-7480. Saksela, K., Bergh, J., and Nilsson, K. (1986).Submitted for publication. Santos, E., Martin-Zanca, D., Reddy, E. P., Pierotti, M. A., Della Porta, G., and Barbacid, M. (1984). Science 223,661-664. Sarasin, A. (1985).Cancer Inuest. 3, 163-174. Schechter, A. L., Stem, D. R., Vaidyanathan, L., Decker, S . J., Drebin, J. A., Greene, M. I., and Weinberg, R. A. (1984).Nature (London)312,513-516. Schimke, R. T. (1982). “Gene Amplification.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Schimke, R. T., Brown, P. C., Kaufinan, R. J., McGrogan, M., and Slate, D. L. (1981). Cold Spring Harbor Symp. Quant. Biol. 55,785-797. Schimke, R. T., Shenvood, S. Q., Hill, A. B., and Johnston, R. N. (1986).Proc. Natl. Acad. Sci. U S A . 83,2157-2161. Schlehofer, J. R., Matz, B., Gissmann, L., Heilborn, R., and zur Hausen, H. (1984).In “Genes and Cancer” (J. M. Bishop, J. D. Rowley, and M. Greaves, eds.), pp. 185190. Alan R. Liss, Inc., New York.

280

KARI ALITALO AND MANFRED SCHWAB

Schwab, M., Alitalo, K., Varmus, H. E., Bishop, M., and George, D. (1983a). Nature (London)303,497-501. Schwab, M., Alitalo, K., Klempnauer, K.-H., Varmus, H. E., Bishop, J. M., Gilbert, F., Brodeur, G., Goldstein, M., and Trent, J. (198313).Nature (London)305,245-248. Schwab, M., Alitalo, K., Varmus, H. E., and Bishop, J. M. (1984a). In “Cancer Cells” (G. F. Vande Woude, A. J. Levine, W. C. Topp, and J. D. Watson, eds.), pp. 215-220. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Schwab, M., Ellison, J., Busch, M., Rosenau, W., Varmus, H. E., and Bishop, J. M. (1984b). Proc. Natl. Acad. Sci. U S A . 81, 4940-4944. Schwab, M., Varmus, H. E., Bishop, J. M., Grezeschik, K.-H., Naylor, S. L., Sakaguchi, A. Y., Brodeur, G., and Trent, J. (1984~). Nature (London)308, 288-291. Schwab, M., Ramsay, G., Alitalo, K., Varmus, H. E., Bishop, J. M., Martinsson, T., Levan, G., and Levan, A. (1985). Nature (London) 315,345-347. Scott, J,, Urdea, M., Quiroga, M., Sanchez-Pescador, R., Fong, N., Selby, M., Rutter, W. J., and Bell, G. I. (1983). Science 221,236-240. Seeger, R. C., Brodeur, G. M., Sather, H., Dalton, A., Siegei, S. E., Wong, K. Y., and Hammond, D. (1985). N . Eng1.J. Med. 313, 1111-1115. Selden, J. R., Emanuel, B. S., Wang, E., Cannizzaro, L., Palumbo, A., Erikson, J., Nowell, P. C., Rovera, G., and Croce, C. M. (1983).Proc. Natl. Acad. Sci. U.S.A. 80, 7289-7292. Semba, K., Kamata, N., Toyoshima, K., and Yamamoto, T. (1985).Proc. Natl. Acad. Sci. U.S.A.82,6497-6501. Shapiro, J. R., Yung, W.-K. A., and Shapiro, W. R. (1981). Cancer Res. 41, 2349-1359. Shibuya, M.,Yokota, J., and Ueyama, Y. (1985). Mol. Cell. Biol. 5, 414-418. Shiloh, Y.,Shipley, J., Brodeur, G. M., Bruns, G., Korf, B., Donlon, T., Schreck, R. R., Seeger, R., Sakai, K., and Latt, S. A. (1985).Proc. Natl. Acad. Sci. U.S.A. 82,37613765. Shimizu, K., Goldfarb, M., Perucho, M., and Wigler, M. (1983). Proc. Natl. Acad. Sci. U.S.A.80, 383-387. Shimizu, N., Kondo, I., Gamou, S., Behzadian, M. A., and Shimizu, Y. (1984). Somatic Cell Mol. Genet. 10, 45-53. Shtromas, I., White, B. N., Holden, J. J. A., Reimer, D. L., and Roder, J. C. C. (1985). Cancer Res. 45,642-647. Siebenlist, U., Hennighausen, L., Battey, J., and Leder, P. (1984). Cell 37,381-391. Spandidos, D. A., and Wilkie, N. M. (1984). Nature (London)310,469-475. Spriggs, A., and Boddington, M. M. (1962). Br. Med. J . 2, 1431-1435. Spun, N. K., Solomon, E., Jansson, M., Sheer, D., Goodfellow, P. N., Bodmer, W. F., and Vennstrom, B. (1984). EMBOJ. 3, 159-163. Stanton, L. W., Schwab, M., and Bishop, J. M. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 1772-1776. Stark, G. R., and Wahl, G. M. (1984).Annu. Reo. Biochem. 53,447-491. Steffen, D. (1984). Proc. Natl. Acad, Sci. U S A . 81,2097-2101. Stewart, T.A., Pattengale, P. K., and Leder, P. (1984a). Cell 38,627-637. Stewart, T. A., BellvB, A. R., and Leder, P. (198413).Science 226,707-710. Siimegi, J., Hedberg, T., Bjarkholm, M., Godal, T., Mellstedt, H., Nilsson, M. G., Perlman, C., and Klein, G. (1985). Int. J . Cancer 36,367-371. Taparowsky, E., Shimizu, K., Goldfarb, M., and Wigler, M. (1983). Cell 34,581-586. Taya, Y.,Hosogai, K., Hirohashi, S., Shimosato, Y.,Tsuchiya, R.,Tsuchida, N., Fushimi, M., Sekiya, T., and Nishimura, S. (1984). E M B O J . 3,2943-2946. Thiele, C. J., Reynolds, C. P., and Israel, M. A. (1985).Nature (London)313,404-406.

ONCOGENE AMPLIFICATION IN TUMOR CELLS

281

Thompson, Z. B., Challoner, P. B., Neiman, P. E., and Groudine, M. (1985). Nature (London) 314,363-336. Tlsty, T. D., Brown, P. C., and Schimke, T. R. (1984).Mol. Cell. Biol. 4, 1050-1056. Trent, J., Meltzer, P., Rosenblum, M., Harsh, G., Kinzler, K., Mashal, R., Feinberg, A., and Vogelstein, B. (1986).Proc. Natl. Acad. Sci. U S A . 83,470-473. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A. W., Lee, J., Yarden, Y., Libermann, T. A., Schlessinger, J., Downward, J., Mayes, E. L. V., Whittle, N., Waterfield, M. D., and Seeburg, P. H. (1984).Nature (London) 309, 418-425. Van der Bliek, A. M., Van der Velde-Koerts, T., Ling, V., and Brost, P. (1986).Mol. Cell. Biol. (in press). Varmus, H. (1984).Annu. Reu. Genet. 18,553-612. Varshavsky, A. (1981a).Cell 25,561-572. Varshavsky, A. (1981b).Proc. Natl. Acad. Sci. U.S.A. 78,3673-3677. Vennstrdm, B., Kahn, P., Adkins, B., Enrietto, P., Hayman, M. J., Graf, T., and Luciw, P. (1984).EMBO J . 3,3223-3229. Wahl, G. M., de Saint Vincent, B. R., and DeRose, M. L. (1984).Nature (London)307, 516-520. Watt, R. A., Shatzman, A. R., and Rosenberg, B. (1985).Mol. Cell. Biol. 5,448-456. Westaway, D., Payne, G., and Varmus, H. E. (1984).Proc. Natl. Acad. Sci. U.S.A. 82, 843-847. Westin, E. H., Gallo, R. C., Arya, S. K., Evan, A., Souza, L. M., Baluda, M. A., Aaronson, S. A., and Wong-Staal, F. (1982).Proc. Natl. Acad. Sci. U S A . 79, 2194-2198. Whang-Peng, J., Bunn, P. A., Kao-Shan, C. S., Lee, E. C., Carney, D. N., Gazdar, A., and Minna, J. D. (1982a).Cancer Genet. Cytogenet. 6, 119-134. Whang-Peng, J., Kao-Shan, C. S., Lee, E. C., Bunn, P. A., Carney, D. N., Gazdar, A. F., Portlock, C., and Minna, J. D. (1982).In “Gene Amplification” (R. Schimke, ed.), pp. 107-113. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Wigley, C. B., and Cowell, J. K. (1984).JNCI, J . Natl. Cancer Inst. 73,219-226. Winqvist, R., Saksela, K., and Alitalo, K. (1984). EMBO J . 3,2947-2950. Winqvist, R., Knuutila, S., Leprince, D., Stehelin, D., and Alitalo, K. (1985). Cancer Genet. Cytogenet. 18,251-264. Winqvist, R., Makela, T.P., Seppanen, P., Janne, 0.A,, Alhonen-Hongisto, L., Janne, J., Grzeschik, H. J., and Alitalo, K. (1986).Cytogenet. Cell Genet. (in press). Winter, E., Yamamoto, F., Almoguera, C., and Perucho, M. (1985).Proc. Natl. Acad. Sci. U.SA. 82,7575-7579. Woodcock, D. M., and Cooper, I. A. (1981).Cancer Res. 41,2483-2490. Xu, Y., Ishii, S., Clark, A. J. L., Sullivan, M., Wilson, R. K., Ma, D. P., Roe, B. A., Merlino, G. T., and Pastan, I. (1984a).Nature (London)309,806-810. Xu, Y., Richert, N., Ito, S., Merlino, G. T., and Pastan, I. (1984b).Proc. Natl. Acad. Sci. U S A . 81,7308-7312. Yancopoulos, G. D., Nisen, P. D., Tesfaye, A., Kohl, N. E., Goldfarb, M. P., and Alt, F. W. (1985).Proc. Natl. Acad. Sci. U.S.A.82,5455-5459. Yokota, J., Yamamoto, T., Toyoshima, K., Terada, M., Sugimura, T., Battifora, H., and Cline, M. J. (1986).Lancet 1, 765-767. Yunis, J. J. (1981).Hum. Pathol. 12,540-549. Zabel, B. U., Naylor, S. L., Grezeschik, K.-H., and Sakaguchi, A. Y. (1984).Somatic Cell Mol. Genet. 10, 105-108. Zachary, I., and Rozengurt, E. (1985).Proc. Natl. Acad. Sci. U S A . 82,7616-7620. Zullo, J. N., Cochran, R. H., Huang, A. S., and Stiles, C. D. (1985).Cell 43, 793-800.

This Page Intentionally Left Blank

TRANSCRIPTION ACTIVATION BY VIRAL AND CELLULAR ONCOGENES Joseph A. Nevins The Rockefeller University, New York.

New York 10021

I. Introduction

The mechanisms by which certain genes can alter the growth regulation of cells and lead to transformation to the oncogenic state are of obvious importance. One class of oncogenes are the viral genes that have the ability to immortalize certain primary cells and, in combination with various other oncogenes, elicit the fully transformed phenotype (for review, see Land et d.,1983). In many instances, these immortalizing genes have been shown to function as transcriptional regulatory genes during lytic viral infection. The suggestion has thus been made that the ability of these genes to alter cellular growth regulation is due in part to their ability to control transcription. Two aspects of this process have been the subject of many investigations. First, how do such oncogenes induce transcription? By what specific mechanisms do these regulatory gene products increase the frequency of initiation of transcription at a given promoter? This question is obviously of importance beyond the realm of oncogenesis. That is, the mechanisms of transcriptional control are central to an understanding of a diverse group of cellular processes. Second, if transcriptional activation is indeed important to oncogenesis, then what are the cellular genes that are activated by these oncogenes and how do these activated genes contribute to the oncogenic state? The direct evidence for a role of transcription activation in oncogenesis is lacking but a variety of indirect evidence has suggested that this may be a contributing factor. In addition, the study of these genes, apart from their role in oncogenesis, has provided valuable insight into mechanisms of transcription control. This review will focus on these two subjects: our current understanding of how such genes control transcription initiation and the possible involvement of such a mechanism in oncogenesis. 283 ADVANCES IN CANCER RESEARCH, VOL. 47

Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

284

JOSEPH R. NEVINS

11. Transcription Control by Viral Oncogenes

A. ADENOVIRUS EIA GENE Perhaps the best studied and understood of the viral genes that control transcription is the adenovirus EIA gene. The EIA transcription unit is one of six viral transcription units active during an early infection, that is, prior to viral DNA replication (for review, see Tooze, 1981). The EIA gene along with the ELB gene, which map to the left end of the viral chromosome, are responsible for oncogenic transformation by adenovirus (Graham et al., 1974; Gallimore et al., 1974; Flint et al., 1976; van der Eb et al., 1977; Frost and Williams, 1978; Graham et al., 1978; Jones and Shenk, 1979a). In the absence of the E I B gene, the ELA gene can convert primary rat fibroblasts into immortal cell lines with unlimited growth potential (Houwelling et al., 1980; Ruley, 1983). During a lytic infection of permissive cells the EIA gene product is required for the expression of each of the other early viral transcription units (Berk et al., 1979; Jones and Shenk, 1979b). In the absence of a functional E1A protein, the only gene expressed is EIA (Berk et al., 1979; Nevins, 1981).The mechanism for ElA-mediated activation is at the level of transcription (Nevins, 1981). Thus, a trans-acting transcriptional regulatory gene was identified. The EIA region is complex in that three distinct mRNAs are produced by differential splicing (Berk and Sharp, 1978; Chow et al., 1979; Kitchingman and Westphal, 1980). Two of the RNAs (a 13 S RNA and a 12 S RNA) maintain the same reading frame and produce proteins of 289 amino acids and 243 amino acids, respectively, that are identical in sequence except for 46 amino acids (Perricaudet et al., 1979; van Ormondt et al., 1980). Splicing to produce the other E1A RNA (a 9 S species) alters the reading frame and thus would produce a distinctly different protein (Dijkema et al., 1982). This 9 S E1A RNA and its product are only produced late in lytic infection and are not found in transformed cells (Spector et al., 1978). A function for this product is unknown. Through the use of mutants that selectively affect the production of one or the other of these mRNAs or proteins, it has been shown that the product of the longest mRNA (13 S), the 289 amino acid protein, is primarily responsible for transcription control (Ricciardi et al., 1981; Monte11 et al., 1984; Carlock and Jones, 1981). The smaller, related protein of 243 amino acids does appear to be able to activate viral transcription, but inefficiently when compared to the larger protein, at least when assayed in viral infection (Winberg and

TRANSCRIPTION ACTIVATION

285

Shenk, 1984; Montell et al., 1984; Leff et al., 1984).A role for the 243 amino acid protein in the activation of viral DNA replication during lytic infection of certain growth-regulated cells has been suggested (Montell et al., 1984; Spindler et al., 1985). Of course, one could imagine that such a function might contribute to transformation and, indeed, the expression of the 243 amino acid protein is required for the fully transformed state (Montell et al., 1984). Both of the major E1A proteins are apparently required for full transformation. Viruses that express only the 13 S product or only the 12 S product are able to generate partial transformants but both products are required for the full range of transformation specific properties (Montell et al., 1984; Winberg and Shenk, 1984). It has been argued that since partial transformation can be obtained in the absence of the protein that activates transcription (13 S product), transcriptional induction may not be a critical aspect of adenovirus transformation (Montell et al., 1984). However, given the fact that the 12 S product retains some transcription-inducing activity, it is impossible to eliminate a contribution of transcriptional control for oncogenesis. Obviously, one does not know what level of activating function is sufficient for generating the transformed phenotype.

Mechanism of E l A Action One might consider two broad mechanisms by which the E1A protein (specifically the 289 amino acid protein, hereafter simply referred to as E1A) could activate transcription. The protein could act directly, by binding to DNA sequences, to facilitate specific transcription initiation. Alternatively, the protein might act indirectly as a regulatory activity, to modify some component of the transcriptional machinery to allow increased transcription from the viral promoters. There is as yet no direct evidence to differentiate these two possibilities, but the available evidence suggests the latter mechanism. If the former mechanism were the correct one, then the activation process might be expected to be very specific. This has not been found to be the case. First of all, there clearly appears to be a cellular involvement. In the complete absence of ElA, early viral genes are transcribed, although inefficiently (Nevins, 1981; Shenk et al., 1979; Gaynor and Berk, 1983).This inefficient transcription can be increased by the inhibition of cellular protein synthesis, suggesting the involvement of a negative-acting cellular component (Nevins, 1981; Katze et al., 1981). Thus, there is transcription in the absence of ElA, but in the presence of E1A the rate increases substantially. The extent of this E1A-independent transcription depends on the host cell, suggesting specificity

286

JOSEPH R. NEVINS

to the role of cellular components. One such example is the murine teratocarcinoma cell line F9. This cell line supports E lA-independent transcription of the early viral genes (Imperiale et al., 1984). However, when the cells are induced to differentiate in culture, this “E 1A-like” activity disappears. The differentiated cells are capable of transcribing the early genes since if E1A is present (wild-type infection) there is efficient transcription (Imperiale et al., 1984). Furthermore, it has been shown that the transcriptional regulatory gene of an unrelated virus, in this case pseudorabies virus (a herpesvirus) can efficiently activate adenovirus genes and replace E1A (Feldman et al., 1982; Imperiale et al., 1983). If direct DNA binding by this regulatory protein was the mechanism for induction, then it would be difficult to imagine how the herpes protein could recognize and interact with the heterologous adenovirus promoters just as efficiently as does E1A. In addition to the various ways that the early adenovirus genes can be activated by ElA, it has been found that E1A can activate a variety of unrelated genes when cotransfected into cells. The first such example was the human p-globin gene that normally requires an enhancer element for efficient expression in HeLa cells. In the presence of E1A or the pseudorabies immediate early gene, p-globin transcription was stimulated and became enhancer independent (Green et al., 1983). Similar results have been found by other groups for a variety of promoters (Gaynor et al., 1984; Svenson and Akusjarvi, 1984; Alwine, 1985);the general conclusion is that E1A can activate a wide variety of promoters, both viral and cellular, when they are assayed by transient transfections. Finally, it has recently been demonstrated that E1A can activate promoters utilizing RNA polymerase I11 (pol 111). Transfection experiments have demonstrated an induction of certain pol 111 promoters, including the adenovirus VA gene promoter and tRNA promoters (Berger and Folk, 1985; Hoeffler and Roeder, 1985; Gaynor et al., 1985). The induction has also been demonstrated in cell-free extracts of adenovirus-infected cells where there is a higher rate of pol I11 transcription than in extracts from uninfected cells (Hoeffler and Roeder, 1985; Gaynor et al., 1985). Fractionation of the extracts has shown that there is an increased amount of a factor that coelutes with the polymerase I11 “C” factor (Segall et al., 1980). All of the above results point to an indirect mechanism of activation of transcription by the E1A protein. How might this occur? Some insight has been gained through the analysis of essential DNA sequences in viral promoters that are stimulated by E1A. The most

TRANSCRIPTION ACTIVATION

287

extensive analyses have involved the E2 promoter where both deletion mutagenesis and linker scanning mutants have been employed (Elkaim et al., 1983; Imperiale and Nevins, 1984; Imperiale et al., 1985; Kingston et al., 1984a; Zajchowski et al., 1985; Murthy et al., 1985). The results of various studies generally agree that sequences between -80 and -50 relative to the transcription initiation site ( + 1) are important for the activity of the promoter, in addition to the pseudo-TATA sequence between -21 and -28. One study also suggested that sequences between -30 and -40 may play a role (Zajchowski et al., 1985). However, in all of this work there was no indication of a specific sequence requirement for E1A induction. The same sequences required for uninduced transcription are also required for E1A stimulation. The same situation appears to be true for the E 3 promoter (Leff et al., 1985). Upstream sequences are required for full activity but no unique sequence is required for E1A stimulation. In contrast, analysis of the E4 promoter indicated that there was a distinct site involved in E1A stimulation, separate from a site necessary for basal, uninduced transcription (Gilardi and Perricaudet, 1984). Far upstream sequences between -158 and -179 appear to constitute a regulatory site, since in the absence of this sequence E1A-induced transcription was reduced to 15-fold whereas E 1A-independent transcription was not affected. What can b e proposed as a mechanism based on all of these previous results? As detailed above, many lines of evidence argue against a direct role for E1A as a DNA-binding transcription factor. It seems more likely that cellular factors are involved in promoter recognition. There is clear precedent for such a mechanism because the early SV40 promoter very likely makes use of a cellular transcription factor (Dynan and Tjian, 1983a,b). This is further supported by the findings that the same sequences are required in the E 2 and the E 3 promoter in the presence or absence of E1A. A simple mechanism consistent with all of the data would be one in which the E1A protein modified a cellular transcription factor so as to enhance its binding to the viral promoter or increased the amount of the factor. Possibly such a factor is limiting in the cell, thus restricting the efficient transcription of the viral genes, but then through the action of E1A the factor becomes nonlimiting. In the case of the E4 promoter the situation becomes somewhat more complex since more than one factor may be involved. However, this could still maintain the same general mechanism as suggested above, yet involving additional factors. Recent experiments addressing this question have provided sup-

288

JOSEPH H. NEVINS

port for the scenario described above. The interaction of proteins with the adenovirus E 2 promoter in virus-infected cells was examined using an in uioo exonuclease I11 mapping procedure (Wu, 1984). In an infection with wild-type virus, thus in the presence of ElA, a protein DNA interaction was observed in the region of the promoter previously deemed critical for activity (Kovesdi et al., 1986a). In the absence of E1A (d1312 infection), no such interaction was detected. However, if cells were infected with a high multiplicity of dl312 for an extended length of time, conditions that allow early transcription in the absence of E1A (Nevins, 1981; Gaynor and Berk, 1983), the same E 2 promoter-protein interaction was detected. Thus, it appears that a protein binds to the E 2 promoter under circumstances in which the promoter is active. The protein is not likely to be E1A itself, certainly not in the d1312 infection, but the interaction is enhanced in the presence of E1A. In addition, the putative factor mediating this interaction has been detected in nuclear extracts of infected cells (Kovesdi et al., 1986b). The factor binds in a sequence-specific manner to the E2 promoter between -33 and -74 upstream of the transcription initiation site. The factor was detected in extracts of uninfected cells, although at much reduced levels than in infected cells. Furthermore, the increased amount of the factor in infected cells requires the E1A gene. Thus, it would appear that E1A stimulation of the E2 promoter involves an increase in the amount of the binding activity of a cellular transcription factor. Finally, the induction of pol I11 transcription by E1A could be the result of either of two mechanisms, either of which would be consistent with the above model. E1A might act on a polymerase I11 factor, possibly the pol I11 C factor, in a manner analogous to the action on a pol I1 factor, as suggested above, so as to increase its activity. Alternately, E1A might stimulate the pol I1 gene specifying a pol I11 factor, similar to the stimulation of the early viral genes or the cellular hsp70 gene, and thus increase the actual amount of the pol I11 factor. Clearly, to elucidate the actual mechanism will require the identification of the factors involved. What are the proteins that interact with critical sequences at E lA-inducible promoters? If the recognition proteins are cellular transcriptional factors, then it must be determined if the same protein(s) recognize these sequences in the absence of E1A as in the presence of E1A. And, if so, what is the nature of the E1Ainduced change? Furthermore, what is the complexity of the regulation? If cellular transcription factors are involved, then how many are used by the E1A-induced promoters?

TRANSCRIPTION ACTIVATION

289

B. SV40 LARGET ANTIGEN A variety of recent experiments have demonstrated that the large T antigen of SV40 can activate transcription in trans. Initially this was demonstrated as the ability of SV40 large T antigen to activate the late SV40 promoter in transient transfection assays (Keller and Alwine, 1984; Brady et al., 1984). More recent data have shown that the SV40 T antigen could activate a variety of other promoters, including certain cellular promoters (Alwine, 1985). Thus, this activator may fall into the general class of trans-activators such as E1A that display a certain wide range of activating capacity. In fact, in these experiments it appeared that the large T antigen might have a broader specificity than E1A. Much less is known about the mechanism of papovavirus large T trans-activation. Sequences in this late SV40 promoter that are necessary for trans-activation have been analyzed. It appears that the T antigen binding sites as well as the enhancer element are required for activation (Brady and Khoury, 1985). A requirement for T antigen binding was also indicated by the fact that T antigen mutants that could no longer bind DNA were deficient in trans-activation (Keller and Alwine, 1984). Although these results argue for binding of T antigen to promoter sequences as a mechanism for activation, nevertheless it remains possible that T antigen might function indirectly. Indeed, the fact that T antigen can stimulate heterologous promoters, such as the adenovirus E2 promoter, where there is no apparent affinity of the protein for the DNA sequence suggests an indirect mechanism (Alwine, 1985). In addition, there may be negative factors involved in regulating transcription from the late SV40 promoter, as suggested by DNA competition experiments (Brady and Khoury, 1985).

C. T LYMPHOTROPHIC VIRUSTRANS-ACTIVATORS Recent data have demonstrated that the HTLV I and I1 viruses as well as the related bovine leukemia virus encode an activity that can induce transcription from the viral long terminal repeat element (LTR).This was initially suggested by the fact that transcription from the viral LTR was much higher in cells transformed by the virus than in uninfected celis (Sodroski et al., 1984; Derse et al., 1985; Rosen et aZ., 1985). The sequences encoding this activity, which map to the 3’ end of the viral genome, have now been isolated and shown to function as a transcriptional activator in transfection assays (Sodroski et al., 1985b; Felber et al., 1985).

290

JOSEPH R. NEVINS

In addition to the HTLV I-like viruses that possess a trans-activator, the virus associated with the acquired immune deficiency syndrome (AIDS) (HTLV 111or LAV) also encodes a trans-activator. Again, cells infected with this virus are more efficient in transcription from the LTR than uninfected cells (Sodroski et al., 1985a).Very recent reports have described the isolation of a trans-acting gene from HTLV I11 (Sodroski et al., 1985c; Arya et al., 1985). This gene is distinct in nature and genome localization from the trans-activator of HTLV I or 11. Furthermore, recent results suggest that trans-activation may involve translational control in addition to transcriptional stimulation (Rosen et al., 1986).

D. HERPESVIRUS IMMEDIATE EARLYGENES Although the various herpesvirus immediate early genes apparently do not act as oncogenes, certain of these genes are transcriptional activating genes. The basis for the phenomenon of sequential viral gene control was in fact established with the herpesvirus (Honess and Roizman, 1975). Mutants subsequently allowed the definition of the a 4 gene of HSV I as an early regulatory gene whose expression was required for both early and late genes (Watson and Clements, 1980; Preston, 1979; Dixon and Schaeffer, 1980). More recent experiments have indicated that the a0 gene of HSV can also trans-activate (Everett, 1984). In addition to HSV I, the other herpesviruses including pseudorabies virus, cytomegalovirus, and varicella-zoster virus possess regulatory genes that control the transcription of other viral genes (Everett, 1984; Tevethia and Spector, 1984). As described for the adenovirus promoters, there does not appear to be a specific sequence element in herpesvirus promoters that responds to immediate early induction (Everett, 1984; El-Kareh et al., 1985). It was from experiments using pseudorabies virus that the phenomenon of general control of transcription by this gene was demonstrated. Specifically, it was shown that the pseudorabies immediate early gene could efficiently activate early adenovirus genes, in fact just as efficiently as E1A (Feldman et al., 1982).This was also demonstrated in transfection assays using the cloned pseudorabies virus immediate early gene (Imperiale et al., 1983). Furthermore, it was shown that the pseudorabies immediate early gene could activate not just viral promoters but also apparently unrelated cellular promoters; the human p-globin gene could be stimulated by the immediate early gene in transfection assays (Green et al,, 1983). The fact that these herpes genes do not act as oncogenes or immortalizing genes may not be an indication of a distinction from those

TRANSCRIPTION ACTIVATION

29 1

genes that do (for example, ElA). The pseudorabies immediate early gene is in fact extremely toxic to cells. When the immediate early gene is cotransfected with a selectable marker (for instance, the neo gene), there is a large reduction in the number of surviving colonies (M. J. Imperiale and J. R. Nevins, unpublished); in fact, it is nearly impossible to obtain a surviving colony although it does occur as a rare event (L. Feldman, personal communication). Possibly, the herpes genes are too vigorous in activity, causing a lethal alteration in transcriptional regulation within the cell. It would be of interest to investigate the properties of attenuated immediate early genes that might produce much less of the protein or a protein with reduced efficiency to trans-activate. Would such a protein now have the ability to immortalize cells in culture and/or to transform in conjunction with the other oncogenes? 111. Transcription Control by Cellular Oncogenes

One might suspect that if viral oncogenes can mediate transformation by transcriptional activation, then there will likely be cellular genes that will perform the same function. Of course, one must be cautious in drawing analogies to the retrovirus/proto-oncogene situation since the transacting genes identified to date that act as oncogenes are all from the DNA tumor viruses. For instance, it has not been proved that the HTLV I “X”gene, the trans-activator, can transform or immortalize cells. These genes are essential for the lytic growth of these viruses and, if they have a cellular counterpart, evolution has intervened so as to render them unrecognizable. This may also be the case for the HTLV trans-activator because this gene is also essential for efficient replication of the virus. Nevertheless, there are candidates for analogous genes among the identified cellular oncogenes. Principally, these are the genes whose products are localized in the nucleus and include the myc, myb,fos, and p53 oncogenes. In fact, there is now direct evidence for myc possessing trans-activating activity. Cotransfection of a CAT gene under the control of a DrosophiZa hsp7O promoter with the myc oncogene resulted in a 10-fold increase in CAT production (Kingston et al., 198413). Although this does not prove that E l A and myc perform the exact same function, nevertheless it does suggest that they may be part of the same pathway. IV. Activation of Cellular Transcription by Viral Oncogenes

If indeed the transcriptional activation function of certain oncogenes is critical to cellular transformation, which is yet to be proved,

292

JOSEPH R. NEVINS

then the next question becomes the identification of the genes that are induced. How many genes are induced, what are the products of these genes, and what do they have in common?

A. INDUCTION OF hsp70 BY E1A Analysis of proteins synthesized in early adenovirus infected HeLa cells revealed a 70-kDa protein whose synthesis was increased in wild-type infected cells but not in E1A mutant-infected cells (Nevins, 1982). This protein is identical to one that is induced by heat shock of HeLa cells and is related to the well-characterized family of proteins induced by heat shock in Drosophila (Ashburner and Bonner, 1979). The induction of the hsp70 gene by E1A is transcriptional and the kinetics of induction follow those of the early viral genes induced by E1A in a lytic infection (Kao and Nevins, 1983). The transcription of the hsp70 gene is also increased in 293 cells, human cells transformed by adenovirus that express E1A (Kao and Nevins, 1983).Finally, the hsp70 gene is also induced by SV40 infection (Khandjian and Turler, 1982) as well as by herpesvirus infection (Notarianni and Preston, 1982) although in neither of these cases has a specific viral gene been identified that is responsible for the induction. Furthermore a high level of hsp70 expression in the absence of a heat shock was found in many tumor cell lines, suggesting the presence of an activity similar to E1A (Imperiale et al., 1984). Evidence for this was provided by the observation that in these cells where hsp7O expression was high there was partial activation of early adenovirus genes in the absence of E1A. A possible role for such a cellular transcriptional activator was suggested by studies of the expression of the hsp7O gene in HeLa cells. The expression of the hsp70 gene in asynchronously growing HeLa cells is high. This expression drops to undetectable levels when cells are synchronized at the GI/S border. Upon entry into the cell cycle, the expression increases dramatically, peaking at the late S or early Gz phase, and then declines (Kao et al., 1985). Furthermore, the control of this expression is transcriptional. In 293 cells, human cells transformed by Ad5, there is a very high expression of hsp70 (Nevins, 1982; Kao and Nevins, 1983). The hsp7O gene is also cell cycle regulated in 293 cells (Kao et al., 1985). Furthermore, the levels of E1A mRNA also fluctuate during the cell cycle and in fact peak somewhat prior to hsp70 expression. Although there is as yet no proof, the strong implication is that the E1A gene product regulates the hsp70 gene in 293 cells and in a cell cycle-specific manner. The further suggestion is that

TRANSCRIPTION ACTIVATION

293

there is a cellular activity similar to E1A in HeLa cells that performs the same function.

B. INDUCTION OF OTHERGENESBY E1A In addition to the stimulation of the hsp70 gene, E1A is also responsible for the increase in expression of the p-tubulin gene (Stein and Ziff, 1984). Although the magnitude of the stimulation is not as great as that of the hsp70 gene, nevertheless, the kinetics of induction are similar to those of hsp70 or the early viral genes and the induction is transcriptional. There are also examples of cellular enzyme activities that are increased by adenovirus infection (see review by Maltzman and Levine, 1981). An increase in thymidine kinase activity may be due to the action of E 1A since there is no stimulation in an E 1A-mutant infection (Braithwaite et al., 1983). However, since under the conditions of this experiment none of the early genes would be expressed, it was not possible to state whether the involvement of E1A is direct or indirect. Furthermore, it was not determined if the induction is transcriptional or not. C. TRANSFORMATION-SPECIFIC GENEINDUCTION

A number of studies have approached the question of differential gene expression in normal versus transformed cells. In each case, the induction of cellular genes in transformed cells was examined in contrast to possible repression of key cellular genes. A number of clones of cellular genes activated as a result of SV40 transformation have been isolated (Schutzbank et aZ., 1982; Scott et d.,1983).These generally detect mRNAs that are present at high levels in SV4O-transformed cells, as well as in many other transformed cells, but are present at very low levels or are undetectable in normal cells. SV40 large T antigen is involved, since a shift of cells transformed by a temperature-sensitive T antigen to nonpermissive temperature lowered the abundance of the transcripts. Relative to the topic of this review is the question of whether these genes are directly controlled by SV40 large T antigen and whether this control is transcriptional. This question has not been resolved. First of all, it has not yet been demonstrated that the control of these cellular genes is transcriptional. Indeed, a similar procedure aimed at the isolation of 293 cell-specific cellular transcripts revealed that most of the major changes were posttranscriptional (Kao and Nevins, 1985).

294

JOSEPH R. NEVINS

V. Summary and Perspectives

Two issues are central to the study of transcriptional control by the viral oncogenes: the mechanisms by which these gene products stimulate transcription and the extent to which cellular transcription is affected. The first issue is conceptually straightforward although experimentally difficult. The resolution of the mechanisms requires the isolation of the proteins involved and an analysis of their nature before and after transcriptional induction. If a specific transcription factor that is utilized by an E1A-inducible promoter is identified, then is this factor present in uninfected cells? If it is, then is it modified in some manner after viral infection and what is this modification? Finally, does the same modification occur in uninfected cells under certain conditions (for instance, cell cycle)? The second issue is more complex and likely will never be fully resolved. It is clear that the viral trans-activators can stimulate cellular transcription. The adenovirus EIA gene and the hsp70 gene are cases in point. However, it is not clear whether this is the only cellular gene that is activated (probably not since the P-tubulin gene is induced, although to a lower level) and, more importantly, it is not clear if this activation is important in oncogenesis or immortalization. Perhaps the only clear way to document the last point is to block the activity of an induced gene (possibly using an antibody or alternatively an antisense RNA) and ask if this action prevents transformation. That is, is an induced cellular gene required for transformation? Only then would it be possible to say that trans-activation was a critical event in oncogenesis.

REFERENCES Alwine, J. C. (1985). Mol. Cell. Biol. 5, 1034-1042. Arya, S. K., Guo, C., Josephs, S. F., and Wong-Staal, F. (1985). Science 229, 69-73. Ashburner, M., and Bonner, J. J. (1979). Cell 17,241-254. Berger, S. L., and Folk, W. R. (1985). Nucleic Acids Res. 13, 1413-1428. Berk, A. J., and Sharp, P. A. (1978). Cell 14, 695-711. Berk, A. J., Lee, F., Harrison, T., Williams, J., and Sharp, P. A. (1979). Cell 17,935-944. Brady, J., and Khoury, G. (1985). Mol. Cell. Biol. 5, 1391-1399. Brady, J., Bolen, J., Radonovich, M., Salzman, N., and Khoury, G. (1984). Proc. Natl. Acad. Sci. U S A . 81,2040-2044. Braithwaite, A. W., Chettham, B. F., Li, P., Parish, C. R., Waldron-Stevens, L. K., and Bellett, A. J. D. (1983).J . Virol. 45, 192-199. Carlock, L. R., and Jones, N. C. (1981).J . Vlrol. 40,657-664. Chow, L. T., Broker, T. R., and Lewis, J. B. (1979).J. M o l . Biol. 134,265-303. Derse, D., Caradonna, J. J., and Casey, J. W. (1985). Science 227,317-320. Dijkema, R., Dekker, B. M. M., and van Ormondt, H. (1982). Gene 8, 143-156.

TRANSCRIPTION ACTIVATION

295

Dixon, R. A. F., and Schaeffer, P. A. (1980).J. Virol. 36, 189-203. Dynan, W. S., and Tjian, R. (1983a).Cell 32,669-680. Dynan, W. S., and Tjian, R. (1983b).Cell 35, 79-87. Elkaim, R., Goding, C., and Kedinger, C. (1983).Nucleic Acids Res. 11,7105-7111. El-Kareh, A., Murphy, A. J. M., Fichter, T., Efstratiadis, A., and Silverstein, A. (1985). Proc. Natl. Acad. Sci. U S A . 82, 1002-1006. Everett, R. D. (1984).EMBOJ. 3,3135-3141. Felber, B. K., Paskalis, H., Kleinman-Ewing, C., Wong-Staal, F., and Pavlakis, G. N. (1985).Science 229,675-677. Feldman, L. T., Imperiale, M. J., and Nevins, J. R. (1982).Proc. Natl. Acad. Sci. U S A . 79,4952-4956. Flint, S. J., Sambrook, J., Williams, J. F., and Sharp, P. A. (1976).Virology 72,456-470. Frost, E., and Williams, J. F. (1978).Virology 91, 39-50. Gallimore, P. H., Sharp, P. A., and Sambrook, J. (1974).J . Mol. Biol. 89,49-72. Gaynor, R. B., and Berk, A. J. (1983).Cell 33,683-693. Gaynor, R. B., Hillman, D., and Berk, A. J. (1985). Proc. Natl. Acad. Sci. U . S A . 81, 1193-1197. Gilardi, P., and Perricaudet, M. (1984).Nucleic Acids Res. 12,7877-7888. Graham, F. G., Harrison, T., and Williams, J. F. (1978).Virology 86, 10-21. Graham, F. L., Abrahams, P. J., Mulder, C., Heijneker, H. L., Warnaar, S. O., de Vries, F. A. J., Fiers, W., and van der Eb, A. J. (1974). Cold Spring Harbor Symp. Quant. Biol. 39, 637-650. Green, M. R., Treisman, R., and Maniatis, T. (1983).Cell 35, 137HoeflIer, W. K., and Roeder, R. G. (1985).Cell 41,955-963. Honess, R. W. and Roizman, B. (1975).Proc. Natl. Acad. Sci. U S A . 72, 1276-1280. Houweling, A., van den Elsen, P. J., and van der Eb, A. J. (1980). Virology 105, 537550. Imperiale, M.J., and Nevins, J. R. (1984).Mol. Cell. Biol. 4,875-882. Imperiale, M. J., Feldman, L. T., and Nevins, J. R. (1983).Cell 35, 127-136. Imperiale, M. J., Kao, H.-T., Feldman, L. T., Nevins, J. R., and Strickland, S. (1984). Mol. Cell. Biol. 4, 867-874. Imperiale, M. J., Hart, R. P., and Nevins, J. R. (1985).Proc. Natl. Acnd. Sci. U.S.A. 82, 381-385. Jones, N., and Shenk, T. (1979a).Cell 17,683-689. Jones, N., and Shenk, T. (1979b).Proc. Natl. Acad. Sci. U S A . 76,3665-3669. Kao, H.-T., and Nevins, J. R. (1983).Mol. Cell. Biol. 3,2058-2065. Kao, H.-T., and Nevins, J. R. (1985).Submitted for publication. Kao, H.-T., Capasso, O., Heintz, N., and Nevins, J. R. (1985).Mol. Cell. Biol. 5,628-633. Katze, M. G., Persson, H., and Philipson, L. (1981).Mol. Cell. Biol. 1, 807-813. Keller, J. M., and Alwine, J. C. (1984).Cell 36, 381-389. Khandjian, E. W., and Turler, H. (1982).Mol. Cell. B i d . 3, 1-8. Kingston, R. E., Kaufman, R. J., and Sharp, P. A. (1984a).Mol. Cell. Biol. 4, 1970-1977. Kingston, R. E., Baldwin, A. S., and Sharp, P. A. (198413).Nature (London)312,280-282. Kitchingman, G. R., and Westphal, H. (1980).J . Mol. Biol. 13, 23-48. Kovesdi, I., Reichel, R., and Nevins, J. R. (1986a).Science 231, 719-722. Kovesdi, I., Reichel, R., and Nevins, J. R. (1986b).Cell 45,219-228. Land, H., Parada, L. F., and Weinberg, R. A. (1983).Science 222, 771-778. L e e T., Elkaim, R., Goding, C. R., Jalinot, P., Sassone-Corsi, P., Perricaudet, M., Kedinger, C., and Chambon, P. (1984).Proc. Natl. Acad. Sci. U.S.A.81,4381-4385.

296

JOSEPH R. NEVINS

Leff, T., Corder, J., Elkaim, R., and Sassone-Corsi, P. (1985).Nucleic Acids Res. 13,

1209- 1221.

Maltzman, W., and Levine, A. J. (1981).Adu. Virus Res. 26,65-116. Montell, C., Courtois, G., Eng, C., and Berk, A. (1984).Cell 36,951-961. Murthy, S. C. S., Bhhat, G. P. and Thimmappay, B. (1985).Proc. Natl. Acad. Sci. U.S.A.

82,2230-2234.

Nevins, J. R. (1981).Cell 26,213-220. Nevins, J. R. (1982).Cell 29, 913-919. Notarianni, E. L., and Preston, C. M. (1982).Virology 123, 113-122. Perricaudet, M., Akusjarvi, G., Virtanen, A,, and Pettersson, U. (1979).Nature (London)

281,694-696.

Preston, C. M. (1979).Virology 29,275-284. Ricciardi, R. P., Jones, R. L., Cepko, C. L., Sharp, P. A., and Roberts, B. E. (1981).Proc. Natl. Acad. Sci. U.S.A. 78,6121-6125. Rosen, C. A., Sodroski, J. G., Kethnan, R., Burny, A., and Haseltine, W. A. (1985). Science 227,320-322. Rosen, C. A,, Sodrowski, J. G., Goh, W. C., Dayton, A. I., Lippke, J., and Haseltine, W. A. (1986).Nature (London)319,555-559. Ruley, H. E.(1983).Nature (London)304,602-606. Schutzbank, T., Robinson, R., Oren, M., and Levine, A. J. (1982).Cell 30,481-490. Scott, M. R. D., Westphal, K.-H., and Rigby, P. W. J. (1983).Cell 34,557-567. Segall, J., Matsin, T., and Roeder, R. G. (1980).J . Biol. Chem. 225, 11986-11991. Shenk, T., Jones, N., Colby, W., and Fowlkes, D. (1979).Cold Spring Harbor Symp. Quant. Biol. 44,367-375. Sodroski, J. G., Rosen, C. A., and Haseltine, W. A. (1984).Science 225,381-385. Sodroski, J. G., Rosen, C. A., Wong-Staal, F., Salahuddin, S. Z., Popovic, M., Arya, S., Gallo, R. C., and Haseltine, W. A. (1985a).Science 227,171-173. Sodroski, J. G., Rosen, C. A., Goh, W. C., and Haseltine, W. A. (198513).Science 228,

1430-1434.

Sodroski, J. G., Patarca, R., Rosen, C. A., Wong-Staal, F., and Haseltine, W. A. (1985~). Science 229,74-77. Spector, D. J., McGrogan, M., and Raskas, H. J. (1978).J. Mol. Biol. 126,395-414. Spindler, K. R., Eng, C. Y.,and Berk, A. J. (1985).J . Virol. 53, 742-750. Stein, R., and Ziff, E. B. (1984).Mol. Cell. Biol. 4,2792-2801. Svenson, C., and Akusjarvi, G. (1984).EMBOJ. 3,789-794. Tevethia, M. J., and Spector, D. J. (1984).Virology 137,428-431. Tooze, J., ed. (1981).“DNA Tumor Viruses,” 2nd ed. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Watson, R. J., and Clements, B. (1980).Nature (London)285,329-330. Winberg, G., and Shenk, T. E. (1984).EMBOJ. 3, 1907-1912. Wu, C. (1984).Nature (London)309,229-234. van der Eb,A. J., Mulder, C., Graham, F. L., and Houweling, A. (1977).Gene 2, 115-

132.

van Ormondt, H., Maat, J., and van Beveren, C. P. (1980).Gene 11,299-309. Zajchowski, D. A., Boeuf, H., and Kedinger, C. (1985).EMBOJ. 4, 1293-1300.

EPIDEMIOLOGY AND EARLY DIAGNOSIS OF PRIMARY LIVER CANCER IN CHINA Yeh Fu-Sun and Shen Kong-Nien” Quangxi Medical College. Nanning, Guangxi, People’s Republic of China

I. Introduction

Liver cancer is one of the most common cancers in the world, particularly prevalent in Africa and Asia. In China, liver is the third most common site, after stomach and esophagus, in the “National Mortality Survey by Cause 1973-1975” (Li and Li, 1980).The world standardized mortality rate for primary liver cancer (PLC) per 100,000 was 19.96 for males and 8.1 for females. The well-known marked geographic variation of mortality rate of this cancer is also evident in China and in its counties and even villages. The study of contrasts of high- and low-risk factors may lead to the control of the disease. The large amount of research work done all over the world has been very rewarding during the last two or three decades in enabling us to learn far more about the pathogenesis, diagnosis, prevention, and treatment of the disease than was known before. Much work has also been done by Chinese scientists under a national health policy which places emphasis on the control of diseases that are common and seriously detrimental to human health. This is a review of research on epidemiology and early diagnosis which has been done in medical centers in this country, particularly of the extensive and prolonged studies done in several high-incidence areas. II. Distribution

A. GEOGRAPHIC DISTRIBUTION Details of the geographic distribution of cancer in China have been published (National Cancer Control Office of the Ministry of Health and Nanjing Institute of Geography of Academia Sinica, 1981; Li and

* The epidemiology sections of this article were prepared by Dr. Yeh Fu-Sun and the diagnostic section (Section VIII) was contributed by Dr. Shen Kong-Nien. 297 ADVANCES IN CANCER RESEARCH. VOL. 47

Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

298

YEH FU-SUN AND SHEN KONC-NIEN

Li, 1981) and are well illustrated in maps. The counties and provinces or autonomous regions with higher mortality rates (MR) are arranged in a band running from north to south along the southeastern seacoast. They are Jiangsu, Zhejiang, Fujien, Guangdong Provinces, and the Guangxi Zhuang Autonomous Region. Very high mortality rates of PLC are found in Qidong County of Jiangsu Province, 96.67 and 27.91 per 100,000 for males and females, respectively. In Fusui County of the Guangxi Zhuang Autonomous Region, which has a world standardized mortality rate (WSMR) per 100,000 of 111.75 for males and 25.04 for females, PLC accounts for 70% of all male and 57% of all female cancer deaths (Li, 1982). Even in the same high-risk region, for instance, Guangxi, the mortality rate shows marked differences between different prefectures and counties: in Guilin Prefecture, whose counties are located over the northern part of the region, the WSMR of PLC per 100,000 is 15.43 for males and 4.74 for females, whereas in Nanning Prefecture, with its counties located over the southern part of the region, the WSMR of PLC per 100,000 is 52.17 for males and 11.55 for females. In general, the mortality rate of PLC is higher near the seacoast and lower inland; that of the southeastern and northeastern parts is higher than the northwestern part. The climate of the highincidence area is warmer and more humid and altitude is lower. In Qidong County the average temperature is 16°C with a relative humidity of 82-84% and precipitation of 1000-1100 mm. In Fusui County, another high-incidence county, the average temperature is 22°C with a relative humidity of 78%and 1176 mm precipitation.

B. DISTRIBUTION OF POPULATION Age and sex. As in other parts of the world, PLC occurs at an earlier age in high-incidence areas. The peak age group in Fusui County is 40 years and in Qidong, 50 years. Also the ratio of males to females is greater. The sex ratio of mortality is 2.59 in China, 5.46 in Fusui County, 3.46 in Qidong County, and 4 in Shunde County (SMR, 26.16 per 100,000) (Hu, 1983). In lower incidence areas, the sex ratio tends to be lower, e.g., 2.3 for Hupei and 1.6 for Gansu Provinces. Occupation. There is no significant difference in the mortality rate among different occupation groups, although peasants and fishermen of the seacoast have a higher mortality rate (Yu, 1981). FamiZy aggregation. A family history of PLC can frequently be elicited from PLC patients, especially in high-incidence areas. A study in Qidong County showed that of 259 cases of PLC, 15.4%had two or more cases of PLC in the family history. In Fusui County, 24.3%of

PRIMARY LIVER CANCER IN CHINA

299

906 PLC cases were found to have two or more cases in the family history. In one family in a high-incidence village, of 21 people in three generations, 9 died of PLC (Guangxi Tumor Mortality Coordinating Study Group, 1978). Migrant population. A number of reports outside China have indicated (Shanmugaratnam, 1965; Ong et al., 1976; King and Haenszel, 1973) that China-born Chinese showed higher risk for PLC than the indigenous inhabitants or their descendants in lower risk countries to which their ancestors had migrated. Similar conditions have been observed in studies in China. In Nantong County (MR 23.6 per 100,000)of Jiangsu, there is a river which sharply divides the northern part (MR 20.2 per 100,000), which consistently has a lower PLC mortality rate, from a southern part, which has a higher PLC mortality rate (31.2 per 100,000). It was found that southerners who migrated to the north showed a drop in the PLC mortality rate to 29.74 per 100,000, whereas that of northerners who migrated to the south rose to 36.36 per 100,000 (Yu, 1977). Also, in Nanhui County of Shanghai, a highrisk area, it was found that (Zhuan et al., 1984) migrants from very high-risk areas, including Qidong, maintained their high mortality rate for PLC, whereas migrants from lower risk areas had a marked rise of mortality rate after migration to Nanhui County. These studies strongly suggest that environmental factors must be operative in the prevalence of PLC. Ill. Environmental Factors

A. HBV INFECTION The evidence is overwhelming in the world literature in favor of the major role played by HBV infection in the development of PLC. Long before the discovery of HBV antigen, scientists noted that chronic hepatitis and cirrhosis of the liver were predetermining factors of PLC. Even with the earlier insensitive tests, it was repeatedly found that the HBsAg antigen was serologically positive in PLC cases in far greater percentage than in the controls. The improvement of the sensitivity of the serological test for HBV markers further confirmed the assumption that HBV infection is closely related to the development of PLC. In Jiangsu Province (Ye et al., 1981), the R-PHA test was used for HBsAg, PHA for anti-HBs, AGD for HBeAg and anti-HBc, and IAHA for HBcAg. If any one of the tests became positive, the case was

300

YEH FU-SUN AND SHEN KONG-NIEN

TABLE I CORRELATION OF HBsAg CARRIER STATUS IN THE GENERAL POPULATION WITH THE INCIDENCE OF LIVER CANCER IN QIDONC COUNTY~ ~~

Carrier status

Number of person-years

Number of PLC cases

PLC incidence rate (per lo5)

HBsAg positive

14,080

33

234.38

HBsAg negative

66,757

9

13.49

RR

P

17.38

cO.01

~~

From Lu et al. (1983), Qidong Liver Cancer Institute, with permission.

included in the calculation of the prevalence rate of HBV infection. The prevalence of HBV infection in the 216 PLC cases tested was 92.59%,distinctly higher than the controls. The relative risk (RR) was calculated to be 8.24; i.e., the incidence was 8.24 times that of the nontumor patients. A nationwide coordinating group of liver cancer pathology (Ying et al., 1984), using the orcein stain technique, reported that 460 of the 757 cases of cirrhosis of the liver had various degrees of chronic active hepatitis, with 82.63%positive for HBsAg. Of the 500 cases of liver carcinoma, 81.2%were positive for HBsAg. Of those accompanied by cirrhosis, 83.69%were HBsAg positive, and of those not accompanied by cirrhosis, 61.04%were positive. A matched prospective study on chronic carriers of HBsAg by Lu and associates (1983)was particularly illuminating. In Qidong County in one commune, 2560 chronic carriers of HBsAg and 12,314 HBsAgnegative adults selected from a survey of the general population were followed from September 1976 to May 1982. The incidence rate of primary liver cancer was found to be 234.38 and 13.5 per 100,000 in the two groups, respectively (Table I). The RR was 17.38. In a matched prospective study of 1236 HBsAg-positive adults and 1611 negative controls 14 PLC cases occurred in the carriers and 4 in the control group. Thus, there was a significant difference between the incidence rate of PLC among the carriers and the noncarriers, and the RR was 4.56 (Table 11).Furthermore, the four PLC cases in the noncarrier group had become HBsAg positive before cancer became apparent. It was noted that dynamic changes occurred in the carrier state of HBsAg: it could convert from negative to positive or vice versa or could persistently be positive or negative. In this same overall study (Lu et al., 1983),a comparison of all those who had a history of a positive test or were presently positive with those who were persistently negative found that the incidence rate of the former was 202 times the latter; i.e., the RR was 202.27. Other

30 1

PRIMARY LIVER CANCER IN CHINA

TABLE 11 CORRELATION OF MATCHEDHBsAg CARRIER STATUS WITH THE INCIDENCE OF PRIMARY LIVERCANCER IN QIDONG COUNTY~ Number of person-years

Number of PLC cases

PLC incidence rate (per lo5)

HBsAg carriers

6798

14

205.94

Controls

8861

4

41.14

RR

P

4.56

<0.01

From Lu et al. (1983),Qidong Liver Cancer Institute, with permission.

malignant tumors also developed among the population under study, but no correlation was found between the carrier status of HBsAg and the incidence rate of cancer of other sites (Table 111; Lu et al., 1983). Another matched prospective study was conducted in Guangxi (Mo et al., 1984) correlating the status of HBsAg carriers living in various aflatoxin contaminated areas with the prevalence of liver cancer. The study began with surveys from 1974 to 1977 of hepatitis and liver cancer by clinical examination and serological screening for HBsAg and AFP by the R-PHA method. The subjects were studied at random from village (brigade) communes of four counties, representing highand relatively low-incidence areas. HBsAg-positive cases above 20 years of age and an equal number of sex- and age-matched controls (551 cases each) were selected and followed at 1-, 3-, and 5-year intervals. The study ended in December of 1982, with periods of observation of 5-8 years. Fourteen cases of PLC developed in the HBsAgpositive group, at a rate of 398 per 100,000, as compared with two cases of PLC or 56.2 per 100,000in the HBsAg-negative group; the RR was 7.1 (Table IV) (Mo et al., 1984).The above studies in Jiangsu and Guangxi strongly suggest that long-standing infection of HBV exerts a specific effect on human liver cells and is conducive to the development of malignant changes. TABLE I11 CORRELATION OF HBsAg CARRIER STATUS IN THE GENERAL POPULATION WITH THE INCIDENCE OF OTHER MALIGNANCIES" Carrier status

Number of person-years

HBsAg positive HBsAg negative

14,080 66,737

Number of cancer cases (incidence per 105) Stomach

Lung

Esophagus

Other

4 (28.41) 2 (14.20) 4 (28.41) 3 (21.31) 14 (20.98) 14 (20.98) 7 (10.49) 28 (41.96)

From Lu et al. (1983),Qidong Liver Cancer Institute, with permission.

302

YEH FU-SUN AND SHEN KONG-NIEN

TABLE IV CORRELATION OF MATCHED HBsAg CARRIER STATUS WITH THE INCIDENCE OF PRIMARY IN GUANGXI” LIVERCANCER HBsAg-positive group

Person-years studied Number of PLC cases PLC incidence (per lo5) RR

HBsAg-negative group

Male

Female

Total

2051.5

1467.5

3519

2074

10

4

14

2

487.4

272.6

397.8

96.4

7.1

1.0

5.1

-

Male

Female

Total

1485.5

3559.5 2

0

56.2

-

1.o

From Mo et al. (1984), Guangxi Medical College, with permission.

In Guangxi, studies in Longan County (Li et al., 1981)using the RPHA method for HBsAg and PHA for anti-HBs survey showed a higher number of HBsAg positives in the high-incidence communes than in the low-incidence ones (Table V). Studies of members of “PLC families” (immediate families of PLC cases) and matched control families showed significantly higher HBsAg occurrence in the members of the former than in those of the latter (24.6 and 12.0%,respectively; Table VI). The differences in occurrence of anti-HBS between the high- and low-incidence villages and between the PLC and control families TABLE V COMPARISON OF HBsAg AND ANTI-HBsOCCURRENCE IN THE POPULATION >20 YEARS OLDIN HIGH-AND LOW-RISKVILLAGESIN LONCAN COUNT+

GroupC

Number examined

HBsAg(+)

(%I

Anti-HBs(+)

X2

X2 ~

Male High risk Low risk Female High risk Low risk Total High risk Low risk

490 49 1

14.9 12.63

1.06

14.29 11.41

1.82

528 60 1

15.53 9.81

8.40

16.29 13.81

1.35

1018 1092

15.23 11.08

7.96

15.32 12.71

2.94

From Li et al. (1981), Guangxi Health and Antiepidemic Station, with permission. SMR for 1969-1978 was 50.8/100,000. c High-risk villages had 2.4 cases of PLC (1969-1978) and low-risk villages had <2 cases of PLC. (1

PRIMARY LIVER CANCER IN CHINA

303

TABLE VI HEPATITISB INFECTION RATE IN LONCAN Cowryo Number examined

HBsAg(+)

Anti-HBs(+)

Group

Infection rate (%)

PLC families Control families Other inhabitants

370 366 699

24.6 12.0 10.3

11.6 10.1 8.9

36.2 22.1 19.2

(%I

From Guangxi Health and Antiepidemic Station (1978), with permission.

were not significant statistically. But it was found (Li et al., 1981) that the differences in occurrence of anti-HBs among the PLC family members were marked between males and females above 20 years of age. Thus in the PLC families, anti-HBs was present in only 1.6%of the males as compared with 9.4%of the females. In contrast, 18.0%of the males were positive for HBsAg, and 7.5%of the females. The rise of PLC incidence in adults of advancing age is in accord with the steady increase in the rate of HBV infection after 20 years of age. The studies in Chongming County of Shanghai by Gao and associates (1981) are very impressive in the evaluation of the different risk factors in PLC. The county is an island at the mouth of Changiiang River with a population of 780,000. It has the highest PLC mortality rate of the 10 urban counties of Shanghai. Within the island, the eastern part has a consistently higher PLC mortality rate than the western part. From 1974 to 1978 the SMR of PLC in the eastern part, in two communes selected by Gao et al., was 83.2 per 100,000 for males and 24.1 per 100,000 for females. The SMR of PLC of two selected communes in the western part was 27.5 and 15.1per 100,000 for males and females, respectively. From the eastern group, 2804 people (1290 males, 1514 females) were sampled and bled, and 2018 (917 males, 1101 females) were sampled and bled from the western group. R-PHA was used for HBsAg, PHA for anti-HBs, and the IAHA method for antiHBc. The results are shown in Table VII. Both in the high- and low-incidence groups, HBsAg-positive rates for males were slightly higher than for females. Comparing the highwith the low-incidence group, HBsAg-positive rates for both males and females were higher, but the difference was statistically insignificant. Both in the high- and low-incidence groups, anti-HBs-positive rates for females were significantly much higher than for males. Also, the anti-HBs-positive rate within females was especially higher in the low-incidence group than in the high-incidence group. Although the rate of anti-HBs positives was significantly higher in the low-

304

YEH FU-SUN AND SHEN KONG-NIEN TABLE VII COMPARISON OF HBV INFECTION STATUS IN HIGH-AND LOW-INCIDENCE ARE AS^ Percentage positive High-incidence area

Low-incidence area

Markers

Males

Females

Males

Females

HBsAg Anti-HBsb Anti-HBcC

11.6 5.0 13.0

9.1 7.3 11.6

9.9 9.8 13.6

8.4 16.3 14.5

From Gao et al. (1981),Shanghai Tumor Institute, with permission. Within the same area, differences are marked between males and females. These differences are significant in both high-incidence and low-incidence areas. c Note the marked difference of rates in females in high- versus lowincidence areas. 0

b

incidence group than in the high-incidence group, taking all the markers of active HBV infection into consideration, the difference between high- and low-incidence groups became insignificant. Gao and colleagues suggested that although no marked increase of HBsAg carriers in the high-incidence area over the low-incidence area could be seen, it was remarkable that anti-HBs occurred much less frequently in the high PLC incidence area. It was suggested that there might be another factor present in the environment of the high-incidence area which effectively impaired the function of the inhabitant’s immunity and eventually led to liver cancer. Also, a hypothesis was proposed that HBV might have acted with another factor in causing PLC. Since HBsAg occurred more frequently in younger age groups and PLC occurred at a younger age in Chongming County compared to elsewhere, it seems likely that the inhabitants probably suffered from HBV infection and liver damage at a young age and the additional presence of a carcinogen then accelerated the development of PLC.

B. AFLATOXINCONTAMINATION OF FOODS Aflatoxin is one of the most potent carcinogens in nature. There are a number of epidemiological studies supporting aflatoxin as a cause of the prevalence of PLC in Africa and Asia. However, many investigators are still skeptical about the role of aflatoxin in PLC development in human beings, especially after the discovery of HBV and its relation to PLC. Since 1964, Chinese scientists working in the high PLC

PRIMARY LIVER CANCER IN CHINA

305

incidence areas have collected different foodstuffs and repeatedly induced liver cancer by feeding foods that were AFBl contaminated to animals, including rats, ducks, dogs, and pigs (Yen et al., 1975). A nationwide survey of food contamination with AFBl was sponsored by the Health Science Institute in Beijing, followed by the establishment of standards of maximum allowances of AFBl content in peanuts, corn, rice, and other foods (Xu, 1981). I t was found that peanuts and corn are far more easily contaminated than other foodstuffs. The percentage of corn samples from Fusui County exceeding the national maximum allowance of AFBl (20 ppb) was 25.5 times that of rice (Tumor Research Group, Guangxi Medical College, 1984). Since corn is consumed as a staple food, it is far more important than peanuts, which are consumed in small quantities and irregularly. Epidemiological studies in Fusui County and later in other counties in Guangxi showed, with few exceptions, correlation of the percentage composition of corn in the staple food and the mortality rate of liver cancer in the same population. A study in another high PLC incidence area, Qidong County in Jiangsu, showed similar results. In this county corn, wheat, cotton, and peanuts are produced and corn is consumed as staple food in large proportions. Corn is the most common aflatoxin-contaminated staple food. The mortality rate of PLC in any particular commune or village correlates well with the percentage of corn in the staple food, and with the severity of contamination of the staple food. Studies of PLC in domestic animals may also shed light on a possible role of aflatoxin in PLC. In a 1973 survey, of 1321 ducks 3.3% had PLC, and of 81 old, female swine, 5 had PLC (Z. Y. Tang, personal communication, 1983). It is noteworthy that the rate of duck liver cancer correlated with the incidence of human PLC in the same commune (Table VIII). Since domestic animals were usually fed corn in these areas, the role played by aflatoxin appeared to be particularly important. In Fusui County in Guangxi, 2171 domestic animals werk examined in 1974. Liver cancer was found in 6.5% of 46 ducks, 1.8%of 105 dogs, and 0.3%of 1388 hens and cocks (Tumor Research Group, Guangxi Medical College, 1975). Here also, corn was the main feed of these animals. In Chongming Island, Gao and associates in 1980 (Gao et al., 1981) studied the relationship of aflatoxin and PLC. From each of 79 highand low-incidence villages (brigades) five families were randomly sampled. In June 1980 cooked staple food was collected once on the same day from each family. The staple food in the high-incidence villages was mainly corn and rice and that of the low-incidence area

306

YEH FU-SUN AND SHEN KONG-NIEN

TABLE VIII GEOGRAPHIC DISTRIBUTION OF INCIDENCE OF LIVERCANCER IN DUCKS AND HUMANSIN QIDONG COUNTY~

Districts

Detected rate of liver cancer detected in ducks in 1973 (%)

Human liver cancer mortality in 1972 (per 105)

Lusi Wangbao Qi Xi Weidong Jianghai Heidong

1.33 3.00 3.05 6.11 5.36 2.94

36.58 63.73 55.47 62.92 60.33 50.75

From Z. Y. Tang (personal communication, 1983).

only rice. Altogether 395 samples were collected, and AFBl content determined was by thin-layer chromatography. In December, another sampling was done from the same family once on the same day. The intake of AFBl was calculated to be 31 ngkg body weightlday in the high-incidence villages and 9 ngkg body weightlday in the lower incidence villages in the first half year. In the second half year, the respective figures were 79 and 61 ngkg body weighuday; the highincidence group again showed higher intake than the lower incidence group. In Chongming County, those areas producing corn and wheat had the highest SMR for PLC (49.1 per 100,000),areas producing rice and wheat (SMR 28.9 per 100,000) and new farm areas (SMR 25.9 per 100,000)had lower mortality rates, while areas producing a mixture of different grains showed an intermediate SMR for PLC (37.1 per 100,000).This suggests an important role for AFBl in PLC. Although inhabitants of Chongming City proper always consumed rice in the past, the SMR of PLC was as high as 24.3 per 100,000. Accordingly, the authors suggested that AFBl alone is inadequate to explain the high prevalence of PLC in Chongming County, just as HBV infection alone is inadequate to explain the same condition. One has to consider the coordinating action of the two factors as a better explanation. W. G. Li and associates (1983)in 1976 made an extensive survey of epidemiological factors in 30 communes selected from 15 counties located near the estuary of Changjiang extending over Jiangsu, Shanghai, and Zhejiang. These counties are, in general, high PLC incidence areas with marked differences of distribution among them. Interviewers (24 in three groups) were assigned to collect information based on

PRIMARY LIVER CANCER IN CHINA

307

a designed form. The data were studied by multivariate statistical analysis. It was found that the mildew of grains, especially that of corn, the pollution of drinking water, viral B hepatitis, and nitrosamines are potentially the most risky factors for liver cancer. Y.B. Wang and associates (1983) studied the correlation of the geographic distribution of aflatoxin B1 and climate to determine if it related to the occurrence of PLC in China. The study was based on the number of days suitable for the growth of mold (principally AspergilZuspauus). The suitable day was defined as having an average relative humidity <80%, with temperature highs of 30-38°C on the same day. The toxin-producing day was defined as the day with an average humidity of 85% and a temperature high of 28-32°C. A map was drawn of the climate of the whole country. Based on a nationwide survey of AFBl content in grains and oils from 22 provinces and metropolises, a map of the rate of AFBl distribution was also drawn. The map showed that the distribution of corn and peanut AFBl contamination was largely consistent with the distribution of liver cancer. According to Wang et al., this is not a mere coincidence since the high incidence of PLC occurred chiefly in areas in which the climate was suitable for the growth of Aspergillusjlauus. Wang et al. claimed that they did not mean to explain PLC solely on the basis of AFBl contaminations. In Longan County of Guangxi, a high PLC incidence area, a study was made on the inhabitants’ intake of AFBl and the urinary excretion of AFM1. W. J. Hu and associates (1981) reported that of 37 inhabitants consuming corn meal, 34 had an intake of 66 pg AFBl per day, and their urinary excretion of AFMl was 1.38% of their AFBl intake. The concentrations were determined with thin-layer chromatography. In contrast to areas in which corn is the predominant food, bringing an increased risk of aflatoxin contamination, Shunde County (SMR 26.16 per 100,000), as one of the highest incidence counties in Guangdong (SMR 70.73 per 100,000), has been studied. The staple food of the whole county is rice. Corn is scarce and used only as animal feed. In 1981-1982 a survey was made by Jiang et al. (1983) of the Guangdong Provincial Food Hygienic Surveillance Institute. Samples of rice, peanuts, peanut oil, fish, pork, and animal feed were collected from the market and families of the high- and low-incidence villages four times a year. AFBl and sterimatocystin were assayed by thin-layer chromatography. The results showed that with the exception of animal feed, which was heavily contaminated, all samples examined were within the national allowance limit. Comparison of the high PLC incidence villages with the low-incidence ones showed no

YEH FU-SUN AND SHEN KONG-NIEN

308

TABLE IX CORRELATION OF STATUS OF HBsAg AND DEGREE OF AFBl EXPOSURE WITH INCIDENCE OF PRIMARY LIVERCANCER" Group

Person-years observed

Number of PLC cases

PLC incidence (per lo5)

13 2 15

649.35 98.57 372.12

1 0 1

65.92 0 32.80

RR

~

ABFl heavily contaminated area HBsAg(+) HBsAg(-) Total AFBl lightly contaminated area HBsAg(+) HBsAg(-) Total

2002 2029 4031

1517 1530.5 3047.5

9.85

-

11.3

1.0

-

1.0

From Mo et al. (1984),Guangxi Medical College, with permission.

difference in AFBl content and percentage of contamination of the foods. Shunde, a high PLC incidence area, showed no evidence of excessive AFBl exposure within the county itself and in comparison with two high and low PLC incidence counties. The cause of the prevalence of PLC in this county remains to be determined. Pollution of water appeared to be an important risk factor in Guangdong. The coordinated action of HBV and AFBl may be observed from the study of HBsAg carriers and noncarriers in areas with various degrees of AFBl exposure. The previous section on HBV described a prospective study of HBsAg carriers done by Mo et al. (1984).In that same study, areas in which the inhabitants' staple food consisted of up to 20-40% corn, having an average content of AFBl of 53.8-303 ppb, were considered to be heavily contaminated areas. The lightly contaminated areas were those in which rice was exclusively consumed as the staple food; the average AFBl content was less than 5 ppb. The results of a 5-to 8-year follow-up study are shown in Table IX. There were 15 cases of PLC that developed in the area heavily contaminated with aflatoxin and only one in the lightly contaminated area; the RR was 11.3. More specifically, 13 HBsAg carriers developed PLC in the former area and only one HBsAg carrier developed PLC in the latter area, giving an RR of 9.85. The study suggests that there is a coordinating action of HBV infection and aflatoxin exposure in the perpetuation of a high PLC incidence area. The study supported the concept of

309

PRIMARY LIVER CANCER IN CHINA TABLE X CORRELATION OF PLC INCIDENCE WITH SOURCE OF DRINKING WATER,QIDONCCOUNTY, JIANCSU (1971-1972)" Drinking water source

Number of inhabitants

Number of PLC cases

MR (per lo5)

Home ditch Village ditch River Well

28,614 37,941 11,727 5,798

58 49 10

101.35 64.57 42.64 0.86

Total

84,080

4

Ob -

117

69.58

From Su (1980), with permission. To avoid zero, use 0.1 as a substitute in calculations.

coordinating factors proposed by Gao and associates (1981) based on their study on Chongming Island.

C. POLLUTION OF DRINKING WATER It has been repeatedly reported by Chinese investigators that inhabitants using water from ditches or pools had a high PLC mortality rate. This is particularly evident in high-incidence areas in Jiangsu, Guangxi, and Guangdong (Tables X-XII). Typically, in the high-incidence areas there is a pond or ditch in front of a village or a house. Wells were not dug either because the TABLE XI CORRELATION OF MORTALITY RATEOF PLC WITH WATERSOURCE,FUSUI,LONCAN,AND WUMIN COUNTIES OF GUANCXI PLC MR (per lo5) Source of water Pool Shallow well River Stream Deep well

Wuminn

Fusuib

Longan"

71.7 64.21 53.0 34.5 27.58

53.12 42.58 37.94 22.92

50.13

-

-

47.16 43.83 23.86d

Mo et al. (1977). Tumor Research Group, Guangxi Medical College (1974). Hu et al. (1981). d City running water.

310

YEH FU-SUN AND SHEN KONG-NIEN

TABLE XI1 CORRELATION OF PLC WITH WATER SOURCE,SHUNDE COUNTY, SAJIAOCOMMUNE"

MR

Village

of water

Number of inhabitants

PLC deaths (1975-1981)

(per 105)

High

Siao-chong Yang-jiao

Mainly wells Reservoir

2984 1342

15 9

71.82 95.83

Low

Sa-bian Da-zhe

Mainly wells Reservoir

2604 2989

1 2

5.49 9.36

Incidence area

Source

From Zhao (1983), with permission.

water is too salty as at the seacoast or because underground water is too deep to be accessible. Therefore, water supply is dependent on rainfall which drains into pools near the village. Shen and associates (1985),reporting the results of a study in Qidong, concluded that the greater the degree of pollution of the drinking water, the higher the risk of PLC. The degree of pollution and risk from drinking water sources is in the following descending order: house ditch, field ditch, river, shallow well, and deep well. The respective incidence rates of PLC were 141.40,72.32,43.45,22.26,and 11.70per 100,000.It may be expected that one or more carcinogenic substances and/or other cancer promotors of PLC may be present in the polluted drinking water in the Qidong area. In Jiangsu and Guangdong, those counties located near the estuary of the Changjiang or Pearl Rivers are lowlands. Streams running to a river are closed by dams to maintain a steady level. The waters are stagnant most of the year and are much the same as ponds: they are heavily polluted. Su (1980) has reviewed this problem comprehensively using an analytical epidemiological approach. He pointed out that the correlation of PLC with drinking water pollution from different parts of China has been so consistent that some factors in the drinking water must be the major cause of PLC. The most convincing evidence supporting the concept that drinking water is the key factor is the significant drop in the PLC mortality rate in Qidong County after campaigns to improve the quality of drinking water by construction of thousands of wells and the establishment of running water systems in the villages. About 60% of the inhabitants responded to the appeal and switched to wells or river water instead of the ditches and pools. A survey of a commune in 1972-1973 with a population of 84,080 showed a PLC mortality rate of 69.58 per 100,000 (Table X). The

PRIMARY LIVER CANCER IN CHINA

311

mortality rate of the same commune in 1974-1978,5 years later and after the campaign to improve water sources, showed a drop to 56.16 per 100,000. The difference was significant. Su’s analysis of the death rate in population groups with a different water source showed that those who switched to well or river water maintained as low an incidence rate as those consuming well water 5 years earlier, whereas for those continuing to use ditch and pond water the incidence rate was high. The general drop in the PLC incidence rate can only be attributed to the increase in the proportion of the population using wells and river water. The author calculated that if the proportion of the population using different water sources had been unchanged, the expected number of PLC cases 5 years after the second survey in 1978 would be 338.3. However, the actual number of PLC cases that developed was only 247, or 18 cases less than the expected number each year. Thus if the 1.08 million people of the county were to use well water, the expected number of PLC deaths would be 13.6. The county would be no longer be a high PLC incidence one. Intensive studies have been made in China by geologists, soil chemists, environmental health specialists, epidemologists, etc. on the risk factors in the polluted waters of high PLC incidence areas, and have found the following: (1)The polluted water in the highincidence areas is in general below the national health standard for human consumption, with much biological pollution: excessive bacterial count, ammonia nitrogen, nitrites and nitrates, increase of oxygen consumption, etc. (2) Detailed analyses of trace elements in the soil and water and in the body tissue and blood of inhabitants of high- and low-risk villages, between Qidong and Shunde Counties have not yielded clear-cut and consistent results. In Sajiao Commune, a highincidence area of Shunde County, like Qidong County, chemical elements were found to be rich in soil deposits. Guangdong investigators (Li and Shu, 1983) reported lower values of Fe, Ni, Mn, and Cu in the high-risk village soil than in low-risk areas. Analysis of the river water and reservoirs showed (Hu and Chang, 1981) markedly excessive Fe in locations correlating with PLC mortality rates in different communes in Shunde Country. (3) On the other hand, Qidong investigators (Jiangsu Research Group, 1973) reported higher Cu in water, human hair, and blood. There was a positive correlation between the copper concentration in water and mortality rate. Mn was lower and correlated negatively with PLC. In Shunde County human hair showed low Mn values (Z. S. Li et al., 1983) as was true of soil and water, a result consistent with the Qidong findings in drinking water.

312

YEH FU-SUN AND SHEN KONG-NIEN

Discrepancies in the other findings await further confirmation and elucidation.

D. PESTICIDE CONTAMINATION There has been a growing tendency to use modern pesticides in China since 1949. Whether these chemicals have anything to do with the prevalence of PLC is a moot question. An extensive collaborative study in Qidong County was conducted by the Shanghai Jiangsu Health Bureau, Shanghai No. 1 Medical College, beginning in 1972 and continuing for 5 years. Samples of drinking water, foodstuffs such as vegetables and fruits, oil products, and human blood, fatty tissue, etc. were assayed for residues of pesticides 223 and 666. The results may be briefly described as follows. There was no consistent difference between the high-risk and low-risk areas in the concentrations of pesticide residues. The study showed no significant difference between samples of staple foods from different areas. There was some increase of organochlorine in human fatty tissue than that of the Qidong (Z. Y. Tang, personal communication, 1983), but no difference was found in tumor and nontumor subjects. In Fusui County, more pesticides were used in the rice-growing than in the corn-growing areas, yet the incidence of PLC was found to be far higher in the corngrowing areas. There is at present no definite correlation observed epidemiologically between pesticides and PLC prevalence in China.

E. NITROSAMINECONTAMINATION Detailed investigation has been done by the Qidong Liver Cancer Institute in collaboration with the Nanjing Soil Institute of Academia Sinica, the Jiangsu hydrogeology team, and others. It was found that the soil in Qidong was rich in total nitrogen. In 1974, a positive correlation was found between the soil content of nitrites and nitrates and the PLC mortality rate in five counties, ranging from 8 to 61 per 100,000 per year (Hu, 1976). Similar significant differences in total nitrogen and nitrates were found between Tongxin and Xinin Communes (mortality rate 45.27 and 7.31 per 100,000,respectively). These compounds were considered important as precursors of nitrosamines. Analysis of samples of salted vegetables collected from PLC families and controls showed significant differences in the content of nitrosamines. In 1976 nitrosamines were found in 53%of 285 samples from Jiangsu, Zhejiang, and Shanghai. The most common type of nitrosamine found was diethylnitrosamine. There is still insufficient epi-

PRIMARY LIVER CANCER IN CHINA

313

demiological data to ascertain the role of nitrosamines in high PLC areas; additional better controlled studies of this risk factor are needed.

F. PARASITIC INFECTION Earlier researchers indicated that Clonorchis sinesis is a cause of PLC (Hou, 1956).I t is now known that by far the most common form of PLC in China is hepatocellular carcinoma (Li, 1963). Clonorchis sinesis may be correlated with cholangiocellular carcinoma in some populations consuming raw fish food only. Epidemiological studies showed that this parasite had no correlation with hepatocellular carcinoma in most high-incidence areas (Yeh, 1984).

IV. Family Factors

Familial aggregation is common in all high-incidence areas. How much of this is due to exposure to common environmental carcinogens and how much to genetic factors is uncertain. The following studies are helpful. The fact that HBV infection and HBsAg carriers are more prevalent in families of liver cancer patients has repeatedly been confirmed in high-incidence areas. The family aggregation of HBV infection should account for the family aggregation of PLC (Y. C. Li et al., 1981). Yu and associates (Yu et al., 1981)reported that “liver cancer families’’ were more susceptible to aflatoxin mutagenicity as tested by the sister chromatid exchange technique (SCE). Of 109 members in four generations of a liver cancer family in Qidong County, 7 probands and 9 controls were chosen from the same village and tested for SCE. Spontaneous SCEs of cultured peripheral blood lymphocytes from the two groups showed no differences. After the lymphocytes were treated with AFB1, 0.001 pg/ml, the number of SCEs observed in lymphocytes from members of the liver cancer family was significantly higher than in the controls (F = 15.06, p < 0.001). Similar techniques were used (Lou et al., 1982) in Fusui County, another high PLC incidence area. One family with more than two cases of PLC in three generations was chosen for investigation. Another family in the same village with no history of PLC was chosen as the control. Of 29 members of the PLC family and 28 of the control tested for SCE, higher rates of SCEs were observed in the former.

3 14

YEH FU-SUN AND SHEN KONG-NIEN

V. Immunosuppression

As discussed in the previous section, Gao and associates (1981) in Chongming Island found no significant differences in the HBsAgpositivity rate among inhabitants of the PLC high- and low-risk areas, whereas the anti-HBs was significantly lower in the high-risk than in the low-risk areas. It was proposed that immunosuppression of young inhabitants exposed to a toxic environmental substance might be crucial for the perpetuation of a carrier state for HBsAg and for the acceleration of the development of PLC. Aflatoxin was the toxic substance to which the population was most heavily exposed in the high-risk areas of Chongming County. Aflatoxin B1 has been shown to be an extremely potent immunosuppressive agent by Herbert Save1 and associates (1970). Similar studies have been carried out by Chinese investigators in laboratory experiments and in the field. Liu and associates (1981) showed that in vitro human lymphocytes were significantly inhibited by AFBl in Ea rosette formation and that there was a reduction in their mitotic rate. W. L. Wang and Xu (1983) showed that in rats AFBl significantly inhibited the production of antibody-forming cells, the proliferative reaction of the germinal centers in the splenic lymph follicles to the stimulation of antigen, the production of antibody (hemolysin), the dinitrochlorobenzene skin hypersensitivity reaction, and the total complement activity (Xu et aZ., 1983). Tests of the immunity of the inhabitants of higher and lower risk areas were conducted in Qidong and Fusui Counties. 0.T. was tested in Qidong in the high PLC risk and low-risk villages. It was positive in 51% of the former villages and 68% of the latter. In Fusui County, lymphocyte transformation tests were done on family members of persons with liver cancer and controls in the same high-incidence area (Tumor Research Group, Guangxi Medical College, 1977). The positive rates were significantly lower in the families of liver cancer cases than the controls. It is suggested, therefore, that there was a general depression of immunity in the liver cancer families and in the inhabitants of the PLC high-incidence area, presumably as a result of exposure to aflatoxin-contaminated foods. VI. Other Factors

Nutrition. Case control studies of nutrition (Yeh, 1963) showed that there was no significant difference in the principal nutrients of the diet. AZcoho2. Studies showed that there was no significant difference

PRIMARY LIVER CANCER IN CHINA

3 15

between the case and control subjects. No difference could be found between the high- and low-risk populations (Tumor Research Group, Guangxi Medical College, 1972). Herbs and wi2d vegetables. It has been postulated that herbs and wild vegetables might be important, but no carcinogenic plants of any kind were found. Smoking. Case control studies have not shown significant differences (Tumor Research Group, Guangxi Medical College, 1972). VII. Discussion and Summary

There is evidence for and against the etiological factors proposed for PLC, and controversies still prevail among some of the investigators of this country. Even if etiological factors could not definitely be ascertained, preventive measures could be taken by recognizing the risk factors involved. The three most important risk factors of PLC are becoming so evident that preventive measures have been initiated in high-incidence areas. A. HBV INFECTION With few exceptions, the distribution of PLC incidence is parallel geographically to that of HBsAg carrier rate. The demonstration of a high percentage of HBV infection in patients with cirrhosis of the liver and PLC has generally been accepted. Family aggregation of HBV infection is prominent in PLC families, and a mother-to-infant transmission is found to be important in high PLC incidence areas (Li et al., 1981).A peak carrier state in young children seems to be the basis for high PLC incidence in an area. B. AFLATOXINAND NITROSAMINECONTAMINATION Ubiquitous AFBl contamination of certain foods in some high PLC incidence areas has been observed. In spite of the lack of accurate measurements of AFBl intake in high-incidence areas, the predominant contamination by AFBl of corn and peanuts to the exclusion of almost any other food product simplified the estimation of its intake by a population for surveillance of the contamination of corn, peanuts, and peanut oil. In two widely separated high-incidence areas, Fusui and Qidong Counties, in which corn was one of the staple foods, it was found that the intake of AFBl correlated with the prevalence of PLC in a given village or commune. Correlation of AFBl

316

YEH FU-SUN AND SHEN KONG-NIEN

contamination of food with PLC incidence rates was demonstrated between counties and even provinces. Rice is only rarely and very slightly contaminated even in high-incidence areas. As previously described in Section IVYYu and associates showed marked individual differences of susceptibility to the mutagenic effect of AFB1, and PLC family members may be affected by doses below the national health standard of maximum allowance. It is not impossible that some populations with a high PLC rate may be at fault on the host side when AFBl exposure does not seem to be excessive for the general population. Nitrosamines are strongly mutagenic, and preliminary epidemiological studies showed that they are undoubtedly important as a cause of PLC (Hu, 1976). More work should be done to establish their correlation with PLC. C. DRINKINGWATER Evidence is abundant and convincing, as pointed out by Su de-long, that some factors in polluted water must be a contributing factor to the development of PLC, especially in high-incidence areas. Trace elements Fe, Cu, Se, Mn, Ni, and Mo deserve further investigation. Pesticide contamination of food and drink has been demonstrated, but their correlation with the prevalence of PLC requires further investigation. Since chronic hepatitis and hepatic enlargement were common in populations consuming polluted drinking water and since many were non-B serologically, they might be water-borne non-A, non-B hepatitis viral infections as reported in the literature (Spertini and Frei, 1982; Wang and Purcell 1980). D. PREVENTIVE MEASURES In the light of the knowledge accumulated in the world literature and in the studies in China, several measures have been taken by health authorities in the high-incidence areas:

1. First, improvement of the quality of drinking water was undertaken. In Qidong County, with the cooperation of the inhabitants, tens of thousands of new wells were dug and running water was established in many villages. Sixty percent of the population no longer obtains their drinking water supply from ditches and ponds but from wells or river water. In a few years the incidence rate of PLC has dropped significantly. In Fusui County measures were also taken to

PRIMARY LIVER CANCER IN CHINA

317

improve the drinking water supply, but great difficulty was encountered since the underground water was far too deep to utilize. 2. Second, aflatoxin contamination of corn was much reduced following a careful study by a team led by the Food Hygiene Institute from Beijing, and effective measures were taken to minimize the molding of grains during harvest and storage by the farmers. Studies several times a year by the County Liver Cancer Institute showed a steady decline in the severity and extent of AFBl contamination in food in recent years. 3. A Chinese-made HBsAg vaccine had been tried on several thousand inhabitants, which effectively raised the anti-HBs level of those inoculated. Production of the new vaccine on a scale large enough to meet the needs of the people, especially in the high-incidence areas, is expected. 4. Reduction in the area allotted to corn farming with an increase in the area for rice farming has been encouraged by the construction of irrigation systems. A change in the composition of the staple food is expected to occur gradually. 5. The use of antioxidants such as butylated hydroxyanisole (BHA) and herbal drugs to counteract the mutagenicity of aflatoxin exposure has been investigated in experimental animals with success. There is hope that they may be used as treatment particularly for populations at high risk, i.e., inhabitants with positive HBsAg, with a family history of PLC, and living in areas with heavy exposure to AFBI. With all these measures, it is reasonable to expect that the incidence rate of PLC will decrease. Since chronic hepatitis and liver enlargement were common in inhabitants consuming polluted drinking water and since they were non-B serologically, they might well be victims of a kind of waterborne non-A, non-B infection recently reported in the literature (Spertini and Frei, 1982; Wang and Purcell, 1980). This is a subject that should be studied in high PLC incidence areas, but HBV infection is by far the more important cause of PLC, for which the use of HBsAg vaccine should reduce the incidence rate. The risk factors and perhaps the etiologic factors for PLC are probably many rather than just one. One or more factors may be of major importance in one place and absent or of minor importance in another. Thus removal of any of the known risk factors, whichever may be feasible, would help lower the incidence rate of PLC. In conclusion, there is reason to believe that the future will show a lowering of the prevalence of PLC in highincidence areas.

318

YEH FU-SUN AND SHEN KONG-NIEN

VIII. The Early Diagnosis of Primary Liver Cancer

Early diagnosis of primary liver cancer is rather difficult because the disease is usually obscure in its early stage. It became possible to make an early qualitative diagnosis only since the discovery of alphafetoprotein (AFP).

A. QUALITATIVE DIAGNOSIS WITH AFP Since no definite symptoms and signs can be found in the early stage of PLC, the detection of AFP in the serum has been a most important diagnostic procedure.

1 . Detection of AFP AFP is quite specific for the diagnosis of PLC, a positive rate of 5090%being reported in patients with the disease (National Collaborating Group, 1973). When a dynamic quantitative examination of AFP was made in combination with other highly sensitive assays, even higher reliability of diagnosis (over 90%)could be obtained (Tang and Qian, 1977). So far except for histopathological methods the detection of AFP is the most specific in the diagnosis of PLC.

2. The Evaluation of AFP in Early Diagnosis of PLC The detection of AFP in the serum is the only way to diagnose early PLC without symptoms and signs. Since 1971 the AFP assay has been widely employed in our country in mass surveys in both natural and liver-diseased populations, and hundreds of asymptomatic cases of PLC have been detected. AFP assay in the early diagnosis of PLC can be applied to (1)natural populations in areas of high PLC incidence; (2) patients with liver diseases, including hepatomegaly, chronic hepatitis, and liver cirrhosis; (3) populations with a persisting low level of positive AFP, especially those with positive HBsAg; (4) populations with a history of liver disorder for 5-10 years, with SGPT stable and AFP increased. Mass surveys performed twice a year would reveal more cases of PLC. Usually the hemagglutination test with finger blood is used for preliminary screening, and positives are subjected to further testing by counterelectrophoresis, rocket-electrophoresis, and SGPT assay. Diagnosis can be made on the basis of a quantitative and dynamic AFP assay combined with a SGPT test.

PRIMARY LIVER CANCER IN CHINA

319

3. How Early Can the Diagnosis of PLC by AFP Screening Be Accomplished in Mass Suroeys?

The diagnosis of PLC based on detection of AFP by the hemagglutination test may be established on an average of 8 months (Tang et al., 1978) and by counterelectrophoresis 3 months (National Collaborating Group, 1973) before the onset of clinical symptoms. Of patients with PLC who were found by coming to a clinic, only 0.4% were subclinical, whereas in a mass survey of 1.96 million persons conducted in Shanghai in 1971-1976, 300 patients were diagnosed, of whom 44.7% were subclinical (196 out of 300 cases) (Shanghai Coordinating Research Group, 1978). The patients found by mass survey were all positive for AFP, but only one-fifth to one-third were positive for other tests. Of the cancer masses removed from early-diagnosed patients in mass surveys 72.7% were of a diameter less than 5 cm (Tang et al., 1978). The percentage of positive patients found by mass survey at an early enough stage permitting surgical removal of the cancer was three times that of the patients who came to the clinics (56.1% compared to 17.7%). The immune status of the patients found by mass survey was much better than those coming to the clinics ( p < 0.01). 4. Reliability of Diagnosing PLC by Detection of AFP Based on detecting AFP by counter-electrophoresis or a quantitative determination of AFP >500 ng/ml serum persisting for more than 1 month, and excluding pregnancy, acute hepatic diseases, and embryoplastic tumors of genital glands, the reliability of diagnosing PLC was as high as 98.2% in a mass survey of a population of 1.96 million in Shanghai (Shanghai Coordinating Research Group, 1978), and 99% in Guangxi and Jiangsu Provinces. The reliability of diagnosing PLC by detecting AFP with less sensitive methods has been generally recognized, and with highly sensitive methods, it was assumed that specificity would be lowered with increasing sensitivity. However, early diagnosis of PLC is possible only when highly sensitive methods are used for preliminary screening. In cases of PLC accompanied with increased SGPT, the reliability of diagnosis by AFP detection was lowered since it was necessary to differentiate from active hepatitis.

5. Clinical Significance of AFP in Early Diagnosis of PLC If early diagnosis of PLC can be accomplished, it becomes possible to start treatment earlier and thus improve the survival rate. A followup of the survival rate of 300 cases of PLC found by mass survey indicated that it was better for cases with early PLC than for patients

320

YEH FU-SUN AND SHEN KONG-NIEN

at the middle stage of the disease, and the survival rate of the latter was in turn better than those at an advanced stage. A comparison of the long-term survival rates of patients found by mass survey with those coming to the clinic clearly showed the survival rate of the former to be much better. The survival rate was much higher in those patients in whom the cancer mass was removed than in those without surgical treatment, both groups being at the same early stage of the disease. Also, patients with early PLC detected in mass survey apparently had a higher survival rate than those coming to the clinic, although they were given similar surgical treatment (Shanghai Coordinating Research Group, 1978). 6. Relationship of Persisting Low Levels of Positive AFP to PLC A certain amount of AFP, usually not over 400 ng/ml, may be detected in patients with active hepatitis or in the active phase of liver cirrhosis, but it is usuaIly of short duration. A persisting Iow IeveI of positive AFP, especially in the presence of positive HBsAg, suggests the possible existence of PLC, and in such cases AFP examination should be done at regular intervals. In 456 cases of persisting low levels of positive AFP followed up by the Qidong Liver Cancer Institute, the subsequent incidence of PLC was 10.7%within half a year, 26.3%in 1 year, and 40.9%in 1.5 years. A similar observation of 51 cases performed by the Shanghai Institute of Biochemistry showed an incidence of 11.8%,24.5%, and 29.4%, respectively. Thus patients with a persisting low level of positive AFP should be followed up carefully. Another 6-year follow-up of 420 subjects with persistingly low levels of positive serum AFP detected during a screening of the general population from 1974 to June 1976 by the same institute (Zhang, 1986) showed that of 420 cases, 249 (59.0%)were diagnosed to have hepatitis or cirrhosis and 138 (32.9%)to have PLC. Of the latter 75 (54.3%) were proved to have subclinical PLC.

B. ENZYMOLOGICAL ASSAYS Although there are now many serological and biochemical methods which may reflect the existence of liver cancer, they lack the significance of early and specific diagnosis. So far we do not have any enzymological criteria which alone can give a diagnosis of PLC as AFP does, but they are helpful in AFP-negative patients. y-Glutumyltranspeptidase. Abnormal levels of y-glutamyltranspeptidase (7-GT) may be detected in about 90%of patients with PLC. Of

PRIMARY LIVER CANCER I N CHINA

321

17 cases of PLC with negative AFP and diagnosed on a histopathological basis in the Teaching Hospital of Guangxi Medical College in 1984, 58.8%were positive for y-GT, and only 34.6%in cases of early PLC. A dynamic observation showed that the rise and fall of y-GT levels were largely in accordance with the status of the disease. Since y-GT is not specific for liver cancer, it may be abnormal in patients with hepatic cell damage or other liver diseases, while in AFP-positive cases or cases with localized liver cancer it may remain unelevated. However, rising y-GT is still of diagnostic value if nonobstructive or other liver diseases can be excluded. Lactate dehydrogenase and its isoenzymes. While LDH3 increases in the serum of patients with tumors, LDH5 increases when they metastasize to the liver. In the light of a study at Sichuan Medical College on serum LDH and its isoenzymes in 45 cases of PLC and 150 normal individuals, the investigators suggested that the determination of these isoenzymes may serve as a supplement to AFP assay and reduce misdiagnoses in AFP-negative patients. PLC is characterized enzymologically by an increase of LDH3, LDH4, and LDH5, especially of LDH5, which may be 10-22 times higher than in normal individuals. The positive rate may be as high as 89%.In patients with PLC, LDH5 was higher than LDH4, while in those with metastatic liver cancer LDH4 was usually higher than LDHs (Department of Cancer Research, Sichuan Medical College, unpublished, 1984). In patients with early PLC of small size, the positive rate was low because of low specificity. Serum alkaline phosphatase. The positive rate of serum alkaline phosphatase (ALP) in patients who came to the clinic, in those found by mass survey, and in those with small-sized PLC was 54.6-89.4%, 44.7%,and about 25%, respectively. A marked increase of ALP may also be found in secondary liver cancer, other space-occupying lesions, extrahepatic obstructive jaundice, and metastatic bone cancer. A recent report by the Shanghai Institute of Cancer Research indicated that the positive rate of ALP (placenta type) was 66.7% in patients with PLC, was still higher in AFP-positive cases, and may aid in the differential diagnosis of PLC and hepatitis. Serum a-antitrypsin (a-AT). a-AT is an important serum proteinase inhibitor which is synthesized and decomposed in the liver. Its increase may be regarded as a criterion of latent liver cancer; in PLC patients its positive rate is 83.9%and of considerable diagnostic value. However, 6.06%of liver cirrhosis cases and 13.5%of those with hepatitis were also found to be positive for a-AT (Guan, 1983). 5'-Nucleotide phosphodiesterase isoenzyme V . The positive rate of

322

YEH FU-SUN AND SHEN KONC-NIEN

5'-nucleotide phosphodiesterase isoenzyme V in AFP-positive and -negative groups was found to be 87.7 and 87.5%, respectively, and thus was of value in the diagnosis of PLC, especially in AFP-negative cases (Tumor Group, Beijing Clinical Research Institute, 1978). Enzyme spectrum assay. Seven serum enzymes, i.e., glutamic pyruvic transaminase (GPT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), leucine aminopeptidase (LAP), phosphohexose isomerase (PHI), P-glucuronidase (P-G), and y-glutamyltranspeptidase (y-GT), were determined simultaneously. The enzyme spectrum showed y-GT >lo0 pl, PHI >200 plyand LDH >500 pl in more than 50% of patients with PLC, whereas the normal limits of the three enzymes are below 100,200, and 500 pl, respectively. When the activity of these enzymes exceeded the levels mentioned above and GPT was within normal limits, diagnosis of PLC was facilitated. With these standards, diagnosis is made in 92% of patients with PLC and in 73.3%of patients with hepatic metastasis. The enzyme spectrum also has some significance for the diagnosis of diffuse types of PLC (Lu, 1974).

C. OTHERS X-Ray examination of patients with liver cancer at its middle or late stage revealed an elevation or partial elevation of the right diaphragm and limitation of diaphragmatic movement, but it was of no value in the diagnosis of early, small-sized liver cancers. The leukocyte adherence inhibition test was found to have definite organ and cancer specificity for PLC; its positive rate was 76.7%(Liu and Sha, 1984). The diagnostic method of microneedle aspiration and cytological examination has been used since 1980 by the Qidong Institute of Liver Cancer, and a positive rate of 89.5%was reported. It was safe and never caused any complications, but was less useful for diagnosing early or deep-localized liver cancer (Wang and Zhang, 1982).

D. LOCALIZATION DIAGNOSIS With the advance in the qualitative diagnosis of liver cancer, it is now possible to diagnose a very early liver tumor, sometimes one that is less than 1-2 cm in diameter. Consequently, the need for localization diagnosis now becomes more and more urgent. Although in recent years diagnostic techniques such as ultrasonography and radionuclide scanning have been upgraded, it is still unlikely that they will meet our needs for localizing early microsized liver cancer.

PRIMARY LIVER CANCER IN CHINA

323

Ultrasonography. On the basis of typical wave patterns, PLC in middle or late stages may be diagnosed without difficulty by ultrasonography type A, its positive rate being 60%, but it is less useful in the diagnosis of early cases because the wave patterns produced are not characteristic (Shen and Yan, 1981). Ultrasonography type B may reveal lesions 2-3 cm in size. Ultrasonography may have a unique function in differentiating cystic or substantial lesions in the liver. Scintiscanning of liuer. Scintiscanning of liver has a positive rate of detection of 81.5% (Shen and Yan, 1981) in middle and late stages of PLC, 24.7% for PLC cases with tumors 3-5 cm in diameter, and nothing in asymptomatic micro-PLC with positive AFP. Liver scanning with indium-113 may help distinguish PLC from liver cysts and abscesses. Hepatophlebography via umbilical uein. Displacement or obstruction of portal branches are seen in the venous phase. Filling defects and a difference of density are seen in the capillary phases. This type of hepatic angiography can reveal tumors of 2 cm in diameter. Using this technique, we examined seven patients, who then had surgery. Findings by angiography were similar to what was found on surgery. Selective celiac arteriography. The major abnormalities in PLC seen by this method are tumor vessels, portal thrombus, arteriovenous shunts and tumor stain. Some indirect signs include dilatation of hepatic artery, occupying displacement of blood vessels, arterial erosion, and pooling and lake of the contrast material. A ring shape is also significant in the diagnosis of PLC. This method can diagnose 90% of PLC patients, and can reveal tumors about 1cm in diameter. In recent years, a blended type of catheter has been applied to the common hepatic artery in hepatic arteriography, and even to superselective arteriography of the right proper hepatic artery (Lu et al., 1983). Computerized tomography. Computerized tomography has not been used widely in China. Its positivity rate in routine clinical diagnosis and localization is high. For subclinical PLC, according to studies in Shanghai Chong-Shan Hospital (Z. Y. Tang, personal communication, 1983), it is inferior to hepatoangiography and even type B ultrasonography . Peritoneoscopy. Peritoneoscopy permits an accuracy of 74.3% (Chen, 1965) in the diagnosis of PLC, but fails to reveal tumors in the deep part of the liver, on the superior surface adjacent to the diaphragm, in the bare area of the liver, or in the early stages. Rheohepatogram localization of PLC. Of 120 cases of PLC rheohepatograms studied in four hospitals in Jilin city and others, the posi-

324

YEH FU-SUN AND SHEN KONG-NIEN

tive rate was similar to that of AFP detection, and no less than that found on nuclide scanning (Chen et al., 1984).

E. DIAGNOSTIC CRITERIA The diagnostic criteria established at the National Conference of Liver Cancer Control Collaboration in 1977 (Tang and Cao, 1981) are as follows: Pathological diagnosis. Histopathological confirmation of PLC. Clinical diagnosis. The diagnosis of PLC may be established on the basis of one of the following: A positive AFP found by counterelectrophoresis or an AFP level >5OOng/d which persists for over a month (determined by radioimmunoassay) without other evidence of PLC, and where pregnancy, active hepatitis, and embryoplastic tumors of genital glands can be excluded. Clinical manifestations of PLC with additional positive results on three of the following assays: liver isotope scanning (or angiography), ultrasonography (clump or obtuse waves), X ray (localized bulging of the diaphragm), enzymological examinations (two of the three itemsALP, y-GT, LDH-are positive), and where secondary liver cancer and benign liver tumors can be excluded. Clinical manifestations of PLC with additional distant metastatic foci (such as lungs, bone, and supraclavicular lymph nodes), bloody ascites (visible to the naked eye), or cancer cells in ascitic fluid, and where secondary liver cancer can be excluded.

The clinical use of AFP assay has played an important role in the early diagnosis of PLC and has greatly improved its accuracy. Nevertheless, there are still the 15% of PLC patients who are AFP negative or have a low level of AFP for whom there are no specific diagnostic methods as yet. Thus further study is needed for diagnosis of patients who are asymptomatic or AFP negative with early microsized PLC.

F. DIFFERENTIAL DIAGNOSIS The early diagnosis of PLC now depends mainly on AFP determination, but AFP may also be present in a few patients with tumors of the alimentary tract or other diseases, especially hepatitis and liver cirrhosis in its active phase. These conditions may be confused with PLC and cause misdiagnosis. Chronic hepatitis and liver cirrhosis. The liver is usually enlarged,

PRIMARY LIVER CANCER I N CHINA

325

firm in consistency, and nodular on palpation. Differential diagnosis is quite difficult, so it is necessary to do quantitative assays of AFP at regular intervals and watch carefully for the development of symptoms and signs. Persistent pain in the hepatic region and rapid emaciation or local enlargement of the liver within a short period may be warning signs of PLC. In addition, in hepatitis and liver cirrhosis AFP synthesis may take place in the regenerated hepatic cells, but the appearance of AFP in the serum of patients is usually transient (less than 2 months). The fluctuation of AFP in cases of hepatitis is usually accompanied by a simultaneous rise or fall of SGPT. If SGPT is normal or decreases while AFP is persistently positive or even increases, that is, the two dynamic curves exhibit a state of dissociation, PLC should be suspected. Secondary liver cancer. There is often a history of primary cancer, but when it is small and latent, differentiation is difficult. Generally speaking, in cases of secondary liver cancer, the disease may progress slowly and the tumor mass may exist as scattered nodules. There is no complication of cirrhosis, and HBsAg and AFP are usually negative. Enzymological determination shows LDH4 > LDH5. Angiography may also aid in the differential diagnosis. Liver abscess. The disease is characterized by acute onset, high fever, hepatomegaly, manifestations of systemic infection, and tenderness and edema in the hepatic region. Ultrasonography shows fluid level waves. Diagnosis is not difficult. However, if the abscess is located deep in the liver and the disease takes a chronic course with mild fever, loss of appetite, emaciation, and an enlarged and tender liver, and if radionuclide scanning demonstrates a space-occupying lesion or ultrasonography shows an atypical wave pattern due to the presence of necrotic tissue and thick pus, differential diagnosis would be difficult. Some rapidly developing liver cancers of the huge-mass type, as a result of central necrosis or hemorrhage, may produce irregular fever, leukocytosis, and pain in the hepatic region, highly resembling liver abscess. If AFP is positive, diagnosis of PLC may be established; when negative, diagnosis is uncertain, and it is necessary to review carefully the disease history, study its mode of onset, and do repeated X-ray examinations, ultrasonography, radionuclide blood pool scanning, etc. Tentative treatment with antiamebic drugs, especially metranidazole, should be tried. Liver biopsy may be recommended if deemed necessary. Congenital liver cyst. The disease progresses slowly, and the AFP test is negative. Qualitative diagnosis and localization of the cyst may be established by ultrasonography and radionuclide scanning.

326

YEH FU-SUN AND SHEN KONG-NIEN

Angioma. The disease also progresses slowly, and the AFP test is negative. Small angioma is usually found when ultrasonography type B is performed. Blood pool scanning may demonstrate a higher concentration of radionuclide than in normal tissues. Liver sarcoma. The disease is rare in occurrence, is AFP negative, and is usually not associated with cirrhosis. It is difficult to differentiate from cancer unless liver biopsy is performed. False-positiue AFP. A positive AFP, especially with less sensitive tests such as agar diffusion and counterelectrophoresis, would be strong evidence for a diagnosis of PLC. But embryoplastic tumors, some of the tumors of the alimentary tract, hepatitis, and pregnancy may also lead to the presence of AFP. and in such cases a dynamic change of AFP should be noted during diagnosis. In hepatitis and pregnancy AFP is of low level and exists transiently. In patients with positive AFP but without definite symptoms and signs, a routine genital examination is necessary in order to exclude the possibility of embryoplastic tumors. In a few patients with tumors of the alimentary tract, the primary focus may be so small that it cannot be found and diagnosed, and in these cases laparotomy should be done if AFP determination shows that it meets the diagnostic criteria for PLC. CZinicaZ types and stages of PLC. According to the criteria established at the National Conference of Liver Cancer Control Collaboration in 1977, the clinical types and stages of PLC are classified as follows: SimpZe type: PLC without clinical and biochemical evidence of cirrhosis. Cirrhotic type: PLC with apparent clinical and biochemical findings of cirrhosis. Inflammatory type: PLC rapidly progressing, accompanied by continuous high fever or by persistent elevation of SGPT by an amount more than double the normal value. Stage Z : PLC without definite symptoms and signs. Stage ZZ: PLC over the criteria of stage I but without evidence of stage 111. Stage ZZZ: PLC with one of the following: cachexia, jaundice, ascites, or distant metastasis. IX. Summary and Conclusion

Epidemiological studies in different areas in China have revealed several outstanding risk factors of PLC, i.e., HBV infection, pollution

PRIMARY LIVER CANCER IN CHINA

327

of drinking water, contamination of food by AFBl and/or nitrosamines, and family predisposition. Accordingly, a program of HBV vaccination, improved supply of drinking water, better preservation and storage of food, and possibly chemoprevention for high-risk populations should be effective preventive measures. Studies have shown that frequent AFP screening in high-risk populations is highly recommended to detect early cases of PLC. According to research in Qidong, careful follow-up of the dynamic changes of AFP in individuals with persistent low levels of positive AFP is important for distinguishing other conditions from true PLC. Newer means for the localization of small-size PLC (under 5 cm), such as type B ultrasonography, nuclide scanning, computerized tomography, and hepatoangiography, represent remarkable progress in improving markedly the success of surgery and hence the survival rate of PLC patients. The advances in knowledge of PLC have been encouraging. Although much work remains to be done on the etiological agents and the mechanism of oncogenesis, it is time that larger scale control measures be put into effect in high-incidence areas to discover if one of the most common cancers in the world can be controlled. NOTEADDEDIN PROOF Recent work at Qidong County (personal communication) showed a consistently negative correlation between regional distribution of liver cancer incidence and the selenium content of blood and grains of the inhabitants. Another important advance is the development of a new system of technology by the Cancer Institute of the Chinese Academy of Medical Science which permits simple and accurate estimation of individual intake of aflatoxins by concentration and examination of 24-hour urine. This will be crucial in the establishment of a causal relationship between aflatoxin and primary liver cancer and hence the design of proper preventive strategies.

ACKNOWLEDGMENT We are grateful to Drs. R. Q. Yan and C. C. Mo for the critical reading of this review paper. We also appreciate the valuable help of the staff of the Guangxi Medical College, particularly Drs. Z. G. Xu and Y. D. Wu, in the preparation of the manuscript.

REFERENCES Chen, Y. X., Gu, Q. K., and Zheng, H. Y. (1984). Chin. J . Oncol. 6(2), 109. Gao, R. N., Tu,J. T., and Gao, Y. T. (1981). Tumor 1,4. Gu, S . Y., et al. (1976). Qidong Liver Cancer Research (1972-1976). Qidong Tumor Control Office (unpublished), Gum, S. F. (1983). Cancer Control Res. 10(3), 174. Guangxi Health and Antiepidemic Station (1978).Nut. Med. J . China.

328

YEH FU-SUN AND SHEN KONG-NIEN

Guangxi Tumor Mortality Coordinating Study Group (1978). Selected Papers of Liver Cancer Research. Fusui Tumor Control Office (unpublished). Hu, C. S., and Chang, T. T. (1981). Enuiron. Sci. China 1(5), 68. Hu, W. J., Ding, Z. R., and Nong, Z. Y. (1981). Guangxi Health and Antiepidemic Station (unpublished). Hu, Y. M. (1976). “Qidong Cancer Research (1972-1976)” (unpublished). Jiang, J. F., Hu, J. K., Li, H., Liang, C. D., Zang, Y. S., and Dai, Y. S. (1983). “The Seventh Symposium on Liver Cancer of Guangzhou Area.” The Liver Cancer Research Collaborating Group (unpublished). King, H., and Haenszel, W. (1973).J . Chronic Dis. 26, 632. Li, C. S. (1963). Chin. J . Pathol. 7, 188. Li, J. Y. (1982). Natl. Cancer Inst. Monogr. 62, 17. Li, P., and Li, J. Y. (1980). Chin. J . Oncol. 2, 1. Li, P., and Li, J. Y. (1981). “Epidemiology, Causation and Approaches to Therapy” (P. A. Marks ed.) pp. 43-64. Grune & Stratton, New York. Li, Q. J., and Shu, F. (1983). “The Seventh Symposium of Liver Cancer Research of Guangzhou Area” (unpublished). Li, Y. C., Chen, K. L., Au, A. P., and Huang, N. C. (1981). “Guangxi Health and Antiepidemic Station,” p. 93. Li, Z. S., Chen, S. Y., and Shen, S. T. (1983). “The Seventh Symposium on Liver Cancer of Guangzhou Area.” The Liver Cancer Research Collaborating Group (unpublished). Liu, Q. Q., and Sha, W. (1984). Chin. I . Intern. Med. 23(2), 87. Lou, S., Huang, W. J., Mo, C. C., Su, C. K., Chang, J. J., and Yeh, F. S. (1982). Guangxi Med. Coll. Bull. No. 4, p. 14. Lu, J. H., Li, W. G., Jiang, T. Y., Ni, J. P., Huang, F. (1983). Chin. J . Oncol. 5(6), 406. Lu, J. Z. (1974). Tumor 8(5), 231. Mo, C. C., Cong, W. H., Zhou, C. C., Cheng, C. C., Su, C. K., and Wei, J. Y. (1984). Guangxl Med. Coll. Bull. No. 1, p. 11. National Cancer Control Office of the Ministry of Health and Nanjing Institute of Geography of Academia Sinica (1981). Atlas of Cancer Mortality in the People’s Republic of China. China Map Press. National Collaborating Group (1973). Paper presented at the 2nd International Congress of Early Diagnosis and Prevention of Cancer, Bologna, Italy. Ong, G. B., Patrik, K. W., and Chan (1976). Surg. Gynecol. Obstet. 143, 31. Qian, S. Q., and Tang, Z. Y. (1977). Jiangsu Med.J. No. 1, p. 501. Savel, H. et al. (1970). Proc. SOC.Exp. Biol. Med. 134, 1112. Shanghai Coordinating Research Group for Carcinoma of Liver (1978). Nut. Med. J . China 58,589. Shanmugaratnam, K. (1965). Br. J . Cancer 10,232. Shen, J. T., Li, W. G., Chen, J. G., and Xie, J. R. (1985).JiangsuMed.J. No. 1, pp. 28-30. Shen, K. N., and Yan, Z. B. (1981). Guangxi Med. Coll. Bull., Suppl. 96. Skinhoj, P., Hansen, J. P. H., and Mikkelsen, F. (1978). Am. J . Epidemiol. 108, 121. Tang, Z. Y. (ed.) (1981). “Primary Liver Cancer.” Shanghai Science and Technology Publishing House, Shanghai. Tang, Z. Y., and Cao, Y. J. (1981). In “Primary Liver Cancer” (Z. Y. Tang, ed.), pp. 226229. Shanghai Science and Technology Publishing House, Shanghai. Tang, Z. Y., and Qian, S . Q. (1977).Jiangsu Med. 3,501. Tang, Z. Y., and Qian, X. G. (1977).Jiangsu Yiyue 11, 501.

PRIMARY LIVER CANCER IN CHINA

329

Tang, Z. Y., Yu, Y. Q. et al. (1978). Nut. Med. J . China 58,608. Tang, Z. Y., Yu, Y. Q., Zhou, X. D., et al. (1978).Acta Acad. Med. Primae Shanghai 5, 118-125. Tang, Z. Y., Yu, Y. Q., Liu, Z. Y., et at. (1983). Chinese Med. J . (Engl.)96, 147-150. Tumor Group, Beijing Clinical Research Institute (1978)J.Tumor Control Res. No. 4, p. 72. Tumor Research Group, Guangxi Medical College (1972).Guangxi Med. Coll. Med. In$ 1, 1. Tumor Research Group, Guangxi Medical College (1974).Guangxi Med. Coll. Med. Znf. 1, 1. Tumor Research Group, Guangxi Medical College (1975).Guangri Med. Coll. Med. Znf. 3,33. Tumor Research Group, Guangxi Medical College (1977a).Cuangri Med. Coll. Bull. 1, 73. Tumor Research Group, Guangxi Medical College (197713).Cuangxi Med. Coll. Bull. 4, 123. Tumor Research Group, Guangxi Medical College (1978).Guangxi Med. Coll. Med. In$ pp. 1-7. Wang, D. C., and Purcell, R. H. (1980). Lancet 2, 876. Wang, N. J., and Zhang, B. C. (1982). Tianjin Med. J . Suppl . Tumor 9(3), 165. Wang, W. L., Xu, F. N., Tang, D. P., and Liu, Y. K. (1983). Guangxi Med. Coll. Tumor Symp. pp. 88-94. Wang, Y. B., Lan, L. T., Yeh, B. F., Zhu, Y. Z., Liu, Y. Y., and Li, W. G. (1983).Sci. Sin. B No. 5, p. 432. Xu, D. D. (1981).I n “Primary Liver Cancer” (Z. Y. Tang, ed.), p. 108. Shanghai Science and Technology Publishing House, Shanghai. Xu, F. N., Wang, W. L., Tang, D. P., and Liu, Y. K. (1983).Guangxi Med. Coll. Tumor Symp. pp. 84-87. Yan, R. Q., et al. (1975). Tumor Preuent. Treat. Studies 2, 13. Ye, B., Shu, Y. C., Li, W. G., and Wang, H. Y. (1981).Chin.J . Epidemiol. 1, 117. Yeh, F. S. (1984). Guangri Med. J . 6(5), 226. Ying, Y. Y., Yan, R. Q., Xu, B. D., Wang, I. L., and Qian, Y. L. (1984). Chin. Med. J . 97(lo), 758-764. Yu, S. J. (1981). In “Primary Liver Cancer” (Z. Y. Tang, ed.), pp. 55-84. Shanghai Science and Technology Publishing House, Shanghai. Yu, S. J. (1983).“Etiologyand Control of Liver Cancer,” Workshop Lect. Notes, Shanghai No. 1 Medical College (unpublished). Yu, X. S., Xu, X., He, F. F., Yao, L., and Zhu, X. L. (1981). Tumor 1(1), 11. Zhang, B. C. (1985) Chin. J . Oncol. 7(1), 26. Zhuan, C. K., Gao, R. N., Gu, Y. J., Yan, S. G., Feng, L. F., and Wu, Z. Y. (1984). Tumor 4(1), 9.

This Page Intentionally Left Blank

INDEX

A Adenovirus, E l A gene, transcription activation, 284-288 AEV, see Avian erythroblastosis virus Aflatoxin B1,food contamination, PLC and, 304-309,315-316 corn contamination, 305-307 correlation with hepatitis B virus antigen carriers, 308 peanut contamination, 305,307 rice contamination, 305-307 wheat Contamination, 305-306 Alkaline phosphatase, PLC diagnosis and, 321 Amplification, oncogenic in DMINs, 235,239-244 expression elevation, 261 c-Ki-ras in Y1 cells, 261-262 c-myb, mechanism of, 263 c-myc, 261,263-264 N - ~ v c261-263 , in HSR, 235,239-244 mechanisms of, 247-249 model, 249 in multistep carcinogenesis multiple c-onc lesions and, 272-273 preexisting mutations and, 270-271 role in leukemia, 271-272 AMV, see Avian myeloblastosis virus Angioma, distinction from PLC, 326 Antibodies monoclonal to TdT, mouse, 41 polyclonal to TdT, rabbit, 39-41 cross-reactivity, 40-41 58-kDa peptide detection, 41 Antigens hepatitis B virus, carriers and PLC incidence, 299-304 coordination with aflatoxin B1exposure, 308 large T of SV40, transcription activation, 289 a-Antitrypsin, serum, PLC diagnosis and, 321

Arteriography, selective celiac, PLC localization, 323 Avian erythroblastosis virus (AEV) AEV-H strain v-erbB allele, 140-144, 147 AEV-R strain v-erbA allele, 141, 143-144, 147 v-erbB allele, 140-142, 144-147 strains, oncogenes, properties, 101103 Avian leukemia viruses acute, oncogene-containing AEV subgroup, 101-103; see also Avian erythroblastosis virus AMV subgroup, 101-104; see also Avian myeloblastosis virus MC29 subgroup, 101-102, 104; see also Myelocytomatosis virus lymphatic (leukosis), lacking oncogenes, 100-101, 105-106; see also Avian lymphoid leukosis virus Avian lymphoid leukosis virus B-cell lymphoma induction, 128-129, 265-266; see also Avian leukemia viruses, lymphatic Avian myeloblastosis virus (AMV) AMV strain, v-myb allele, 152-157 E26 strain v-ets allele, 152-156 v-myb allele, 152-156 strains, oncogenes, properties, 101103

B Blood, Tdt+ lymphocytes neonatal in humans, 45 transient in rodents, 42-43 Bombesin-like peptides, small-cell lung cancer, 259 Bone marrow, TdT+ lymphocytes, ontogenY in humans, 44-45 in rodents, 42-44 331

332

INDEX

Breast cancer glutamate-pyruvate transaminase locus and, 26 predisposition in families, 8-12, 16-17 segregation analysis, 22 Burkitt lymphomas, human c-myc activation without IgH locus, 212 gene product, 204 recombination with IgH, 189-191, 197-198 breakpoints, 198-201 expression deregulation and, 207210 mechanism of, 203-204 translocation, 130-131,266-267 c-myc mRNA transcription, 205-207 translation, 267-268 IgH locus, translocation breakpoints, 201-202 mechanism of, 203-204 variant translocations, 212-215

C Cancer, familial aggregation 1900-1930 assays epidemiology, 5-6 genetics, 6-7 statistics, 5 1930-1970 assays breast cancer, 8-12 leukemia, 12-13 population-based survey, 10-11 site of cancer, 8 current assays breast cancer, 16-17 children with cancer, 17 genetics, 18-19 statistics, 14-15, 18-19 genetic epidemiology, 19-28; see also Segregation analysis, Linkage analysis PLC in China, 313 reports through nineteenth century, 25 Carcinogenesis, multistep c-onc amplification and, 270-273

retroviruses with two v-onc and, 165168 Carcinogens, Xiphophorus sensitivity to, 76-81 Cell cycle, c-myc mRNA during, 195, 196 Cellular oncogenes (c-onc) activation by amplification, 111,231-273; see also Amplification, oncogenic mechanisms of, scheme, 238-239 by multiple lesions, 272-273 by mutation, 107, 109-110,270-271 lym gehes from B- and T-lymphomas, 109-110 rus gene family, 109 by translocation, see Translocation, chromosomal c-ubl, translocation, 111 in chronic myeloid leukemia, 220223 c-bcl-1, translocation in B-cell tumors, 217-218 c-bcl-2, translocation in B-cell tumors, 217-2 18 c-bcr, translocation in chronic myeloid leukemia, 220-223 c-erbA, chicken, human function, 151-152 structure and expression, 150 c-erbB, chicken EGF receptor encoding by, 150 structure and expression, 148 c-erbB, human amplification, 251 EGF receptor coding by, 150-151 homology to EGF receptor gene, 148-150,260 c-ets, chicken function, 159 structure and expression, 158 c-Ki-rus, amplification and expression, 261-262 c-mil, chicken homology to v-mil, chicken, 132133, 137 oncogenic activation, 139-140 protein product of, 138-139 transduction, recombination in, 161162

333

INDEX

c-myb, chicken function, 158-159 structure and expression, 157-158 c-myb, murine, amplification, 263 c-myc activation without IgH, 211-212 amplification in DMINs and HRS, 241-244 expression elevation, 261, 263264 in promyelocytic cells, 271-272 rearrangement and, 246 in small-cell lung cancer, 257-259 in B-cell lymphomas, 128-129,265266 CAT gene transcription activation, 291 conformation, 194 functions, 126-131 growth factor and, 195-196 homology to N-myc, neuroblastoma, 251-252 lability, 194 recombination with IgH, 189-191, 197-199 expression deregulation and, 207210 mechanism of, 203-204 structure, chicken, human, murine, 124-125,191-193 in T-cell lymphomas, 129, 266 transcription control and, 196 transduction, recombination in, 161162 translational products, 126 translocation, 110; see also Translocation, chromosomal c-put-1, variant translocation in plasmacytoma, 215-217 c-ruJ human, in small-cell lung cancer, 259 c-ruf, murine homology to v-raJ 138 oncogenic activation, 139-140 protein product of, 138-139 c-ras mutant alleles during tumorigenesis, 273 distinction from retroviral v-onc, 105107, 163-165

multistep carcinogenesis and, 165-166 N-myc in neuroblastoma, 251-257, 261, 263; see also Neuroblastomas N-rus in neuroblastoma, 255 transcriptional activation, 110 transduction by retroviral vectors, 105-109 proviral integration, 160-161 recombination, 161-163 Chronic hepatitis, distinction from PLC, 324-325 Clonorchis sinensis, PLC in China and, 313

D DMINs, see Double minute chromosomes DNA amplification, see Amplification, oncogenic N region coding for Ig heavy chains, in TdT+ cell lines, 50-51 polymorphism, RFLP, 25-26 translocation, see Translocation, chromosomal Double minute chromosomes (DMINs) amplified c-onc, location, 242-243 (table) C - ~ V C ,241-244 in cell cultures, 239-240 DNA amplification in tumor cells, 235, 239-241 rearrangements and, 245-246 in drug-resistant tumor cells, 235, 240-241 in metaphases, 239 Drinking water, sources in China, PLC and, 309-312 biological and mineral pollutions, 311

E EGF, see Epidermal growth factor EGF receptor coded by c-erbB gene in A431 tumor cells, 251 avian, human, 150-151 structure, human, 149

334

INDEX

Electron microscopy macromelanophores and melanoma cells, Xiphophorus, 82-83 TdT in lymphocytes, 53-54 Epidermal growth factor (EGF) c-myc and, 195-196 distribution, 259 physiological functions, 259-260 Erythroblastosis, induction by AEV strains, 101-103

F False-positive diseases, distinction from PLC, 326 a-Fetoprotein, in PLC early diagnosis clinical significance, 319-320 detection in serum, 318 mass surveys, 318-319 persisting low levels and PLC, 320 reliability of, 319

G Gene products c-ets, identification, 158 c-Ki-ras, expression in YI cells, 261262 c-mil(raf), identification, 137-139 c-myc identification, 126 in tumors and normal cells, 204, 268-269 EIA, adenoviral, transcription activation cellular factor and, 285, 287, 288 E2 promoter and, 286-288 E3 promoter and, 287 v-erbB, identification, 146-147 v-ets, identification, 156 v-mil, structure and function, 136-137 v-myb, identification, 156 v-myc, properties, 123 v-raj, structure and function, 136-137 Genes activation in transformed cells, 283, 293

amplification in normal cells, 270 CAT, transcription activation by c-myc, 29 1 Diff (differentiation), anti-oncogenic, Xiphophorus hybrids, 84-86 EGF receptor, human homology to c-erbB, 148-150,260 malignancy and, 260 enu, weakly oncogenic retrovirus, 105- 106 Est-1 (esterase-1), in benign melanomas, Xiphophorus hybrids, 84-85, 88 g (golden), anti-oncogenic, Xfphophorus hybrids, 84,88-89 gag, weakly oncogenic retrovirus, 105-106 glutamate-pyruvate transaminase, breast cancer and, 26 HLA region Hodgkin’s disease and, 27 malignant melanoma and, 26-27 hsp70, induction by adenovirus ElA in HeLa and 293 cells, 292-293 IgH (immunoglobulin heavy chain) formation by somatic recombination, 197 recombination with c-myc, 189-191, 197-199 atypical in two plasmacytomas, 210-21 1 c-myc expression and, 207-210 mechanism of, 203-204 translocation breakpoints, B-cell tumors, 201-202 Ig light chain loci, translocation and, 214-2 15 immediate early of herpesvirus, transcription activation, 290-291 macromelanophore loci, Xiphophorus, 65-69 carcinogen-induced changes, 76-81 in hybrids with melanomas, 69-75 oncogenes, see Oncogenes pol, weakly oncogenic retrovirus, 105106 retroviral transforming, homologs in Xiphophorus, 90-92 TdT, evolutionary structure, 55-57 B-tubulin, induction by adenovirus EIA, 293

335

INDEX

Genetics, cancer heredity assays, human current state, 17-18 during early twentieth century, 6-7 linkage analysis, 23-28 segregation analysis, 20-23 Glioblastoma multiforme, human c-erbB amplification, 261 characteristics, 260-261 EGF receptor kinase content, 260 y-Glutamyltranspeptidase, PLC diagnosis and, 320-321 Growth factor, see Epidermal growth factor (EGF)

H Hepatitis B virus, PLC in China and, 299-304,315 antigen carriers and PLC incidence, 299-304 Hepatophlebography via umbilical vein, PLC localization, 323 Herpesviruses, immediate early genes as transcriptional activators, 290-291 HSV I and, 290 pseudorabies and, 290 gene toxicity to cells, 290 Hodgkin’s disease, HLA region and, 27 Homogenously staining chromosome regions (HSR) amplified c-onc, location, 242-243 (table) c-myc, 241-244 in cell cultures, 239-240 DNA amplification in tumor cells, 235, 239-241 rearrangements and, 245-246 in drug-resistant tumor cells, 235, 240-24 1 HSR, see Homogenously staining chromosome regions

I Immunoperoxidase, TdT in lymphocytes and, 52-53 Immunosuppression, in PLC, 314 datoxin B1 effect, 314

L Lactate dehydrogenase and its isozymes, PLC diagnosis and, 321 Leukemia acute myeloid chromosome abnormalities, 224225 TdT+ cells and, 47-48 chronic granulocytic, c-myc amplification and, 271-272 chronic myeloid, Philadelphia chromosome, 9;22 junction, 220-223 predisposition in families, 12-13 promyelocytic, c-myc amplification and, 271-272 TdT as marker of, 46-49 pre-B cell lines and, 50 pre-T cell lines and, 50 Linkage analysis, human application, 27-28 breast cancer, 26 DNA, RFLP, 25-26 polymorphic markers, 25 Hodgkin’s disease, 27 malignant melanoma, 26-27 recombination frequencies in pedigrees, 23-24 computation, 24 Liver abscess, distinction from PLC, 325 cancer in ducks and humans, geographical distribution in China, 306 primary in humans, see Primary liver cancer secondary, see Secondary cancer congenital cyst, distinction from PLC, 325 sarcoma, distinction from PLC, 326 TdT+ lymphocytes, ontogeny in humans, 44 in rodents, 42-43 Long terminal repeat (LTR), retroviral in B-cell lymphomas, 128-129,265 in T-cell lymphomas, 129, 266 transcription induction by HTLV I and I1 viruses, 289 LTR, see Long terminal repeat Lung, TdT+ lymphocytes, transient, in rodents, 42

336

INDEX

Lymphocytes, TdT+ populations in acute leukemia, human, 46-49 morphology, 51-55 ontogeny in blood, 42-43,45 in bone marrow, 42-45 in liver, 42-44 Lymphomas B-cell, avian lymphoid leukosis virusinduced c-myc role, 128-129,265-266 viral LTR and, 128-129, 265 Burkitt, see Burkitt lymphomas T-cell, retrovirus-induced, c-myc and, 129,266

Macromelanophores, Xiphophoncs incomplete differentiation, melanoma and, 82 melanomas in hybrids and, 69-75 spot pattern, 65-68 MC29, see Myelocytomatosis 29 Melanoma, malignant, human HLA region and, 26 linkage analysis, 26-27 Melanomas, Xiphophorus (fish) benign esterase-1 gene and, 84-85, 88 phenotype, 84-86 in hybrids, 69-75 hereditary changes and, 81 macromelanophore loci and, 71, 7374 morphology, 72 reversibility, 70-71 macromelanophore lineage, 82 anti-oncogenes and, 84-89 differentiation gene effect, 84-86 golden gene effect, 84, 88-89 ultrastructure, 82-83 malignant, phenotype, 84, 87 spot pattern and, 64-68 spot pattern loci and, 65-69 N-Methyl-N-nitrosourea (MNU), carcinogenesis in Xiphophoncs hybrids and, 76-80,87-88

Mitogenic response, c-myc mRNA and, 195,269-270 MNU, see N-Methyl-N-nitrosourea MSV, see Murine sarcoma virus Murine sarcoma virus (MSV), v-raf allele in 3611-MSV strain, 132-137 Myeloblastosis, induction by AMV strains, 101-104 Myelocytomatosis, induction by MC29 strains, 101-102,104 Myelocytomatosis virus 29 (MC29) MH2 strain carcinogenesis and, 167 v-mil allele, 132-137 v-myc allele, 114-116, 119-121 strains, oncogenes, properties, 101102,104 v-myc alleles CMII strain, 114-118 functions, 121-124 MC29 strain, 114-117 OK10 strain, 114-116,118-119

N Neuroblastomas N-myc gene amplification, 252-256 DNA structure of, 256 translocation and, 254 tumor progression and, 255 copy number, prognosis and, 256257 expression elevation, 263 homology to c-myc, 251-252 N-ras gene, 255 Nitrosamine contamination, PLC in China and, 312-313 5’-Nucleotide phosphodiesterase isozyme V, PLC diagnosis and, 321322

0 Oncogenes activation from proto-oncogenes, 63 basic definition, 111-113

337

INDEX

cellular, see Cellular oncogenes (cone)

future research, 168-170 identification in Xiphophoms by DNA-mediated transfer, 89-90 homologs to retrovirus transforming genes, 90-92 neoplasia and, evidence for connections, 94 retroviral, see Viral oncogenes (v-onc) in retroviruses, 236-237 (table) in tumor cells, 237 (table)

P Peritoneoscopy, PLC localization, 323 Pesticide contamination, PLC in China and, 312 Plasmacytomas, murine c-myc activation without IgH locus, 212 gene product, 204 mRNA transcription, 205-207 translocation, 189-191, 197-198,266 IgH locus, translocation atypical, 210-211 breakpoints, 201-202 c-myc expression and, 207-210 mechanism of, 203-204 variant translocations, pot-1 and, 215217 PLC, see Primary liver cancer Primary liver cancer (PLC), in China adatoxin B1 food contamination, 304309, 315-316 clinical types and stages, 326 Clonorchis sinensis infection and, 313 distribution geographical, 297-298 population, 298-299 drinking water pollution and, 309-312, 316 early diagnosis clinical, 324 distinction from other liver diseases, 324-326 enzymological assays, 320-322 a-fetoprotein in serum, 318-320

localization, 322-324 pathological, 324 familial aggregation, 313 hepatitis B virus infection and, 299304,315 immunosuppression and, 314 pesticide contamination and, 213-313, 316 preventive measures, 316-317

R Restriction fragment length polymorphism (RFLP), human genome, 2526 Retinoblastomas, N-myc expression, 261, 263 Retroviruses oncogenes, 236-237 (table); see also Viral oncogenes (v-onc) with two oncogenes, carcinogenesis and, 165-168 RFLP, see Restriction fragment length polymorphism Rheohepatogaphy, PLC localization, 323-324 RNA, messenger (mRNA) in Burkitt lymphoma, 267-268 c-myc during cell cycle, 195, 196 during embryogenesis, 194 mitogenic response and, 195,269270 transcription in tumors and normal cells, 205-207 EIA, adenoviral, three species, 284 Rous sarcoma virus (RSV),src oncogene, 106 RSV, see Rous sarcoma virus

S Scintiscanning, PLC localization, 323 Secondary liver cancer, distinction from PLC, 325 Segregation analysis, human application, 27-28

338

INDEX

cancer transmission in pedigrees, 2023

breast cancer, 22 models, 20-21 soft tissue sarcoma, 22-23 Sex chromosomes, Xiphophorus macromelanophore loci, 65-69 in hybrids, 67-81 Simian virus 40 (SV40), large T antigen transcription activation indirect mechanism of, 289 promoters and, 289 transformation-specific gene induction, 293

Small-cell lung cancer, human bombesin-like peptides, 259 characteristics, 257-258 c-myc amplification, 258 copy number, prognosis and, 258 expression elevation, 258 c-raf, 259 Soft tissue sarcoma, human, segregation analysis, 22-23 Spleen, TdT+ lymphocytes, transient, in rodents, 42 Squamous cell carcinomas, human c-erbB, expression and amplification, 260

EGF receptor kinase content, 260

T T-cell antigen receptor, recombination with oncogenes a-subunit, 218-220 p-subunit, 220 TdT, see Terminal deoxynucleotidyltransferase Terminal deoxynucleotidyltransferase (TdT) DNA N region production and, 50-51 earlier reviews, 37 enzymatic properties, 38-39 as leukemia marker, human acute myeloid leukemia and, 47-48 in circulation and bone marrow, 46

in immature lymphocyte populations, 48 in pre-B leukemic cell lines, 50 in pre-T leukemic cell lines, 50 in thymus, 49 in lymphocytes future research, 58 localization, 51-55 electron microscopy, 53-54 immunoperoxidase staining, 52-53 ontogeny in birds, 45-46 early studies, 41-42 in humans, 44-45 in rodents, 42-44 protein structure, human amino acid composition, 39-40 amino acid sequence, 39-40 intracellular transport and, 53, 55 DNA binding site, 39 reactions with antibodies monoclonal from mouse, 41 polyclonal from rabbit, 39-41 Thymus, TdT+ lymphocytes, ontogeny in birds, 45-46 in humans, 44 in rodents, 42-44 T lymphotrophic viruses HTLV I and 11, transcription activation from viral LTR, 289 HTLV 111, transcription activation, 290 Tomography, computerizing, PLC localization, 323 Transcription, cellular, activation by adenovirus E l A gene, 284-288 c-myc, 291 herpesvirus immediate early genes, 290-291

SV40 large T antigen, 289 T lymphotrophic viruses, 289-290 Translocation, chromosomal in acute myeloid leukemia, 224-225 atypical between chromosomes 15 and 12,210-211

in chronic myeloid leukemia c-abl on chromosome 9 and, 220223

c-bcr on chromosome 22 and, 221223

339

INDEX

Philadelphia chromosome 9;22 junction and, 220-222 c-myc in Burkitt lymphoma, 130-131,266267 in plasmacytomas, 189-191, 197198,266 future research, 225-226 myclIgH in B-cell tumors, 189-191 breakpoint location c-myc, 198-201 IgH locus, 201-202 c-myc expression and, 204-210 interchromosomal recombination, 197-198 mechanism of, 203-204 oncogenic amplification and, 245247 T-cell tumor-specific, T-cell antigen receptor and, 218-220 variant in B-cell tumors bcl-1 and bcl-2 oncogenes, 217218 breakpoints far from c-myc, 212214 Ig light chain loci and, 214-215 put-1 locus on plasmacytoma chromosome 15 and, 215-217 Tumor cells 293, human, hsp70 induction by adenoviral ElA, 292-293 A431, EGF receptor coded by c-erbB gene, 251 HeLa, human, hsp70 induction by adenoviral ElA, 292-293 oncogene amplification, 235-273; see also Amplification, oncogenic oncogenes, 237 (table) Y1, rat, c-Ki-ras amplification and expression, 261-262 Tumors B-cell, see also Lymphomas, B-cell leukemic pre-B-cell lines, TdT and, 50 translocations involving IgH locus, bcl-1, and bcl-2, 217-218 genetic origin, plant, animal, 91-93 MNU-induced, Xiphophorus hybrids epidermal carcinoma, 80

experimental strategy, 76-77 fibrosarcoma, 79 retroocular neuroblastoma, 78 T-cell, translocation, T-cell antigen receptor and, 218-220

U Ultrasonography, PLC localization, 323

v Viral oncogenes (v-onc) c-onc transduced mutant alleles, 105109 ElA, adenoviral production of three mRNAs, 284 transcription activation E l A protein indirect action, 285288 hsp70 gene induction in HeLa and 293 cells, 292-293 p-tubulin gene induction, 293 src of RSV, 106 transcription activation, 283-294; see also Transcription, cellular v-erbA allele in AEV-R strain function, 147 structure and expression, 141, 143144 v-erbB allele in AEV-H strain function, 144, 147 structure and expression, 140-143 v-erbB allele in AEV-R strain function, 144-147 structure and expression, 140-142 v-mil allele in MH2 strain, avian structure and expression, 132-134 v-myb allele in AMV strain function, 156-157 structure and expression, 152-155 v-myb and v-ets alleles in E26 strain function, 156 structure and expression, 152-156

340

INDEX

v-myc, in myelocytomatosis virusinfected cells, 264 v-myc alleles in MC29 subgroup CMII strain, 114, 118 functions of, 121-124 MC29 strain, 114-117 MH2 strain, 114-116, 119-121 OK10 strain, 114-116, 118-119 v-rufallele in 3611-MSV strain, murine oncogenic function, 136-137 structure and expression, 132-135

W Wilms’ tumors, N-myc expression, 261, 263

X Xiphophorus (teleost) biology, 65 pigment cell spots, 64 selective crossing breeding strategy, 69-70 melanoma formation and, 69-75 spot loci without melanomas, 73, 75 spot pattern loci, melanoma and, 6566 tumor gene identification, 89-92 X. hellerd (swordtail) spot pattern “dubbed” locus, 68-69 X. rnuculutus (platyfish) spot pattern loci, melanoma and, 6769 X rays, carcinogenesis in Xiphophorus hybrids and, 76-77,81