Advances in Cancer Research Volume 47

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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.


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


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.


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


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-


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


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-


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


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.)


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


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-


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)

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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


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


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-


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


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


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.

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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-


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.


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


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


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


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-


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


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-


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.

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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-


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


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-


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


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


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 (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


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

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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

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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‘


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

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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


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

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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


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

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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).

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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


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

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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

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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

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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

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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.,

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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


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

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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


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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.

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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


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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

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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.

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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


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


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.


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


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).


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


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.


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


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).


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


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


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


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


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.


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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-

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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


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


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.


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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

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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-


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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

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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


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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

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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


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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

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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


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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-

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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

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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.

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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.

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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

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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

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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


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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-

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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?


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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).

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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


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,

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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


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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).

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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.

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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.

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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

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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

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