Advances in Cancer Research Volume 50

ADVANCES IN CANCER RESEARCH VOLUME 50 This Page Intentionally Left Blank ADVANCES IN CANCER RESEARCH Edited by GEO...

Author: George Klein | Sidney Weinhouse

23 downloads 976 Views 17MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

ADVANCES IN CANCER RESEARCH VOLUME 50

This Page Intentionally Left Blank

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLElN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

SIDNEY WEINHOUSE Fels Research Institute Health Sciences Center Temple University Philadelphia, Pennsylvania

Volume 50

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

San Diego New York Berkeley Boston London Sydney Tokyo Toronto

COPYRIGHT

0 1988 BY ACADEMICPRESS, INC.

ALL RIGHTS RESERVED. NO PART OF 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 . 1250 Sixth Avenue San Diego, California 92101

United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road. London NWl 7DX

LIBRARYOF CONGRESS

CATALOG CARD

ISBN 0-12-006650-5 (alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA 88

89 90 91

9 8 7 6 5 4

3 2

1

NUMBER:52- 13360

Biological Markers of Cell Proliferation and Differentiation in Human Gastrointestinal Diseases

MARTINLIPKINAND PAULHIGGINS I. Introduction . . . . . .............................................. 11. Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Stomach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................................... 7 V. Antigenic Determinants of Gastrointestinal Cell Differentiation ..... and Proliferation . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2 3

22

Chemical Carcinogenesis: From Animal Models to Molecular Models in One Decade STUART

H.

YUSPA AND

MIRIAMc.

POIRIER

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Biological Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Cellular and Molecular Mechanisms of Carcinogenesis. . . . . . . . . . . . . . . . . . . . . . . V. Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . ......... .. .............................................. 61 I.

11.

25 26 33 39 54 59

Oncogenes and the Nature of Malignancy

IAN BUCKLEY .......................................................... 11. 111. IV. V.

Basic Phenomena of Malignancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Malignant Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T h e Malignant Genotype . ........................ General Synopsis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... .................................... References . . . . . . . . . . . . . . ........................ V

72 74 79 87 93

71

vi

CONTENTS

The Epstein-Barr Virus Proteins JOAKIM

DILLNER A N D BENCT KALLIN

1.

Biology of EBV Infection ............................. I1 . EBV-Associated Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11 . T h e EBV Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. EBV-Encoded Proteins in EBV-Transformed Cells . . ...... V. Viral Proteins in Virus-Producing Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V1 . Summary and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

95 96 100 104 138 149 151

Stromal Involvement in Malignant Growth

A . VAN

DEN

HOOFF

I . Introduction . . . . . . . . 11. Stromal Reactions to I11 . I v. V. VI . VII . VIII .

.... . . . . . . . . . . . . . . . 159 Growth . . . . . . . . . . . . . . . . . . 160 The Roles Played by Stromal Adhesive Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . T h e Role of the Basement Membrane in Invasiveness . . . . . . . . . . . . . . . . . . . . . . . 173 Characteristics of Fibroblasts in Malignant Conditions . . . . . . . . . . . . . . . . . . . . . . . 177 Primary Stromal Disorders as Putative Factors in Carcinogenesis . . . . . . . . . . . . . . 178 Stromal Alterations Preceding Invasive Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 186 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

170

MICHAEL F. A . WOODRUFF I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Origin and Diversity of Cells in Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. I v. V. VI . VII . VIII .

197 198 ..................................... 200 Definition of Clonality . . . . Distinguishing between Mono- and Pleoclonal Tumors . . . . . . . . . . . . . . . . . . . . . . . Analysis of Pleoclonal Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 216 Observations o n Clonality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 Factors Influencing Clonality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................ 224 Conclusions and Perspcctives . . . . . . . . . 227 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

202

CONTENTS

vii

Newborn Macrosomy and Cancer

LEV M . BERSTEIN I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Basic Evidence and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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

231 235 239 272 273

Louvain Rat lmmunocytomas

.

HERVE BAZINWARREN S.

PEAR.AND JANOS SUMEGI

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... I1 . Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Incidence of the Ileocecal Lymphoid Tumors in Rats . . . . . . . . . . . . . . . . . . . . . . . . I V Macroscopic and Microscopic Description of the IR Tumors or RIC . . . . . . . . . . . V Biosynthesis of Immunotglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . In Vzvo Transplantation, in Vitro Cell Culture, and Storage of the IR cccRIC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 VII . Etiology of the IR Tumors or RIC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

279 280 282 283 287

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

311

. .

294 305 307


BIOLOGICAL MARKERS OF CELL PROLIFERATION AND DIFFERENTIATION IN HUMAN GASTROINTESTINAL DISEASES

Martln Lipkin* and Paul Higginst 'Memorial Sloan-KetterlngCancer Center, New York, New York 10021 tVeterana Admlnlstratlon Medlcal Center, Albany, New York 12208

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

I. 11.

111. Normal Stomach . . . . . . . . . . . . . . . . . . . . B. Diseases of the Stomach. . . . . . . . . . . . . . . . . . . IV . LargeIntestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. NormalColon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . DiseasesoftheColon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Modulation of Colonic Cell Proliferation and Differentiation . . . . . . . . . . . . V. Antigenic Determinants of Gastrointestinal Cell Differentiation and Proliferation . . . . ................... References. .............. A.

1 2 2 2 3 3 4 7 7 9 13

15 22

I. Introduction Advances in our understanding of the proliferation and differentiation of gastrointestinal cells have come from studies of cell proliferation kinetics and from biochemical and immunologic measurements defining parameters related to cell division and maturation. New approaches have been developed to analyze molecular aspects of cell proliferation and differentiation in normal cells, and modifications occurring in various stages of abnormal cell development during the evolution of diseases. Results of recent studies also have increased our understanding of how gastrointestinal cells respond to a variety of stimuli that modify their growth and development. This article will summarize some earlier studies and in particular newer measurements on the proliferation and differentiation of normal and abnormal cells in the esophagus, stomach, and large 1 Copyright 0 1988 by Academic Press, Inc.

ADVANCES IN CANCER RESEARCH, VOL. 50

AU rightsofreproduction in any form reserved.

2

MARTIN LIPKIN AND PAUL HIGGINS

intestine of humans and rodent species, and will describe modifications that occur in gastrointestinal cells during various stages of abnormal development.

II. Esophagus

A. NORMAL ESOPHAGUS In the esophagus of both rodents and humans, the epithelial cell lining is squamous, unlike the epithelial lining in the lower regions of the gastrointestinal tract. Proliferating cells normally are situated in a single basal layer beneath the surface of the esophageal mucosa and are covered with stratified migrating and nondividing cells; these differentiating and maturing cells move toward the lumen and eventually are extruded from the mucosal surface. All basal cells can be labeled with tritiated thymidine ([3H]dThd)and are presumed to proliferate. As in the epithelial lining of the normal stomach and small intestine (Lipkin and Bell, 1968), esophageal cells that synthesize DNA are normally seen randomly among all the basal layer cells (Cameron el al., 1965). The renewal of epithelial cells in the basal layer of rodents occurs approximately every 4-5 days, and the entire rodent epithelium is replaced in about 7-8 days. Renewal in humans is several times slower (Lipkin and Bell, 1968; Lipkin, 1971). As in other regions of the gastrointestinal tract, extensive morphological and functional changes normally develop in cells of the esophagus after they proliferate, as they become well differentiated and migrate to the lumenal surface. After cell division, two daugther cells may stay basal and redivide, or one may remain in the basal layer while the other differentiates, or both may leave to differentiate.

B. DISEASES OF THE ESOPHAGUS In diseases of the esophagus, as in other regions of the gastrointestinal tract, the rate of proliferation of epithelial cells increases. For example, in patients with reflux esophagitis, measurements in organ-cultured biopsy specimens have shown that increased numbers of cells incorporate [3H]dThd compared to normal esophageal epithelium (Livestone et al., 1976); earlier findings had shown a high frequency of mitotic figures in the basal layer of diseased specimens (Ismail-Beigi et al., 1970, 1974). In the esophagus of patients with Barrett’s epithelium, a disease leading to increased incidence of esophageal cancer, cell proliferation also was increased (Herbst et al., 1976). [3H]dThd-labeled cells have been observed in characteristic villuslike epithelium in this disease, with an enlarged proliferative compartment of epithelial cells. In esophageal biopsy samples

CELL PROLIFERATION AND DIFFERENTIATION

3

from individuals in Linxian, China, an expansion of the proliferative compartment of epithelial cells also was found in individuals whose risk for cancer of the esophagus was markedly increased (Fig. 1) (Munoz et al., 1985). This is similar to the ectopic migration of proliferative cells near the crypt surfaces, in colonic micosa adjacent to polyps (Bleiberg et al., 1972; Lipkin and Bell, 1968), and in atrophic intestinalized gastric mucosa (Winawer and Lipkin, 1969).

Ill. Stomach A. NORMAL STOMACH Mucus-secreting epithelial cells normally cover the surface of the body and fundus of the stomach and extend down into the gastric pits; beneath these are long glands that extend deeper into the gastric mocosa. These glands are lined by acid and intrinsic factor-secreting parietal cells and pepsinogen-secreting chief cells, which empty into the gastric pits within the mucosa. Below the body of the stomach, the mucosa is pyloric in type; here mucus-secreting cells also normally line the gastric pits, while glandular cells underneath have a alkaline secretion that contains pepsinogen. Endocrine cells that synthesize, store, and secrete the various hormones (Greider et al., 1972; Johnson and Guthrie, 1974; McGuigan and Grieder, 1971) are located in both types of mucosae, and mucous-neck cells are believed to function as stem cells for these endocrine cells. The latter are normally

Percentage of labeled cells at level 3 or above FIG.1. Histogram of percentage of [SHIdThd-labeled cells at esophageal cell levels of 3 and above (expansion of proliferative compartment of basal layer cells) in normal and precancerous human esophageal biopsy specimens. Empty bars, low-risk area (Jiaoxian);filled bars; high-risk area (Linxian). Categories of the abscissa refer to percentages up to the following category. (From Muaoz ef QI., 1985.)

4


intermingled with other mucous cells and are located mainly in the midzone of the gastric glands. The endocrine cells are believed to have originated from the neural crest and to respond to autonomic, mechanical, and intralumenal stimuli by discharging their granules into the circulation. Although there appear to be at least 11 different endocrine cell types in the gastrointestinal mucosa, the primary cells in the gastric fundus are the A cell or (enteroglucagon-secreting cell), the G cell (or gastrin cell), and the argentaffin cell, which secrete serotonin and histamine. The mucosa of the pyloric antrum normally has gastric pits that are deeper than in the fundus, lined mainly with epithelial cells that are similar to the mucous neck cell. The parietal acid secretory cells and the G cells, which secrete gastrin, are also present in this region; the most concentrated gastrinsecreting cell population is in the middle third of the antral mucosa. Gastric mucous and specialized glandular cells renew at rates that differ markedly, and the specialized cells renew more slowly. In rodents and in humans, mucous epithelial cells in the stomach have rates of cell proliferation that are similar to epithelial cells of the colon, while cell renewal in the small intestine is slightly more rapid (Lipkin, 1971; MacDonald et a l . , 1964; Shorter et al., 1964). In humans the proliferative cell cycle for gastric mucous epithelial cells is 2-3 days, and mucosal replacement takes 4-8 days (Lipkin et al., 1963a,b; MacDonald et al., 1964), while it is slightly more rapid in rodents.

B. DISEASES OF THE STOMACH The effects of hormones on gastric epithelium are better understood now than formerly. These include the effects of gastrin, produced by gastrin cells in normal mucosa and by gastrinomas, on cell proliferation in the stomach. The proliferation of gastrin cells in antrum following vagotomy in rats was studied as a function of the total number of antral gastrin cells, the antral surface area, and the serum gastrin concentration (Delince et a l . , 1978). Three weeks after vagotomy and pyloroplasty, or pyloroplasty alone, the number of gastrin cells in the stomach was significantly elevated in vagotomized animals, and both the mucosal surface of the antrum and the concentration of gastrin cells were significantly higher. Serum gastrin values also were elevated after vagotomy. The hypergastrinemia observed after vagotomy could be explained in part by vagotomy inducing an antral gastrin cell hyperplasia. When the antrum was excluded, however, elevated antral pH was not sufficient to produce proliferation of gastrin cells (Alumets et al., 1980), suggesting the stimulation of high antral p H and passage of food contribute to gastrin cell proliferation. The effects of fasting and refeeding on proliferation of antral gastrin cells also has been studied in rats, by measuring the number of gastrin cells with injections of [3H]dThd and microautoradiography, thus quantitating newly


5

formed gastrin cells (Bertrand and Willems, 1980). The total number of gastrin cells decreased 68% after a 4-day period of fasting, whereas refeeding of rats for 6 days after a 4-day fasting period resulted in a significant (79%) increase in gastric cell mass. Thus, after refeeding, the [SH]dThd-labelingindex of gastrin cells was significantly increased compared to controls, and the degree of proliferation during refeeding was comparable to the increase in number of gastrin cells during refeeding. Current findings therefore indicate that a new population of gastrin cells is formed in the antral glands of the stomach when rats are refed after fasting. When bombesin tetradecapeptide was administered chronically, the antral gastrin content of rats also significantly increased with stimulation of antral gastrin cell proliferation, following which the antral gastrin cell population increased significantly (Lehy et al., 1983). Infusion of somatostatin transiently reduced [3H]dThd incorporation and cell division both in fundic and in antral progenitor cells (Lehy et al., 1979). In stomach stimulated by gastrin plus somatostatin, DNA synthesis was lower than the gastrin-stimulated stomach. Thus, somatostatin appears to inhibit-perhaps indirectly-cell proliferation, and to antagonize the trophic activity of gastrin in fundic and antral mucosae. Both endogenous gastrin and exogenous pentagastrin protected against damage induced in gastric cells, and the protective effect of gastrin was directly related to the balance between cell production and cell loss (Takeuchi and Johnson, 1982). The mucosal epithelial cells that line the gastric pits and that cover the surface of the stomach are prone to develop inflammatory diseases and malignancy in certain populations. The overall rate of development of precancerous diseases in the stomach varies markedly according to nutritional intake (Correa et al., 1976). It has been postulated that conversion of precursor nitrogenous compounds of some foods to nitrosamines, mutagens, and carcinogens contributes to the development of precancerous diseases in the stomach (Mirvish, 1981; Tannenbaum and Corea, 1985). In normal stomach, microautoradiographic studies have shown that DNA, RNA, and protein synthesis are most active in the proliferating mucous epithelial cells at the base of the gastric pits. However, with the development of diseases that lead to increased frequencies of cancer, such as atrophic gastritis, and intestinalization of the gastric mucosa, increased cell proliferation has been found to occur and to increase in mucous epithelial cells with an expansion of the compartment of proliferating cells including gastric surface cells (Deschner et al., 1972). Later studies have confirmed and extended these findings, with comparisons of cell proliferation in gastric epithelium of patents with various degrees of superficial to severe atrophic gastritis, peptic ulcer disease, and adenomatous polyposis of the stomach (Sizikov and Azykbekov, 1981). Levels

6


of cell proliferation in patients with peptic ulcer disease and polyps were simiar to nonatrophic gastritis and mild atrophy of the gastric mucosa. However, cell proliferation increased in advancing atrophy and reached a peak in severe atrophic gastritis. Thus, a hyperproliferative state developed in gastric mucosa having chronic gastritis with marked atrophy of the mucosa. Further findings in a recent study of a population with chronic atrophic gastritis (CAG) at markedly increased risk for gastric cancer (Lipkin et a l . , 1985) also showed an expansion of mucous epithelial cell proliferation in the gastric pits (Fig. 2). Additional measurements in normal human subjects and in patients with different degrees of gastritis revealed no significant differences in the duration of the S phase of the proliferative cell cycle in normal mucosa, superficial gastritis, mild atrophic gastritis, and severe atrophic gastritis (Hart-Hansen et al., 1979), suggesting that measurements of the labeling index can be used to assess that rate of cell proliferation in human gastric mucosa. In atrophic gastritis, more mitotic activity also occurred in intestinal metaplastic glands than in normal gastric pits (Liavig, 1968); the mitotic index doubled (1 YY to 2.3 %) and the labeling index increased, together with the appearance of small intestinal cells in the stomach. Recent studies have analyzed the distribution of DNA content as a potential marker of precancerous or early malignant changes in digestive cells (Capurso et al., 1982). Samples were derived from stomach, duodenum, arid colon of ENTIRE

LABELING INDEX

GASTRIC PIT VALUES ~-

.20

1

--f. .20

.18

.18

,.

.I6

.16

.14

.14

.14

.12

.12

.12

.10

.10

.10

.m

.on .06

f_

.m

t

.06

.06

.M

3-

1 AND OVER

0

31 AND OVER

2 ',* 1 -5

(SURFACE)

FIG. 2 . In the mucosa of the corpus of the stomach: comparison of labeling-index statistics (mean 1 SE) between a population with chronic atrophic gastritis (clear areas) and a normal population (diagonal shading) for individual gastric pit compartments and for the gastric pit as a whole. Symbols **p < 0.006; 'p < 0.02; tp < 0.07 forcompatment 7; lp = 0.12 for compartment 3 ; no symbol, 20% < p < 3 3 % . (From Lipkin ct a [ . , 1985.)

*


7

normal donors, and from patients affected with ulcer disease, atrophic gastritis, chronic gastritis, and polyps. Normal samples showed the usual DNA distribution in GI,,,, S , and GZ/M compartments when stained for flowcytometric analysis using ethidium bromide and mithramycin. In gastritis, a subpopulation of aneuploid cells was found similar to that of gastric adenocarcinoma, with DNA index ranging from 1.1 to 1.4 in comparison to 1.0. In tumors, however, the percentage of aneuploid cells was generally higher than in normal cells or gastritis. Polyps also exhibited an aneuploid peak, whereas ulcers were characterized by a normal, diploid DNA content. These findings supported the possibility that aneuploidy in these lesions indicated a significant degree of abnormal cell development in premalignant conditions. Previous studies of gastric mucosa have shown a general association between degrees of metaplasia and the histologic characteristics of gastric cancer, with greater metaplasia occurring in more differentiated carcinomas; poorly differentiated adenocarcinomas occur with less metaplasia. Metaplasia accompanies or precedes adenocarcinoma in 90% of gastric specimens studied. A higher degree of intestinal metaplasia is present in the gastric mucosa of patients with cancer than with gastric ulcer, implying again that metaplasia is an indicator of precancerous disease (Morson, 1955; Oota, 1967; Stemmermann and Hayashi, 1968). Further studies have shown that as precancerous gastric cells progress to malignancy, cells have increased mitotic indexes, paralleling changes that include increased diameter, staging, and degree of invasion (Tabuchi et aE., 1980). The average mitotic index of early cancers was lower than in advanced cancers, while poorly differentiated adenocarcinomas had a high mitotic index than well-differentiated adenocarcinomas. Metastatic lesions also showed a higher mitotic index than primary lesions. There was, however, no correlation between the mitotic index and the location, the degree of the stromal reaction and the type of infiltration, or the age and sex of the patients.

IV. Large Intestine A. NORMALCOLON The proliferative region of the large intestine normally occupies the basal three-quarters of the crypts. Cells migrate toward the surface of the colonic crypts and are extruded from the mucosal surface. The large intestinal crypts are more closely spaced than either in stomach or small intestine, and the surface of the large intestine is flat. The colonic epithelial cells, columnar, mucous, and entroendocrine, most likely originate in the crypt base (Chang and Leblond, 1971). Migration of cells to the crypt surface takes 3-8 days in humans (Cole and McKalen, 1961;

8


Lipkin et al., 1963a,b, 1971; MacDonald et a l . , 1964; Shorter et al., 1964) and 2-3 days in rodents (Chang and Leblond, 1971; Messier and Leblond, 1971; Messier and Leblond, 1960). Argentaffin (enteroendocrine) cells of the human rectal mucosa undergo slow renewal in 35-100 days (Deschner and Lipkin, 1966). Several more recent studies have analyzed cell proliferation in the colonic crypts at several sites within the large intestine, or along the length of the crypt in rodents. In rat (Sunter et al., 1979), major differences were found in the size and shape of the crypts at different sites within the large intestine. The distribution of the proliferating cells within the crypts also varied. The average cell cycle duration ranged from 58 hr in the descending colon to 25 hr in the cecum; this variation appeared to be brought about largely by changes in the duration of the GI phase of the proliferative cell cycle, the durations of the other phases remaining relatively constant. There also was variability in the cell cycle duration and growth fraction at different levels within the crypts; throughout, the colonic epithelial cells appeared to cycle more slowly at the very bottom of the crypt. Modifications of cell proliferation previously have been demonstrated with hormonal and nutritional treatment (Hoff et al., 1981). Thus, estrogen decreased the incorporation of [SH]dThdinto colonic epithelial cell DNA most markedly 4 hr after injection. With refeeding of male mice after 48 hr of fasting, however, the colonic mucosa was not affected when mice were treated with estrogen implants for up to 4 days. Estrogen treatments caused no significant change in the DNA, RNA, or protein content of the colonic mucosa. Further studies have indicated diverse effects of modulating factors. Thus, dibutyryl cyclic adenosine monophosphate (dibutyryl-CAMP) also inhibited cell proliferation in colonic crypts and in colonic adenocarcinomas in rodents (Tutton and Barkla, 1980). In jejunum, dibutyryl-CAMP at high doses inhibited crypt cell proliferation, but lower doses was reported to accelerate jejunal crypt cell proliferation. Neither bilateral adrenalectomy nor chemical sympathectomy abolished the ability of dibutyryl-CAMP to stimulate jejunal crypt cell proliferation. An ornithine decarboxylase (ODC) inhibitor (difluoromethylornithine, DFMO) inhibited cell proliferation in colon of primary colonic tumors (Tutton and Barkla, 1986). Testosterone treatment accelerated cell proliferation in colonic tumors; however, following castration, cell proliferation was retarded in colonic tumors (Tutton and Barkla, 1982a). Similarly, in colonic tumors cell proliferation also was inhibited by the antiandrogenic drug, flutamide. In contrast, in the colonic crypt epithelium of both normal and carcinogen-treated animals, testosterone and castration each failed to influence cell proliferation. Comparative measurements of oophorectomy and treatment with ovarian hormones on cell proliferation have also been made (Tutton and Barkla,


9

1982b). The proliferation of colonic tumors decreased following oophorectomy, and the decrease was reversed by the administration of estradiol but not by the administration of progesterone. However, oophorectomy did not retard cell proliferation in normal colonic crypts. The current findings noted above have suggested that in colonic crypts, cell proliferation is under both endocrine and autonomic neural control, whereas in colonic tumors it is subject to endocrine regulation alone. In the development of human colonic tumors age-dependent variations are large, and sex differences also are found that are associated with an increased incidence of human colonic cancer; therefore, as indicated above, hormonal factors that increase the proliferation of abnormal or damaged colonic cells could influence cancer rates. The possible role of pericryptal fibroblasts in regulating intestinal cell proliferation has been studied for many years. Relationships in rat colonic mucosa after in vivo incorporation of [SH]dThd (Maskens el al., 1979) have been analyzed. The labeling index of pericryptal fibroblasts at 1 hr after injection was only 2.4%, and labeled fibroblasts were slightly predominant along the lower two-thirds of the crypts; within 24 hr most underwent at least one cell division. Cell migration was not observed and a significant fraction of the labeled fibroblasts was still present after 3 weeks, indicating that the fibroblasts constituted a slowly renewing cell population. Thus findings failed to confirm the hypothesis of an “en bloc” migration of fibroblasts in synchrony with the epithelial cells.

B. DISEASES OF THE COLON Relationships of cell proliferation and neoplasia have been further evaluated in several studies. Thus, an expansion of the proliferative compartment of epithelial cells again was found in colonic mucosa of individuals with diseases that predispose to colon cancer (Lipkin et al., 1983, 1985; Terpstra et al., 1986) (Fig. 3). Chronic ulcerative colitis is one of the diseases that is accompanied by an increased incidence of colonic cancer. In ulcerative colitis in remission, a spatial pattern of proliferating cells in colonic mucosa was found similar to that seen both in regenerating and in precancerous mucosa (Serafini et al., 1981). Patients with a short history of colitis had an abnormal proliferative pattern as frequently as others with a long history, indicating that proliferative activity itself does not signify impending malignant change. With ulcerative colitis in remission, the colonic mucosa also had increased epithelial cell proliferation similar to that seen in active colitis, suggesting the abnormal pattern observed in remission is the pattern of an actively regenerating mucosa. The high rate of mucosal turnover, observed not only during clinically active disease but

10


Ew cc n o

9;

010

zg

Colonic crypt compartments FIG.3 . Comparison of population occupancy fractions of [3H]dThd-labeled epithelial cells in the colonic crypt compartments of a high-risk group with symptomatic familial polyposis (A),a high-risk group previously affected with familial colon cancer ( O ) ,and a low-risk group from the general population ( 0). Abscissa: colonic crypt-height compartments. Ordinate: fraction of a given population's labeled cells that occupy any specified height compartment in the colonic crypts. (From Lipkin et d.,1983.)

throughout remission, may contribute to the increased incidence of carcinoma in this disease; increasing the proliferation of damaged cells may increase the rate of development of neoplasms, functioning in a phase of tumor promotion. In further studies of experimental damage to nuclear DNA, cell proliferation was studied in colonic adenocarcinomas of the rat after administration of the chemical carcinogen 1,2-dimethylhydrazine (DMH), and findings were compared to normal colon (Pozharisski et al. , 1980). Microautoradiographic observations revealed that the distinct normal zone of crypt cell proliferation was not present in the tumors, and cells replicated in nearly all segments of the neoplasms. Tumor cells also had a longer proliferative cell cycle, again due to an extension of the GI cell cycle phase duration. They also had more heterogeneity in the durations of S and G2 phases. Adenocarcinoma cells resembled basal crypt cells of control animals, and the latter had subpopulations with varying durations of the proliferative cell cycle. When colonic epithelial cells became premalignant and then malignant, impaired differentiation also led to decreased cell shedding into the intestinal lumen as benign and malignant tumors arose. In human populations and in rodent exposed to chemical carcinogens, carcinomas arise mainly in the distal colon. This was seen further in a sequential study of the changes developing in epithelial cell proliferation in the descending colon during tumor induction after D M H treatment (Pozharisski et al., 1982). Expansion of crypt epithelial cell proliferation with migration of dividing cells as far as the crypt mouth occurs in


11

the early stages of D M H exposure, suggestive of impaired epithelial cell differentiation. The crypt cells mainly proliferated through a cell cycle whose mean duration was slightly greater than in normal intestinal cells. Reduced epithelial cell loss and resulting disturbance induced by D M H led to the accumulation of great numbers of atypical cells in the superficial layers of the crypts and formation of carcinomas in situ in the descending colon. Bile acids also are believed to function as tumor-promoters in the colon. In the rat, bile acid concentrations were altered by intrarectally injecting either deoxycholic or lithocholic acid for 4 weeks or by increasing the dietary fat or fiber intake (Glauert and Bennink, 1983). Over the course of D M H administration, bile acids, dietary agar, and wheat bran contributed to a slight hyperplasia in the colon. Intrarectal instillation of bile acids or the tumorpromoting agent (TPA) resulted in early, transient induction of ODC and an increase in [3H]dThd uptake (Takano et al., 1982). In explant cultures of rat colon, bile acids or TPA stimulated ODC activity and DNA synthesis. This stimulation of ODC activity and DNA synthesis was greater with secondary bile acids than primary ones, further suggesting that TPA and secondary bile acids exert promoting effects in colon carcinogenesis. Nutritional elements and total pa re nt e d nutrition also influence both cell proliferation and carcinogenesis. The oral administration of solid and liquid diets, regardless of chemical constituents, contributed to DMH-induced increased colonic epithelial cell proliferation; however, a DMH-induced increase in epithelial cell proliferation was not observed in rats maintained on total parental nutrition (Heitman et al., 1983). Thus, the route of administration has a significant influence on epithelial cell proliferation on colonic epithelium of DMH-treated rats. The preneoplastic state is a committed state that appears not to be dependent on the continued presence of a carginogen; in the D M H models all crypt epithelium is preneoplastic, although not all cells progress to the transformed state. It is important that total parental nutrition inhibited the expression of DMH-induced neoplasia together with decreased epithelial cell proliferation. As colon tumors grow there is a small decrease in cell loss, rather than an increased rate of cell proliferation (Camplejohn, 1982). [3H]dThd microautoradiography again demonstrated that carcinoma cells have a longer generation time than normal cells (Hattori, 1981). The cell generation time was about 3-7 days for cancer cells compared to 1 day for normal cells. There is an increase in the life of cancer cells rather than extreme variation in the growth fraction. Volume-doubling time for human cancers in general is about 1-3 months, and about 1 year to several years for colonic cancers. In a unique in vivo analysis of human premalignant adenomas, characteristics of both normal cells and premalignant cells were observed, the latter in the benign neoplasms. The in vivo measurements of proliferation

12


kinetics of the epithelial cells were made following intravenous pulse injection of [3H]dThd into a patient with familial polyposis and Gardner syndrome (Lightdale et al., 1982). Results demonstrated both in the adenomas and in adjacent flat mucosa that cells completed only a single proliferative cycle during 4 days of observation. Durations of Gz and S phases of the cell cycle were approximately 5 hr and 15 hr, respectively, both in adenomas and in flat mucosa. After pulse injection, the locations of [3H]dThd-labeled epithelial cells within the adenomatous crypts showed a distribution with maximum proliferation near the lumenal surface (Fig. 4); However, in flat mucosa, maximum proliferation of labeled epithelial cells occurred in the lower portion of the colonic crypts. Migration rates were similar to earlier studies at 0.4 cell positions per hour in crypts of adenomas, and 0.3 cell position per hour in crypts of adjacent flat mucosa. In the study of Lightdale et al. (1982) it was of particular interest that the direction of migration differed in adenoma and flat mucosal cells; in flat mucosa migration of epithelial cells occurred toward the lumenal surface of the crypts, while in adenomas a markedly abnormal retrograde average migration away from the surface of the mucosa was observed as occurs with malignant cells (Fig. 5 ) . However, the adenoma cells had not developed the properties of malignant cells that enable them to invade through the submucosa. CfYPI Lwngn

L

I

10

2 u

" 'E1

0 2o

9 Y

-

10 c

u c

p

o 20

10

0 I50

120 90

60

M

0

100

I50

120

90

60

30

0

Cell position no

FIG. 4. Distribution of labeled cells within adenomatous crypts at different times after [3H]dThdlabeling by pulse injection of an individual with familial polyposis; distributions of cells in sections (A-F) correspond to intervals from 12 to 86 hr shown in Fig. 5. Cell position zero is at the lumenal surface of the crypt, and cell position numbers increase away from the surface and deeper into the crypt. (From Lightdale el al., 1982.)


10

-

13

A

L

l oO

L-

0

C w )

Bore

10

20

30 40

50

60 70 00 90

Hours after I3H1dThd

FIG.5. Mean position oflabeled cells versus time of assay for adenomas (A) and flat mucosa (B), in an individual with familial polyposis after pulse injection of [%H]dThd. (From Lightdale et al., 1982.)

In understanding mechanisms of carcinogenesis, studies also have shown that carcinogens were activated to carcinogen-DNA adducts by cultured human tissues including esophagus and colon (Harris d al., 1982). Marked differences in carcinogen metabolism among people also have been found. The magnitude of differences found ranged from 50- to 150-fold, similar to that found in pharmacogenetic studies of drug metabolism. Further rate-limiting enzymatic reactions could also differ among human subjects and in different tissues.

C . MODULATION OF COLONIC CELL PROLIFERATION AND DIFFERENTIATION It is now also possible to study the effects of growth factors and other modulators of cell proliferation and differentiation on colonic epithelial cells in vitro.

14


Short-term tissue culture of colonic epithelial cells has been successfully applied to the in vitro study of colonic adenomas and normal colonic cells. With this approach (Friedman et al., 1981), premalignant colonic epithelial cells from benign human tumors were reproducibly grown in tissue culture. The cells grew as tightly packed colonies from small explants free of fibroblasts and remained viable for up to 9 weeks. Cells were identified as epithelial with electron microscopy, and the living cells transported water and salt. Adenoma cells were cultured with approximately equal frequency from three premalignant classes: tubular (low malignant potential), villotubular (intermediate malignant potential), and villous (high malignant potential). This model has permitted further evaluations of the stages of abnormal colonic cell development, and the beginning of measurements to determine the effects of physiological growth factors and tumor promotors on colonic cells. For example, the effect of exogenous epidermal growth factor (EGF) on cultured colonic epithelial cells was measured by assaying the fraction of cells incorporating [ 3H]dThd under conditions of continuous labeling. EGF increased the fraction of replicating cells cultured from tubular adenomas, while it had no effect on cells cultured from villous adenomas (Friedman et a l . , 1981). EGF has a strong structural homology to human urogastrone, a tropic hormone in the intestine, suggesting the in vitro results may mirror the in vivo growth modulation of benign tumors. In further measurements, tissue culture studies made possible classification of the colonic cells into a series based on stages of premalignancy determined by tumor-associated antigen expression. An antisera raised against human second-trimester fetal tissue and absorbed with pooled adult tissues was found to react with cultured adenoma cells (Friedman et a l . , 1981; Higgins et a l . , 1983). Thus, when cells were cultured from adenomas of different histopathologic classes and from colon carcinomas, fetal-associated antigen was found most frequently in carcinoma cells, and the frequency decreased in parallel with a decrease in malignant potential of the adenomas; antigen expression appeared in 71 % of carcinomas, 43% of villotubular adenomas, and 18% of tubular adenomas. Many tumors synthesize proteins whose expression is otherwise limited to a particular period in fetal development, and benign adenoma cells were found to express this property with increasing frequency as they progressed toward malignancy. Further measurements using tissue culture were carried out using two agents, TPA and deoxycholic acid (DOC), that act as tumor promotors in the gastrointestinal tract of experimental animals; their effects were compared in primary cultures of human premalignant colonic adenoma cells (Friedman, 1981). DOC affected only early-stage premalignant cells; among DOC, TPA, and EGF, DOC had the greatest stimulatory effect on DNA replication. The findings thus suggested that the major role of DOC in tumor progression


15

in vivo could be to enlarge the proliferative cell population of normal colon and early-stage premalignant cells, but not late-premalignant or tumor cells. TPA, on the other hand, differed from DOC with less stimulation of cell growth than DOC. The most pronounced effect of TPA was on intermediate and late-stage premalignant cell cultures, where it induced cell clustering, multilaying, and concomitant release of a protease with many properties similar to a plasminogen activator. Thus, with the development of new techniques for the tissue culture ofhuman colonic epithelial cells, it was possible to observe that as colonic cells progress to progressively abnormal stages of growth and differentiation, they gain or lose responsiveness to different growth factors and tumor-promoting agents (Fig. 6).

V. Antigenic Determinants of Gastrointestinal Cell Differentiation and Proliferation Further biomarkers of abnormal gastrointestinal cell differentiation and proliferation also have been studied and include specific gene products. Quantitative changes in gene expression that accompany transformation of gastrointestinal cells could reflect expansions of the stem cell compartment (Goldenberg, 1981) with concomitant arrested differentiation or aberrant clonal proliferation of specific cell types. Investigations of these properties of gastrointestinal cells have utilized immunochemical probes, which provide a wide range both of sensitivity and specificity. Certain antigens associated with human tumors have a relatively restricted distribution among normal tissues; they are broadly classified as organ, tissue, or site specific. In some cancers, particularly those involving the gastrointestinal tract, antigens have been described that have a more or less restricted site specificity [e.g., the mucusassociated “M” series (Bara et al., 1980) and second-trimester fetal antigen or TPA

DOC TPA

-* -

PLASMINOGEN ACTIVATOR

ENHANCE0 DNA REPLICATION

FIG.6. Diagram illustrating the differential and changing actions of tumor-promoting agents during stages of normal and abnormal growth of colonic epithelial cells. Deoxycholic acid (DOC) stimulated cell proliferation in normal and early-stage premaligmant (tubular) adenoma cells, but not in late-stage (villous) adeonoma cells. TPA stimulated cell proliferation in tubular adeonomas, and stimulated production of plasminogen activator in villous adeonomas and in carcinomas but not in tubular adenomas. (From Lipkin et ~ l . 1983.) ,

16


STFA (Higgins et a l . , 1984)l. Some, such as intestinal mucosal-specific glycoprotein (IMG), colonic mucoprotein antigen (CMA), colon-specific antigen(s) (CSA), sulfated glycopeptidic antigen (SGA), goblet cell antigen (GOA), and colon-specific antigen-p (CSAp), appear to be cell specific; none of these serologically defined markers, however, is cancer specific (reviewed in Goldberg, 1981). STFA, for example, has been found in colonic epithelial cells of patients with chronic ulcerative colitis whose [3H]dThd-labeling patterns indicated an expansion of the proliferative compartment of epithelial cells toward the lumenal surface of the crypts (Biasco et a l . , 1984). Similar to the circumstances of excess sialomucin production in ulcerative colitis with no apparent dysplasia, it is not yet clear if the correlation of STFA expression with expansion of the proliferative compartment in specific subpopulations of cells is reflective of a predisposition eventually to develop dysplasia. The inappropiate or ectopic expression of particular epithelial-specific antigens (at anatomic sites in the adult gastrointestinal tract at which they are not usually expressed) has provided an additional measurement for tumor clasification (Bara et a l . , 1981). The mucus-associated antigens M1, M2, and M3 occur in three major groups of nucus-secreting cells in the human gastrointestinal tract: M1 is associated with columnar cells of the gastric epithelium, M2 with the mucus cells of gastric Brunner’s glands, and M3 with intestinal goblet cells (Bara et a l . , 1980). The gastric M1 antigen occurs in %29% of colonic adenocarcinomas and in certain benign villous adenomas but is not present in normal adult colon (Bara et a l . , 1980). While the mechanism involved in the expression of M1 in colonic disease is not known, goblet cells of the second-trimester human fetus have been shown to contain both M1 (gastric) and M3 (colonic) antigens (Bara et al., 1980). The detection of M1-type determinants in the adult colon, therefore, appeared to reflect an altered pattern of expression of tissue-type components attendant to the development of particular premalignant and malignant lesions at this organ site. As detection and screening methods become more refined, previous putative “tumor-specific” antigens have become reclassified as “tumorassociated” or “differentiation” antigens due, in large measure, to quantitative differences in the level of their expression in normal and transformed cells. This trend is likely to continue, as it is apparent that gene activity may vary not only among individual subsets of cells within a tissue but also as a function of population growth status and individual cell cycle phase. An example of such heterogeneity in gene expression in differentiating gastrointestinal epithelial cells as a function of the stage of population growth, is provided by analysis of the cellular content of a single gene product (albumin) in individual cells of a clonal line of murine hepatic tumor cells propagated under low (exponential phase growth) and high (near plateau phase


17

growth) cell density culture conditions (Ryan and Higgins, 1988). Similar cell-to-cell variations in the content of carcinoenbryonic antigen occur during the transmission of some human colon carcinoma cell lines from low to high culture population densities (unpublished observations). It is clear that considerable cell-to-cell variability in the expression of a single gene product characterizes even clonally derived gastrointestinal tumor cell populations in vitro. Problems of tumor cell heterogeneity and intralesional variability of biomarkers have been discussed previously (Kerbel, 1984). These data illustrate the difficulty in applying one biomarker (be it at the morphologic protein, or nucleic acid levels) as a monitor of tumor cell “maturation,” or stage of differentiation, and underscore the importance of multiparametric profiling of pretumor or tumor cell populations. Nevertheless, the use of such antigens, particularly those that can be monitored in blood (such as a-fetoprotein, carcinoembryonic antigen, pancreatic oncofetal antigen, galactosyltransferaseisoenzyme, basic fetoprotein, TennaGen, and CSAp) (Goldenberg, 1981), can be relevant as associated parameters to evaluate loci of abnormal cell differentiation, and for prognostic assessments of tumor development. The distribution of these and other polyclonal antibody-defined antigens and/or monoclonal antibody-defined epitopes on human gastrointestinal epithelial cells, both normal and transformed, has recently been reviewed (Arends et al., 1985). Rapid progress is being made in the identification of specific molecular markers of differentiation in gastrointestinal cells. Such markers include, but are not restricted to, certain differentiation-dependent (and transformation-associated) changes in cellular enzyme systems (Colony, 1984; Balisetal., 1984), mucincomposition (Goldand Shochat, 1984; Baraetal., 1980), cell surface glycoconjugates (Kim et al., 1984), and monoclonal antibodydefined antigens (Steplewski and Koprowski, 1983; Hellstrom et al., 1984). Monoclonal antibody cloning criteria, screening strategies, and problematic aspects involving determination of tumor specificity relevant to construction of hybridomas with activity to human gastrointestinal antigens have been described in recent compendia (Arends et al., 1985; Atkinson et al., 1982; Raux et al., 1983; Steplewski and Koproski, 1983). It is clear from the present data that, at least for tumors arising in humans, a tumor-specific epitope has yet to be unequivocally demonstrated in abnormal gastrointestinal cell differentiation or in carcinomas (Summarized in Arends et a l . , 1985). Several promising candidate antigens, however, are currently under study (reviewed in Hellstrom et al., 1984). Since abnormally differentiating cells that develop into more aggressively growing tumor cells generally show loss of expression of tissue-related differentiation antigens (Arends et al., 1985), it would be important additionally to identify specific cellular antigens expressed mainly to very immature tumor cell types. The work of Quaroni et al. (1986) is illustrative of this last point. Using a panel of monoclonal antibodies prepared

18


to surface membrane components of the CaCo-2 human colon tumor cell line,

certain developmentally regulated “oncofetal” crypt cell antigens were identified in all poorly and moderately differentiated adenocarcinomas induced in the rat small and large bowel by DMH. Well-differentiated tumors reacted less intensely with these antibodies, with approximately half of such tumors exhibiting negative responses. A similar pattern of expression was seen in all human adenocarcinomas examined (Quaroni et al., 1986). Immunochemical approaches to the identification of human gastrointestinal tumor-associated antigens have rather obvious potential applications in diagnosis, follow-up, and patient management. Such directions would, most importantly, include the broadly classified areas of immunodiagnosis, radioimmunodetection (and tumor imaging), and immunotherapy (Zamchech, 1983; Begent and Bagshawe, 1983). Whether conventional polyclonal or defined monoclonal antibodies prove to be the most effective mediators for critical in situ imaging of gastrointestinal neoplasms or cytotoxic drug delivery remains to be determined (e.g., Lane and Koprowski, 1982). Many of the currently available monoclonal antibodies that define tumorassociated epitopes in gastrointestinal neoplasms have been found to be directed to specific carbohydrate or glycolipid moieties, the expressions of which appear to be developmentally regulated (Quaroni et al., 1986; Kim et a l . , 1984), supporting their tentative classification as “oncofetal” determinants (Quaroni et a l . , 1986; Bara et al., 1981). Other cellular components also change during carcinogenesis, however. The altered morphology that typifies certain transformed cells in vivn and in vitro appears to be reflected particularly in changes in the composition and organization of specifc cytoskeletal elements. Several recent reviews have focused on a comparison of cytoskeletal networks and their associated proteins in both normal and transformed cells (e.g., Brinkley and Chafouleas, 1984; Weber and Osborn, 1985). It is not intended to reiterate these data here, but rather to describe particular aspects of cytoskeletal protein expression as they relate to stages of cell transformation within the human gastrointestinal tract. Human colon carcinomas, both as primary cultures and as established cell lines, were found to be completely lacking (or possess poorly developed) actin stress fibers (Friedman et al., 1984). The SW480 colon tumor cell line, for example, is devoid of F-actin microfilament structures (Friedman et al., 1984). This is of particular interest in that SW480 carcinoma cells contain an activated c - r d - 2 oncogene (Cunningham and Werinberg, 1984), a sequence that when transfected (as a calcium phosphate precipitate of high molecular weight DNA isolated from SW480 cells) into NIH 3T3 cells results in aquisition of altered cell morphology, expression of transformed growth characteristics, and loss of actin cable structure (Wahrman et al., 1985). Disruption of actin organization was reflected in changes in the expression and cellular content of specific


19

tropomyosinlike isoforms (Wahrman et al., 1985), proteins that are thought to act to stabilize actin microfilament structures in situ. Altered expression of tropomysin isoforms appears to be a general property of cells transformed by oncogenic retroviruses (Hendricks and Weintraub, 1984; Lin et a l . , 1985), and occurs in cells transformed by various structually diverse oncogenes (Cooper et al., 1985). In addition to the tropomyosins, the cytoskeletal-associated protein p35 (Higgins et a l . , 1986) is also altered both in quanity and subcellular distribution in transformed cells from diverse species. p35 is a 35-kDa singlepolypeptide chain protein which has been extensively characterized (Higgins et al., 1979, 1986) and localized to the cytoskeletal matrix of at least some cell types in vitro, including Friend erythroleukemia (FL), hamster fibrosarcoma (Pizzi and Higgins, 1985) and bovine endothelial cells, either by the criteria or resistance to detergent extraction or by direct visualization on cellular filimentous elements (predominantly within the region of the membrane cytoskeleton) by immunofluorescence microscopy (Pizzi and Higgins, 1985). This protein is expressed at high levels in cells within the adult bone marrow, and its synthesis is maintained by FL cells in vitro. Studies have clearly indicated that the cellular content of p35 directly reflects the proliferative status of individual cells within a given population (Higgins et al., 1986) (Fig. 7). A human homolog of murine p35 has been identified by virtue of crossreactivity with anti-p35 serum (Higgins et al., 1984). Immunohistochemical analysis of the distributionin humans focused initially on its distribution in the fetus, since late-gestation fetal tissue is an enriched source of p35 in the mouse (Higgins et a l . , 1979, 1986). Since human cells did express a p35-like protein in vivo and (whereas in FL cells) the levels of p35 expression were linked to specific proliferative states, the incidence of p35 expression was examined in particular disease conditions of the human gastrointestinal tract that are associated with altered proliferative kinetics. Here the distribution of p35-positive epithelial cells was studied in the gastric mucosa of patients with chronic atrophic gastritis (CAG), a condition accompanied by changes in gastric epithelial cell proliferation and epidemiologically predispositional to the development of gastric carcinoma, as noted above. Both in corpus and in antrum of the stomach, mean numbers of [3H]dThd-labeled cells per gastric pit column and labeling index were almost twice as large for the CAG population compared to controls. In the antrum of the stomach in CAG, where most carcinomas arise, p35-positive lesions had an expanded proliferative compartment with labeling indices significantly greater than those of p35-negative lesions (Fig. 8). These findings indicated that a hyperproliferative state existed in CAG compared to the normal gastric mucosa, and in gastric antrum the hyperproliferation was accompanied by an increased incidence of p35-positive cells (Lipkin etal., 1985). Present data, however, cannot distinguish between an increased cellular content or

20

MARTIN LIPKIN AND PAUL HIGGINS Exponential Control

,-[Lv

400 c

Iz'

,

z' 0

so0

DNA Content

1000

0

p35

SO0 Content

loo0

FIG. 7. Histograms illustrating the cell cycle phase distributions (left panels) and the p35 content of GI-phase cells (right panels) in control, retinoic acid, and dimethyl sulfoxide (DMS0)-treated populations of FL cells. Equal numbers of control and treated FL cells were collected by centrifugation at 900 g. After two washes in Hanks balanced salt solution (HBSS) cell pellets were resuspended in 1 ml HBSS and forcefully ejected into 9 ml of ice-cold acetone-ethanol (1 : 1) for fixation at - 20°C for 24 hr. Fixed cells were collected by centrifugation at 900 g, rehydrated by two washes in HBSS, and treated with ribonuclease A (bovine pancreas, 200 U/ml) for 30 min at 37OC. Following ribonuclease digestion, FL cells were collected by centrifugation and resuspended in 1 ml of rabbit anti-p35 serum (1 : 50 in HBSS) or in normal rabbit serum (1 : 50) for a 1-hr incubation on ice. The cells were then pelleted at 900 g, washed twice in HBSS, and incubated for 1 hr with fluorscein isothiocyanate (F1TC)-conjugated IgG fraction of goat anti-rabbit I& in HBSS. After a final series of washes, cells were stained with propidium iodide (PI; 20 pglml in HBSS) for 30 min at room temperature prior to analysis with the use of an Ortho System 50H flow cytometer equipped with a 488-nm argon ion laser. Relative cellular DNA content was obtained on a cell-by-cell basis as the integrated red PI fluorescence signal. The pulse width of the PI signal was measured separately and used to get out unwanted cell doublets and aggregates. A third parameter (either integrated green fluorescence or forward-angle light scatter) was measured and correlated with the red fluorescence (DNA content) of each cell. The integrated green (FITC) fluorescence was proportional to cellular p35 content (Higgins ct al.,


21

increased immune reactivity of p35 in CAG tissue relative to p35 in the normal gastric mucosa. Whereas p35 is down-regulated in dimethylsulfoxide (DMS0)differentiated FL cells (Higgins et al., 1986) (Fig. 7), subsequent evidence indicates that this reduction in cellular p35 content is a function of entrance into a quiescent substate and is independent of the capacity for subsequent terminal differentiation. This conclusion is supported by several experimental observations: (1) FL cells exposed to all-trans retinoic acid, which results in the establisment of a quiescent substate without associated terminal differentiation (Traganos et al., 1984), have down regulated p35 contents similar to fully differentiated DMSO-stimulated FL cell populations (Fig. 7) (Traganos et al., 1984; Higgins et a l . , 1986), and (2) in both DMSO- and retinoic

FIG. 8. In the mucosa of the antrum of the stomach, comparison of labeling-index statistics (mean 1 SE) between CAG subgroup having positive ./-FA reactivity (clear areas) and the CAG subgroup having negative y-FA reactivity (diagonal shading) for individual gastric pit compartments and for the gastric pit as a whole. Symbols: "'p < 0.006; 'p < 0.02; tp < 0.07. (From Lipkin el al., 1985.)

*

1986). The mean fluorescence values for the G I compartment, based on DNA content, were computer-isolated and recorded for anaylsis with an Ortho 2150 minicomputer. A minimum of 5000 cells were measured for each sample. All values are in arbitrary units. Note the substantial S-phase (DNA-synthesizing) fraction in the control cell population, indicating a high level of proliferation. All histograms were normalized. Evident is the accumulation of DMSOdifferentiated hemoglobin-containing (Higgins et al., 1986) and retinoid-treated undifferentiated (Traganos ef al., 1984) cells in G I and the associated shift to lower cellular p35 content.

22


acid-treated FL cells, p35 reduction occurs early after exposure to inducer and is maximal within 48 hr of accumulation of cells into GI phase (Higgins et al., 1986). Release of FL and murine hepatic tumor cells from retinoid-induced (Traganos et a l . , 1984) and DMSO-induced (Higgins et a l . , 1986) quiescence is consisently associated with modulated p35 expression as is in vivo growth of hepatic adenoma cells, which have no defined quiescent substate (Higgins, 1985), 1986). The molecular basis governing the regulation of p35 expression in human gastrointestinal epithelial cells is currently under study.

ACKNOWLEDGMENTS This work was aided by Grant SIC-7 from the American Cancer Society, Grants CA 40876 and 08748 from the National Cancer Institute (ML), and a Merit Review Grant from the Veterans Administration (PH). We thank Mrs. P. Monaghan and Rosemary Petras for assistance in carrying out this work.

REFERENCES Aluments, J , , El Munshid, H . A,, Hakanson, R . , Hedenbro, J., Lieberg, G . , Oscarson, J., Rehfeld, J . F., Sundler, F . , and Vallgren, S. (198O).J. Physiol (London) 298, 557-569. Arends, J. W., Bosman, F. T . , and Hilgers, J. (1985). Biochim Eiophys. Acta 780, 1-19. Atkinson, B. F., Ernst, C . S., Herlyn, M . , Steplewski, Z . , Sears, H . , and Koprowski, H. (1982). Cancer Res. 42, 4820-4823. Balis, M . E., Yip, L . C., Rinaldy, A , , Relyea, N . M . , Higgins, P. J . , and Kemeny, N. E. (1984). frog. Cancer Res. Ther. 29, 295-308. Bara, J., Loisillier, F . , and Burtin, P. (1980). Br. J. Cancer 41, 209-221. Bara, J., Hanielin, L., Martin, E . , and Burtin, P. (1981). f n t . J . Cancer 28, 711-719. Begent, R . H . J , , and Bagshawe, K . D. (1983). “Oncodevelopmental Markers: Biologic, Diagnostic, and Monitoring Aspects” (W. H . Fishman, ed.), pp. 167-188. Academic Press, New York. Bell, B., Almy, T . P . , and Lipkin, M . (1967).J Nall. Cancer fnst. 38, 615-623. Bertrand, P . , and Willems, G. (1980). Gastroenterology 78, 918-24. Biasco, G . , Lipkin, M . , Minarini, A , , Higgins, P., Miglioli, M., anmd Barbara, L. (1984). Cancer ReJ. 44, 5450-5454. Bleiberg, H . , Mainguet, P., and Galand, P. (1972). Gastroenferology 63, 240-245. Brinkley, B. R., and Chafouleas, J . G. (1984). frog. Cancer Res. Ther. 2 9 , 67-90. Cameron, I. L . , Gosslee, D. G., and Pilgrim, C . (1965).J. Cell. Comp. Physiol. 6 6 , 431-435. Camplejohn, R. S. (1982). Recent Results Cancer Res. 83, 21-30. Capurso, L . , Teodori, L., DeVita, R . , Galloni, L., Tarquini, M . , Mauro, F., Nerui, C . , and Pallone, F. (1982). Gastroenterology 82, 1029 (Abstr). Chang, W . W . L., and Leblond, C . P. (1971). A m . J . Anat. 131, 73-100. Cole, J . W . , and McKalen, A. (1961). Gastroenterology 41, 122-125. Colony, P. C . (1984). frog. Cancer Res. Ther. 29, 91-111. Cooper, H . L . , Feuerstein, N., Noda, M . , and Bassin, R. H. (1985). Mol. Cell €501. 5 , 972-983. Correa, P., Cuello, C . , Duque, E., et al. (1976).J. Natl. Cancer f n s t . 5 7 , 1027-1035. Cunningham, J. M . , and Weinberg, R . A. (1984). B o g . Cancer Res. Ther. 29, 403-411. Delince, P . , Willems, G . , and deGraef, J . (1978). Digestion 18, 27-34. Deschner, E. E., and Lipkin, M . (1966). Exp. Cell Res. 43, 661-65.


23

Deschner, E. E., and Lipkin, M . (1978).Gastrointestinal Tract. 3-24. Deschner, E. E.,Winawer, S. J., and Lipkin, M . (1972).J. Natl. Cancer Inst. 48, 1567-74. Friedman, E. (1981).Cancer Res. 41, 4588-99. Friedman, E. A., Higgins, P. J., Lipkin, M . , Shinya, H . , and Gelb, A. M . (1981).In Vitro 17,

632-44. Friedman, E., Verderame, M . , Winawer, S., and Pollack, R . (1984). Cancer Res. 44,3040-3050. Glauert, H. P.,and Bennink, M . R. (1983).J. Nutr. 113,475-482. Gold, D.V., and Shochat, D. (1984).Prog. Cancer Res. Ther. 29, 159-167. Goldenberg, D. M . (1981). In “Gastrointestinal Cancer”. 0 . R. Stroehlein and M . M . Romsdale, eds.), pp. 65-81. Raven, New York. Greider, M. H., Steinberg, V . , and McGuigan, J . E. (1972).Gastroenterology 63, 572-583. Hansen, P. H . , Peterson, T . , and Larsen, J. K . (1975).Gut 16,23-27. Harris, C . C . , Trump, B. F., Grafstrom, R . , and Autrup, H. (1982).J . Cell Biochem. 18,

285-294. Hart-Hansen, O., Johansen, Aa., Larsen, J. K., and Svendsen, L. B. (1979).Acta Pathol. Microbiol. Scand. Sect. A 87, 217-222. Hattori, T. (1981).Nippon Rinsho 39, 36-41. Heitman, D. W., Grubbs, B. G., Heitman, T. O . , and Cameron, I. L. (1983).Cancer Res. 43, 1 153-1 162. Hellstrom, I., Hellstrom, K. E., and Brown, J . P. (1984).Prog. Cancer Res. Ther. 29, 185-195. Hendricks, M . , and Weintraub, H . (1984).Mol. Cell. Biol. 4, 1823-1833. Herbst, J. J., Berenson, M . M., Wiser, W . C . , and Freston, J . W. (1976). Clin. Res. 24, 168A (Abstr). Higgins, P. J. (1985).Age 8, 122-126. Higgins, P. J. (1986).Age 9, 71-78. Higgins, P. J., Tong, C . , Borenfreund, E., Okin, R. S., and Bendich, A. (1979).Br. J . Cancer

40, 253-259. Higgins, P. J . , Friendman, E., Lipkin, M . , Hertz, R . , Attiyeh, F., and Stonehill, E. H. (1983). Oncology 40, 26-30. Higgins, P. J., Murakami, S . , and Cardon-Cardo, C . (1984).Abstr. B i d . Med. Proc. 4th. Higgins, P. J . , Silverstone, A. E . , Bueti, C . , Pizzi, V. F . , Melamed, M . R . , Lipkin, M . , and Traganos, F. (1986).J.Natl. Cancer Inst. 76, 885-893. Hoff, M . B., Chang, W. W. L., and Mak, K. (1981). Virchorvs Arch. Cell Pathol. 35, (3). Ismail-Beigi, F., and Pope, C . E., I1 (1974).Gastroenetrology 66, 1109-13. Ismail-Beigi, F . , Horton, P. F., and Pope, C . E. I1 (1970).Gastroenterology 58, 163-174. Johnson, L. R.,and Guthrie, P. D. (1974).Gastroenterology 67,601-606. Kerbel, R . S. (1984).Prog. Cancer Res. Ther. 29, 197-201. Kim, Y.S . , Boland, C . R., and Mcintyre, L. J. (1984).Prog. Cancer Res. Ther. 29, 221-236. Lane, D.,and Koprowski, H . (1982).Nature (London) 296, 200-202. Lehy, T.,Dubrasquet, M . , and Bonfils, S. (1979).Digestion 19, 99-109. Lehy, T., Accary, J. P . , Labeille, D . , and Dubrasquet, M . (1983).Gastroenterology 84,914-919. Liavig, I. (1968).Acta. Pathol. Micurobiol. Scand. 72, 43-63. Lightdale, C . , Lipkin, M . , and Deschner, E. (1982).Cancer Res. 42, 4280-4283. Lin, J . J. C . , Helfman, D. M . , Hughes, S. H., and Chou, C:S. (1985).J . Cell Biol. 100,

692-703. Lipkin, M . (1971).In “Cell Cycle and Cancer,” pp. 1-26.Decker, New York. Lipkin, M., and Bell, B. (1968).Hand. Physiol. 5, 2861-79. Lipkin, M . , Bell, B., and Sherlock, P. (1963a).J. Clin. Invest. 42, 767-776. Lipkin, M.,Sherlock, P., and Bell, B. (1963b).Gastroenterology 45, 721-729. Lipkin, M . , Blattner, W . E. et al. (1983).Cancer Res. 43, 1899-1904. Lipkin, M . , Correa, P., Mikol, Y .B., Higgins, P. J , , Cuello, C . , Zarama, G . , Fontham, E . , and Zavala, D. (1985).J.Natl. Cancer Inst. 75, 613-619.

24


Livstone, E., Sheahan, D. G., and Behar, J. (1976). Gasfromferology 70, 909 (Abstr). MacDonald, W. C., Trier, J. S . , and Everett, N. B. (1964).Gastroenferology 46, 405-17. McGuigan, J. E., and Greider, M. H. (1971).Gastroenferology 60, 223-236. Maskens, A. P., Rahier, J . R., Meersseman, F. P., Dujardin-Loits, R., and Haot, J . G. (1979). Gut 20, 775-79. Messier, B., and Leblond, C. P. (1960.Am. J. A n d . 106, 247-294. Mirvish, S. S. (1981). In “Inhibition of Tumor Induction and Development” (M. Zedeck and M. Lipkin, eds.), pp. 101-126. Plenum, New York. Morson, B. (1955).Br. J. Cancer 9,377-385. Muiioz, N., Lipkin, M . , Crespi, M., Wahrendorf, J., Grassi, A., and Hsien, L. S. (1985).Inf. J. Cancer 36, 187-189. Oota, K. (1967). In “Carcinoma of the Stomach in Early Phase.” (T. Kurokowa, T. Kajitani, and K. Oota, eds.), pp. 1-266. Nakayama Shoteu Co., Tokyo, Japan. Pizzi, V. P., and Higgins, P. J. (1985).J. Cell Biol. 101,416a. Pozharisski, K. M., Klimashevski, V. F., and Gushchin, V. A. (1980).Exp. Pafhol. 18,407-413. Pozharisski, K.M . , Klimashevski, V. F., and Gushchin, V. A. (1982).Exp. Pafhol. 21, 165-179. Quaroni, A,, Weiser, M. M., Lee, S . , and Amodeo, D. (1986).J. Nafl. Cancer Inst. 77,405-415. Raux, H., Labbe, F., Fondanneche, M. C., Koprowski, H., and Burtin, P. (1983). In.J . Cancer 32, 315-319. Ryan, and Higgins, P. (1988).Submitted. Serafini, E. P., Kirk, A. P., and Chambers, T. J. (1981).Gut 22, 648-652. Shorter, R. G., Moertel, C . G . , Titus, J. L., and Reitemeier, R . J . (1964).Am. J . Dig. Dis. 9, 760-763. Sizikov, A. I., and Azykbekov, R. (1981). Vopr. Onkol. 27, 19-22. Stemmerman, G. N., and Hayashi, T. (1968).J.Natl. Cancer Insf. 4, 627-634. Steplewski, Z . , and Koprowski, H . (1983).“Oncodevelopmental Markers: Biologic, Diagnostic, and Monitoring Aspects” (W. H. Fishman, ed.), pp. 155-166.Academic Press, Inc., New York. Steplewski, Z., and Koprowski, H . (1983).“Oncodevelopmental Markers: Biologic, Diagnostic, and Monitoring Aspects” (W. H. Fishman, ed.), pp. 167-188.Academic Press, New York. Sunter, J. P., Watson, A. J., Wright, N. A,, and Appleton, D. R . (1979).Virchows Arch. Cell Pafhol. 32, 75-87. Tabuchi, Y., Inoue, K., Takiguchi, Y., Kawaguchi, K., and Mitsuno, T . (1980). Gann 71, 84-93. Takano, S.,Akagi, M . , Misumi, A., Matsuda, M . , Inamori, Y., Araki, K., and Shegaki, N. (1982). Proc. Jpn. Cancer Assoc. Annu. Meet. I l s f , Osaka p. 425. Takeuchi, K., and Johnson, L. (1982).Am. J. Physiol. 243, G463-68. Tannenbaum, S. R. and Corea, P. (1985). Nature 317, 675-676. Terpstra, 0. T . , van Blankenstein, M., Dees, J . , and Eilers, G. (1986). Gustromefrology 90,1662. Traganos, F., Higgins, P. J., Bueti, C., Darzynkiewicz, Z., and Melamed, M. R. (1984).J . Nafl. Cancer Insf. 73, 205-218. Tutton, P. J. M., and Barkla, D. H. (1980).Cancer 45, 1172-1177. Tutton, P. J. M., and Barkla, D . H . (1980). Clin. Ewp. Pharmucol. Physiol. 7, 275-80. Tutton, P. J., and Barkla, D. H. (1982a).Anticancer Res. 2, 199-202. Tutton, P. J. M., and Barkla, D. H. (1982b). Virchows Arch. Cell Pafhol. 38, 351-355. Tutton, P. J . M., and Barkla, D. H . (1986).Cancer Res. 46, 6091-6094. Wahrman, M. Z., Gagnier, S. E., Kobrin, D. E., Higgins, P. J., and Augenlicht, L. H . (1985). Tumour Bid. 6, 41-56. Weber, K., and Osborn, M., (1985). Sci. Am. 253, 110-121. Winawer, S. J., and Lipkin, M . (1969). J . NaL Cancer Insf. 42,9-17. Zamcheck, N. (1983).In “Oncodevelopmental Markers: Biologic, Diagnostic and Monitoring Aspects” (W. H . Fishman, ed.), pp. 333-349.Academic Press, New York.

CHEMICAL CARCINOGENESIS: FROM ANIMAL MODELS TO MOLECULAR MODELS IN ONE DECADE Stuart H. Yuspa and Miriam C. Poirier Laboratory of Cellular Carclnogenasls and Tumor Promotion Dlvlslon of Cancer Etlology, National Cancer lnstltute Betheeda, Maryland 20892

I.

Introduction

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

A. AnimalModels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cell Culture Models. . . . . . . . . . . . . . . . . . . . . 111. Biological Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Multistage Tumor Development .................... B. Clonality of Tumors. . . . . . . . . . . ............... C . Number of Genetic Changes Required for Cancer Development . . . . . . . . . D. Tumor Promotion . . . . . . . . . . . . . . . . . . . . . ........... IV. Cellular and Molecular Mechanisms of Carcinogenesis . . . . . . . . . . . . . . . . . . . . . A. Carcinogen Metabolism . . . . . . . . . . . . . ............ B. Carcinogen-DNA Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Activation of Oncogenes by Chemical Carcinogens .................... D. Phenotype of Initiated Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Cellular Basis for Tumor Promotion . ....................... 1 Ester Tumor Promoters F. Molecular Mechanism of Action of Ph V . Future Directions ............................................. Human Population for Carcinogen Exposure . . . . . . . B. Transgenic Mice and Cancer Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary and Conclusions . . . . . . ............................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25 26 26 30 33 33 35 36 38 39 39 41 45 48 49 51 54 54 58 59 61

I. Introduction During the last decade, chemical carcinogenesis research has substantially broadened our understanding of the cellular and molecular changes involved in cancer development and provided new information on numerous questions in cell biology. The association of chemicals as causative factors in human cancers, the development of in vivo and in vitro experimental models, and technical advances facilitating a molecular approach to problems in cell biology have provided the impetus for prbgress in this research area. It is remarkable that the initial observations of the pioneers in carcinogenesis research, now more than 40 years old, have remained valid and relevant. 25 ADVANCES IN CANCER RESEARCH, VOL. 50

26

STUART H . YUSPA AND MIRIAM C . POIRIER

The newer experimental models and techniques have built on these early observations to enhance our understanding of the process of cancer development at the molecular level. The purpose of this review is to focus on conceptual advances in chemical carcinogenesis research and describe, to a limited extent, the experimental data upon which they are founded. It is not intended to be a comprehensive review of research details but rather to provide an overview of the status of the field and its future directions. For the purposes of this review, carcinogenesis is evaluated as a multistage process in which the summation of events is required to produce a cancer. The first stage, initiation, is defined as the consequences of the initial interaction of a tissue with carcinogens. While pathological changes may not be apparent, the tissue is irreversibly altered and contains cells (initiated cells) which are the precursor to the future tumor. Promotion, the second stage, is confined to those processes which facilitate the expression of the initiated phenotype at the tissue level. Promotion may result from the influence of promoting agents on the entire tissue or on the initiated cells. Distinct from tumor promotion is the process of tumor progression. The changes in this stage of carcinogenesis include further phenotypic alterations in the initiated cell population, including conversion from a premalignant to a malignant cell type. While progression beyond malignant conversion to produce tumor heterogeneity and the metastatic phenotype is an important aspect of tumor biology, our review is confined to those changes which lead to the first detectable measure of malignancy. The ability to differentiate these stages and analyze their biology and biochemistry is possible through the use of specialized animal and cell culture model systems.

II. Experimental Models A.

ANIMAL MODELS

Chemical carcinogenesis research during the decade of the 1970s focused on the development or refinement of animal models for chemically induced cancers at specific organ sites in which the pathogenesis of benign and malignant neoplasms closely resembled tumor formation in the homologous human tissue (Farber and Cameron, 1980; Yuspa and Harris, 1982). These are outlined in Table I. Virtually every major form of human cancer can be reproduced in an animal species by exposure to specific chemical carcinogens, and, in many cases, cancer development is modified by additional exposure to tumor promoters. The development of skin cancer in rodents after systemic or topical exposure to chemical carcinogens has been widely used to define the stages in

27

CHEMICAL CARCINOGENESIS TABLE I ANIMALMODELSFOR CHEMICAL CARCINOGENESIS" Tumor type Skin

Mouse, rat

Nitrosamines, alkylating agents, aromatic amines, PAH

Skin

Hamster, guinea pig

DMBA

Melanoma

Liver

Mouse, rat

Nit rosamines, aromatic amines, vinyl chloride, PAH

Hepatocellular carcinoma, angiosarcoma

Lung

Mouse, rat, hamster, dog

Nitrosamines, asbestos, PAH

Adeno and squamous cell carcinoma, mesothelioma

Breast

Mouse, rat, dog

NMU, aromatic amines, DMBA

Adenocarcinoam

Colon

Mouse, rat

Nitrosamines, D M H

Adenocarcinoma

Pancreas

Rat, hamster, guinea pig

Azaserine. nitrosamines

Adenocarcinoma, ductal carcinoma

Bladder

Mouse, rat, hamster

Aromatic amines, nitrosamines

Urothelial carcinoma

Squamous cell and basal cell carcinomas

"Other target sites for chemical carcinogenesis include cervix, endometrium, esophagus, kidney, and brain.

carcinogenesis (Yuspa, 1986). The predominant lesions developing in carcinogen-exposed mouse skin are squamous papillomas and squamous carcinomas. Rats develop basal cell carcinomas at low carcinogen doses and squamous cell carcinomas from higher exposures (Rasmussen et a!. , 1983). Topical exposures of carcinogens and tumor promoters to the skin of hamsters and guinea pigs yields malignant melanomas which mimic the human lesion with regard to histological findings and pattern of metastatic spread (Pawlowski and Lea, 1983). Mouse strains vary widely in their sensitivity to skin cancer development after exposure to carcinogens and promoters (Slaga and Fischer, 1983), and this variability has been used as a model to explore the biological basis for cancer susceptibility (Strickland et a l . , 1982). This approach has indicated that the genetic composition of the experimental subject may determine sensitivity to tumor initiators (Stenback, 1980; Legraverend et al., 1980) or tumor promoters (Poland et al., 1982; Reiners et a l . , 1984). Liver cancer in rodents is commonly induced by aromatic amines, aflatoxin B,, and N-nitrosamines, and the predominant lesion is hepatocellular carcinoma (Farber, 1980; Peraino et a!., 1983). Proliferative stimuli such as partial hepatectomy enhance the sensitivity of the liver to carcinogen exposure.

28

STUART H. YUSPA AND MIRIAM C . POIRIER

When proliferative stimuli accompany or precede carcinogen exposure, liver tumors may develop after exposure to carcinogens that do not normally affect that site or after a single exposure to agents which usually require multiple exposures (Columbano et a l . , 1981). Inhalation of vinyl chloride by rodents produces hepatic angiosarcomas similar to tumors occurring after vinyl chloride exposure in human populations (Popper el a l . , 1977). The development of hepatic carcinomas is commonly preceded by numerous cellular foci which demonstrate aberrant expression of common hepatic enzymatic markers (Pitot et a l . , 1978). A fraction of these progress to premalignant hepatic nodules of which a few convert to cancer (Goldfarb and Pugh, 1982; Peraino et a l . , 1983). Lung cancer can be induced in mice, rats, hamsters, and dogs by systemic administration of certain N-nitroso carcinogens and by topical or inhalation exposure to a variety of other carcinogenic agents (Nettesheim and Marchok, 1983). Nonspecific irritation (e.g., saline lavage) leading to proliferation of the respiratory epithelium, in conjnction with carcinogen exposure, enhances pulmonary carcinogenesis (Shami et a l . , 1982). Asbestos induces lung carcinomas and mesotheliomas by intratracheal instillation and intrapleural administration. Asbestos also enhances the induction of bronchogenic carcinomas when combined with exposure to benzo[a]pyrene (Shabad et al., 1974; Stelle and Nettesheim, 1983). The induction of mammary cancers in mice, rats, and dogs, after exposure to a variety of carcinogenic agents, is dependent on the hormonal status of the host (Welsch and Nagasawa, 1977). Ovariectomy or hypophysectomy prior to carcinogen administration eliminates the tumor response. Similar ablations at later times increase the latency period for tumor formation or cause regression of preexisting mammary tumors. Likewise, adrenalectomy or elevation of prolactin or ovarian hormones enhances tumorigenicity of subcarcinogenic doses of chemicals or radiation (Clifton and Crowley, 1978; Welsch and Nagasawa, 1977). The mammary tumors derived from carcinogen-exposed animals are usually adenocarcinomas of ductal or alveolar origin and are generally preceded by preneoplastic hyperplastic alveolar nodules, ductal hyperplasias, or ductal papillomas (Medina, 1976). The metastatic pattern of chemically induced experimental mammary cancers is also characteristic of human breast cancer (Gullino et al., 1975). Animal age and prior mating history at the time of carcinogen exposure are important determinants of the tumor yield. In most strains of rats, a single carcinogen exposure around the time of sexual maturity produces a high incidence of tumors, but if carcinogen exposure occurs in older animals or in animals which had experienced pregnancy and lactation, the mammary tumor incidence is low (Russo et a l . , 1983).

CHEMICAL CARCINOGENESIS

29

Colon cancer can be specifically induced in mice and rats by systemic or topical administration of several classes of carcinogens, particularly aromatic amine, hydrazine, and N-nitroso carcinogens (Weisburger et al., 1977). The entire spectrum of changes seen in human colon cancer are reproduced in experimental rodent models, including abnormal crypt architecture with disrupted proliferation, papillary and adenomatous polyps, carcinoma in situ, and adenocarcinoma (Thurnherr et al., 1973). In the animal models, as in human cancer, the distal colon is affected with a greater frequency than other regions of the intestine. From comparative studies in germ-free animals, it appears that the intestinal flora play a role in carcinogen metabolism and may in part be responsible for the activation of certain previously metabolized forms of carcinogens which reach the colon via bile flow (Reddy et al., 1975). Pancreatic cancer can be induced in rats, guinea pigs, and hamsters by specific N-nitroso carcinogens (Moore d al., 1983; Roebuck et al., 1983; Safiotti, 1975). Oral administration of specific N-nitrosamines to hamsters produces ductal atypia, papillary hyperplasia, carcinoma in situ, and ductal or adenocarcinoma. Clinical symptoms and metastases develop in a pattern similar to those seen in human pancreatic cancer (Takahashi et al., 1977). Azaserine administration produces a high incidence of pancreatic adenocarcinomas (Roebuck d al., 1983). Bladder cancer occurs in rodents exposed by intravesicular instillations or systemically to N-nitrosamines, polycyclic aromatic hydrocarbons (PAH),’ or aromatic amines including 2-naphthylamine, a known human bladder carcinogen (Cohen et al., 1983a,b; Hicks et al., 1978). Carcinogen administration produces multiple papillary hyperplastic lesions of the bladder as well as transitional and squamous cell carcinomas. The incidence of bladder cancer is increased if subcarcinogenic exposure of a bladder carcinogen is followed by dietary administration of saccharin, cyclamate, or tryptophan (Cohen et al., 1983a,b; Hicks et al., 1978). Adenocarcinoma of the prostate gland occurs spontaneously in older males of certain rat strains. This process can be accelerated by chronically adminstering testosterone beginning at the time of sexual maturity (Pollard and Luckert, 1985). Systemic carcinogens which induce or accelerate prostrate carcinogenesis have not been identified. Other animal models exist for the production of cervical carcinoma (Park and Koprowska, 1968), endometrial Abbreviations used: 2-AAF, 2-acetylaminofluorene;2-AF, 2-aminofluorene;BHBN, N-butyl-N(4-hydroxybuty1)nitrosamine; BPL, 0-propiolactone; CO, croton oil; DDT, dichlorophenyltrichloroethane; DES, diethylstilbestrol; DMBA, 7,12-dimethylbenz[a]anthracene, DMCC, dimethylcarbamyl chloride; DMH, dimethylhydrazine; ELISA, enzyme-linked immunosorbent assay; GCMS, gas chromatography-mass spectrometry;GPI, glucose-phosphateisomerase; GTP, guanosine triphosphate; Hb, hemoglobin; MCA, 3-methylcholanthrene;MFO, mixed-functionoxidase; MMS, methyl methanesulfonate;NBC, nucleated blood cell; NMU, N-methyl-N-nitro.uourea; N-OH-AAF, N-hydroxy-2-acetylaminofluorene; 06-Me-dG, 06-methyldeoxyguanosine;PAH, polycyclic aromatic hydrocarbons; PGK, 3-phosphoglycerate kinase; SC, subcutaneous; SFS, synchronous fluorescence spectroscopy;TPA, 12-0-tetradecanoylphorbol-13-acetate.

30


carcinoma (Baba and von Hamm, 1967), esophageal and kidney cancers (Hard, 1979; Magee et al., 1976; Mirvish et al., 1985; Shirai et al., 1984), and brain tumors (Goth and Rajewsky, 1974). In addition, the transplacental or neonatal administration of DES to mice results in vaginal cancer in the offspring (Bern et a l . , 1976), an observation made well before the association was noted in humans.

B. CELLCULTURE MODELS About two decades ago, the first reports that carcinogens induced malignant transformation of cultured cells (Berwald and Sachs, 1965) provided impetus to explore the process of carcinogenesis at the cellular level. Cultured mesenchymal cells in early passage, or mesenchymal cell lines, were first used because they grew well under the relatively simple culture conditions which were standard at that time. These models confirmed the in vivo observations that malignant transformation was an inductive process and proceeded through multiple stages (Barrett and Ts’o, 1978; DiPaolo and Casto, 1977, 1978; Mondal and Heidelberger, 1970). The data demonstrated that proliferation after carcinogen exposure was required for transformation (Kakunaga, 1974), that the effectiveness of exposure was cell-cycle specific (Bertram and Heidelberger, 1974), and that genotoxic effects of exposures, including mutagenesis, could be measured directly and in conjunction with cell transformation (Barrett and Ts’o, 1978; Gehley et al., 1982; Huberman et al. , 1976). Together, results from these studies dissociated cytotoxicity from carcinogenicity and supported a somatic mutation mechanism for transformation, but indicated that the process was more complex than a single gene mutation. The more recent development of specialized cell culture techniques to support the growth of epithelial cells has been important for the establishment of the cellular criteria for transformation in specific target tissues (Borek, 1983). Currently, four culture models for the maintenance of epithelial cells from skin, liver, trachea, and breast have yielded substantial insights into unique changes which are intrinsic to cancer development in each cellular target site. Of interest for all models has been the consistent finding that early carcinogen-induced changes in epithelial cells are subtle, usually unrecognizable by most criteria studied, but become obvious when selective pressures for cell growth or survival are applied to the culture system. The basis or selection rests on the discovery that carcinogen-altered epithelial cells, long before they are tumorigenic, deviate in their response to normal signals for differentiation or normal requirements for growth. Each cell type has its own characteristic alteration, and common markers of carcinogenic changes among specialized cell types ( e . g . , chromosomal aberrations, anchorage-independent growth) occur much later in the process of carcinogenesis.


31

Culture methods specific for clonal or mass culture of epidermal cells from mouse, hamster, rat, and human have been developed (Yuspa, 1985). Epidermal cells from all of these species metabolize a variety of skin carcinogens (Hosomi et a l . , 1982; Kuroki et a l . , 1982). Metabolically activated carcinogens bind to mouse epidermal DNA to a similar extent in vivo and in vitro (Nakayama et a l . , 1984). At least three kinds of DNA repair processes have been identified in epidermal cells (Bowden and Yuspa, 1979; Hennings et a l . , 1974; Nakayama et a l . , 1984), and carcinogens can induce mutations in human and mouse epidermal cell cultures (Allen-Hoffman and Rheinwald, 1984; Reiners, 1985). Following carcinogen exposure some epidermal cells deviate from their normal program of differentiation or have altered growth requirements and can survive under culture conditions where normal cells slough from the culture dish because they terminally differentiate or senesce (Kulesz-Martin et a l . , 1980; Sina et al., 1982). Cell lines, derived from carcinogen-exposed cells after several subpassages, produce squamous cell carcinomas when transplanted to syngeneic or immunosuppressed hosts in vivo but other wise retain many characteristics of normal epidermal cells (Fusenig et a l . , 1979; Yuspa et a l . , 1980b). Cultured epidermal cells are responsive to a variety of skin tumor promoters and have been essential for delineating the biological heterogeneity of epidermal subpopulations which appears to be required for the promoting action of the phorbol ester class of tumor promoters (Yuspa et a l . , 1982) (see Section IV,E and F). Cultured liver cells of rats and mice have been a useful resource for analysis of carcinogen metabolism, which is qualitatively similar to liver metabolic activity in vivo (Suolinna, 1982). Mutation assays in rat hepatocyte cultures have provided a method to classify agents which are associated with liver carcinogenesis in vivo as genotoxic or nongenotoxic (Telang et d.,1982). Furthermore, ethionine is carcinogenic when added chronically to rat hepatocyte culture medium, suggesting a direct hepatocyte action of this dietary factor in liver carcinogenesis (Brown et a l . , 1983). Cultured hepatocytes have an inducible DNA repair system, and the activity of this system is related to the level of DNA synthesis in target cells (Zurlo and Yager, 1984), a finding which may be relevant to the enhanced carcinogenicity of several agents for proliferating liver cells in vivo. As with cultured epidermal cells cultured liver cells are not easily distinguished by cytological criteria as a predictor for the ability to produce tumors when transplanted in vivo (Montesano et a l . , 1977). However, selective culture methods can distinguish neoplastic cells from normal cells (San et al., 1979a,b), and carcinogen exposure in vivo yields cultured liver cells which are more resistant to the cytotoxic effects of several drugs than cells cultured from unexposed livers (Carr and Laishes, 1981). Methods for the cultivation of mammary epithelial cells from mouse, rat, and human sources in both cell and organ culture have privided new

32


information on the biology of the mammary gland and on the process of carcinogenesis in that organ. Subpopulations of mammary cells have been identified which show differential abilities to metabolize specific carcinogens (Gould, 1982). Mammary organ cultures are induced to undergo squamous differentiation by cyclic nucleotides and prostaglandins, a response which is maintained even by cultured anaplastic mammary carcinoma cells (Schaefer et al., 1980, 1985). Subpopulations of mammary cells with unique growth properties which influence the behavior of one another have been isolated from mammary tumors (Bano et al., 1984). In response to carcinogen exposure, both human and rodent mammary cells exhibit an extended life span or hormone independence in culture. These cells can be selected from normal cells, which senesce spontaneously and require continuous in vitro hormonal stimulation for survival (Chatterjee and Banerjee, 1982b; Ethier, 1985; Stampfer and Bartley, 1985; Tonelli et a l . , 1979). Carcinogen-exposed rodent mammary cells produce hyperplastic nodules in organ culture or when transplanted into the cleared mammary fat pad in vivo (Richards and Nandi, 1978; Tonelli et al., 1979). Selenium and retinoids, both agents known to inhibit mammary tumorigenesis in vivo, supress the development of carcinogeninduced preneoplastic cells in culture (Chatterjee and Banerjee, 1982a,b). In vitro techniques have facilitated the discovery of multiple preneoplastic stages during chemical carcinogenesis of airway epithelium. Cultured tracheal and bronchial cells of rat, hamster, rabbit, and human have the capacity to metabolize carcinogens (Cohen et al., 1979; Harris et al., 1982). In clonal assays in which carcinogen exposure occurs in vitro, or when tracheas are exposed to carcinogens in vivo and the epithelial cells are isolated and cultured clonally, the frequency of carcinogen-induced cell variants showing altered growth characteristics can be as high as l o - * (Pai et al., 1983; Thomassen et al., 1983). Altered growth variants are distinguished by their prolonged survival in vitro, while normal cells spontaneously die. Growth variants can be selected by using culture medium suboptimal for normal cells. These variants are considered the earliest manifestation of preneoplasia in the trachea, and dose-response characteristics suggest a one-hit phenomenon (Marchok et al., 1977; Pai et al., 1982; Thomassen et al., 1983). During the progression from variant growth control to malignant transformation, respiratory epithelial cells undergo a series of sequential phenotypic changes including immortality in vitro, anchorage independence, and tumorigenicity (Nettesheim and Barrett, 1984; Steele and Nettesheim, 1983). The predominant tumors derived from respiratory epithelial cells which undergo malignant transformation in vitro are squamous cell carcinomas; adenocarcinomas occur less commonly. The development of culture methods for cells from specific human tissues has provided the methodology required to extrapolate experimental animal data to human disease. Studies with human tissues, some of which have been


33

described above and will be discussed further (Section IV,A), have established that rodent and human cells from the same organ site are similar with regard to growth requirements, metabolism of carcinogens, and some carcinogeninduced biological changes (Harris, 1987). However, human epithelial cells and human fibroblasts have been extremely resistant to malignant transformation by chemicals (Benedict et al., 1975; Kakunaga, 1978) and demonstrate a high level of chromosomal stability in vitro (DiPaolo, 1983). The high stability of the human genome with regard to spontaneous or induced aneuploidy is an essential difference between human and rodent cells, and delineation of its underlying mechanisms has become a major challenge for research in carcinogenesis .

111. Biological Concepts

A. MULTISTAGE TUMOR DEVELOPMENT The multistage development of cancer, as defined by Berenblum and Schubik over 40 years ago, has become a commonly accepted concept. The linear dose-response curve for initiation of tumorigenesis, the irreversibility of the initiated state, and the persistence of initiated cells have been confirmed in a variety of target sites and indicate a genetic basis for initiation. In contrast, tumor promotion is reversible and requires repeated exposures of promoting agents with a defined frequency, suggesting that promotion is epigenetic. When one examines a list of model systems in which tumor promotion has been experimentally demonstrated (mouse skin, rat liver, rat esophagus, rat colon, rat bladder, rat mammary gland, mouse and rat stomach, rat trachea, mouse lung), it is clear that the phenomenon occurs in the epithelium of complex tissues. Most commonly, target epithelial cells are organized in a stratifying or maturing arrangement, frequently (but not exclusively) in a terminally differentiating lining epithelium. Such epithelia are often composed of more than one cell type or of cells in differing states of maturation. It is not clear if tissue complexity contributes to a requirement for a promotion stage in carcinogenesis or if other less complex organ sites have simply not been adequately explored. Nevertheless, these kind of epithelia and these organ sites represent the major target tissues for cancer in humans. The development of multiple experimental model systems to study initiation and promotion has expanded our awareness of the variety of pathways which ultimately produce a cancer (Fig. 1). In most model systems, the standard initiation and promotion protocols involving many classes of initiators and promoters primarily produce benign tumors and preneoplastic changes (Burns et al., 1983; Kaufmann et a l . , 1985; Peraino et a l . , 1975). T h e number of premalignant lesions which develop is dependent on the dose of intiator and

34

S T U A R T H . YUSPA AND MIR IAM C. PO IRIER Initiation linitiatorol

1

initialed cell

Progression

Conversion (promoters, initiators, others)

Promotion (promoters)

benign tumor

carcinoma

metastasis

carcinoma

metastasis

carcinoma

metastasis

1 2 initiatedcell a

benign tumor a

initiatedcell b

benign tumor b

3

initiatedcella

I

I

benign tumor a

benign tumor b

FIG.^. Stages in chemical carcinogenesis. Figure reflects experimental results using a variety of protocols to produce benign and malignant tumors with carcinogens a nd tumor proniotors in several experimental animal models. Ascending and descending arrows, initiator; vertical bars, promoters.

promoter. The frequency of conversion to malignancy is low (1-5% of the benign tumor yield), and carcinomas which occur late arise from benign tumors (Scherer, 1984; Taguchi et af., 1984) (Fig. 1, row 1). Most tumor promoters are not detectably genotoxic (Table 11). Putative nongenotoxic promoters do not enhance the number of carcinomas which develop, even after prolonged exposure (Hennings et af., 1983; Kaufmann et al., 1985; Verma and Boutwell, 1980), and do not alter the preneoplastic phenotype (Goldsworthy and Pitot, 1985). Thus nongenotoxic promoters do not appear to influence neoplastic progression. In contrast, repeated exposure to genotoxic initiating agents and certain tumor promoters with demonstrable genotoxicity (such as benzoyl peroxide on mouse skin) enhances the yield of malignant tumors (Hennings et al., 1983; Reiners et al., 1984; Williams et al., 1981) (Fig. I , rows 3 and 4). This implies that multiple genetic changes may be required to induce the malignant phenotype. The potential for individual benign tumors to become malignant spontaneously in the absence of further treatment beyond promotion is variable, and many benign tumors are essentially at no risk while others are at high risk, as suggested in Fig. 1, row 2 (Becker, 1985; Burns et af., 1976; Hennings et al., 1985; Peraino et al., 1983; Scribner et af., 1983). In the mouse skin model,


35

TABLE I1 EXAMPLES OF EXPERIMENTAL PROMOTING AGENTS Tissue Mouse skin Rat liver Hamster or rat trachea Rat colon Rat bladder Hormone-dependent tissues

Promoting agents Phorbol esters, anthralin, wounding, benzoyl peroxide. cigarette smoke condensate Phenobarbital, DDT, polychlorinated byphenyl Chronic irritation, phorbol esters Bile acids, cholestyramine, wounding Saccharin, cyclamate, DL-tryptophan, MMS Hormones, diet

this has been established through the application of weak promoters or limited applications of stronger promoting agents to intiated mouse skin. Such protocols produce fewer benign tumors than protocols with stronger promoters applied for longer periods, but similar numbers of carcinomas. The benign tumors which develop with weak promoters must therefore be highly sensitive to promoting agents and at high risk for malignant conversion. The determinants for these phenotypes were likely to occur at the time of initiation, perhaps as a consequence of the number of genes affected by the initiator (e.g, due to variable carcinogen delivery to individual target cells) or to the cellular function of a single genetic target of the initiator (Fig. 1, rows 2 and 3 ) (Hennings et al., 1985; Peraino et al., 1984; Scribner et al., 1983). Several studies have indicated that further genetic changes in a premalignant cell are responsible for conversion from a benign to a malignant tumor. Animals bearing benign tumors of the skin as a result of initiation-promotion protocols develop a high incidence of carcinomas with a short latency period when reexposed to initiating agents (Hennings et al., 1983). Administration of a single initiating dose of a genotoxic liver carcinogen to rats which had previously been exposed to a subcarcinogenic dose of a different carcinogen, resulted in a high proportion of hepatic nodules and carcinomas, whereas the secondary treatment caused no tumors in animals without prior exposure (Becker, 1975). A second exposure to initiating agents in rats bearing preneoplastic liver foci results in neoplastic progression via an intratumoral conversion of cells in hyperplastic nodules to neoplastic cells (Scherer et al., 1984). These studies imply that two or more genetic changes are required for carcinoma formation in the liver and skin models of carcinogenesis.

B . CLONALITY OF TUMORS Fundamental to interpreting biological data in carcinogenesis is an assessment of the clonal origin of tumors. Epithelial and mesenchymal tumors have

36


been chemically induced by a number of protocols at several organ sites in chimeric mice or in mosaic mice which express distinct isozymes of GPI or PGK (Table 111). These studies uniformly indicate that both benign and malignant tumors arise from cells of one isozyme type and are therefore primarily monoclonal. In two cases where exceptionally high doses of chemicals were used (Reddy and Fialkow, 1979, 1983), polyclonal tumors were detected, probably reflecting the coalescence of adjacent primary tumors, Dose-response studies indicate that a lower exposure dose favors monoclonality (Tanooka and Tanaka, 1984). When progression of individual monoclonal skin papillomas was monitored to the carcinoma stage (Taguchi et al., 1984), the malignant tumors always had the same isozyme pattern as their benign precursor lesions, confirming a precursor-product relationship. Similar monoclonal tumor patterns have been found in the vast majority of human tumors studied in mosaic individuals (Fialkow, 1976). The monoclonality of chemically induced tumors speaks against a cancer induction mechanism involving the release of a transforming factor into the tumor enviroment which would recruit normal cells into the transformed phenotype. For this mechanism to play a role in carcinogenesis, a preferential response of neoplastic cells to the effects of such a factor would have to be postulated (Sporn and Todaro, 1980).

C. NUMBEROF GENETIC CHANGES REQUIRED FOR CANCER DEVELOPMENT A variety of hypotheses have been proposed to define the number of genetic changes required to produce a malignant cell (Armitage, 1985; Moolgavkar and Knudson, 1981). Recent cell culture experiments support a two-hit requirement for genetic alterations in cancer development consistent with in vivo experiments involving initiation-promotion-initiation (see Section 111,A). Transfection of a single activated oncogene into primary low-passage diploid cells produces an altered cell phenotype, but in most cases the cells are not tumorigenic (Land et al., 1983; Ruley, 1983; Thomassen et al., 1985). In contrast, transfection of the same activated oncogenes into premalignant (nontumorigenic) cultured cells results in their malignant (tumorigenic) conversion (Newbold and Overell, 1983; Storer et al., 1986; Thomassen et al., 1985). Similarly, the cotransfection of two oncogenes into primary diploid cells is sufficient to produce the tumorigenic phenotype (Land et al., 1983; Newbold and Overell, 1983; Ruley, 1983). However, the combinations of activated oncogenes required are specific, suggesting an action of each individual oncogene which complements the action of the corresponding member of the pair for the test system studied. If chemical carcinogens activate or otherwise mutate cellular genes to produce active oncogenes, then a two-hit theory for

TABLE I11 CLONAL ORIGIN OF CHEMICALLY INDUCED TUMORS Tissue

Marker"

Chemicals

Results

References

1. Mouse skin

GPI chimera

DMBA and CO; MCA and TPA; MCA

Monoclonal papilloma and carcinoma

Iannaccone et al. (1978)

2. Mouse skin

PGK mosaic

DMBA and TPA; DMBA

Monoclonal papilloma; mono- and polyclonal papilloma

Reddy and Fialkow (1983)

3. Mouse skin

PGK mosaic

DMBA and TPA; DMBA

Monoclonal papilloma and carcinoma

Taguchi el al. (1984)

4. Mouse liver

PGK mosaic

2-AAF

Monoclonal preneoplastic nodules

Rabes

5. Mouse bladder

PGK mosaic

BHBN

Monoclonal bladder cancers

Kakizoe et al. (1983)

6. Mouse subcutis

PGK mosaic

> 200 pg MCA

Multiclonal fibrosarcomas

Reddy and Fialkow (1979)

7. Mouse subcutis

PGK mosaic

5 pg MCA

Monoclonal fibrosarcomas

Tanooka and Tanaka (1982)

8. Mouse subcutis

PGK mosaic

2.5 pg MCA

Monoclonal fibrosarcomas

Deamant and Iannaccone (1985)

9. Mouse skin

PGK mosaic

UV light

Monoclonal carcinomas

Burnham cf al. (1986)

%PI, glucose phosphate isomerase; PGK, 3-phosphate kinase.

ct

al. (1982)

38


cancer development is supported by the transfection studies. However, the genetic damage would have to be highly selective, resulting in the activation of complementary gene pairs for the production of cancer for that organ site. In a recent cytogenetic analysis of the tumors derived from normal cells transformed by the action of two cotransfected oncogenes (Oshimura et al., 1985), a nonrandom loss of chromosome 15 (Syrian hamster cells) was consistently identified. Likewise, preneoplastic Syrian hamster cell lines required the integration and expression of an exogenous oncogene for the transformed phenotype, but additional changes were required to produce rapidly growing malignant tumors when the cells were tested in viuo (Thornassen et al., 1985). In these studies, the additional genetic alterations may have resulted as an artifact of the transfection procedure, or from genetic instability of host cells receiving the two complementing oncogenes. Whether additional genetic alterations are required for malignant transformation or simply provide a growth advantage to the affected cell type during the process of tumor formation in vivo is not clear. However, results of all studies are consistent with two or more genetic changes being required for cancer induction.

D. TUMOR PROMOTION With the expanding array of experimental models to study carcinogenesis at specific target sites, the number of agents recognized as tumor promoters has increased (Table 11). These agents have been identified based on their capacity to increase the number and/or reduce the latency period of both preneoplastic and neoplastic lesions after limited exposure to initiators. Most promoting agents are tissue specific, do not require metabolic activation, and are nonmutagenic (Diamond et al., 1980). Mutagenic agents such as ultraviolet (UV) light and bromomethylbenzanthracene may also have tumor-promoting activity, although they are weak complete carcinogens (Scribner and Scribner, 1980). Promoting agents are structurally specific so that minor changes in chemical structure markedly affect promoting activity (Diamond et al., 1980; Hecker, 1978; Peraino et al., 1975). Most promoters induce proliferation in target cells; yet, a number of agents which induce Proliferation in specific target tissues are not active as promoters (Raick, 1974b; Slaga et al., 1976). Many of the benign lesions induced by initiation-promotion protocols are reversible upon withdrawal of the promoting agent (Burns et al., 1976; Pitot et al., 1985). While the precise mechanisms of action for all promoters are not understood (see Section IV,E and F), the likeliest common action of these agents is to cause a selective clonal expansion of the initiated cell population resulting in a clinically evident premalignant lesion and increasing the number of cells at risk for further changes in neoplastic progression (Burns et al., 1983; Farber, 1984; Moolgavkar and Knudson, 1981; Yuspa et a / . , 1981).

39


Experimental data suggest that the tissue-specific action of tumor promoters may play an important role in determining the target site for tumor formation (Table IV). When rodents are systemically exposed to certain widely acting initiating agents, initiating mutations occur in multiple organ sites. Subsequent administration of tissue-specific promoting agents determines the organ where tumors develop. In some models, e.g., rat liver and bladder, administration of a particular bladder tumor promoter depresses the induction of liver tumors while enhancing the number of bladder tumors in rats exposed to several different initiating agents (Williams et al., 1983; Imaida et al., 1983). These models may be extremely important in determining the cellular and molecular mechanism of action of tissue-specific promoting agents. The concept that the promoting agent is a major determinant in site-specific development of cancer has important implications for human carcinogenesis.

IV. Cellular and Molecular Mechanisms of Carcinogenesis

A. CARCINOGEN METABOLISM The elucidation of specific pathways involved in carcinogen metabolism and the identification of covalently bound adducts have been major accomplishments in the field of chemical carcinogenesis (Dipple et al., 1985; Miller, 1970). Precise delineation of the metabolic activation of many PAH (Dipple et al., 1984; Gelboin and Ts’o, 1978), aromatic amines (Garner et a l . , 1984; Miller, 1970), N-nitrosamines (Preussman and Stewart, 1984; Singer and Grunberger, 1983), aflatoxins (Busby and Wogan, 1984), and other compounds in animal models has constituted a large portion of the chemical carcinogenesis research effort during the past several decades. Many of these compounds are metabolized initially by the complex M F O system of

TABLE IV PROMOTING AGENTSDETERMINE TARGET SITEFOR TUMORS Initiation

Promotion

Target site

Reference

2-AAF or BHBN

Phenobarbital Saccharin

Liver Bladder

Nakanishi et al. (1982)

NMU NMU

Phenobarbital Saccharin

Liver and thyroid Bladder

Tsuda

4-Acetylaminostilbene 4-Acetylaminostilbene 4-Acetylaminostilbene 4-Acetylaminostilbene

None Phenobarbital DDT DES

Sebaceous gland Liver Liver Liver and breast

Hilpert el al. (1983)

N-Nitrosodiethylamine N-Nitrosodiethylamine

Phenobarbital Barbital

Liver and thyroid Liver and kidney

Diwan

el al. (1983)

el al.

(1985)

40


microsomal enzymes (Eisen et al., 1983; Lu and West, 1980; Singer and Grunberger, 1983, Chapter V). These enzymes, and subsequent enzymatic pathways which further modify metabolic products of the MFO, are ultimately responsible for the generation of DNA-bound moieties (Beland and Kadlubar, 1985; Miller, 1978; Singer, 1985; Stowers and Anderson, 1985). Additional pathways produce carcinogen metabolites which may be directly removed from cells and excreted (Singer and Grunberger, 1983,Chapter V; Yang et al., 1978)or which covalently bind to proteins (Neumann, 1984;Tannenbaum and Skipper, 1984). These metabolites are presumed not to be directly involved in carcinogenesis, but definitive evidence is lacking. Figure 2 provides an example of one of the major achievements in the field of carcinogen metabolism, the elucidation of the activation of benzo[a]pyrene by the P-450class of MFOs (Yang et al., 1978). A great number of metabolites of this carcinogen were identified, but none were specifically associated with the carcinogenic process until the late 1970s when several laboratories simultaneously focused on a pathway leading to the production of anti-benzo[a]pyrene-7,8-diol-9,10-epoxide, which proved to be the major mutagenic and carcinogenic metabolite of this molecule (Huberman et al., 1976;Phillips, 1983;Sims etal., 1974;Yang et al., 1977, 1978). Simultaneously, this metabolite was shown to interact almost exclusively with the 2-amino position of deoxyguanosine (Koreeda et al., 1978;Jeffrey et al., 1976;Phillips, 1983) (see Fig. 2), and one of the four possible stereoisomeric DNA adducts

FIG.2 . Metabolic activation of benzo[a]pyrdne diol epoxides. Solid triangles and dashed lines indicate that substituents are toward and away from the viewer, respectively. MFO, Mixedfunction oxidase; EH, epoxide hydrase.


41

was again associated uniquely with mutagenesis and carcinogenesis (Buening et al., 1978; Burgess et al., 1985; Levin et a l . , 1977; Pelling et al., 1984). This example illustrates the high degree of specificity necessary for some carcinogen metabolism pathways to yield tumor-initiating products. Recently developed expertise in culture of human cells and explants (Harris et al., 1982) has made it possible to study carcinogen metabolism in human tissues and test the validity of using animal models to study this aspect of carcinogenesis. Metabolic profiles generated by human tissues from specific organ sites have been compared to profiles from the analogous site in experimental animals and found to be qualitatively similar (Daniel et al., 1983). However, among individuals in the human population there is a 100-fold variation in quantitative capacity to metabolize a specific carcinogen (Harris et al., 1982). These studies are important both for predicting the likelihood that compounds known to cause tumors in other species will be human carcinogens, and for identifying cohorts of potentially high cancer susceptibility within the human population (Autrup and Harris, 1983; Harris, 1987). Human cell culture studies have identified distinct polymorphic phenotypes for chemical carcinogen metabolism, some of which appear to be associated with altered cancer susceptibility. For example, deficiency of a P-450 isoenzyme, debrisoquine 4-hydroxylase, is responsible for the poor metabolism of debrisoquine and approximately 14 other drugs observed among 5-10% of humans (Wolff et al., 1985). This phenotype, which appears to be associated with slow metabolism of PAH and aflatoxins, may also be associated with decreased incidence of liver and lung cancer in humans (Ritchie and Idle, 1982). In addition, measurement of specific enzyme activities has demonstrated slow and fast aromatic amine-metabolizing phenotypes in 28 samples of human liver microsomes exposed to 2-AAF in vitro (Minchin et al., 1985). In these samples polymorphism was exhibited for metabolic pathways involved in both N-hydroxylation (activation) and ring-hydroxylation (detoxification). A polymorphism for slow and fast aromatic amine acetylation has been demonstrated experimentally in bladder epithelium of rabbits (Weber and Hine, 1985). Slow acetylation is associated with higher DNA damage and greater susceptibility to bladder cancer in humans (Mommsen et al., 1985). The existence of polymorphisms for carcinogen metabolism in humans and the capability to screen for this variability provide an approach to evaluate individuals for cancer susceptibility with the ultimate goal of cancer prevention.

B . CARCINOGEN-DNA INTERACTIONS The formation of DNA adducts of chemical carcinogens provides one testable mechanism for induction of a permanent genetic change which could lead to the heritable lack of growth or differentiation control exhibited by

42

STUART H . YUSPA A N D MIRIAM C . POIKIER

cancer cells (Magee, 1983). Covalent DNA adducts of many carcinogens have been synthesized in vitro (Hemminki, 1983) and characterized in different tissues of animal species (Hemminki, 1983; Wogan and Gorelick, 1985). In general, there is an overall positive correlation between the extent of DNA adduct formation and the number of tumors which form in a specific tissue, and tumors rarely form in tissues which do not contain adducts. DNA adduct formation is considered to be a necessary, but not sufficient, condition for initiation of tumorigenesis (Wogan and Gorelick, 1985), since tumors do not always form in tissues containing adducts. For example, of five aromatic amines which induce rat liver DNA adducts, only two induce rat liver tumors (Neumann, 1983). Whether or not a tumor eventually forms in an initiated tissue may depend on many factors including the fixation of mutations in the DNA during replication (Grisham et al., 1983), and the clonal expansion of the mutated cells through tumor promotion (Sections IV,E and F). A specifictissue may exhibit varied susceptibility to the carcinogenic action of members of a class of compounds, while another tissue in the same animal may show an entirely different susceptibility pattern for the same compounds (Neumann, 1983). In part, these phenomena may be due to the tissue-specific metabolism of individual chemicals and demonstrate an important conceptual contribution of metabolic analysis (Miller, 1970). Mutagenicity, which occurs as a direct result of damage to DNA, is a shortterm surrogate of tumorigenesis which directly reflects the biological consequences of DNA adduct formation (Singer and Kusmierek, 1982). For this reason, mutagenicity assays have been employed to screen for carcinogens and reduce the time and expense involved in long-term tumor induction studies in animals (Poirier and DeSerres, 1979; Rinkus and Legator, 1979). For mammalian cells exposed under the same conditions, the frequency of neoplastic transformation is higher than the frequency of single-gene mutations, suggesting that carcinogenesis may involve damage at any one of multiple genetic sites (Huberman et al., 1976; Parodi and Rrambilla, 1977). Qualitatively, most carcinogens are mutagenic, but there are notable quantitative discrepancies among results for mutagenesis and carcinogenesis assays. For example, the mutagenic potency of a series of aromatic amines which formed adducts at the C-8 position of deoxyguanosine correlated neither with the quanitity of C-8 adducts formed in bacterial or mammalian mutagenesis assays, nor with the overall potency of the same compounds for liver tumor formation in the rat (Beland et al., 1983; Heflich et al., 1986). These studies imply that not only are the amount of DNA adducts and the structure of the lesions important, but their genomic location in the DNA is also biologically relevant. In order to focus on the types of genomic changes required for mutagenesis and carcinogenesis, specific adducts have been introduced into small, welldefined pieces of DNA and the biological consequences of these lesions explored. For example, the tetracycline resistance locus on plasmid pBR322 was

43


mutated by exposure in uitro to 2-AF or 2-AAF and passaged through Escherichia coli. AAF produced twice as many tetracycline-sensitive mutants per adduct as AF. Sequence analysis of a 276-base pair carcinogen-targeted restriction fragment in the tetracycline resistance gene indicated that frameshift mutants commonly followed AAF adduction, whereas G T transversions were common after AF exposure. These experiments demonstrate that particular adducts produce specific gene changes and that the type of gene change produced affects the frequency of the expression of the mutation (Bichara and Fuchs, 1985). In another study, sequence specificity for the occurrence of base transitions and transversions following UV light exposure was demonstrated within a suppressor tRNA gene carried by an SV40 shuttle vector (Hauser et al., 1986). In these experiments thymine dimers were less mutagenic than other kinds of DNA damage, suggesting that the replication of DNA past dimers may be less error prone than replication past other UV lesions. Site-directed mutagenesis, using a specifically placed carcinogen-DNA adduct, has also contributed to our understanding of the biological consequences of a particular type of DNA adduction (Singer and Grunberger, 1983, Chapter IX). For example, insertion of an 06-Me-dG at a specific site in a plasmid genome resulted exclusively in G A transitions upon passage through E. coli (Essigman et al., 1985). This observation is important in light of recent oncogene studies which have demonstrated that mammary gland tumors of rats administered NMU, which forms 06-Me-dGin mammalian cells, contain a G - A transition at the r d gene twelfth codon. This mutation is known to activate the rmH protooncogene into a transforming oncogene (Zarbl et a / . , 1985). This topic will be detailed in Section IV,C. The importance of DNA adducts in the carcinogenic process has been emphasized by investigations of adduct removal, which occurs spontaneously due to adduct instability, by dilution during normal cell turnover, and enzymatically by DNA repair processes. In most cases, enzymatic adduct removal is a crucial protective mechanism acting to offset the carcinogenic insult (Magee, 1983; Singer and Grunberger, 1983, Chapter VIII.) When repair mechanisms are lacking, such as in the human disease xeroderma pigmentosum (XP), tumorigenesis is accelerated (Knudson, 1977). XP patients develop multiple skin cancers relatively early in life because they lack the capacity to repair DNA lesions including those produced by exposure to sunlight (Cleaver, 1968). Comparative studies of carcinogen-exposed cultured fibroblasts from normal and X P patients indicate that efficient removal of carcinogen-DNA adducts protects against cytotoxicity, mutagenesis, and the carcinogen-induced phenotype of anchorage-independent growth (McCormick and Maher, 1983). Complex mechanisms for repair of carcinogeninduced DNA damage (Fig. 3) have been identified through analysis of carcinogen-DNA interactions (Kraemer and Slor, 1984; Roberts, 1982). These

-

-

44

S T U A R T H . YUSPA A N D MIRIAM C . POIRIER

BAsf UCISKm REPllR DAMAQEDDIU

r

L*u nnnn

1

wcmonoE utusnm WlR

nnnnn ~ENDONUCLEASE

GLYCOSYLASE

nnnn

INSERTASE

uuuu

nnnn

REPAIRED DNA IOAMAGE REMOVE01

WIT E P t I u T I o N REPAIR

-lnTmmT

-0-umJ-u

nnnnn -O-u

REPLICATION WITH DAMAGE IN PLACE

I1Iu1,IuuL

GAP FILLING IWLYMERASE OR RECOMBlNATlONl

t

nnnnn ~KXVMERASE

TT7J-rrmr

nnnnn p s E

Qn-u-uu

nnnnnn +

-uuuuuu

nnnnnn REKICATED DNA IDAMAGE PRESENT1

nnnnn

REPAIRED DNA IDAMAGE REMOVED)

FIG.3. Schematic diagram of D N A repair pathways in mammalian cells. (Modified from Kraemer and Slor, 1984.)

pathways are expressed to various extents in cells of different species and in response to particular classes of carcinogens (Roberts, 1982; Singer and Grunberger, 1983, Chapter VIII). Base excision repair involves removal of a few nucleotides, including the specific modified base, usually by glycosylase enzymes (Roberts, 1982). Removal of small alkyl moieties from the O6position of deoxyguanosine is accomplished by 06-alkylguanine-DNA transferase (Pegg, 1984); in this case the base is undisturbed. Larger adducts require removal of about 100 nucleotides adjacent to the damaged site and subsequent replacement of the excised sequences using the opposite strand as template in the nucleotide excision repair pathway. Cells in S phase can also replicate DNA strands around a carcinogen-DNA adduct, leaving a gap in the newly replicated strand which is filled in at a latter time (postreplication, Fig. 3). It is not clear that all of these processes are error free, and in some experimental systems mistakes made during the course of repair of carcinogen damage have been demonstrated (Razzaque et al., 1983). While repair processes are often efficient at removing DNA adducts, they are rarely complete, and the harmfulness of persistent adducts may also contribute to the carcinogenic process (Wogan and and Gorelick, 1985). For example, in mouse skin topically exposed to radiolabeled PAH, 10-15% of the total DNA adducts adducts formed within 24 hr persist for many weeks after exposure (Randerath et a l . , 1985a; Shugart, 1985a), suggesting that, in the epidermis, adduct persistence occurs in dormant subpopulations of cells. Autoradiographic studies have confirmed the prolonged retention of radiolabeled benzo[a]pyrene in the nuclei (but not necessarily the DNA) of a


45

slowly cycling subpopulation of skin basal cells which are located in the center of the epidermal proliferative unit (Morris et al., 1986). Application of a hyperplastic agent to mouse skin results in the rapid loss of persistent DMBA adducts (Randerath et al., 1985b), suggesting that the same slowly cycling cells were induced to enter the cell growth cycle, resulting in adduct removal or dilution. The predominant intrastrand DNA adducts of the chemotherapeutic agent cisplatin, which is also an experimental carcinogen, persist in tissue DNA of cancer patients for weeks after cisplatin treatment (Pokier et al., 1987). The presence of these adducts may be related to the chemotherapeutic efficacy of the drug but, in addition, could be a risk factor for potential cisplatin-induced second malignancies. Highly sensitive and specific procedures are now available to measure persistent adducts in both experimental animals and in the human population (see Section V,A).

c. ACTIVATIONOF O N C O G E N E S BY C H E M I C A L CARCINOGENS The foregoing analysis of carcinogen metabolism and DNA modification has not addressed the critical question of genes involved in tumorigenesis. If DNA damage constitutes the mechanism of initiation, one could postulate the existence of specific genomic regions which must be altered if initiation is to occur. Several years ago it was discovered that the transfection of DNA from tumor cells into nonmalignant NIH 3T3 cells could transmit the malignant phenotype, producing 3T3 cell tumors which could be passaged in nude mice (Shih et al., 1979; Shilo and Weinberg, 1981). Expansion of these studies to include DNA from human tumor cell lines and tumor biopsies demonstrated that up to 30% of human tumor cell lines and 5-10% of human solid tumors contain DNA sequences, or oncogenes, able to transform NIH 3T3 cells after transfection (Fujitaetal., 1985; Yokotaetal., 1986). Simultaneously, it was shown that the malignant phenotype in a number of animal and human tumors is associated with either increased expression of an otherwise-normal gene product or expression of an altered gene product (Balmain, 1985; Hunter, 1981). The identification of these sequences as activated cellular homologs of retrovirzl transforming genes was unexpected (Hunter, 1981). It should be noted that many primary animal tumors do not appear to contain oncogenes which can be detected by transfecting tumor DNA into NIH 3T3 cells (Garte et al., 1985; Guerrero et al., 1985). T o date, the predominant oncogenes isolated from solid tumors of the type experimentally induced by chemicals (Balmain et al., 1984; Balmain and Pragnell, 1983; Beer et a l . , 1986; Yaswen et al., 1985) are activated forms of individual members of a family of genes designated ras. Members of this gene family all encode a 2 1,000 MW membrane-bound protein (p2 1) which binds G T P and has intrinsic GTPase activity, although the exact cellular function of this protein has not been determined. The possibility that in uitm carcinogen modification of ras DNA could result in activation of the ras gene product by

46


mutagenesis was first supported by studies in which a cloned, plasmid-borne cellular ras gene was modified with the anti benzo[a]pyrene-7,8-diol 9,lO-epoxide in uitro and transfected into NIH 3T3 cells (Marshall et al., 1984). Transformation was obtained in these cells at plasmid modification levels between 0.08and 0.4% (one or two carcinogen molecules per coding sequence) and 5 of 17 secondary transfectants derived from separate foci exhibited loss of restriction-enzyme recognition sites at codon 12. Thus, a mutation had occurred at this site. In uiuo exposure to chemical carcinogens and consequent tumor development have also been associated with mutation-mediated ras gene activation (Table V). Mammary tumors were induced in three different rat strains by a single exposure to NMU or DMBA (Sukumar et al., 1983;Zarbl et al., 1985). Of 48 NMU-induced carcinomas, 36 contained transforming DNA, and in each of these the ras gene was activated by a point mutation in codon 12 resulting in a G 4 A transition. This is the mutation commonly induced by 06-Me-dG formed as a result of NMU exposure (Pegg, 1984; Singer and Kusmierek, 1982). In the same study, 3 of 14 DMBA-induced mammary tumors yielded transforming DNA activity, and all three contained an activated rm gene but none had mutations at codon 12 (Zarbl et al., 1985). More recently the mechanism of rm activation by DMBA initiation in mouse skin has been postulated to involve a mutation at codon 61. When NIH 3T3 cells were transfected with DNA from DMBA-induced skin papillomas and carcinomas, a high percentage produced transformed foci (Balmain et al., 1984;Balmain and Pragnell, 1983;Quintanilla et al., 1986). All of these foci contained an activated form of ru", and >90% were activated by an A T transversion in codon 61. Since DMBA has a high propensity to produce deoxyadenosine adducts (Dipple et al., 1983), a mutation has been postulated to occur at the deoxyadenosine residue in mouse rmHcodon 61 resulting from DMBA binding to that site at the time of initiation (Quintanilla et al., 1986). These data as well as other examples of mutational M S activation shown in Table V (Garte et al., 1985;Guerrero et al., 1985;Wiseman et al., 1986), suggest that carcinogens produce site-specific mutations based in part on nucleoside selectivity of the ultimate carcinogenic metabolite. Furthermore, the sequence of a particular cellular gene, as well as the role of the amino acid in the gene product which is encoded by the target codon, will determine if a carcinogen-induced mutation will lead to activation of the gene product. Thus, in human populations sequence polymorphisms in potential oncogenes could determine susceptibility to carcinogenesis (Krontiris et al., 1985). ras codons 12 and 61 are sites where mutation causes a change in the gene product associated with oncogenic potential (McCormick et al., 1985;Jurnak, 1985). Mutations at these sites induce conformational changes which alter nucleotide binding to the p2 1 protein so that there is a reduction in p2 1 GTPase activity.

-

TABLE V ACTIVATION OF ONCOGENES BY CHEMICAL CARCINOGENS

Target tissue

Carcinogen exposure

Tumor (number studied)

Number of tumors with 3T3 transforming DNA

Transfectants containing activated rmH (%)

Codon

Molecular change Mutation

Rat breast

NMU

Carcinoma (48)

36

100

12

G

Mouse skin

DMBA and TPA

Papilloma or carcinoma (33)

26

92

61 12

A G

Mouse liver

Vinyl carbarnate

Hepatoma (7)

7

100

61

AT AT

1 '-OH-2 ',3 '-dehydroestragole

Hepatoma (1 1)

11

Hepatoma (7)

7

N-OH-AAF

91a

AT AT

100

CG 61

Mouse thymus

Rat and mouse skin

?-Ray NMU

Thymic lymphoma (15) Thymic lymphoma (7)

MMS BPL DMCC

Rat nasal carcinoma (8) Mouse skin carcinoma (5) Mouse fibrosarcoma (10) or carcinoma

12 7

8 2 0

Ob Ob 0 100

-

A transition

Zarbld al. (1985)

T transversion, 89%

Quintandla ct al. (1986)

TA transversion, 85% GC transition, 15%

Wiseman et al. (1986)

- A transition, 3%

-

Reference

TA transversion, 50% GC transition, 50%

- AT transversion, 100% C - A transversion

58 % 57 %

Guerrero el al. (1985)

Unknown

Garte d 01. (1985)

-

"One t u m o r contained nuKi. bAll of the NMU-induced transfectants contained msK,a n d all of the y-ray-induced transfectants contained rasN.

48


This, or some other associated change in the function of the gene product, ultimately affects the growth characteristics of the cell. Other studies suggest that rus activation is associated not only with initiation, but with malignant conversion as well. Transfection of an activated human rus oncogene, containing a codon 12 mutation, into cell lines derived from mouse skin papillomas, which do not contain a constitutively activated rm gene, causes malignant progression (Harper et ul., 1986). These studies suggest that a defined temporal sequence for the activation of a specific oncogene may not be required, and that mutations may occur at a single genomic locus early or late in carcinogenesis and contribute to phenotypic progression,

D. PHENOTYPE OF INITIATED CELLS A large data base has now been established regarding the molecular interactions of initiators with cells and the activation of specific genes which contribute to the cancer phenotype, but the biological changes which define initiated cells or more advanced premalignant cell types have only recently been characterized. A detailed analysis of the initiated phenotype has come from studies of cultured mouse epidermal cells (Yuspa, 1985a,b). In mouse skin, papillomas are the neoplastic manifestation of the initiated phenotype. The papilloma is characterized by a high proliferation rate and the presence of proliferating cells in the tissue strata normally confined to postmitotic terminally differentiating cells (Yuspa, 1985b). In cell culture, it was discovered that carcinogen exposure resulted in clonal foci of proliferating cells which grew selectively under culture conditions which favored terminal differentiation of normal epidermal cells (Kulesz-Martin et al., 1980) (see Section 11,B). Similar differentiation-resistant foci formed in cultures from carcinogeninitiated mouse skin but not from normal mouse skin (Kawamura et al., 1985; Yuspa and Morgan, 1981). The number of foci was proportional to the potency of the initiator and the dose of initiator, and was related to the extent of DNA binding at the time of exposure (Nakayama et al., 1984). Differentiation-altered foci could be isolated in culture from mouse skin which was initiated 10 weeks prior to cell isolation (Kawamura et ul., 1985). This observation is consistent with the previously demonstrated persistence of the initiated state. These experiments, and other supporting data, have led to the conclusion that an altered response to signals for terminal differentiation is associated with the initiated cell phenotype in epidermis. The frequency of this event in initiated skin (10-5-10-fi)is consistent with a mutation at a large or multiple genetic site (Yuspa and Morgan, 1981). Defective terminal differentiation is also a characteristic of mouse epidermal cells transfected with an exogenous mutated sus oncogene (Yuspa et al., 1985) and of human skin


49

carcinomas which otherwise behave as normal cells in culture (Rheinwald and Beckett, 1980). Results with hematopoietic cells or adipocytes, model systems which express differentiated function in vitro, indicate that changes in the differentiation program may be an early and common change in carcinogenesis of these cell types (Sachs, 1980; Scott and Maercklein, 1985). Other epithelial cells, when exposed to carcinogens in vivo or in vitro, acquire an altered phenotype which may be recognized by selectivecriteria in vitro (as reviewed in Section 11,B). To reiterate, the life span of carcinogen-exposed rat tracheal epithelial cells is extended in culture, and the altered cells become less sensitive to growth factor or feeder layer requirements for clonal growth. An altered mammary epithelial cell phenotype, resulting from limited carcinogen exposure, is manifested in cells from mouse, rat, and human samples as a focal growth of cells with an extended life span in vitro, the ability of cells to form alveolar nodules in the absence ofexogenous hormone in vitro, or the ability to form hyperplastic nodules when transplanted to cleared mammary fat pads in vivo. Comparative analysis of hepatocytes from normal rat liver and from preneoplastic nodules indicates that the preneoplastic cells are resistant to the cytotoxic effects of certain liver carcinogens and toxins (Novicky et d . , 1985). It has been proposed that resistance to cytotoxicity is associated with y-glutamyltranspeptidase activity, which is frequently elevated in preneoplastic mouse and rat liver foci (Hanigan and Pitot, 1985). Furthermore, compared to normal liver cells, preneoplastic liver foci have reduced levels of enzymes required for drug metabolism (Pitot et a l . , 1985) and a reduced capacity to form DNA adducts of 2-AAF during continuous 2-AAF feeding (Huitfeldt et al., 1986). Taken together, current results in four model systems indicate that early carcinogen-induced lesions are focal and their phenotypic character is tissue specific, but generally involves an altered response to specific differentiation stimuli, altered growth requirements, or resistance to cytotoxic substances. More limited analysis suggests that similar changes occur in esophageal and lung cells from rats exposed to carcinogens in vivo (Terzaghi et al., 1981). At present, these subtle and tissue-specific changes must constitute our definition of the cellular phenotype of initiation.

E. CELLULAR BASISFOR TUMOR PROMOTION It is unlikely that the molecular mechanism of action of promoters is common for all agents in all tissues. However, a common cellular basis for promotion is evolving from recent in vivo and in vitro experiments which have analyzed responses of carcinogen-altered (initiated), preneoplastic or benign, and malignant tumor cells to promoter exposure. Normal cells from the same tissue were similarly evaluated. The data obtained from four tissues (skin, liver, colon, and bronchus) of several species indicate that tumor promoters are often cytostatic or cytocidal to normal cells whereas altered cells are resistant. Alternatively, certain tumor promoters are mitogens for their

50


target tissues, but altered cells are more sensitive to their growth-stimulatory effects. For example, normal mouse epidermal cells terminally differentiate in response to phorbol ester tumor promoters, while initiated epidermal cells and cells lines from benign skin tumors do not (Hartley et al., 1985; Yuspa et al., 1983, 1986). A similar differential response pattern was observed among normal and neoplastic human epidermal cells (Parkinson et al., 1984). Cells lines derived from benign skin tumors are highly sensitive to the mitogenic properties of phorbol esters (Yuspa et al., 1986). Other studies have shown that initiated epidermal cells are resistant to cytotoxic effects of benzoyl peroxide, a skin tumor promoter which is highly toxic to normal epidermal cells (Hartley et al., 1985). Differences analogous to the skin model have been observed between preneoplastic and normal rat liver cells exposed to liver tumor promoters. A variety of initiation protocols yielded preneoplastioc cellular foci which could be promoted by virtue of their resistance to a promotion protocol utilizing agents which are cytostatic or cytotoxic to surrounding normal liver cells (Tsuda et al., 1980). Preneoplastic hepatic foci, visualized following carcinogen exposure, are more sensitive than normal liver cells to the mitogenic effects of a variety of noncytotoxic liver promoters (Schulte-Hermann et al., 1981, 1983). Furthermore, cells in these foci may undergo a programmed cell death which is specifically prevented by certain liver tumor promoters (Bursch et al., 1984). Normal, preneoplastic, and neoplastic human colon cells in culture respond differentially to phorbol ester tumor promoters, although these agents are not known to promote colon carcinogenesis. Preneoplastic colonic cells from patients with familial polyposis and cells from villous adenomas are highly sensitive to the mitogenic effects of phorbol esters, whereas normal colonic cells, including those from colon cancer patients, are insensitive (Friedman et al., 1984). Interestingly, phorbol esters suppress the growth of skin fibroblasts from normal human donors, but fibroblasts from familial polyposis patients are insensitive to this cytostatic effect. This has prompted the suggestion that a systemic alteration in response to promoting agents may exist in this autosomal dominant cancer-susceptibility syndrome (Kopelovich and Gardner, 1983). Cultured human bronchial epithelial cells like epidermal cells, are induced to differentiate by phorbol ester tumor promoters, while cell lines derived from bronchogenic carcinomas are resistant, even at high promoter concentration (Willey et al., 1984). These examples of differential response patterns to promoters, though specific for each agent and diverse in specific responses, would all produce the same biological effect, the selective growth of cells with the carcinogen-altered phenotype. In essence, the promoting environment in vivo would be similar to the selective growth conditions in vitro, which have been successful in


51

elucidating the initiated phenotype (see Section IV,D) (De Luca, 1983). Although other cellular mechanisms for tumor promotion may become obvious, current data suggest that the selective clonal expansion of the initiated cell population is the predominant action of tissue-specific tumor promoters.

F. MOLECULAR MECHANISM OF ACTIONOF PHORBOL ESTERTUMOR PROMOTERS Of the known promoting agents, phorbol esters and particularly TPA have been most extensively studied. These plant-derived diterpene esters are extremely potent in mouse skin and produce a number of biological and biochemical changes in cultured cells (Blumberg, 1980, 1981; Diamond et al., 1980; Hecker et al., 1982; Slaga, 1983). Studies with these agents have formed the basis for virtually all of the current hypotheses on the molecular events in tumor promotion. The reversibility of individual promoter exposures, the requirement for repeated, frequent phorbol esters exposures to accomplish promotion, and a likely mechanism of promotion based on selective clonal expansion of initiated cells suggest that the mechanism of phorbol ester action is epigenetic. Pharmacologically, phorbol esters demonstrate a strict specificity of structure, pleiotropic effects at nanomolar concentrations, and effectiveness after brief exposures, all suggesting the presence of a specific cellular receptor. The presence of cellular receptors with high affinity for phorbol esters was first documented in 1980 by Driedger and Blumberg. Subsequently, receptors have been identified on nucleated cells of all vertebrate and most invertebrate species studied, and receptor number is particularly high in brains of several species (Blumberg et a l . , . 1982, 1984; Shoyab and Todaro, 1980). The high degree of receptor conservation, its ubiquitous presence, its tissue distribution, and the very high number of binding sites per cell, suggested that it might be a structural protein or an enzyme (Blumberg et al., 1982, 1984). Recently, the phorbol ester receptor has been identified as the calcium phospholipid-dependent protein kinase C (Ashendel, 1985; Castagna et al., 1982; Leach et al., 1983; Niedel et a l . , 1983; Sando and Young, 1983). Phorbol esters not only bind to this enzyme with structural specificity, but also activate its kinase function. The binding affinity and enzyme-activating potency of a series of phorbol esters parallel their promoting potency (Blumberg et al. , 1984, 1985; Leach and Blumberg, 1985). The central importance of protein kinase C in tumor promotion was demonstrated by the discovery of potent promoting agents, structurally dissimilar from phorbol esters, which also bind to protein kinase C with high affinity and activate its kinase function (Arcoleo and Weinstein, 1985; Fujiki and Sugimura, 1987). Furthermore, the proposed endogenous ligand for protein kinase C, diacylglycerol, competitively

52


inhibits phorbol ester binding to the enzyme (Sharkey et a l . , 1984) and reproduces many of the pharmacological effects of phorbol esters (Ebeling et a l . , 1985; Jeng et al., 1985; Jetten e t a l . , 1985; Rozengurt et al., 1984). Protein kinase C is implicated as a second messenger in the actions of several hormones or other exogenous stimuli which function through membrane receptors (Nishizuka, 1986; Takai et al., 1982, 1985), suggesting that a common primary action may exist for several classes of tumor promoters. The activation of a protein kinase and subsequent phosphorylation of critical substrates provides a framework to explain the cascade of biochemical changes produced in vivo and in vitro by phorbol esters and some other promoters. However, the ensuing cellular changes which are critical to an epigenetic mechanism of tumor promotion remain to be defined. A number of cell surface changes have been observed in cultured cells exposed to phorbol esters. Among these, alterations in ion transport (Rosoff et al., 1984; Sussman et a l . , 1985), alterations in hormone binding (Lee and Weinstein, 1978), and inhibition of cell-to-cell communication (Enomoto and Yamasaki, 1985; Trosko and Chang, 1983; Yotti et a l . , 1979) are likely to have broad biological significance. Activation of the arachidonic acid cascade, with subsequent generation of prostaglandins, may also mediate some of the events required for promotion (Fischer, 1983; Marks, 1983). In most cell systems, two general classes of cellular responses have been evident, the modification of cellular differentiation or the stimulation of cellular proliferation (Blumberg, 1980, 1981; Diamond et al., 1980). These responses vary with the cell type under study. Phorbol esters can block or induce differentiation or can modify the differentiation pathway, for example, by channeling promyelocytic leukemia cells into macrophage differentiation (Yamasaki, 1983). Both in vivo and in vitro, phorbol esters can modify the differentiation of epidermal cells, their primary target cell for promotion (Raick, 1974a; Yuspa et al., 1980a). Phorbol esters act differentially on epidermal cell subpopulations, inducing differentiation in one cell class and stimulating cell proliferation in another (Reiners and Slaga, 1983; Yuspa et al., 1982). This differential response pattern may be associated with heterogeneity in expression of protein kinase C species among epidermal subpopulations, since at least two receptor classes have been observed in normal mouse and human skin in vivo and in vitro (Dunn and Blumberg, 1983; Dunn et a l . , 1985; Greenebaum et a l . , 1983), and multiple protein kinase C genes and mRNA species have been identified in mammalian tissues (Coussens et a l . , 1986; Knopf et al., 1986). At the molecular level, the resistance of initiated or preneoplastic epidermal cells to the induction of terminal differentiation by phorbol esters (see Section IV,E) could be related to a change in expression of a particular species of protein kinase C which is required in the transduction of signals for terminal differentiation. Such a change would likely be an indirect consequence of the


53

initiating event if it were consistently found to occur. In a limited study of initiated epidermal cell lines, a single class of phorbol ester receptors predominated and was similar in binding parameters to the putative receptor associated with the proliferative response in normal cells (Dunn et a l . , 1985). This information, though currently confined to the epidermal cell model, provides a molecular basis for the differential response patterns to phorbol esters observed among normal and carcinogen-modified cells originating from several target sites for carcinogenesis (see Section IV,E). It is important to consider alternative mechanisms for phorbol ester action, in particular, that promoter-induced changes in genetic information or expression in initiated cells would facilitate the phenotypic manifestation of an otherwise suppressed tumorigenic genotype. Several reports have indicated that phorbol esters can induce sister chromatid exchanges or chromosomal aberrations in cultured cells, but these effects have been variable depending on the cell types studied or culture conditions (Dzarlieva-Petrusevska and Fusenig, 1985; Kinsella and Radman, 1978; Nagasawa and Little, 1981). Since chromosomal changes can be inhibited by antioxidants or superoxide dismutase, and phorbol esters can generate the release of oxygen radicals from appropriate target cells (Copeland, 1983; Emerit and Cerutti, 1982; Troll et a l . , 1982), activated oxygen species have been proposed to mediate tumor promotion by phorbol esters (Cerutti, 1985; Kensler and Trush, 1984; Troll and Wiesner, 1985). DNA strand breaks, observed after phorbol ester exposure, have also been associated with the release of active oxygen species (Cerutti, 1985), but phorbol esters have not been shown to be mutagenic in most bacterial or mammalian test systems. Thus, it is unlikely that oxygen radicals generated by phorbol ester exposure would contribute to tumor promotion by permanently altering the initiated cell phenotype via a genotoxic action. However, free radicals could contribute to tumor promotion indirectly by producing a selective cytotoxic effect. Alternatively, release of free radicals could play a role in altering gene expression in phorbol ester-treated cells, and thus contribute to an epigenetic process (Friedman and Cerutti, 1983). Phorbol esters are known to induce a variety of cellular functions, presumably by a transcriptional activation mechanism (Blumberg, 1980, 1981; Diamond et al., 1980). Of particular interest are reports of the activation of latent viral genomes of oncogenic papilloma and Epstein-Barr viruses (Amtmann and Sauer, 1982; Zurhausen et al., 1978) to produce replicating and transforming viruses. Phorbol esters also enhance the transforming activity of certain defective DNA viruses (Fisher et al., 1979). Phorbol esters have a variable effect on the expression of retroviral oncogenes or cellular protooncogenes and suppress oncogene expression in some model systems while enhancing expression in others (Coughlin et al., 1985; Craig and Bloch, 1984). The transcription of a series of protooncogenes was not altered in mouse

54


epidermis after single or multiple applications of TPA (Toftgard et al., 1985), although chronic exposure tended to reduce expression of c-abl. The expression of the transformed phenotype induced by a mutated c-ras" gene or certain viral oncogenes was enhanced in cultured fibroblasts upon exposure to phorbol esters (Connan et a l . , 1985; Dotto et al., 1985; Hsaio et al., 1984). Furthermore, the growth of epidermal cells expressing a u-rusH oncogene was selectively stimulated over normal cells when the cells were exposed to TPA in uitro (Yuspa et al., 1985). Certain cell lines contain specific endogenous gene sequences which make them highly susceptible to phorbol esters, particularly with regard to inducing the transformed phenotype (Colburn, 1987). Alteration in expression or structure of such genes could contribute to susceptibility differences for tumor promoters among mouse strains or to the differential responses noted among cell types. Elucidation of the structure and function of the promoter-sensitivity gene products could enhance understanding of the molecular events beyond protein kinase C activation in phorbol estermediated tumor promotion.

V. Future Directions A . BIOMONITORING OF T H E HUMAN POPULATION FOR CARCINOGEN EXPOSURE Carcinogenesis studies in experimental animals have provided much information concerning carcinogen metabolism, macromolecular binding and other aspects of the interaction between a carcinogen and host tissue, and the relationship of this interaction to tumor formation. In uitro experiments confirm that similar interactions occur in human cells and tissues. However, until recently it was not possible to study such interactions in uiuo in the human population. Development of highly sensitive methods to measure attomole (10 - I 8 M) quantities of carcinogen bound to cellular macromolecules has made possible the monitoring of human tissues for evidence of biologically relevant chemical carcinogen exposure (Harris, 1985; Poirier and Beland, 1987). Carcinogen-DNA adducts and carcinogen-protein adducts, which parallel the extent of DNA adduction in animal models given single-dose exposures (Neumann, 1984; Shugart 1985b; Tannenbaum and Skipper, 1984), are being monitored in human populations, and the data are being evaluated for possible contributions to human cancer epidemiology. Measurement of carcinogen modification of DNA and protein has been achieved with a high degree of sensitivity by immunoassays utilizing adduct-specific antibodies (Poirier, 1984), 32P-postlabeling of adducts (Randerath et a l . , 1985c), and various chemical detection methods including SFS (Vahakangas et al., 1985),


55

GCMS (Tannenbaum and Skipper, 1984), and high-pressure liquid chromatography (HPLC), which can be used alone or in combination with other methods (Groopman et al., 1985; Umbenhauer et al., 1985). The first application of DNA adduct detection methods for human exposure to carcinogens utilized antibodies specific for benzo [alpyrene-modified DNA in a pilot study to probe for adducts in tissues of lung cancer patients (Perera et al., 1982). Since then, similar studies have been initiated with other human cohorts (Table VI), including smokers (Everson et al., 1986; Santella et al., 1987); coke oven, roofing, and foundry workers (Harris et al., 1985; Haugen et al., 1986; Shamsuddin et al., 1985); Chinese living in areas of either high aflatoxin (Groopman et al., 1985) or high N-nitrosamine exposure (Umbenhauer et al., 1985); and cancer patients receiving either cisplatin (Poirier et al., 1985, 1987) or 8-methoxypsoralen (Santella et al., 1987) chemotherapy. The polyclonal and monoclonal antibodies used in these studies were elicited against either individual adducts or modified DNAs, and were employed in either radioimmunoassays or ELISAs with sufficient sensitivity to measure attomoles of adducts per microgram of DNA. This corresponds to one adduct in lo8 nucleotides. The assays are specific, quantitative, and inexpensive, and they can be performed on the DNA obtained from nucleated blood cells using 35-50 ml of blood. Variability among replicate assays at low adduct levels requires repetitive analysis of individual samples for validation. It has been shown that the anti-benzo[a]pyrene DNA is specific for adducts of other hydrocarbons which are structurally similar to the major adduct of benzo[a]pyrene (Weston et al., 1987). This broad specificity is actually advantageous in the monitoring of human samples, but its discovery suggests the necessity to characterize each antiserum thoroughly. 32P-Postlabeling(Table VI) has also been utilized for detection of DNA adducts in placenta from smokingmothers (Everson etal., 1986,1987), and in buccal mucosaof Indians who chew tobacco or betel nut, or Filipinos who practice inverted smoking (Dunn and Stich, 1986). The postlabeling procedure involves hydrolysis of DNA to nucleosides, enzymatic phosphorylation to mononucleotides with 32P-ATP,and separation of normal nucleotides from adducted nucleotides by their altered migration on thin-layer chromatography (Randerath et al., 1985c). The labeled, modified nucleotides are localized by fluorography . This procedure is capable of detecting one adduct in lo9 bases, and requires only microgram amounts of DNA. However, the small quantities of adducts which are measured precludes the structural characterization of an unknown adduct (Everson et al., 1986). It is auseful screen for the presence of DNA adduction in a biological sample when the nature of the adduct or the exposure is unknown. A variety of chemical procedures have been used to enhance the detection of DNA and protein adducts. In one approach, DNA is first hydrolyzed and fractionated by HPLC, and presumptive adduct peaks are quantitated by

TABLE VI INDUCED DNA AND PROTEINADDUCTSIN MONITORINGOF CHEMICALLY

Assay

THE

HUMAN POPULATION

Estimated adduct concentration (finol/pg DNA)

Human cohort

Source of human tissue

ELISA

Lung cancer patients

0.08-0.115

Perera ef QI. (1982)

PAH

ELISA

0.04-2.4

Shamsuddin ct af. (1985)

PAH

ELISA

Roofers and foundry workers Coke oven workers

Normal lung and lung tumor NBCa NBC

0.10-34.0

PAH

ELISA

Smokers

NBC, placenta

Cisplatin 8-Methoxypsoralen PAH

ELISA ELISA

SFS

Cancer patients Cancer patients Coke oven workers

NBC NBC NBC

Anatoxin

Chinese in highexposure area Chinese in highexposure area Smokers

Urineb

Unknown

HPLC and ELISA HPLC and ELISA ”P Postlabeling

Harris cf af. (1985); Haugen d af. (1986) Santella cf a1 (1985); Everson ct al. (1986) Pokier d al. (1987) Santella et d. (1987) Vahakangas d af. (1985); Weston d al. (1987) Groopman ef d.(1985)

Unknown

32PPostlabeling

Betel nut and tobacco chewers

Ethylene oxide

GCMS

4-Aminobiphenyl

GCMS

Sterilization plant workers Smokers

Exposure

DNA d u PAH

Reference

d s

Nitrosamines

Stomach and esophagus Placenta, buccal smears Buccal smears

0.4-1.6 0.025-0.40 1.6-2.2 0.4-26.5 7

0.02-0.16

Umbenhauer ct d. (1985)

0.03

Everson ef af. (1986, 1987); Durn and Stich, (1986) Dunn and Stich, (1986)

(0.3

Protein adducts

“Nucleated blood cells.

‘Adducts expressed as ng/ml urine.

Hemoglobin‘ Hemoglobin‘ “pmol adduct/g Hb.

50-1 3,000 0.02-1.25

Calleman d af. (1978); Farmer ef QI. (1986) Tannenbaum ef af. (1986)


57

immunoassay. This method, which removes unmodified bases prior to immunoassay, has enhanced the sensitivity for detection of 06-Me-dG (Umbenhauer at al., 1985) and aflatoxin and its adducts (Groopman et a l . , 1985) in human tissues and urine, respectively. SFS (Vahakangas et a l . , 1985) is a chemical method used to detect DNA adducts of carcinogens, such as benzo[a]pyrene and aflatoxins, which have intrinsic fluorescent properties. DNA samples are scanned at a fixed difference between excitation and emission wavelengths in a fluorescence spectrophotometer. The method is accurate in the range of one adduct in lo7nucleotides and has been used to confirm results with immunoassays (Weston et al., 1987). The most sensitive chemical method used for detection of carcinogen bound to protein (primarily Hb) is GCMS, in which the chemical, released from the protein by hydrolysis, is derivatized to pentafluoropropionic anhydride, and then analyzed by gas chromatography coupled to a mass spectrometer. The ability to scan at a particular molecular weight eliminates background signals contributed by other compounds. This method provides absolute chemical characterization and is sensitive in the range of 10 fmol/g Hb, where it has been used to detect adducts of 4-aminobiphenyl in the blood of smokers (Tannenbaum et al., 1986). The same method, without the derivatization, is less sensitive, but has been used to detect hydroxyethylvaline and hydroxyethylhistidine adducts in the blood of ethylene oxide-exposed workers (Farmer et al., 1986). Sufficient experience in the measurement of DNA and protein adducts in human samples has accumulated to allow preliminary conclusions to be drawn concerning the feasibility and usefulness of the approach. Biomonitoring requires readily available human tissues, and current protocols have successfully used red blood cells (Farmer et al., 1986; Tannenbaum et af., 1986), nucleated blood cells (Harris et al., 1985; Haugen et al., 1986; Poirier et al., 1985; Shamsuddin et al., 1985), placenta (Everson et al., 1986), exfoliated bladder cells (Groopman et al., 1985), and buccal smears (Dunn and Stich 1986), suggesting that the obtainment of tissue by relatively harmless techniques is readily accomplished. Precise determination of dose-response is not possible for environmentally ubiquitous agents (Perera et al., 1982), even though human cohorts with higher than average exposure, e.g., coke oven workers, or people who live in high-exposure areas, do tend to have higher adduct levels than the population at large (Haugen et al., 1986; Shamsuddin et al., 1985; Tannenbaum et af., 1986; Umbenhauer et al., 1985). In addition, significant levels of PAH, nitrosamine, and 4-aminobiphenyl adducts are present in tissues of individuals not obviously subjected to unusually high exposure. For example, postlabeling methods have detected as many total DNA adducts in buccal smears from laboratory workers as in those from inverted smokers and betel nut or tobacco chewers (Dunn and Stich, 1986). To avoid the problems of incidental exposures and to accumulate dose-response data in

58


a controlled human setting, recent experiments have utilized DNA samples from patients receiving cancer chemotherapeutic agents which bind to DNA and may also be experimental carcinogens. In a study with nucleated blood cell DNA samples from patients on cisplatin therapy, adducts increased in a dose-related fashion and unexposed controls showed no evidence of adduct formation (Poirier et al., 1987). The data in Table VI indicate that, following incidental or iatrogenic exposures, DNA adducts are formed in human tissues in concentrations of one adduct in between lo6 and lo8 nucleotides, or 110-1 1,000 adducts per cell. This range is similar to adduct concentrations in experimental animals exposed to tumorigenic dosages of carcinogenic chemicals (Nakayama et al., 1983; Wogan and Gorelick, 1985). DNA adduct formation in humans occurs in a variety of organ sites, again similar to the situation in animal models. H b adducts in humans range between 0.02 and 13,000 fmol/mg Hb, while H b adducts in exposed animal models are ususally 100-fold higher. Many of the human determinations have been made near the limits of sensitivity of current assays. The development of more sensitive assay procedures will allow confirmation of previous determinations and assessment of adducts in currently unmeasureable target sites. The measurement of carcinogen adducts in samples from humans receiving incidental exposures provides evidence that exposure has occurred and has had a biological effect on the exposed individual. Present experience indicates that the quantity of adduct measured will be a function of multiple factors in addition to exposure dose. Insufficient data exist to assess the influence of factors which modulate the extent of adduct formation or the long-term biological consequence of these adducts (Poirier and Beland, 1987). Although the value for risk assessment remains to be determined, these techniques provide a unique opportunity to extrapolate experimental concepts to the human population and a potentially valuable approach for biochemical epidemiology of cancer.

B. TRANSGENIC MICEAND CANCER GENES The construction of transgenic mice by injecting, into the fertilized embryo, a single extrinsic gene construct which is then present in all somatic and germ cells, promises to be the technological advance which may ultimately provide the best data on cancer genes. Recently, genetically modified mice have been produced which carry in their genome an oncogene linked to a mammalian gene sequence with a controllable or tissue-specific regulatory region (Table VII). These transgenic mice and their progeny have been observed for spontaneous tumor development. In all cases, tumors developed within 6 months in a single target site which was associated with the specificity of the regulatory

59


TABLE VII TRANSGENIC MICEAS INDICATORS OF GENETIC DETERMINANTS OF CANCER Exogenousgene

Tumor

Modifiying factor

Reference

MTV/myc fusion gene SV40 T-antigen gene

Mammary carcinomas Choroid plexus tumors

Stewart et al. (1984) Brinster et al. (1984)

Rat insulin II/SV40 T-antigen fusion gene

Pancreatic &cell tumors

Required pregnancy Amplification of SV40 genes in the tumor Other islet cells destroyed prior to tumor formation

Immunoglobulin enhancerhyc gene

Lymphoma

Long latency preceded by lymphadenopathy

Hanahan (1 985)

Adams et al. (1985)

element or with amplification of the extrinsic gene (Brinster et al., 1984). However, the biology of tumor development in all transgenic constructs suggested that, while expression of the oncogene was required in the target tissue, additional changes in target cells must occur before tumors develop. In the case of mammary tumors (Stewart et al., 1984) or pancreatic tumors (Hanahan, 1985), all of the mammary gland cells or islet cells of the pancreas expressed the exogenous oncogene, but only a few developed into tumors. Mammary tumors required pregnancies in the affected animals. Many pancreatic @ cells were destroyed prior to tumor formation in only a few islets in the insulin II/SV40 transgenic mice. Furthermore, in the mice constructed with a mydimmunoglobulin enhancer gene complex (Adams et al., 1985), a long latency period was followed by the emergence of one or two clonal lymphoma lines, suggesting that additional cellular changes were required in individual lymphocytes prior to malignant tumor formation. These results are consistent with a multistage process for cancer development. The production of transgenic mice for use in combination with carcinogen or promoter exposure, could elucidate the precise role of individual genes or modifying factors in each stage of tissue-specific carcinogenesis.

VI. Summary and Conclusions During the last decade, progress in chemical carcinogenesis research has been substantial, and understanding the cellular changes and molecular causes of initiation, promotion, and malignant conversion appears to be within reach. Cancer begins as a carcinogen-induced genetic change in a single cell. The interaction of a particular carcinogen with specific genetic sites results, in part, from selectivity of metabolically activated carcinogens for particular nucleosides or gene sequences. In turn, modification of the molecular structure at specific genetic loci will have tissue-specific and species-specific

60


consequences dependent on the expression of a particular gene, its sequence, and the function of the gene product in the target cell. It is likely that inactivation of regulatory regions, genomic rearrangements, and point mutations in coding sequences all can result in an altered cell phenotype. The r d gene (and perhaps other members of the ~(1sgene family) appears to be a common target for coding sequence mutations in the initiation of carcinogenesis in several organ sites and species by specific carcinogens. Whatever genetic mechanisms are involved, an initiated cell phenotype commmon to many epithelial cell types is observed. Initiated cells have an altered program of terminal differentiation, are resistant to cytotoxic substances or show altered requirements for specific growth factors or nutrients. These cells would have a selective growth advantage in cytostatic or cytotoxic situations or under conditions favoring terminal differentiation. Tumor promoters, some acting through specific cellular receptors, produce a tissue environment conducive to the selective clonal outgrowth of the initiated cell population resulting in a clinically evident premalignant lesion. The tissue specificity for most promoters depends on the ability of a particular agent to produce the selective conditions required for the initiated phenotype of that organ. At the molecular level, phorbol ester tumor promoters bind to and activate protein kinase C and transduce signals through this second-messenger pathway. Heterogeneity in the species of protein kinase C molecule expressed by normal and initiated epidermal cells could account for the differential response pattern observed in these cell types during skin tumor promotion. Malignant conversion of benign tumors requires further genetic changes in the tumor cell. Such changes could result from inherent instability in the genome of initiated cells, from spontaneous mutations more likely to occur in the expanding population of proliferating benign tumor cells, or by additional exposure to exogenous genotoxic agents. The ~(1sgene family may also be a target for the conversion event. Little is known regarding the frequency of the conversion step relative to that of initiation, yet it is this change which ultimately produces the greatest threat to the viability of the organism. The production of transgenic mice with constitutively initiated cells should provide the proper model to study the molecular changes associated with malignant conversion. With this technological advance, the field of experimental carcinogenesis has come full circle. Prodded by the development of animal models and advanced by refinements in cell biology, carcinogenesis research has been more recently illuminated by the power of molecular biology. It is ironic that the most specific answers in carcinogenesis, at the single gene level, will apparently come from future studies in whole animals constructed transgenically to suit the most precise question.

ACKNOWLEDGMENT The authors are grateful for the many helpful comments of Dr. Curtis Harris in review of this manuscript.


61

REFERENCES Adams, J. M., Harris, A. W., Pinkert, C. A., Corcoran, L. M., Alexander, W. S., Cory, S., Palmiter, R. D., and Brinster, R . L. (1985). Nafure (London) 318, 533-538. Allen-Hoffman, B. L., and Rheinwald, J . G. (1984). Proc. Nod. A d . Sci. U.S.A. 81, 7802-7806. Amtmann, E., and Sauer, G. (1982). Nafure (London) 296, 675-677. Arcoleo, J. P., and Weinstein, I. B. (1985). Carcinogenesis 6, 213-217. Armitage, P. (1985). Enuiron. HeaffhPerspect. 63, 195-201. Ashendel, C . L. (1985). Biochim. Biophys. Acfa 822, 219-242. Autrup, H., and Harris, C. C. (1983).In “Human Carcinogenesis” (C. C. Harrios and H . N. Autrup, eds.), pp. 169-194.Academic Press, New York. Baba, N., and von H a m , E. (1967). Rag. Exp. Tumor Res. 9, 192-260. Balmain, A. (1985).Br. J . Cancer. 51, 1-7. Balmain, A., and Pragnell, I. B. (1983).Nature (London) 303, 72-74. Balmain, A., Ramsden, M., Bowden, G. T . , and Smith, J. (1984). Nafure (London) 307, 658-660. Bano, M . , Lewko, W. M., and Kidwell, W. R . (1984). Cancer Res. 44, 3055-3062. Barrett, J. C., and Ts’o, P. 0. P. (1978). Proc. Nad. Acad. Sci. U.S.A. 75, 3761-3765. Becker, F. F. (1975). Cancer Res. 35, 1734-1736. Becker, F. F. (1985).Cancer Res. 45, 768-773. Beer, D. G., Schwarz, M., Sawada, N., and Pitot, H . C. (1986). Cancer Res. 46, 2435-2441. Beland, F. A,, and Kadlubar, F. F. (1985).Enuiron. HeaffhPerspecf. 62, 19-30. Beland, F. A,, Beranek, D. T . , Dooley, K. L., Heflich, R . H., and Kadlubar, F. F. (1983). Enuiron. HeaW Perspecf. 49, 125-134. Benedict, W. F., Jones, P. A,, Laug, W. E., Igel, W. J . , and Freeman, A. E. (1975).Nature (London) 256, 322-324. Bern, H . A., Jones, L. A., and Mills, K. T. (1976). J. Toxicol. Enuiron. Heaffh (Suppl.) 1, 103-116.

Bertram, J. S., and Heidelberger, C . (1974). Cancer Res. 34, 526-537. Berwald, Y., and Sachs, L. (1965).J.Nafl. Cancer Insf. 35, 641-661. Bichara, M., and Fuchs, R . P. P. (1985).J.Mol. B i d . 183,341-351. Blumberg, P. M. (1980). CRC Crit. Reu. Toxicol. 8, 153-197. Blumberg, P. M. (1981).CRC Crif. Reu. Toxicol. 9, 199-234. Blumberg, P. M.,Delclos, K. B., Dunphy, W. G., and Jaken, S. (1982). Carcinogencsis 7, 519-535. Blumberg, P. M., Jaken, S., Konig, B., Sharkey, N. A., Leach, K . A.,Jeng, A. Y., and Yeh, E. (1984).Biochem. Pharmacol. 33,933-940. Borek, C. (1983).Ann. N . Y. Acad. Sci. 407, 284-290. Bowden, G. T.,and Yuspa, S. H. (1979).Biochim. Biophys. Acfa 565,67-83. Brinster, R. L., Chen, H . Y., Messing, A., van Dyke, T., Levine, A. J., and Palmiter, R . D. (1984).Celf 37, 367-379. Brown, J. D., Wilson, M. J., and Poirier, L. A. (1983).Carcinogenesis 4, 173-177. Buening, M.K., Wislocki, P. G., Levin, W., Yagi, H., Thakker, D. R., Akagi, H.,Koreeda, M., Jerina, D. M., and Conney, A. H. (1978).Proc. Natf. Acad. Sci. U . S . A . 75,5358-5361. Burgess, J. A., Stevens, C. W., and Fahl, W. E. (1985).Cancer Res. 45, 4257-4262. Burnham, D. K., Gahring, L. C., and Daynes, R. A. (1986).J. Naff.Cancn Insf. 76, 151-158. Burns, F., Albert, R., Altshuler, B., and Morris, E. (1983).Enuiron. Health Perspecf. 50, 309-320. Burns, F.J . , Vanderlaan, M., Sivak, A,, and Albert, R . E. (1976).Cancer Res. 36. 1422-1427. Bursch, W., Lauer, B., Timmermann-Trosiener, I., Barthel, G., Schuppler, J . , and SchulteHermann, R. (1984).Carcinopenesis 5, 453-458.

62


Busby, W. F., and Wogan, G. N. (1984). In “Chemical Carcinogens” (C. E. Searle, ed), pp. 945-1136. American Chemical Society, Washington, D.C. Calleman, C. J., Ehrenberg, L . , Jansson, B., Osterman-Golkar, S., Segerback, D., Svensson, K., and Wachtmeister, C . A. (1978).J . Enuiron. Pafhol. Toxicol. 2, 427-442. Carr, B. I., and Laishes, B. A. (1981). Cancer Res. 42, 1715-1719. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982).J. Biol. Chtm. 257, 7847-7851. Cerutti, P. A. (1985). Science 227, 375-381. Chatterjee, M., and Banerjee, M. R. (1982a). Cancer Letf. 17, 187-195. Chatterjee, M . , and Banerjee, M. R. (198213).Carcinopenesis 3 , 801-804. Cleaver, J . E. (1968). Nafure (London) 218, 652-656. Clifton, K. H., and Crowley, J. J. (1978). Cancer Res. 38, 1507-1513. Cohen, G. M., Metha, R . , and Meredith-Brown, M. (1979). Inf. J . Cancer 24, 129-133. Cohen, S. M., Greenfield, R . E., and Ellwein, L. B. (1983a). Enuiron. Healfh Perspect. 49, 209-215. Cohen, S. M., Murasaki, G., Ellwein, L. B., andGreenfield, R. E. (198313).In “MechanismsofTumor Promotion” (T.J . Slaga, ed.), Vol. 1, pp. 131-149.CRC Press, Boca Raton, Florida. Colburn, N. (1987). In “Mechanisms of Environmental Carcinogenesis” fJ.C . Barrett, ed.). CRC Press, Boca Raton, in press. Columbano, A , , Rajalakshmi, S., and Sarma, D. S. R . (1981). Cancer Res. 41, 2079-2083. Connan, G., Rassoulzadegan, M., and Cuzin, F. (1985).Nafure (London) 314, 277-279. Copeland, E. S. (1983). Cancer Res. 43, 5631-5637. Coughlin, S. R., Lee, W. M. F., Williams, P. W., Giels, G. M., and Williams, L. T. (1985). Cell 43, 243-251. Coussens, L., Parker, P. J., Rhee, L., Yang-Feng, T. L., Chen, E., Waterfield, M . D., Francke, U., and Ullrich, A. (1986). Science 233, 859-866. Craig, R . W., and Bloch, A. (1984).Cancer Rcs. 44, 442-446. Daniel, F.B., Schut, H. A. J., Sandwisch, D. W., Schenck, K. M . , Hoffman, C. O., Patrick, J . R., and Stoner, G . D. (1983). Cancer Res. 43, 4723-4729. Deamant, F. D., and Iannaccone, P. M . (1985).J . Nafl. Cancer Insf. 74, 145-150. DeLuca, L. M. (1983).J . Natl. Cancer Insf. 70, 405-407. Diamond, L., O’Brien, T. G., and Baird, W.M. (1980). Adu. Cancer Res. 32, 1-74. DiPaolo, J. A. (1983).J . Natl. Cancer Insf. 70, 3-8. DiPaolo, J. A., and Casto, B. C . (1977). In “Recent Advances in Cancer Research: Cell Biology, Molecular Biology and Tumor Virology” (R. C. Gallo, ed.), pp. 17-47. CRC Press, Cleveland. DiPaolo, j.A., and Casto, B. C . (1978). Nafl. Cancer Insf. Monogr. 48, 245-257. Dipple, A., Sawicki,J. T . , Moschel, R . C . , and Bigger, C. A. H. (1983).In “Extrahepatic Drug Metabolism and Chemical Carcinogenesis” 0 . Rydstrom, J. Montelius, and M . Bengtsson, eds.), pp. 439-448.Elsevier, Amsterdam. Dipple, A,, Moschel, R . C., and Bigger, C. A. H . (1984). In “Chemical Carcinogens” (C. E. Searle, ed.), pp. 41-163.American Chemical Society, Washington, D.C.) Dipple, A , , Michejda, C. J , , and Weisburger, E. K. (1985). Pharmacol. Ther. 27, 265-296. Diwan, B. A , , Palmer, A. E., Oshima, M . , and Rice, J. M. (1985).J. NufL Cancer Inst. 75, 1099-1105. Dotto, G. P . , Parada, L. F., and Weinberg, R. A. (1985).Nafure (London) 318, 472-474. Driedger, P. E.,and Blumberg, P. M. (1980).Roc. Natl. Acad. Sci. U . S . A . 77, 567-571. Dunn, J. A,, and Blumberg, P. M . (1983). Cancer Rcs. 43, 4632-4637. Dunn, B. P., and Stich, H. F. (1986). Carcinopcnesis 7, 1115-1120. Dunn, J. A . , ,Jeng, A. Y., Yuspa, S. H., and Blumberg, P. M. (1985). Cancer Res. 45, 5540-5546.


63

Dzarlieva-Petrusevska, R . T . , and Fusenig, N. E. (1985). Carcinogenesis 6 , 1447-1456. Ebeling, J. G., Vandenbark, G. R . , Kuhn, L. J . , Ganong, B. R . , Bell, R . M., and Neidel, J . E. (1985). Proc. Natl. Acad. Sci. U . S . A . 82, 815-819. Eisen, H. J., Hannah, R. R . , Legraverend, C., Okey, A. B., and Nebert, D. W. (1983). Biochcm. Action Horm. 10, 228-257. Emerit, I . , and Cerutti, P. A. (1982). Proc. Natl. Acad. Sci. U . S . A . 79, 7509-7513. Enomoto, T., and Yamasaki, H . (1985). Cancer Res. 45, 2681-2688. Essigman, J. M., Fowler, K. W . , Green, C . L., and Loechler, E. L. (1985). Enuiron. Health Perspect. 62, 171-176. Ethier, S. P. (1985). J . Natl. Cancer Inst. 74, 1307-1318. Everson, R. B., Randerath, E., Santella, R . M., Cefalo, R . C., Avitts, T. A,, and Randerath, K. (1986). Science 231, 54-57. Everson, R . B., Randerath, E., Schut, H . A,, and Randerath, K. (1987). Prog. Exp. Tumor Res. 31, 86-103. Farber, E. (1980). Biochim. Biophys. Acta 605, 149-166. Farber, E. (1984). Biochim. Biophys. Acta 738, 171-180. Farber, E., and Cameron, R . (1980). Adu. Cancer Res. 31, 125-226. Farmer, P. B., Bailey, E., Gorf, S. M., Tornqvist, M . , Osterman-Golkar, S., Kautiainen, A,, and Lewis-Enright, D. P. (1986). Carcinogenesis 7 , 637-640. Fialkow, P. J . (1976). Biochim. Biophys. Acta 458, 283-321. Fischer, S. M . (1983). In “Mechanisms of Tumor Promotion” (T. J. Slaga, ed.), Vol. 2, pp. 113-126. CRC Press, Boca Raton, Florida. Fisher, P. B., Bozzone, J. H., and Weinstein, I. B. (1979). Cell 18, 695-705. Friedman, E., Urmacher, C., and Winawer, S. (1984). Cancer Res. 44, 1568-1578. Friedman, J., and Cerutti, P. (1983). Carcinogenesis 4 , 1425-1427. Fujiki, H . , and Sugimura, T . (1987). Adu. Cancer Rcs.. in press. Fujita, J . , Srivastava, S. K., Kraus, M. H., Rhim, J. S . , Tronick, S. R., and Aaronson, S. A. (1985). R o c . Natl. Acad. Sci. U.S.A. 82, 3849-3853. Fusenig, N. E., Breitkreutz, D., Boukamp, P., Lueder, M., Irrnscher, G., and Worst, P. K. M. (1979). In “Neoplastic Transformation in Differentiated Epithelial Cell Systems in Vitro” (L. M. Franks and C. B. Wigley, eds.), pp. 37-98. Academic Press, New York. Garner, R. C . , Martin, C . M., and Clayson, D. B. (1984). In “Chemical Carcinogens” (C. E. Searle, ed.), pp. 175-276. American Chemical Society, Washington, D. C. Garte, S. J., Hood, A. T., Hochwalt, A. E., D’Eustachio, P., Snyder, C . A,, Segal, A,, and Albert, R . E (1985). Carcinogeneszs 6, 1709-1712. Gehley, E. B., Landolph, J . R., Heidelberger, C . , Nagasawa, H., and Little, J. B. (1982). Cancer Res. 42, 1866-1875. Gelboin, H. V., and Ts’o, P. 0. P. (1978). “Polycyclic Hydrocarbons and Cancer. Vol. 1 . Environment, Chemistry, and Metabolism. ” Academic Press, New York. Goldfarb, S., and Pugh, T . D. (1982). J . Am. Colloq. Toxicol. 1, 119-144. Goldsworthy, T. L., and Pitot, H. C. (1985). Carcinogenesis 6, 1261-1269. Goth, R . , and Rajewsky, M. F. (1974). Proc. Natl. Acad. Sci. U.S.A. 71, 639-643. Gould, M. N. (1982). Carcinogenesis 3, 667-669. Greenebaum, E., Nicolaides, M., Eisinger, M., Vogel, R . H., and Weinstein, I . B. (1983). J . Natl. Cancer Inst. 70, 435-441. Grisham, J. W., Kaufmann, W. K . , and Kaufman, D. G . (1983). In “Survey and Synthesis of Pathology Research” (‘J. M. Cruse, ed.), pp. 49-66. Karger, Basel. Groopman, J . D., Donahue, P. R., Zhu, J., Chen, J., and Wogan, G. N . (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 6492-6496. Guerrero, I., Villasante, A,, Corces, V., and Pellicer, A. (1985). Proc. Natl. Acad. Sci. U . S . A . 82, 7810-78 14.

64


Gullino, P . M., Pettigrew, H . M., and Grantham, F. H. (1975). J . Natl. CancnwInst. 54, 401-41 4. Hanahan, D. (1985). Nature (London) 315, 115-122. Hanigan, M.H., and Pitot, H . C . (1985). Carcinogenesis 6, 165-172. Hard, G. C . (1979). Cuncer R e f . 39, 4965-4970. Harper, J . R., Roop, D. R., and Yuspa, S. H . (1986). Mol. Cell. Bid. 6, 3144-3149. Harris, C. C. (1985). Enuiron. Healfh Pnspect. 62, 185-191. Harris, C. C. (1987). Cancer Res. 47, 1-10, Harris, C. C., Trump, B. F., Grafstrom, R., and Autrup, H . (1982). J . Cell. Biochem. 18, 285-294. Harris, C. C., Vahakangas, K., Newman, M. J., Trivers, G. E.,Shamsuddin, A., Sinopoli, N., Mann, D. L., and Wright, W. E. (1985). Roc. Natl. Acad. Sci. U.S.A. 82, 6672-6676. Hartley, J. A,, Gibson, N. W., Zwelling, L. A., and Yuspa, S. H . (1985). Cancer Res. 45, 4864-4870. Haugen, A , , Becher, G., Benestad, C., Vahakangas, K., Trivers, G . E . , Newman, M. J . , and Harris, C . C. (1986). Cancer Res. 46, 4178-4183. Hauser, J . , Seidman, M. M., Sidur, K., and Dixon, K. (1986). Mol. Cell. Bid. 6 , 277-285. Hecker, E. (1978). In “Carcinogenesis-A Comprehensive Survey. Vol. 2. Mechanisms of Tumor Promotion and Cocarcinogenesis” (T. J . Slaga, A. Sivak, and R . K. Boutwell, eds.), pp. 11-48. Raven, New York. Hecker, E., Fusenig, N. E., Kunz, W., Marks, F., and Thielman, H. W. (1982). “Carcinogenesis-A Comprehensive Survey. Vol. 7. Cocarcinogenesis and Biological Effects of Tumor Promoters.” Raven, New York. Heflich, R . H., Morris, S. M., Beranek, D. T . , McGarrity, L. J . , Chen, J. J . , and Beland, F. A. (1986). Mutagcncsis 1, 201-206. Hemminki, K. (1983). Arch. Toxicol. 52, 249-285. Hennings, H., Michael, D. M., Eaton, S. D. A., and Morgan, D. L. (1974).J. Invest. Derrnutol. 62,480-484. Hennings, H., Shores, R . , Wenk, M. L., Spangler, E. F., Tarone, R., and Yuspa, S. H . (1983). Nuture (London) 304, 67-69. Hennings, H., Shores, R . , Mitchell, P., Spangler, E. F., and Yuspa, S. H . (1985). Carcinogenesis 6, 1607-1610. Hicks, R. M., Chowaniec, J , , and Wakefield, J. (1978). In “Carcinogenesis-A Comprehensive Survey. Vol. 2. Mechanisms of Tumor Promotion and Cocarcinogenesis” (T. J . Slaga, A. Sivak, and R. K. Boutwell, eds.), pp. 475-489. Raven, New York. Hilpert, D., Romen, W., and Neumann, H. G . (1983). Carcinogenesis 4, 1519-1525. Hosomi, J., Nemoto, N., and Kuroki, T . (1982). Gann 73, 879-886. Hsaio, W. L.W., Gattoni-Celli, S., and Weinstein, I. B. (1984). Science 226, 552-555. Huberman, E., Sachs, L., Yang., S. K., and Gelboin, H . V. (1976). Roc. Natl. Acad. Sci. U.S.A. 73, 607-611. Huitfeldt, H . S., Spangler, E. F., Hunt, J. M . , and Poirier, M. C. (1986). Carcinogenesis 7, 123- 129. Hunter, T. (1981).J. Natl. Cancer Insf. 73, 773-786. Iannaccone, P. M., Gardner, R. L., and Harris, H. (1978). J . Cell Sci. 29, 249-269. Imaida, K., Fukushima, S., Shirai, T., Ohtani, M . , Nakanishi, K . , and Ito, N. (1983). Carcinogcnesis 4, 895-899. Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinstein, I. B., Beland, F. A,, Harvey, R . G., Miura, I., Nakaniski, K., and Kasai, H . (1976).J. Am. Chem. Soc. 98, 5714-5715. Jeng, A. Y., Lichti, U., Strickland, J. E., and Blumberg, P. M. (1985). Cancer Res. 45, 57 14-572 1. Jetten, A. M . , Ganong, B. R., Vandenbark, G . R . , Shirley, J. E., and Bell, R . M. (1985). hot. Natl. Acad. Sci. U.S.A. 82. 1941-1945.


65

Jurnak, F. (1985). Science 230, 32-36. Kakizoe, T . , Tanooka, H . , Tanaka, K., and Sugimura, T. (1983). Gann 74, 462-465. Kakunaga, T . (1974). Znf. J . Cancer 14, 736-742. Kakunaga, T . (1978). Roc. Natl. Acad. Sci. U . S . A . 75, 1334-1338. Kaufmann, W. K., Mackenzie, S. A,, and Kaufman, D. G. (1985). Am. J . Pafhol. 119, 171-1 74.

Kawamura, H . , Strickland, J . E., and Yuspa, S . H. (1985). Cancer Res. 45, 2748-2752. Kensler, T. W., and Trush, M. A. (1984). Enuiron. Mufagenesis 6, 593-616. Kinsella, A. R . , and Radman, M . (1978). Proc. Nafl. Acad. Sci. U . S . A . 75, 6149-6153. Knopf, J. L., Lee, M . H . , Sultzman, L. A., Kritz, R. W., Loomis, C. R., Hewick, R . M., and Bell, R. M. (1986). Cell 46, 491-502. Knudson, A. G. (1977). Cold Spring Harbor ConJ Cell Prolif. pp. 45-52. Knutson, J . C., and Poland, A. (1982). Cell 30, 225-234. Kopelovich, L., and Gardner, E. J . (1983). Cancer 51, 716-720. Koreeda, M., Moore, P. D., Wislocki, P. G . , Levin, W., Conney, A. H., Yagi, H . , and Jerina, D. M. (1978). Science 199, 788-791. Kraemer, K. H . , and Slor, H. (1984). Clin. Dmnafol. 2, 33-69. Krontiris, T. G., DiMartino, N. A . , Colb, M . , and Parkinson, D. R. (1985). Nature (London) 313, 369-374.

Kulesz-Martin, M . , Koehler, B., Hennings, H., and Yuspa, S . H. (1980). Carcinogenesis 1, 995- 1006.

Kuroki, T . , Hosomi, J., Munakata, K., Onizuka, T., Terauchi, M . , and Nemoto, N. (1982). Canccr Res. 42, 1859-1865. Land, H., Parada, L. F., and Weinberg, R . A. (1983). Nafure (London) 304, 596-602. Leach, K. L., and Blumberg, P. M . (1985). Cancer Res. 45, 1958-1963. Leach, K. L., James, M . L., and Blumberg, P. M. (1983). Proc. Natl. Acad. Sci. U . S . A . 80, 4208-42 12.

Lee, L. S., and Weinstein, I. B. (1978). Science 202, 313-315. Legraverend, C . , Mansour, B., Nebert, D. W., and Holland, J . M. (1980). Pharmacolou 20, 242-255.

Levin, W., Wood, A. W., Chang, R. L., Slaga, T. J , , Jerina, D. M., and Conney, A. H . (1977). Cancer Rcs. 37, 2721-2725. Lu, A. Y. H . , and West, S. B. (1980). Phannacol. Rev. 31, 227-295. McCormick, F., Clark, B. F. C . , LaCour, T . F. M., Kjeldaared, M., Norskov-lauritsen, L., and Nyborg, J . (1985). Science 230, 78-82. McCormick, J . J . , and Maher, V. M. (1983). In “Development in the Science and Practice of Toxicology” (A. W. Hayes, R . C. Schnell, and T . S. Miya, eds.), pp. 153-162. Elsevier, Amsterdam. Magee, P. N. (1983). In “Organ and Species Specificity in Chemical Carcinogenesis” (R. Langenbach, S. Nesnow, and J. M. Rice, eds.), pp. 497-508. Plenum, New York. Magee, P. N., Montesano, R., and Preussmann, R . (1976). In “Chemical Carcinogens” (C. E. Searle, ed.), pp. 491-625. American Chemical Society, Washington, D.C. Marchok, A. C . , Rhoton, J . C., Griesemer, R . A , , and Nettesheim, P. (1977). Cancer Res. 37, 1811-1821.

Marks, F. (1983). Carcinogenesis 4 , 1465-1470. Marshall, C. J . , Vousden, K. H . , and Phillips, D. H . (1984). Nature (London) 310, 586-589. Medina, D. (1976). Cancer Res. 36, 2589-2595. Miller, E. C. (1978). Cancer Res. 38, 1479-1496. Miller, J . A. (1970). Cancer Res. 30, 559-576. Minchin, R . F., McManus, M . E., Boobis, A. R., Davies, D. S . , and Thorgeirsson, S. S. (1985). Carcinogenesis 6, 1721-1724.

66

STUART H . YUSPA AND MIRIAM C. POIRIER

Mirvish, S. S., Salmasi, S., Lawson, T . A , , Pour, P., and Sutherland, D. (1985).J.Nafl. Cancer Inst. 74, 1283-1290. Mommsen, S., Barfod, N. M., and Aagaard, J. (1985).Carcinogenesis 6, 199-201. Mondal, S., and Heidelberger, C . (1970).Roc. Nad. Acad. Sci. U . S . A . 65, 219-225. Montesano, R., Drevon, C . , Kuroki, T . , St Vincent, L., Handelman, S., Sanford, K. K., Defeo, D., and Weinstein, I. B. (1977).J.Natl. Cancer Znst. 59, 1651-1658. Moolgavkar, S. H . , and Knudson, A. G. (1981).J. Natl. Cancer Insf. 66, 1037-1052. Moore, M . A , , Takahashi, M., Ito, N., and Bannasch, P. (1983). Carcinogenesis 4, 431-437. Morris, R. J., Fischer, S. M . , and Slaga, T . J. (1986). Cancer Res. 46, 3061-3066. Nagasawa, H . , and Little, J. B. (1981).Carcinogenesis 2, 601-607. Nakanishi, K., Fukushima, S., Hagiwara, A., Tamano, S., and Ito, N. (1982).J.Nad. Cancer Insf. 68, 497-500. Nakayama, J., Yuspa, S. H., and Poirier, M. C. (1984). Cancer Res. 44, 4087-4095. Nettesheim, P., and Barrett, J. C . (1984). CRC Crit. Reu. Toxicol. 12, 215-239. Nettesheim, P., and Marchok, A. (1983). Adv. Cancer Res. 39, 1-70. Neumann, H. G. (1983).Recmf Resubs Cancer Res. 84, 77-89. Neumann, H . G. (1984).Arch. Toxicol. 56, 1-6. Newbold, R. F., and Overell, R. W. (1983).Nature (London) 304,648-651. Niedel, J. E., Kuhn, L. J., and Vandenbark, G. R . (1983).Roc. NatL A d . Sci. U.S.A. 80, 36-40. Nishizuka, Y. (1986). Science 233, 305-312. Novicki, D. L., Rosenberg, M. R., and Michalopoulos, G. (1985).Cancer Res. 45, 337-344. Oshimura, M., Gilmer, T. M . , and Barrett, J. C. (1985). Nafure (London) 316, 636-639. Pai, S. B., Steele, V. E., and Nettesheirn, P. (1982).Carcinogencsis 3 , 1201-1206., Pai, S. B., Steele, V. E., and Nettesheim, P. (1983).Carcinogmesis 4, 369-374. Park, H. Y., and Koprowska, I. (1968). Cancer Res. 28, 1478-1489. Parkinson, E. K., Pera, M. F., Emmerson, A,, and Gorman, P. A. (1984). Carcinogenesis 5, 1071-1077. Parodi, S., and Brambilla, G. (1977). Mufat. Res. 47, 53-74. Pawlowski, A,, and Lea, P. J . (1983).J.Cutan. Patho/. 10, 81-110. Pegg, A. E. (1984).Cancer Invest. 2, 223-231. Pelling, J. C., Slaga, T . J., and DiGiovanni, J. (1984).Cancer Res. 44, 1081-1086. Peraino, C., Fry, R . J. M., Staffeldt, E., and Christopher, J. P. (1975). Cancer Res. 35, 2884-2890. Peraino, C . , Richards, W. L., and Stevens, F. J. (1983). In “Mechanisms of Tumor Promotion” (T. J. Slaga, ed.), Vol. 1, pp. 1-53.CRC Press, Boca Raton, Florida. Peraino, C . , Staffeldt, E. F., Carnes, B. A., Ludeman, V. A,, Blomquist, J . A , , and Vesselinovitch, S. D. (1984).Cancer Res. 44, 3340-3347. Perera, F. P., Poirier, M. C., Yuspa, S. H., Nakayama, J . , Jaretzki, A,, Curnen, M. M., Knowles, D. M . , and Weinstein, I. B. (1982). Carcinogcnesis 3, 1405-1410. Phillips, D. H.(1983). Nature (London) 303, 468-472. Pitot, H . C., Barsness, L., Goldsworthy, T . , and Kitagawa, T. (1978).Nature (London) 271, 456-458. Pitot, H. C., Glauert, H. P., and Hanigan, M. (1985).Cancer Leff. 29, 1-14. Poirier, L. A , , and deSerres, F. J. (1979).J . N d . Cancer Insf. 62, 919-925. Poirier, M. C . (1984). Enuiron. Mutagen. 6, 879-887. Poirier, M . C., and Beland, F. A. (1987). Prog. Exp. Tumor Res. 31, 1-10, Poirier, M. C., Reed, E., Zwelling, L. A , , Ozols, R. F., Litterst, C. L . , and Yuspa, S. H. (1985). Enuiron. Health Perspect. 62, 89-94. Poirier, M. C., Reed, E., Ozols, R. F., Fasy, T., and Yuspa, S. H. (1987).Rog. Exp. Tumor Res. 31, 104-113.


67

Poland, A., Palen, D., and Glover, E. (1982). Nature (London) 300, 271-274. Pollard, M . , and Luckert, P. H . (1985). Prostate 6, 1-5. Popper, H., Selikoff, I. J., Maltoni, C . , Squire, R . A,, and Thomas, L. B. (1977). In “Origins of Human Cancer” (H. H . Hiatt, J . D. Watson, and J. A. Winsten, eds.), pp. 1359-1382. Cold Spring Harrbor Laboratories, Cold Spring Harbor, New York. Preussman, R., and Stewart, B. W. (1984). In “Chemical Carcinogens” (C. E. Searle, ed.), pp. 643-828. American Chemical Society, Washington, D. C. Quintanilla, M . , Brown, K., Rarnsden, M . , and Balmain, A. (1986). Nature (London) 322, 78-80. Rabes, H . M . , Bucher, T . , Hartmann, A,, Linke, I., and Dunnwald, M. (1982). Cancer Res. 42, 3220-3227. Raick, A. N. (1974a). Cancer Res. 34, 2915-2925. Raick, A. N. (1974b). Cancer Res. 34, 920-926. Randerath, E., Agrawal, H P., Weaver, J. P., Bordelon, C. B., and Randerath, K. (1985a). Carcinogenesis 6, 1117- 1126. Randerath, K., Agrawal, H . P., and Randerath, E. (198513). Cancer Lett. 27, 35-43. Randerath, K . , Randerath, E., Agrawal, H . P., Gupta, R. C., Schurdak, M. E., and Reddy, M. V. (1985~).Enuiron. Health Perspect. 62, 57-65. Rasmussen, K. S., Glenthoj, A., and Arffmann, E. (1983). Acta Pathol. Microbiof. Zmmunol. Scand. Sect. A 91, 445-455. Razzaque, A., Mizusawa, H., and Seidman, M . M. (1983). Proc. Natl. Acad. Sci. U . S . A . 80, 3010-3014. Reddy, A. L., and Fialkow, P. J. (1979).J. Exp. Med. 150, 878-887. Reddy, A. L., and Fialkow, P. J. (1983). Nature (London) 304, 69-71. Reddy, B. S., and Narisawa, T . , Wright, P., Vukusich, D., Weisburger, J. H., and Wynder, E. L. (1975). Cancer Res. 35, 287-290. Reiners, J. J. (1985). Cancer Lett. 26, 215-220. Reiners, J . J., and Slaga, T . J. (1983). Cell 32, 247-255. Reiners, J. J . , Nesnow, S., and Slaga, T. J. (1984). Carcinogenesis 5, 301-307. Rheinwald, J. G., and Beckett, M . A. (1980). Cell 22, 629-632. Richards, J., and Nandi, S. (1978). Proc. Natl. Acad. Sci. U . S . A . 75, 3836-3840. Rinkus, S. J., and Legator, M . S. (1979). Cancer Res. 39, 3289-3314. Ritchie, J. C., and Idle, J . R. (1982). In “Host Factors in Human Carcinogenesis” (H. Bartsch and B. Armstrong, eds.), pp. 381-394. International Agency for Cancer Research, Lyon. Roberts, J . J. (1982). In “Biological Reactive Intermediates-11, Part B” (R. Snyder, D. V. Parke, J . J. Kocsis, D. J . Jollow, C. G. Gibson, and C. M . Witmer, eds.), pp. 1367-1387. Plenum, New York. Roebuck, B. D., Longnecker, D. S., and Yager, J. D. (1983). In “Mechanisms ofTumor Promotion” (T. J . Slaga, ed.), pp. 151-171. CRC Press, Boca Raton, Florida. Rosoff, P. M . , Stein, L. F., and Cantley, L. C. (1984).J. Biol. Chem. 259, 7056-7060. Rozengurt, E., Rodriguez-Pena, A,, Coombs, M., and Sinnett-Smith, J. (1984). Proc. Nad. Acad. Sci. U . S . A 81, 5748-5752. Ruley, H . E. (1983). Nature (London) 304, 602-606. Russo, J., Tay, L. K . , Ciocca, D. R., and Russo, I. H . (1983). finuiron. Health Perspect. 49, 185- 199. Sachs, L. (1980). Proc. Natl. Acad. Sci. U . S . A . 77, 6152-6156. Saftiotti, U . (1975). Cancer Res. 35, 2223-2294. San, R . H . C., Laspia, M. F., Soiefer, A. I., Maslansky, C . J . , Rice, J. M., and Williams, G . M. (1979a). Cancer Res. 39, 1026-1034. San, R . H . C . , Shimada, T . , Maslansky, C. J., Kreiser, D. M., Laspia, M . F., Rice, J. M . , and Williams, G. M. (1979b). Cancer Res. 39, 4441-4448.

68


Sando, J. J., and Young, M. C. (1983).Proc. Natl. Acad. Sci. U . S . A . 280, 2642-2646. Santella, R . M., Hsieh, L. L., Lin, C . D., Viet, S., and Weinstein, I. B. (1985).Enuiron. Healfh Perspecf. 62, 95-99. Santella, R . M . , Gasparo, F., and Hsieh, L. L. (1987). Rog. Exp. Tumor Res. 31, 63-75. Schaefer, F. V., Custer, P. R . , and Sorof, S. (1980). Nafurc (London) 286, 807-809. Schaefer, F. V.,Custer, R. P., and Sorof, S. (1985). Cancer Res. 45, 1828-1833. Scherer, E. (1984).Biochim. Biophys. Aca 738, 219-236. Scherer, E.,Feringa, A. W., and Emrnelot, P. (1984).In “Models, Mechanisms and Etiology of Tumour Promotion” (M. Borzsonyi, K. Lapis, N. E. Day, and H . Yamasaki, eds.), pp. 57-66.International Agency for Cancer Research, Lyon. Schulte-Hermann, R . , Ohde, G., Schuppler, J . , and Timmermann-Trosiener, I. (1981).Cancer RCS.41, 2556-2562. Schulte-Hermann, R.,Schuppler, J., Tirnmermann-Trosiener, I., Ohde, G., Bursch, W., and Berger, H . (1983).Enoiron. Health Perspect, 50, 185-194. Scott, R . E., and Maercklein, P . B. (1985).Proc. (Nafl.Acad. Sci. U . S . A . 82, 2995-2999. Scribner, J . D., Scribner, N. K., McKnight, B., and Mottet, N. K. (1983). Cancer Res. 43,

2034-2041. Scribner, N. K., and Scribner, J. D. (1980).Carcinogenesis 1, 97-100. Shabad, L. M., Pylev, L. N., Krivosheeva, L. V., Kulagina, T. F., and Nemenko, B. A. (1974).J.Null. Cancer Inst. 52, 1172-1187. Shami, S. G . , Thibodeau, A,, Kennedy, A. R., and Little, J. B. (1982). Cancer Res. 42, 1405-1411. Shamsuddin, A. K. M., Sinopoli, N. T., Hemrninki, K., Boesch, R. R . , and Harris, C. C . (1985). Cancer Res. 45, 66-68. Sharkey, N. A,, Leach, K. L., and Blumberg, P. M . (1984).Roc. Natl. Acad. Sci. U . S . A . 81,

607-610. Shih, C., Shilo, B. Z . , Goldfarb, M. P., Dannenberg, A., and Weinberg, R. A. (1979). Proc. Nafl. Sci. U . S . A . 76, 5714-5718. Shilo, B. Z . , and Weinberg, R. A. (1981).Nature (London) 289, 607-609. Shirai, T . , Ohshima, M . , Masuda, A., Tamano, S . , and Ito, N. (1984).J.Natl. Cancer Inst. 72,

477-482. Shoyab, M., and Todaro, G. J. (1980). Nature (London) 288, 451-455. Shugart, L. (1985a).J . Toxicol. Environ. Healfh 15, 255-263. Shugart, L. (1985b).Toxicology 34, 211-220. Sims, P.,Grover, P. L., Swaisland, A., Pal, K., and Hewer, A. (1974). Nature (London) 252,

326-327. Sina, J. F., Bradley, M . O., and O’Brien, T. G . (1982). Cancer Res. 42, 4116-4123. Singer, B. (1985).Enuiron. Healfh Perspect. 62, 41-48. Singer, B., and Grunberger, D. (1983). “Molecular Biology of Mutagens and Carcinogens.” Plenum, New York. Singer, B., and Kusmierek, J. T . (1982).Annu. Rev. Biochm. 52, 655-693. Slaga, T. J. (1983). “Mechanisms of Tumor Promotion,” Vols. 1-4.CRC Press, Boca Raton, Florida. Slaga, T. J., and Fischer, S. M. (1983).R o g . Exp. Tumor Res. 26, 85-109. Slaga, T. J., Scribner, J. D., Thompson, S., and Viaje, A. (1976).J . N a L Cancer Inst. 5 7 , 1 145-1 149. Sporn, M. B., and Todaro, G . J. (1980).New Engl. f.Mcd. 303, 878-880. Stampfer, M . R., and Bartley, J. C . (1985). Roc. Nafl. Acad. Sci. U . S . A . 82, 2394-2398. Steele, V.E.,and Nettesheim, P. (1983).In “Mechanisms of Tumor Promotion” (T.J. Slaga, ed.), Vol. 1, pp. 91-105.CRC Press, Boca Raton, Florida. Stenback, F. (1980).Acfa Pharmacol. Toxicol. 46, 89-97.


69

Stewart, T. A., Pattengale, P. K., and Leder, P. (1984). C e l l 3 8 , 627-637. Storer, R. D., Stein, R . B., Sina, J. F., DeLuca, J. G . , Allen, H . L., and Bradley, M. 0. (1986). Cancer Res. 46, 1458-1464. Stowers, S. J., and Anderson, M. W. (1985). Environ. Health Perspect. 62, 31-40. Strickland, J. E., Hennings, H., Jetten, A. M., and Yuspa, S. H. (1982). In “Host Factors in Human Carcinogenesis” (B. Armstrong and H . Bartsch, eds.), pp. 259-268.International Agency for Cancer Research, Lyon. Sukumar, S., Notario, V., Martin-Zanca, D., and Barbacid, M . (1983). Nature (London) 3 0 6 , 658-661. Suolinna, E. M. (1982). Med. Eiol. 60,237-254. Sussman, I., Prettyman, R., and O’Brien, T . G . (1985).J. CeN Biol. 101,2316-2323. Taguchi, T . , Yokoyama, M., and Kitamura, Y. (1984).Cancer Res. 44, 3779-3782. Takahashi, M., Pour, P.,Althoff, I., and Donnelly, T. (1977). Cancer Res. 3 7 , 4602-4607. Takai, Y . , Kishimoto, A., and Nishizuka, Y. (1982). In “Calcium and Cell Function” (W. Y. Cheung, ed.), pp. 385-412.Academic Press, New York. Takai, Y., Kaibuchi, K., Tsuda, T . , and Hoshijima, M . (1985).J.Cell. Eiochem. 29, 143-155. Tannenbaum, S. R . , and Skipper, P. L. (1984). Fundam. Appl. Toxicol. 4, S367-S373. Tannenbaum, S. R . , Bryant, M. S., Skipper, P. L., and Maclure, M. (1986).In “Mechanisms in Tobacco Carcinogenesis” (D. Hoffman and C. C. Harris, eds.), pp. 63-75.Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Tanooka, H . , and Tanaka, K. (1982). Cancer Rcs. 42, 1856-1858. Tanooka, H . , and Tanaka, K. (1984).Cancer Res. 44, 4630-4632. Telang, S., Tong, C., and Williams, G. M . (1982). Carcinogenesis 3, 1175-1178. Terzaghi, M., Nettesheim, P., Yarita, T . , and Williams, M. L. (1981).J . Natl. Cancer Imt. 67, 1057-1061. Thornassen, D. G., Gray, T. E., Mass, M. J., and Barrett, J. C. (1983). Cancer Res. 43, 5956-5963. Thomassen, D. G., Gilmer, T. M., Annab, L. A,, and Barrett, J. C. (1985). Cancer Res. 45, 726-732. Thurnherr, N., Deschner, E. E., Stonehill, E. H., and Lipkin, M. (1973). Cancer Res. 33, 940-945. Toftgard, B., Roop, D. R., and Yuspa, S. H . (1985). Carcinogenesis 6,655-657. Tonelli, Q. J., Custer, R . P., and Sorof, S. (1979). Cancer Res. 39, 1784-1792. Troll, W., and Wiesner, R. (1985). Annu. Rev. Pharmucol. Toxicol. 25, 509-528. Troll, W., Witz, G., Goldstein, B., Stone, D., and Sugimura, T . (1982). Carcinogenesis 7, 593-597. Trosko, J. E., and Chang, C. C. (1983). In “Mechanisms of Tumor Promotion” (T.J. Slaga, ed.), Vol. 4, pp. 120-139. CRC Press, Boca Raton, Florida. Tsuda, H., Lee, G., and Farber, E. (1980). Cancer Res. 40, 1157-1164. Tsuda, H., Fukushima, S., Imaida, K., Kurata, Y., and Ito, N. (1983). Canccr Res. 43, 3292-3296. Umbenhauer, D., Wild, C . P., Montesano, R., Saffhill, R . , Boyle, J. M., Huh, N., Kirstein, U., Thomale, J., and Rajewsky, M . T . (1985). Int. J. Cancer 36, 661-665. Vahakangas, K . , Trivers, G., Rowe, M., and Harris, C. C . (1985).Environ. Health Perspecf. 62, 101-104. Verma, A. K., and Boutwell, R. K. (1980).Curcinogenesis 1, 271-276. Webber, W. W., and Hine, D. W. (1985). Pharmntol. Rev. 37, 25-79. Weisberger, J. H., Reddy, B. S., and Wynder, E. L. (1977). Cancer 409, 2414-2420. Welsch, C. W., and Nagasawa, H . (1977). Cancer Res. 37, 951-963. Weston, A,, Trivers, G., Vahakangas, K., Newman, M., Mann, D., and Harris, C. C . (1987). Prog. Exp. Tumor Res. 31, 76-85.

70

STUART H . YUSPA AND MIRIAM C. POIRIER

Willey, J. C., Moser, C. E., Lechner, J. F., and Harris, C . C. (1984). Cancer Res. 4 4 , 5124-5126. Williams, G. M . , Katayama, S., and Ohmori, T. (1981). Carcinogenesis I , 1111-1117. Williams, G. M., Maeura, Y., and Weisburger, J. H . (1983). Cancer Lett. 1 9 , 55-60. Wiseman, R . W., Stowers, S.J., Miller, E. C., Anderson, M. W., and Miller, J. A. (1986). A o c . Natl. Acad. Sci. U.S.A. 8 3 , 5825-5829. Wogan, G . N., and Gorelick, N. J. (1985).Environ. Health Perspect. 6 2 , 5-18. Wolff, T.,Distlerath, L. M . , Worthington, M. T., Groopman, J. D., Hammons, G. J., Kadlubar, F. F., Prough, R . A,, Martin, M. V., and Guengerich, F. P. (1985). Cancer Res. 4 5 , 2116-2122. Yamasaki, H . (1983).In “Mechanisms of Tumor Promotion” (T. J. Slaga, ed.), Vol. 4, pp. 1-26. CRC Press, Boca Raton, Florida. Yang, S. K., McCourt, D. W., Leutz, J. C., and Gelboin, H. V. (1977).Science 1 9 6 , 1199-1201. Yang, S. K., Deutsch, J., and Gelboin, H . V. (1978). In “Polycyclic Aromatic Hydrocarbons and Cancer” (H. V. Gelboin and P. 0. P. Ts’o,eds.), pp. 205-231.Academic Press, New York. Yaswen, P., Goyette, M., Shank, P. R., and Fausto, N. (1985).Mol. Cell. Biol. 5 , 780-786. Yokota, J., Tsunetsugu-Yokota, Y., Battifora, H., LeFevre, C . , and Cline, M . J . (1986).Science 2 3 1 , 261-264. Yotti, L.P., Chang, C. C . , and Trosko, J. E. (1979). Science 206, 1089-1091. Yuspa, S. H. (1985a).In “Methods in Skin Research” (D. Skerrow and C. Skerrow, eds.), pp. 213-249.Wiley, Sussex. Yuspa, S. H . (1985b).In “The Role of Chemicals and Radiation in the Etiology of Cancer” (E. Huberman and S. Barr, eds.), pp. 201-209.Raven, New York. Yuspa, S. H . (1986).J . Am. Acad. Dmnatol. 1 5 , 1031-1044. Yuspa, S. H., and Harris, C. C. (1982). In “Cancer Epidemiology and Prevention” (D. Schottenfeld and J. F. Fraumeni, eds.), pp. 23-43. Saunders, Philadelphia. Yuspa, S. H . , Ben, T., Hennings, H . , and Lichti, U. (1980a). Biochem. Biophys. Res. Cornrnun. 9 7 , 700-708. Yuspa, S. H., Hawley-Nelson, P., Koehler, B., and Stanley, J. R. (1980b). Cancer Res. 40, 4694-4703. Yuspa, S. H., Hennings, H., and Lichti, U. (1981).J. Cell Biochem. 1 7 , 245-257. Yuspa, S. H . , Ben, T . , Hennings, H . , and Lichti, U. (1982). Cancer Res. 4 2 , 2344-2349. Yuspa, S. H . , Kulesz-Martin, M., Ben, T., and Hennings, H. (1983).J . Invest. Dermatol. ‘ 8 1 , 162s-168s. Yuspa, S . H., Kilkenny, A. E., Stanley,J . , and Lichti, U. (1985).Nature(London)314, 459-462. Yuspa, S. H., Morgan, D., Lichti, U., Spangler, E. F., Michael, D., Kilkenny, A,, and Hennings, H. (1986).Carcinogenesis 7,949-958. Zarbl, H., Sukurnar, S., Arthur, A. V., Martin-Zanca, D., and Barbacid, M. (1985). Nature (London) 3 1 5 , 382-385. Zurhausen, H., O’Neill, F. J., Freese, U. K., and Hecker, E. (1978). Nature (London) 2 7 2 , 373-375. Zurlo, J., and Yager, J. D. (1984).Carcinogenesis 5, 495-500.

ONCOGENES AND THE NATURE OF MALIGNANCY Ian Buckley Department of Experlmental Pathology, John Curtln School of Medlcal Research, Australlan Natlonal University, Canberra, ACT 2601, Australia

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Basic Phenomena of Malignancy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The Malignant Phenotype . . . . . . . . . . . . . . . . . . . . . . . . A. Lack of Positional Control .................... B. Lack of Growth Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . The Malignant Phenotype-A Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Malignant Genotype. . . . . ............. A. General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Dominant Oncogenes . . . . . . . . . .......... C . Recessive Oncogenes . . . . . . . . . . .......... D . The Malignant Genotype-A Summary V. Generalsynopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Appendix.. . . . . . . . . . . . . . . . . .......... A. Cellular Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Maturation Arrest . . . . . . . . . . . . . . . . . . . . . . . . ........... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71 72 74 74 76 79 79 79 81 85 86 87 89 89 92 93

I. Introduction In recent years major discoveries concerning viral oncogenes and their cellular counterparts have generated much interest and excitement in the prospect that all malignancy might ultimately be understood in terms of derangements of oncogene function (Bishop, 1983). Indeed, much has been revealed about the dominant mode of action of these genes, and detailed structural studies have demonstrated that some exhibit close homologies with growth factors or their receptors (e.g., Waterfield et al., 1983; Downward et al., 1984). Nevertheless we still do not know just what roles these genes play either physiologically in normal development or pathologically in malignancy. Moreover, it is not known how they function in relation to a separate class of malignancy-determining genes which act recessively (Gateff, 1982; Knudson, 1985). Accordingly, our understanding of the malignant genotype is still limited. At another level of understanding, it is apparent that we do not yet have a full appreciation of the essential features of the malignant phenotype. That is, 71 ADVANCES IN CANCER RESEARCH, VOL. 50

Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

72

IAN BUCKLEY

we have yet to identify within malignant cell heterogeneity, those particular phenotypic features that determine malignant behavior (Ruddon, 1981). It can be said, therefore, that despite great gains in knowledge, we do not have a clear picture of the underlying nature of malignancy at either the genotypic or the phenotypic level. Nevertheless, when a number of recent findings are considered in relation to earlier knowledge, it seems that some valuable insights could emerge. Beginning with a brief review of the well-established structural and behavioral features of malignancy, an attempt is made to define the nature of those differentiation aberrations affecting malignant cells that lead them to lose control over both mitotic and locomotory activity. Then knowledge of gene control of differentiation is reviewed in relation to recent discoveries concerning the two major classes of oncogenes. Evidence is adduced to support the proposal that whereas the recessively acting oncogenes code directly for key surface differentiation antigens involved in the control of cell movement and growth, the dominantly acting oncogenes are regulatory genes that modulate the expression of these same antigens. According to this view, a malfunction of either class of oncogenes will result in malignancy whenever the end result is lack of adequate expression of the key cell surface antigens. In outlining the present proposals, no attempt has been made to review all of the relevant literature, citations being limited to the minimum necessary to support the ideas presented.

II. Basic Phenomena of Malignancy As is generally understood, although malignant cells are derived from host cells, they are highly aberrant both in structure and behavior. At the individual cell level their most striking structural abnormality is lack of differentiative features, the extent of which broadly parallels the degree of malignancy. At the tissue level this lack of differentiation is further displayed as a failure of normal histological organization, i.e., absence of the normal cellular patterns characteristic of the tissue of origin. Again, the degree of disorganization parallels the degree of malignancy (Willis, 1948; Ruddon, 1981). O n the behavioral side, two features stand out. One is an augumented rate of cell proliferation; the other is “invasion,” malignant cells penetrating adjacent normal tissues. At an early stage in the development of malignancy these changes may be minor, cell proliferation rates being only slightly augumented and the cells departing from their original positions but little. This is in line with the knowledge that most naturally occurring malignancies have long histories extending over many years, even decades (Willis, 1948; Ruddon, 1981).

ONCOGENES AND THE NATURE OF MALIGNANCY

73

It must be emphasized, however, that by itself cell proliferation is not a great hazard because the resulting tumor remains localized. Indeed, being localized, such tumors are treatable and therefore “benign. ” The failure to maintain normal cell position that results in invasion is therefore the cardinal feature of malignancy. This is understandable because malignant cell invasion leads to progressive disruption and destruction of surrounding tissues and, ultimately, by invasion of regional blood vessels and lymphatics, to tumor cell embolization, secondary (metastatic) growths in distant organs, and death. That is not to underrate the importance of cell proliferation, however, for without that there would be no tumor mass and no threat to life; indeed, its importance is that, when coupled to invasive behavior, proliferation continually amplifies the numbers of invading cells and thereby the total destructive effect. Many experimental studies have emphasized cell proliferation, regarding it as the central problem. However, in the discussion which follows on the possible roles of oncogenes in malignancy, equal emphasis will be placed on the need to account for the loss of control over cell position that we see as invasion. Indeed, since most malignant tumors are derived from stem cells the proliferative rates of which are commonly greater than those of malignant cells (Cairns, 1975; Markert, 1978), the task of explaining tumor cell proliferation is not likely to represent the more difficult aspects of the problem. Malignant cell populations are also noted for their great heterogeneity (Foulds, 1969). That is, both structurally and functionally, the cells of malignant tumors exhibit considerable cell-to-cell variation, the extent of which roughly parallels the degree of malignancy. However, since degrees of heterogeneity occur also in many nonmalignant conditions and since the essential lethal-determining features of malignancy are a constant feature of the condition, cellular heterogeneity will be considered separately (see Section V1,A). In attempting to characterize the features of malignancy outlined above, it seems reasonable to interpret them broadly as “lack of control.” Such lack is, of course, not absolute but proportional to the degree of malignancy. That is, the more malignant the tumor’s behavior, then the greater the loss of cell differentiation, the less controlled the proliferative rate, the more disordered the tissue architecture, and the greater the intrusion of tumor cells into adjacent normal tissues. If we think of malignancy in terms of “lack of control,” then it seems logical to consider the idea that this lack of control could stem directly from an absence of key differentiative features. Indeed, it will be argued here that malignancy might most satisfactorily be explained in terms of specific dejicits of key differentiation antigens the normal functions of which are to limit cell proliferation and to maintain the proper relative positioning of cells (cf. Mintz and Fleischman , 1981; Buckley , 1985).

74

IAN BUCKLEY

This approach represents a major shift in emphasis from a more traditional one (sometimes stated but more often implied), namely, that malignancy represents a positive trait, i.e., that malignant behavior stems from positive malignant cellular attributes (e.g., Wallach, 1968; Ruddon, 1981; Oettgen and Hellstrom, 1982). For long such a view seemed to gain support from the finding of various tumor-specific ‘heoantigens. ” It now appears, however, that rather than having either malignancy or tumor type specificity, these antigens are recognized as “tumor associated,” since they are attributed to various stem cell-derived “developmental” antigens or, in the case of experimental systems, to particular carcinogens or oncogenic viruses (Greaves et a l . , 1981; Lennox, 1982; Sulitzeanu, 1985). Thus, to date, despite prolonged intense research efforts, it has not been possible to demonstrate the occurrence of unique malignancy-associated antigens which might have the potential for determining malignant behavior. At this juncture, therefore, it appears reasonable to explore the abovementioned converse possibility, namely, that malignant behavior might derive directly from specific molecular deficits. In this regard it seems logical to give prime consideration to those molecules that come under the general heading of “differentiation antigens,” especially those located at the cell surface. The following sections attempt to account for both the malignant phenotype and the malignant genotype in these terms.

111. The Malignant Phenotype As already indicated, the principal concern here must be to account for the twin lethal features of malignancy that derive from the loss of proper control over cell proliferation and cell position (i.e., “growth” and “invasion”).

A. LACKOF POSITIONAL CONTROL For reasons given above, it seems that the more crucial of the twin problems is to account for the loss of control over cell position that allows malignant cells to invade surrounding tissues. However, before considering the possible nature of the differentiation antigens, loss of which might lead to invasion, one must ask: what sorts of cellular processes are involved in invasion? As with all migrating cells, protease production is an important mechanism, reducing extracellular barriers, but direct microscopic observations have made it clear that the prime effector of invasion is active cellular movement (Wood, 1958; Strauli, 1980). That being so, it might be thought that some positive stimulus to cellular movement would be required. However, all tissue cells are not only capable of locomotion but, as seen whenever transferred to culture, they are always “primed,” ready to move as soon as normal constraints are lifted


75

(e.g., Buckley and Porter, 1967). Indeed, this “readiness to move” is an essential condition for organismal survival, since it is crucial for the prompt closure of wounds (Alberts et al., 1983). Accordingly, we do not need to ask: what stimulates the movement of malignant cells? Instead it is more useful to ask: what phenotypic controls normally constrain tissue cell movements? It is argued here that if we understood these controls and their potential deficiencies, we would understand the basis of malignant cell invasion. According to this view, an early task should be to discover the molecular cues that determine the correct mutual positioning of tissue cells. Historically this problem was seen as an embryological one, i.e., how to account for the fact that during development there are extensive migrations of embryonic cells which, in the process, reassort themselves and form tissue-specific histological arrays. Experimentally, Holtfreter (1948) approached the problem by disassociating the cells of young amphibian embryos, then allowing them to reassociate in culture. He then observed that the separated cells continually moved about over one another until, following some form of mutual recognition, they stopped moving, having re-formed stable tissue arrays. The same result occurred also with the disassociated tissue cells of chick and mouse embryos (Moscona, 1962). One conclusion from these results which should be emphasized in the present context is that in the absence ofthe cell recognition events that result in stable histological arrays, continual cell movement is an altogether normal phenomenon. The other general conclusion is that the specific cell-cell associations characteristic of tissues can be ascribed to tissue-specific cell surface recognition molecules (Moscona, 1962; Alberts et al., 1983). This general conclusion has been greatly strengthened through studies from Goodman’s laboratory (cf. Bastiani et al., 1985a,b). Working on the nervous systems of early grasshopper and Drosophila embryos, it has been possible to define accurately a number of neurons and to plot their axon migrations and subsequent fates in considerable detail. In this way it has been demonstrated that the growth cone of any given neuron always follows a set migratory pathway, that this pathway is defined by a series of “landmark” cells (both neuronal and nonneuronal), and that in each case intimate cell-cell contacts via fdopodial probing movements are involved. The growth cone comes to rest finally only after it has, by further fdopodial probing of many cells, positively identified its predetermined “target” cell. That such intimate cell-cell contacts are an essential feature of neuronal pathfinding has been further demonstrated by elegant cell ablation experiments, these revealing that when landmark or target cells are destroyed, the migrating growth cone, having lost direction, continues to wander on, never establishing a stable cellular contact. These results thus demonstrated highly specific cell-cell recognition events as the basis of the mutual disposition and interconnection of cells within the nervous system. Similar results have been obtained from studies with Cuenorhabditis

76

IAN BUCKLEY

elegans (Sulston, 1983). To date it has not been possible to identify the particular molecules involved in the recognition events, but results of monoclonal antibody studies indicate that the number of recognition molecules may be quite large (Bastiani et a l . , 1985b). Of course the logical extension of these results to the fully developed nervous or immune systems of higher animals could indicate that the number of identifying cell markers would need to be vary large indeed (i.e., millions). In a sense that may be true, but it should be borne in mind that it has been calculated that, providing each cell is identified by a set combination of surface markers chosen from a limited range (e.g., 20), then very large numbers of cells can be individually specified by a relatively small number of individual markers (Gierer, 1974). Indeed, indicating that this combinatorial way of cell identification may be general, such a mode of cell specification according to cell type and differentiation stage has already been demonstrated for the immune system (Boyse and Cantor, 1978). Overall, then, although the molecular details have still to be elucidated, the above findings strongly support the long-held view that the extensive migrations, reassortments, and final tissue-specific arrangements of differentiating embryonic cells require the presence at the cell surface of the appropriate tissue-specific recognition molecules (Holtfreter, 1948; Moscona, 1962; Alberts et a l . , 1983). If, as the above evidence indicates, cell movement is a normal occurrence in the absence of mutual cell recognition events (involving as-yet-undefined cell recognition molecules), it follows that serious defects in a cell’s complement of recognition molecules would lead to the release of normal constraints on movement and hence to random wandering, i.e., to invasion. Of course, whereas a total deficit of recognition molecules could be expected to lead to rapid movement, reduced levels of expression or loss of only a subset of identity markers are likely to result in slower movement, a situation in accord with the slow rate of invasion of most naturally occurring malignancies (Buckley, 1985). According to this concept of the basis of malignant cell invasion, it will be important to try to determine with as much precision as possible the entire spectrum of cell surface markers that characterize particular cell types and differentiation stages in the hope that the same sort of analysis applied to their malignant counterparts will reveal specific deficits, either qualitative or quantitative.

B. LACKOF GROWTH CONTROL The foregoing discussion on failure of positional control as a basis for malignant cell invasion has stressed the possibility that such failure could stem directly from defects in the expression of particular cell surface differentiation antigens. Although this concept of malignancy is speculative, it is supported


77

by evidence demonstrating that accurate cell positioning during embryonic development is critically dependent on the occurrence of specific cell surface recognition molecules (Alberts et al., 1983; Bastiani et al., 1985b). With respect to growth control, however, although much has been established concerning the intracellular control signals for mitosis (Alberts et al., 1983), little is known of the molecular control mechanisms used to regulate numbers of different cell types within living tissues. However, since in normal developmental processes, control over proliferation, differentiation, and cell position are highly coordinated, it seems likely that the differentiation molecules controlling cell position will also be involved in the control of proliferation. Two broad lines of evidence support this suggestion. One relates to the well-studied phenomena of epithelial cell replacement, the other to wound healing and regeneration in experimentally extirpated and grafted tissues. Indeed, there is considerable circumstantial evidence that points to the controlling influence on proliferation of cell position in tissues, the end result being a proper balance between stem cell production on the one hand and differentiation, cessation of mitosis, and programmed cell death on the other. Within the epithelial lining of the small intestine, for example, stem cells located deep within the crypts proliferate at a rate which nicely balances the rate of cell differentiation and migration toward (and loss from) the villus tip (Potten et al., 1979). Thus, despite the very high proliferative rates of the stem cells, tissue cells do not accumulate because the direction of cell migration is strictly controlled (cells never migrate beneath the basement membrane) and cell production exactly matches cell loss into the gut lumen (Cairns, 1975). Likewise, in the renewal of epidermis, the extremely rapid stem cell proliferation (all but residual stem cells “turning over” in 3 weeks) is nicely balanced by differentiation and loss by desquamation (Alberts et al., 1983). Again, the direction of cell movement is strictly controlled (normal stem cells do not penetrate the basement membrane) and there are similarly strict controls as to which stem cell progeny may divide. Hence, although all cells within the most basal layer (i.e., those with stem cell neighbors and in contact with the basement membrane) are “eligible” to divide, mitosis is normally limited to about 1 in 10 basal cells, this indicating that the signal to proliferate is not “broadcast” but highly selective. Moreover, although all epidermal cells are stem cell progeny, even the daughter cells of stem cell mitosis are limited in their mitotic potential, depending on their particular position within the epidermis. Thus whereas if both daughter cells remain within the most basal layer, both retain “eligibility” for further mitosis, once a daugther cell moves away from the basement membrane and comes into contact with the more mature “prickle” cells, its further mitosis is prevented. That strongly suggests that (1) absence of contact with basement membrane and neighboring stem cells and/or (2) contact with more mature cells, induces the

78

IAN BUCKLEY

beginnings of differentiation which, among other things, precludes further mitosis. Accordingly, regardless of the precise cellular and molecular signals involved, it seems clear that it is the change in cell position which, altering particular cell contacts, has determined the change in mitotic behabior. In the healing of wounds of the epidermis, there is a coordinate response such that surviving stem cells at the wound margin undergo augumented rates of proliferation and these and progeny cells migrate across the wound surface until they meet their “opposite numbers’’ from the other side. At this point migration stops, but augumented proliferation and coordinate differentiation continue until the normal epidermis is reconstituted. Again, the direction of migration is strictly controlled (epidermal cells do not wander into the underlying dermis) and proliferation is limited so that once lost tissue is replaced, its rate reverts to normal. Moreover, whenever still-viable epidermal cells are physically embedded in the dermis at the time of wounding, they are inhibited from further mitosis. Thus, as in the normal process of epidermal cell renewal, the relative positioning of stem cells clearly has a controlling influence over their proliferative behavior, turning it on or turning it off (Alberts et a l . , 1983). Even more dramatic illustrations of the significance of cell position in controlling cell proliferation come from studies of the patterns of regeneration of insect tissues. By careful study of larval cockroach limbs subjected to segmental resections (with and without subsequent homologous tissue transplantations), it has been possible to demonstrate that the pattern of tissue regeneration proceeds in accord with a preexisting biaxal coordinate cell position plan (French et al., 1976). For example, removal of a narrow longitudinal strip of epidermis from the circumference of the femur results in closure of the cut edges, thereby bringing together populations of cells which are normally nonadjacent. This is followed by regeneration of the epidermal strip and restoration of a normal-appearing limb. Grafting a strip of epidermis to an abnormal position around the circumference (without changing its proximal-distal level) again confronts normally nonadjacent cell populations, i.e., at the perimeter of the graft. However, since this stimulates growth of structures that normally lie between the newly apposed populations of misplaced cells, the result is not simply restoration of a normal-appearing limb but intercalation of additional regenerated tissue that accords with reconstitution of the original coordinate cell position plan. These and many other experimental variations make clear that cells not only “know” exactly where they are in the plan but, reading off the positional information from neighboring cells and “checking” it against their own, they respond by proliferating and differentiating to reconstitute the original “ground plan.” In summary, detailed studies of the spatial and temporal patterns of mitoses which occur within living tissues during normal regeneration and repair


79

indicate that such mitoses are closely controlled, not only according to cell type and lineage stage, but according to the cell’s relative position within its tissue. This has suggested that the tissue-derived cues for mitosis must be closely coupled to the molecular cues for cell position. Although we do not yet know just what mechanisms are involved in this control, it nevertheless seems likely that a form of cell surface molecular signaling must be used and that there will be at least some overlap here with the molecular signals employed in the control of cell position.

C . THEMALIGNANT PHENOTYPE-A SUMMARY Restating the main feature of the malignant phenotype, it can be said that malignant cells are characterized in a number of ways, all essential elements of which represent aspects ofdefective differentiation and control. Thus, structurally malignant cells appear poorly differentiated not only individually but in the societal sense, the cells failing to associate together normally either with one another or with their nonmalignant neighbors, this resulting in abnormal cell separations and the disorganization of histological patterns. Functionally such cells exhibit many deficiencies, but most significantly, they fail to limit their growth to that appropriate for the tissue and, moving from their normal positions, they “invade,” disrupt, and destroy surrounding normal tissues. Hence both structurally and behaviorally malignant tissues display a lack of normal control. In analyzing the factors which seem to control normal cell position and growth within living tissues, it has been emphasized that these are likely to be closely related to the cell’s state ofdifferentiation and in particular to the set ofdifferentiation molecules by which cells are believed to identify one another. Accordingly, it is proposed that the malignant phenotype depends on the occurrence of particular differentiation antigen deficits, i.e., deficits affecting one or more of the complement of surface recognition molecules that control a cell’s position and growth. This view of malignancy as a form of differentiation deficiency is consistent with the knowledge that to date it has not been possible to demonstrate malignancyspecific neoantigens which might have the potential to account for malignant cell behavior. It is also consistent with the knowledge that malignant cells are in general poorly differentiated and that their lack of differentiation is roughly proportional to the degree of malignancy.

IV. The Malignant Genotype A.

GENERAL CONSIDERATIONS

Here it is considered how the above concept of the malignant phenotype (as a cell defective in surface molecules required for control ofboth position and growth) might fit in with what is known of the genetics and epigenetics of malignancy.

80

IAN BUCKLEY

Malignancy has for long been recognized as a “disease of differentiation,” the general concept being that the differentiation of malignant cells is not only deficient but deranged (Markert, 1968, 1978; Pierce and Cox, 1978; Mintz and Fleischman, 1981). In this regard it is relevant to consider recent discoveries concerning the two known classes of oncogenes in relation to what we know about the genetic control of cell differentiation. Unfortunately, despite recent intriguing findings concerning gene control of development (Manly and Levine, 1985), our understanding of this area is limited. However, although the hierarchical mechanisms of gene control in development are still anything but clear, some broad features of the end results of such control are well established (Alberts et al., 1983). Thus we know that for any cell type only limited portions of the genome are expressed, the remainder being suppressed (i.e., nonderepressed). In addition to expression of the usual array of “housekeeping” gene products, each cell expresses its special range of products, i.e., those characteristic of its type and stage of differentiation. Among these are a complement of cell surface differentiation molecules a subpopulation of which are believed to be engaged in the recognition of other cells (Gierer, 1974; Boyse and Cantor, 1978). Experimentally, it is known that each cell is identified by a considerable variety of such molecular markers and, although some are unique to a particular cell type, others are shared with cells of other types. Thus it is the combination of all of its surface markers that specifies the cell type and its place in a lineage (Boyse and Cantor, 1978). Although in humans, for example, there are some 200 major cell types and many times that number of subtypes and differentiation stages (Alberts et a l . , 1983), the combinatorial way in which markers are used to specify cells means that very large numbers of cells can be individually specified by combinations selected from a relatively limited range of separate markers. As pointed out by Gierer (1974) in his valuable theoretical treatment of the topic, selection from as few as 20 individual markers would suffice to identify lo6 separate states of differentiation; indeed, considering the degrees of fine-tuned differentiation likely to be needed within lymphoid and nervous tissues, for example, such large numbers of individually specified differentiation states are likely to be required. In the course of differentiation within a lineage, selective expression and suppression of cell surface markers is an ongoing process. That is, as new markers are being expressed, others, previously expressed, are being suppressed, the net result characterizing the particular stage of differentiation (Boyse and Cantor, 1978; Bastiani et al., 1985b). Obviously proper control of these processes is crucial for correct development. The converse of this is that incorrect patterns of expression and suppression have the potential to result in cells with inadequately expressed markers and, according to the above reasoning (cf.


81

Section 111), to the antisocial proliferative and invasive behavior characteristic of malignancy (c.f. Buckley, 1985). Now although comparatively little is known about gene control of differentiation programs and although the molecular details of how cells are specified phenotypically in different tissues are incomplete, it nevertheless seems worthwhile to examine how present knowledge of oncogenes could fit in with the model of the malignant phenotype suggested (c.f. Section 111). As indicated at the outset, the occurrence of malignancy is profoundly influenced by two separate classes of oncogenes, one class acting dominantly (Bishop, 1983), the other recessively (Gateff, 1982; Knudson, 1985).

B. DOMINANT ONCOGENES As now well known, these genes were first identified as malignancy-coding portions of the genome of oncogenic viruses, viruses capable of rapidly inducing malignancy in susceptible cells (Bishop, 1983). Although a great deal is known about the viral oncogene products, including their intracellular distributions and some biochemical effects, not enough is known to indicate just how malignancy is brought about and how it is that only some cell types are susceptible. It has been discovered that the genomes of all eukaryotic cells from yeast to human, contain homologous genes termed cellular (or proto) oncogenes (Bishop, 1983). Thus, corresponding to the various viral oncogenes, some 30 cellular oncogenes have been identified. It is widely believed that the normal function of cellular oncogenes is in some way concerned with developmental processes, but it has yet to be discovered what this function is and how their control is exerted. Of course, if we could understand the physiological role of these oncogenes, that would not only provide insights into the control of normal development but it could suggest ways in which aberrations of that control might lead to malignancy. To date direct experimental approaches to the functions of cellular oncogenes have been limited, but some useful information has emerged and important clues have come also from investigation of the structural properties and cellular effects of viral oncogenes and their products.

1. Oncogenes and Growth Factors One important indication of oncogene function has come from the discovery that a number of oncogenes exhibit structural homologies with either growth factors or their receptors. For example, the protein product of the simian sarcoma viral oncogene (v-xis) shows significant homology with platelet-derived growth factor (PDGF) (Waterfield et al., 1983). Similarly, the product of the viral oncogene V-erbB exhibits close homology with the cell

82

IAN BUCKLEY

receptor for epidermal growth factor (EGF) (Downward et al., 1984). Such findings have been interpreted to indicate that the malignancy-promoting effect of viral oncogenes might be ascribed entirely to stimulation of cell proliferation. Although growth control is part of the explanation, it must be stressed that growth factors have significant effects on cell differentiation as well. Earlier failure to appreciate this may be due to the fact that many procedures for evaluating the effects of oncogenes and/or growth factors have involved the use of cultured cell lines where the most obvious criterion of change is growth. However, whenever the test system has involved differentiating cells, the differentiation-controlling effect has become apparent. For example, when administered to newborn rats, EGF greatly modifies the patterns of differentiation of the still-developing hair follicles and, under these conditions, it actually reduces growth rates (Moore et al., 1983). Again it has been found that the granulocyte-macrophage colony-stimulating factor applied to hematopoietic cell cultures is required not only for stem cell proliferation but for subsequent differentiation of both cell types (Metcalf, 1985). Thus it is clear that, depending on the type of cell tested, EGF, PDGF, and other growth factors have profound effects not only on proliferation but on differentiation as well. This evidence is now complemented by the recent finding of significant structural homologies between EGF and both L i n - 1 2 and Notch, developmentcontrolling homeotic genes of C. elegans and Drosophila, respectively (Bender, 1985). Overall, therefore, it can be appreciated that “growth factor” effects should be reinterpreted as “growth factor/differentiation-modulating” effects.

2. Oncogenes and Cell Diferentiation In addition to the above considerations, there is other evidence to indicate a role for oncogenes in the control of cell differentiation. This role has been

revealed whenever viral oncogene influence has been studied in defined celldifferentiating culture systems. Thus, in a variety of in vitro systems involving the differentiation of bone marrow, cartilage, muscle, and retina, it has been shown that infection with Rous sarcoma or simian virus 40 had the effect of holding stem cells in a proliferative phase and preventing their further differentiation (Pacifici et al., 1977; Boettiger and Durban, 1979; Graf et al., 1980). Moreover, use of temperature-sensitive mutants showed that these oncogene effects were reversible. Over succeeding years these findings have been confirmed and extended to include the effects of other viral oncogenes, largely in culture systems designed to study hematopoietic cell differentiation. For example, differentiation-controlling effects have been noted for the oncogene of Fujinami sarcoma virus (v-fps) (Carmier and Samarut, 1986) and for the oncogenes of the Abelson murine leukemia virus (v-abl) and Harvey murine

ONCOGENES AND T H E NATURE OF MALIGNANCY

83

sarcoma virus (v-Ha-ras), which exhibit mutual dependence in their influence (Waneck et a l . , 1986). Then, in a study of the combined effects of avian myeloblastosis virus (AMV) and avian myelocytomatosis virus (MC29) on developing cells of the myelomonocytic lineage, Symonds et al. (1986) found that the expression of V-myb (AMV) and v-myc (MC29) coordinately regulated the phenotype, a high level of v-myb expression favoring an immature phenotype, high v-myc expression promoting a more mature phenotype; an intermediate phenotype resulted when both oncogenes were expressed in roughly equal amounts. However, such effects do not apply only to viral oncogenes. There are now a number of reports indicating that a general function of cellular oncogenes may be to control cell differentiation (Coll et al., 1983; Gonda and Metcalf, 1984; Duprey and Boettiger, 1985; Muller et al. (1985). For example, Muller et al. (1985) have shown that early expression of c-fos correlates with macrophage differentiation. In another study of macrophage differentiation, Duprey and Boettiger (1985) found that, whereas in a subpopulation (5%) of yolk sac hematopoietic cells, c-myb was expressed at a very high level, the level fell 100-fold abruptly as cells differentiated to the promonocyte stage. Similarly it was shown that during hematopoiesis, c-myb, c-myc, and c-erb were differentially expressed in the course of differentiation (Coll et al., 1983). Although in none of these cases is it certain that the changes in cellular oncogene expression are driving rather than responding to cell differentiation, these results, considered in conjunction with those derived from viral oncogenes, strongly indicate a differentiation-controlling effect. In this regard, a specially significant study is that of Symonds et al. (1986) on the effects of the viral oncogenes v-myb and v-myc on the differentiation of cells of the myelomonocytic series. Here it was found that although by controlling the relative levels of v-myb and v-myc expression, cells of differing degrees of apparent maturity could be induced (as described above), close analysis of phenotypic features expressed (e.g., Fc, C3 receptors, membrane ATPases) demonstrated that none of the resulting cells corresponded exactly to any normal phenotype. That is, each resultant type, whether “immature,” “intermediate, ” or “mature, ” always showed either positive or negative anomalous phenotypic features. Now from the earlier results with temperaturesensitive mutants (e.g., Pacifici et al., 1977), it was demonstrated that, since the cell proliferating/differentiation-blockingactions of oncogenes were reversible at the nonpermissive temperature, then the oncogene effect was not to alter the differentiation program as such but rather to prevent its implementation. The knowledge that viral oncogenes influence the expression of differentiation programs (rather than altering or destroying the program itself) is most important, but it should be emphasized that such influence does not necessarily involve complete suppression. That is made clear in the above-cited results

84

IAN BUCKLEY

of Symonds et al. (1986), where the oncogene effect was not to block differentiation totally but instead to distort the normal pattern of expression of individual phenotypic features, presumably by anomalous gene activations and suppressions. With the viral oncogene as a model, one can suggest that cellular oncogenes (alone or in combination) might function similarly, that is. that they will modulate the expression (or nonexpression) of a set of individual phenotypic features which together go to make up a particular cell phenotype. In that case normal cell differentiation will depend on the overall pattern of expression conforming to a strict standard. Should that be so, then the potential for viral oncogene products with known truncated or substituted sequences to alter differential gene expression seems obvious. Of course, there is no commonly agreed mode by which oncogene products exert their effects on gene expression. Nevertheless, an attractive idea is that this could be by differential (i.e., site specific) binding within the genome. However, oncogene products are known to exert several biochemical effects, and fluorescent-antibody studies have shown that while some bind to particular bases and concentrate within the nucleus, others concentrate at the cell periphery (Bishop, 1985). Such differential concentration has suggested that there may be separate sites and modes of action for the various cellular oncogene products. The possibility should be considered, however, that regardless of sites of greatest concentration, the site of oncogenic action might nevertheless be closely related to the genes coding for the various differentiation antigens (possibly at the relevant enhancer regions). If that were so, such an effect could be induced at each controlling site by as few as one molecule per site, in which case the aggregate number of oncogene-coded molecules involved may be below the threshold for fluorescence detection. Should this mode of oncogene action apply generally, then the altered structure of viral oncogenes (cf. cellular oncogenes) suggests that the distortions of normal oncogene modulation patterns induced by altered oncogenes are likely to result from their modified DNA-binding characteristics, Some examples of low molecular weight site-specific DNA-binding proteins capable of differentially activating or repressing genes in eukaryotic (yeast) cells were given b y Keegan et al. (1986). It may be that a similar mode of action could apply with respect to the proposed differential gene-controlling effects of oncogene products. As suggested above, such a model of oncogene action could account for an altered pattern of tissue-specific gene expression whenever the coded oncogene product differs qualitatively from that of the normal cellular oncogene. Of course, on the face of it, it might seem that the model would not fit in with the proposal that malignancy could result solely from overexpression of normally constituted cellular oncogenes (Waterfield et a l . , 1983). However, if the end result of “overexpression” is not only augmented expression of some genes


85

but exaggerated suppression of others, there is not necessarily any contradiction. In any case, there is continuing doubt as to whether merely heightened expression of otherwise normal cellular oncogenes is a sufficient basis for malignant transformation (Parker et al., 1984; Duesberg, 1985). For example, Parker et al. (1984) have shown that whereas rat cells transfected with v-ssc were transformed at high efficiency, the same cells transfected with c-src (expressed at three times the level of the v-SIC)were not transformed.

C. RECESSIVE ONCOGENES As mentioned above, these represent a separate class of malignancydetermining genes quite distinct from viral oncogenes and their cellular counterparts. The distinction is based on the finding in both Drosophila (Gateff, 1982) and humans (Knudson, 1985) that, in contrast to the dominant effect of viral oncogenes which can act heterozygously, the recessive oncogenes exert their malignancy-promoting effect only when affected homozygously . Moreover, in acting in this fashion, it is clear that the malignancydetermining effect is most strongly exerted when the gene concerned shows complete deletion (Gateff, 1982; Knudson, 1985). The information on this point is well established, being based on some 25 recessive mutants in Drosophila (Gateff, 1982) and less extensive but equally hard data derived from human patients with retinoblastoma and Wilms’ tumor (Knudson, 1985). As pointed out by bath authors, since all of these mutational deficiencies result in tumors that are not only cell type specific, but stage specific, there is strong reason to believe that the loci concerned are somehow involved in coding for tissue-specific cell differentiation. This conclusion is supported by results derived from the temperature-sensitive mutants available in Dsosophila, which demonstrate that the deletion of a given key locus causes malignancy in a particular tissue immediately following a known differentiation step (Gateff, 1982). These discoveries raise a number of questions. For example: What is the physiological role of the recessive oncogenes? How could their absence determine malignancy? What relationship might the recessive oncogenes have to the dominantly acting ones? Although Knudson (1985) recognized that the physiological functions of neither class of oncogenes could be the causation of cancer, he nevertheless posed the question as to whether the normal role of the recessive oncogenes might be to counteract the otherwise dominantly acting cellular oncogenes, the idea being that the latter would normally promote a level of cell proliferation that requires termination by a gene controlling cell differentiation. However, as discussed above, there is reason to believe that the dominantly acting oncogenes play a vital role in controlling differentiation as well as cell proliferation. Moreover, there is no compelling evidence to

86

IAN BUCKLEY

indicate that the recessive oncogenes are involved in the control of cell differentiation rather than, say, coding for the key differentiation antigens the expression of which must be controlled. Indeed the available data appear consistent with this latter possibility. That is because (1) the number of recessive oncogenes is very large, (2) each one exhibits extremely narrow tissue specificity (in contrast to the broader tissue specificities exhibited by the dominant oncogenes), and (3) because the maximum malignancy-promoting effect occurs when the locus is totally deleted (Gateff, 1982; Knudson, 1985). Thus the recessive oncogene loci could code for specific key differentiation antigens, the aggregate expression of which would be responsible for normal tissue-specific histological organization, the nonexpression of which could lead directly to that antisocial “uncontrolled” behavior we know as “malignancy. ” Although it is envisaged that all recessive oncogene products would fall into the category of differentiation antigens, the converse would, of course, not apply, since there would be many tissue-specific antigens that were not concerned with the maintenance of proper intercellular spatial and growth relationships. Nevertheless, since the number of cell surface markers for any given tissue is likely to be large, it seems that the total number of recessive oncogenes for any given species could turn out to be very large. Accordingly, the number of potential mutation sites would be equally numerous, this representing an extensive “target” for mutating agents of all kinds. However, the coding system for such genes is highly protected in the sense that mutations will not be “expressed” as malignancy unless two conditions are met. First, mutations must involve both alleles equally, i.e., at least two “hits” required and-considering the efficiency of genetic repair processes-probably many more (Alberts et a l . , 1983). Second, both mutational events must result in significant alteration of the gene loci such that the expression of both is simultaneously grossly distorted, reduced, or deleted.

D. THEMALIGNANT GENOTYPE-ASUMMARY There are two classes of genes that have the potential to induce malignancy, the one acting recessively, the other dominantly. The class which acts recessively appears to code for a wide spectrum of tissue-specific differentiation antigens, and malignancy occurs (in the matching tissue) whenever expression is, homozygously , seriously distorted or completely lacking. The other class, that which acts dominantly, represents the cellular oncogenes, counterparts of the well-known viral oncogenes. It is suggested that the normal role of cellular oncogenes is to modulate expression of the abovementioned recessive oncogenes (i.e., those coded for by the various cell differentiation programs), this being effected through the combined actions of the protein products of one or more of the cellular oncogenes; here the mode


87

of action may be by site-specific DNA binding, the end result being differential selective expression and suppression of the individual genes within the differentiation programs so that the appropriate stage-specific composite phenotypes result. It is further suggested that malignancy ensues from a malfunction of these cellular oncogenes whenever there is inadequate expression of key cell surface differentiation antigens, i.e., those responsible for controlling cell growth and position. Thus it is proposed that malignancy can arise in either of two general ways. The most “direct” and irretrievable way is by homozygous deletion of a recessive oncogene, resulting in nonexpression of a key antigen (or antigens). The other is by significant alteration of a dominant oncogene protein product such that its expression-modulating action is distorted, especially when such distortion gives rise to inappropriate suppression. It should be emphasized that regardless of which class of oncogenes is involved, the end result would be the same, i.e., failure to express one or more of the key differentiation antigens required for proper control of cell growth and position. However, it can be expected that whereas complete homozygous deletion of a recessive oncogene would give rise to the most malignant kind of tumor, more subtle alterations of these genes (homozygously affected) could result in less malignant behavior. Likewise, one can envisage many subtle alterations in the protein products of the dominantly acting oncogenes that would give rise to only slightly modified patterns of expression of a given phenotype-this, too, helping to account for the commonly occurring malignancies that are extremely slow both in their growth and rate of invasion.

V. General Synopsis Despite recent advances in knowledge, our understanding of both the malignant phenotype and the malignant genotype is limited. However, it is well established that general features of malignancy include (1) failure of normal cell differentiation, (2) abnormal histological organization, (3) exaggerated cellular heterogeneity, (4) increased cell proliferation, and (5) malignant cell invasion of normal tissues. All of these features indicate a lack of proper control over cell differentiation, but because it is malignant cell invasion and augmented proliferation which together lead to tissue destruction, metastasis, and death, it is important to focus attention on those aberrations of differentiation which might shed light on these two. The broad approach taken is to consider the likelihood that the cell’s lack of control over its mitotic and locomotory activities could stem directly from lack of expression of those differentiation antigens normally involved in such controls (cf. Section 11).

88

IAN BUCKLEY

A considerable amount of circumstantial evidence indicates that within living tissues both cell position and proliferation are controlled by sets of cell surface differentiation antigens the role of which is mutual cell recognition. At the same time it is known that, as in the cell culture situation, absence of normal tissue constraints leads to unrestrained cell proliferation and wandering. Accordingly, it is proposed that the critical feature of the malignant phenotype is a differentiation failure which includes deficits in the expression of one or more of the cell’s tissue-specific surface recognition molecules, such deficits leading directly to inadequate control over cell proliferation and to random cell wandering (i.e., to “invasion”) (cf. Section 111). In attempting to reconcile this view of the malignant phenotype with recent discoveries concerning the malignant genotype, a number of points can be made. There are two known classes of genes with malignancydetermining potential (i.e., “oncogenes”), the one class acting dominantly, the other recessively, The dominantly acting class of oncogenes, termed “cellular oncogenes,” correspond to the well-known viral oncogenes. It appears likely that the physiological role of cellular oncogenes (alone or in combination) is to modulate the expression of tissue-specific differentiation molecules, including those that code for the range of antigenic markers which characterize a particular cell type and stage of differentiation. Viral oncogenes have a similar mode of action but, due to their altered coding sequences, the end result is distortion of the pattern of expression of the differentiation molecules, this resulting in aberrations of the phenotype. T o account for the malignancy-determining influence of deranged cellular oncogenes, it is suggested that this occurs whenever their differentiationmodulating effect is such as to suppress the expression of a key surface antigen, the normal function of which is to control cell position and proliferation (cf. Section IV,A,B). The other class of oncogenes, that which acts recessively, is also known to play a vital role in differentiation, since-for a given affected locus-malignancy occurs in a particular tissue at a predictable differentiation switch time. With these genes, however, malignancy does not occur unless both alleles are equally affected, and the most malignant tumors occur when the locus is completely deleted. Moreover, in contrast to the dominantly acting oncogenes, which are moderate in number and have overlapping tissue specificities, the recessive oncogenes exhibit extremely narrow tissue specificities and are much more numerous. This suggests the possibility that the recessive oncogenes code directly for those tissue-specific cell differentiation molecules which must be adequately expressed on a cell surface for the proper control of cell position and growth, i.e., for the same set of surface antigens, the expression of which is modulated by the cellular oncogenes (cf. Section IV,C).

ONCOGENES AND T H E NATURE OF MALIGNANCY

89

Accordingly, the overall proposal is that defects in oncogenes of both classes have the potential to result in inadequate expression of key cell surface differentiation antigens the normal role of which is “cell recognition” and, through that, the proper control of cell position and proliferation. The general concept appears to be consistent with what has long been known about faulty cell differentiation and behavior in malignancy, with current knowledge concerning the probable nature of tissue controls over cell position and mitosis and with recent discoveries concerning the mode of action and general effects of the two known classes of oncogenes (cf. Section IV,D).

VI. Appendix A. CELLULAR HETEROGENEITY No discussion of the nature of malignancy would be adequate without an attempt to account for tumor cell heterogeneity, i.e., the great variety of cellular phenotypes that occur within any malignant tumor (Foulds, 1969; Buick and Pollak, 1984; Heppner, 1984). Although some sources of heterogeneity are obvious, others require closer consideration because they are still speculative. Clearly one source is the presence within invasive tumors of many different types of nontumor cells, some resident in the region, others migratory. A second major source is the occurrence of tumor-derived cells in many different stages of differentiation (Pierce and Cox, 1978; Buick and Pollak, 1984; Heppner, 1984). That is, in addition to the tumor stem cells (which may appear undifferentiated), all but the most malignant of tumors contain a proportion of progeny cells in various stages of differentiation, some even forming quasinormal histological structures such as the glandlike arrays of adenocarcinomas. Indeed, in many tumors a proportion of these progeny cells differentiate to the point that they are not themselves malignant. Thus the more differentiated portions of experimental epitheliomas in rats were shown by Pierce and Cox (1978) to be no longer malignant when dissected out and transplanted to a new host animal. As pointed out by Heppner (1984), cell differentiation-derived heterogeneity is common to all tissues (normal as well as malignant) and degrees of differentiation (even though abnormal in character) account for much tumor cell heterogeneity. A third source of heterogeneity in malignancy is differently based, however, since it is known to occur in highly malignant tumors in which cell differentiation is minimal or absent. Indeed, characteristically the more malignant the tumor’s behavior, the greater is this heterogeneity which is seen as variations in cell size, shape, cytoplasmic staining, nuclear size, and karyotype (Willis, 1948; Ruddon, 1981). This aspect of heterogeneity is more difficult to explain. Clearly it is not simply the result of rapid cell proliferation, since normal stem

90

IAN BUCKLEY

cells, which also have extremely high proliferative rates, do not exhibit anomalous karyotypes or extreme variations in cellular form. One possible source of this form of heterogeneity stems from observations of Mintz (197 l ) , who demonstrated with allophenic (but otherwise normal) mice, that clones of, for example, epidermal cells develop which differ in certain phenotypic characteristics from the cells of neighboring clones. A similar phenomenon appears to affect human skin, as evidenced by the occurrence (especially in later life) of well-defined patches which vary in their pigmentation and other characteristics. Although the basis for the development of Mintz’s variant clones (termed “phenoclones”) is not fully understood, it is known that it does not involve gene mutation but rather some epigenetically controlled variation in the regulation of gene expression (Mintz, 1971). Under normal conditions, where tissues are allowed to develop undisturbed, variations are limited in extent: clonal cells always remain within normal structural and physiological limits and so are commonly indistinguishable from one clone to the next. However, it is known that greater variations are induced in tissues by many pathological disturbances (e.g., following wounding or infection). Even wider variations may be found when previously normal tissue cells are set up in primary culture. Although initially such cells may resemble those of the tissue of origin, variations from these appear within days and subsequently considerable cell-to-cell variations develop. This situation is in marked contrast to that in true tissue culture, where the constituent cells maintain their normal tissue characteristics. A reasonable inference from this is that cell variation is not induced by culture as such but rather by the separation of cells from their normal tissue context. Since the variant primary culture cells do not behave malignantly following animal inoculation, the mechanism of variation may be thought of as an exaggeration of the mechanisms involved in the formation of phenoclones, induced by removal of the constraints on such variation due to intercellular contacts within a normal tissue environment and perhaps aided by other “stress” conditions of cell culture. It may be said, of course, that none of this defines the epigenetic mechanisms responsible and that even the possible connection between defects in intercellular communication and the occurrence of augmented cellular heterogeneity (implied by the above-cited observations) is highly speculative. This is so, and only painstaking experimental work will eventually test such a proposal and identify other possible sources. However, whatever its basis, the occurrence of cellular heterogeneity should not be allowed to dominate ideas about the nature of malignancy. This is for the following reasons. First, as indicated above, a degree of cellular heterogeneity is a regular feature of all normal tissues. Second, greater than normal heterogeneity is a common feature whenever tissues are disturbed, being found not only in malignancy but under in uitw cell culture conditions and


91

in many nonmalignant (e.g., inflammatory) as well as premalignant pathological conditions in vivo. Finally, it does not seem possible to envisage cell-to-cell variation as the basis for a type of cell behavior which is a constant feature of malignancy. It seems rather that such behavior must be sought in cellular features (whether positive or negative attributes) which, while they can be expected to vary in magnitude according to the degree of malignancy, are constant in their occurrence, not variant (Ruddon, 1981; Buckley, 1985). Of course, none of the above should be construed to mean that cellular heterogeneity is irrelevant to malignancy. Indeed it is highly relevant for at least two different sorts of reasons. The first is that it acts as a great “confusion factor, ’’ making it extremely difficult to recognize cellular characteristics that are the essential features of malignancy. The second is that although most new variations occurring within a malignant cell population will have little or no influence on malignant behavior, over extended periods of time, some variations may occur that do affect key phenotypic features which determine malignant behavior for the worse, this no doubt helping to account for tumor progression (Foulds, 1969). In summary, this analysis of the place of cellular heterogeneity in malignancy suggests the following. Within normal tissues a degree of heterogeneity occurs between cells of the same type, but its extent is limited by continuous controls mediated by cell-cell communication systems. Anything which disturbs proper intercellular communication induces abnormally high degrees of phenotypic variation, and the greater the disturbance, the greater the extent of variation. This is illustrated by the differences in phenotypic variation occurring within tissue cultures (where communication is minimally disturbed), compared to cell cultures where communication is grossly disturbed. Malignant cell populations, which also exhibit considerable phenotypic variations, may, in a sense, do so for similar reasons, namely, that in that case too there is a serious disturbance to intercellular communication (see Section 111). However, whereas the disturbance in cell culture is based on physical separation, that in malignancy is based on a special form of differentiation defect, affected cells having specific deficits in their surface recognition molecules. From this it would follow that the greater the deficit, the greater would be the degree of heterogeneity; certainly, it is well established that the more malignant the tumor, the greater is the degree of heterogeneity. Notwithstanding this, heterogeneity should be regarded not as a prime cause of malignancy but as a by-product which, in the course of time, may or may not give rise to variants exhibiting greater malignant behavior. Of course those variants showing more malignant behavior may selectively come to predominate.

92

IAN BUCKLEY

B. MATURATION ARREST Especially in the hematological field, a prevalent view is that malignancy is due to arrest of cell maturation within the relevant lineage, so that while differentiation is blocked, growth continues (Greaves et al., 1981; Sachs, 1984; Metcalf, 1985). This view appears to be based on two broad considerations. First, despite diligent searching, it has not been possible to identify any leukemia-specific antigens which might account for malignant behavior (Greaves et a l . , 1981). Second, it has seemed that the major cell behavioral problem is one of cell growth, the most striking examples of which involve blast cells of apparent stem cell origin that, in crises, come to dominate the hematological picture. Since the normal levels of stem cells and differentiated cells are set by an appropriate balance between cell generation on the one hand and proper maturation followed by cell death on the other, it can be agreed that there is a serious flaw in the regulation of this balance. But can such faulty regulation in malignancy be adequately described in terms of maturation arrest or differentiation block? The concept of maturation arrest as a sufficient explanation for hematological (or other) malignancy appears inadequate for several reasons, First, even in the worst leukemic crises, a fraction of malignant stem cell progeny continue to exhibit some differentiative features, this indicating that these is no absolute block to differentiation, Second, the idea of differentiation block implies that the malignant stem cells are normal up to the point of the block. However, this cannot be so because, whereas normal stem cells remain within their usual tissue locations, leukemic blast cells (and their anomalously differentiated progeny) invade the vascular spaces and colonize distant tissues. Further, unlike normal stem cells, leukemic blast cells exhibit excessive cellular heterogeneity and karyotypic variation. The observation that the differentiation of virally induced leukemic cells is distorted such that none of the apparently mature progeny cells corresponds exactly to any normal phenotype indicates that differentiation is not just blocked but seriously deranged (Markert, 1968; Symonds et al., 1986). The fact that no leukemia-specific antigens are found associated with these abnormal phenotypes is consistent with the idea that the abnormality may consist of specific antigen dejicits (rather than additions), these deficits leading directly to malignant behavior, as proposed above (see Section 111).

ACKNOWLEDGMENTS It is a pleasure to thank Dr. Terry Walsh for careful reading of the manuscript and for many helpful suggestions. Thanks are due also to Mr. Colin McLachlan for assistance in “taming” the word processor.

ONCOGENES AND T H E NATURE O F MALIGNANCY

93

REFERENCES Alberts, B., Bray, D., Lewis, J., Raff, M . , Roberts, K . , and Watson, J . D. (1983). “Molecular Biology of the Cell.” Garland, New York. Bastiani, M. J . , Du Lac, S . , and Goodman, C . S. (1985a). In “Model Neural Networks and Behavior” (A. I. Selverston, ed.), pp. 149-174. Plenum, New York. Bastiani, M. J., Doe, C. Q., Helfand, S. L., and Goodman, C. S. (198513). Trends NeuroSci. 8, 1-10,

Bender, W. (1985). Cell 43, 559-560. Bishop, J. M. (1983). Annu. Reu. Biochem. 52, 301-354. Bishop, J . M. (1985). Cell 42, 23-38. Boettiger, D., and Durban, E. M. (1979). Cold Spring Harbor Symp. Qwnt. Biol. 44, 1249-1254. Boyse, E. A , , and Cantor, H . (1978). In “The Molecular Basis of Cell-Cell Interaction. Birth Defects: Original Article Series’’ (R. A. Lerner and D. Bergsma, eds.), pp. 249-283. Liss, New York. Buckley, I. K. (1985). Cell Bid. Int. Rep. 9, 23-30. Buckley, I. K . , and Porter, K. R. (1967). Protoplasma 64, 349-380. Buick, R . N., and Pollak, M. N. (1984). Cancer Res. 44, 4909-4918. Cairns, J. (1975). Nature (London) 255, 197-200. Carmier, J. F., and Samarut, J . (1986). Cell 44, 159-165. Coll, J., Saule, S., Martin, P., Raes, M. B., Lagrou, C . , Graf, T . , Beug, H., Simon, I. E., and Stehelin, D. (1983). Exp. Cell Res. 149, 151-162. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A,, Schlessinger, J., and Waterfield, M . D. (1984). Nature (London) 307, 521-527. Duesberg, P. H . (1985). Science 228, 669-677. Duprey, S. P., and Boettiger, D. (1985). Proc. Natf. Acad. Sci. U.S.A. 82, 6937-6941. Foulds, L. (1969). “Neoplastic Development.” Academic Press, London. French, V., Bryant, P. J . , and Bryant, S. V. (1976). Science 193, 969-981. Gateff, E. (1982). Adu. Cancer Res. 37, 33-73. Gierer, A. (1974). Cold Spring Harbor Symp. Quanl. Bid. 38, 951-961. Gonda, T . J., and Metcalf, D. (1984). Nature (London) 310, 249-251. Graf, T., Beug, H . , and Hayman, M . J. (1980). Proc. Nad Acad. Sci. U.S.A. 77,389-393. Greaves, M. F., Delia, D., Robinson, J., Sutherland, R . , and Newman, R . (1981). Blood Cells 7, 257-280. Heppner, G. H . (1984). Cancer Res. 44, 2259-2265. Holtfreter, J. (1948). Ann. N . Y. Acad. Sci. 49, 709-760. Keegan, L., Gill, G., and Ptashne, M. (1986). Science 231, 699-704. Knudson, A. G., Jr. (1985). CancerRes. 45, 1437-1443. Lennox, E. S. (1982). In “Hybridomas in Cancer Dianosis and Treatment” (M. S. Mitchel and H. F. Oettgen, eds.), pp. 5-13. Raven, New York. Manly, J. L., and Levine, M. S. (1985). Cell 43, 1-2. Markert, C. (1968). Cancer Res. 28, 1908-1914. Markert, C. (1978). In “Cell Differentiation and Neoplasia” (G. F. Saunders, ed.), pp. 9-22. Raven, New York. Metcalf, D. (1985).Cell 43, 5-6 Mintz, B. (1971). In “Control Mechanisms of Growth and Differentiation” (D. D. Davies and M . Balls, eds.), pp. 345-370. Cambridge Univ. Press, London. Mintz, B., and Fleischman, R . A. (1981). Adu. Cancer Res. 34, 211-277. Moore, G . P . M . , Panaretto, B. A., and Robertson, D. (1983). Anat. Rec. 205, 47-55. Moscona, A. (1962). J. Cell. Comp. Physiol. Suppl. 60, 65-80. Miiller, R., Curran, T . , Burckhardt, J., Ruther, U., Wagner, E. F., and Bravo, R . (1985). In

94

IAN BUCKLEY

“Growth Factors and Transformation” (J. Feramisco, B. Ozanne, and C . Stiles, eds.), pp. 289-300. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Oettgen, H . F., and Hellstrom, K. E. (1982). In “Cancer Medicine” (J. F. Holland and E. Frei, 111, eds.), pp. 1092-1067. Lea & Febiger, Philadelphia. Pacifici, M., Boettiger, D., Rohy, K . , and Holtzer, H . (1977). Cell 11, 891-899. Parker, R . C . , Varrnus, H. E., and Bishop, J . M . (1984). Cell 37, 131-139. Pierce, G . B., and Cox, W. F., Jr. (1978). In “Cell Differentiation and Neoplasia” (G. F. Saunders, ed.), pp. 57-66. Raven, New York. Potten, C . S., Schofield, R . , and Lajtha, L. G . (1979). Biochim. Biophys. Acfa 560, 281-299. Ruddon, R . W. (1981). “Cancer Biology.” Oxford Univ. Press, New York and London. Sachs, L. (1984). In “Mediators in Cell Growth and Differentiation” (R. J . Ford and A. L. Maizel, eds.), pp. 341-360. Raven, New York. Strauli, P. (1980). In “Cell Movement and Neoplasia” (M. De Brahander, M. Mareel, and L. De Ridder, eds.), pp. 187-191. Pergarnon, Oxford. Sulitzeanu, D. (1985). Adu. Cancer Res. 44, 1-42. Sulston, J . E. (1983). Cold Spring Harbor Symp, Quanf. Biol. 48, 443-452. Syrnonds, G . , Klernpnauer, K.-H., Snyder, M., Moscovici, G., Moscovici, C . , and Bishop, J . M. (1986). Mol. Cell. Biol. 6 , 1796-1802. Wallach, D. F. H. (1968). Proc. Nat. Acud. Sci. U.S.A. 61, 868-874. Waneck, G. L., Keyes, L., and Rosenberg, N. (1986). Cell44, 337-344. Waterfield, M. D . , Scrace, G. T., Whittle, N . , Stroobant, P., Johnsson, A , , Wasteson, A , , Westerrnark, B., Heldin, C.-H., Huang, J . S., and Duel, T. F. (1983). Nature (London) 304, 35-39. Willis, R . A. (1948). “Pathology of Tumours.” Butterworths, London. Wood, S., Jr. (1958). Am. Med. Assoc. Arch. Pafhol. 6 6 , 550-568.

THE EPSTEIN-BARR VIRUS PROTEINS Joaklrn Dillner’ and Bengt Kallln Department of Tumor Blology, Karollnska Instltute, 5.104 01 Stockholm, Sweden

I. Biology of EBV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Viral Tropism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Lymphoblastoid Cell Line . . . . . . . . . . . . . . . . . . . . . . . 11. EBV-Associated Diseases . . . . . . . . . . . . . . . . , , , , , . , . , . , . . . . . . . . , . , . . A. Burkitt’s Lymphoma . . . . . . . . . . . . . . . . . . . , . , . , . , , , . , , . , . . , , . . . . B. Nasopharyngeal Carcinoma . . . . . . . . . . . . . , , . . , . , . , , , , , . . . . . . . . C . Acute Primary EBV Infection: Infectious Mononucleosis . . . . . . . . . . . . . , . D. EBV Genome Carrying Lymphoproliferative Diseases in Immunodeficient Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . , . . . . . , , , , , , 111. TheEBVGenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General Structure of EBV DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transcription ofthe EBV Genome in Transformed Cells, . . . , . . . . . . , , , , . IV. EBV-Encoded Proteins in EBV-Transformed Cells . . . . . . . . . . . . . . , . . . . . . . , , A. EBNA ............................... B. EBNA.................................... C . EBNA............................... D. EBNA-3 . . . . . . ............................... E. EBNA-4 . . . . . . . . . . . . . . . .................... F. EBNA-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . G. The Latent Infection Membrane Protein . . . . . . .... .. , H . The Differential Expression of the Transformation-Associated Proteins. . . . I. Are There More Transformation-Related Proteins Yet to Be Identified? K. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Viral Proteins in Virus-Producing Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Detection, Classification, and Nomenclature of the Productive CycleProteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. T h e E B V G e n e M a p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Early Genes and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Proteins of the Virus Particle. . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , , , VI. Summary and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

95 95 96 96 96 98 98 99 100

100 102 104 104 104 113 121 124 127 131 134 136 137 138 139 140 141 147 149 151

I. Biology of EBV Infection A. THEVIRALTROPISM EBV is spread by oral contact, usually by the ingestion of saliva from an EBV-infected individual. The virus replicates in epithelial cells of the upper ‘Present address: Department of Molecular Biology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037.

95 ADVANCES IN CANCER RESEARCH. VOL 50

Copyright 0 1988 by Academic Press, In‘ All rights ofreproducttan in any farm rerervd

96

JOAKIM DILLNER AND BENGT KALLIN

respiratory tract, in particular in the epithelial cells of the parotid gland (Morgan et al., 1979; Wolf et al., 1981, 1984). The virus persists in the infected individual throughout life, and infectious virus is intermittently released in the saliva of almost all healthy EBV-infected individuals (Gerber et a l . , 1972; Yao et al., 1985). In the replicative site in epithelial cells, the virus is believed to infect the B lymphocytes, which are found massively infiltrating the epithelial tissue of the upper respiratory tract. The infectability by EBV is restricted to B lymphocytes, due to the fact that the EBV receptor is the B-cell-specific receptor for the third component of complement (C3D) (Yefenof et al., 1976; Fingeroth et a l . , 1984; Frade et al., 1985). It is clear that EBV infects epithelial cells in vivo and this can under certain experimental conditions be accomplished in vitro (Sixbey et al., 1983). Recent evidence indicates that the C3D receptor may also be expressed on epithelial cells of the nasopharynx (Young et al., 1986).

B. THELYMPHOBLASTOID CELLLINE The in vitro EBV-infected B lymphocytes are transformed into lymphoblastoid cell lines (LCL). Compared to the normal B lymphocyte, the LCL has acquired the ability to produce its own B-cell growth factor (BCGF) (Blazar et al., 1983; Gordon et al., 1984a,b) and possesses the ability to grow indefinitely in culture. Whereas the LCL are for their growth dependent on BCGF, the Burkitt’s lymphoma (BL)-derived lines may or may not produce and respond to BCGF, but are in no instance dependent on BCGF (Gordon et al., 1985). Compared to the BL-derived cell line, the LCL is regarded as lowmalignant, since it does not form tumors in nude mice and shows a low cloning frequency in soft agar (Nilsson, 1979). After inoculation with EBV, polyclonal malignant lymphomas with phenotypic characteristics similar to the LCL arise in several species of New World monkeys, such as cottontop marmosets, owl monkeys, and tamarins (Shope et al., 1973; Miller et al., 1977; Cleary et al., 1985). Similarly, B-lymphocytic tumors resembling the LCL may occur in severely immunodeficient patients (Purtilo et a l . , 1977a,b). It is evident that the virus has evolved a delicate virus-host balance that, unless disrupted, will protect the host from disease and allow the further spread of the virus.

II. EBV-Assoclated Diseases A. BURKITT’SLYMPHOMA EBV was first discovered by electron microscopy in a cell line derived from a Burkitt’s lymphoma (BL) (Epstein et al., 1964). BL is a lowdifferentiated non-Hodgkin B-lymphocytic lymphoma. The disease accounts for

THE EPSTEIN-BARR VIRUS PROTEINS

97

nearly one-half of all cancer in children in the tropical regions of Africa (Burkitt, 1962, 1963). The high endemic occurrence of BL is across tropical Africa and in Papua New Guinea, but only at altitudes < 1500 m (Burkitt, 1962). BL does also occur outside the endemic areas, but very rarely, and the sporadic form of BL differs from the endemic form in several phenotypic aspects (Rowe et a l . , , 198513). The location of the endemic form of BL is also usually in the jaws, whereas the sporadic form frequently is found in other locations, such as the long bones, kidneys, adrenals, thyroids, ovaries, or testes. The peak incidence of endemic BL is 6-10 years of age, whereas the sporadic form usually occurs after 15 years of age (Burkitt, 1962; Ziegler et a l . , 1976; Pagano et al., 1973). Some 98 % of endemic BL carry the EBV genome, often in multiple copies per cell (zur Hausen and Scholte-Holthausen, 1970; zur Hausen et a l . , 1972). All of the cells in an EBV-carrying BL express the EBV-determined nuclear antigen (EBNA) (Reedman and Klein, 1973; Lindahl et al., 1974). Other EBV-determined antigens first found to be expressed in these tumors are the viral capsid antigen (VCA) (Henle and Henle, 1966), early antigen (EA) (Henle et al., 1970), and membrane antigen (MA) (Klein et al., 1966). In 100% of cases, BL patients have elevated antibody titers against the VCA (Henle and Henle, 1979). The geometric mean anti-VCA titer is 8-fold higher than a control group with other neoplasms (Henle and Henle, 1979). If an African child has an elevated VCA titer by two or more dilutions, he has an elevated risk to develop BL of > 30 times that of the normal population (de-The et al., 1978). Moreover, BL patients exhibit antibody titers to the restricted type of the early antigen (EA-R) and to the MA, which are not normally seen among healthy EBV-seropositive donors (Henle and Henle, 1979; Klein et al., 1966). The EA-R and MA titers are correlated with the clinical activity of BL, high titers of anti-MA being observed in long-term survivors and dramatic rises in anti- EA-R levels correlating with disease onset or relapse (review by Henle and Henle, 1979). The sporadic cases of BL which occur outside the endemic area are only EBV-carrying in ~ 2 0 %of the cases (Pagano et al., 1973; Ziegler et al., 1976). It thus seems that some other factor, specific to the endemic area, must play a role in the etiology of BL as well. Klein (1979, 1983) has suggested that BL develops in three phases in the endemic regions. Phase 1 is the primary infection with EBV. While uninfected B lymphocytes differentiate toward the plasma cell end stage, EBV-infected B lymphocytes are arrested in an early differentiation stage and are only stimulated to divide. These EBV-infected cells may risk chromosomal damage in direct proportion to the number of cell divisions. Phase 2 involves an environmental cofactor, most likely holoendemic malaria. A recent study has shown that infection with Plasmodium falciparum leads to an impairment of the ability of cytotoxic T cells to control the proliferation of the EBV-infected B cells (Whittle et al., 1984), thereby further increasing the “pool” of EBV-infected B cells.

98

JOAKIM DILLNER A N D BENGT KALLIN

Phase 3 would involve the translocation of the distal part of chromosome 8 to chomosome 14 (or 2 or 22), leading to the constitutive activation of the c-myc oncogene and subsequent monoclonal B-lymphocyte proliferation, resulting in BL.

B. NASOPHARYNGEAL CARCINOMA Nasopharyngeal carcinoma (NPC) is the most common tumor in the densely populated areas of southern China. The tumor is also common in East and North Africa and in Arctic Eskimo populations. Irrespective of its geographic origin, 100% of anaplastic NPC tumors carry multiple copies of EBV (zur Hausen et a l . , 1970; Nonoyama and Pagano, 1973; Wolf et al., 1973; Saemundsen et al., 198213). In the tumor, both EBV DNA and EBNA can be detected in the epithelial cancer cells (Klein et al., 1974). As in BL, antibody titers to VCA are elevated (\lO-fold) (Henle and Henle, 1979). Antibodies against EA are regularly present in NPC patient sera, but unlike in BL, they are mainly directed against the diffuse subspecificity (EA-D) (Henle and Henle, 1979). Characteristically, these patients develop IgA antibodies to VCA, even before a detectable tumor mass has developed (Henle and Henle, 1976). Since IgA-VCA is not seen in other conditions, the IgAVCA test can be used routinely for early detection of NPC. In the provinces Zang-Wu and Laucheng in southern China, the test has been employed on 185,000 inhabitants, resulting in the early diagnosis of 132 NPC cases (Zeng and de The, 1985). The 100% ’ correlation between EBV and NPC indicates that EBV has an etiological role in this tumor, although the restricted geographic occurrence of the tumor indicates that genetic and/or environmental cofactors also contribute to its etiology. First-generation immigrants of southern Chinese origin maintain a high frequency of NPC (Ho, 1975). Offspring of mixed marriages between southern Chinese and non-Chinese groups show an intermediate frequency (Shanmugaratnam and Muir, 1967; Muir, 197 1). Several reports also speak of familial aggregation of NPC (Nevo et al., 1971; Ho, 1972; Williams and de The, 1974). A correlation between NPC and a certain HLA phenotype has been claimed (Lanier et a!., 1980; Simmons et al., 1976). Thus, while in BL there is evidence for an environmental cofactor, the epidemiology of NPC speaks more in favor of genetic cofactors.

C. ACUTEPRIMARY EBV INFECTION: INFECTIOUS MONONUCLEOSIS Infectious mononucleosis (IM) is a self-limiting disease characterized by polyclonal proliferation of EBV-infected B cells, followed by the appearance of characteristic, atypical cytotoxic T cells. The disease has a peak incidence between 17 and 25 years of age, but can also occur in children and older adults. In

T H E EPSTEIN-BARR VIRUS PROTEINS

99

lower socioeconomic groups, infection with EBV occurs during the early years of life and is usually not accompanied by clinical illiness, but results in permanent seroconversion that induces immunity to EBV for life. Previously uninfected adolescents develop IM in %50% of cases when they are exposed to EBV. I M is usually transmitted by kissing, presumably as a result of ingestion of viral particles shed in the saliva of I M patients during the inoculative period, or of healthy seropositive individuals. Prodromal symptoms of IM are headache, chills, and lassitude. The typical IM syndrome lasts for \3 weeks and consists of fever, lymphadenopathy, skin rashes, pharyngitis, splenomegaly , and some hepatocellular dysfunction. After 3-4 weeks the symptoms resolve without treatment and complete recovery almost always ensues. Recurrences have not been documented (reviewed in Chang, 1980). During the early stages of I M the patients develop the chacteristic heterophile antibodies that agglutinate sheep erythrocytes and are used in quick test for the diagnosis of IM. A heterogeneous population of antibodies are induced by the EBV infection of B cells (Kirchner et al., 1979), and a similar phenomenon is seen during the early stages of I M . At this stage up to 18 % of circulating B cells may express EBNA (Robinson et a l . , 1980), but this is already diminished to some 0.1 % ’ by the second week of illness. Virus-specific antibodies that arise early in the course of IM are IgM antibodies to the VCA and antibodies to the EA, mainly the EA-D. The IgMVCA antibodies give way to IgG-VCA after the first weeks, whereas EA-D antibodies are usually not seen in healthy seropositive individuals. Antibodies to EBNA appear during convalescence, some 30-50 days after onset of disease (Henle et al., 1974).

Chronic Mononucleosis This syndrome is characterized b y crippling lassitude and recurrent opportunistic infections. Antibody titers to VCA are elevated and high EA-antibody titers are also seen. The syndrome ususally starts as a normal IM, which does not disappear in the usual self-limiting course (Jones et al., 1985). In an extreme case of chronic IM the antiviral drug Acyclovir has brought about almost complete recovery (Schooley et a l . , 1986).

D. EBV GENOME CARRYING LYMPHOPROLIFERATIVE DISEASES IN IMMUNODEFICIENT HOSTS 1. Fatal Infectious Mononucleosis Although I M is characterized by B-cell proliferation and occasionally presents with severe clinical symptoms, lethal cases of I M are very rare.

100


Typical features of fatal IM are depletion of T cells, bone marrow plasmacytosis, and infiltration of brain and viscera by lymphocytes. The patients are heterophile antibody positive (Purtilo et a l . , 1977a,b). In typical cases, a widely disseminated proliferation of EBNA-positive B cells has been found (Crawford et al., 1979). The syndrome frequently has a familial setting (Bar et a l . , 1974) and may be linked to the X-linked lymphoproliferative syndrome (XLP) (Puritilo et al., 1977a). This syndrome is characterized by defective proliferation of B lymphocytes, X-linked inheritance, and immunodeficiency to EBV (Purtilo et al., 1977a). Approximately 40% of patients develop fatal IM, 40% develop malignant B-cell lymphoma, and 20% develop dysgammaglobulinemia, often associated with chronic EBV infection (Purtilo et a l . , 1979).

2. EB V-Carrying Lymphoproliferative Disorder EBV DNA-positive lymphoproliferative disorders of B-cell origin have been found in a number of conditions associated with immune deficiencies, notably ataxia telangiectasia (AT) (Saemundsen et al., 1981a), X LP (Saemundsen et al., 1981b), renal transplant recipients (Hanto et al., 1981), and cardiac transplant recipients (Saemundsen et al., 1982a). These proliferating lymphocytes are usually polyclonal (Reece et al., 1981). Monoclonal EBVcarrying lymphomas are frequently seen in transplant recipients, but these lymphomas do not carry specific karyotypic alterations (Hanto et al., 1982; Cleary et al., 1984). The EBV-carrying lymphoproliferative disorders of transplant recipients, whether polyclonal or monoclonal, usually show complete regression once the immunosuppressive therapy is removed (Starzl et al., 1984). EBV has also been implicated to play a role in several other diseases, notably thymic and salivary gland carcinomas, the Guillain-BarrC syndrome, and the Chediak-Higashi syndrome. For a comprehensive review, see Purtilo et al. (1985).

111. The EBV Genome A. GENERAL STRUCTURE OF EBV DNA The EBV genome of the virus particle is a linear double-stranded DNA molecule of 173,000 base pairs (Fig. 1). At each end of the molecule there are 4-12 copies of a 500-bp terminal repeat (TR). A recognized role of the T R is to facilitate circularization of EBV DNA following infection (Lindahl et al., 1976). Inside the infected cell, the viral DNA becomes covalently linked and the genome persists as multiple covalently closed circular episomes. Multiple tandem repeats of a 3071-bp internal repeat (IR) sequence separate the

20

kbo

I

us

I

OR1 P

Transformationassociated proteins

40

IR

60

80

I

-

120

140

160 TR

II

UL

BYRFl k EBNA-2

BKRFl

BLRF3 +BERFl

r-

BNLFla-c

P

4

EBNA.3 BERF2.3.4 EBNA-1 t+F

EBNA.5

Putative immediate early and transactivating proteins

100

LMP

FRNA.4

BMRFl

BZLFl 4

I,

~

BOW1 BaRFl

BXLFl 4 70K TK

+I-

Early proteins

RRs

BALE

cl

1lOK BALFZ POL 4

l&?f

~

Major Late proteins

BNRF1 145' MA

44

gp 3501220

MA

BcLFl

4 155K VCA

~~~

BAN4

+I

gp a5? gB Homolog.

FIG. 1. The EBV DNA and the coding sequences of the transformation-associated proteins and the major lytic cycle proteins. (Top) Relative size in kilobase pairs (kbp). (Upper bar) Organization of the genome in short unique sequence (US), internal repeat (IR), long unique sequence (UL), and terminal repeats (TR). (Lower bar) Positions of the BamHI restriction fragments (Skare and Strominger, 1980). (Gene map) Size, position, and direction of transcription are given by arrows. Horizontal lines denote exons. The names of the open reading frames are given above each transcript, according to the system of Baer et ul. (1984). For example, BYRFl stands for BumY Rightward open reading Frame number 1.EBNA, EBV-determined nuclear antigen; LMP, latent membrane protein, pp, phosphoprotein; R3, a monoclonal antibody used to define these proteins, gp, glycoprotein; IE, immediate early protein; K, kilodalton; RRs, ribonucleotide reductase; TK, thymidine kinase; POL, EBV DNA polymerase; DBP, major DNA-binding protein (homologous to HSV ICP 8); MA, membrane antigen; VCA, viral capsid antigen; gB homolog, a predicted protein showing homology to the HSV glycoprotein B, probably identical to gp 85 MA.

102


genome into two domains: a short unique region (Us)of 15,000 bp and a long unique region U, of 150,000 bp (Fig. 1). Several other smaller repeat units have been found (Baer et al., 1984). The third internal repeat region (IR3) is most peculiar. It is a 700-bp sequence composed of only three nucleotide triplets, GGG, GCA, GGA (Heller et a l . , 1982a,b). The EBV DNA has been cloned as a set of overlapping restriction endonuclease fragments of the restriction enzymes EcoRI and BamHI (Skare and Strominger, 1980; Dambaugh et al., 1980; Arrand et a l . , 1981), and a position in the EBV DNA is usually defined by the name of the EcoRI or BamHI fragment. The complete nucleotide sequence of EBV has been determined (Baer et al., 1984).

B. TRANSCRIPTION OF THE EBV GENOME IN TRANSFORMED CELLS In the virus nonproducer growth-transformed cells at least five distinct regions are transcribed. The most abundantly transcribed region is in the EcoRI J fragment and encodes two small nonpolyadenylated RNAs (Rymo, 1979) transcribed by RNA polymerase I11 g a t and Arrand, 1982). These RNAs do not code for protein but exist in the cell complexed with a host cell protein termed “La” (Lerner et al., 1981). They have been designated as EBERs, for EBV-encoded RNAs, and show structural similarities to the VA RNAs of adenovirus (Arrand and Rymo, 1982). The EBERs can functionally substitute the adenovirus VA RNAs and facilitate the translation of adenovirus proteins (Bhat and Thimmappaya, 1983). It is therefore highly likely that the EBERs facilitate translation of EBV mRNAs. The BamWYH region corresponds to the IR and the adjacent 3 ’ region. The principal transcript from this region is a 3.0-kb mRNA that has a 1.6-kb exon toward its 3 ‘ end. This RNA includes multiple copies of internal repeats. There is a promoter in each copy of the IR that could be the promoter for this mRNA and a probable poly(A) addition site immediately 3 ‘ of the 1.6-kb exon. Approximately three copies per cell of this mRNA are found in the cell cytoplasm (review by Kieff et al., 1984). The 1.6-kb exon contains a 1.5-kb long open reading frame, termed BYRFl , which encodes a nuclear protein termed EBNA-2 (Hennessy and Kieff, 1985; Dillner et al., 1985a,b). Bodescot et al. (1984) reported the isolation of a cDNA clone from the BamWYH region, which contained a long open reading frame spliced together of several small open reading frames. This long open reading frame encodes another nuclear protein, termed EBNA-5 (Dillner et a l . , 1986b). Both the EBNA-1 mRNA (Speck and Strominger, 1985), the EBNA-2 mRNA (Sample et al., 1986b), and a BamE mRNA (Bodescot et a l . , 1986) include the EBNA-5 coding sequences in their 5 ‘ parts. Transciption of mRNAs encoding EBNA-2 and EBNA-5 are initiated at a promoter in BamW (Sample et


103

al., 1986b; Speck et al., 1986). A tissue-specific enhancer in the BamW fragment is likely to be involved in the transcription of these mRNAs (L. Rymo, personal communication). The BamWYH region is associated with EBV transformation, since two viruses, P3HR-1 and Daudi, with deleted BamWYH regions are incapable of transformation (Menezes et a l . , 1975; B. Griffin, personal communication; I. Ernberg, unpublished data). Superinfection of the nonproducer line Raji results in the release of recombinant viruses with regained transforming ability (Fresen et al., 1978). All such recombinants were shown to have acquired the BamWYH region (Fresen et al., 1980; Skare et al., 1985). It has been suggested that the association of the BamWYH region and EBVmediated transformation is due to the BamYH-encoded EBNA-2 protein (Hennessy and Kieff, 1985), but the discovery of EBNA-5 challanges this assumption. The BamE fragment has been reported to hybridize to a 4.5-kb mRNA (Hennessy et al., 1985). Two cDNA clones (T2 and T4) map to this region and they both contain long open reading frames, predicted to code for %100,000-Da polypeptides (Bodescot et al., 1986; Bodescot and Perricaudet, 1986). The BamE fragment contains three long open reading frames, not utilized in lytic infection (Baer et al., 1984; Seibl and Wolf, 1985b), each one with a coding capacity of %lo0 kDa. The leftmost of these open reading frames [also contained in the T 2 cDNA clone (Bodescot et al., 1986)l has been shown to encode a part of a nuclear antigen (EBNA-3) (Hennessy et al., 1986). The BamK fragment hybridizes to a 3.7-kb mRNA that has a 2.0-kb exon in this region. This exon contains a single long open reading frame and a short untranslated 3 ' tail. There are approximately three copies of this mRNA in each latently infected cell (review by Kieff et al., 1984). This open reading frame, termed BKRFl , contains the complete coding sequence for a nuclear antigen termed EBNA-1 (Summers et al., 1982; Hennessy and Kieff, 1983; Dillner et al., 1984; Weigel and Miller, 1985). The 5 ' end of the 3.7-kb EBNA-1 mRNA contains several exons both from the BamWY region and from the BamE fragment (Speck and Strominger, 1985). The BamN,,, fragment gives rise to a 2.9-kb spliced, leftward mRNA which is present in at least 60 copies in each growth-transformed cell. The mRNA contains a 1.2-kb open reading frame spliced together from two short and one longer exon. This open reading frame encodes a membrane protein expressed during latent infection, termed LMP. After the end of this open reading frame, the mRNA has a 1.7-kb-long, untranslated 3 ' tail (Fennewald et al., 1984).

104


IV. EBV-Encoded Proteins In EBV-Transformed Cells A. EBNA All latently infected cells contain a nuclear antigen (EBNA) detected by anticomplement immunofluorescence (ACIF) (Reedman and Klein, 1973). After primary EBV infection of B lymphocytes, EBNA appears between 12 and 24 hr postinfection. Cellular DNA synthesis is initiated %24 hr later, followed by mitosis (Einhorn and Ernberg, 1978; Robinson and Smith, 1981). EBNA is known to be a heat-stable protein with DNA-binding ability (Baron and Strominger, 1978), and is found associated with the chromosomes in metaphase (Reedman and Klein, 1973). EBV-related simian lymphotropic herpesviruses all express virus-determined nuclear antigens cross-reactive with and similar to EBNA (Ohno et al., 1977, 1979; Gerber et al., 1976). A protein kinase activity copurifies with EBNA (Kamata et al., 1981), and EBNA has been found to stimulate template activity of chromatin (Kamata et al., 1979) and to stimulate new initiation points for cellular DNA replication (Oppenheim et al., 1981). Thus, there have been several indications that EBNA may be implicated in EBV-mediated tumorigenesis, but the main reason for this suggestion was the impression that this was the only protein detected in all cells in EBV-transformed cell lines (Reedman and Klein, 1973). Recent studies have shown that there are at least five different EBVdetermined nuclear antigens (EBNA) in EBV-transformed cells. As will be discussed, the EBNA ACIF reaction discussed in this section primarily measures EBNA-1, but a significant part of the reaction is due to EBNA-2 and in some cases it may be due to other EBNA (Dillner et al., l984,1985a, 1986a).

B. EBNA-1 EBNA- 1 was identified by immunoblotting and radioimmunoelectrophoresis in four EBV-positive but not in three EBV-negative cell lines, utilizing four EBV-positive human sera in comparison with two EBV-negative human sera (Strnad et al., 1981). The antigens identified had different molecular weights in the different cell lines analyzed, ranging from 65,000 to 73,000. A complement-fixing antigen had been partially purified more than 200 times and was found to copurify with the 65-kDa EBNA identified by immunoblotting. Since EBNA is defined by ACIF, it was suggested that the 65-kDa antigen was a major component of EBNA (Strnad et al., 1981). The EBNA gene was mapped by transfecting mouse cells with the cloned BamHI K restriction enzyme fragment of EBV DNA. The transfection of amouse fibroblast line with this fragment, together with a dominant selectable marker, led to the stable expression of a nuclear antigen identified in ACIF with EBNApositive, but not EBNA-negative human sera (Summers et al., 1982). In a


105

subsequent study it was found that BamK-transfected cells expressed a 78-kDa polypeptide that comigrated with the EBNA-1 polypeptide of B95-8 cells (Fischer et al., 1984). A rabbit serum was produced against a fusion protein between /3-galactosidase and a part of the BamK fragment. It reacted with an EBV-specific polypeptide that varied in molecular weight between different cell lines in a similar manner as detected by Strnad et al. (Hennessy and Kieff, 1983). Comparison of EBV-negative BL line converted with different strains of EBV, showed that the size variation of EBNA-1 is determined by the viral genome (Gergely et al., 1984; Sculley et al., 1984a), and particularly by the length of the third internal repeat array (IR3), included in the BamK fragment (Hennessy et a l . , 1983). We produced synthetic peptides deduced from three possible open reading frames in the IR3 repeat unit. Monospecific antipeptide antibodies were obtained by rabbit immunization and subsequent affinity purification of peptidespecific antibodies (Dillner et al., 1984). One antiserum, antipeptide 107 (a 22-residue copolymer of glycine and alanine), gave an EBNA-specific staining in the ACIF test and reacted in immunoblots with a 70- to 92-kDa polypeptide that varied in molecular weight between different cell lines as described by Strnad et al. (1981) (Fig. 2a). The development of the anti-107 antiserum thus provided direct evidence that the 78-kDa polypeptide (B95-8 cells) detected by immunoblotting was responsible for EBNA-specific ACIF staining. The EBNA staining pattern of the 107 antiserum was discretely punctate (Fig. 2b), in contrast to the finely dispersed EBNA cells (Fig. 2c) (BamKtransfected cells were a gift of T. Dalianis). A subsequent comparison of the “EBNA” titers of human sera as measured on EBV-carrying lymphoid lines and as measured on Bum K-transfected fibroblasts, showed that EBNA-1 is the EBV-encoded protein that bears the responsibility for the main part of the EBNA ACIF reaction (Hearing et al., 1984). The anti-107 sera are the first (and so far only) sera produced in experimental animals that give a specific EBNA staining. These antibodies also reacted with EBNA-1 in its native form in solution. We could utilize them both as catching antibodies in EBNA-1 quantification assays (Sternas et al., 1986; Dillner et al., 1985c) and for immunoprecipitation of EBNA-1 from 32P-labeledcells (Dillner et al., 1987a). Two other antipeptide sera (106 and 108) were directed against the same open reading frame in the Bum K fragment, but did not react .in the ACIF test. Both sera did, however, react specifically with the EBNA-1 polypeptide on immunoblots (Fig. 2d) (Dillner et al., 1985~).The 107 peptide reacted with 19 of 22 tested EBNA-positive human sera in a direct-binding enzyme-linked immunosorbent assay (ELISA). The three EBNA-positive sera that failed to react were derived from BL patients. Seven EBNA-negative sera did not react or gave very weak reactions. Titers of up to 10.240 were found among healthy seropositive individuals, which was equal to or better than the titers of

106


FIG. 2. (a) Immunoblotting with an IR3-Gly-Ala antiserum. Immunoblot of a 10% polyacrylamide gel stained with affinity-purified antipeptide 107 serum, diluted 1 : 10. DNA-cellulose-binding proteins corresponding to lo7 cells have been applied to each lane. EBVnegative BL-derived lines: BJAB and Loukes. EBV-carrying lymphoid lines: B95-8, Cherry, P3HR-1, and Raji. Inhibition with the corresponding synthetic peptide showed that both the EBVspecific 70- to 92-kDa protein (EBNA-I) and the cellular 44- and 49-kDa proteins were inhibited. The cellular 31- to 34-kDa proteins were not inhibited. A faint 62-kDa protein was seen in all lines, somewhat stronger in Loukes cells. The faintness of this reaction did not allow determination of whether it was inhibitable or not. (Reproduced from Dillner el al., 1984.) @) EBNA-1 Gly-Alaspecific fluorescence. Anticomplement immunofluorescence (ACIF) on EBV-positive P3HR- 1 cells stained with afinity-purified rabbit antipeptide 107 serum, diluted 1 : 4. Eight EBV-positive lines gave similar reactions, whereas five EBV-negative lines were negative. The reaction could be inhibited by addition of free peptide. Note the characteristic “speckled” staining morphology. (Reproduced from Dillner et al., 1984.)

immunized rabbit sera. The peptide-specific antibodies were purified from two human EBNA-positive healthy donor sera. They gave a similar EBNA-specific speckled staining pattern, as did the rabbit peptide-specific antibodies (Fig. 2e). The ACIF titers of these two sera were only moderately reduced by the peptide immunosorbent purification, implying that this particular peptide


107

Fig. 2 (continued). (c) Ordinary EBNA-1 fluorescence. ACIF staining with a human EBNA-positive serum on Earn K-transfected EBNA-1-expressing Donna Martin fibroblasts. Mock-transfected fibroblasts were negative. Note the dispersed staining throughout the nucleus, similar in morphology to an ordinary EBNA staining (Reedman and Klein, 1973). (d) EBNA-1-specific irnmunoblotting. Immunoblot ofa 10% gel stained with antipeptide 106 serum (directed against the junction of IR3 and the 3 'unique region). EBV-negative cell lines: BJAB and Loukes. EBV-carrying cell lines: Raji, B95-8, Jijoye, and P3HR-1. Note the typical EBNA-1 size variation and the abscence of cross-reactive proteins.

sequence is responsible for a significant proportion ofthe EBNA reactivity of these sera (Dillner et al., 1984). In further studies, four synthetic peptides deduced from the EBNA-1 sequence were all shown to be recognized by a proportion of human EBV-positive sera in ELISA. The 107 Gly-Ala copolymer was found to be the most immunogenic. It was recognized by 89 of 114 sera with a geometric mean titer (GMT) of > 600. Several disease-specific reactivities were noted. Thus, titers against peptides from the Gly-Ala repeat were increased >20-fold in nonHodgkin lymphoma, whereas titers against a carboxy-terminal synthetic peptide were increased in NPC and BL sera (Dillner et al., 1987b). Milman et al. (1985) produced a bacterial fusion protein containing a carboxy-terminal part of

108


Fig. 2 (continued) (e)Naturally occurring human antipeptide antibodies give Gly-Ala-specific fluorescence. ACIF on P3HR-1 cells with peptide 107-specific human antibodies (Dilution 1 : 4) affinity-purified from a human EBNA-positive serum (B. Kallin). Eight EBV-positive lines gave similar reactions, whereas five EBV-negative lines were negative. The reaction could be completely blocked by addition of free peptide 107. Note the similar staining morphology compared to the rabbit antipeptide antibodies. (Reproduced from Dillner et al., 1984.)

EBNA- 1, and likewise found that antibody titers against this protein were elevated in BL and NPC sera. With our data, these findings suggest that the antibody response to EBNA-1 in BL and NPC, as compared to healthy individuals, shows a “shift” from the N-terminal (Gly-Ala) to the C-terminal epitopes.

1. The Third Internal Repeat Array (IR3) The third internal repeat array (IR3), in the BumK fragment, is a simple repeat array of only three triplet codons, GGG, GCA, and GGA, repeated in


109

an irregular fashion (Heller et al., 1982a,b). The length of the IR3 is different in different EBV substrains. Since EBNA-1 is in part encoded by IR3, the molecular weight of EBNA-1 varies accordingly (Heller et a l . , 1982b; Hennessy et a l . , 1983). The human chromosomes contain multiple sequences homologous to 1R3 (Heller et al., 1982a). Such sequences could be detected in all human chromosomes, with the possible exception of the Y chromosome. One of these homologous sequences has been cloned and sequenced. It was found to have a high degree of homology to the IR3 of EBV, although the cellular sequence was not composed of a perfect triplet repeat array (Heller et al., 1985). Heller et al. (1982a) speculated that EBV may have acquired the IR3 repeat sequence from the cellular genome in a similar manner as transforming retroviruses have acquired oncogenes from the cellular genome. The IR3 repeat could in this model be a candidate for an EBV-carried oncogene involved in the transforming effect of EBV. We found that affinity-purified rabbit antipeptide 107 antibodies, directed against the IR3 repeat sequence, were able to identify two cellular DNA-binding proteins of molecular weights 44,000 and 49,000, in addition to their reactivity with EBNA-1. The reaction could be blocked by addition of an excess of peptide, showing that it was an antigenic cross-reaction due to the peptide 107 (Fig. 2a; Dillner el al., 1984). The reaction was seen with affinity-purified antipeptide sera from three different rabbits, but was not seen on acetone-methanol-fixed cells in the ACIF test. Whereas a peptide-specific reaction with EBNA-1 was seen in both on immunoblots of whole-cell lysates (Dillner et al., 1987a) and on immunoblots of DNA-binding proteins (Dillner et al., 1984), the 44- and 49-kDa proteins were detected only on immunoblots of DNA-cellulose-binding proteins (Dillner et al., 1984), implying a comparatively lower affinity for the antipeptide antibodies. In subsequent studies we isolated five monoclonal antibodies specific for peptide 107. None of these were reactive either with EBNA-1 or with the 44-and 49-kDa proteins, but instead with two 75- and 80-kDa cellular, nuclear DNA-binding proteins and one 120-kDa cytoplasmic protein. Several additional minor reactivities were also detected (Dillner, Kallin, and Rosen, unpublished observations). Two other studies have reported cellular proteins, which could be possible candidates for cellular proteins encoded by IR3 homologs. Seibl and Wolf, (1985b) used cloned EBV DNA fragments for in vitro-translation studies. Only one EBV DNA fragment, BamK, was able to select mRNAs from EBV-negative cells, Two proteins of molecular weights 92,000 and 84,000 were seen in this in uitro translation with the BamK fragment. No cellular homologies apart from the IR3 have been detected in the BumK fragment (Heller et al., 1982a,b). Luka et al. (1984) analyzed a rabbit antiserum against a fusion protein of P-galactosidase and the IR3. The antibodies were not affinity-purified and were reactive on immunoblots with

110


many proteins both in EBV-infected and EBV-negative cells and stained EBV-negative and EBV-positive cells equally well in the ACIF test. For the immunobloting test, it was possible to absorb the non-EBNA-related antibodies with EBV-negative cells and obtain a serum specific for EBNA-1 on immunoblots (Hennessy and Kieff, 1983). In the ACIF test, however, all fluorescence-reactive antibodies were absorbed upon treatment with EBVnegative cells. This was shown to be due to an antigenic cross-reaction with a 62-kDa cellular protein. Since the 107 peptide sequence is contained within this hybrid protein, our ability to produce EBV-specific antibodies reactive in fluorescence and in native form in solution is in apparent contradiction to the results of Hennessy and Luka. A possible explanation is that the 0-galactosidase part of the hybrid protein influences its protein folding, resulting in that the 107 EBNA-specific Gly-Ala determinant is altered. In summary, three independent studies have reported several apparently different, possibly IR3-related cellular proteins. In view of the presence of one homology on each human chromosome, this is not surprising. An elucidation of whether the immunologically cross-reactive proteins are indeed encoded by homologous sequences has not been performed, and the functional significance of the cross-reactions and homologies is unclear today.

2. Structure and Function of EBNA-1 The complete sequence of EBNA-1 is shown in Fig. 3 . It has been conclusively shown that EBNA-1 is encoded by this particular open reading frame (BKRF1) in the BumK fragment (Hennessy and Kieff, 1983; Dillner et ul., 1984). Analysis of the EBNA-1-coding mRNA by S1 nuclease protection mapping has shown that the EBNA-1 mRNA has a 2.0-kb exon which encompasses the entire BKRFl open reading frame (Weigel and Miller, 1985). Exons of the EBNA-1 mRNA located upstream of the BumK fragment can thus not be represented in the EBNA-1 protein, due to a stop codon early in the 2.0-kb exon. Since there is only one possible initiator codon in this reading frame before the Gly-Na copolymer, this initiator codon must necessarily be the N-terminus of EBNA- 1. The mRNA and the 2.0-kb exon end at a poly(A) addition site a few hundred base pairs 3 ' of the BKRF1 reading frame, indicating that this is the carboxy-terminus of EBNA-1 (Kieff et ul., 1984). Antisera to a 22-amino acid synthetic peptide corresponding to the carboxyterminus of the BKRF1 open reading frame, were reactive with EBNA-1 on immunoblots (J, Dillner and R. Lerner, unpublished observation), confirming that this is the carboxy terminus of EBNA- 1. The structure of the EBNA-1 protein could thus be described as a short N-terminal sequence, followed by a 20- to 45-kDa glycine-alanine copolymer,


111

MET S E R A S P G L U GLY P R O G L Y T H R G L Y P R O GLY A S N GLY L E U GLY G L U L Y S GLY A S P THR S E R GLY P R O G L U GLY S E R GLY G L Y S E R G L Y P R O G L N ARG ARG GLY GLY A S P A S N H I S GLY ARG GLY ARG G L Y ARG G L Y ARG G L Y ARG G L Y GLY GLY ARG P R O G L Y A L A P R O GLY G L Y S E R GLY S E R GLY P R O ARG H I S ARG A S P GLY V A L ARG ARG P R O G L N L Y S ARG P R O S E R C Y S I L E GLY C Y S L Y S G L Y T H R H I S GLY G L Y T H R G L Y A L A G L Y A L A G L Y A L A G L Y G L Y A L A G L Y ALA GLY GLY A L A G L Y A L A G L Y GLY G L Y ALA G L Y A L A G L Y GLY GLY A L A GLY G L Y A L A G L Y GLY ALA G L Y GLY A L A GLY A L A GLY G L Y GLY A L A GLY A L A GLY GLY G L Y A L A GLY GLY A L A GLY GLY ALA GLY A L A GLY G L Y G L Y I A L A GLY A L A GLY G L Y GLY ALA G L Y GLY ALA GLY A L A GLY GLY GLY A L A GLY GLY A L A GLY I G L Y ALA G L Y ALA GLY GLY GLY I A L A GLY A L A GLY GLY GLY ALA GLY GLY ALA GLY A L A GLY G L Y GLY A L A GLY G L Y ALA G L Y l G L Y A L A GLY ALA G L Y GLY GLY ALA GLY ALA GLY GLY ALA G L Y GLY A L A GLY G L Y ALA GLY ALA GLY GLY A L A GLY ALA GLY GLY GLY A L A GLY G L Y A L A GLY GLY A L A GLY A L A GLY GLY ALA GLY ALA GLY GLY ALA GLY ALA GLY GLY ALA G L Y A L A GLY GLY A L A GLY GLY A L A GLY A L A GLY GLY ALA GLY GLY ALA GLY A L A GLY GLY A L A GLY G L Y IALA G L Y ALA G L Y GLY GLY A L A GLY GLY A L A GLY ALA

I

GLY GLY GLY A L A GLY GLY ALA GLY ALA GLY GLY A L A GLY GLY ALA GLY A L A GLY GLY ALA GLY GLY ALA GLY ALA G L Y GLY A L A GLY GLY ALA GLY ALA GLY GLY GLY ALA GLY ALA GLY GLY ALA GLY A L A GLY GLY GLY GLY ARG GLY ARG GLY GLY S E R GLY GLY ARG GLY ARG GLY GLY S E R GLY GLY ARG G L Y ARG GLY GLY S E R GLY G L Y ARG ARG GLY ARG GLY ARG G L U ARG ALA ARG GLY GLY S E R ARG GLU ARG ALA ARG GLY ARG GLY ARG GLY ARG GLY GLU L Y S ARG P R O ARG S E R P R O S E R S E R GLN S E R S E R S E R S E R G L Y S E R P R O P R O ARG ARG P R O P R O PRO GLY ARG ARG P R O P H E P H E H I S P R O VAL GLY GLU A L A A S P T Y R P H E G L U T Y R H I S GLN GLU GLY GLY P R O A S P GLY GLU P R O A S P V A L P R O P R O GLY ALA I L E GLU G L N GLY P R O ALA A S P A S P P R O GLY GLU GLY P R O S E R T H R GLY P R O ARG G L Y GLN GLY A S P GLY GLY ARG ARG LYS L Y S GLY GLY T R P P H E GLY L Y S H I S ARG GLY GLN GLY GLY S E R A S N P R O L Y S P H E GLU ASN I L E A L A GLU GLY L E U ARG A L A L E U L E U A L A ARG S E R H I S V A L GLU ARG T H R T H R A S P GLU GLY T H R T R P V A L A L A GLY V A L P H E V A L T Y R GLY GLY S E R L Y S T H R S E R L E U T Y R ASN LEU ARG ARG GLY T H R ALA L E U ALA I L E P R O G L N C Y S ARG L E U T H R P R O L E U S E R ARG L E U PRO P H E GLY MET A L A P R O GLY P R O G L Y P R O G L N P R O G L Y P R O L E U ARG GLU S E R I L E VAL CYS T Y R P H E MET V A L P H E L E U GLN T H R H I S I L E P H E A L A G L U V A L L E U L Y S A S P ALA I L E LYS A S P L E U V A L MET T H R L Y S P R O A L A P R O T H R C Y S A S N I L E ARG V A L T H R V A L C Y S S E R PHE A S P A S P GLY V A L A S P L E U P R O P R O T R P P H E P R O P R O MET V A L G L U GLY A L A ALA ALA GLU GLY ASP ASP GLY ASP A S P ~ G L YASP GLU GLY GLY ASP GLY ASP GLU GLY GLU GLU GLY G L N

3 FIG.3. Amino acid sequence of EBNA-1 (Baer et al., 1984). The IR3-encoded glycine-alanine copolymer is underlined. The sequences of peptide 107 (major epitope) and peptide 135 (epitope mainly seen in NPC sera) are boxed. Note the highly charged acidic carboxy terminus and the arginine-rich region immediately following the repeat structure.

112


flanked by basic arginine-rich sequences and finally a highly charged acidic carboxy-terminal sequence. The arginine-rich sequence contains many potential cleavage sites for trypsinlike proteases. Proteolytic cleavage has been a major problem in many studies on partial purification ofEBNA-1 (e.g., Sculley et al., 1983). A recent study (Milman et al., 1985)has synthesized a 28-kDa protein in bacteria, corresponding to the highly charged, acidic carboxy-terminal part of EBNA- 1. The protein could be purified to homogeneity by chromatography on phosphocellulose, These authors subsequently showed that this carboxy-terminal protein had three sequence-specific binding sites on the EBV genome (Rawlins et al., 1985). Two of these sites were localized in the BamC fragment, in a region that has been shown to be necessary for EBV plasmid maintenance and is termed oriP (Yates et al., 1984). The oriP consists of two elements located some 900 bp apart. The 3 ‘element contains a characteristic dyad symmetry, whereas the 5 ‘element contains inverted repeat sequences. The oziPrequires the EBNA-1 gene to be also present in order for it to function in the maintenance of the EBV plasmids (Yates et al., 1985), a function that thus apparently is mediated by sequence-specific DNA binding of the EBNA- 1 protein, The oriP has been reported to have enhancer activity, which is dependent on the EBNA-1 protein. The enhancer activity is contained within a 1.6-kb sequence, which contains the inverted repeat but not the dyad symmetry (Reisman et al., 1985; Lupton and Levine, 1985). The EBNA-1 carboxy-terminal part had one binding site in the dyad symmetry and one binding site in the inverted-repeat region. The third EBNA-1 binding site is situated in the EcoRI G 1 fragment. No proteins or DNA-regulatory elements have hitherto been reported in this region. Presumably, each one of the EBNA-1 binding sites on the EBV genome represents a function of EBNA-1. One of these functions is apparently the regulation of plasmid maintenance, probably exerted by the binding to the inverted repeats and the dyad symmetry. It is possible that the requirement of EBNA-1 for the function of the enhancer element is its binding to the inverted-repeat element. In these two respects EBNA-1 seems to be very similar to the SV40 large T antigen, a multifunctional protein that binds to and regulates the SV40 origin of replication and the SV40 enhancer. If the DNA- binding functions of EBNA-1 are mediated by its carboxy-terminal sequence, the function of the huge EBNA-1 glycine-alanine copolymer is puzzling. Recently, several proteins have been described that contain repeated structures, including Plasmodiumfalciparum proteins (Berzins et al., 1986) and eukaryotic RNA polymerase I1 (Corden et al., 1985). The main feature of these repeated structures is that they are highly antigenic. This is in line with our findings on the EBNA-1 repeat structure. A unique feature of the EBNA-1 protein is that it binds diffusely to all the chromosomes, as can be seen by ACIF staining of mitotic EBV-carrying cells. This phenomenon was first described for “EBNA” with EBNA-positive sera (Reedman and Klein, 1973). Utilizing the EBNA-1-specific antipeptide 107


113

antibodies we were able to show that this is specifically valid for the EBNA-1 protein (Dillner et al., 1985a), but not for EBNA-2 (Dillner et al., 1985a) or for EBNA-3 (unpublished observation of J , Dillner and B. Ehlin-Henriksson, utilizing the EBNA-3 staining described in Dillner et al., 1986a). Nothing similar has been reported with other tumor antigens and not even a suggestion has been made as to the functional significance of the EBNA-1 chromosome binding. According to a recent electron-microscopic study, the EBV episomes are associated with the human chromosomes, in random pattern (Harris et a l . , 1985). This could conceivably be mediated by the EBNA-1 protein, e.g., by the binding of its C-terminal part to the EBV genome and the simulataneous binding of an EBNA-1 N-terminal part to a structural chromosomal protein. The chromosomal association of the episomes may be important in securing equal distribution of episomes to the daughter cells.

C. EBNA-2 The first evidence for more than a single nuclear antigen was reported by Strnad et al. (1981). It was noted that two of four EBNA-positive sera detected an additional polypeptide in two of four EBV-carrying cell lines. The polypeptide had an apparent molecular weight of 81,000 and was biochemically distinct from EBNA-1, although both EBNA-1 and the 81-kDa protein bound to dsDNA-cellulose. Hennessy and Kieff (1983) detected an 82-kDa polypeptide in five EBV-positive cell lines, but not in one EBV-negative line. Subcellular fractionation showed that it was a mainly nuclear polypeptide. The coding region for EBNA-2 was originally assigned to the EcoRI Dhetfragment (Hennessy and Kieff, 1983). In a further characterization of this peptide by immunoblotting, we found that EBNA-2-positive sera could be demonstrated in a proportion of EBV-immune sera derived from patients with different EBV-associated diseases (Dillner et al., 1985a). Particularly strong EBNA-2 reactions were found in three acute mononucleosis sera, where maximum activity was detected between 40 and 50 days after onset of disease. We consistently found that three EBV-positive cell lines were negative for the EBNA-2 polypeptide (P3HR-1, Daudi, and Jijoye) (Dillner et al., 1985a). Both P3HR-1 and Daudi carry EBV substrains with 6-kb deletions in their BamWYH fragments (Delius and Bornkamm, 1978; M. Jones et a l . , 1984). The Jijoye cell line has a 2-kb nonhybridizable (with the B95-8 prototype virus) region in the BamYH fragments (King et al., 1982). T o address the question whether the 86-kDa EBNA-2 polypeptide may be partly responsible for the EBNA reaction in the ACIF test, we absorbed EBNA-2-positive sera with the EBNA-2-negative cell lines. All three EBNA2-positive sera were found to contain an ACIF-reactive EBNA antibody which

114


could only be absorbed with EBNA-2-expressing cell lines, but not with EBNA-2-negative cell lines. This second EBNA antibody could stain EBNA-2-expressing cell lines, but not EBNA-2-negative cell lines. Two sera that did not contain antibodies to the EBNA-2 polypeptide in immunoblotting also lacked the ACIF-reactive second EBNA antibody. Several lines of evidence thus indicated that the second EBNA detected by ACIF with preabsorbed sera and the 86-kDa EBNA polypeptide were identical. Since we could not conclusively show this, we suggested the designation “EBNA-2 nuclear antigen” for the second EBNA detected by ACIF and referred to the 86-kDa immunoblotting-detected polypeptide as the “EBNA-2 polypeptide. ” Whereas the staining morphology of EBNA-1-nonGly-Ala (Fig. 2c) is described as “smooth” or “dispersed throughout the nucleus, ” that of EBNA-1-Gly-Ala as “discretely punctate” or “speckled” (Fig. 2b,f), the EBNA-2 staining morphology is best described as “finely granular” (Fig. 4a). The complete DNA sequence of the EBNA-2-associated BamWYH region has been determined (Baer et a l . , 1984). It contains two long (1.1 and 1.6 kb) open reading frames, BWRF12 and BYRF1. A likely promoter sequence was situated shortly 5 ’ of the BWRF12 reading frame, and a likely poly(A) addition site was positioned immediately 3 ’ of the BYRFl reading frame. We selected two \20-residues-long amino acid sequences from each of these open reading frames and synthesized peptides accordingly. Antisera to two of these peptides were found to react with the EBNA-2 polypeptide on immunoblots (Fig. 4b; Dillner et a l . , 1985b). Since these peptides (1 15 and 116) were deduced from the BYRFl open reading frame, the finding provided conclusive evidence that BYRFl encoded EBNA-2 (Dillner et al., 198513). Within a short time span, several articles assigned EBNA-2 to the BamWYH region. Sculley et al. (198413) and Dambaugh et al. (1984) reported the absence of EBNA-2 in P3HR-1 and other BamWYH-deleted cell lines. Hennessy and Kieff (1985) made a heteroantiserum in rabbits against a bacterial fusion protein containing the BYRFl open reading frame, which was shown to be specific for the EBNA-2 polypeptide on immunoblots. Rymo et al. (1985) and MuellerLantzch et al. (1985) reported the production of stably BamWYH-transfected cell lines expressing the EBNA-2 polypeptide and an ACIF-detected EBNA-2 nuclear antigen. The amino acid sequence of the BYRF1 open reading frame predicts a 55-kDa protein with an extremely high proline content (28%). High proline content is known to increase the apparent molecular weight of proteins on SDS gels (Perricaudet et a l . , 1979). The isolation of two BYRF1-containing cDNA clones has shown that the entire EBNA-2 protein is encoded within BYRFl (Sample et a l . , 1986b; Speck et al., 1986). Interesting features of the EBNA-2 amino acid structure, shown in Fig. 5a, include a 26-amino acidslong proline polymer, a 12-amino acids glycine-arginine repeat, and a highly charged, acidic carboxy terminus.


115

FIG 4. (a) EBNA-2 (type A)-specific immunofluorescence. A human EBNA-positive serum has been absorbed with PSHR-1 cells that carry an EBNA-2 deletion mutant strain of EBV. ACIF on Raji cells. The absorbed serum did not stain 4 cell lines carrying EBV with deleted EBNA-2 regions, nor did it react with 12 BL-derived cell lines carrying EBV with the variant EBNA-2 sequence (EBV type B). Twenty-seven lymphoblastoid cell lines were all found to carry EBNA-2 type A and also showed nuclear staining with this antiserum. (Reproduced, with permission, from Dillner et al., 1985a.) (b) Detection of EBNA-2 by immunoblotting. A rabbit antiserum against the carboxy terminus of EBNA-2 (22 residues peptide n : o 115) has been used for staining a 7.5% gel of nuclear extracts from 3 x 10’ cells. EBV-positive cell lines: B95-8 and Raji. EBV-positive cell lines containing deleted BurnWYH regions: P3HR-1 and Daudi. EBV-negative cell line: Ramos. The reaction with the 87-/or 86-kDa polypeptide could be inhibited by addition of free peptide, whereas the reaction with the 135-kDa cellular protein was not inhibited. Four monoclonal antibodies directed against this peptide reacted exclusively with the EBNA-2 polypeptide. The small size variation is due to differences in the length of the EBNA-2 polyproline repeat. (Reproduced, with permission from Dillner et u l . , 1985b.) (c) EBNA-2 is a phosphoprotein. A monoclonal antibody against the EBNA-2 carboxy terminus bas been used for immunoprecipitation of 32P-labeledcell extracts and subsequent autoradiograFhy . EBV-positive cell lines: Raji and B95-8. EBV-negative cell line: Loukes. Note that EBNA-2 migrates as a 103/104 kDa protein on gels with low cross-linking.

116


Fig. 4 (continued)

The synthetic peptide 115, deduced from the BYRF1 carboxy-terminal sequence, was used for mouse immunization and subsequent hybridoma production (Dillner et d.,submitted for publication). Five peptide-specific monoclonal antibosies were isolated. Four of these were reactive with EBNA-2 on immunoblots, but not with EBNA-2 in solution. The fifth monoclonal antibody was only reactive with EBNA-2 in its native form in solution and could be used to immunoprecipitate EBNA-2 from [Y3]methionine or [32P]orthophosphate-labeled cells (Fig. 4c). EBNA-2 was coprecipitated with a 32-kDa protein from 35S-Met-labeledEBV-positive Raji cells, but not from EBV- negative Loukes cells. This finding is of potential interest, since the transforming function of several tumor antigens is associated with the


117

a MET PRO THR PHE TYR LEU ALA LEU H I S GLY GLY GLN THR TYR H I S LEU I L E VAL A S P THR A S P S E R LEU GLY ASN PRO S E R LEU S E R VAL I L E PRO S E R ASN PRO TYR GLN GLU GLN LEU S E R A S P THR PRO LEU I L E PRO LEU THR I L E PHE VAL GLY GLU ASN THR GLY VAL PRO PRO PRO L E U l P R O PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO S E R PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO] GLN ARG ARG A S P ALA T R P THR GLN GLU PRO S E R PRO LEU A S P ARG A S P PRO LEU GLY TYR A S P VAL GLY H I S GLY PRO LEU ALA S E R ALA MET ARG MET LEU T R P MET ALA ASN TYR I L E VAL ARG GLN S E R ARC GLY A S P ARG GLY LEU I L E LEU PRO GLN GLY PRO GLN THR ALA PRO GLN ALA ARC LEU VAL GLN PRO H I S VAL PRO PRO LEU ARC PRO THR ALA PRO THR I L E LEU S E R PRO LEU S E R GLN PRO ARG LEU THR PRO PRO GLN PRO LEU MET MET PRO PRO ARG PRO THR PRO PRO THR PRO LEU PRO PRO ALA THR LEU THR VAL[PRO PRO ARG PRO THR ARG PRO THR THR LEU PRO PRO THR PRO LEU LEU T H R ~ V A L LEU GLN ARG PRO THR GLU LEU GLN PRO THR PRO S E R PRO PRO ARG MET H I S LEU PRO VAL LEU H I S VAL PRO A S P GLN S E R MET H I S PRO LEU THR H I S GLN S E R THR PRO ASN A S P PRO A S P S E R PRO GLU PRO ARG S E R PRO THR VAL PHE TYR ASN I L E PRO PRO MET PRO LEU PRO PRO S E R GLN LEU PRO PRO PRO ALA A I A PRO ALA GLN PRO PRO PRO GLY VAL I L E ASN A S P GLN GLN LEU H I S H I S LEU PRO S E R GLY PRO PRO T R P T R P PRO PRO I L E CYS A S P PRO PRO GLN PRO S E R LYS THR GLN GLY GLN S E R ARG GLY GLN S E R ARG GLY ARC GLY ARG GLY ARG GLY ARG GLY ARG GLY LYS GLY LYS SER ARG A S P LYS GLN ARG LYS PRO GLY GLY PRO T R P ARG PRO GLU PRO ASN THR S E R S E R PRO

S E R MET PRO GLU LEU S E R PRO VAL LEU GLY LEU H I S GLN GLY GLN GLY ALA GLY A S P S E R PRO THR PRO GLY PRO S E R ASN ALA ALA PRO VAL CYS ARG ASN S E R H I S THR ALA THR PRO ASN VAL S E R PRO I L E H I S GLU PRO GLU S E R H I S ASN S E R PRO GLU ALA PRO I L E LEU PHE PRO A S P A S P T R P TYR PRO PRO S E R I L E A S P PRO ALA A S P LEU A S P GLU S E R T R P A S P TYR I L E PHE GLU THR THR GLU S E R PRO S E R S E R l A S P GLU A S P TYR VAL GLU GLY PRO S E R L Y Y ~

ARG PRO ARG PRO S E R I L E GLN]

FIG.5. (a) The amino acid sequenceof EBNA-2, type A (B95-8) (Baer et al., 1984).The polyproline repeat, an internal peptide sequence, and the carboxy-terminal sequence used for generation of monoclonal antibodies are boxed. Note especially (1) the highly charged, acidic carboxy terminus that contains a typical nuclear localization signal (KRPR), (2) the Gly-Arg repeat situated 5 ’ of the carboxy-terminal peptide, and (3) the generally high proline content (28%). @) The amino acid sequence of EBNA-2, type B (AG876) (Dambaugh ct al., 1984). Note that the polyprolinerepeat is much shorter. The region 5 ‘ofthe polyprolinerepeat has good homology to the EBNA-2, type A sequence, as has the carboxy-terminal sequence. The putative nuclear localization signal is conserved.

formation of complexes with cellular proteins, such as the SV40 large T-p53 complex and binding of the polyoma middle T to pp60 “c-m.”

1. Genetic Variation of EBNA-2: EBV Types A and B Two laboratories have reported molecular analyses of the nonhybridizable region in the BamYH fragments. Dambaugh et al. (1984) reported the cloning

118


b MET PRO THR TYR TYR LEU ALA LEU H I S GLY GLY GLN S E R TYR ASN LEU I L E VAL A S P THR ASP

MET S E R GLY ASN PRO S E R LEU S E R VAL I L E PRO THR ASN PRO TYR

GLN

GLU

GLN

LEU S E R ASN ASN PRO LEU I L E GLN LEU GLN I L E VAL VAL GLY GLU ASN THR GLY ALA PRO ALA PRO PRO GLN PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO PRO GLU ARG ARG A S P ALA T R P THR GLN GLU PRO LEU PRO LEU A S P MET ASN PRO LEU GLY S E R A S P ALA S E R GLN GLY

PRO LEU ALA S E R S E R I L E ARG MET LEU CYS MET ALA GLN TYR LEU LEU ARG ASN ALA ARG GLY GLN GLN GLY LEU LEU ARG PRO LEU GLY PRO GLN THR ARG S E R GLN VAL THR LEU GLU ARG GLN PRO VAL H I S ASN PRO ARG GLN GLU ALA PRO I L E I L E LEU LEU GLN S E R PRO ALA PRO PRO ARG PHE THR PRO VAL PRO MET VAL ALA LEU GLY H I S THR LEU GLN PRO THR PRO

PRO PRO ARG PRO THR LEU PRO GLN PRO ARG I L E PRO LEU I L E I L E PRO PRO ARG H I S THR A S N GLN PRO ALA THR THR PRO PRO THR ALA PRO GLN ARG LEU THR

LEU GLY H I S GLN LEU

S E R LEU PRO PRO H I S PRO PRO PRO H I S GLN S E R THR PRO H I S CYS S E R S E R A S P S E R THR GLY LEU PRO PRO PRO PRO THR S E R TYR S E R I L E PRO S E R MET THR LEU S E R PRO GLU PRO LEU PRO PRO PRO ALA ALA PRO ALA H I S PRO LEU PRO GLY VAL I L E TYR A S P GLN GLN ALA LEU PRO PRO THR PRO GLY PRO PRO T R P T R P PRO PRO VAL ARG A S P PRO THR PRO THR THR CLN THR PRO PRO THR ASN THR LYS GLN GLY PRO A S P GLN GLY GLN GLY ARG GLY ARG T R P ARG GLY ARG GLY ARG SER LYS GLY ARG GLY ARG MET H I S LYS LEU PRO GLU PRO ARG ARG PRO GLY PRO A S P THR S E R S E R PRO S E R MET PRO GLN LEU S E R PRO VAL VAL S E R LEU H I S GLN GLY GLN GLY PRO GLU ASN S E R PRO THR PRO GLY PRO S E R THR ALA GLY PRO VAL CYS ARG VAL THR PRO S E R ALA THR PRO A S P I L E S E R PRO I L E H I S GLU PRO GLU S E R S E R A S P S E R GLU GLU PRO PRO PHE LEU PHE PRO S E R A S P T R P TYR PRO PRO THR LEU GLU PRO ALA GLU LEU A S P GLU S E R T R P GLU GLY I L E PHE GLU THR THR GLU S E R H I S S E R S E R A S P GLU GLU ASN VAL GLY GLY PRO S E R LYS ARG PRO ARG THR S E R THR GLN

Fig. 5 (continued)

and DNA sequencing of the BamWYH region of the AG876 virus. The nonhybridizable region of the B95-8 and AG876 viruses had only -25% homology. Both sequences were found to contain a single long open reading frame, BYRF1. The predicted translation products had -50% amino acid homology to each other (Fig. 5a,b). The BYRF1 open reading frame from AG876 hybridized to a similar sequence in the genome of the Herpesuiruspapio, a B-lymphotropic herpesvirus of baboons. The BYRF1 region was also cloned from the EBNA-2-negative Jijoye virus (Adldinger el al., 1985). The DNA sequence of this region was found to be slightly different from that of AG876, but the predicted protein had an identical amino acid sequence (B. G . Barrel1 and G . W . Bornkamm, personal communication). Based on the hybridization of the BYRFl region, Zimber et al. (1986) have proposed that EBV strains be subtyped into type A and type B strains. B95-8 and M-ABA were nominated as prototypes for type A viruses, AG876 and Jijoye for type B viruses.


119

When we used the specific EBNA-2 staining on a total of 27 BL-derived cell lines, we were surprised to find that only 10 lines were EBNA-2 positive. In contrast, all of 27 tested EBV-transformed LCL expressed EBNA-2. An analysis of the DNA of the EBNA-2-negative cell lines showed that they either lacked 6 kb of this region or had a full-sized BamWYH region, but a deletion of the BamHI site between BamY and BamH (Ernberg et al., 1986). Type A-and B-specifc probes were obtained from Dr. G. W. Bornkamm and used to type our BL-derived cell lines; 12 cell lines were found to carry B-type virus. They were all EBNA-2-negative. Zimber et al. (1986) have described 3 additional BL-derived cell lines that carry B-type viruses. The B-type virus has thus so far been detected in 10 isolates from the BL-endemic region of Africa, 2 from Reunion Island, 1 from New Guinea, 1 from North Africa, and 1 from the United States. Zimber et al. pointed out that the distribution of B-type virus correlates roughly with the occurrence of endemic BL. The occurrence of the EBV type B only in BL-derived cell lines and its occurrence in the area endemic for BL, raises the question of whether individuals infected with EBV type B would be at increased risk of developing BL. However, the data so far obtained do not indicate whether or not this is the case, for two reasons: The number of EBV-carrying BL-derived cell lines from nonendemic regions is very small, and no nonmalignant LCL from the BL-endemic region have been available for study. The protein that would be predicted from the B-type (Jijoye) BYRF1 reading frame, i.e., a B-type EBNA-2, has been reported to be a 78- to 80-kDa doublet polypeptide (Rowe et al., 1985). We have recently been able to confirm this finding, using monospecific antipeptide antibodies directed against the predicted Jijoye EBNA-2 protein sequence (Kallin and Dillner, unpublished observations).

2. The Function of EBNA-2 The EBNA-2 coding region BamWYH has been shown to be necessary for EBV transformation. Superinfection of the Raji nonproducer cell line with the nontransforming P3HR-1 virus results in release of recombinant viruses with transforming ability (Fresen et ul., 1978, 1980). All transforming viral isolates had acquired the BurnWYH region (Skare d al., 1985). At least four different EBVcarrying BL-derived lines (PSHR-1, Daudi, Jijoye nude, and Naliaka) have acquired very similar deletions in the BarnWYH region (Delius and Bornkamm, 1978;Jones et a l . , 1984; Ernberg et al., 1986). This finding is interpreted to show that the BamWYH region is necessary only for the initiation of transformation, but not for its maintenance. Since all LCL that we have studied (N = 27) contained an intact BamWYH region (Ernberg et al., 1986), it is conceivable that the BarnWYH region is required only for the proliferation of low-malignant LCL, but

120


that the region is not necessary for the growth of the highly malignant BLderived lines carrying the activated c-myc oncogene. The deletion of this region not only is unnecessary but may impose a selective disadvantage to the BL cell. The P3HR-1 deletion has occurred in vitro, the deletion of Jijoye nude either in vitro or during nude mouse passage, whereas the deletions of Naliaka and Daudi could have occurred either in vitro or in vivo (Ernberg et a l . , 1986). When the BamWYH region is used for transfection of lymphocytes and subsequent EBNA-2 induction, it can be toxic for the recipient cells both in transient and long-term assays (P. Aman, personal communication; J. Dillner, unpublished data). It is conceivable that the BamWYH region may be required for the growth of the primarily infected B lymphocyte, but when the virus-host cell interaction is altered, e.g., after conversion to high-malignant phenotype of the host or in experimental systems such as transfection, the effect of the BamWYH region may be turned into selective disadvantage. One study has indicated that EBNA-2 may confer to transfected rodent fibroblasts the ability to grow in low serum concentration (Dambaugh et a l . , 1986). Since EBV never infects fibroblasts or rodents in vivo it is difficult to interpret these data. In a later study, these authors have transfected human BL-derived cells with a retroviral vector containing an EBNA-2 insert and shown that EBNA-2 induces expression of the B-cell activation-related surface antigen CD-23 (Wang et a l . , 1987). Taken together with the concomitant finding that CD-23 is related to the receptor for the BCGF (Gordon et a l . , 1986a), these experiments have provided the first fundamental piece of evidence as to how EBV immortalizes human B cells,

3. Nuclear Antigens Determined by Simian EBV-like Viruses EBV-like viruses have been isolated from four Old World primate species, namely, Herpesvirus gorilla from gorillas (Gorilla gorilla), H . pan from chimpanzees (Pan troglodytes), H . pongo from orangutans (Pongo pygmaeus), and H . papio from baboons (Papio hamadyas). They are all B-lymphotropic and are able to transform B cells into LCL. They all share 30-40% DNA sequence homology to EBV. They express VCA and EA that are highly cross-reactive between the different species. They also express nuclear antigens (NA), which, however, are only cross-reactive in certain combinations (Gerber et a l . , 1976; Rabin et a l . , 1978, 1980). We have identified the NA of these viruses by immunoblotting (Dillner et a l . , 1987~).Herpesvirus gorilla determines two NA (GONA-1 and GONA-2), none of which is cross-reactive with the EBNA. Herpesvirus pan determines two NA, one of which is present as a doublet (PANA-1A and 1B). Chimpanzee sera did not react with the EBNA, but human EBV-immune serum crossreacted with the higher molecular weight antigen (PANA-2). Herpesvirus pongo


121

determines one NA of 74 kDa (PONA-2). A PONA-2-positive orangutan serum cross-reacted with EBNA-2 (Fig. 6), and a human EBNA-positive serum reacted with PONA-2. In addition, PONA-2 reacted with monospecific antipeptide sera directed against the EBNA-2 carboxy terminus. Herpesvirus pupio determines two NA (HUPNA-1 and HUPNA-2). A HUPNA-2-positive baboon serum cross-reacted with EBNA-2 of both types A and B. In summary, the simian EBV-like viruses also express multiple NA, like EBV. A surprising finding was that the serological cross-reactions were only found with the NA corresponding to EBNA-2. As referred to above, the EBNA-2 coding sequence shows extensive variation between the different species, as evidenced by a low extent of hybridization (Dambaugh et u l . , 1984). In contrast, the IR3 repeat sequence, which for EBV encodes a major epitope of EBNA-1 (Dillner et al., 1984), is highly conserved between EBV and the simian viruses (Heller and Kieff, 1981; Heller et al., 1981). Although we detected NA of molecular weights similar to EBNA-1, we failed to show any evidence of cross-reactivity, neither of simian sera with EBNA-1, nor of human sera with the lower molecular weight simian antigens. In addition, monospecific antipeptide sera directed against the IR3-encoded glycine-alanine copolymer failed to react with the simian antigens. The evolutionary conservation of EBNA-2 epitopes in the absence of conservation of the corresponding DNA sequence indicates that these epitopes of the protein may be functionally important. The evolutionary conservation of the I R 3 repeat region in the absence of conserved epitopes suggests that the I R 3 region has a function primarily at the DNA level.

D. EBNA-3 Miller et al. (1985) reported that three EBNA-positive sera from chronic I M patients lacked antibodies to EBNA-1 as determined by ACIF on Bum K-transfected cells. Immunoblotting showed that these sera did not contain antibodies to the EBNA- 1 polypeptide, but other EBNA-related polypeptides could not be seen. We analyzed these sera for the presence of EBNA antibodies on Raji cells, and for EBNA-1 and EBNA-2 antibodies on transfected lymphoid lines that expressed EBNA-1 or EBNA-2. Two of these sera contained non-EBV-specific antinuclear antibodies and were not studied further. The third serum (WC) was analyzed in detail (Dillner et ul., 1986a). Comparison between EBV-negative cell lines and their in uitro-converted counterparts demonstrated the EBV specificity of the nuclear staining. The WC serum had no EBNA-1 antibodies, and only a faint anti-EBNA-2 reaction was obtained at a 1 : 2 dilution. In contrast, the EBNA staining had a 1 : 80 titer aganst both EBNA-2-negative and EBNA-2-positive cell lines. The ACIF reaction could therefore not be due to the previously described EBV NA. The

122


FIG 6. A nuclear antigen (PONA-2) determined by the orangutan lymphotropic herpesvirus H. pongo: Cross-reaction with EBNA-2. An orangutan serum (1 : 10) has stained an irnmunoblot of a 7.5% gel, low cross-linking, to which ssDNA-binding proteins corresponding to lo’ cells have been applied to each lane. Loukes: EBV-negative cell line. 733/EBV: EBV-carrying cell line, 733/pongo and CP81: H. pongo-carrying cell lines.

new EBNA was designated EBNA-3 nuclear antigen (Fig. 7a). The EBNA-3 staining morphology resembled the ‘‘finely granular” EBNA-2 staining. In addition, some large globular structures were seen (Fig. 7a). Immunoblotting with the WC serum revealed that it reacted mainly with an EBNA-associated 143-kDa (Raji) or 157-kDa (IB4) polypeptide (Fig. 7b). Since we could not conclusively show that the prominent EBNA polypeptide detected in immunoblotting was the same as the EBNA nuclear antigen seen in the ACIF test, we refer to these entities as the EBNA-3 nuclear antigen and the EBNA-3 polypeptide, respectively. Under optimal conditions, the EBNA-3 polypeptide


123

FIG. 7. (a) ACIF for EBNA-3. A human serum (WC) lacking antibodies to EBNA-1 has been used to stain an EBNA-2-negative cell line (P3HR-I). (See text fordetails.) Note that the staining morphology resembles the finely granular EBNA-2 staining. (Reproduced, with permission, from Dillner et a l . , 1986a.) (b) EBNA-3 polypeptide seen by immunoblotting. The WC serum (diluted 1 : 20) has been used for immunoblot of a 7.5% gel, low cross-linking. Raji and IB4, Latently infected EBV-carrying cell lines. BL2 and BL32, EBV-negative BL-derived cell lines. Note that neither EBNA-1 nor EBNA-4 is seen with this serum, and that EBNA-3 is a doublet polypeptide. (Reproduced with permission from Dillner et al., 1986a.)

was resolved into two polypeptides that differed slightly in molecular weight (Fig. 7b) (Dillner et al., 1986a). This may be due to posttransitional modifications. The EBNA-3 polypeptide appeared 2 days after the primary infection of tonsillar B cells (Kallin et al., 1986). It was detected in all 25 EBV-carrying cell

124


lines studied, but not in any of 6 EBV-negative cell lines (Kallin et a l . , 1986; Dillner et al., 1986a). Two EBV-carrying cell lines (Solubo and Rael) were originally recorded as negative for the EBNA-3 polypeptide (Dillner et al., 1986a), but subsequent experiments have shown that they do express the polypeptide, at very low levels (Dillner, unpublished observation). A total of 17 EBV-positive sera and 7 EBV-negative sera were tested for the presence of EBNA-3 antibodies; 8 EBNA-3-positive sera were found. They were all EBVpositive. The size variation of the polypeptide (140-157 kDa) is determined by the infecting strain of EBV (Kallin et al., 1986). The nuclear localization of the antigen was shown both by subcellular fractionation (Kallin et a l . , 1986) and immunofluorescent staining (Dillner et al., 1986a). Its association with the latent transforming infection is shown by the fact that it was detected in cell lines with no or only very low proportion of lytically infected cells (20% virus-producing cells (Kallin et al., 1986), and because it was detected in 100% of cells by immunoflourescence (Dillner et al., 1986a). Hennessy et al. (1985), reported a new EBNA species with a molecular weight of 140,000. Since these authors used the same WC serum as we did, they have clearly detected the same polypeptide. In another study, these authors reported the production of a fusion protein of P-galactosidase and part of an open reading frame (BERFl) from the BamE fragment. The rabbit antiserum against this protein was specific for EBNA-3 on immunoblots (Hennessy et al., 1986), and thus shows that a part of EBNA-3 is encoded by BERFl . Bodescot et al. (1986) have isolated a cDNA clone, T2, which starts in BamC, continues through BamW and Y (Where it contains the EBNA-5 coding sequences), makes a long splice to the BamL fragment, splices into BamE where it stops at a poly(A) site shortly after BERFl . A continuous long open reading frame with an initiator codon at the beginning, starts in the BamL fragment (BLRF3 open reading frame) and splices into the BERFl open reading frame in BamE. The BLRF3 + BERFl open reading frame would be predicted to code for a 110-kDa protein, which is in reasonably good agreement with the observed molecular weight of EBNA-3 of 143,000. It cannot, however, be excluded that BERFl might encode several transcripts, and it is therefore not conclusively shown that the BLRF + BERF1-encoded protein is identical to EBNA-3.

E. EBNA-4 Optimization of the immunoblotting conditions for high molecular weight proteins led to the discovery of a fourth EBNA polypeptide with a molecular weight between 148,000 and 180,000 (Fig. 8; Kallin et al., 1986). The same lines of evidence that permitted the identification of the EBNA-3 polypeptide


125

FIG. 8. Detection of EBNA-1, 2, 3, and 4 in 10 infectious mononucleosis-derived lymphoblastoid cell lines. Human EBNA-positive serum K F used for immunoblotting of a 9 % polyacrylamide gel, low crosslinking. Note that the size of all four EBNAs varies between different cell lines. EBNA-3 is defined as the polypeptide which reacts with the WC serum, and is in this blot the lower of the high-molecular-weight bands. Note that EBNA-4 is frequently present in two molecular weight species, i.e., the 166,000 and 180,000 species present in C123 and M105. Also note the high level of expression of EBNA-3 and EBNA-4 in the lymphoblastoid cell lines. (Reproduced from Kallin ct al., 1986.)

as a new EBNA could also be applied for the EBNA-4 polypeptide (Kallin et al., 1986). Several lines of evidence indicate that EBNA-3 and EBNA-4 are not related. The apparent molecular weight (148,000-180,000 for EBNA-4; 140,000-157,000 for EBNA-3) varied independently for EBNA-3 and EBNA-4. The opposite would have been expected if they had variable regions in common. Two sera reacted well with EBNA-3 but not at all with EBNA-4, showing that EBNA-3 contains epitopes different from those of EBNA-4 (Kallin d al., 1986). An antiserum to a BERFl-Ogalactosidase fusion protein

126


reacted with EBNA-3 but not with EBNA-4 on immunoblots, showing that these BERFl epitopes are not included in EBNA-4 (Hennessy et a l . , 1986). EBNA-4 is expressed as two molecular-weight species, 180,000 and 166,000 in B95-8 virus-carrying cell lines. (Kallin et a l . , 1986). None of > 40 EBNA-4positive sera was specific for either component, suggesting that the two EBNA-4 species are immunologically related. IB4 cells carry the B95-8 genome but express only the 166,000 MW EBNA-4 species, suggesting that the expression of the two EBNA-4 species may be influenced by the host cell (Kallin et a l . , 1986). We were recently able to produce a cell line stably expressing EBNA-4, by transfection of Cos cells with a pSVgpt vector with an insert containing the right-hand part of BarnE, containing the long open reading frames BERF2a, BERFILb, BERF3, and BERF4. The transfected cell line expresses a full-size EBNA-4 protein of 180 kDa (A. Ricksten, L. Rymo, and B. Kallin, unpublished observations). Interestingly, only the upper molecular weight species of EBNA-4 was expressed. Immunoaffinity-purified antibodies to the 180-kDa EBNA-4 protein specifically reacted with the 180-kDa comigrating protein expressed in cells transfected with the right-hand partial fragment of BamE (Kallin, unpublished). Conversely, antibodies to a bacterial fusion protein encompassing >50% of the BERF4 open reading frame identified the 166-kDa EBNA-4 protein in IB4 cells (Fincke and Kallin, unpublished). Bodescot and Perricaudet (1986) have isolated a cDNA clone, T4, that contains a continuous long open reading frame spliced together from the BERF3 and BERF4 open reading frames. This cDNA clone may thus be related to either of the two EBNA-4 molecular-weight species. In an effort to identify a rheumatoid arthritis NA, Sculley et al. (1984) reported the existence of two high-molecular-weight proteins present in EBVtransformed cell lines and detected only with rheumatoid arthritis patient sera. Although we had used sera from healthy EBV-immune donors for the detection of EBNA-3 and EBNA-4, an exchange of reference sera showed that the two rheumatoid arthritis-high-molecular-weight antigens are identical to EBNA-3 and EBNA-4 (Kallin et al., 1986).

In Vitro DNA-Binding Properties of the EBNAs The affinity of EBNA 1-4 for double- and single-stranded DNA was studied by DNA-cellulose chromatography. EBNA-1 had a strong affinity for ssDNA, in accordance with another study (Hearing and Levine, 1985). The binding of EBNA-1 to dsDNA-cellulose was heterogeneous. The bulk of EBNA-1 showed an affinity similar to ssDNA, but a small proportion of EBNA-1 had low or no affinity to dsDNA. This suggests that EBNA-1 is present in two different modified forms in the cell, and that this modification


127

affects its dsDNA-binding ability, but not its affinity for ssDNA. It is known that EBNA-1 is phosphorylated (Hearing and Levine, 1985; Dillner et al., submitted. Under optimal conditions for gel electrophoresis and immunoblotting, it is possible to resolve the EBNA-2 band into three different subspecies that differ slightly in molecular weight (Kallin et al., 1986). Interestingly, the two subspecies with somewhat lower molecular weights bind more weakly to ssDNA than the EBNA-2 species of the highest molecular weight. The two smaller subspecies were eluted from dsDNA-cellulose with 0.25 M NaCl, whereas the larger subspecies was eluted with 0.6 M NaCl (Kallin et al., 1986). This suggests that the DNA-binding ability of EBNA-2 is regulated by some posttranslational modification that affects its molecular weight slightly. We have been able to show that EBNA-2 is phosphorylated (Fig. 4c; Dillner et al., 1987a). Phorphorylation has been reported to be important for DNA-binding ability and gene regulation in several other systems (Krebs and Beavo, 1979). EBNA-3 had poor affinity to dsDNA but bound to ssDNA. EBNA-2 and EBNA-4 bound to both dsDNA and ssDNA, but had an increased affinity for ssDNA. ssDNA-binding proteins are frequently involved in DNA replication and genetic recombination (Kornberg, 1980). It is conceivable that these EBNA proteins may play a functional role in the replication of viral DNA.

F. EBNA-5 Bodescot et al. (1 984) isolated a cDNA clone, T1, from the latently infected Raji cell line (Bodescot et al., 1984). Its sequence was compared to the DNA sequence of the B95-8 virus (Baer et al., 1984). It was found to contain one exon from the BamH fragment, three exons from the BamY fragment, and two exons from each of the internal repeats (BamW). The cDNA clone contains a long open reading frame which, if translated, would be predicted to code for a 261-amino acid polypeptide with a repetitive element of 66 amino acids from each internal repeat. The 5 ' part of this mRNA was not included in the cDNA clone, and it was therefore not known how may repetitive elements would be included in the protein or if it would contain unique sequences 5 ' of the internal repeat. The predicted amino acid sequence is rich in arginine, glycine, and proline. The basic p l and high proline content of the predicted polypeptide would be in line with the likely properties of a DNA-binding protein. In order to investigate the possible existence of this protein, we synthesized four peptides from the deduced amino acid sequence, immunized rabbits, and isolated the corresponding peptide-specific antibodies (Dillner et al., 1986b). Three different antipeptide antibodies reacted with a 46-kDa nuclear polypeptide present in the latently infected IB4 cell line (EA expression 0.05% of cells) (Fig. 9a). Among 20 EBNA-positive human sera, 3 contained

128


66-

*

-92

92m 0

*

-70 -64--

-

-66

45-

FIG. 9. (a) Identification of a fifth EBNA by irnrnunoblotting with antisera against three different peptides deduced from the EamWYH region. IB4, FEBM, PJ, M , M108, E95-C139, and M3-JIJ, EBV-carrying lyrnphoblastoid cell lines (LCL). Raji, Bja-B95-8, Ramos-HRlK, E95-D-Ramos, Namalwa, and AG876, EBV-carrying BL-derived cell lines. Bjab, EBV-negative BL-derived cell line. (Blot A) Antipeptide 186 serum; (Blot B) antipeptide 188 serum; (Blot C) antipeptide 189 serum. (Reproduced from Dillner et al., 1986b.) @) Induction of multiple EBNA-5 polypeptides by EBV infection of tonsillar B cells. Tonsillar B cells were infected with B95-8 EBV and harvested at different times after infection. Immunoblot of a 10% gel, stained with antipeptide 186 serum. IB4, EBNA-5-positive LCL. (Reproduced from Dillner et al., 1986b.)

antibodies against this polypeptide. It is a nuclear polypeptide that binds more strongly to the nucleus than any of the other EBNA. It is not even solubilized by salt concentrations as high as 2 M NaCI. Many BL-derived cell lines appear to be negative for this polypeptide. Of 11 tested BL-derived cell lines only in 1 line (Namalwa) could an EBNA-5 protein be demonstrated. Ten of 11 LCL tested contained EBNA-5 polypeptides varying in molecular weight between 22,000 and 70,000 (Fig. 9A); Dillner et d.,1986b). Six EBNA-5 proteins, ranging in molecular weight between 41,000 and 70,000 are induced 1-2 days after infection of tonsillar B cells with the B95-8 strain of EBV (Fig. 9b). It is known that the B95-8 strain of virus is heterogeneous with respect to the number of internal repeats (Heller et al., 1981). Since EBNA-5 is predominantly encoded by the internal repeats, the induction of multiple molecular-weight forms could be due to this heterogeneity. Another possibility is that an active EBNA-5 promoter is present in each of the internal repeat


b

129

DAYS POST INFECTION

Fig. 9 (continued)

fragments. The EBNA-1 mRNA has recently been found to be partially homologous to the T1 cDNA clone of Bodescot et al. The peptide sequences used for identification of EBNA-5 are present also in this cDNA clone (Speck and Strominger, 1985). The designation “EBNA-5” is used to refer to the EBV-encoded proteins identified by our three antisera, directed against peptide sequences deduced from the cDNA clone of Bodescot et al. Since the EBNA-5 coding sequence is contained within the transformation-associated B a m W Y H region of EBV DNA, it is conceivable that this polypeptide is involved in the process of EBV transformation. The existence of EBNA-5 has recently been confirmed by other laboratories. Speck et al. (1986) translated a B a m W Y H region cDNA clone in vitro

130

JOAKIM DIL,LNER AND BENGT KALLIN

and showed that the translated product was precipitated by an EBV-immune serum, and Wang el al. (in press) made a heteroantiserum against a 6-galactosidase-EBNA-5 fusion protein and showed that this serum recognized EBNA-5 on immunoblots. Both Sample et al. (1986b) and Speck d al. (1986) have isolated putative EBNA-5-encoding cDNA clones that continue to the 5 ' end of the message. The complete EBNA-5 amino acid sequence, as predicted from the Speck et al. cDNA clone, is given in Fig. 10. The transcript comes from a promoter in the BamW (internal repeat) region. The first splice generates an ATG, which is the only ATG present in the whole EBNA-5 open reading frame. After the EBNA-5 stop codon, the message splices to the BYRF1 open reading frame, where it possibly encodes EBNA-2 As has been discussed, potential EBNA-5-coding sequences are present both in the EBNA-1, EBNA-3, and EBNA-4 messages (Bodescot and Perricaudet, 1986). However, in these instances the message originates in BamC , and no ATG has been found in the putative EBNA-5-encoding open reading frame of these messages. The BamW contains an enhancer element that is only functional in lymphoid cells (L. Rymo, persofial communication). The data thus V

MET GLY A S P ARG S E R GLU GLY PRO GLY PRO THR ARG PRO GLY PRO PRO GLY I L E GLY PRO GLUVGLY PRO LEU GLY GLN LEU LEU ARG ARG H I S ARG S E R PRO S E R PRO THR ARG GLY GLY

GLN GLU PRO ARG ARG VAL ARG ARG ARG VAL LEU VAL G L N ~ G L N GLU GLU GLU VAL VAL SER GLY SER PRO SER GL$PRO

ARG GLY ASP ARG SER GLU GLY PRO GLY PRO THR ARG PRO GLY

PRO PRO GLY I L E GLY PRO GLUvGLY PRO LEU GLY GLN LEU LEU ARG ARC H I S ARG S E R PRO S E R PRO THR ARG GLY GLY GLN GLU PRO ARG ARG VAL ARG ARG ARG VAL LEU VAL GLN(GLN GLU GLU GLU VAL VAL SER GLY SER PRO SER GL+PRO

ARG GLY ASP ARG SER GLU GLY PRO

GLY PRO THR ARG PRO GLY PRO PRO GLY I L E GLY PRO GLUVGLY PRO LEU GLY GLN LEU LEU ARG ARG H I S ARG S E R PRO S E R PRO THR ARG GLY GLY GLN GLU PRO ARG ARG VAL ARG ARG ARG VAL LEU VAL G L N [ G L N GLU GLU GLU VAL VAL SER GLY SER PRO SER GL$PRO

ARG GLY

A S P ARG S E R GLU GLY PRO GLY PRO THR ARG PRO GLY PRO PRO GLY I L E GLY PRO GLUVGLY PRO LEU GLY GLN LEU LEU ARG ARG H I S ARC S E R PRO S E R PRO THR ARG GLY GLY GLN GLU PRO ARG ARG VAL ARG ARG ARG VAL LEU VAL GLN IGLN GLU GLU GLU VAL VAL S E R GLY S E R PRO SER GL+PRO

LEU ARG PRO ARG PRO ARG PRO PRO AIA

ARGVSER

LEU ARG GLU TRP LEU

LEU ARG I L E ARG ASP H I S PHE GLU PRO PRO THR VAL _TH-R-TER-GY-ARRG-GLJ _SE< VAL-

_ I L-E

_GLu _GLu _CL_V_CLJ

_AsP-GLI

rYR_

ASP

FIG. 10. The EBNA-5 amino acid sequence (IB4 cells). v, Splice sites. Note especially that ( 1 ) almost the entire protein is made up of a repeat unit, (2) the initiator methionine is spliced together from two different exons, (3) the long stretches of basic residues that could conceivably be involved in ensuring nuclear localization, and (4) the highly charged, acidic carboxy-terminus that is strikingly resernblant to the acidic carboxy termini of EBNA-1 and EBNA-2 (cf. Figs, 3 and 5). The positions of three peptide sequences used to generate EBNA-5-specific antibodies are marked by boxes (n:o 186), underlining (n:o 188), or a dotted line (n:o 189).


131

suggest that only when the BamW enhancer is used can the EBNA-5 ATG be spliced together and EBNA-5 be translated.

G. THELATENT INFECTION MEMBRANE PROTEIN (LMP) The first demonstration of the existence of an EBV-determined MA in virally transformed, virus nonproducer cells came from the demonstration that EBNA-free extracts of membranes from EBV-positive, but not from EBV-negative, cells were able to trigger the release of lymphokines from leukocytes of EBV-seropositive, but not from EBV-seronegative donors, as measured by a leukocyte migration inhibition (LMI) assay (Szigeti et al., 1984). Antibodies to the LMP were regularly seen in sera from NPC and BL patients, but not regularly in the sera of healthy EBV-seropositive donors. The mRNA from the BamN fragment is the most abundant EBV-specific mRNA in the growth-transformed cell. It has been estimated that virus nonproducer IB4 cells contain 50-60 copies per cell of this mRNA, in contrast to only 3 copies of EBNA-1 mRNA per cell. This allowed detailed S1 nuclease mapping of this mRNA. Three potentially protein-coding exons have been mapped (Fennewald et al., 1984). A specific serum was produced by Hennessy et al. (1984) by the fusion of the third exon of the BamN gene to /3-galactosidase and immunization of rabbits with the fusion protein. The serum identified a 58-kDa protein on immunoblots. Subcellular fractionation showed that it was present in the plasma membrane fraction. The same molecular weight, 58,000, was obtained by in vitro translation of the BamN mRNA, indicating the absence of major posttranslational modifications. There are no sites for N-linked glycosylation in the predicted amino acid sequence. Utilizing the fusion protein and the corresponding antiserum, Sulitzeanu et al. (1986) were able to show that the MA detected by LMI and the M W 58,000 BamNencoded membrane protein were identical. LMP contains a hydrophilic N-terminus, followed by six strongly hydrophobic regions interspersed with short hydrophilic regions, followed by a long hydrophilic carboxy terminus. It was proposed that the LMP spans the plasma membrane six times and is positioned with the N- and C-termini on the outside of the plasma membrane. The latter suggestion was based on the finding that the monospecific antisera to the C-terminus reacted with the LMP only on fixed, not on living cells (Hennessy et al., 1984). The model was confirmed by the finding that chymotrypsin treatment of living cells cleaved the LMP only in a chymotrysin cleavage site predicted to be on the outside of the plasma membrane, but not in the cleavage sites predicted to be on the inside of the membrane (Liebowitz et a l . , 1986). A monoclonal antibody (S12) to the LMP-/3-galactosidase fusion protein has been isolated (Mann et a l . , 1985). This antibody detected LMP both in the

132


plasma membrane and the mitochrondria. Upon immunofluorescence on fixed cells, it appeared that at least the main proportion of the LMP is present on the plasma membrane. The LMP immunofluorescence shows a typical “patching” pattern (Liebowitz et al., 1986). The S12 monoclonal antibody detects the LMP as a doublet protein in most cell lines, in some cell lines even as a triplet protein (Fig. 11; M. G. Masucci, personal communication). Two monospecific antipeptide sera directed against the carboxy- terminus of LMP and against an internal hydrophilic sequence react only with the highest molecular weight species (Fig. 11) (Dillner, unpublished). Transfection of Rat1 fibroblasts with a retroviral vector containing the LMP gene induced the ability of this cell line to form tumors in nude mice (Wang et a l . , 1985). This tumorigenic conversion was not seen on NIH 3T3 fibroblasts, and the natural host for EBV, the B lymphocyte, was not analyzed.

136

138

s12

s12

FIG. 11. Detection of L M P by immunoblotting. KK and IARC 171, EBV-carrying lymphoblastoid cell lines. Daudi, EBV-carrying, but LMP-negative BL-derived cell line. Ramos, EBV-negative BL line. Immunoblots of 10% gels stained with (left to right) antipeptide 136 serum, directed against the L M P carboxy terminus; antipeptide 138 serum, directed against an internal hydrophilic L M P sequence; and (two right-handed panels) monoclonal antibody S12 (Mann et a l . , 1985) directed against an L M P fusion protein. Note that the 60- and 55-kDa species are only detected with the S12 monoclonal antibody.


133

Hudson et al. (1985a) reported that a partially homologous mRNA, including only the third LMP exon, was expressed late during the viral lytic cycle of EBV. This lytic protein was also predicted to be a membrane protein. This could have interesting implications for the humoral and cellular immune recognition of LMP, since antibodies to the lytic MA are known to be a characteristic finding of BL patient sera (Klein et al., 1966). Indeed, the original LMI article reported that antibody levels to LMP were higher in BL patient sera than in healthy seropositive individuals (Szigeti et a l . , 1984). Modrow and Wolf (1986) synthesized a peptide corresponding to the N-terminal part of LMP. Antisera to this peptide immunoprecipatated LMP from lymphoblastoid cell lines, but not from most BL cell lines. It was suggested that LMP would exist in a truncated form in BL-derived cell lines. Another explanation for these findings is that LMP is down-regulated in most BL lines. The latter interpretation is supported by a recent immunoblotting study (Rowe et a l . , 1986) and by studies in our own laboratory. Two antipeptide sera against carboxy-terminal sequences are only able to see the LMP in LCL and only the upper (major) species (Fig. 1 1 ; Dillner, unpublished).

The L YDMA Problem The name ‘‘EBV-determined lymphocyte-detected membrane antigen” (LYDMA) was proposed for the antigen(s) expressed on EBV-infected B lymphocytes, that serve as a target for EBV-specific lysis mediated by cytotoxic T cells (Svedmyr and Jondal, 1975). The initial experiments were performed with lymphocytes from I M patients and the targets were cultured cell lines. It was proposed that in the primary EBV infection, IM, the proliferative capacity of EBV-infected B cells is controlled by cytotoxic T cells. Subsequently, the cytotoxic experiments used T cells derived from EBV- infected cultures in which B cells were present, or T cells stimulated by autologous LCL. These T cells, and clones derived from them, often showed an M H C class I-restricted selectivity for lysis of LCL, but not mitogen-induced B blasts (review by Rickinson and Moss, 1983). However, it is not known whether the LCL are lysed on the basis of recognition of an EBV-encoded antigen. Following the discovery of the LMP, it was suggested that this protein may be identical to the LYDMA (Kieff et al., 1984). Thorley-Lawson and Israelsohn (1987) synthesized three peptides from the LMP sequences predicted to be on the outside of the plasma membrane. One of these (a 10-residue peptide) was able to induce EBV-specific cytotoxic T cells in an in vztro-stimulation assay. It can be concluded that this peptide sequence contains a determinant that can induce LYDMA reactivity, at least in uitro. However, in an effort to correlate the ability of BL-derived cell lines to be lysed by

134

JOAKIM DILLNER AND RENGT KALLIN

cytotoxic T cells with the expression of latent proteins in these cell lines, Rowe et al. (1986) found that expression of L M P did not correlate with the expression of LYDMA of these cell lines. Sulitzeanu et al. (1986) showed that an LMP-0galactosidase fusion protein was capable of eliciting an EBV-specific LMI, an assay which measures the release of lymphokines by sensitized T cells. Similar results could be obtained with two 22-amino acid synthetic peptides deduced from the L M P amino acid sequence (Dillner et al., 1987b). It must be emphasized that even though this assay does demonstrate a T-cell response to the LMP, the sensitization of the T lymphocytes is likely to occur after lysis of EBV-infected cells. Both the L M P fusion protein and the LMI-reactive synthetic peptides correspond to regions of L M P which are supposed to be on the inside of the cell membrane. Similarly, an EBV-specific T-cell response can also be triggered by two latently expressed nuclear proteins, both in the form of purified EBNA-1 (Sulitzeanu et al., 1987), and in the form of synthetic peptides deduced from the EBNA-1 or EBNA-2 amino acid sequences (Dillner et al., 1987b). The data on the influenza virus nucleoprotein of Townsend et al. (1985) have proven that an antigen need not to be exposed on the surface of the cell in order to evoke a cytotoxic T-cell reaction. Considering this fact, the data on the identity of LYDMA suggest that it may be a composite of several of the proteins of the transformed cell, with the amendment that the L M P is the only protein so far shown to be able to take part in this reaction.

H. THEDIFFERENTIAL EXPRESSION OF T H E TRANSFORMATION-ASSOCIATED PROTEINS During the course of I M , antibodies to “EBNA” and to both EBNA-1 and EBNA-2 appear 13-2 months after onset of clinical IM symptoms (Henle et al., 1974, Henle et al., 1986). However, whereas antibodies to EBNA-1 show a continuous increase and remain at their maximum titer throughout the life of the EBV-infected individual, the antibodies to EBNA-2 (Henle et al., 1986) show a maximum titer during the acute stage of I M some weeks after onset of disease. These findings not only may be diagnostically useful; they also suggest that the expression of EBNA-2 is up-regulated during primary infection of B cells, after which it is maintained at a steady, much lower level. Similarly, we have found that the expression of EBNA-5 is higher during primary infection than in established LCL (Dillner et al., 1986b). The expression of EBNA-5 also appears to be higher in LCL than in most BL-derived lines (Dillner et al., 1986b). Using a quantitative radioimmunoassay based on a monospecific 0-galactosidase-LMP fusion protein antiserum, Hatzubai et al.


135

(1987) were able to show that the expression of LMP is 810-fold elevated in B1-derived cell lines as compared to LCL (with few exceptions). Similarly, as determined by immunoblotting, the expression of EBNA-2, -3, and -4 is frequently lower in BL-derived cell lines than in LCL (Kallin et a l . , 1986; Ernberg et a l . , 1986; Dillner et al., 1986a). Similar conclusions have been arrived at by immunoblotting studies from other laboratories (Rowe et a l . , 1986). An explanation for these findings is not obvious, but we will suggest a hypothesis. EBNA-1 seems to be different from the other proteins of the transformed cell. It is not expressed differentially in different types of cell lines. This contrasts to the other proteins of the transformed cell for which the rule of expression (though not without exceptions) can be summarized as follows: primary infection > LCL > BL-derived cell line. The function of EBNA-1 has been determined. Its role is the maintenance of the EBV genome in its nonintegrated episomd from (Yates et al., 1984), 1985; Rawlins et a l . , 1985), a function that obviously is vital to EBV and could not possibly be down-regulated. At least in some in uitro systems, LMP and EBNA-2 can have transforming functions (Wang et a l . , 1985; Dambaugh et al., 1986). An everintriguing question of EBV tumor virologists over the years has been, How can EBV, which has such potent in uitro-transforming abilities, persist for many years in the majority of humanity without causing disease? During acute mononucleosis, up to 18% of the infected individual’s B cells express EBNA and have proliferative capacity (Robinson et a l . , 1980), but no EBVinfected B cells have ever been detected in the blood of healthy carriers. The question we want to pose is, Could it be that EBV-infected cells are transformed only during primary infection when they express all the transformationassociated proteins? The attack of sensitized T cells or humoral interfering factors may trigger a down-regulation of the transformation-associated proteins and the return of the infected cell to a nontransformed, truly latent state where only viral genome maintenance (EBNA-1 expression) is required, and at a minimal level. Some support of this hypothesis is derived from the study of the expression of transformation-associated proteins in BL cell lines (Kallin et al., 1986; Dillner et al., 1986a,b; Ernberg et a l . , 1986; Rowe et a l . , 1986; Kallin and Dillner, unpublished). This can be staged in three different classes: class 1, expression of EBNA-1 only (with minimal or no expression of the other transformation-associated proteins); class 2, expression of EBNA- 1 and some other transformation-associated protein at LCL-like levels, the other proteins being low in expression, and class 3, full LCL-like expression of all transformation-associated proteins. Class 1 is seen only in a few select BL-derived cell lines such as Rael. According to our hypothesis, such a cell line would not be transformed, were it not for the 8:14 translocation and the c-myc activation, and this class is thus comparable to the EBV-negative BL in etiology. Class 2 covers many of the

136


BL-derived cell lines, An example would be the Salim Mwalim cell line, which has full expression of EBNA-4 and EBNA-1, but comparatively low levels of the other proteins, or Jijoye, which has full expression of EBNA-1 and LMP, but low expression of the other proteins. This type would represent a partial escape from the presumed nontransformed, resting state, and EBV may or may not contribute to the growth potential of those cell lines. Class 3 would represent the true EBV-transformed state seen in the LCL and is among BL lines exemplified by Namalwa or Raji. In our hypothesis this state would only exist in uivo during the primary infection or in immunodeficient individuals, such as in XLP or chronic malaria infection. Rickinson and co-workers have studied the surface marker characteristics of BL-derived cell lines (Rowe et al., 1985). While BL biopsies and newly established cell lines show surface markers reminiscent of resting B cells, the cell phenotype of EBV-carrying lines changes toward a more activated state after in vitro propagation. This phenotypic change is sometimes accompanied by an increase in expression of transformation-associated proteins, from only EBNA-1 in the biopsies and newly established cell lines to a complete LCLlike expression in long-term established cell lines (Rowe et al., 1986; M . Rowe and A. B. Rickinson, personal communication). In this context, it should be noted that an LCL is established by in uitro EBV infection of B cells, in the absence of sensitized T cells or humoral interfering factors. “Spontaneous outgrowth” from the blood of EBV-seropositive individuals does not occur in the presence of virus-neutralizing antibodies, and is therefore postulated to occur by a two-step mechanism. After in uitro cultivation of B cells from EBV-seropositive donors, infectious virus is released which infects and transforms previously uninfected B cells (Rickinson et al., 1974, 1977). It is therefore uncertain whether the “latent, growth-transforming” infection of the LCL type is existent in uivo in healthy carriers. A confirmation of this hypothesis would require the identification of EBVinfected B-cells in uzuo, a laborious task for which there has not been sufficient interest in the past. It is our hope that this hypothesis will promote such studies. I.

ARETHERE MORETRANSFORMATION-ASSOCIATED PROTEINS YETTO BE IDENTIFIED?

Speck and Strominger (1985) have isolated a cDNA clone corresponding to the EBNA-1 mRNA. The mRNA was found to have several exons, localized widely (> 70 kilobases) apart on the EBV genome. In the BumW repeat region it has exons in common with the EBNA-5 mRNA. Two 5 ‘exons in the Bum E fragment were found to contain two open reading frames with initiator codons close to their 5 ’ ends. It was pointed out that for polycistronic mRNAs where the 3 ’ end is translated, the 5 ’ initiator codons were also utilized, as a


137

rule. The molecular weights of the two predicted polypeptides would be 8000 and 14,000, respectively. Hudson et al. (1985b) reported two new latent state mRNAs mapping to the BamC fragment. An analysis of the DNA sequence of this fragment revealed that it was possible that highly spliced mRNAs are transcribed from this region that contain long open reading frames. Both predicted proteins exhibit several N-linked glycosylation sites and hydrophobic regions in line with their being membrane proteins. Early studies by Kieff and colleagues reported latent mRNAs mapping to this fragment (Kieff et a l . , 1982), but these have not been reported in more recent reviews (Kieff et a l . , 1984). Bodescot and Perricaudet (1986) reported the isolation of a cDNA clone with similar 5 ' structure in the BamWY region as the EBNA-1, EBNA-2, EBNA-3, and EBNA-5 mRNAs. This mRNA continues to the BamH fragment, where it contains a long open reading frame. Since this cDNA clone was isolated from B95-8 cells that express early and late EBV antigens in %2 % of the cells, it is not known if this predicted protein is latent or lytic. Two large fragments, Bum0 and BamP, together comprizing 10% of the EBV genome, contain two long open reading frames with coding capacity of 350 and 130 kDa, respectively (Baer et al., 1984). Although it seems obvious that these open reading frames must code for protein, no protein, either lytic or latent, has been found which could match these two open reading frames. A possible explanation is that they represent low-abundance latent-state mRNAs.

K. SUMMARY The demonstration of multiple different proteins encoded by EBV in the transformed B cell and the characterization of these proteins has evoked many questions and some intriguing insights. The evidence linking the BamWYH region to EBV transformation (Skare et al., 1985) and the link between EBV transformation and the BCGF (Gordon et a l . , 1984a) may both be elucidated by the induction of the BCGF receptor-related antigen CD-23 by the BamWYH-encoded EBNA-2 protein (Wang et al., 1986). The evidence that a first activating signal is transmitted by the virion particle (Walker et al., 1986; H u et al., 1986) may have an explanation in the finding of the LMP protein in the virion (Mann et al., 1985) and the finding of transforming ability of the LMP protein in a fibroblast system (Wang et al., 1985). The fact that EBV persists as episomes in EBV-transformed cells (Lindahl et a l . , 1974) has been elucidated in molecular terms with the finding that EBNA-1 regulates the oriP plasmid maintenance region and actively prevents integration (Yates et a l . , 1984, 1985; Rawlins et al., 1985). However, the many findings on the EBV proteins of the transformed cell leave many questions unanswered. What is the

138


role of the Gly-Ala copolymer of EBNA-1 and its homologs? The EBNA-1 chromosome binding? The type A and type B EBNA-2 proteins and their relation to EBV-associated diseases? EBNA-3 and EBNA-4? The bicistronic EBNA-5 and EBNA-2 message? The host cell phenotype-dependent expression of several of these pro.teins? Clearly, the fundamental characterization of these proteins has paved the way for the study of their interaction with the host cell genes and an understanding of the transforming ability of a unique human tumor virus.

V. Viral Proteins In Virus-Producing Cells Study of the viral productive cycle and its proteins has been hampered by the lack of permissive cell system and the difficulty in obtaining virus preparations free of cellular contaminants. Although there is compelling evidence that EBV in vivo replicates in epithelial cells, it has not been possible to study the replication of EBV in this type of cells. Knowledge of EBV replication and the proteins of the replicative cycle has been derived from the study of a few BL cell lines and the EBV-transformed marmoset B-cell line B95-8. In these cell lines a fraction of the cells, usually 1-5 '70,express early and late viral antigens. The proportion of virus-producing cells can be significantly increased by any of three procedures: superinfection of Raji cells with virus from P3HR-1 cells (Bayliss and Nonoyama, 1978), or treatment with tumor promoters (zur Hausen et al., 1978; Eliasson et al., 1983), or by n-butyrate (Luka et al., 1979). Thus, in contrast to most other mammalian viruses, replication can only be observed upon infection of cells already containing the viral genome. Superinfection-induced virus replication is completed within 24 hr (Bayliss and Wolf, 1981; Feighny et a l . , 1980, 1981). Reduction of the multiplicity of the infecting virus leads to ineffective induction of late antigens and prolongation of the cycle completion (Kallin and Klein, 1983). Induction of virus replication by tumor promoters or n-butyrate leads to less synchronous induction involving u p to 80% of the cells, depending on the type of cell line, culture conditions, and type of inducer used. The viral cycle is completed within 2-5 days, as a rule (Fig. 12). Both types of systems produce modest amounts of infectious virus. The complexity of viral proteins detected in the respective systems is similar. There are only modest strain variations and, with few exceptions, different viral isolates express an identical set of viral proteins. The relative lack of genetic variation in the early and late viral proteins is in sharp contrast to the proteins expressed in transformed cells where virtually each virus isolate can be distinguished from others on the basis of the size of the viral proteins. Most proteins in transformed cells are in part encoded by repeat sequences. Superinfection is well suited for study of the coordination of viral protein synthesis, whereas induction by chemical means provides a


139

FIG.12. Time course of the EBV viral cycle in n-butyrate-induced PSHR-1 cells. At indicated time points, 5 x lo6 cells were removed and labeled with [3sS]methionine.Viral polypeptide synthesis was monitored by precipitation with the EBV-immune serum used in Fig. 13. Lane C is a control precipitate from uninduced cells.

reliable and inexpensive source of virus mRNA and proteins for biochemical studies.

A.

DETECTION, CLASSIFICATION, AND NOMENCLATURE OF THE PRODUCTIVE CYCLE PROTEINS

The serological classification of virus antigens in the productive cycle of

EBV is based on different fluorescent techniques. In virus-producing cells human sera recognize three classes of antigen: the VCA, the EA, and the MA. By variation of the fixation conditions, the EA can be subdivided into two subclasses: EA-D (Diffuse) and EA-R (restricted to the cytoplasm). The MA can be demonstrated on the surface of virus-producing cells. This simplified antigen classification served extremely well for the analysis of disease-related

140


patterns regarding predominant antibody specificity and antibody subclasses (for review see Henle and Henle, 1979). Today fluorescent-antibody techniques still are important in clinical practice. Biochemical analysis of viral proteins in virus-producing cells has mainly been studied by radioimmunoprecipitation of proteins from extracts of cells treated with chemical inducers (Kallin et a l . , 1979; Mueller-Lantzsch et al., 1980). Alternatively, immunoblotting or direct analysis with metabolic labeling followed by gel electrophoresis of whole-cell extracts could be applied to monitor the synthesis of viral antigens (Sculley et al., 1985; Bayliss and Wolf, 1981). Some 35 viral proteins in the size range from 350,000 to 18,000 Da have been identified in infected cells by any of the three methods described. By the application of inhibitors of DNA synthesis, such as araC or PAA, a segregation of early and late proteins could be achieved. The majority of proteins detected in virus-producing cells are early, although the antibody titers of human sera usually have far higher antibody titers to the VCA (late) antigens (Fig. 12). However, two of the five most abundant proteins detected are late. The different viral polypeptides are designated by their molecular weight, and no common nomenclature has been established except for the virus envelope proteins (Thorley-Lawson et al., 1982). Comparison of data from different laboratories is therefore difficult. In the following we focus on proteins which are associated with two important steps in the life cycle of EBV: the viral genes involved in the activation of the viral cycle, and the early genes associated with replication of the viral genome and the nucleic acid metabolism. For other groups of proteins there is either a scarcity of data or, as in the case of the virus envelope proteins, the area has been extensively reviewed previously (Thorley Lawson et al., 1982).

B. THEEBV GENEMAP Analysis of the EBV DNA sequence has resulted in the identification of > 80 major open reading frames. Fifteen of these reading frames were classified as coding for late proteins (Baer et a l . , 1984). The virus particle consists of %30 different proteins (Dolyniuk et a l . , 1976). With few exceptions, structural polypeptides belong to the group of late proteins. Efforts to obtain gene maps for the early and late genes have been carried out by selection of mRNA by immobilized EBV DNA fragments and subsequent translation of selected mRNA in vitro. Up to 100 viral proteins have been mapped by this procedure (Hummel and Kieff, 1982; Cohen et al., 1984; Seibl and Wolf, 1985). With respect to the major proteins the three reports are consistent, but for the majority of proteins significant discrepancies are observed, both regarding the coding capacity of the different fragments, and the molecular weights of proteins coded for by the fragments. The gene map (Fig. 1) represents an attempt to compile data from a large number of reports and to


141

assign the gene to a specific open reading frame (Baer et al., 1984) where this has been feasible. In some cases detailed mapping of small genome fragments has been done by various gene transfer experiments and transcription analysis. The application of monoclonal antibodies has also contributed to make mapping data consistent. A significant number of viral genes, particularly those of viral enzymes, have been identified by their homology with genes of herpes simplex virus, or HSV (Baer et al., 1984). In three instances, naturally occurring mutants of EBV have been helpful in the definition of functional regions of the viral genome. Even if there is a modest number of genes identified so far, it is very clear that early and late genes are interspersed throughout the viral genome. In most cases the productive-cycle genes are constituted by a single open reading frame with the promoter region closely upstream of the structural gene. Interestingly, the right-hand part of the genome contains a cluster of genes homologous to genes of HSV (Fig. 1).

C. EARLYGENESAND PROTEINS 1. Viral Genes and Proteins Related to Activation of the Productive Cycle Immediate early (IE) genes are operationally defined as a subset of early viral genes expressed in the absence of ongoing protein synthesis. They can be studied by application of reversible inhibitors of protein synthesis such as cycloheximide. Following the removal of cycloheximide, actinomycin D is added to block further transcription. Under these conditions, IE gene products accumulate whereas early and late genes are inactive. IE proteins of HSV are required for the coordinate expression of early and late genes. At least one of the five HSV-1 IE proteins has been shown to be a transcriptional activation factor (Beard et al., 1986). Since EBV replication in BL cells only takes place in cells already expressing significant numbers of genes, definition of IE genes involves some difficulties on the level of terminology. Superinfection of Raji cells in the presence of cycloheximide has been studied in several reports. Following release of the cycloheximide block, Feighny et al. (1981) detected three polypeptides (1 17, 62, and 48 kDa, respectively), whereas Bayliss and Wolf (1981) detected seven polypeptides (120, 110, 105, 102, 63, 54, 49, and 48 kDa), of which the 120 and 102 kDa species were induced without superinfecting virus. In the latter report, all polypeptides but the 120-kDa species could be immunoprecipitated with polyvalent human serum. Due to the presence of the resident genome, Bayliss and Wolf used the nomenclature “primary phase” for the proteins detected following cycloheximide treatment. The primary phase proteins could be subdivided into two classes by the addition of the arginine analog canavanine. In the presence of this drug, only three proteins (120, 102, and 49 kDa) were detected.

142


From these studies it is clear that the class of IE proteins of EBV may be as complex as in HSV-infected cells (Honess and Roizman, 1974). The molecular weights of these potential IE antigens are different from those of the six proteins expressed in transformed cells. The only exception would be LMP, which has a molecular weight close to 63,000. Data on transcription of the viral genome in cycloheximide-treated Raji cells suggests that the BamF and M fragments are potential coding regions for immediate early genes. Also, in early superinfected cells abundant transcripts are encoded by BamM (Sample et al., 1984). Further transcriptional analysis of the BamM fragment reveals five transcripts. The three leftward transcripts (4, 2.65, and 1.9 kb) have a common 3 ’ terminus but different cap sites. One of the two rightward transcripts is a 1.9-kb spliced message, whereas the other 2.0-kb message is unspliced (Sample et al., 1986a). This fragment has three major open reading frames (Baer et al., 1984). The monoclonal antibody R 3 precipitates 44- and 47-kDa polypeptides following in vitro translation of mRNA selected by hybridization with BamM DNA (Pearson et al., 1983). The 47-kDa protein appears to be a presursor for the 48- to 56kDa ladder of phosphoproteins, whereas the 44-kDa protein does not seem to be posttranslationally processed, as demonstrated by a pulse-chase experiment (Fig. 13). The proteins precipitated by the R 3 antibody have been mapped by gene transfer to the BMRFl reading frame (Cho et al., 1985b). The BMRFl reading frame encodes a major 60-kDa and two minor 45- and 50-kDa proteins (Cho et al., 1985a). The nature of the posttranslational modification resulting in the extensive size heterogeneity is unknown. The promoters for the BMRFl and the BMLFl genes are both activated by treatment with butyrate in transfected cells (Cho et al., 1985a). It is likely that the 48-49 and 62-63 kDa proteins classified as IE gene products (Feighny et al., 1981; Bayliss and Wolf, 1981) are encoded by these two open reading frames in BamM. It is notable that at least the heterogeneous proteins encoded by BMRFl are abundantly expressed and these proteins, as analyzed with biochemically purified proteins reactive with the R 3 antibody, consitute the major component of EA-D (Dolken et al., 1986). These proteins are perhaps the most immunogenic of all early and late proteins and it is possible that the modification leading to the size heterogeneity may be related to the immunogenic properties. The R 3 antibody cross-reacts with a family of cellular proteins around 7OkDa (data not shown). Two groups of cellular proteins known to have a similar magnitude of size variation have been described. One group is a family of 24- to 30-kDa ssDNA-binding proteins from calf thymus which stimulate the activity of DNA polymerase a. The other group is a family of 30- to 40-kDa proteins that are constituents of 40s heterogeneous nuclear RNA particles, The two groups of proteins share antigenic determinants (Valentini et al., 1985).

T H E EPSTEIN-BARR V I R U S PROTEINS

143

FIG 13. T h e R 3 family of proteins, the BamM-encoded major immunogenic protein of the diffuse early antigen complex (EA-D), is a phosphoprotein that is posttranslationally modified from a 48-kDa precursor molecule. (Left panel) Immunoprecipitates obtained with human EBVimmune serum (lanes A1-A4) and R 3 monoclonal antibody (lanes B1-B4) from induced P3HR-1 cells labeled with [35S]methionine.Precipitates in lanes A1 and B1 were from cells labeled from 20 to 26 hr postinduction. Lanes A2 and B2 were from cells pulse-labeled for 15 min. Lanes A3, B3, A4, and B4 were labeled as for A2 and B2, but chased for 1 hr (A3 and B3) or for 4 hr (A4 and 84). (Right panel) Lanes A l , B1, and B2 are the same as in left panel. Lanes AP and BP were from induced P3HR-1 cells labeled with [3*P]orthophosphate and precipitated with human serum (AP) or R 3 monoclonal antibody (BP).

Another line of investigation originates from studies of defective DNA from P3HR-1 virus and the ability of this DNA to activate the replication of viral DNA in trans. Subclones of P3HR-1 cells produce virus containing heterogeneous DNA. This type of defective viral genomes is much more efficient in the induction of EA expression in Raji than are subclones lacking defective DNA (Rabson et al., 1983). These defective genomes include sequences from three areas of the standard EBV P3HR-1 genome. Sequences from the BamW fragment have been rearranged to generate a palindrome Uenson et al., 1986; Sample et al., 1986a). It has been concluded that the sequences from BamM, which are represented in the rearranged DNA, are not likely to be involved in the transactivation process (Sample et al., 1986a).

144


Instead, sequences from the BamZ fragment, which are present in the rearranged DNA, have been strongly implicated for role in the activation of virus replication in Raji cells (Countryman and Miller, 1985; Takada et al., 1986). The latter observation fits very well with the observation that the EBV genome of Raji cells has a part of the BamZ fragment deleted (Polack et a l . , 1984). In this context it should be recalled that efficient chemical induction of EA in Raji cells requires a combination of both n-butyrate and tumor promoters (Eliasson et a l . , 1983). Thus, whatever viral genes are required to bypass the restrictions of expression of early viral genes in Raji cells, these functions could most likely be provided by cellular genes induced by the two drugs. Countryman and Miller (1985) have been able to demonstrate that transfection of an EBV-carrying cell line with rearranged viral DNA including sequences from the BamW and Z fragments results in the induction of EA expression from the resident viral genomes. The transfected fragment was found to encode a 33-kDa protein. Based upon similar experiments, Takada et al. (1986) concluded that the BZLFl open reading frame encoded the protein needed for transactivation. In virus-producing cells a 32-kDa protein has been mapped to the BamZ fragment (Seibl and Wolf, 1985a). Even if the exact number and genomic localization of IE EBV genes still is a controversial matter, the three genes encoded by the BamM and Z fragments all encode transactivators which are likely to be functionally related to activation of virus replication in one way or another (Countryman and Miller, 1985; Lieberman et al., 1986; Wong and Levine, 1986). The biological effect of the BamW + Z fragment strongly suggests that the most important IE gene is encoded by BamZ. It is therefore surprising that it has not been possible to demonstrate expression of this gene product in cycloheximide-treated cells, either on the level of transciption or on the protein level.

2. Viral Enzymes and Proteins Associated with Replication Ofthe Viral DNA This group of viral proteins includes EBV proteins with sometimes very close functional and genetic homology with proteins of other herpesviruses, particularly HSV-1. O n the level of genetic homology, the EBV DNA polymerase and the EBV ribonucleotide reductase (RR) exhibit the strongest homologies with HSV (Baer et a / . , 1984). The genes for the EBV thymidine kinase (TK) and the EBV alkaline exonuclease show less overall homology but with clusters of conserved sequences (Littler et al., 1986). The major DNA-binding protein of EBV is an early 135-kDaprotein (review by Ernberg and Kallin, 1984; Figs. 12 and 13), mapped to the BALF2 reading frame (Hummel and Kieff, 1982; Cohen etal., 1984; Seibl and Wolf, 1985a) and is homologous with the major DNA-binding protein (ICP 8) of HSV-1 (Quinn and McGeoch, 1985).


145

a. Viral Enzymes. The best-studied of the EBV enzymes is the DNA polymerase. The EBV DNA polymerase gene is encoded by a single open reading frame: BALF5 (Baer et al., 1984). From the size of the homologous sequence the predicted size of the protein is 113.4 kDa (Baer et al., 1984; Quinn and McGeoch, 1985), whereas a major protein which copurifies with the enzyme has an amino acid composition very similar to the predicted protein and migrates as a 110-kDa protein (Kallin et al., 1985). Certain areas of the EBV DNA polymerase gene are homologous to the DNA polymerase locus of pox, adeno, and cytomegalovirus (Earl el a l . , 1986). A protein with a molecular weight of 105,000-110,000 has also been mapped to the BamA fragment by hybrid-select translation in vitro (Seibl and Wolf, 1985b). The biochemical properties of the EBV DNA polymerase are readily distinguished from its cellular counterparts. Thus identification of the EBV enzyme in vitro is based mainly on the following three characteristic properties: (1) stimulation by ammonium sulfate, (2) preferential utilization of synthetic templates, and (3) the differential sensitivity to certain drugs like the pyrophosphate analogs, PAA and PFA, and to acyclovir triphosphate (Miller et al., 1977; Ooka et al., 1979; Kallin et al., 1985). With respect to these three properties, the EBV and the HSV DNA polymerases are very similar. The HSV enzyme is associated with a 150-kDa protein (Powell and Purifoy, 1977), whereas the EBV counterpart is associated with a 110-kDa major protein and 66- and 51-kDa minor proteins (Kallin et al., 1985). The latter enzyme has not been obtained homogeneously purified and thus it has not been possible to determine if the enzymatic activity resides within a single polypeptide chain. Exposure to high-salt conditions or pH > 8 usually results in rapid inactivation of the enzyme, suggesting that the enzyme stability may be associated with a dissociable protein. A 45-kDa protein stimulating the enzymatic activity of the EBV DNA polymerase has been isolated (Chiou et al., 1985). The HSV DNA polymerase is associated with 3 ' to 5 ' exonuclease. A similar exonuclease has not been described for the EBV enzyme. However, it is likely that the EBV DNA polymerase has an associated exonuclease, as several reports show that the EBV enzyme copurifies with significant nuclease activities (Ooka et al., 1979, 1984; Kallin et al., 1985). As the viral DNA polymerase is a prime target for antiviral therapy, further investigations are clearly required to find out more about its function. The most abundant nuclease of EBV-producing cells is associated with a 70-kDa molecule (Ooka et al., 1984; Kallin et a l . , 1985). This enzyme has properties very similar to the HSV alkaline exonuclease (Costa et d . , 1983). Its function is unclear, but it is possible that it provides nucleotide precursors by degradation of cellular DNA (Feighny et a l . , 1981). The alkaline exonuclease may also be involved in genetic recombination. The gene encoding the EBV

146


exonuclease may be found in the BamB fragment (J. Arrand, personal communication). Antibodies to the EBV alkaline exonuclease are preferentially found in sera of NPC patients (Ooka et al., 1984; Chen, 1985). It is notable that the numerous proteins homologous to HSV are clustered at the righthand part of the EBV genome. It has been shown that the BALF4 open reading frame, which is flanked by the gene for the major DNA-binding protein and the DNA ploymerase gene, is homologous to the HSV glycoprotein B gene (Pellett et al., 1985). Ribonucleotide reductase (RR) catalyzes the conversion of ribonucleotide diphosphates to deoxynucleoside diphosphates by substitution of the 2 '-hydroxyl group of the ribose moiety for a hydrogen atom. This enzyme is responsible for the synthesis of precursors of DNA and is in all cells a tightly regulated enzyme (for a review, see Thelander and Reichard, 1979). Cellular enzymes of both bacteria and higher cells contain two subunits responsible for catalysis and regulation, respectively. Two open reading frames (BORF2 and BcRF1) have been shown to be homologous to the two subunits of HSV ribonucleotide reductase (Baer et a l . , 1984). The predicted sizes of the EBV proteins are 93 and 34 kDa, respectively. The EBV ribonucleotide reductase has not been isolated and nothing is known about how it is expressed. The most well studied of all HSV enzymes is the thymidine kinase (TK). It has been very difficult to show convincingly that EBV-infected cells express a T K that is readily distinguishable from cellular enzymes (Roubal et al., 1981). The strongest indication of the existence of a virus-coded T K has been the fact that EBV replication is sensitive to drugs that require a viral T K for their phosphorylation-activation (Ooka et al. , 1984; Anderson et al., 1986). Recently, gene transfer experiments have verified that the EBV genome encodes a T K gene. This enzyme is encoded by the BXLF1 open reading frame, has a good overall homology with the HSV-1 T K gene, and cross-reacts with monoclonal antibodies to HSV T K (Littler et al., 1986). This reading frame has a coding capacity for a 67-kDa molecule (Baer et al., 1984). The EBV protein detected following in vitro transcription-translation had a molecular weight of 70,000 (Littler et a l . , 1986). The HSV T K has a molecular weight around 40,000. A fusion protein of the EBV gene expressed in Escherichia coli could complement a T K deficiency in a strain of E. coli (Littler et a l . , 1986). 6. DNA Replication Proteins. The major early protein has a molecular weight of 135,000 and binds well to both ssDNA and dsDNA in vitro (Roubal et a l . , 1981; Sugawara et a l . , 1982). Apart from being the major DNA-binding protein, the 135-kDa early protein has numerous similarities with the ICP 8 major DNA-binding protein of HSV-1, as discussed previously (Kallin and Klein, 1983; Kallin, 1983; Ernberg and Kallin,


147

1984). Both the EBV 135-kDa protein and ICP 8 of HSV-1 seem to be required for replication of the viral DNA. In Raji cells, where part of its structural gene (BALF2) and some of the upstream sequences are deleted (Polack et al., 1984), synthesis of this protein cannot be induced by chemicals and neither late proteins nor virus DNA synthesis can be demonstrated (Kallin and Klein, 1983). Recently the concept that EBV 135-kDa early protein is homologous to ICP 8 was confirmed by DNA sequence analysis (Quinn and McGeoch, 1985). Although the function of ICP 8 is not completely clarified, it is known to keep DNA in an extended configuration (Ruyechan, 1983). Most likely ICP 8 and EBV 135-kDa are functionally related to the E. coli ssb protein and the bacteriophage T 4 p32 gene product (Kornberg, 1980).

3. Major Immunogenic Early Proteins Although EBV-producing cells contain an abundance of early proteins, very few have been studied in detail. Several of the early proteins are very abundant in virus-producing B cells. In particular the 135-, 85-, and 48- to 56-kDa proteins are of interest for the EBV serology since they are the major antibody targets of human EBV-immune sera. The 48- to 55-kDa family of early polypeptides discussed above constitutes a major component of the diffuse subspecificity of the EA complex (EA-D). These proteins can also readily be detected by immunoblotting with human sera (Sculley et al., 1985). The 85-kDa early protein, which forms filament structutes in the cytoplasm of infected cells, is the main target for EA-R antibodies (Luka et al., 1986). The gene encoding this polypeptide has not been identified. The 135-kDa early protein that constitutes 1% or more of the total proteins of virus-producing cells seems to contribute little to either EA-D or EA-R staining, although this antigen is the major EA detected by immunoprecipitation with human sera (Kallin et al., 1979; Figs. 12 and 13).

D. PROTEINS OF THE VIRUS PARTICLE There are less than a handful of reports on the polypeptide composition of the EB virion. Dolyniuk et al. (1976) described a total of 33 polypeptides associated with virions purified from P3HR-1 cells. Three polypeptides (160, 152, and 47 kDa) were especially abundant. Differential solubilization with detergents suggested that the nucleocapsid was composed of seven polypeptides. It is now well established that the major late antigen of virusproducing cells is a polypeptide of about 155 kDa (Fig. 12). The 145-kDa virus envelope protein and the 155-kDa polypeptide constitute the VCA complex in virus-producing cells (Kallin et a l . , 1979). The 155-kDa

148


polypeptide has been assigned to the BCRFl reading frame of the BamC fragment (Hummel and Kieff, 1982; Cohen et al., 1984; Seibl and Wolf, 1985b; Baer et al., 1984). Monoclonal antibodies to the 155-kDa major capsid protein are available (Vroman et al., 1985).

Virion Envelope Proteins Proteins of the virion envelope are natural targets of neutralizing antibodies. The restricted host range of the virus is intimately associated with the properties of the envelope proteins. As a result, the virion envelope proteins have been intensely studied. The developments until 1981 have been excellently reviewed by Thorley-Lawson et al. (1982), and we will only briefly discuss recent developments. The most abundant antigen is two immunologically cross-reactive glycoproteins gp 350/250 (Thorley-Lawson et a l . , 1982; Kallin et al., 1979). These proteins are both encoded by the BamL fragment (Hummel et al., 1984; Fig. 12). Translation of differentially spliced transcripts yields two polypeptides with molecular weights of 135,000 and 100,000 (Beisel et al., 1985). Following translation, N-linked and 0-linked sugars are sequentially added to the molecule, to yield two large glycoproteins with molecular weights of %350,000 and 220,000 (Edson and Thorley-Lawson, 1983). Glycosylation intermediates can sometimes accumulate in infected cells. In marmosets gp 350 is the predominant species, whereas in human cells gp 220 is the most abundant (Edson and Thorley-Lawson, 1981). Due to differential splicing, an I R sequence is only present in the larger 350-kDa glycoprotein (Beisel et al., 1985). Numerous studies have demonstrated that antibodies to gp 350/220 are neutralizing (Thorley-Lawson et al., 1982; Hoffman and Cheng, 1979; Wells et al., 1982). A somewhat less abundant glycoprotein (gp 85) is unrelated to the gp 350/220 glycoproteins and seems to be derived from a 75-kDa precursor molecule (Edson and Thorley-Lawson, 1981, 1983). The function of this protein and its coding sequence has not been defined. The BALF4 open reading frame may encode a protein that is structurally related to the glycoprotein B of HSV-1 (Pellett et al., 1985). The HSV gpB is implicated in the internalization of HSV virions and thus it is possible that the EBV BALF4 protein has a similar function. The predicted size of the BALF4 glycoprotein is similar to that of the gp 85 precursor; there is no experimental evidence for an identity between these two proteins, however. A 140-kDa unglycosylated protein is a major constituent of the virus particle (Dolyniuk et al., 1976; Edson and Thorley-Lawson, 1981). This protein appears to be encoded by the BCRFl open reading frame of the BamC fragment (Hummel and Kieff, 1983; Cohen et al., 1984; Seibl and Wolf, 1985b). Recently it has been shown that the cell surface protein of transformed cells


149

(LMP) is present in the virion envelope (Mann et a l . , 1985). This protein is the only one of the proteins of transformed cells known to be affected by inducers of virus replication. The expression of LMP is increased manyfold in TPAtreated cells (M. Rowe, personal communication). The presence of LMP on the surface of recently infected cells may be important for the immunoresponse to EBV infected cells.

VI. Summary and Future Perspectives The rapidly increasing knowledge about molecular biology of EBV and in particular expression of EBV in B lymphocytes evokes many questions. The number of proteins expressed in transformed cells far exceeds our previous expectations. Genes expressed in the virus-transformed B cells are usually referred to as “latent.” The origin of this designation probably relates to a comparison with Herpes simplex virus, which establishes latency in neurons. However, this comparison seems increasingly irrelevant, particularly as it is by no means certain that latency of EBV infection takes place in B cells. The complexity of expression with well over half of the viral genome transcribed also does not justify the present terminology, The complex expression of the EBV genome in transformed B cells is likely to reflect a functional diversity of the various expressed proteins. Unlike most other transforming DNA viruses, EBV transforms resting cells, and for this reason, multiple functions may have to be provided by the virus that would otherwise be present in proliferating cells. The transformation of B lymphocytes bypasses several normal restriction points in B-cell growth control (Gordon et al., 1986b). Binding of the virus to the C3D receptor in itself is able to transduce a first signal sufficient to activate the B cell, but not to force the cells to replicate (Hu et al., 1986; Walker et al., 1986). This first activation step is seen with UV-inactivated virus. The nontransforming virus (P3HR-1) also induces cellular RNA synthesis. For the transition from G I to S phase an intact transforming virus is required. For the latter step the area of the viral genome encompassing the EBNA-2 and EBNA-5 coding sequences has a key function (Skare et al., 1985). The observation that EBNA-2 can induce expression of the CD-23 growth factor receptor (Wang et al., 1987) seems to fit well with the previous observation that EBV-transformed cells are dependent of an autocrine growth factor for their in uitro growth (Blazar et al., 1983; Gordon et a l . , 1984). This finding fits well with the finding that only receptor-expressing cells become immortalized (Thorley-Lawson and Mann, 1985). Apart from the function of EBNA-1 and EBNA-2, we have very few clues to the function of the other genes expressed in transformed cells. One or more of these proteins may function in keeping the B cell frozen in a stage of differentiation that makes it suitable as a host for the virus. One or more of the NA may be functionally associated with the

150


turning on of the cellular DNA replication. The intriguing observation that LMP is able malignantly to transform rat fibroblasts (Wang et al., 1985) is difficult to ascertain, since fibroblasts (and rodents) are not susceptible to EBV infection. The progress made in our understanding about the EBV-B-cell relations is in sharp contrast to the nearly complete lack of data about the interaction between EBV and epithelial cells as well as the in viuo interaction between virus production in various epithelial tissues and generation of infected B cells. EBV has a dual, very narrow tropism, dictated by the availability of the C3D receptor on the target cell. The life of EBV in epithelial cells might be very similar to that of papilloma viruses. It is possible that EBV replication is associated with the differentiation stage of the epithelial cell (Wolf et al., 1973, 1981). Since the virus can be routinely isolated from throat washings of infected persons lacking symptoms of active infection (Yao et a l . , 1985), it is likely that epithelial cells are the exclusive source of virus production. Thus, the function of B cells in the life cycle of EBV and the transformation by EBV of these cells is hard to understand, since there is no obvious selective force involved for the establishment of the B-cell tropism. Yet, the interaction between EBV and B cells bears every sign of being evolved under very stringent conditions. In spite of the tremendous amount of studies, we know nothing about the role of the B-cell tropism in the life cycle of EBV. This question may be too Aristotelian to promote relevant experiments, but it clearly exposes the limitations of our knowledge of the biology of EBV. The life of EBV in epithelial cells is intimately associated with a field of study initiated almost 15 years ago. The role of EBV in NPC has not been elucidated further since it was first demonstrated that EBV plays a leading role in the development of the disease (Klein, 1979). Although NPC is a major health problem in many parts of the world, little has emerged that defines the nature of interaction between the virus and the tumor cells. The viral genes expressed in NPC are likely to be different from those of transformed B cells. In part, the lack of data is due to the fact that epithelial cells are difficult to grow in uitro. As has been pointed out by Baer et al. (1984), two large open reading frames (BPLF1 and BOLF1) that together constitute nearly a tenth of the viral genome, do not seem to be associated with virus production or with transformation of B cells. This white spot on the EBV gene map may be related to the much-neglected life of EBV in epithelial cells.

ACKNOWLEDGMENTS We are grateful to Drs. Perricaudet, Kieff, Strominger, Thorley-Lawson, and Zeuthen for communication of preprints, and to Dr. G . Klein for comments on the manuscript. This work was supported by National Cancer Institute Grant 5RO1-CA 28380-03 and by the Swedish Cancer Society. The authors are recipients of fellowships from the Cancer Research Institute and from the Concern Foundation.


151

REFERENCES Adldinger, H . K., Delius, H., Freese, U., Clark, J., and Bornkarnm, G . W. (1985). Virology 141, 221-234. Andersson, J . , Britton, S., Ernberg, I., Andersson, U., Henle, W., Skoldenberg, B., and Tisell, A. (1986).J.Z n ~ c t .Dis. 153, 283-290. Arrand, J . R . , and Rymo, L. (1982).J . Virol. 41, 376-389. Arrand, J. R . , Ryrno, L., Walsh, J. E., Bjork, E., Lindahl, T . , and Griffin, B. E. (1981).Nucleic Acids Res. 9, 2999-3014. Baer, R . , Bankier, A. T., Biggin, M. D., Deininger, P. L., Farrell, P. J., Gibson, T. J., Hatfull, G., Hudson, G . S., Satchwell, S. C., Seguin, C., Tuffnell, P. S., and Barrell, B. G. (1984). Nature (London) 310, 207-21 1. Bar, R. S., Delor, J., Clausen, K. P., Hurtubise, P., Henle, W., and Hewetson, J. F. (1974). New Engl. J . Med. 290, 363-367. Baron, D.,and Strominger, J. L. (1978).J.Biol. Chem. 253, 2875-2881. Bayliss, G. J., and Nonoyama, M. (1978). Virology 87, 204-207. Bayliss, G . J., and Wolf, H . (1981).J. Cen. Virol. 56, 105-118. Beard, P., Taber, S . , Wilcox, K. W., and Pizer, K. J . (1986). Roc Nutl. Acud. Sci. U.S.A. 83, 4016-4020. Beisel, C., Tanner, J., Matsuo, T . , Thorley-Lawson, D., Kezdy, F., and Kieff, E. (1985).J. V i ~ o l .54, 665-674. Berzins, K., Perlmann, H., Wohlin, B., Carlsson, J., Wahlgren, M . , Udomsangpetch, R., Bjorkman, A,, Patarroyo, M. E., and Perlmann, P. (1986).Roc. Nufl.Acud. Sci. U . S . A . 83, 2677-2681. Bhat, R., and Thimmappaya, B. (1983). Proc. Nutl. Acud. Sci. U.S.S. 80, 4789-4793. Blazar, B. A., Sutton, L. M., and Strome, M. (1983).Cancer Res. 43, 4562-4568. Bodescot, M., and Perricaudet, M. (1986). Nucleic Acids Res., 14, 7103-7114. Bodescot, M., Brinson, O., and Perricaudet, M. (1986). Nucleic Acids Res. 14, 2611-2620. Burkitt, D. P. (1962).Nature (London) 194, 232-234. Burkitt, D. P. (1963).Int. Rev. Exp. Puthol. 2, 67-138. Chang, R. S. (1980). “Infectious Mononucleosis.” G Hall, Boston. Chen, J. Y. (1985).J . Med. Virol. 16, 99-105. Chiou, J . T., Li, J. K . K., and Cheng, Y. C. (1985).Proc. Nutl. Acud. Sci. U . S . A . 82,5728-5731. Cho, M.S.,Jeang, K. T . , and Hayward, D. S. (1985a).J. Virol. 56, 852-859. Cho, M.S.,Milrnan, G., and Hayward, S. D. (1985b).J. Virol. 56, 860-866. Cleary, M. L., Warnke, R . , and M a r , J. (1984). New Engl. J. Med. 310, 477. Cleary, M. L.,Epstein, M . A., Finerty, S . , Dorfman, R. F., Bornkamrn, G. W., Kirkwood, J. K., Morgan, A. J., and Sklar, J . (1985). Science 228, 722-724. Cohen, L. K. , Speck, S. H., Roberts, B. E., and Strominger, J. L. (1984).Proc. Nutl. Acud. Sci. U . S . A . 81, 4183-4187. Corden, J. L., Cadena, D. L., Ahearn, J . M., and Dahmus, M. E. (1985). Roc. Nufl.Acud. Sci. U . S . A . 82, 7934-7938. Costa, R. H., Draper, K. G., Banks, L., Powell, K. L., Cohen, G., Eisenberg, R., and Wagner, E. K. (1983).J. Virol. 48, 591-603. Countryman, J., Miller, G. (1985). Roc. Nutl. Acud. Sci. U . S . A . 8 2 , 4085-4089. Crawford, D. H . , Epstein, M. A., Achong, B. G., Finerty, S., Newman, J., Liversedge, S., Tedder, R . S., and Stewart, J. W. (1979).J.Znzect. 1, 37-48. Dambaugh, T . , Beisel, C., Hummel, M., King, W., Fennewald, S., Cheung, A,, Heller, M., Raab-Traub, N., and Kieff, E. (1980). Proc. Nud. Acud. Sci. U.S.A. 77, 2999-3003. Dambaugh, T . , Hennessy, K., Charnnankit, L., and Kieff, E. (1984). R o c . Nutl. Acud. Sci. U . S . A . 81, 7632-7636.

152


Dambaugh, T . , Wang, F., Hennessy, K., Woodland, E., Rickinson, A,, and Kieff, E. (1986).J. Virol. 59, 453-462. De The, G., Geser, A,, Day, N . E., Tukei, P. M., Williams, E. H., Beri, D. P., Smith, P. G., Dean, A. G., Bornkamm, G. W., Feorino, P., and Henle, W. (1978). Nature (London) 274, 756-761. Delius, H . , and Bornkamm, G . W. (1978).J. Virol. 27, 81-89. Dillner, J , , Sternas, L., Kallin, B., Alexander, H., Ehlin-Henriksson, B., Jornvall, H., Klein, G., and Lerner, R . (1984). Roc. Natl. Acad. Sci. U . S . A . 81, 4652-4656. Dillner, J , , Kallin, B., Ehlin-Henriksson, B., Timar, L., and Klein, G. (1985a). Znt. J. Cancer 35, 359-366. Dillner, J., Kallin, B., Klein, G., Jornvall, H., Alexander, H . , and Lerner, R . (1985b). EMBO J . 4, 1813-1818. Dillner, J., Eliasson, L., Sternas, L., Kallin, B., Klein, G . , and Lerner, R. A. (1985~).In “EBV and Associated Diseases” (P. Levine, G . Pearson, D. Ablashi, and D. Kottaridis, eds.), pp. 446-455. Nijhoff, Boston. Dillner, J., Kallin, B., Ehlin-Henriksson, B., Rymo, L., Henle, G., Henle, W . , and Klein, G . (1986a). Znt. J . Cancer 37, 195-200. Dillner, J . , Kallin, B., Alexander, H., Ernberg, I., Uno, M., Ono, Y., Klein, G., and Lerner, R . A. (1986b). ROC. N d . Acad. Sci. U . S . A . 83, 6641-6645. Dillner,J., Wendel-Hansen, V., Kjellstrorn, G., Kallin, B., andRosen, A. (1987a). Purificationand characterization of EBV nuclear antigen using monoclonal antipeptide antibodies. Submitted. Dillner, J , , Szigeti, R., Henle, W., Henle, G . , Klein, G . , and Lerner, R. (1987b). Int.J. Cancer, 1587-1 596. Dillner, J., Rabin, H . , Letvin, N., Henle, W., Henle, G., and Klein, G. (1987c).J. Gen. Virol. 68, 1587-1596. Dolken, G . , Hecht, T., Weitzmann, U., Wagner, M., Lohr, G. W., and Hirsch, F. W. (1986). Virology 148, 58-73. Dolyniuk, M. R . , Pritchett, R . , and Kieff, E. (1976).J. Virol. 17, 935-949. Earl, P. L., Jones, E. V., and Moss, B. (1986). Proc. Natl, Acad. Sci., U.S.A. 83, 3659-3663. Edson, C . M., and Thorley-Lawson, D. A. (1981).J. Virol. 39, 172-184. Edson, C . M., and Thorley-Lawson, D. A. (1983).J. Virol. 46, 547-556. Einhorn, L., and Ernberg, I. (1978). Int. J . Cancer 21, 157-160. Eliasson, L., Kallin, B., Patarroyo, M., Klein, G., Fujiki, H., and Sugimura, T . (1983). Int. J . Cancer 31, 7-11. Epstein, M. A,, Achong, B. G., and Barr, Y. M . (1964). Lancef 1, 702-703. Ernberg, I., and Kallin, B. (1984). Cancer Surv. 3, 51-89. Ernberg, I., Kallin, B., Dillner, J., Falk, K., Ehlin-Henriksson, B., and Klein, G. (1986). Znt.J. Cancer 38, 729-737. Feighny, R. J . , Farrell, M. P., and Pagano, J . S. (1980). J . Virol. 34, 455-463. Feighny, R . J., Henry, B. E., and Pagano, J. S. (1981).J. Virol. 39, 651-655. Fennewald, S., van Santen, V., and Kieff, E. (1984).J. Virol. 51, 411-419. Fingeroth, J. D., Weis, J. J., Tedder, T. F., Strominger, J . L., Biro, P. A,, and Fearon, D. T . (1984). R o c . Natl. Acad. Sci. U . S . A . 81, 4510-4514. Fischer, D. K . , Robert, M . F., Shedd, D., Summers, W. P . , Robinson, J . E., Wolak, J., Stefano, J. E., and Miller, G. (1984). Proc. Nad. Acad. Sci. U . S . A . 81, 43-47. Frade, R., Barel, M . , Ehlin-Henriksson, B., and Klein, G. (1985). Proc. Natl. Acad. Sci. U . S . A . 82, 1490-1494. Fresen, K. O., Cho, M . S., and zur Hausen, H . (1978). Znt. J . Cancer 22, 378-383. Fresen, K. O., Cho, M. S., and zur Hausen, H . (1980). Cold Spring Harbor Cony. Cell Prolif. 7, 35-44. Gerber, P., Nonoyama, M . , Lucus, D., Perlin, E., and Goldstein, L. I. (1972). Lancet 2, 988-989.


153

Gerber, P . , Pritchett, R . F., and Kieff, E. (1976).J. Virol. 19, 1090-1099. Gergely, L., Sternas, L., Dillner, J., and Klein, G. (1984). Zntcrviroloa 22, 85-96. Gordon, J., Ley, S. C . , Melamed, M. D., English, L. S., and Hughes-Jones, N. C. (1984a). Nature (London) 310, 145-147. Gordon, J . , Ley, S. C., Melamed, M. D., Jman, P., and Hughes-Jones, N. C. (1984b).J. Exp. Med. 159, 1554-1559. Gordon, J., Aman, P., Rosen, A,, Ernberg, I., Ehlin-Henriksson, B., and Klein, G. (1985).Znt. J . Cancer 35, 251-256. Gordon, J . , Rowe, M., Walker, L., and Guy, G . (1986a).Eur. J . Zmmunol. 16, 1075-1080. Gordon, J., Walker, L., Guy, G., Brown, G., Rowe, M . , and Rickinson, A. (1986b).Zmrnunologv 58, 591-595. Hanto, D., Frizzera, D . , Purtilo, D., Sakamoto, K., Sullivan, J., Saemundsen, A,, Klein, G., Simmons, R.,and Najarian, J. (1981). Cancer Res. 41, 4253-4261. Hanto, D., Frizzera, G., Gajl-Peczalska, K. J., Sakamoto, K., Purtilo, D. T . , Balfour, H . H . , Simmons, R . L.,and Najarian, J. S. (1982). New Engl. J . Med. 306, 913-918. Harris, A,, Young, B. D., and Griffin, B. E. (1985).J . Virol. 56,328-332. Hatzubai, A., Anafi, M . , Masucci, M. G., Dillner, J., Lerner, R., Klein, G., and Sulitzeanu, D. (1987).Down-regulation of the EBV-encoded membrane protein in Burkitt lymphomas. Znt. J. Cancer, in press. Hearing, J. C., and Levine, A. J . (1985). V i r o l o ~145, 105-116. Hearing, J. C., Nicolas, J . C. and Levine, A. J. (1984).Roc. Natl. Acad. Sci. U . S . A . 81, 4373-4377. Heller, M., and Kieff, E. (1981).J. Virol. 37, 821-826. Heller, M., Dambaugh, T . , and Kieff, E. (1981).J. Virol. 38, 632-648. Heller, M., Henderson, A,, and Kieff, E. (1982a).Proc. Nutl. Acad. Sci. U . S . A . 79,5916-5920. Heller, M . , van Santen, V . , and Kieff, E. (1982b).J . Virol. 44, 311-320. Heller, M . , Flemington, E., Kieff, E., and Deininger, P. (1985). Mol. Cell. Biol. 4, 425-445. Henle, G., and Henle, W. (1986).J.Bacteriol. 9,1248-1256. Henle, G . , and Henle, W. (1976). Znf.J . Cancer 17, 1-7. Henle, W, and Henle, G. (1979). In “The Epstein-Barr Virus” (M. A. Epstein and B. G. Achong, eds.), pp. 61-78.Springer-Verlag, Berlin. Henle, W., Henle, G., Ho, H . C., Burtin, P., Cachin, Y., Clifford, P., De Schryver, A., deThe, G . , Diehl, V., and Klein, G . (1970).J.Natl. Cancer Znsf. 44, 225-231. Henle, W., Henle, G., and Honvitz, C. A. (1974).Hum. Pathol. 5, 551-565. Henle, W., Henle, G., Andersson, J., Ernberg, I., Horwitz, C. A,, Marklund, G., Rymo, L., Strauss, S. E., and Klein, G . (1987). Proc. Nutl. Acad. Sci. U . S . A . 84, 570-574. Hennessy, K., and Kieff, E. (1985).Science 227, 1238-1240. Hennessy, K., and Kieff, E. (1983).Proc. Natl. Acud. Sci. U . S . A . 80, 5665-5669. Hennessy, K., Heller, M., van Santen, V., and Kieff, E. (1983).Science 220, 1396-1398. Hennessy, K . , Fennewald, S., Hummel, M., Cole, T., and Kieff, E. (1984).Roc. Natl. Acud. Sci. U . S . A . 81, 7207-7211. Hennessy, K., Fennewald, S., and Kieff, E. (1985).Proc. Nafl. Acud. Sci. U . S . A . 82, 5944-5948. Hennessy, K., Wang, F . , Bushman, E. W., and Kieff, E. (1986).Roc. Nutl. Acud. Sci. U.S.A. 83, 5693-5697. Ho, H . C. (1975).J. R . Coll. Surg. Edinborough 20, 223-235. Ho, J. H . C . (1972).Adu. Cancer Res. 15, 57-92. Hoffman, P. J., and Cheng, Y. C . (1979).J. Virol. 32, 449-457. Honess, R . W., and Roizman, B. (1974).J . Virol. 14, 8-19. Hu, C. P., Aman, P., Masucci, M. G . , Klein, E., and Klein, G . (1986). Eur. J. Zmmunol. 16, 841 -845. Hudson, G. S., Farrell, P. J., and Barrell, B. G. (1985a).J. Virol. 53, 528-535.

154


Hudson, G . S . , Bankier, A. T., Satchwell, S. C . , and Barrel], B. G . (1985b). Virology 147, 81-98. Hummel, M., and Kieff, E. (1982). R o c . Natl. Acad. Sci. U . S . A . 79, 5698-5702. Hummel, M., Thorley-Lawson, D., and Kieff, E. (1984).J. Virol. 49, 413-417. Jat, P., and Arrand, J . R. (1982). Nucleic Acids Res. 10, 3407-3425. Jenson, H., Rabson, M. S . , and Miller, G. (1986). J . Virol. 58, 475-486. Jones, J. F., Ray, C. G., Minnich, L. L., Hicks, M. J., Kibler, R., and Lucus, D. 0. (1985). Ann. Intern. Med. 102, 1-7. Jones, M. D., Foster, L., Sheedy, T . , and Griffin, B. (1984). EMBO J. 3 , 813-821. Kallin, B. (1983). Doctoral thesis, Karolinska Institute, Stockholm. Kallin, B., and Klein, G. (1983). Intervirology 19, 47-51. Kallin, B., Luka, J . , and Klein, G . (1979).J. Virol 32, 710-716. Kallin, B., Sternas, L., Saemundsen, A. K . , Luka, J . , Jornvall, H., Eriksson, B., Tao, P. Z . , Nilsson, M., and Klein, G. (1985). J . Virol. 54, 561-568. Kallin, B., Dillner, J., Ernberg, I . , Ehlin-Henriksson, B., Rosen, A., Henle, W . , Henle, G . , and Klein, G. (1986). Roc. Nutl. Acad. Sci. U.S.A. 83, 1499-1503. Kamata, T . , Tanaka, S., Aikawa, S., Hinuma, Y., Watanabe, Y. (1979). Virology 95, 222-226. Karnata, T . , Takahi, K., Hinurna, Y., and Watanabe, Y. (1981). Virology 113, 512-520. Kieff, E., Dambaugh, T . , Heller, M., King, W., Cheung, A , , van Santen, V., Hummel, M . , Beisel, C., Fennewald, S . , Hennessy, K., and Heineman, T . (1982). J . Infect. Dis. 146, 506-523. Kieff, E., Dambaugh, T . , Hennessy, K., Fennewald, S., Heller, M., Matsuo, T., and Humrnel, M . (1984). In “The Role of Viruses in Human Cancer” (G. Giraldo and E. Beth, eds.), Vol. 11, pp. 103-118. King, W., Dambaugh, T . , Heller, M . , Dowling, J., and Kieff, E. (1982). J . Virol. 43, 979-986. Kirchner, H., Blaese, R., Tosato, G . , Broder, S., and Magrath, I. (1979). J . Immunol. 122, 1310-13 13. Klein, G . (1979). Roc. N a L Acad. Sci. U . S . A . 76, 2442-2446. Klein, G . (1983). Cell 32, 311-315. Klein, G., Clifford, P., Klein, E., and Stjernsward, J. (1966). Roc. Natl. Acad. Sci. U . S . A . 5 5 , 1628-1 635. Klein, G . , Giovanella, B. C., Lindahl, T . , Fialkow, P. J . , Singh, S . , and Stehlin, J . S. (1974). Proc. NaL Acad. Sci. U . S . A . 71, 4737-4741. Kornberg, A. (1980). “DNA Replication,” pp. 277-317. Freeman, San Francisco. Krebs, E. G . , and Beavo, J. A. (1979). Annu. Rev. Biochm. 48, 923-959. Lanier, A., Bender, T . , Talbot, M., Wilmeth, S . , Tschopp, C., Henle, W., Henle, G., Ritter, D., and Terasaki, P. (1980). Cancn 46, 2100-2105. Lerner, M. R., Andrews, N. C., Miller, G., and Steitz, J . A. (1981). Proc. Natl. Acad. Sci. U . S . A . 78, 805-809. Lieberman, P. M . , O’Hare, P., Hayward, G . S . , and Hayward, S. D. (1986). J . Virol. 60, 140-148. Liebowitz, D., Wang, D., and Kieff, E. (1966). J . Virol. 58, 233-237. Lindahl, T . , Klein, G . , Reedman, B. M., Johansson, B., and Singh, S. (1974). Znt.f.Cancer 13, 764-772. Lindahl, T., Adams, A., Bjursell, G., Bornkamm, G. W., Kaschka-Dierich, G., and Jehn, U. (1976).J. Mol. Biol. 102, 511-530. Littler, E., Zeuthen, J . , McBride, A. A., Frost-Sorensen, E., Powell, K. L., Walsh-Arrand, J . E., and Arrand, J . R . (1986). EMBOJ. 5 , 1959-1966. Luka, J., Kallin, B., and Klein, G. (1979). Virology 94, 228-231. Luka, J . , Kreofsky, T., Pearson, G . R., Hennessy, K., and Kieff, E. (1984). f. Virol. 52, 833-838. Luka, J., Miller, G . , Jornvall, H . , and Pearson, G . R. (1986). J . Virol. 58, 748-756.


155

Lupton, S., and Levine, A. J. (1985).Mol. Cell. Biol. 5 , 2533-2542. Mann, K. P., Staunton, D., and Thorley-Lawson, D. A. (1985).J. Virof. 55, 710-720. Menezes, J., Leibold, W., and Klein, G. (1975).Exp. Cell Res. 92, 478-484. Miller, G., Shope, T . , Coope, D., Waters, L., Pagano, J., Bornkamm, G., and Henle, W. (1977).J.Exp. Med. 145, 948-967. Miller, G., Grogan, E., Fischer, D. K., Niederman, J. C., Schooley, R. T., Henle, W., Lenoir, G., and Liu, C. R . (1985).New Engl. J . Mcd. 312, 750-755. Miller, R . L., Glaser, R . , and Rapp, F. (1977). Virology 76, 494-502. Milman, G., Scott, A. L., Cho, M. S., Hartman, S. C . Ades, D. K., Hayward, G. S., Ki, P.F., August, J. T . , and Hayward, S. D. (1985).R o c . Natf. Acad. Sci. U.S.A. 82, 6300-6304. Modrow, S., and Wolf, H . (1986). Roc. Nafl. Acad. Sci. U.S.A. 83, 5703-5707. Morgan, D. G., Niederman, J. C., Miller, G., Smith, H . W., and Dowaliby, J. M. (1979). Lanccf 1, 1154-1157. Mueller-Lantzsch, N., Georg, B., Yamamoto, N., and zur Hausen, H. (1980). Virology 102, 231-233. Mueller-Lantzsch, N., Lenoir, G. M., Sauter, M., Takaki, K., Bechet, J. M., KuWik-Roos, C., Wunderlich, D., and Bornkamm, G. W. (1985). EMBOJ. 4, 1805-1811. Muir, C. S. (1971). Znf. J. Cancn 8, 351-363. Nevo, S., Meyer, W., and Altman, M. (1971). Cancer 28, 807-809. Nilsson, K. (1979). Zn “The Epstein-Barr Virus” (M. A. Epstein and B. G. Achong, eds.). Springer-Verlag, Berlin. Nonoyama, M., and Pagano, J. S. (1973). Nature (London) 242, 44-47. Ohno, S., Luka, J., Falk, L., and Klein, G. (1977). Znf. J. Canccr 20, 941-946. Ohno, S., Luka, J., Klein, G., and Daniel, M. D. (1979). Roc. Natl. Acad. Sci. U.S.A. 76, 2042-2046. Ooka, T., Lenoir, G., and Daillie, J. (1979).J. Virol. 29, 1-10. Ooka, T., de Tarenne, M., de The, G., and Daille, J . (1984).J. Virol. 49, 626-628. Oppenheim, A , , Shlomai, Z., and Ben-Bassat, H. (1981). Mol. Cell. Bid. 1, 753-762. Pagano, J. S., Huang, C. H., and Levine, P. H . (1973). Ncw Engl. J. Med. 289, 1395-1399. Pearson, G. R . , Vroman, B., Chase, B., Sculley, T., Hummel, M., and Kieff, E. (1983).J . Virof. 47, 193-201. Pellett, P., Biggin, M. D., Barrell, B. G., and Roizman, B. (1985).J. Virof. 56, 807-813. Perricaudet, M., Akusjarvi, G., Virtanen, A., and Peterson, U. (1979). Naturc (London) 281, 694-696. Polack, A , , Delius, H., Zimber, U., and Bornkamm, G . W. (1984). Virology 133, 146-157. Powell, K. L.,and Purifoy, D. J . M. (1977).J. Virol. 24, 618-626. Purtilo, D. T . , De Florio, D., Hut, L. M., Bhawan, J., Yang, J. P. S., Otto, R., and Edwards, W., (1977a).New Engf.J. Mcd. 297, 1077-1081. Purtilo, D. T., Yang, J. P. S., Allegre, S . , De Florio, D., Hutt, L. M., Soltani, M., and Vawter, G. (1977b).Am. J. Med. 62, 225-233. Purtilo, D. T., Paquin, L., De Florio, D., Virzi, F., and Sakhuja, R. (1979).Scmin. Hmtafol. 1, 309-343. Purtilo, D. T . , Manolov, G., Tatsumi, E., Manolova, Y., Harada, S., Lipscomb, H., and Kruger, G. (1985).Znt. Rev. Exp. Pafhol. 27, 113-183. Quinn, J. P., and McGeoch, D. J . (1985). Nucleic Acidr Res. 13, 8143-8163. Rabin, H., Neubauer, R. H., Hopkins, R. F., and Nonoyama, M. (1978).Znt. J. Cancer 21, 762-767. Rabin, H . , Strnad, B. C . , Neubauer, R. H., Brown, A. M., Hopkins, R . F., and Mazur, R. A. (1980).J . Vir01. 28, 265-272. Rabson, M., Heston, L., and Miller, G. (1983).Roc. Nafl. Acad. Sci. U.S.A. 80, 2762-2766. Rawlins, D. R . , Milman, G., Hayward, S. D., and Hayward, G. S. (1985). Ccll42, 859-868.

156


Reece, R . , Gartner, J . G . , Scemayer, T. A., Joncas, J., and Pagano, J . (1981). Cancer Res. 41, 4424-4428. Reedman, B. M . , and Klein, G. (1973). Int. J . Cancer 11, 499-520. Reisman, D., Yates, J . , and Sugden, B. (1985). Mol. Cell. Biol. 5 , 1822-1832. Rickinson, A. B., and Moss, D. J . (1983). Adu. Viral Oncolo. 3, 213-228. Rickinson, A. B., Jarvis, J . , Crawford, D., and Epstein, M. (1974). Int. J . Cancer 14, 704-715. Rickinson, A. B., Finerty, S . , and Epstein, M. (1977). Int. J . Cancer 19, 775-782. Robinson, J., and Smith, D. (1981). Virology 109, 336-343. Robinson, J . , Smith, D., Niederman, N., and Miller, G. (1980). Nafure (London) 287, 334-335. Roubal, J., Kallin, B., Luka, J., and Klein, G. (1981). Virololzy 113, 285-292. Rowe, D., Heston, L., Metlay, J . , and Miller, G. (1985a). Proc. Natl. Acad. Sci. U . S . A . 82, 7429-7433. Rowe, D. T . , Rowe, M., Evan, G. I., Wallace, L. E., Farrell, P. J . , and Rickinson, A. B. (1986). EMBOJ. 5 , 2599-2607. Rowe, M . , Rooney, C . M., Rickinson, A. B., Lenoir, G. M., Rupani, H . , Moss, D. J . , Stein, H., and Epstein, M. A. (198513). Inf. J . Cancer 35, 435-441. Ruyechan, W. T. (1983). J . Virol. 46, 661-666. Rymo, L. (1979).J. Virol. 32, 8-18. Rymo, L., Klein, G . , and Ricksten, A. (1985). Roc. Nafl. Acad. Sci. U . S . A . 82, 3435-3439. Saemundsen, A. K . , Berkel, I., Henle, W., Henle, G., Anvret, M., Sand, O., Ersoy, F., Cagal, M., and Klein, G. (1981a). Br. J . Med. 282, 425-427. Saemundsen, A. K . , Purtilo, D. T . , Samamoto, K., Sullivan, J . L., Synnerholm, A. C., Hanto, D. C., Simmons, R . , Anvet, M., Collins, R . , and Klein, G. (1981b). Cancer Res. 41, 4237-4240. Saemundsen, A. K., Klein, G., Cleary, M., and Warnke, R. (1982a). Lancef 2 , 158. Saemundsen, A. K., Albeck, H . , Hansen, J . P. H., Nielsen, N. H., Anvret, M., Henle, W . , Henle, G., Thomsem, K. A., Kristensen, H . K . , and Klein, G. (1982b). Br. J . Cancer 46, 721-727. Sarnple,J., Tanaka, A., Lancz, G., and Nonoyama, M. (1984). Virology 139, 1-10, Sample, J . , Lancz, G., G., and Nonoyama, M. (1986a).J. Virol. 5 7 , 145-154. Sample, J . , Hummel, M., Braun, D., Birkenbach, M . , and Kieff, E. (1986b). Proc. Natl. Acad. Sci. U . S . A . 83, 5096-5100. Schooley, R . T . , Carey, R . W., Miller, G., Henle, W . , Eastman, R . , Mark, E. J . , Kenyon, K . , Wheeler, E. O., and Rubin, R . H . (1986). Ann. Intern. Med. 104, 636-643. Sculley, T. B., Kreofsky, T . , Pearson, G. R., and Spelsberg, T. C. (1983).J. Biol. Chem. 258, 3974-3982. Sculley, T. B., Spelsberg, T . C., and Pearson, G . R . (1984a). Intervirology 22, 191-200. Sculley, T . B., Walker, P. J., Moss, D. J., and Pope, J . H . (1984b).J. Virol. 5 2 , 88-93. Sculley, T . B., Sculley, D. G., and Pope, J. H . (1985).J. Gen. Virol. 66, 1113-1122. Seibl, R., and Wolf, H . (1985a). Int. Herpesvirus Workshop, loth, Ann Arbor, MI. Abstr. 33. Seibl, R., and Wolf, H. (1985b). Virology 141, 1-13. Shanmugaratnam, K., and Muir, C. S. (1967). In “Cancer of the Nasopharynx” (C. Muir, ed.), pp. 47-53. Munksgaard, Copenhagen. Shope, T., Dechairo, D., and Miller, G. (1973). Proc. Natl. Acad. Sci. U . S . A . 70, 2487-2491. Simmons, M. J . ,Wee, G. G., Goh, E. H . , Chan, S. H . , Shanmugaratnam, K., Day, N. E., and de-The, G . B. (1976).J. Natl. Cancer Inst. 57, 977-980. Sixbey, J . W., Vesterinen, E. H., Nedrud, J. G., Raab-Traub, N., Walton, L. A,, and Pagano, J . S. (1983). Nature (London) 306, 480-481. Skare, J . , and Strominger, J . L. (1980). Proc. Nafl. Acnd. Sci. U . S . A . 77, 3860-3864. Skare, J., Farley, J., Strominger, J. L., Fresen, K. O., Cho, M . S . , and zur Hausen, H . (1985). J . Virol. 5 5 , 286-297. Speck, S . , and Strominger, J . L. (1985). Proc. Natl. Acad. Sci. U . S . A . 8 2 , 8305-8309.


157

Speck, S. H., Pfitzner, A., and Strominger, J. L. (1986). Roc. N d l . Acad. Sci. U . S . A . 83, 8305-8308. Starzl, T. E., Nalesnik, M . A., and Porter, K. A. (1984). Lancet 1, 583-587. Sternas, L.,Eliasson, L., Lerner, R., and Klein, G. (1986).J . Znimunol. Methods. 89, 151-154. Strnad, B. C., Schuster, T . , Hopkins, R. F., Neubauer, R. H., and Rabin, H . (1981).J.Virol. 38, 996-1004. Sugawara, K., Kawanishi, M . , and Ito, Y. (1982). Virology 116, 354-358. Sulitazeanu, D., Szigeti, R., Klein, G., Hennessy, K., and Kieff, E. (1986).J . Virol. 58, 230-232. Sulitzeanu, D . , Szigeti, R . , Hatzubai, A,, Dillner, J . , Hennessy, K., Kieff, E., and Klein, G . (1987). Antibodies in human sera against an EBV-coded cell membrane Protein, detected by the blocking of the EBV-specific leukocyte migration inhibition reaction. Submitted. Miller, G. (1982).Proc. Nod. Acad. Sci. U . S . A . 79, 5688-5692. Svedmyr, E., and Jondal, M. (1975). Proc. Nod. Acad. Sci. U . S . A . 72, 1622-1626. Szigeti, R., Sulitzeanu, D., Henle, G., Henle, W., and Klein, G. (1984). Proc. Natl. Acad. Sci. U . S . A . 81, 4178-4182. Takada, K., Shimizu, N., Sakuma, S., and Ono, Y. (1986).J. Virol. 57, 1016-1022. Thelander, L., and Reichard, P. (1979).Annu. Rev. Biochcm. 48, 136-158. Thorley-Lawson, D. A,, and Israelsohn, E. S. (1987).Proc. N d l . Acad. Sci. U . S . A . 84, in press. Thorley-Lawson, D. A., and Mann, K. (1985).J.Exp. Med. 162, 45-49. Thorley-Lawson, D. A., Edson, C . M., and Geilinger, K. (1982).Adv. Cancer Rex. 36, 295-346. Townsend, A . R. M., Gotch, F. M., and Davey, J. (1985).Cell 42, 457-462. Valentini, O., Biamonti, O., Pandolfo, M., Morandi, C., and Riva, S. (1985). Nucleic Acidr Res. 13, 337-346. Vroman, B., Luka, J . , Rodriquez, M., and Pearson, G. R. (1985).J. Vcrol. 53, 107-113. Walker, L., Guy, G . , Brown, G., Rowe, M., Milner, A. E., and Gordon, J. (1986). Irnmunolou 58, 583-589. Wang, F.,Gregory, C . D., Rowe, M., Wang, D., Rickinson, A. B., and Kieff, E. (1987).Roc. Natl. Acad. Sci. U . S . A . 84, 3452-3456. Wang, D., Liebowitz, D., and Kieff, E. (1985).Cell 43, 831-840. Wang, F., Petti, L., Braun, D., Seung, S., and Kieff, E. A. (1988).Bicistronic EBV mRNA encodes two nuclear proteins in latently infected, growth transformed lymphocytes. J . Virol., in press. Weigel, R . , and Miller, G. (1985).J. Virol. 54, 501-508. Wells, A , , Koide, N., and Klein, G. (1982).J. Virol. 46, 286-297. Whittle, H . C., Brown, J . , Marsh, K., Greenwood, B. M., Seidelin, P., Tighe, H . , and Wedderburn, L. (1984).Nature (London) 312, 449-450. Williams, E. H., and de The, G. (1974). Lancet 2, 295-296. Wolf, H., zur Hausen, H . , and Becker, V. (1973). Nature (London) 244, 245-247. Wolf, H., Bayliss, G. J., and Wilmes, E. (1981).In “Cancer Campaign: Nasopharyngeal Carcinoma,” pp. 101-109.Fiescher, Stuttgart. Wolf, H . , Haus, M., and Wilmes, E. (1984).J. Virol. 51, 795-798. Wong, K. M., and Levine, A. J . (1986).J. Virol. 60, 149-156. Yao, Q. Y., Rickinson, A. B., and Epstein, M. A. (1985). Int. J . Cancer 35, 35-42. Yates, J., Warren, N., Reisman, D., and Sugden, B. (1984). Proc. Natl Acad. Sci. U.S.A. 81, 3806-3810, Yates, J . L., Warren, N., and Sugden, B. (1985).Nature (London) 313, 812-815. Yefenof, E., Klein, G., Jondal, M . , and Oldstone, M . B. A. (1976).Int. J . Cancer 17, 693-700. Young, L. S.,Clark, D . , Sixbey, J. W., and Rickinson, A. B. (1986).Lancet 1, 240-242. Zeng, Y., and de The, G. (1985). In “EBV and Associated Diseases” (P. Levine, G. Pearson, D. Ablashi, and D. Kottaridis, eds.), pp. 151-163. Nijhoff, Boston. Ziegler, J . L., Andersson, M . Klein, G., and Henle, W. (1976).Znt. J. Cancer 17, 701-706.

158


Zimber, U.,Adldinger, H . K.,Lenoir, C. M., Vuillaume, M., von Knebel-Doeberitz, M . , Laux, G., Desgranges, C., Wittman, P., Freese, U. K., Schneider, U., and Bornkamm, G. W. (1986). Virolou 154, 56-66. Zur Hausen, H . and Schulte-Holthausen, H . (1970). Nature (London) 227, 245-248. Zur Hausen, H . , Schulte-Holthausen, H . , Klein, G., Henle, W., Henle, G., Clifford, P., and Santesson, L. (1970). Nature (London) 228, 1056-1058. Zur Hausen, H . , Diehl, V., Wolf, H . , Schulte-Holthausen, H., and Schneider, U . (1972). Nature (London)237, 189-190. Zur Hausen, H . , O’Neill, F., and Freese, U. K. (1978). Nature (London) 272, 373-375.

STROMAL INVOLVEMENT IN MALIGNANT GROWTH A. van den Hooff Laboratory of Histology and Cell Biology, Unlverslty of Amsterdam, Academic Medlcal Center 1105 A2 Amsterdam, The Netherlands

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Stromal Reactions to Manifest Malignant Epithelial Growth . . . . . . . . . . . . . . . . . A. Regressive Collagen Changes in Malignant Growth B. Collagenases . . . . . . . . . . . . . . . C . Other Collagenolytic Enzymes D. Plasminogen Activator. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Regulatory Mechanisms in Collagenolysis F. The Significance of Collagenolysis in Malig .......... G. Productive Changes in Malignant Growth H. Desmoplasia . . . . . . . . . . . . . . . I. Qualitative Alterations in Collagen Synthesis during Malignant Growth . . . J . Elastogenesis and Elastolysis in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Stromal Proteoglycans in Tumor Growth ........ .. L. Observations on the Variety of Stromal Reactivity ..................... 111. The Roles Played by Stromal Adhesive Glycoproteins ...................... A. Fibronectins in Malignant Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Laminin in Malignant Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Role of the Basement Membrane in Invasiveness ...................... V. Characteristics of Fibroblasts in Malignant Conditions . . . . . . . . . . . . . . . . . . . . . A. Localchanges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. GeneralizedChanges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Primary Stromal Disorders as Putative Factors in Carcinogenesis A. Wounding and Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . B. Fibrosis and Scar Cancer. ........................... VII. Stromal Alterations Preceding I VIII. Concluding Remarks . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

159 160

162 164

167 168 168 169 170 170 172 173 177 177 177

183 188

I. Introduction The most obvious way connective tissue stroma seems to be involved in malignancy is its breakdown by lytic processes as a crucial element in invasive growth. This looks relatively simple, but data have emerged that demonstrate that the stroma is not just a passive, but rather an active participant. Recent advances in the borderland between cancer research and 159 ADVANCES IN CANCER RESEARCH, VOL. 50

Copyright 0 1988 by Academic Press, Inc. All rights ofreproduction in any form reserved.

160

A. VAN DEN HOOFF

connective tissue research have increasingly made it clear that the relationship between malignant tissue and its stroma is a very intricate one. Gradually the picture emerges of an integrated system consisting of tissues that are intimately connected by a multitude of interactions, only a fraction of which have so far come to light. The intention of this contribution is to review and discuss recent data pertaining to various aspects of this system of interactions.

II. Stromal Reactions to Manifest Malignant Epithelial Growth Constituents of the extracellular matrix of the stroma are (1) the fibrous components collagen and elastin; (2) proteoglycans, a heterogeneous group of large molecules with specialized distributions in different types of stroma; and (3) glycoproteins, among which fibronectin and laminin are most extensively studied. The three classes of molecules by links and interactions form the insoluble and more or less firm extracellular matrices. A concise review on the composition of connective tissues is given by Hukins and Aspden (1985). The reactions of the connective tissue stroma around invasive tumors can be utterly dissimilar. In many cases regressive changes i.e., lysis of stromal components, dominate the picture. This seems to be a logical course of events: the way has to be cleared for the expanding and infiltrating tumor, which as it were lyses itself through its surroundings. However, the reaction of the stroma can be quite different. Production of collagen and the other constituents of the connective tissue may dominate the picture: ‘‘desmoplasia. ’ ’ These remarkable, seemingly conflicting effects of a tumor on its stroma raise doubts as to the concept of a tumor “lysing its way” through the surrounding mesenchymal tissues. This section will include (1) data on regressive stromal alterations accompanying malignant growth, (2) data on productive stromal changes accompanying malignant growth, and (3) observations on the variety of stromal reactivity.

A. REGRESSIVE COLLAGEN CHANGES IN MALIGNANT GROWTH Malignant epithelial growth is often, but not always (see Section II,F), accompanied by apparent lysis of the adjacent stroma including most constituents of the extracellular matrix. Since the mechanical quality of the extracellular matrix is mainly determined by the properties of its collagenous component, it is the presence of collagen that is considered the main barrier to be cleared away to make room for the infiltrating cell mass. Enzymatic degradation of collagen during tumor growth has been a subject of numerous investigations.

STROMAL INVOLVEMENT IN MALIGNANT GROWTH

161

B. COLLACENASES The collagens form a heterogeneous class of strong fibrous proteins. At least 12 types have been discovered so far. The collagen molecule is a triple helix of peptide chains of specific amino acid composition. At the ends each molecule has nonhelical regions, telopeptides, which are the sites of crosslinking. Collagens differ in amino acid composition and the lengths of their component chains. Fiber-forming collagens are the types I, 11, and 111. Type I, the most abundant, is found in skin, tendons, and bones, and in the stroma of most organs. Type I1 is the main fibrous constituent of cartilage. Type I11 is also present in a variety of tissues, mostly in association with type I; it probably forms reticulin fibers. Type IV collagen forms an open nonfibrillar network and is specific to basement membranes. Of the other collagens, type V is worth mentioning, since it is a prominent constituent of fibrotic lesions. For concise reviews on the collagens the reader is referred to papers by Mayne (1984) and Martin et al. (1985). It is the specific mammalian collagenases, metalloproteinases with a neutral pH optimum, that are especially suited to the degradation of collagen in the extracellular matrix. The enzymes cleave the triple helix of the collagen molecule at a single locus into two fragments of three-quarters and onequarter length, rending the collagen fiber susceptible to attack by other proteases (review by Sellers and Murphy, 1981). The enzyme is synthesized in a proenzyme form, which can be activated by a variety of pathways, including proteolytic activation. Collagenase activity is regulated at a variety of levels, including proenzyme activation, interaction with inhibitors, and de nouo synthesis (Goldberg et al., 1986, referring to the relevant publications). The obvious assumption that mammalian neutral collagenase plays a predominant role in collagenolysis facilitating malignant growth seems amply borne out in a great number of studies employing biochemical and immunolocalization techniques and dealing with various types of clinical and experimental cancer (reviews by Pauli et al., 1983; van den Hooff, 1983; Woolley, 1984; Liotta et al., 1982, Liotta, 1985). Depending on the type of cancer, the source of collagenase can be attributed either to the tumor cells themselves or to stromal cells, especially activated fibroblasts, that are stimulated to produce the enzyme under inductive influences from the tumor cells. Often latent and active forms of collagenase are present simultaneously. Activation can be brought about by various proteolytic enzymes, e.g., plasminogen activator (see Section I1,D). The importance of inhibitors is illustrated by Hicks et al. (1984), who, working with a highly invasive mouse fibrosarcoma model, could not demonstrate collagenase but instead detected a 10- to 20-fold decrease in secretion of metalloproteinase inhibitor.

162

A . VAN DEN HOOFF

C. OTHER COLLAGENOLYTIC ENZYMES Evidence is accumulating that the activity of neutral mammalian collagenases is not necessarily implicated in collagenolysis during tumor growth. Collagen is also digested by lysosomal enzymes at low p H , specifically acidic cathepsins (Burleigh et al., 1974), and by the leukocyte enzymes elastase and cathepsin G at neutral p H (review by Sellers and Murphy, 1981). Cathepsins attack the collagen fibrils at the nonhelical telopeptide regions. Increased activities of cathepsin B have been demonstrated in a number of cancers (reviewed by van den Hooff, 1983). Phagocytosis of collagen by stromal cells has been described in malignant conditions (van den Hooff, 1983), subsequent breakdown having to be effected by lysosomal enzymes. Graf et al. (1981), who demonstrated cathepsin B in host cells (fibroblasts and leukocytes) at the invasion front of rabbit V2 carcinoma, ascribe a dual function to the enzyme: intracellular degradation (in host cells) of endocytosed protein and extracellular activation of collagenase. In explants from malignant breast tumors, cathepsin B-like enzymes active at neutral pH have been described (Poole et al., 1980; Recklies et al., 1982) that could unfold their activities in the extracellular compartment. Collagenase levels were low. Similar enzyme activities have been found in murine melanoma cells (Sloane et al., 1982; Sloane and Honn, 1984) and-besides collagenase-in rabbit V2 carcinoma (Baici et al., 1984). The enzyme may act directly on components of the extracellular matrix (e.g., collagen) or, alternatively, activate latent collagenase (Baici et al., 1984). Pauli et al. (1986) have presented evidence of a broadly active protease in invasive rat bladder carcinomas, capable of breaking down native collagen in the absence of a vertebrate type of collagenase. In invasion assays the levels of enzyme activities correlated well with the depth of invasion. According to the authors, the enzyme’s activities may be initiated by degradation of the telopeptides, the regions that contain the crosslinks, or, alternatively, one may deal with a bacterial collagenaselike activity.

D. PLASMINOGEN ACTIVATOR Plasminogen activator (PA) is correlated with a number of destructive processes. A controversial issue is its role in destruction of the extracellular matrix during malignant growth. Two types of PA are known so far: the urokinase type (uPA) and the tissue type (tPA). uPA is hypothesized to play a role in degradation of stroma by cancer cells via the formation of plasmin. Although in many systems correlations between PA activity and malignant growth were found, in other studies no such correlation was apparent. For a recent extensive review on PA and its role in tissue degradation and cancer, the reader is referred to a publication by Dan0 et al. (1985).


163

Plasmin is a trypsin-like enzyme of broad specificity. The major and possibly only proteins in the extracellular matrix that are resistant to degradation by plasmin are the type I and type I1 collagens. PA could nonetheless play a role in the breakdown of these collagens via plasmin-mediated activation of latent collagenases. Weighing all the evidence, Dan0 et al. (1985) consider it reasonable to conclude that none of the experimental evidence contradicts the view that the release of PA from cancer cells plays a causal role in tissue destruction. A substantial body of evidence, most of it circumstantial, in their view supports the assumption that uPA indeed plays a key role in tissue degradation in cancer. It may be of significance that some evidence points to a crucial role of PA in metastasis, but not in local invasiveness (Ossowski and Reich, 1983). This finding could point to a fundamental difference between invasive growth at the primary site that occurs after an extended period of gradual alterations (see also Section VII) and ingrowth that starts abruptly by breaching a vessel wall elsewhere.

E. REGULATORY MECHANISMS I N COLLAGENOLYSIS O n the regulatory mechanisms of production of collagenolytic enzymes during tumor growth, significant data have come to light recently. Wirl(l984) and Wirl et al. (1984), working with DMBA-induced mammary tumors of the rat, found that collagenase production by tumor cells in uitro was markedly increased when exogenous collagen was provided, In nonmalignant systems epithelial cytokines have been found that modulate synthesis. A 20-kDa factor was demonstrated in rabbit corneal epithelium (Johnson-Wint and Bauer, 1985). A number of factors with different molecular weights were found in cultured human epidermis cells. Basal cell carcinomas elaborate a factor (MW 19,000) that induced collagenase synthesis in skin fibroblasts (Goslen et al., 1985). Biswas (1984, 1985) has described interactions between cocultured tumor cells and fibroblasts resulting in the production by fibroblasts of collagenase against type I. In systems containing human tumor cell lines and human fibroblasts it was found that the tumor cells secrete a factor which stimulates collagenase synthesis by fibroblasts. In a system consisting of cocultured mouse B16 melanoma cells and rabbit fibroblasts the pattern of interactions was rather intricate. Tumor cells appeared to release a factor that stimulated the fibroblasts to produce collagenase. This activity in its turn was found to be influenced by the matrix deposited by the fibroblasts. A finding of great potential significance has been reported by Goldberg et al. (1986). The authors determined the complete primary structure of a human

164

A. VAN DEN HOOFF

fibroblast collagenase and observed a homology to an oncogene-induced rat protein, whose function is unknown. Woolley (1984) has argued that free cells present in the stroma (macrophages and mast cells) could possibly contribute to the regulation of collagenolysis during tumor growth. He postulated an intricate pattern of interactions in which products from macrophages and mast cells stimulate the expression of collagenolytic enzymes by tumor cells or stromal cells. Macrophages have been shown to cooperate with tumor cells in collagen degradation (Henry et al., 1983). The impressive increase in mast cell numbers in the stroma of incipient and manifest cancers has puzzled investigators from the time the phenomenon was first described by Ehrlich in 1879. It has been shown that mast cell products have a stimulatory effect on mononuclear cells (Yoffe et a l . , 1985) and can also stimulate collagenase production by fibroblasts (Yoffe et al., 1984). It has earlier been demonstrated (Birkedal-Hansen et a l . , 1976) that proteases produced by mast cells are capable of activating fibroblast procollagenase. Another factor in collagen loss during primary malignant growth may be decreased collagen synthesis by mesenchymal cells that are somehow under the influence of carcinogenic factors (see also Section VII). Although these data on factors that regulate collagenase production in malignant conditions do not add up to a coherent picture, they are indicative of complicated patterns of interactions between malignant tumors and their stroma resulting in collagen degradation.

F. THESIGNIFICANCE O F COLLAGENOLYSIS IN MALIGNANT GROWTH in Viuo It appears that, depending on the type of tumor, one is confronted with a number of possible modes of collagen degradation rather than with a single, clearly defined mechanism. There is no absolute agreement among authors as to the supposed crucial roles of collagenolytic enzymes in invasiveness. Strauli (1980) has made the point that the advance of cancer cells within the extracellular matrix does not necessitate total lysis of opposing structures. Lysis may be nondestructuive when it consists in the temporary loosening of tissue texture which facilitates the access of cancer cells to deeper strata. Gabbert (1985) has also expressed doubt as to the significance of proteolytic enzymes in in vitro malignancy. Although positive correlations have been demonstrated between the amounts of collagenolytic enzymes in vitro and the metastatic potential of particular tumors in vivo (Sloane et a l . , 1982; Tarin et al., 1982), he considers it uncertain whether the enzymes are also functionally active in vivo where natural protease inhibitors are present. The only in vivo


165

evidence in his view comes from ultrastructural investigations, which he considers equivocal, although some electron-microscopic data seem to point to replacement of regular collagen fibrils by amorphous or filamentous material (Hashimoto et a l . , 1972; Tarin, 1972; van den Hooff and Tigchelaar-Gutter, 1983a). Gabbert stresses the role of interstitial edema of host tissue for tumor invasion. Accumulation of certain proteoglycans may be of importance (see also Section 11,K). Gabbert concludes that confrontation of the host tissue with the invading tumor cells results by no means in its immediate destruction. A phase of coexistence precedes the subsequent phase of progressive host tissue atrophy. This atrophy (“melting”) he considers a poorly understood phenomenon. This view concurs to a certain extent with the one proposed earlier by Roos et al. (1978, 1981), who in metastases visualized a similar coexistence based on interactions between invading cells and parenchymatous host cells. G.

PRODUCTIVE CHANGES IN MALIGNANT GROWTH

Certain cancers are characterized by their propensity to form dense masses of stroma around and between the invading malignant growths. This desmoplasia is especially obvious in scirrhous malignancies of the breast an in certain cancers of the stomach (linitis plastica). Desmoplastic growth always contains enhanced amounts of collagen. In breast cancers elastosis is also a common feature, Productive changes of the stroma are also described in other less common types of cancer.

H. DESMOPLASIA The origin of desmoplasia is controversial. As to the cells which produce the excessive stroma, views diverge. A plausible surmise is that stromal components in desmoplasia are synthesized by the cells whose physiological task it is to provide the stroma with its fibrillar and nonfibrillar elements, the fibroblasts. Lane (1957) and Goellner et al. (1973) reported on a number of cases in which large amounts of stroma were associated with the carcinomatous component of squamous cell carcinoma of the mouth and larynx. They considered this “pseudosarcoma” a bizarre reactive nonneoplastic phenomenon. Kao et al. (1986) observed that the extracellular matrix formed by cultured human breast tumor cells stimulated growth of fibroblasts in vitro and also stimulated collagen and elastin synthesis. Barsky et al. (1982) demonstrated an increased content of type V collagen in desmoplasia of human breast carcinoma, possibly produced by host stromal fibroblasts recruited in response to the invasive carcinoma. Ban0 et al. (1983) found that tumor cells in rat mammary

166

A . VAN DEN HOOFF

adenocarcinoma contained factors that stimulate collagen type IV production. Dvorak et al. (1984) and Form et al. (1984) expressed the opinion that in desmoplasia accompanying bile duct carcinoma in the rat, interstitial collagens are fibroblast products whereas carcinoma cells themselves produce primarily basement membrane collagens. They argued that desmoplasia is analogous to wound healing. A number of authors (Tremblay, 1979; Seemayer et a l . , 1979; Barsky et al., 1982; Lagack et al., 1985) have proposed that it is stromal myofibroblasts which, induced by tumor cells, synthesize the surplus amounts of collagen. These remarkable cells are mainly characterized by their contractility, which may explain retraction phenomena of the skin (dimpling) in breast cancer (Tremblay , 1979). Contrary to these views, stressing stimulation of stromal cells by tumor cells, are data that point to a radical metaplasia of epithelial tumor cells themselves. Earlier authors (Krompecher, 1900; Martin and Stewart, 1935) entertained the possibility of the transformation of epithelium into mesenchyme. Langness and Udenfriend (1974) have shown that cloned untransformed nonfibroblastic cell lines indeed may have the capacity to produce collagen which under physiological conditions is totally or almost totally repressed. It has been reported that the “pseudosarcomatous” components of spindle cell carcinomas derived from squamous cells described by Lane (1957) and mentioned above do indeed metastasize (Mincklar et a l . , 1970; Leifer et al., 1974; Battifora, 1976). Metastases were found to contain both carcinomatous and sarcomatous elements and to produce collagen fibers. These observations argue in favor of the malignant nature of the extracellular matrix-producing cells and fail to support the concept of reactive fibroblasts, at least in cases of spindle cell carcinomas. Synthesis of collagen and/or prolyl hydroxylase (an enzyme necessary for collagen synthesis) by tumor cells themselves has been described in human scirrhous breast cancer (Al-Adnani et al., 1975; Al-Zuhair et a l . , 1986), in cloned mouse mammary tumor cells (Roesel et al., 1978), and in cloned human gastric carcinoma cells (Sakakibara et al., 1982). Harris (1982) has put forward a “highly speculative” hypothesis that seems worth mentioning: In the course of spindle cell squamous carcinoma, in vivo fusion between squamous cells and fibroblasts or myofibroblasts would result in cells with properties of both cell types. Most data presented in this section-though prima facie controversial-seem convincing. It seems probable that malignant cells may be metaplastic to such an extent that the capacity to produce stromal components is expressed. It seems not less probable that in other malignant conditions fibroblasts appear to be stimulated by malignant epithelial cells to produce excessive amounts of stroma.


167

Summarizing these data on desmoplasia in cancer, one has to conclude that a variety of stroma-producing mechanisms may be operative, depending on the type of cancer. Seemingly contradictory to the findings on desmoplastic processes are in vitro data on the production of collagen by transformed fibroblasts and other collagen-producing cells, which consistently point to a decreased synthesis. The reader is referred to Section VII where this point is elaborated. How desmoplasia may figure in the condition called “scar cancer” is discussed in Section V1,B.

I. QUALITATIVE ALTERATIONS IN COLLAGEN SYNTHESIS DURING MALIGNANT GROWTH In a number of neoplasms qualitative changes in collagen production have been described. Shifts from one type of collagen to another have been observed by a number of authors (e.g., Bateman and Peterkofsky, 1981; Carter, 1982; Triieb et al., 1985). The most striking and consistent change is the production of an unusual type of collagen, the so-called type I trimer. The triple helix of the regular collagen type I molecule consists of two identical at chains and one a2 chain. The type I-trimer molecule has three a1chains of somewhat different composition. Synthesis of the type I trimer may represent the expression of an embryonic phenotype (Bornstein and Sage, 1980). The trimer has been demonstrated in in vivo malignant conditions and in a number of in vitro systems derived from clinical and experimental cancers. Moro and Smith (1977) demonstrated the type I trimer in a virus-induced mouse tumor, and Little et al. (1977) found it in a mouse teratoma-derived cell line. Since then the type I trimer has been detected in various systems and conditions; in virally transformed human fibroblasts (Krieg et al., 1980), in human osteosarcoma (Shapiro and Eyre, 1982), in chemically transformed mouse epithelial cells (Keski-Oja et al., 1982a, 1984), in virus-induced mouse osteosarcoma (Yamagata and Yamagata, 1984), in chemically transformed rat liver epithelial cells (Marsilio et a l . , 1984), in ductal infiltrating carcinoma of the human breast (Minafra et al., 1984; PucciMinafra et a l . , 1985), and in certain pediatric tumors (De Clerck et al., 1985). The presence of the molecule in tumors may reflect a potential that is lost by most cells during the normal development of tissues (Bornstein and Sage, 1980).

168

A. VAN DEN HOOFF

J. ELASTOGENESIS AND ELASTOLYSIS IN CANCER As already mentioned in Section II,H, in some cancers, especially of the mammary gland, elastosis in the majority of cases is part of the desmoplastic reaction (Lundmark, 1972; Azzopardi and Laurini, 1974; Tremblay, 1976, 1979; Hornebeck et al., 1980; Kao and Stern, 1986b). Probably elastin is synthesized by connective tissue cells that may be activated by a factor secreted by cancer cells (Azzopardi and Laurini, 1974; Tremblay, 1976; Hornebeck et al., 1980). According to Ghosh et al. (1980), the carcinoma cells themselves can also produce elastin. Hornebeck et al. (1980) could demonstrate elastolytic activities in extracts of all the breast cancers they studied. The authors considered various cell types as possible sources of the enzyme: tumor cells, fibroblasts, macrophages, leukocytes. Kao and Stern (1986b) examined elastases of human fibroblast and breast carcinoma cell lines. The tumor cell lines had 10- to 30-fold higher elastase activity than did the fibroblasts. Three separate elastinolytic activities were observed. The elastases in some cell lines were inhibited by steroids.

K. STROMAL PROTEOGLYCANS IN TUMOR GROWTH Pathologists have frequently described an increase in metachromatic staining of tumor stroma with basic dyes, presumably caused by enhanced presence of glycosaminoglycans, sulfated and nonsulfated carbohydrate polymers as building blocks of proteoglycans. Biochemical data on these alterations in cancer are given in a great number of reports. For detailed information the reader is referred to several reviews (Chiarugi, 1982; Iozzo, 1985a). Although data on the glycosaminoglycan profiles of different tumors are very variable, there is a consistent tendency to increased production of (nonsulfated) hyaluronic acid over sulfated glycosaminoglycans (Iozzo, 1985b). Accumulation of hyaluronic acid in the extracellular matrix is considered to be conducive to proliferation and invasion, since the resulting hydration of the extracellular matrix is supposed to create pathways for cell migration (Toole et al., 1984; Turley and Tretiak, 1985). Takeuchi (1971) injected the glycosaminoglycan chondroitin sulfate subcutaneously into the backs of mice and observed enhanced growth of inoculated tumors. Hyaluronic acidcontaining pericellular coats are further considered to inhibit adhesion to other structures preventing immobilization (Toole et al. , 1984). Changes in stromal proteoglycan composition in at least some cancers are induced by mediators derived from tumor cells. In colon carcinoma proteoglycan metabolism in stromal fibroblasts in uitm is modulated by polypeptides released by tumor cells (Iozzo, 1985b). Intraperitoneal implantation of


169

V2 carcinoma cells in the rabbit stimulated fibroblasts in the mesentery to produce increased amounts of collagen fibers and proteoglycans (Iozzo and Muller-Glauser, 1985). Extracts of a number of murine tumors and of human lung carcinoma are found to stimulate the production of hyaluronate by fibroblasts in vitro (Knudson et a l . , 1984; Knudson and Toole, 1985). Productive proteoglycan alterations in malignant growth are only part of a disturbed tissue remodeling. Reports on various malignant conditions make mention of enhanced activities of glycosaminoglycan-degrading enzymes. Hyaluronidase activity was found in interstitial fluid of rat Walker carcinoma (Fiszer-Szafarz and Gullino, 1970), and proteoglycan-degrading neutral protease in Lewis lung carcinoma cells (Emonds-Alt et al., 1980). Heparan sulfate degradation was found to correlate with mouse B16 melanoma invasive and metastatic properties (Nakalima et al., 1983). The involvement of heparan sulfate degradation in lysis of the extracellular matrix by highly metastatic mouse lymphoma cells was observed by Bar-Ner et al. (1985). A variety of glycosidases, catalyzing the hydrolysis of carbohydrate moieties from proteoglycans, glycoproteins, and other complex carbohydrates have been demonstratesd in the interstitial fluids of various cancers. Increased levels are correlated with metastatic potential (review by Bernacki et al., 1985). One has to conclude that the behavior of proteoglycans in malignancy, apart from the tendency to a shift in production from sulfated glycosaminoglycans to hyaluronic acid, does not follow a well-defined pattern. Proteoglycans are subject to both productive and regressive alterations. In this respect there lies a parallel with other stromal constituents, collagen and elastin.

L. OBSERVATIONS ON THE VARIETY OF STROMAL REACTIVITY The data presented in this section do not add up to a coherent picture of the involvement of the extracellular matrix in malignant growth. The extracellular matrix may react in quite different ways. Changes may be of either a degradative or a productive nature. They may even vary in seemingly conflicting ways as a function of time and localization. In experimental carcinoma of the mouse skin, loss of collagen may in the course of transformation turn into excessive production (van den Hooff, 1962). In the rabbit mesentery invaded by V2 carcinoma cells, productive and regressive changes occur side by side in the same area (Miiller-Glauser et al., 1984). In human breast carcinoma cell lines, the production of collagen and elastin and of enzymes which degrade these components may take place concurrently. The equilibrium can be shifted toward desmoplasia or lysis under the influence of hormones (Kao and Stern, 1986a,b).

170

A . VAN DEN HOOFF

An explanation can be suggested along the following lines. The extracellular matrix in adult organs normally is in a steady state: synthesis and breakdown of its constituents are in equilibrium, a consequence of the built-in “system of checks and balances that allows cellular activities to be closely regulated by opposing stimulatory and inhibitory influences” (Marx, 1986). This equilibrium can only be maintained if a constant flow of feedback signals emanating from the extracellular matrix components or the production of degradative agents, and the cells respond in a well-defined way. It is these feedback loops that may be disturbed either way, resulting in desmoplasia or regressive changes. Fibroblasts carry several putative membrane-associated collagen receptors (references summed up by Nagata and Yamada, 1986). After malignant transformation a type I collagen-binding protein is found to be altered in both quantity and degree of phosphorylation (Nagata and Yamada, 1986). The speculation seems warranted that it plays a role in information transduction, linking the effects of collagen as a signal substance to eventual effects on the genome, similar to the role of protein kinase C (Nishizuka, 1986).

111. The Roles Played by Stromal Adhesive Glycoproteins During recent years insights have been gained into the nature of adhesive glycoproteins as dynamic constituents of the stroma. It seems clear that as tumor cells spread, they have to break old adhesions and yet be capable of forming new adhesions at secondary sites (McCarthy et al. , 1985). Most extensively studied are the extracellular adhesive glycoproteins fibronectin and laminin. Both affect the adhesion of cells to substrates and have effects on migration, morphology, growth, and differentiation of cells.

A. FIBRONECTINS IN MALIGNANT GROWTH The structure and physiological roles of fibronectins are reviewed in papers by McCarthy et al. (1985), Yamada et al. (1985), and Hynes (1986). Fibronectin molecules are dimers consisting of two similar subunits which are joined at one end by disulfide bonds. Each protein chain (MW %250,000) is subdivided in a series of domains, within each of which the protein chain is tightly folded. Each domain can bind specifically either to cell surfaces or to components of the extracellular matrix (collagen, heparin, fibrin), thus binding a cell to the structures of the extracellular matrix. Pericellular insoluble fibronectin plays a role in normal cell adhesion. Fibronectins may polymerize, perhaps after stretching (Hormann, 1982), and form adhesive webs that appear to promote the migration of many kinds of cells during development and in the course of wound healing. Fibronectin interacts across the cell membrane with the intracellular actin filaments, a main constituent of the cell’s


171

locomotory apparatus. This interaction is mediated by the so-called 140K complex, a protein situated in the cell membrane. Many malignant cells synthesize subnormal amounts of fibronectin and moreover fail to deposit it in an insoluble cell coat (Ruoslahti, 1984). However, lack of cell surface fibronectin does not correlate very well with tumorigenicity. Brown and Parkinson (1984, 1985) have observed that various transformed epithelial cells-and most cancers are of epithelial origin-continue to synthesize fibronectin and retain it on their surfaces. They point out that most studies on the effect of transformation on fibronectin have been carried out on fibroblastic cells (Alitalo and Vaheri, 1982). Ruoslahti (1984) nonetheless considers it likely that lack of pericellular fibronectin influences migration and metastatic capacity of malignant cells. Although fibronectin in a polymerized state in the extracellular matrix provides pathways for cell migration in embryogenesis, and during wound healing a similar role in malignant invasion in vivo so far has not been strictly proven. Migration of normal and especially of malignant cells in vitro is enhanced by contact with fibronectin (Ali and Hynes, 1978; Schor et al., 1981; Mensing et al., 1984). In an in vitro study employing metastatic melanoma cells, it was found that the human fibronectin molecule contains two distinct domains with adhesion-promoting qualities, only one of which promotes haptotactic motility of tumor cells. This indicates that haptotatic migration is not due to a simple adhesion gradient of fibronectin (McCarthy et a l . , 1986). It may further be of interest that tumor cells can produce polypeptides which are capable of releasing fibronectin from fibroblasts (Keski-Oja et al., 1979). Stenn et al. (1983) have relativized the role of fibronectin, since they showed that epithelial cells employ several mechanisms of spreading and that none of a series of substances so far studied, including fibronectin, is crucial. In spite of many uncertainties, it is felt that fibronectin in concert with other adhesive glycoproteins probably acts to keep normal cells in place and regulate migration. Alterations in any of these proteins could underlie the vagrant behavior of cancer cells (Hynes, 1986). A quite different function has also been allotted to fibronectin in the extracellular matrix, both in wound healing (Kurkinen et a l . , 1980) and in desmoplastic stromal reaction in carcinoma of the human breast (Lagack et a l . , 1985). Since fibronectin was seen to appear parallel with type I11 collagen, it is felt that fibronectin could function as a primary scaffolding for connective tissue deposition. The association between type I11 collagen and the glycoprotein fibronectin would explain the argentophilia of reticulin, so well known to classical histologists. An intriguing finding, difficult to fit into the picture outlined so far, is that in an in vitro system (chick embryo fibroblasts infected with Rous sarcoma

112

A . VAN DEN HOOFF

virus), gelatin-binding fragments of fibronectin displayed transformationenhancing activity (De Petro et al., 1981).

B. LAMININ IN MALIGNANT GROWTH The structure and biological activities of the other adhesive glycoprotein, laminin, are reviewed by Kleinman et al. (1985) and by McCarthy et al. (1985). Laminin is a cross-shaped glycoprotein (MW lo6)present in basement membranes where it links its constituents, i.e., collagen type IV and heparan sulfate, to one another and to epithelial cells. The binding sites for these tissue constituents have been identified. Laminin probably has a major role in organizing basement membranes, in maintaining their stability, and in anchoring cells to the membrane. In the context of this review, other regulating activities of laminin may be mentioned. It influences cellular morphology, depending on the cell type, and promotes growth. During development it has a key role in differentiation. It promotes cell migration, a property that probably is important during embryogenesis. Like fibronectin, laminin affects the cytoskeleton. A cell membrane-bound protein that may join the contractile cytoskeleton to laminin has been isolated in tumor cells and designated connectin (Brown et al., 1983; Malinoff et al., 1983; Rao et a l . , 1983). The role of the extracellular matrix in tumor invasion and metastasis, stressing the part played by laminin, has also been reviewed by Liotta (1986). The reader is also referred to Section IV on basement membrane involvement in malignancy. The first and perhaps crucial step in invasion of the extracellular matrix is the attachment of the tumor cells via their surface receptors to laminin in the basement membrane. Receptors for laminin may be altered in number, distribution, and degree of occupancy in tumor cells. Terranova et al. (1982, 1984) have shown a correlation between malignancy (metastatic potential) of tumor cells and the number of laminin receptors. At least two mechanisms may explain this correlation (Liotta, 1986). First, unoccupied receptors on the tumor cell may bind directly to host laminin. Second, if receptors are occupied with laminin, this surface laminin can form attachments to other extracellular matrix constituents. It may be of potential importance that a laminin fragment that binds only to the cell’s receptor (understandably) blocks both mechanisms (Barsky et a l . , 1984). Since fibronectin was found to suppress invasiveness (Terranova et al., 1984), it appears that the two adhesive glycoproteins in a way act in opposing fashions. The roles of stromal adhesive glycoproteins in invasiveness summarily sketched in this section probably are just fragments of an intricate system of integrated mechanisms regulating attachment and movement of malignant cells once they are no longer contained and have entered the stromal compartment.


173

IV. The Role of the Basement Membrane in lnvasiveness When an epithelium starts growing invasively into the stroma, the basement membrane is the structure that has to be dealt with first. Recent data seem unequivocal: invasive growth of the majority of epithelial tumors is accompanied by the production of large amounts of lytic enzymes that degrade the constituents of the membranes. Certain data have to be brought up, however, that are difficult to reconcile with the relatively simple concept of a tumor penetrating a thin passive membrane. A number of recent reviews discuss the normal structure, composition and functions of basement membranes (e.g., Risteli and Risteli, 1981; Briggaman, 1982; Linsenmayer et al., 1984). The basement membrane is an epithelial product. Its maintenance may in part be mediated by stromal constituents such as fibrillar collagen (Meier and Hay, 1974; Emerman and Pitelka, 1977; David and Bernfield, 1982). Ultrastructurally the epithelium and the stroma are separated by a basal lamina consisting of a very thin osmiophilic lamina rara (lucida). The basal lamina owes its firmness to the presence of collagen, mainly of type IV, whose molecules form a network. In addition, heparan sulfate-containing proteoglycans and the glycoproteins laminin and fibronectin are present. The membranes are thought to serve as scaffolds for organizing and anchoring epithelia. Besides, quite a different category of functions has gradually come to light: basement membranes may assume active instructive roles in development, regeneration, and tissue homeostasis (Linsenmayer et al., 1984). As regards the basement membrane as the first barrier to be breached when epithelial growth becomes invasive, significant data have been obtained. A collagenase that specifically attacks type IV collagen has been demonstrated in a variety of malignant tumors. This points to lysis of the constituent which imparts firmness to the membrane (Liotta, 1985, 1986). Most morphological studies of the behavior of basement membranes in malignant growth make use of immunocytochemical detection methods of the basement membrane components type IV collagen and laminin. The basement membrane around tumors may be locally absent or fragmented. For a variety of tumors it has been found that the less differentiated the tumor, the more defective the membrane (Albrechtsen et al., 1981; Meyer et al., 1982; Barsky et al., 1983; Cam et al., 1984a,b; Kallioinen et al., 1985a,b). Murine tumor cell hybrids that differ in metastatic potential also differ in the expression of collagenase type IV (Turpeenniemi-Hujanen et al., 1985); the two characteristics were found to be positively correlated. Benign epithelial tumors are surrounded by an intact basement membrane. So malignant tumors obviously differ in their capability enzymatically to degrade basement membranes. It seems plausible that an epithelial tumor that

174

A. VAN DEN HOOFF

synthesizes substantial amounts of collagenase type IV not only penetrates its own local basement membrane, it also has the propensity to erode basement membranes of local blood vessels. When its cells thus enter the bloodstream they may be supposed to penetrate vessel walls elsewhere in the organism, giving rise to metastases. Liotta and his co-workers have proposed a three-step hypothesis describing the sequence of events during breaching of the basement membrane and invasion of the extracellular matrix (Liotta et al., 1977; Liotta, 1986). Tumor cells with their specific receptors bind to laminin of the basement membrane as a first step (Turpeenniemi-Hujanen et al., 1986). This induces the second step, dissolution of the basement membrane by type IV collagenase activity. The third step is tumor cell locomotion. So, as already pointed out in Section III,B, it has become evident that laminin plays a crucial role in invasion and metastasis (Terranova et al., 1982; Barsky et al., 1984). Tumor cell populations are heterogeneous, and Liotta (1986) has argued that in the course of carcinogenesis a more aggressive subpopulation is selected in which the metastatic phenotype is genetically linked to type IV collagenase expression. The concept of the significance of basement membrane penetration for invasiveness has a practical consequence, since it has led to the development of a reconstituent model, consisting of laminin and collagen type IV on a base of type I collagen, to measure invasiveness (Terranova et al., 1986). A surprising fact has come to light (Woodley et al., 1986). Normal adult human migratory keratinocytes cultured in contact with interstitial collagen produce collagenolytic enzymes active against types I and IV collagen. In this respect they appear to be similar to highly metastatic cells. This finding seems to unsettle the notion that collagenase type IV activity is a specific attribute of the metastatic cell. As mentioned, the basement membrane is not merely a mechanical scaffolding. It has other functions as well, and perhaps data which will be presented below and that do not seem to fit in the obvious and welldocumented model described so far, have to do with these functions. Light-microscopic and ultrastructural data suggest that more is going on than a simple piercing of the basement membrane resulting from lytic processes. When invasion is imminent, a number of structural alterations are described by various authors studying the epithelial-stromal junction in different experimental and clinical malignant conditions (Tarin, 1967, 1969; van den Hooff, 1962; van den Hooff and Tigchelaar-Gutter, 1983b; Woods and Smith, 1969; Smith, 1972; Frithiof, 1972; Jao and Gould, 1975; Gould and Battifora, 1976; McNutt, 1976; Pitelka et al., 1980; Otsubo and Kameyama, 1982). See also Section VII. In all these studies, essentially the same


175

phenomena are described. Under the light microscope the membrane looks split up into irregular fibers, often without parallel orientation. O n the electron microscope level, interruptions are frequent but often on a very small scale, making room for only very small cellular projections. Reduplications of the basal lamina are common. Ectopic irregular fragments of basal lamina with masses of attached anchoring fibrils are described, separated from the epithelium by a dense layer of collagen (van den Hooff, 1983). Malignant epithelial cells may continue to synthesize basement membrane material (van Cauwenberge et a l . , 1983); this production is most clearly defined in well-differentiated tumors (Cam et al., 1984a,b). According to Gabbert (1985), loss of tumor basement membrane is the expression of the insufficiently of poorly differentiated tumor cells to synthesize a regular basement membrane rather than the result of destructive tumor growth. Some normal epithelia require contact with collagen for basement membrane assembly to occur. Results obtained with transformed mouse mammary epithelial cells grown on collagen suggest that transformation inhibits this interaction (Warburton et a l . , 1986). In certain neoplasms production of basement membrane material may be qualitatively deranged. David and Bernfield (1982) and David and van den Berghe (1983) observed that transformed mouse mammary epithelial cells grown on collagen synthesize undersulfated basement membrane proteoglycans. The authors postulated that the effect of collagen on basement membrane production by transformed cells is reduced and that as a consequence undersulfation may cause disturbed assembly of the basal lamina. Alterations in the amount and nature of glycosaminoglycans produced were also observed by Luikart et al. (1983) in the case of B16 murine melanoma cells growing on collagen. Data given above point to a radical derangement of the dynamics at the epithelial-stromal junction. The high rate of degradation of the basement membrane in malignant growth may thus be a part of an enhanced and spatially disorganized turnover. It is of significance that lysis of basement membrane is not a sine qua non of invasiveness. Basal cell carcinoma of the skin that is locally invasive but nonmetastatic is surrounded by a continuous basement membrane (van Cauwenberge et al. , 1983; Liotta, 1985). Well-differentiated squamous carcinomas of the larynx (Cam et a l . , 1984a,b) or the skin (Gusterson et al., 1986) also show intact basement membranes, whereas poorly differentiated squamous carcinomas are characterized by discontinuity or loss of the basement membrane.

176

A . VAN DEN HOOFF

The instructive role of basement membranes is crucial in epithelialmesenchymal morphogenetic interaction during embryogenesis (Bernfield, 1983; Bernfield et al., 1984). It is also evident in the development of certain cancers. It has been observed (Lewko et al., 1981; Wicha etal., 1981) that for development of adenocarcinoma of the mammary gland the synthesis of a basement membrane seems necessary, since blocking of basement membrane collagen type IV deposition inhibits the growth of carcinogen-induced rat mammary tumors. The nature of the putative instructive signals is not clear. Signals given by the basement membrane as elements in stromal-epithelial interactions are possibly of a chemical nature. This surmise is supported by the histochemical variability among basement membranes of different organs detected by monoclonal antibody analysis (Fitch and Linsenmayer, 1983; Wan et al., 1984; Hessle et al., 1984). Hessle et al. related these differences to unique components present in addition to ubiquitous constituents such as type IV collagen and laminin. Gerfaux et al. (1979) have postulated that a role has to be attributed to sites of the basement membrane consisting of associations between collagen and glycoproteins. In mammary differentiation it is laminin that plays a role; its effect may be mediated via the cytoskeleton (Blum and Wicha, 1985). It has been argued (Ingber and Jamieson, 1982, 1985; Ingber et al., 1986) that information contained in the basement membrane is not entirely of a chemical nature. Ingber and his coauthors have introduced to biology the concept of the so-called tensegrity system, a spatial arrangement of discontinuous compression-resistant elements held together by a continuous series of tension elements. Such an architectural system of physical forces determines shape and thus acts in an informative manner. If a cell is an tensegrity system, then physical information which is transduced via the cytoskeleton could eventually determine the metabolic condition of the cell. The basement membrane because of its anchoring function may be viewed as part of a larger tensegrity system, providing a continuity between epithelial and mesenchymal societies. This common structural element would serve to distribute equally and communicate physical forces between neighboring tissues. Maintenance of basement membrane integrity thus assures stability of normal epithelial morphology. When this architectural scheme breaks down-i.e., if the continuity of tension transmission is interrupted-information contained in the normal system would be disturbed and malignancy may ensue. In trying to summarize the data presented in this section, the conclusion is inescapable that we have no satisfactory picture of the manifold and intricate functions of the basement membrane. The current view of the membrane’s role in malignant growth may underestimate its dynamic and instructive nature. Fundamental knowledge is lacking on its role in development in the normal adult organism and during malignant growth.


177

V. Characteristics of Fibroblasts in Malignant Conditions Under this heading two quite different aspects may be discussed: first, the properties of fibroblasts in the stroma of the primary tumors, and second, deviations of a generalized nature observed in fibroblasts obtained for other localizations, e.g., skin.

A. LOCALCHANGES The fibroblasts in the stroma of primary tumors have been observed by a number of authors to exhibit features reminiscent of their fully malignant neighboring cells. Delinassios et al. (1981) described “double minutes” in cultured fibroblastlike cells isolated from various human cancers, pointing to properties hitherto considered typical for malignant cells. Nordbye (1969) and Chaudhuri et al. (1974) observed that fibroblasts cultured from stroma of human cervical carcinomas showed the disorganized growth patterns typical of malignant cells. Similar uncontrolled in uitro growth of fibroblasts obtained from benign and malignant tumors of the breast were described by Delinassios et al. (1983); the cells were otherwise normal. Chaudhuri etal. (1975) described a different agglutinability of fibroblasts underlying human cervical carcinomas and their precursor states. Fibroblasts in the stroma of both benign and malignant tunors of the rat thyroid, induced by thyroid-stimulating hormone, showed extended life spans, especially in cases of overt malignancy, presumably as a response to signals from the stimulated thyroid epithelium (Garbi et al., 1986). These data are suggestive of the supposedly benign conditions as premalignant states. Todaro et al. (1980) have found that a number of human tumor cell lines in culture release transforming growth factors that confer the transformed phenotype on untransformed fibroblasts.

B. GENERALIZED CHANGES Still more intriguing than these data pointing to an involvement of stromal cells in the process of local malignant growth, are data pointing to deviations of fibroblasts in localizations unrelated to the tumor. Kopelovich and his coworkers studied the in uitso behavior of skin fibroblasts obtained from patients suffering from hereditary adenomatosis of the colon and rectum (Pfeffer et al., 1976; Kopelovich 1980, 1982a,b; Kopelovich and Gardner, 1983; Kopelovich et al. , 1977, 1979, 1985) and discovered a number of deviations. Actin patterns were disorganized: cytoplasmic cables were replaced by a diffuse matrix. Requirements for in uitro growth were altered 1% fetal calf serum was sufficient versus 15% for normal fibroblasts. Growth patterns were disorganized. It was

178

A. VAN DEN HOOFF

found that tumor promoter alone induced neoplastic transformation, which may indicate that the fibroblasts existed in an initiated state. Some of these alterations were also found in asymptomatic relatives. Azzarone and his co-workers (Azzarone et al., 1976) observed spontaneous transformation in vitro of skin fibroblasts obtained from patents with lung cancer. Cells were characterized by abnormal ploidy, a capacity to form colonies in semisolid agar, the formation of foci of piling up cells among monolayers, and the acquisition of infinite life span. The same group (Azzarone et al., 1984) discovered similar abnormal properties in vitro of skin fibroblasts from patients with breast cancer. Whereas an (expected) inverse correlation was found between the doubling potential of fibroblasts and the donor’s age for cells from patients with benign lesions, no such correlation was found with cultures from cancer patients. Dolfini et al. (1976) described how fibroblast-like cells in skin biopsy samples from lung cancer patients transformed spontaneously in vitro losing their fibrin clot retractile activity. Smith et al. (1976) cultured skin fibroblasts in osteosarcoma patients and discovered that the cells showed a greater ability to grow in immunosuppressed mice than did normal fibroblasts, suggesting that all fibroblasts may have an increased propensity for malignant transformation. Schor and co-workers (1985a,b, 1986) studied skin fibroblasts from patients with breast cancer, melanoma, polyposis coli, and retinoblastoma, and observed altered fetal-like migratory behaviors. The phenomenon was more prominent in patients with a positive family history of breast cancer and was also seen in unaffected first-degree relatives of patients. Since the generalized abberration of fibroblasts is also seen in the next-ofkin of patients, an unlikely explanation is the presence of transforming factors produced by overtly malignant cells such as described by Todaro et al. (1980). Two hypotheses on the relationship between malignant growth and generalized fibroblastic abnormalities have been proposed (Schor et a l . , 1985a). The genetic or epigenetic defect expressed by the fibroblasts may be similarly expressed by the target epithelial cell population. Alternatively, it is conceivable that deviant fibroblasts are dysfunctional in the sence that normal inductive interactions with incipiently malignant cells are disturbed. The implication of the second mechanism as regards stromal involvement in malignant growth would be momentous. The propensity to get certain types of cancer would be partly determined by genetic or epigenetic conditions of the mesenchymal tissues.

VI. Primary Stromal Disorders as Putative Factors in Carclnogenesis Considering connective tissue involvement in cancer a pertinent question is which role may possibly be attributed to wounding-or rather tissue damage


179

in general-subsequent healing, and scar formation in the process of carcinogenesis. The information on the supposed causal relationship between wounding and cancer growth is fragmentary. In a paper by Bang (1925) “Le cancer des cicatrices,” mention is made of historical case reports on cancer (epitheliomas) resulting from old scars. Lumikre (1929), in his book “Le cancer, maladie des cicatrices,” considered the presence of longstanding wounds with scar formation an essential condition for the origin of epitheliomas. An additional factor was repeated traumas.

A. WOUNDING AND WOUNDHEALING In 1924 Deelman described his classical experiments in which wounds were inflicted inadvertently upon the skin of mice treated with tar. He noticed how malignant growths made their first appearances at the margins of healing wounds and he concluded that tarring in itself may not be sufficient to elicit cancer and that an indifferent stimulus applied to the epithelial cells contributed to eventual malignancy. The carcinogen in his view resulted in a precarcinomatous condition in which cells still look normal; it was accidental stimuli, e.g., cell damage and regeneration, that led to manifest malignant growth. In the same year Mertens (1924) took issue with Deelman on one point. Studying Deelman’s figures, he concluded that tumors arose outside the wound margins and he proposed that the supporting connective tissue was abnormal and contributed somehow to malignancy. Gillman et al. (1955) adduced evidence that the reactions of the epidermis to injury was profoundly influenced by the state of the connective tissue. Injury to the skin of human volunteers led to “elastotically degenerated’’ collagen which in its turn evoked invasive, albeit benign, growth of epidermis. The authors argued that in clinical skin cancer in humans a similar mechanism involving elastotic collagen is operative. Raeburn and Spencer (1957) put forward an analogous view as regards malignancies of the lung. More recent data are equivocal as regards the supposed role of the stroma during wound healing as a promotional factor in carcinogenesis. Hennings and Boutwell (1970) elicited skin tumors in mice by making skin incisions after initiation with a carcinogen. Their explanation was that wound healing enhanced the mitosis rate, which in its turn had the effect of a tumor promoter. Pozharisski (1975) studied the combined effects of carcinogen administration and chronic injury to the cecal mucosa (caused by the presence of a ligature) in rats. A 4-fold increase of tumor development at the sites of injury was seen. Argyris (1976, 1980) combined initiation by a single application of a carcinogen with abrasion of the epidermis in mice. The idea was to induce hyperplasia of the epidermis, leaving the dermis intact. This treatment resulted in epidermal tumors, which suggested that epidermal hyperplasia is a

180

A. VAN DEN HOOFF

sufficient stimulus for promotion. However, it should be remarked that, although damage to the underlying dermis was minimal and only little bleeding was observed, it cannot be excluded that the stroma may be somehow involved. Konstantinidis et al. (1982) saw a higher incidence of tumors of the soft tissues of the oral cavity of rats as a result of intraperitoneal injection of a carcinogen combined with continuous irritation of the buccal mucosa by a stainless-steel wire. Dolberg et al. (1985) observed that local wounding plays a part in the formation of tumors following intramuscular injection of Rous sarcoma virus in chicks. As the wound heals it loses this ability to act as a cocarcinogenic factor. Manipulation of the epidermis by massage that elicits a proliferation response without wounding is not a tumor-promoting stimulus (Clark-Lewis and Murray, 1978). So it is wounding rather than the proliferation response that seems to be a critical factor. Marks et al. (1982) have reasoned that, since superficial wounding elicits epithelial hyperplasia but does not promote tumor development, whereas deep wounding has an additional tumor-promoting effect, it is the connective tissue damage that presents such an additional stimulus, possibly provided by certain growth factors (“wound hormones”), either blood-borne or of mesenchymal origin.

B. FIBROSISAND

SCAR CANCER

A related problem bears upon the so-called scar cancer described in the lungs. Friedrich (1939) and Rossle (1943) were the first to point out the spatial relationship often seen in histological preparations between peripheral lung cancer and collagenous scars in the vicinity of the pleura. O n the basis of his microscopic findings during postmortem examinations, Friedrich argued that the scar tissue was older than the cancer. Consequently the presence of scar tissue somehow contributed to the generation of malignant growth. The frequent concurrence of fibrotic lesions and adenocarcinomas in the lung now seems definitely established (Raeburn and Spencer, 1957; Carroll, 1962; McDonnell and Long, 1981), but as to the interpretation, opposite views are expressed. Friedrich’s original view was that external unknown influences (tobacco smoke?) resulted in a cancer disposition (Krebsbereitschaft), but that it was the presence of a scar that formed an additional factor that elicits manifest cancer. Until recently this idea was generally accepted but an alternative view has emerged in recent publications, viz., that the causal relationship is the other way around: one is dealing with a desmoplastic reaction in and around certain tumor types. Raeburn and Spencer (1957) felt that scar tissue irrespective of its causative condition (most often tuberculosis; Yokoo and Suckow, 1961) exerts some


181

influence on epithelium, in time leading to malignant transformation. It could be elastotically degenerate collagen that predisposes to the development of cancer. Balo et al. (1956) found epithelium proliferations especially on the borders of old infarcts. They considered these alterations as precancerous lesions and stated that “peripheral stratified epithelial carcinoma can develop at the site of scars originating from infarcts. ” Stanton and Blackwell (1961) produced experimental pulmonary infarcts in rats and administered a carcinogen intravenously. The infarcts were found to be the sites of invasive epidermoid carcinomas. Ohwada et al. (1980) also elicited experimental infarctions of the lung in rats. The lesions resulted in areas of fibrosis. Repeated intratracheal instillations of polonium 210 led to carcinomas in close proximity to the localized pulmonary fibrosis. It may be stressed that these findings on the role of scars originating from infarcts seemingly speak in favor of fibrosis as a causative factor rather than as a consequence of malignancy. McDonald and Long (1981) stated that, although many adenocarcinomas of the lung are scar related, the view is unacceptable that malignancy occurs irrespective of smoking habits. This opinion parallels that of Friedrich (1939), who saw scarring as an additional rather than a primary factor. A similar spatial relationship between fibrosis and malignant growth has been described in cases of diffuse interstitial pulmonary fibrosis (“honeycombing”) (Meyer and Liebow, 1965; Fraire and Greenerg, 1973). These conditions may result from chronic irritation or infections (Scadding, 1960). Interstitial pulmonary fibrosis may be a consequence of diffuse connective tissue diseases, specifically scleroderma. The probable relationship between this type of longstanding pulmonary interstitial fibrosis and cancer of the lung has been discussed by Medsger (1985), who mentions the possibility of a defective cancer surveillance, since the connective tissue conditions are characterized by immune abnormalities. A number of data are suggestive of a pathophysiological relationship between scleroderma and breast cancer (Roumm and Medsger, 1985). A possible involvement of preceding fibrosis in some special types of clinical cancer, uiz., lung cancer resulting from chronic asbestos exposure and cancer of the bladder in bilharziasis following Schistosoma hanatobium infection, has been proposed (van den Hooff, 1984). The data mentioned so far seem to be in accordance with the idea that it is the scar tissue or diffuse fibrosis which somehow contributes to epithelial malignancy. As already touched on, the issue is controversial and recent data pertinent to this disagreement are suggestive of a quite different causative mechanism: the tumor, owing to its desmoplastic nature, allegedly gives rise to the deposition of collagen that impresses one as a scar. Shimosato et al. (1980) have put forward a number of microscopic observations arguing against the scar as a primary

182

A. VAN DEN HOOFF

lesion, i.e., in favor of secondary desmoplasia. Madri and Carter (1982, 1984) examined the types of collagen present in fibrotic parts of adenocarcinomas by immunofluorescent analysis. They found mainly type I11 and some type V. Type I11 is characteristic of immature, young connective tissue. Areas of interstitial fibrosis in the same lungs but not associated with the tumor showed increased staining of types I and V. Their conclusion was that the fibrosis was the expression of a host response to the tumor. El-Torky et al. (1985), using similar techniques, also found higher amounts of collagen type I11 in pulmonary adenocarcinomas, and they also detected type IV. These findings in the authors’ view point to recent formation in areas adjacent to cell nests and in the periacinar area, i.e., a desmoplastic reaction of host tissue. At the periphery of the tumor mass collagen types I and V were increased, a finding consistent with old fibrosis. One wonders whether in both these studies the augmented presence of type I collagen in areas unrelated to the tumor does not point to fibrosis that precedes formation of the tumor, which in it turn elicits the synthesis of collagen type 111. Although these findings based on immunofluorescent analysis of collagen types in pulmonary neoplasms are significant, the wealth of data on cancer arising from scars or diffuse interstitial pulmonary fibrosis cannot be lightly dismissed, The most plausible concept of the course of events that fit all morphological and chemical data available so far, is the following. Excessive fibrosis (diffuse interstitial or in the shape of a scar) somehow has a promotional or progressional effect on epithelial cells that have become initiated by a primary carcinogenic agent. The developing tumor may give rise to excessive collagen production (desmoplasia) as a host response, which obscures the ultimate picture as far as collagen types are concerned. Circumstantial evidence is suggestive of a promoting effect of scars in other organs as well. Cass et al. (1976) observed that local recurrences of tumors following surgery for adenocarcinoma of the colon and the rectum in 92% developed in structures continuous to the operative area of incision. Carcinomas developing in the stomach after partial resection showed a definite predilection for the area of the anastomosis (Morgenstern and Yamakawa, 1973; Domellof et af., 1976; Domellof and Janunger, 1977; Osnes et a l . , 1977; Miederer et al., 1977). If scar cancer develops, it takes a long time. In humans it is seen many years after damage has been inflicted. There is a curious discrepancy between this type of cancer and those in which wounding plays a crucial role. The data presented by Dolberg et al. (1985) point to tumor-eliciting factors which are active during the earliest phases of wound healing, perhaps growth factors that are released after wounding. So it would appear that, although wounding and scar formation are often discussed as being reducible to a common denominator, essentially different mechanisms are operative in the two conditions,


183

Summing up this section, the supposition that it is stromal processes during wounding and the early phase of healing that contribute to the promotion of malignancy has found some support. The concept of scar cancer as a genuine entity may still be considered valid.

VII. Stromal Alterations Preceding lnvasive Growth Numerous investigators studying clinical and experimental cancers, elicited by carcinogen treatment, have described microscopic changes indicating that the stroma undergoes radical changes antecedent to manifest malignant growth (reviewed by van den Hooff, 1983). Light-microscopic changes can be summarized as follows. Collagen seems to disintegrate; fibers take up elastin stains (“elastotic degeneration”). Elastin is lost. Fibroblasts are markedly activated. Metachromasia, pointing to a rise in glycosaminoglycans, is enhanced. The number of mast cells substantially increases. Macrophages and lymphocytes increase in varying degrees. This microscopic picture has been supplemented with ultrastructural and biochemical data. Electron micrographs point to diffuse collagenolysis and phagocytosis of intact collagen fibrils. Quantitative studies have confirmed a gradual loss of stromal collagen in the course of carcinogen treatment. The decrease was not correlated with neutral mammalian collagenase activity (Wirl, 1977). A factor in the loss of collagen in the course of carcinogenesis may also be a decrease in synthetic activity as part of collagen turnover. A great number of findings have been reported that point to a reduction in collagen formation in vitro by fibroblasts and other collagen-producing cells, which are transformed by various means (Green et al., 1966; Howard et al.. 1978; Declos and Blumberg, 1979; Krieg et al., 1980; Bateman and Peterkovsky, 1981; Sandmeyer et al., 1981; Keski-Oja et al., 1982b; Dion et al., 1982; Sobel et al., 1983; Schmidt et al., 1985; Setoyama et al., 1985). Parker et al. (1982) observed that methylation of the procollagen a1 (I) and a2 (I) genes was increased in simian virus 40-transformed human fibroblasts concurrent with the loss of type I collagen synthesis. An inverse correlation of collagen production to anchorage independence and tumorigenicity was found by Smith et al. (1983). It is conceivable that stromal cells in vivo may reduce their collagen production because they are to a certain extent affected by the same carcinogenic influences that eventually result in overt malignancy of the epithelial component (see also Section V,A). Data on the effects of tumor promotion are confusing. Some authors (Declos and Blumberg, 1979; Dion et al. , 1982; Sobel et al., 1983) have found that collagen synthesis could be inhibited in vitro by treatment of fibroblasts with tumor-promoting phorbol esters. Other evidence on the effect of tumor

184

A. VAN DEN HOOFF

promoters seem to be at variance with these data. In mouse skin, initiated with a single dose of methylcholanthrene, treatment with the promoter 12-O-tetradecanoylphorbol13-acetate (TPA) led to a reduction of collagen content. This was the result of an increased collagenolysis, in part directly related to tumor promotion, and a less marked rise in collagen synthesis (Marian and Mazzuco, 1985;Marian, 1986). Treatment of human squamous epidermoid carcinoma cells with TPA in vitro rapidly produced a 6-fold increase in the production of the basement membrane components laminin and type IV collagen (Panneerselvam et al., 1985). Ribbert, as early as 191 1, postulated that at the site of primary cancer the component tissues cooperate in a process that ultimately leads to malignancy of one of the components, the epithelium. Up to the present day Ribbert’s idea runs as a continuous thread through the contemplations on carcinogenesis. Smithers (1962, 1983) has put forward the view that cancer is a disease of tissue organization, that it is organizational breakdown that leads to progressive loss of growth control, producing an excess of tissue no longer coordinated with the whole. He stressed that implant tumors and metastases are unreliable indicators of modes of tumor development. Primary growth is a gradual process restricted to few locations of the total body mass in which environmental influences can occur and where vulnerable control processes necessary for growth, repair, and regeneration normally are operative. This process differs essentially from what happens when an established tumor sheds clonogenic cells into the circulation. It has been speculated that “elastotically degraded’’ collagen exerts a positive tropism at the epithelium (Vernoni, 1933, quoted by Maltoni and Prodi, 1960; Gillman et al., 1955; Mackie and MacGovern, 1958). Other authors (Vasiliev, 1958; Maltoni and Zajdela, 1963) interpreted stromal changes as an inflammatory proliferation of undifferentiated connective tissue, a condition essential for invasive growth. Data that are relevant to the intriguing possibility of a stromal role in carcinogenesis have resulted from transplantation experiments. The idea was to recombine untreated epithelium with stroma from carcinogen-treated skin or mucous membrane. Billingham et al. (1951), Orr (1958,196l), and Orr and Spencer (1972) in a series of studies observed tumor formation when untreated mouse epidermis was transplanted onto carcinogen-treated stroma. Orr (1961) supposed that stromal alterations (“permutation”) plays a primary role in the development of neoplastic qualities of the overlying epidermis. Hodges et al. (1977) did similar transplantations in carcinogen-treated urinary bladder of the rat. This organ has the advantage that epithelium and stroma can be readily separated, so that the criticism that hair follicles are left behind in the stroma is not valid. In this study untreated epithelium, brought together with carcinogen-treated stroma, acquired characteristics of


185

carcinogen-treated stroma. It was concluded by Hodges that the “stroma of the treated bladder has an instructive influence on normal epithelium eliciting the appearance of new behavior resembling that manifested by neoplastic epithelium. ” In a comprehensive review to which the reader is referred for a wealth of details, Hodges (1982) has stated that “stromal influences can be implicated in the carcinogenic process both in a permissive and instructive capacity, although their role at different stages of neoplasia for initiation to overt neoplasm remains to be defined in specific terms. ” It is of interest to consider the data presented so far in this section against a more general background of physiological stromal-epithelial interactions. Embryonic development depends on precisely timed and localized cellular interactions. Stroma possesses qualities that generate specific genetic expression and regional specialization of epithelia (Hodges, 1982). Certain stromal-epithelial regulatory mechanisms persist in adult tissues (Cunha et al. , 1985). The extracellular matrix is postulated to exert chemical and physical influences on the cell via transmembrane receptors and the cytoskeleton (review by Bissell et al., 1982). Most spontaneous tumors arise from epithelia, and it has been argued by Hodges (1982) and van den Hooff (1984) that dysfunction of these mechanisms plays a role in carcinogenesis in the sense that it elicits a crucial step in tumor promotion or progression. In connection with stromal changes concurrent with incipient epithelial malignancy, the ontogenetic aspect of the stromal-epithelial relationship (i.e., inductive influence of fetal mesenchyme) may be further elaborated. The following data seem relevant. First, fetal stroma plays an essential role in embryonic inductive reactions (see Grobstein, 1967, for a review). Second, there is evidence for a role of collagen in these interactions (reviewed by Hay, 1981). Third, evidence has been presented to the effect that interactions between fetal mesenchyme and epithelium influence the development of neoplasms. Dawe (1972) in a series of experiments demonstrated that polyoma virus-induced tumors of salivary gland rudiments of the mouse embryo only developed in the presence of mesenchyme. Sakakura et al. (1981) found that grafted salivary mesenchyme accelerated tumor virusinduced mammary carcinogenesis in the adult mouse. Sakakura (1983) later observed that in the stroma of the adult mammary gland of the mouse “embryonic change’’ may occur and that as a consequence the epithelium proliferates and shows an enhanced tendency to the development of cancer. Since synthesis of type I collagen trimer is thought to represent the expression of an embryonic phenotype (Bornstein and Sage, 1980), it may be speculated that type I collagen trimer, which is also found in the stroma of malignant tumors (see Section II,I), may be instrumental in the induction of tumor promotion (van den Hooff, 1986).

186

A. VAN DEN HOOFF

VIII. Concluding Remarks In the foregoing sections various modes of interaction between malignant cells and stroma have been described: Malignant cells have been shown to produce various lytic enzymes, which attack the stroma or to induce fibroblasts to synthesize collagenolytic, elastolytic, and glycosaminoglycan-degrading enzymes (Sections I1,B-FJ ,K). In some cancers tumor cells stimulate fibroblasts to produce stromal components: collagen, elastin, glycosaminoglycans (Sections 11,G-K). Basement membranes have paradoxical characteristics, since they may as well fall prey to lysis in invasive growth as constitute an instructive element necessary for the growth of certain cancers (Section IV). Migration of tumor cells is partly determined by versatile adhesive glycoproteins in the stroma (Section 111). Wound healing and especially fibrosis (scars) appear to be possible auxiliary factors in carcinogenesis (Section VI). Stromal cells (local or generalized) may exhibit subtle alterations similar to transformed cells (Section V). There is evidence that stromal alterations preceding manifest malignancy contribute to carcinogenesis (Section VII). In vitro data point to intricate interdependencies between cancer cells and fibroblasts resulting in synthesis of collagenolytic enzymes (Section 11,E). Normal ontogenesis depends on a complex of interactions between various tissues among which the stroma plays an essential role. Epithelial-stromal interactions are also important in the adult organism (Section VII). As regards cancer growth, it has been argued that in this condition microenvironmental signals constitute an essential category of influences contributing to malignancy (Cairns, 1981; Farber, 1984; Rubin, 1985). Taking a comprehensive view of the data on the numerous interdependencies between cancer cells and stroma presented above, one has to conclude that a close cooperation exists between the two tissues and that data on the possible involvement of connective tissue in tumorigenesis fit into the last-mentioned view on the significance of environmental influences. So it can be postulated that (1) both during embryogenesis and in the adult organism epithelia and stroma form an integrated system based on a network of cellular and tissue interactions; (2) during embryogenesis the system is in a dynamic state and serves for orderly organogenesis, whereas in the adult organism it is in a steady state; and (3) in malignant growth the system is effective but deranged. One must ask how these postulates fit into the current view on carcinogenesis. This view hinges on two concepts: the role of (proto-) oncogenes


187

and the multistep mechanism which results in the stepwise progression of malignancy. As regards the activation of cellular protooncogenes to active oncogenes, it is believed that the phenotype of malignant cells is largely determined by dysregulation of these oncogenes. Cooperation between several oncogenes seems to be required to elicit tumorigenicity. Carcinogenesis is a multistep process, each step reflecting the activation of another cellular gene (Weinberg, 1984; Klein and Klein, 1985). After initiation, promotion is necessary for cells to transform. Subsequently tumors become more malignant in a stepwise fashion: progression. A significant role in this tumor progression may be played by disruption of local regulatory mechanisms following damage to the microenvironment (Nowell, 1986). It is in this concept of promotion and progression that stromal influences may fit. It is of possible significance that the proteins encoded by the YLZS oncogenes are localized at the inner surface of the plasma membrane and seem to be involved in the transduction of extracellular signals to an intracellular target, setting off mechanisms that contribute to cellular transformation. Here may lie a point of connection between the part played by oncogenes and the view stressing the importance of supposed microenvironmental influences in the stepwise progression of malignancy. Rubin (1984, 1985) has argued that the causative role of oncogenes tends to be overemphasized. Instead he points to the role of the disturbance of normal homeostasis, i.e., the system of microenvironmental signals that form a network between cells and tissues. Oncogene activation in his opinion could be viewed as an adaptive response to this disturbed interrelationship. “Chromosomal and other genetic changes,” he surmises, have to be “considered epiphenomena rather than causative agencies in tumor formation, although it may not be possible to clearly distinguish cause from effect in the dynamic ever changing process. ” Data collected in the present review, pointing to an altered relationship between malignant tissue and its neighboring stroma, can be envisaged as expressions of a disturbed homeostasis. For the time being it has to be left undecided whether it is oncogene activation or this disturbed homeostasis that has to be considered as the prime mover. It seems true that “oncogenes may represent the solution to only a small part of a very large puzzle” (Weinberg, 1983). Although lysis of stroma (basement membrane plus underlying collagenous strata) seems a self-evident prerequisite for invasive growth, doubt has been expressed as to this mechanism (Section 11,F). Lysis may be absent; on the contrary, excessive production of extracellular matrix may prevail. So things could be quite different. A functional relationship between tumor and stroma seems to remain, though radically altered. Data that seemingly indicate lysis as an essential

188

A. VAN DEN HOOFF

requirement for invasive growth could be interpreted as the expression of a radically altered coexistence of tissues rather than mere lysis. The part played by stromal elements in the maintenance or-as the case may be-dysfunction of neighboring cells or tissues could be of a still more generalized nature than sketched in this review. The possible role of bone marrow stroma cells in the normal or abnormal proliferation and differentiation of blood cells may be illustrative. Data presented by Dexter et al. (1985) and by Gaido and Wierda (1985) are highly suggestive of a disturbed interaction of stromal cells with hematopoietic cells as a decisive factor in blood cell malignancy. Certain oncogenes that result in leukemia primarily affect stromal cells, which react by emanating modulated signals affecting hematopoietic cells in an abnormal way (Dexter et a l . , 1985). Bone marrow stromal cells are found to be more sensitive to benzene than hematopoietic precursor cells, which indicates a regulatory role for the stroma in the mechanism of benzene toxicity (Gaido and Wierda, 1985). Another example is the influence of hormones on the proliferation and the metabolism of certain epithelia, specifically of the mammary gland and the prostate, which are mediated by stromal cells. This may imply that the frequent development of cancers in these organs-also multistep processes (Isaacs, 1985) largely determined by the endocrine conditions-is “likely to involve a loss of coordination and alteration in the interactions between epithelial and stromal cells” (Cunha et a l . , 1985). We have to be aware that vast gaps in our knowledge of tissue interactions in carcinogenesis have to be filled. Detailed information on modes of interaction between cells of different types can only be obtained from in vitro systems, i.e., models that represent only parts of the total system of interactions making up the intact organism. Furthermore, these models in their isolated state may differ fundamentally from the complex pattern of in vivo interactions (Weiss, 1971). Consequently information will of necessity be fragmentary and of limited validity. It seems open to doubt whether it is within our capacity to construe an all-inclusive concept of the immensely complicated system of interactions in the intact organism from bits and pieces of fragmentary and disputable data. The essential difficulty is put into words by Smithers (1983): “Reduction gains precision about parts but at each step loses information about the larger organization it leaves behind. ”

REFERENCES Al-Adnani, M . S . , Kirrane, J . A , , and McGee, J. 0. (1975). Br. J . Cancer 31, 653-660. Albrechtsen, R . , Nielsen, M . , Wewer, U . , Engvall, E., and Ruoslahti, E . (1981). Cancer Res. 41, 5076-5081. Ah. I . U.,and Hynes, R. 0. (1978). Cell 14, 439-446. Alitalo, K . , and Vaheri, A. (1982). A h . Cancer Rex. 37, 111-157.


189

AI-Zuhair, A. G. H., Al-Adnani, M . S., AJ-Bader, A. A,, and Abdulla, M. A. (1986).J Submicrosc. Cyfol. 18, 409-416. Argyris, T. S. (1976).Am. J. Pafhol. 83, 329-340. Argyris, T. S. (1980).J. Invesf. Dennatol. 75, 360-362. Azzarone, B., Pedulla, D., and Romanzi, C . A. (1976). Nafure (London) 262, 74-75. Azzarone, B., Mareel, M., Billard, C . , Scernama, P., Chaponnier, C., and Macieira-Coelho, A. (1984). Inf. J . Cancer 33, 759-764. Azzopardi, J. G., and Laurini, R. N. (1974). Cancer 33, 174-183. Baici, A,, Gyger-Marazzi, M., and Strauli, P. (1984). Inuasion Mefastasis 4 , 13-27. Balo, J., Juhasz, E., and Temes, J. (1956). Cancer 9, 918-922. Bang, F. (1925). Bull. Assoc. Fr. Cancer 14, 203-218., Bano, M., Zwiebel, J . A., Salornon, D. S., and Kidwell, W. R. (1983).J. B i d . Chem. 258, 2729-2735. Bar-Ner, M . , Kramer, M. D., Schirrrnacher, V., Ishai-Michaeli, R., Fuks, Z . , and Vlodavsky, I. (1985). Inf. J . Cancer 35, 483-491. Barsky, S. H., Rao, C . N., Grotendorst, G . R., and Liotta, L. A. (1982). Am. J. Pafhol. 108, 276-283. Barsky, S . H., Siegal, G. P., Jannotta, F., and Liotta, L. A. (1983).Lab. Znuesf. 49, 140-147. Barsky, S. H . , Rao, C . N., Williams, J. E., and Liotta, L. A. (1984). J . Clin. Invest. 74, 843-848. Bateman, J. F., and Peterkofsky, B. (1981). Roc. Nafl. Acad. Sci. U . S . A . 78, 6028-6032. Battifora, H . (1976). Cancer 37, 2275-2282. Bernacki, R. J., Niedbala, M. J., and Korytnyk, W. (1985). Cancer Metastaris Rev. 4, 81-102. Bernfield, M .(1983).Eur. J. Cell B i d . Suppl. 3, 4. Bernfield, M . , Banerjee, S. D., Koda, J. E., and Rapraeger, A. C. (1984).In “The Role of Extracellular Matrix in Development” (R. L. Trelstad, ed.), pp. 545-572.Liss, New York. Billingham, R. E., Orr, J. W., and Woodhouse, D. L. (1951). Br. J. Cancer 5 , 417-432. Birkedal-Hansen, H.,Cobb, C . M., Taylor, R. E., and Fullmer, H. M. (1976). Biochim. Biophys. Acfa 438, 273-286. Bissell, M. J., Hall, H. G . , and Parry, G. (1982).J . Theor. B i d . 99, 31-68. Biswas, C. (1984). Cancer Left 24, 201-207. Biswas, C. (1985).J. Cell. Biochem. 28, 39-45. Blum, J . L.,and Wicha, M. A. (1985).J. Cell B i d . 101, 463a. Bornstein, P., and Sage, H . (1980).Annu. Rev. Biochem. 49, 957-1003. Briggaman, R . A. (1982).J.Znuesf. Dennatol. 78, 1-6. Brown, K.W., and Parkinson, E. K. (1984).In!. J . Cancer 33, 257-263. Brown, K.W., and Parkinson, E. K. (1985). Inf.J . Cancer 35, 799-807. Brown, S. S . , Malinoff, H . L., and Wicha, M. S. (1983). Proc. Natl. Acad. Sci. U . S . A . 80, 5927-5930. Burleigh, M . C., Barrett, A. J., and Lazarus, G. S. (1974). Biochem. J . 137, 387-398. Cairns, J . (1981). In “Cancer: Achievements, Challenges, and Prospects for the 1980’s” (J. H . Burchenal and H. F. Oettgen, eds.), Vol. I, pp. 445-453.Grune & Stratton, New York. Cam, Y., Cadet, T . , Bellon, G., Poulin, G., Legros, M., and Pythynska, M. (1984a).J.Pathol. 144, 35-44. Cam, Y., Bellon, G., Poulin, G., Caron, Y., and Birembaut, P. (1984b).Inuasion Mefasfasis 4, 61-72. Carroll, R. (1962).J. Pafhol. Bacferiol. 83, 293-297. Carter, W. G. (1982).J. B i d . Chum. 257, 13805-13815. Cass, A. W., Million, R . R., and Pfaff, W. W. (1976). Cancer 37, 2861-2865. Chaudhuri, S., Koprowska, I., Putong, P. B., and Townsend, D. E. R. (1974). Cancer Res. 34, 1335-1343.

190

A. VAN DEN HOOFF

Chaudhuri, S., Koprowska, I., and Rowinski, J. (1975).Cancer Res. 35, 2350-2354. Chiarugi, V. P. (1982).Anticancer Res. 2, 275-282. Clark-Lewis, I., and Murray, A. W. (1978).Cancer Res. 38, 494-497. Cunha, G . R.,Bigsby, R. M., Cooke, P. S., and Sugimura, Y. (1985). CellDi@. 17, 137-148. Dano, K., Andreasen, P. A,, Grondahl-Hansen, J., Kristensen, P., Nielsen, L. S., and Skriver, L. (1985).Adu. Cancer Res. 44, 139-266. David, G., and Bernfield, M. (1982).J. Cell. Physiol. 110, 56-62. David, G., and van den Berghe, H . (1983).J.Biol. Chem. 258, 7338-7344. Dawe, C . J . (1972). In “Tissue Interactions in Carcinogenesis” (D. Tarin, ed.), pp. 305-358. Academic Press, London. DeClerck, Y. A,, Bogenmann, E., and Jones, P. A. (1985).Cancer Res. 45, 1229-1238. Declos, K. B., and Blumberg, P . M. (1979). Cancer Res. 39, 1667-1672. Deelman, H . T. (1924).2.Krebsforsch. 21, 221-226. Delinassios, J. G . , Pandis, N. B., Margaronis, M., and Kottaridis, S. D. (1981).Experientia 37, 569-570. Delinassios, J. G., Kottaridis, S . D., and Garas, J. (1983). Exp. Cell Eiol. 51, 201-209. De Petro, G., Barlati, S . , Vartio, T . , and Vaheri, A. (1981). Proc. Natl. Acad. Sci. U . S . A . 78, 4965-4969. Dexter, T . M., Whetton, A. D., Spooncer, E., Heyworth, C., and Simmons, P. (1985).J.Cell Sci. Suppl. 3, 83-95. Dion, L. D., Bear, J., Bateman, J., DeLuca, L. M . , and Colburn, N. H . (1982).J.Natl. Cancer Inst. 69, 1147-1154. Dolberg, D. S . , Hollingsworth, R . , Hertle, M., and Bissell, M . J . (1985). Science 230, 676-678. Dolfini, E., Azzarone, B., Pedulla, D., Ottaviano, E., de Gaetano, D., Donati, M . B., and Morasca, L. (1976).Eur. J . Cancer 12,823-825. Domellof, L . , and Janunger, K. G. (1977).Am. J . Surg. 134, 581-584. Domellof, L., Eriksson, S., and Janunger, K. G . (1976).Am. J . Surg. 132, 26-31. Dvorak, H . F.,Form, D. M., Manseau, E. J . , and Smith, B. D . (1984).J.Natl. Cancer Inst. 73, 1195-1206, Ehrlich, P. (1879). Arch. Anat. Physiol. 3, 166-169. El-Torky, M., Giltman, L. I., and Dabbous, M. (1985).Am. J. Pathol. 121, 322-326. Emerman, J . T . , and Pitelka, D. R . (1977).Zn Vitro 13, 316-328. Emonds-At, X.,Quisquater, E., and Vaes, G. (1980). Eur.J. Cancer 16, 1257-1261. Farber, E. (1984).Carcinogenesis 5, 1-5. Fiszer-Szafarz, B., and Gullino, P. M. (1970).Proc. SOC.Exp. Biol. Med. 133, 805-807. Fitch, J. M., and Linsenmayer, T. F. (1983). Deu. Eiol. 95, 137-153. Form, D. M., VandeWater, L., Dvorak, H. F., and Smith, B. D. (1984).J.Natl. CancerZnst. 73, 1207-1214. Fraire, A. E., and Greenberg, S. D. (1973). Cancer 31, 1078-1086. Friedrich, G. (1939). Virchow’s Arch. (Pathol. Anal.) 304, 230-247. Frithiof, L. (1972). In “Tissue Interactions in Carcinogenesis” (D. Tarin, ed.), pp. 161-189. Academic Press, London. Gabbert, H. (1985). Canccr Metastasis Reu. 4,293-309. Gaido, K. W., and Wierda, D. (1985). Toxicol. Appl. Pharmacol. 81, 469-475. Garbi, C., Tacchetti, C., and Wollman, S. H. (1986). Exp. Cell Res. 163, 63-77. Gerfaux, J., Chany-Fournier, F., Bardos, P., Muk, J.-P., and Chany, C . (1979). Proc. Natl. Acad. Sci. U . S . A . 76, 5129-5133. Ghosh, L., Ghosh, B. C . , and Das Gupta, T. K. (1980). Am. J . Surg. 139, 229-232. Gillman, T.,Penn, J . , Bronks, D., and Roux, M. (1955).Br. J . Cancer 9, 272-283. Goellner, J. R.,Devine, K. D., and Weiland, L. H. (1973).Am. J . Clin. Pathol. 59, 312-326.


191

Goldberg, G. I., Wilhelm, S. M., Kronberger, A., Bauer, E. A., Grant, G. A,, and Eisen, A. Z. (1986).J. B i d . Chem. 261, 6600-6605. Goslen, J. B., Eisen, A. Z.,and Bauer, E. A. (1985).J.Invest. Dmnafol. 85, 161-164. Gould, V. E., and Battifora, H. (1976). Pafhol. Ann. 11, 353-386. Graf, M.,Baici, A., and Striuli, P. (1981). Lab. Znvesf. 45,587-596. Green, H., Todaro, G. J., and Goldberg, B. (1966). Nature (London) 209, 916-917. Grobstein, C . (1967). Nafl. Cancer. Znsf. Monogr. 26, 279-299. Gusterson, B. A,, Clinton, S., and Gough, G . (1986). Hisfopafhology 10, 161-169. Harris, M . (1982). Hisfopafhologv 6, 197-210. Hashimoto, K., Yamanishi, Y., and Dabbous, M. K. (1972). Cancer Rcs. 32, 2561-2567. Hauser, C . , Saurat, J. H . , Jaunin, F., Sizonenko, S., and Dayer, J. M. (1985).Biochim. Biophys. Acta 846, 350-355. Hay, E. D. (1981). In “Cell Biology of Extracellular Matrix” (E. Hay, ed.), pp 379-409. Plenum, New York. Hennings, H . , and Boutwell, R. K. (1970). Cancer Res. 30, 312-320. Henry, N., Van Lamsweerde, A. L., and Vaes, G. (1983). Cancer Res. 43,5321-5327. Hessle, H., Sakai, L. Y., Hollister, D. W., Burgeson, R . E., and Engvall, E. (1984).DrJercnfiation 26, 49-54. Hicks, N. J., Ward, R . V., and Reynolds, J. J. (1984).Znf. J. Cancer 33,835-844. Hodges, G. M .(1982). In “Functional Integration of Cells in Animal Tissues” (J. D . Pitts and M. E. Finbow, eds.), pp. 333-356.Cambridge Univ. Press, Cambridge. Hodges, G. M., Hicks, R. M., and Spacey, G. D. (1977). Cancn Rcs. 37, 3720-3730. E r m a n n , H . (1982). Klin. Wochcnschr. 60, 1265-1277. Hornebeck, W., Brechemier, D., Bellon, G., Adnet, J. J., and Robert, L. (1980). In “Proeinases and Tumor Invasion” (P. Striuli, A. J . Barrett, and S. Baici, eds.), pp. 117-141. Raven, New York. Howard, B. H., Adams, S. L., Sobel, M. E., Pastan, J., and De Crombrugghe, B. J . (1978).J. Biol. Chnn. 253, 5869-5878. Hukins, D. W. L., and Aspden, R. M. (1985). Trmh Biochn. Sci. 10,260-264. Hynes, R. 0. (1986). Sci.Am. 254, 32-41. Ingber, D. E., and Jamieson, J. D. (1982).In “Tumor Invasion and Metastasis” (L. A. Liotta and I. R . Hart, eds.), pp. 335-357.Nijhoff, The Hague. Ingber, D. E., and Jamieson, J. D. (1985). In “Gene Expression during Normal and Malignant Differentiation” (L. C. Anderson, C . G. Gahmberg, and P. Ekblom, eds.), pp. 13-32. Academic Press, London. Ingber, D. E., Madri, J. A., and Jamieson, J. D. (1985).Am. J . Pathol. 121, 248-260. Ingber, D. E., Madri, J. A., and Jamieson, J. D. (1986).Am. J. Pafhol. 122, 129-139. Iozzo, R. V. (1985a). Lab. Znucsf. 53, 373-396. Iozzo, R . V. (198513).J. B i d . Chcm. 260, 7464-7473. Iozzo, R . V., and Miiller-Glauser, W. (1985). Cancer Res. 45, 5677-5587. Isaacs, J. T. (1985). Rcu. Endocrine Reluf. Cancer 20, 13-18. Jackson, D. (1984). Cancer 54, 361-366. Jao, W., and Gould, V. E. (1975). Cancer 35, 1280-1292. Johnson-Wint, B., and Bauer, E. A. (1985).J. Bid. Chem. 260, 2080-2085. Kallioinen, M., Autio-Harmainen, H., Dammert, K., Risteli, J., and Risteli, L. (1984a).J. Znvest. D-tol. 82, 248-251. Kallioinen, M., Autio-Harmainen, H., Dammert, K., Risteli, J., and Risteli, L. (1984b).J.Znvest. Dmnafol. 83, 276-280. Kao, R . T . , and Stern, R. (1986a). Cancer Res. 46, 1349-1354. Kao, R . T., and Stern, R . (1986b). Cancer Rcs. 46, 1355-1358. Kao, R . T., Hall, J., and Stem, R . (1986). Connect. Tissue Rcs. 14, 245-255.

192

A. VAN DEN HOOFF

Keski-Oja, J . , Marquardt, H., De Larco, J . E.,and Todaro, G . J. (1979).J . Supramol. Strucf. 11, 217-225. Keski-Oja, J., Gahmberg, C. G., and Alitalo, K. (1982a). Cancer Res. 42, 1147-1153. Keski-Oja, J., Rapp, U. R . , and Vaheri, A. (1982b).J . Cell. Biochtm. 20, 139-148. Keski-Oja, J . , Alitalo, K., Hautanen, A,, and Rapp, U. R. (1984).Biochim. Biophys. Acfa 803, 153-162. Klein, G., and Klein, E. (1985). Nafurc (London) 315, 190-195. Kleinman, H.K., Cannon, F. B., Laurie, G. W., Hassell, J. R., Aumailley, M., Terranova, V. P., Martin, G. R., and DuBois-Dalcq, M. (1985).J.Cell. Biochcm. 27, 317-325. Knudson, W., and Toole, B. P. (1985).J. Cell Biol. 101,339a. Knudson, W., Biswas, C., and Toole, B. P. (1984).J. Cell. Biochem. 25, 183-196. Konstantinidis, A,, Smulow, J. B., and Sonnenschein, C. (1982). Science 216, 1235-1237. Kopelovich, L. (1980). In “Colorectal Cancer: Prevention, Epidemiology and Screening” (S. Winawer, D. Schottenfeld, and P. Sherlock, eds.), pp. 97-108. Raven, New York. Kopelovich, L. (1982a). Cancer Genef. Cyfogcncf.5, 333-351. Kopelovich, L. (1982b).Znf. Reu. Cyfol. 77, 63-88. Kopelovich, L.,and Gardner, E. J. (1983). Cancer 5, 716-720. Kopelovich, L.,Coulon, S., and Pollack, R . (1977).Proc. Natl. h a d . Sci. U . S . A . 74,3019-3022. Kopelovich, L.,Bias, N. E., and Helson, L. (1979).Nature (London) 282, 619-621. Kopelovich, L.,Murray, J. C., Kleinman, H. K., Martin, G. R . , Fukuda, Y., and Rennard, S . I. (1985). Exp. Cell Biol. 53, 314-321. Krieg, T., Aumailley, M., Dessau, W., Wiestner, M., and Muller, P. (1980).Exp. CeMRcs. 125, 23-30. Krompecher, E. (1900). Bcifr. Pafhol. 28, 1-41. Kurkinen, M., Vaheri, A,, Roberts, P. J., and Stenman, S. (1980).Lab. Znuesf. 43, 47-51. Lagad, R., Grimaud, J.-A,, Schiirch, W., and Seemayer, T. A. (1985). Virchows Arch. (Patho[. A d . ) 408, 49-59. Lane, N. (1957). Cancn 10, 19-41. Langness, U.,and Udenfriend, S. (1974). R o c . Nafl. Acad. Sci. U . S . A . 71, 50-51. Leifer, C., Miller, A. S., Putong, P. B., and Min, B. H. (1974).Cancer 34, 597-605. Lewko, W. M., Liotta, L. A,, Wicha, M. S., Vonderhaar, B. K., and Kidwell, W. R . (1981). Cancer Res. 41, 2855-2862. Linsenmayer, T . F., Fitch, J . M., and Mayne, R. (1984).I n “The Role of Extracellular Matrix in Development” (R. L. Trelstad, ed.), pp. 145-172. Liss, New York. Liotta, L. A. (1985).In “Important Advances in Oncology” (V. T . de Vita, S. Hellman, and S . A. Rosenberg, eds.), pp. 28-41. Lippincott, Philadelphia. Liotta, L. A. (1986). Cancer Rcs. 46, 1-7. Liotta, L. A,, Kleinerman, J., Catanzaro, P . , and Rynbrandt, D. (1977).J.Natl. Cancer Znsf. 58, 1427-1431. Liotta, L. A,, Thorgeirsson, U. P.,and Garbisa, S. (1982). Cancer Metastasis Rev. 1, 277-288. Little, C. D., Church, R., Miller, R . A,, and Ruddle, F. H. (1977).Cell 10, 287-295. Luikart, S. D.,Maniglia, C. A., and Sartorelli, A. C . (1983).R o c . Natl. Acad. Sci. U.S.A. 80, 3738-3742. Lumirh, A. (1929). “Le Cancer, Maladie des Cicatrices.” Masson, Paris. Lundmark, C . (1972). Cancer 30, 1195-1201. McCarthy, J . B., Basara, M. L., Palm, S. L., Sas, D. F., and Furcht, L. T. (1985). Cancer Metasfasis Reu. 4, 125-152. McCarthy, J . B., Hagen, S. T . , and Furcht, L. T. (1986).J. Cell Bid. 102, 179-188. McDonnell, L.,and Long, J. P. (1981).J.Clin. Pafhol. 34, 996-999. Mackie, B. S.,and McGovern, V. J. (1958). Am. Mcd. Assoc. Arch. Dermutol. 78, 218-244. McNutt, N. S . (1976). Lab. Znucsf. 35, 132-142. Madri, J., and Carter, D. (1982). Lab. Znuesf. 46, 50A.


193

Madri, J . A,, and Carter, D. (1984). Hum. Pathol. 15, 625-631. Malinoff, H . L., Brown, S. S . , and Wicha, M . S. (1983).J.CellBiol. 97, 122A. Maltoni, C., and Prodi, G. (1960). Turnon‘ Suppl. pp. 49-156. Maltoni, C., and Zajdela, F. (1963).Acta Union Int. Cunnum 19, 584-588. Marian, B. (1986). Carcinogenesis 7, 723-726. Marian, B., and Mazzucco, K. (1985). Curcinogencsis 6, 501-504. Marks, F., Berry, D. L., Bertsch, S., Fiirstenberger, G., and Richter, H. (1982). Curcinogencsis 7, 331-346. Marsilio, E., Sobel, M . E., and Smith, B. D. (1984).J.Biol. Chem. 259, 1401-1404. Martin, H. E., and Stewart, F. W. (1935).Am. J . Cancer 24, 273-298. Martin, G. R., Timpl, R., Muller, P. K., and Kiihn, K. (1985).Trendr Biochem. Sci. 10, 285-287. Marx, J. L. (1986). Science 232, 1093-1095. Mayne, R . (1984). In “The Role of Extracellular Matrix in Development” (R. L. Trelstad, ed.), pp. 33-42. Liss, New York. Medsger, J r . , T. A. (1985).J.Rhcumutol. 12, 1041-1043. Meier, S.,and Hay, E. D. (1974).Dev. B i d . 38, 249-270. Mensing, H., Albini, A,, Krieg, T., Pontz, B. F., and Miiller, P. K. (1984). Int. J. Cancer 33,

43-48. Mertens, V. E. (1924).Z.Krebsforsch. 21, 494-501. Meyer, E. C., and Liebow, A. A. (1965). Cancer 18, 322-351. Meyer, J. R . , Kramer, R . H., Migliorati, C., Greenspan, J. S., Stegman, S., and Tromovitch, T. (1982).J. Cell Bid. 95, 115 A. Miederer, S. E., Muller, R., and Kutz, K. (1977). Endoscopy 9, 50-53. Minafra, S.,Pucci-Minafra, I., Tomasino, R. M., and Sciarrino, S.(1984).CeNBiol. Inf. Rep. 8,

79-85. Minckler, D. S., Meligro, C. H., and Norris, H . T . (1970).Cancer 26, 195-200. Morgenstern, L., and Yamakawa, T . (1973).Am. J. Surg. 125, 29-38. Moro, L.,and Smith, B. D. (1977).Arch. Biochm. Biophys. 182, 33-41. Miiller-Glauser, W., Haemmerli, G., In-Albon, A,, and Strauli, P. (1984).Int. J. Cancer 34,

545-553. Nagala, K., and Yamada, K. M. (1986).J. Biol. C h m . 261, 7531-7536. Nakajima, M., Irimura, T . , DiFerrante, D., DiFerrante, N., and Nicolson, G. I. (1983).Science 220, 611-613. Natali, P. G., Nicotra, M . R., Bellocci, M., Cavaliere, R., and Bigotti, A. (1985). Znt. J . Cancer 35, 461-467. Nishizuka, Y. (1986).J . Nutl. Cancer Inst. 76, 363-370. Nordbye, K. (1969).Actu Obsfet. Gynecol. Scund. Suppl. 3 , 102-109. Nowell, P. C. (1986). Cuncer Res. 46, 2203-2207. Ohwada, H., Hayashi, Y., and Seki, M . (1980). Gunn 71, 285-292. Orr, J. W. (1958). Br. Med. Bull. 14, 99-101. Orr, J . W. (1961). Actu Union Inf. Cancrum 17, 64-71. Orr, J. W., and Spencer, A. T . (1972). In “Tissue Interactions in Carcinogenesis” (D. Tarin, ed.), pp. 291-303. Academic Press, London. Osnes, M., Lotveit, T., and Myren, J. (1977). Endoscopy 9, 45-49. Ossowski, L.,and Reich, E. (1983). Cell 35, 611-619. Otsubo, Y., and Kameyama, Y. (1982).J. Oral Pufhol. 11, 159-173. Panneerselvam, M . , Sahai, A., and Salomon, D. S. (1985). Arch. Dzrmutol. Res. 277, 377-383. Parker, M. I., Judge, K., and Gevers, W. (1982). Nucleic A c i h Res. 10, 5879-5891. Pauli, B. U.,Schwartz, D . E., Thonar, E. J. M., and Kuettner, K. E. (1983). Cuncer Metastasis Reu. 2, 129-152. Pauli, B. U., Arsenis, C., Hohberger, L. H . , and Schwartz, D . E. (1986). Cancer Res. 46,

2005-2012.

194

A. VAN DEN HOOFF

Pfeffer, L., Lipkin, M . , Stutman, O., and Kopelovich, L. (1976).J. Cell. Physiol. 89, 29-37. Pitelka, D. R., Hamamoto, S. T . , and Taggart, B. N. (1980). Cancer Res. 40, 1600-1611. Poole, A. R., Recklies, A. D., and Mort, J. S. (1980).Zn “Proteinases and Tumor Invasion” (P. Strauli, A. J. Barrett, and S. Baici, eds.), pp. 81-95.Raven, New York. Pozharisski, K. M . (1975). Cancer Res. 35, 3824-3830. Pucci-Minafra, I, Luparello, C., Sciarrino, S., Tomasino, R . M., and Minafra, S. (1985). Cell Bid. Znf. Rep. 9, 291-296. Raeburn, C., and Spencer, H . (1953). Thorax 8, 1-10, Raeburn, C., and Spencer, H . (1957).Br. J . Tuberculosis Dis. Chesf 51, 237-245. Rao, N. C.,Barslay, J. H., Terranova, V. P . , and Liotta, L . A. (1983). Biochm. Biophys. Res. Commun. 111, 804-808. Recklies, A . D., Mort, J. S., and Poole, A. R . (1982). CQWURes. 42, 1026-1032. Ribbert, H . (1911).“Das Karzinom des Menschen.” Cohen, Bonn. Risteli, L . , and Risteli, J. (1981).Med. B i d . 59, 185-189. Roesel, R . A , , Cutroneo, K. R., Scott, D. F., and Howard, E. F. (1978).Cancer Res. 38, 3269-3275. Roos, E., Dingemans, K. P., Van de Pavert, I. V., and Van den Bergh-Weerman, M. A . (1978).Br. J . Cancer 38, 74-85. Roos, E., Van de Pavert, I. V., and Middelkoop, 0. P. (1981).J. Cell Sci. 47, 385-397. Rossle, R. (1943).Schweiz Med. Wochenschr. 73, 1200-1203. Roumm, A. D., and Medsger, T . A. (1985). Arthritis Rheum. 28, 344-360. Rubin, H. (1984).Nafure (London) 309, 518. Rubin, H. (1985). Cancer Res. 45, 2935-2942. Ruoslahti, E. (1984). Cancer Mefastasis Reo. 3, 43-51. Sakakibara, K., Suzubi, T., Motoyama, T . , Watanabe, H., and Nagai, Y. (1982). Cancer Res. 42, 2019-2027. Sakakura, T. (1983). In “Understanding Breast Cancer” (M. A. Rich, J. C. Hager, and P. Furmanski, eds.), pp. 261-284.Dekker, New York. Sakakura, T., Sakagami, Y., and Nishizuka, Y. (1981).J.Nafl. Cancer Znst. 66, 953-957. Sandmeyer, S., Smith, R., Kiehn, D., and Bornstein, P. (1981). Cancer Res. 41, 830-838. Scadding, J. G.(1960). Br. Med. J . 1, 443-450. Schmidt, A., Setoyama, C., and De Crombrugghe, B. (1985). Nature (London) 314, 286-289. Schor, S. L., Schor, A. M., and Bazill, G. W. (1981).J. Cell Sci. 48, 301-304. Schor, S. L.,Schor, A. M., Rushton, G . , and Smith, L. (1985a).J . Cell Sci. 73, 221-234. Schor, S. L.,Schor, A. M., Durning, P., and Rushton, G. (1985b).J. Cell Sci. 73, 235-244. Schor, S. L., Haggie, J. A., Durning, P., Howell, A , , Smith, L., Sellwood, R . A. S., and Croether, D. (1986). Znf..I. Cancer 37. 831-836. Seemayer, T . A., Schiirch, W:, LagacC, R., and Tremblay, G. (1979). Am. J . Surg. Pafhol. 3 , 525-533. Sellers, A,, and Murphy, G. (1981). Znt. Rev. Connect. Tissue Res. 9, 151-190. Setoyama, C., Lian, G., and De Crombrugghe, B. (1985). Cell 41, 201-209. Shapiro, F. D., and Eyre, D. R . (1982).J. Nafl. Cancer Znst. 69, 1009-1016. Shimosato, Y., Hashimoto, T . , Kodama, T . , Kameya, T . , Suzuki, A , , Nishiwaki, Y., and Yoneyama, T. (1980).Am. J. Surg. f‘afhol. 4, 365-373. Slamon, D. J., deKernion, J. B., Verma, I. M . , and Cline, M. J. (1984).Science 224, 256-262. Sloane, B. F., and Honn, K. V. (1984). Cancer Metastasis Rcu. 3 , 249-263. Sloane, B. F., Honn, K. V . , Sadler, J. G., Turner, W. A , , Kimpson, J. J., and Tayor, J . D. (1982). Cancer Res. 42,980-986. Smith, B. D., Mahoney, A. P., and Feldman, R . S. (1983).Cancer Res. 43, 4275-4282. Smith, C . J. (1972). Zn “Tissue Interactions in Carcinogenesis” (D. Tarin, ed.), pp. 191-225. Academic Press. London. ~I


195

Smith, H . S., Owens, R . B., Hiller, A. J., Nelson-Rees, W. A., and Johnson, J. 0. (1976).Int. J . Cancer 17, 219-234. Srnithers, D. W. (1962). Lancet 1, 493-499. Smithers, D.W. (1983). Int. J. Radiaf. Oncol. Biol. Phys. 9, 731-738. Sobel, M. E., Dion, L. D., Vuust, J., and Colburn, N. H. (1983).Mol. Cell. Bid. 3, 1527-1532. Stanton, M. F., and Blackwell, R . (1961).J . Natl. Cancer Inst. 27, 375-408. Stenback, F., Wasenius, V.-M., Risteli, J., and Risteli, L. (1985a). Gynecol. Obstet. Znuesf. 20, 158-166. Stenback, F., Risteli, J . , Risteli, L., and Wasenius, V.-M. (1985b). Oncology 42, 370-376. Stenn, K.S . , Madri, J . A., Tinghitella, T . , and Terranova, V. P. (1983).J.Cell Bid. 96, 63-67. Strauli, P. (1980). In “Proteinases and Tumor Invasion” (P. Strauli, A. J. Barrett, and A. Baici, eds.), pp. 1-15. Raven, New York. Takeuchi, J. (1971). Acta Pafhol.Jpn. 21, 1-12. Tarin, D. (1967).Int.J. Cancer 2, 195-211. Tarin, D. (1969).Br. J. Cancer 23, 417-425. Tarin, D. (1972).In “Tissue Interactions in Carcinogenesis” (D. Tarin ed.), pp. 227-290. Academic Press, London. Tarin, D., Hoyt, B. J., and Evans, D. J. (1982). Br. J. Cancer 46, 266-278. Terranova, V. P., Liotta, L. A., Russo, R . G., and Martin, G . R. (1982). Cancer Res. 42, 2265-2269. Terranova, V. P., Williams, J. E., Liotta, L. A., and Martin, G. R . (1984). Science 226, 982-985. Terranova, V. P., Hujanen, E. S., Loeb, D. M., Martin, G. R., Thornburg, L., and Glushko, V. (1986).Proc. Natl. Acad. Sci. U.S.A. 83, 465-469. Todaro, G. J . , Fryling, C . , and Delarco, J. E. (1980). Roc. Nafl. Acad. Sci. U.S.A. 77, 5258-5262. Toole, B. P., Goldberg, R. L., Chi-Rosso, G., Underhill, C. B., and Orkin, R . W. (1984). In “The Role of Extracellular Matrix in Development” (R. L. Trelstad, ed.), pp. 43-66.Liss, New York. Tremblay, G. (1976). Cancer 37, 307-316. Trernblay, G. (1979).Exp. Mol. Pafhol. 31, 248-260. Triieb, B., Lewis, J . B., and Carter, W. G. (1985).J. Cell Biol. 100, 638-641. Turley, E. A., and Tretiak, M. (1985). Cancer Res. 45, 5098-5105. Turpeennierni-Hujanen, T., Thorgeirsson, U. P., Hart, I. R., Grant, S. S., and Liotta, L. A. (1985).J. Natl. Cancer Insf. 75, 99-103. Turpeenniemi-Hujanen, T . , Thorgeirsson, U. P . , Rao, C . N., and Liotta, L. A. (1986).J . Bid. Chm. 261, 1883-1889. Van Cauwenberge, D., Pierard, G. E., Foidart, J . M., and Lapiere, Ch.M. (1983). Br. J. Der~ ~ d o 108, l . 163-170. Van den Hooff, A. (1962).Oncologia 15, 161-179. Van den Hoof, A. (1983). Inf. Reu. Connect. Tissue Res. 10, 395-432. Van den Hooff, A. (1984). Perspect. Biol. Med. 27, 498-509. Van den Hooff, A. (1986). Anticancer Res. 6, 775-780. Van den Hooff, A,, and Tigchelaar-Gutter, W. (1983a).Proc. Kon. Ned. Akad. wct. 86, 179-190. Van den Hooff, A,, and Tigchelaar-Gutter, W. (1983b).Proc. Kon.Ned. Akad. Wef.86, 191-196. Vasiliev, J. M.(1958). Br. J . Cancer 12, 524-536. Vernoni, G. (1933).“Tumori Maligni.” SOC.Ed. Libraria, Milan. Wan, Y. J., Wuh, T . C., Chung, A. E., and Darnjanov, I. (1984).J. Cell Bid. 98, 971-979. Warburton, M. J , , Ferns, S. A., and Hynes, N. E. (1986).Biochem. Biophys. Res. Commun. 137, 161-1 66.

196

A. VAN DEN HOOFF

Weinberg, R. A. (1983).Cancn 52, 1971-1975. Weinberg, R. A. (1984). Trends Biochem. Sci. 9, 131-133. Weiss, P. A. (1971).Pnspccf. Biol. Mcd. 14, 182-205. Wicha, M. S.,Liotta, L. A., Lewko, W. M., and Kidwell, W. R . (1981).Cancer Lett. 12,9-21. Wid, G.(1977).Connccf. Tissue Res. 5, 171-178. Wid, G.(1984).Cancer Metastasis Rev. 3, 237-248. Wid, G.,Kronberger, A., and Langanger, G. (1984).Exp. Cell Rcs. 151, 502-518. Woodley, D.T . , Kalebec, T . , Banes, A. J., Link, W., Prunieras, M . , and Liotta, L. (1986).J. Invest. D m t o l . 86, 418-423. Woods, D. A., and Smith, C . J. (1969).J. Invest. D m t o l . 52, 259-263. Woolley, D. E. (1984).Cancn Metusfaris Rcu. 3, 361-372. Woolley, D.E., Tetlow, L. C., Mooney, C. J., and Evanson, J. M. (1980).In “Proteinases and Tumor Invasion” (P. Strauli, A. J. Barrett, and A. Baici, eds.), pp. 97-115.Raven, New York. Wynford-Thomas, D . , Smith, P., and Williams, E. D. (1986). Cancn Rcs. 46, 3125-3127. Yamada, K. M., Akiyama, S. K., Hasegawa, T., Hasegawa, E., Humphries, M. J., Kennedy, D. W ., Nagata, K., Urushihara, H., Olden, K., and Chen, W.-T. (1985).J.Ccll. Biochem.

28, 79-97. Yamagata, S.,and Yamagata, T. (1984).J . Biochem. 96, 17-26. Yoffe, J. R., Taylor, D. J., and Woolley, D. E. (1984). Biochcm. Biophys. Rcs. Commun. 122,

270-276.

Yoffe, J. R.,Taylor, D. J., and Woolley, D. E. (1985). Biochem. J . 230, 83-88. Yokoo, H., and Suckow, E. E. (1961).Cancn 14, 1205-1215.

TUMOR CLONALITY AND ITS BIOLOGICAL SIGNIFICANCE Michael F. A. Woodruff Medlcal Research Councll Cllnlcal and Populatlon Cytogenetlca Unit, Western General Hospital, Edlnburgh EH4 2XU, Scotland

I. 11. Origin and Diversity of Cells in Tumors 111. Definition of Clonality

IV . Distinguishing between A. X-Linked Markers . . . . . B. Karyotypic and Other Chromosomal Markers

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

V.

VI .

D. Cell Surface Markers Other Than Ig ...... E. Markers in Natural or Artificially Pro ...... Analysis of Pleoclonal Tumors. . . . . . . . . . . ...... A. HowManyClones? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... B. Spatial Distribution of Clones . . . . . . , , . . . . . . . . . . . . . . . . . . , . . . . . . . . . . Observations on Clonality . . . . . . , . . . . . . . , . . , . . , . . . . . . . . . . . . . . , . , . . . . . . A. Human Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... B. AnimalTumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... Factors Influencing Clonality . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . , . . . . . . ,..... A. Some Theoretical Predictions . . . . . . . . . . . . . . . . . . . . . . . . . B. Interpretation of Data and Gaps to Be Filled . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . , . . ...... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VII.

VIII.

.

I

.

.

.

197 198 200 202 203 208 210 211 211 212 212 215 216 216 217 220 220 221 224 227

I. Introduction The main purpose of this review is to assess the contribution that the study of clonality has made, and may be expected to make in future, to our knowledge of carcinogenesis, with particular reference to the provenance and population kinetics of the various cells that are found in tumors. To achieve this we must look critically at the methods used to investigate clonality, and the results of such investigations. In 1965, when Linder and Gartler reported that human uterine leiomyomas appeared to originate from a single cell, few people would have anticipated that within a decade it would be widely accepted that most tumors, whether 197 ADVANCES IN CANCER RESEARCH, VOL. 50

Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved

198

MICHAEL F. A. WOODRUFF

benign or malignant, are monoclonal, and that this proposition would be backed by impressive experimental evidence. Agents such as ionizing radiation, chemical carcinogens, and hormones, which were known to play an etiological role in relation to some tumors, were seen to produce prolonged and widespread effects on tissues, and cancer was thought to develop in multiple foci in a “field” (Willis, 1960) of precancerous cells. How, then, could cancer be monoclonal? This paradox has not gone away. The only way to resolve it, or any other paradox, is to reexamine the meaning of the terms we use, and reappraise the evidence on which our conclusions are based. What, then, do we mean when we say that a tumor is monoclonal, and is the evidence of monoclonality quite as clear-cut as it appears? From the answers to these questions other questions stem. If monoclonality is the rule, why is this so, and why do some tumors (which we shall refer to as pleoclonal) not conform to it? Does the clonal composition of a pleoclonal tumor remain more or less constant? If so, how is this achieved; it not, what factors are responsible for the changes that occur? These questions are important because if we knew the answers we would have a better understanding of cancer and, in particular, of carcinogenesis. This understanding has been greatly deepened in recent years by the application of the methods of molecular genetics to the study of transformation. Defining the steps on the road to transformation and elucidating the mechanisms involved in each step is, however, not enough; we need also to analyze the interactions that occur between various categories of altered cells, and between altered cells and the tumor host, as the population of cells that have taken one or more of these steps expands. Clonal markers, and in particular clonal markers that can be used at the cellular level, are essential for such analysis, but their potential value will not be fully realized until other markers have been developed which recognize cells at different stages on the road to transformation.

II. Origin and Diverslty of Cells in Tumors A tumor, except perhaps at a very early stage, is not just a collection of tumor cells. It is a complex structure whose cell population includes normal leukocytes, macrophages, fibroblasts, and endothelial cells, as well as the descendants of one or more transformed cells. It may conceivably include also cells which have taken one or more, but not all, of the steps on the road to malignancy, which we may call initiated or part-way cells, and hybrid cells, formed by the fusion of two cells of the same or different kinds. There is, as a rule, a supporting framework of vascular connective tissue which, together with the various normal cells, constitutes the strona of the

TUMOR CLONALITY

199

tumor. The leukemias do not constitute an exception to this rule because the leukemic cells in the blood arise from stroma-containing neoplastic tissue in the bone marrow or elsewhere (McGee and Al Adani, 1976). Even in the case of ascites tumors, the ascitic fluid contains leukocytes and macrophages as well as tumor cells, and there are often nodules of stroma-containing neoplastic tissue on the surface of abdominal viscera. The leukocytes and macrophages are derived from bone marrow; fibroblasts may be of local origin, but experiments in bone marrow chimeras suggest that there may also be fibroblast-like cells of bone marrow origin (Barnes et al., 1970, 1971; Ansell et al., 1986), derived presumably from circulating blood monocytes. It is obvious, but important to stress, that the nonneoplastic cells in a tumor are of polyclonal origin. The descendants of transformed cells include proliferating cells in various stages of the cell cycle, so-called Go cells which have stopped cycling temporarily but may later reenter the cycle, and end-state cells which are incapable of further division. Some of the dividing cells must have unlimited proliferative potential if the tumor population is to continue to expand indefinitely. This is not necessarily true of all, however, and it has been suggested (Mackillop et al., 1983) that there is, as a general rule, a hierarchy of neoplastic cells in a tumor, consisting of (1) proliferating self-renewing cells, for which the term stem cell is appropriate; (2) proliferating non-self-renewing cells which are capable of undergoing only a limited number of divisions; and (3) nonproliferating, differentiated end-state cells. The concept of tumor stem cell is useful if the term is used in this way. Unfortunately, the adjective stem is often used loosely, and sometimes misleadingly, as for example, when the term stem line is used (Harnden and Klinger, 1985) to denote the neoplastic cells in a tumor which possess the modal number of chromosomes. It is perhaps less objectionable to refer to cells which form colonies in uitm, or give rise to tumors on transplantation, as stem cells, but it is by no means certain that all such cells possess unlimited proliferative potential in the autochthonous host. The neoplastic cells in a malignant tumor are, as a rule, markedly heterogeneous (see Woodruff, 1983, for review). There would seem to be three possible ways in which this heterogeneity could arise: (1) The tumor develops from more than one transformed cell. It will then be termed T-pleoclonal (Section 111). (2) Phenotypic differences are generated with a clone of transformed cells by mutation or some form of heritable epigenetic change. (3) Spontaneous hybridization occurs between transformed cells, or between a transformed cell and an initiated or normal cell, and this is followed by loss of chromosomes from the hybrid cell without loss of proliferative potential.

200


Many of the biological and clinical consequences of tumor cell heterogeneity (see Schnipper, 1986, for review) are the same irrespective of the underlying cause, the most important of which is undoubtedly the second of those listed above; in this review, however, we will be concerned only with heterogeneity due to pleoclonality.

111. Definition of Clonallty Before discussing the clonal composition of any cell population it is essential to decide what constitutes a clone. The members of a clone are by definition all

descendants of one cell, but how is the cell which constitutes the starting point to be defined? Micklem (1986), in discussing this question in the context of normal hematopoiesis, states that “common usage allows a clone to begin wherever one wishes it to begin; the only restriction is that the founder cell should divide at least once.” We shall accept this proposition with the further proviso that, for a discussion of clonality to be meaningful, the “wishes” of those taking part must be explicitly stated. Unfortunately, this simple rule is often ignored. We may, of course, choose as the founder cell of a particular clone a member of another clone with an earlier starting point. In this case the new clone may be termed a subclone of the earlier clone, and the earlier clone the proximate parent clone of the new clone. In the context of tumor clonality, if carcinogenesis were a one-step process which was completed before the cell divided, it would obviously be appropriate to define the founder cell of a neoplastic clone as an initially normal cell which becomes transformed before it divides. Insofar as carcinogenesis is a multistep process in the course of which cell division occurs, there are three possibilities to consider. First, the founder cell of a neoplastic clone may be defined as an initially normal cell which undergoes one or more of the steps on the way to transformation before it divides, and some of whose descendants become fully transformed. Second, the founder cell may be defined as a cell whose immediate parent had undergone all except the last of the steps on the way to transformation, and which undergoes the last step before it divides. Third, when a stable marker appears for the first time in a cell that lies somewhere between these extremes, it may be convenient to choose this cell as a founder (see Section IV,B). We shall refer to clones as N clones, P clones, or T clones, according to whether the founder cell is a normal cell, a part-way cell, or a transformed cell. These terms have been introduced because there is no existing appropriate nomenclature, apparently because it has not been thought necessary, and this lack has been the source of much confusion. The first definition corresponds to clonality as assessed with X-linked and other genetic markers of normal cells (see Ansell and Micklem, 1986), and is

TUMOR CLONALITY

201

usually (but not always) implied when a tumor is said to be, or not to be, monoclonal. To achieve greater precision, while conforming as far as possible to current usage, we shall, in this review, use the terms monoclonal and pleoclonal without qualification strictly in this sense. When a distinction is required, we shall use the terms N-monoclonal, T-monoclonal, and P-monoclonal to indicate that the tumor cell population consists of a single N clone, a single T clone, or a single P clone, respectively. It is important to note that a tumor which is N-monoclonal may have arisen from more than one transformed cell, because more than one, and conceivably many, of the descendants of the founder cell may, sooner or later, become fully transformed and proliferate. If this happens the tumor will be T-pleoclonal, although this cannot be demonstrated with markers which discriminate only between normal cells of various kinds. The problem of definition does not end here because we must also be clear about what constitutes a tumor-in particular, about whether, as Deamant et al. (1986) have claimed, a valid distinction can and should be drawn between what we may call a single tumor and a conglomerate tumor formed by the coalescence of two or more single tumors. It is, of course, conceivable that tumors which arise at widely separated sites may meet and form what appears to be a single tumor mass; indeed, there is plenty of evidence that this actually happens. It would therefore be of interest, with tumors which express two or more allelic markers, to study the spatial distribution of each clonal population. At present this is possible only on a very limited scale, but even if more widely applicable methods were developed it would be difficult, if not impossible, to devise rigorous, generally valid criteria by which to draw a distinction of the kind proposed. The special case of tumors which initially appear to be widely separated and subsequently coalesce seems clear-cut, but what can be said in the limiting case where two adjacent cells undergo transformation and proliferate to form populations which begin to intermingle after a few generations? It can be argued equally plausibly that the result is a single tumor or that it is a composite tumor, but if it is classed as composite, then there can be no such thing as a pleoclonal single tumor. Moreover, between the extremes we have been considering there is a wide range of possibilities that defy classification. Faced with this dilemma, the policy least open to objection, and which will be adopted in this review, is to classify as pleoclonal all tumors in which two or more neoplastic clones are identified within what is macroscopically a single tumor mass. The pattern of distribution of cells of the clones represented will, no doubt, vary considerably in the group of tumors so classified, but the possibility of clonal interaction by short-range mediators exists in all members of the group. Having defined what we mean by the terms clone and tumor, it remains only to define the clonality of a tumor as the number of distinct neoplastic clones which make up the tumor cell population.

202


IV. Distinguishing between Mono- and Pleoclonal Tumors Given a set (S) of potential founder cells, it is possible in principle to determine whether or not the members of some defined tumor cell population constitute a clone, in the sense that they have all descended from the same founder cell, if, and only if, each member of S carries a unique marker that persists through all subsequent divisions. In practice, it is desirable to have two or more independent markers. There seems no prospect of coming anywhere near meeting this general requirement in the foreseeable future so far as the population of neoplastic cells in a tumor is concerned. Fortunately, however, even with only a single pair of mutually exclusive stable markers, many pleoclonal populations can be recognized as such; moreover, if it can be assumed (1) that the chance of a cell dividing in a given time is independent of the marker it carries, and (2) that the presence of a marker on even one cell in the population can be detected, the likelihood of a population whose cells all carry the same marker being pleoclonal may be sufficiently small to be regarded as negligible. The first assumption seems likely to be true as a general rule, although there may be some exceptions. The second assumption is certainly not true, and the possible existence of small numbers of “foreign” cells in an apparently monoclonal population can never be formally excluded. It is not suggested that we should refrain, on this account, from ever concluding that a particular tumor, or type of tumor, is monoclonal when the evidence points strongly in this direction; excessive concern about theoretical possibilities could prevent us from drawing conclusions about anything. What is suggested is that we should bear in mind the resolving power of whatever method we use to assess clonality, and the possible biological significance of small populations which our procedure may be insufficiently sensitive to detect. The markers that have been used to study the clonality of human and/or animal tumors may be classified as follows: (1) X-linked markers: alloenzymes and restriction fragment length polymorphisms; (2) karyotypic and other chromosomal markers; (3) immunoglobulins (Ig) and Ig-gene rearrangements; (4) cell surface markers other than immunoglobulin; and (5) markers in natural or artificially produced chimeras. In this section we shall discuss how these markers have been used to try to discriminate between monoclonal and pleoclonal tumors. The more difficult problem of how to determine the number and distribution of clones in a pleoclonal tumor will be considered in Section V. Any analysis of clonality can, of course, only provide information about the situation at the time; the possibility that the clonal composition of a tumor may change in the course of its life history must therefore always be borne in mind. Repeated sampling of a primary tumor, though not justifiable in

TUMOR CLONALITY

203

humans, is feasible with animal tumors but may be misleading if the sampling procedure affects the subsequent behavior of the tumor. It is preferable, therefore, to induce a large number of tumors by the same technique in a uniform animal population, and analyze both early and late tumors. A useful method of inducing tumors that can be harvested when they weigh as little as 20 mg is to implant Millipore disks impregnated with a carcinogen (Woodruff et al., 1982, 1986).

MARKERS A. X-LINKED The use of X-linked markers depends on the fact, discovered by Mary Lyon (see Lyon, 1974), that in the somatic cells of mammals only one X chromosome is active, no matter how many are present. The differentiation of a particular X chromosome as active or inactive begins early in embryogenesis, and once the choice is made it is maintained through all subsequent cell divisions. Except in marsupials, and in the extraembryonic tissues of mice (Takagi and Susaki, 1975) and rats, where the paternally derived chromosome (X‘) is preferentially inactivated, the choice of which chromosome (X‘ or the maternally derived X”) is inactivated appears to be random, although in some cases genetic (Cattanach and Isaacson, 1965) or parental (see Forrester and Ansell, 1985) factors may modify the inactivation process in such a way that X‘ and XMhave an unequal probability of being inactivated. In mice, for example, there appears to be an inactivation center on the X chromosome designated the X-chromosomecontrolling element (Xce), and three alleles at this locus affect the probability that the chromosome will be inactivated. Geneticists often refer to this phenomenon as nonrandom inactivation. This terminology is open to criticism because what is happening is random inactivation in which the probability is not the same for each option. The situation is thus comparable to tossing a biased coin, or drawing a card at random from a “doctored” pack in which most of the cards are red, where the colors of the two sets of cards (red and black) correspond to the Xce phenotypes of the two sets of X chromosomes. Two types of X-linked markers have been used to study tumor clonality: X-linked alloenzymes and restriction fragment length polymorphisms (RFLP). Both are ordinarily used to analyze cell digests, but a start has been made in using alloenzymes at the cellular level to study clonality.

1. Alloenzymes In humans the only alloenzymes that have so far proved generally useful are the electrophoretic variants (A and B) of glucose-6-phosphate dehydrogenase (G-6-PD), and even with this system the ethnic and geographical distribution of one form (A) is inconveniently restricted.

204


In principle, the variants are separated by gel electrophoresis of a tumor digest, and enzyme activity is demonstrated chemically (for details see, e.g., De Mars and Nance, 1964; Linder and Gartler, 1965; Fialkow et al., 1970; Fialkow, 1972). It has been reported (Nance, 1964) that in mixtures of A and B the minor component can be detected when it contributes only 10% of the total activity, but this will vary according to the technique used, and in all studies the resolving power of the method used should be determined and clearly stated. There are no known electrophoretic variants of G-6-PD in Mus musculus, although they have been found in a related species, Mus caroli. Mus musculus does, however, possess another X-linked enzyme, phosphoglycerate kinase-1 (PGK-l), for which electrophoretic variants exist. The first studies of mouse tumor clonality using PGK-1 (Reddy and Fialkow, 1979) were undertaken with fibrosarcomas induced chemically in hybrids of laboratory mice (all common strains of which express PGK-1 B) and feral mice expressing PGK-1 A. Subsequently, the problems posed by the use of feral mice have been avoided by using instead backcross CBA mice histocompatible with normal CBA but which express PGK-1 A (Woodruff et al., 1982, 1984). As in the case of G-6-PD, the resolving power of the analysis depends on the precise technique used. Reddy and Fialkow (1979), using a modification of the assay of Chen et al. (1971) in which the formation of glycerate 1,3-diphosphate is associated with the loss of NADPH fluorescence, claim to have detected a minor component in known mixtures which contributed 2-5 % of the total enzyme activity; later, however, in a thorough study of this form of assay, West et al. (1977) found it to be appreciably less sensitive. Bucher et al. (1980) have developed a linkedenzyme assay based on the reverse reaction which is more sensitive, but more complicated because the substrate, glycerate 1,3-diphosphate, being unstable, must be prepared in situ. Woodruff et al. (1982, 1984), using a form of this assay in which the production of NADPH is visualized by the reduction of a tetrazolium dye, thiazolyl blue, to its formazan derivative, obtained linear results when the minor component contributed 10% or more of the total activity, and detected minor components consistently when they contributed only 2 % of the activity provided by lo5 tumor cells. A more sensitive form of Bucher’s assay exists in which the formation of NADPH is monitored by fluorescence; in this way PGK-1 can be detected from a single fertilized ovum (McMahon, 1983), and probably from as few as 15 normal fibroblasts (M. Monk, personal communication, 1986), but the method does not seem to have been used in studies of tumor clonality. Yet another assay, based on the use of [ 14C]glucose and autoradiography , which is intermediate in sensitivity between the last two procedures, is also available (see Ansell and Micklem, 1986).

TUMOR CLONALITY

205

Although, as already mentioned, M. musculus does not appear to possess electrophoretic variants of G-6-PD, some strains are deficient in this enzyme (Charles and Pretsch, 1984).If the deficiency were sufficiently great it should be possible to study the clonality of tumors induced in F1 hybrids of deficient and normal mice at a cellular level by histochemical staining for the enzyme, but is doubtful whether, with the strains currently available, the difference is sufficient to give clear-cut results. There are other X-linked enzymes in mice with known variants which can be demonstrated only in particular tissues. Williams and his colleagues (Williams et al., 1983;Howell et al., 1985)have studied liver tumors induced chemically in mice heterozygous for two forms of ornithine carbamoyltransferase (OCT). The normal form of the enzyme can be demonstrated histochemically by a standard technique which does not show the variant form, so that the normal liver is seen as a mosaic of O C T + and O C T - hepatocytes. In many, but not all, of the tumors the neoplastic cells were either all O C T + or all O C T - , and these were classed as monoclonal. The status of tumors with a mixture of O C T + and OCT - cells was regarded as uncertain because some O C T - cells may be seen also in tumors induced in animals not deficient in O C T .

2. Restriction Fragment Length Polymorphisms A novel strategy for studying the clonality of tumors in women has been developed by Vogelstein et al., (1985),based on the use of a cloned polymorphic X-chromosomal gene (HPRT) and two restriction endonucleases. The first endonuclease (BamHI) distinguishes DNA from Xp and XMby a restriction fragment length polymorphism (RFLP); the second (HhaI) distinguishes active from inactive copies of the gene by differences in methylation of cytosine residues. All human X-chromosomes possess two BamHI sites (B1 and B3) in the H P R T gene, but in 16% there is an additional site (B2),so that 27 % (2 x 0. 16 x 0.84 x 100)ofwomen are heterozygous at this locus. The authors point out that there are many RLFPs in other loci on the human X chromosome at which gene activation is associated with changes in methylation, and suggest that a similar strategy will eventually be applicable to tumors from nearly all women, although aberrant methylation in the genome of cancer cells (Feinberg and Vogelstein, 1983) may impose some limitations.

3. Interpretation of Assays with X-Linked Markers Since the fixed cellular mosaicism on which the use of X-linked markers depends is present before carcinogenesis begins, the evidence such markers provide concerning clonality can relate only to clones whose founder cell is an

206


initially normal cell, as in the first of the definitions discussed in Section 111. Studies with such markers therefore cannot show that a tumor has arisen from a single transformed cell unless, as pointed out in Section 111, the whole process of transformation, once it has begun in a cell, is completed before the cell divides. What such studies may do is to provide evidence that a tumor has, or has not, arisen from a single normal cell. The point is stressed because it does not seem to be generally appreciated (see Alexander, 1985). To decide what weight to attach to such evidence we must consider what factors, apart from the clonality of the tumor, determine whether the studies will reveal a double or a single phenotype. The main reason for observing a double phenotype with a truly monoclonal tumor is the presence of nonneoplastic cells in the tissue used to prepare the tumor digest. Some authors rely on histological evidence to exclude the presence of contaminating normal cells, but this can be fallacious, especially with nonepithelial tumors, and the error may be magnified if the marker used is expressed more strongly by normal cells than by tumor cells. The risk of error can be reduced by preparing a cell suspension from tumor tissue and removing leukocytes and macrophages before making a digest. An effective way of doing this (Woodruff et al., 1982) is to seed tissue culture flasks with the cell suspension, pour off the medium after 18 h (which removes most of the leukocytes) and harvest the weakly adherent tumor cells by brief exposure to a dilute solution of trypsin (which leaves most of the macrophages adhering to the flask). There may still be significant contamination with fibroblasts, and monoclonality can only be excluded with certainty by cloning the suspension in uitro and showing that some clones express one marker and some the other. A further refinement (Woodruff et al., 1982) is to demonstrate that each type of clone gives rise to tumors of the same phenotype when transplanted to a homozygous host of the opposite phenotype. It may still be objected that two tumors might have arisen separately and later coalesced but, for reasons discussed in Section 111, this criticism is itself open to objection. A theoretical possibility which might account for a double phenotype in a cell population from a monoclonal tumor is that, contrary to the general rule enunciated by Lyon (1974), two or more X chromosomes were active in some or all of the tumor cells. It would be unwise to ignore this possibility, particularly in view of the observations of Migeon et al. (1985), which suggest that X reactivation may occur in human trophoblast. With the enzyme G-6-PD, which is in the form of a dimer, if X reactivation occurred in a sufficient number of cells, the gels should show a third electrophoretic band between the bands corresponding to G-6-PD A and G-6-PD B (Yoshida et d., 1967; Silagi et al., 1969); no such band has been reported in human tumor studies, however, or in studies of fibrosarcomas in M . caroli. With PGK-1, which is monomeric, a third band would not be expected, but with both

TUMOR CLONALITY

207

PGK-1 and G-6-PD the existence of cells in which XMand Xpwere both active could be excluded by cloning, or more simply with appropriate DNA probes. The converse situation in which a pleoclonal tumor appears to express only a single phenotype could be due to any of the following causes: 1. Failure to detect a small subpopulation of a different clonal origin may occur if the method of assay is not sufficiently sensitive. 2. Cells expressing Xp and XMare clustered in patches, and the probability of a tumor arising from two or more cells in the same patch is a function of the patch size (s) and the number of cells (n) at risk of transformation. The value of n depends on the tissue and the transforming agent to which it is exposed. If n is large in comparison with s, the effect of patch size is negligible because the probability (p) that the population at risk will include cells of both kinds approaches the value (p = S''-') it would have if the distribution of Xp and XM cells was random instead of being patchy. If, on the other hand, n is of the same order of magnitude as s, the effect of patch size is important and, as n/s decreases, p decreases and eventually becomes vanishingly small. The patch size differs in different tissues and species. In humans it has been reported to range from 950 to 3000 cells in scalp hair follicles (Gartler el al., 1971) to lo5 cells in myometrium (Linder and Gartler, 1965). We shall return to this subject in discussing methods of estimating the number of clones in a pleoclonal tumor (Section V,A). 3. Selection may favor transformation and survival of cells of a particular phenotype. This may depend on the phenotype of the marker used, or on phenotypic differences determined by other loci on the X chromosome. The first type of selection is unlikely to be operating unless the proportion of tumors expressing a particular marker differs significantly from the proportion of normal cells in the tissue in which the tumor originates that express the same marker, but this does not exclude the second type of selection. It may well be that this is rarely, if ever, important with tumors arising in normal hosts, though it has to be borne in mind in interpreting experiments, such as those of Deamant et al. (1986), in which tumors are induced in interspecies chimeras (Section IV,E). 4. X-chromosome loss or anomalous gene expression in neoplastic cells may occur. These factors seem more likely to be an important cause of error when the pair of markers used consists of the presence or absence of a single phenotypic character, as in the experiments of Williams et al. (1983). When two different phenotypes are involved, X-chromosome loss can be excluded, at least in theory, by cytological observations, and anomalous gene expression is unlikely to be important if the relative extent to which each phenotype is expressed is found to be much the same in a population of tumors of the same kind (as distinct from a single tumor), as it is in normal tissues.

208

B.


KARYOTYPIC AND

OTHER CHROMOSOMAL MARKERS

Chromosome abnormalities in cancer may be constitutional or acquired. Constitutional abnormalities occur in the inherited forms of retinoblastoma and Wilms’ tumor. Patients with these conditions are congenitally heterozygous for a specific chromosomal deletion in all of their normal somatic cells (Evans, 1986),and the presence of the deletion in tumor cells gives no information whatever about tumor clonality. Acquired abnormalities include the characteristic microscopically recognizable translocations seen in many patients with chronic myeloid leukemia (CML), Burkitt’s lymphoma (BL), various other leukemias and lymphomas, and small cell carcinoma of the lung (see Evans, 1986), and allelic loss recognizable at the molecular level in many patients with retinoblastoma, Wilms’ tumor, and osteosarcoma. None of these markers per se provide evidence for or against monoclonality as we have defined it, i.e., N monoclonality (Section 111). They do, however, provide evidence about P monoclonality and, particularly if used in conjunction with an X-linked marker, can contribute to the study of changes in clonal composition during carcinogenesis. At present their usefulness is limited by uncertainty about when they first appear. We shall illustrate this by considering first the Philadelphia chromosome and second the phenomenon of allelic loss.

1. The Philadelphia Chromosome The Philadelphia chromosome (Ph’) is found in almost all the mitoses in the bone marrow in > 85 % of patients presenting with C M L (Tough et a l . , 1963). It has been established that the abnormal chromosome is always number 22, and in >90% of cases it results from a reciprocal translocation between chromosomes 22 and 9;moreover, the point of exchange is always in band 22qll (Rowley, 1981), and the amount of material translocated from chromosome 22 is constant (see Evans, 1982). But at what stage in the development of C M L does the abnormality appear? The presence of the Ph’ chromosome before the diagnosis of C M L could be made on other grounds has been reported by Canellos and Whang-Peng (1972)and other authors. Kamada and Uchino (1978)studied 102 patients with CML, 41 of whom had been exposed to radiation in Hiroshima as a result of the atomic bomb. In 16 of these 41 patients, laboratory examinations had been performed twice a year for 5-10 years ‘‘prior to the development of CML,” and the appearance of Ph’ was followed successively by the appearance in the blood of 5% of immature granulocytes, an increase in the serum level of vitamin BIZ,splenomegaly, and the development of subjective

TUMOR CLONALITY

209

symptoms such as fatigue and gastrointestinal disturbances. These results, with others cited by Kamada and Uchino, lend support to the view of Killman (1972) and Pedersen (1973) that the Ph’ chromosome is “a marker of the preleukemic state and not a leukemic process itself.” This begs the question of what criteria should be used to make the diagnosis of CML, but the notion that a chromosomal abnormality should be a marker of what we have called in Section I1 “part-way cells” is not implausible. It may be asked whether the translocation is present in a small proportion of normal cells, and, if so, whether the leukemia originates from one or more such cells. To investigate this thoroughly would require an enormous amount of time and effort; moreover, even if this were established, it would not follow that C M L is N-monoclonal unless it were shown also that (1) the Ph’ translocation is present in all the neoplastic cells and (2) the Ph’ cells are all derived from a single normal cell. There is, in fact, some not very strong evidence from two sources which suggests that this may be the case. Fitzgerald et al. (1971) reported that in a patient with C M L who was an XY/XXY sex chromosome mosaic, the Ph’ translocation was present in all the 46 XY cells which were scored but not in any of the 47 XXY cells; the significance of this seems uncertain, however, because in a similar mosaic reported previously (Tough et al., 1961), some cells of each kind carried the translocation. Gahrton et al. (1974) observed that the Qbanding of the Phl chromosome in one patient resembled that of one of his mother’s normal number 22 chromosomes, and in another patient it resembled that of one of his father’s number 22 chromosomes, and interpreted this as evidence of a clonal origin of the Ph’ chromosome from either the paternal or the maternal chromosome 22. The data we have been considering are thus not inconsistent with the conclusion that C M L is N-monoclonal, but the evidence provided by studies with G-6-PD (Fialkow et al., 1967) is of much greater weight.

2. Allelic Loss Some tumors that arise in children, in particular retinoblastoma, Wilms’ tumor, and some osteosarcomas, appear to result from deletion or inactivation of both copies of a wild-type genetic locus. It has been shown in some patients, by using DNA probes homologous to a variety of arbitrarily chosen loci on chromosome 13 (retinoblasoma and osteosarcoma) or 11 (Wilms’ tumor), that alleles which are present on the patient’s normal cells may not be present on at least some, and conceivably all, of the tumor cells (Cavanee et al., 1983; Solomon, 1984; Dryja et al., 1986). This has led to the hypothesis that an inherited or acquired specific deletion at a locus on one chromosome is followed by a second, less specific, mutational event on the homologous chromosome

210


which is responsible for the observed allelic loss. This second event may take the form of loss of a whole chromosome, loss of one chromosome and reduplication of the other, mitotic recombination, or translocation. The pattern of allelic loss, which may be different in different tumors of the same kind, has been cited as evidence of “a clonal origin” of Wilms’ tumor (Cavanee et al., 1983). It seems to be quite strong evidence, subject to the proviso that it relates to P-clonality, but might be tested further, if the tumor cell population could be cloned, by assaying the resulting cloned cell lines.

C . IMMUNOGLOBULINS AND IG-GENE REARRANGEMENTS Membrane-bound immunoglobulin (Ig) on a normal human B lymphocyte is restricted to one Ig class (and, if this is IgG, to one subclass), one light chain, and one Gm allotype even in Gm heterozygotes (Froland and Natvig, 1972). This fact has prompted the use of these markers to study the clonality of neoplasms whose cells express surface Ig. Findings in patients with chronic lymphatic leukemia (CLL), whose cells nearly always express surface Ig (usually IgM), and BL (Fialkow et al., 1973) are consistent with a monoclonal origin for these neoplasms. This interpretation is open to the objection that malignant transformation of a B lymphocyte may change its surface expression of Ig and that selection may operate to favor the transformation or subsequent growth of cells bearing a particular Ig molecule. The risk of error due to selection is, however, greatly reduced when, as has been found with BL, studies with two independent markers (surface Ig and G-6-PD alloenzymes) yield concordant results. The Ig of the cells of murine plasmacytomas and Ig-secreting lymphomas is similarly restricted to one Ig class and one light chain, and the conclusion that these neoplasms are monoclonal (Potter, 1972) seems plausible, subject to the same caveats as in the case of human lymphomas. The Ig produced by a tumor is usually nonfunctional, in the sense that it does not react with an identified antigen. It may nevertheless possess a unique idiotype, and this provides further evidence of monoclonality. It may be noted in passing that such idiotypes are potential targets for specific immunotherapy (Stevenson et al., 1977; Krolick et al., 1979). Sklar et al. (1984) have investigated the clonality of human B cell lymphomas both by characterizing their surface Ig and by analyzing the configuration of Ig genes in tumor DNA with probes specific for the heavy-chain joining region, the heavy-chain constant region, and the constant regions of the x and X light chains. Four of the lymphomas studied were judged to be biclonal (Section V,A).

21 1

TUMOR CLONALITY

D. CELLSURFACE ANTIGENS OTHER THAN IG Some tumors possess unique surface antigens of the type known as tumorassociated transplantation antigens (TATA) (see Woodruff, 1980, for review). It has been observed with one type of murine tumor that clones which expressed the same PGK-1 alloenzymes had TATA in common, whereas those which expressed different PGK-1 alloenzymes did not (Woodruff el al., 1984), and it would be of interest to examine the feasibility of using TATA to assess the clonality of other experimental tumors.

E. MARKERS IN NATURAL OR ARTIFICIALLY PRODUCED

CHIMERAS

Chimerism may arise naturally in a variety of ways (McLaren, 1975). Naturally occurring human chimeras are rare but may occasionally provide an opportunity for studies of clonality. There seems little point in using naturally occuring animal chimeras for this purpose because animal chimeras can easily be produced artificially. Iannaccone et al. (1978) investigated the clonality of chemically induced epidermal and mesodermal tumors in intraspecific chimeric mice made by amalgamating embryos of two different mouse strains which express different electrophoretic variants of the enzyme glucose phosphate isomerase. Deamant et al. (1986) have since studied chemically induced fibrosarcomas in interspecific chimeras made by amalgamating embryos of M. musculus and M. caroli. Iannaccone et al. point out that differences between the two subpopulations in respect of liability to carcinogenesis are more likely to occur in artificial chimeras than in natural heterozygotes, but argue that this was not a source of error in their experiments. Such commendable caution is less apparent in the later paper, although the likelihood of error due to selection would be expected to be greater with interspecific than with intraspecific chimeras. Iannaccone et al. also discuss what they refer to as the number of clones in a patch of cells of the same enzyme phenotype in their chimeric mice. This is of interest in relation to the study of chimeras but seems irrelevant to the study of tumor clonality because the founder cells for these clones must be cells which existed when the embryos were amalgamated to make the chimeras. If the same starting point were to be taken for neoplastic clones there would be yet another definition to add to those considered in Section 111. This, in the writer’s view, would be unhelpful. Indeed, it may be asked, if one is going as far back as this, why not proceed to the limit and start with the fertilized ovum? Such a reductio ad absurdurn would obviate the necessity for further discussion of tumor clonality, since all tumors would be, by definiton, monoclonal.

212

MICHAEL F. A . WOODRUFF

V. Analysis of Pleoclonal Tumors

A. How MANYCLONES? The number of clones in a pleoclonal tumor could be determined if, and only if, each clone carried a unique, stable, detectable marker. For most categories of tumor no such markers are known, but there are two possible exceptions, namely, tumors which produce Ig (Section IV,C), and tumors which express TATA (Section IV,D). In neither case is there rigorous proof that each clone in a pleoclonal tumor would express a unique marker; this is a more-or-less plausible assumption based on observed differences between different monoclonal tumors, and between clones from the same tumor which are distinguished by some other marker. Should this assumption be false, the presence of only a single marker would not necessarily imply that the tumor was monoclonal. Conversely, for Ig + tumors, the presence of two or more distinct Ig molecules is not necessarily incompatible with monoclonality because Ig-gene rearrangement or class switching may have occurred during or after transformation. With these reservations, studies of the cell surface Ig, and Ig - gene configuration, of human B cell lymphomas ( W a r et al., 1984) point to the conclusion that some of these tumors are biclonal. In studies with murine fibrosarcomas, cloned subpopulations derived from a single tumor were found to possess cross-reacting TATA if, but only if, they were of the same PGK-1 phenotype (Woodruff et al., 1986). This suggests that different clones from the same tumor do not share common TATA, and is what would be expected if the tumors were biclonal. It would, however, be prohibitively costly in time and animals to use TATA as the basis for a thorough study of the number of clones in a tumor unless in vitso assays for TATA could be developed to replace current in vivo assays, or probes were developed which recognized genes coding for TATA. The situation is not quite so bleak if we are asking questions about a population of similar tumors rather than about a single tumor, although it is by no means free from obstacles and traps for the unwary. Fialkow et al. (1971a) found that 14 neurofibromas from two patients with congenital neurofibromatosis who were heterozygous for the A and B forms of G-6-PD, all expressed both alloenzymes, and concluded that these tumors originate from at least 150 cells. They based this conclusion on the following two propositions: 1. If A cells and B cells were present in equal numbers and were equally distributed in the tissues in which the tumors originated, the probability that all of the tumors would contain neoplastic cells of both kinds would be (%)I4, i.e., 400 hepatomas induced with phenobarbitone, alone or in association with N-nitrosodiethylamine, in mice heterozygous for deficiency of OCT. The great majority of the tumors were judged to be monoclonal on the grounds that they contained only OCTpositive or only OCT-negative tumor cells. As discussed previously (Section IV,A, l), however, this evidence cannot be regarded as conclusive because OCT-negative tumor cells have also been found in hepatomas induced in animals not deficient in OCT.

VII. Factors Influencing Clonality Every tumor begins with the transformation of one or more susceptible target cells as a consequence of the action of a set of genetic and environmental factors which constitute what, for want of a generally accepted term, we shall call the oncogenic package. The factors which might be expected to influence tumor clonality are of three kinds: (1) the number and distribution of susceptible target cells; (2) various parameters of transformation which depend on the nature of the oncogenic package and the target cells, including the number and nature of the steps involved, the growth potential of cells at each stage in the process, and the probability that a cell which has reached any given stage will proceed to the next stage; and (3) interactions between different clones of part-way or fully transformed cells, and between such cells and the host (Section I). It would seem premature, when the available data are so meager, to attempt to develop a general theory; instead, we shall consider a number of theoretical possibilities which generate predictions that can usefully be compared with what has been observed, or which point to gaps in our knowledge that need to be filled.

A . SOMETHEORETICAL PREDICTIONS If a given number of cells is exposed to the same oncogenic package, and clones arise and develop independently, the number of cells which become transformed and give rise to a neoplastic clone should be the same irrespective of whether the cells all belong to one individual or are distributed between two or more identical individuals. In consequence, if two identical individuals are exposed to the same oncogenic package, the probability that they will each develop a single monoclonal tumor should equal the probability that one will develop either a biclonal tumor or two monoclonal tumors, depending on the volume of tissue in which the oncogenic package operates and the distribution of susceptible target cells. If the development of one clone inhibits the development of another in the vicinity, the probability of a biclonal tumor will be correspondingly reduced; and if the inhibitory effect extends to clones anywhere in the same individual, the probability of two separate tumors will also be reduced.

TUMOR CLONALITY

22 1

The following predictions are based on the application of these principles to some particular cases. Case 1. Transformation involves only one step and the probability of transformation is low. Prediction: Few tumors will develop, and those that do will nearly all be monoclonal. Case 2. Transformation involves only one step and the probability of transformation is high. Prediction: The number of tumors will depend on the probability that a clone of transformed cells will give rise to a tumor, and this will depend inter aliu on the intrinsic growth potential of the transformed cells and the reaction of the host. If many tumors develop they are likely to be pleoclonal provided that (1) clones arise independently and one does not outgrow all the others as the result of a greater intrinsic growth potential or competitive clonal interaction, and (2) there are regions in which susceptible target cells are densely concentrated. The latter proviso, which we shall refer to subsequently as the target cell concentration condition, is necessary because for the progeny of two or more cells to form a pleoclonal tumor the founder cells must not be too far apart. Cells which fulfill this condition may conveniently be described as adjacent. There are no data from which to estimate how close the cells must be but there is no obvious reason why they should be contiguous, nor indeed why the distance between them should not be considerably greater than the diameter of patches associated with Xp and XMmosaicism (Section IV,A,3). Case 3. Transformation involves two or more steps. The probability of each step from the beginning, or after a certain point, is low. Prediction: As in Case 1. Case 4. Transformation involves two or more steps. The probability of each step up to a certain critical point is low, but thereafter is high. Prediction: The number of tumors will be limited by the number of cells that reach the critical point. If this number is appreciable, and the clonal independence and target cell concentration conditions are fulfilled, the tumors are likely to be pleoclonal. Case 5. Transformation involves two or more steps, the first being either the inheritance of a mutant gene (Knudson et ul., 1973), or some other step which, though not already achieved, is highly probable. Prediction: Any of the results discussed in Cases 1-4, depending on the subsequent steps involved.

B. INTERPRETATIONOF DATAAND GAPSTO BE FILLED 1. Human Tumors Many human tumors are typically solitary, and it seems likely that the probability of transformation is low; the probability that they will be monoclonal is therefore high. Other tumors, including leiomyomas, warts, neurofibromas in patients with congenital neurofibromatosis, and a variety of lymphomas are typically multiple. We may then distinguish between three situations, and suggest an explanation when sufficient data concerning clonality are available.

222

MICHAEL F. A . WOODRUFF

1. Individual tumors are monoclonal but different tumors in the same patient are not all of the same clonal origin, as is the case with uterine leiomyomas. Here, if the patient has a lot of tumors, the probability of transformation is not sufficiently low to account for the monoclonality, and the explanation is probably either wide separation of susceptible target cells or clonal interaction. In the case of uterine leiomyomas the former explanation seems more plausible. If, as has been suggested from a study of six cases (Murray et al., 1971), the common wart is a monoclonal tumor, and if further investigations show that different warts in the same patient are of different clonal origin, warts would fall into the same category as leiomyomas, but clonal interaction would seem the more likely reason for their monoclonality . 2. All the tumors in a patient appear, from studies with X-linked markers, to be of the same clonal origin. Here we must begin by asking whether the appearance of monoclonality is due to selection favoring the transformation of cells of one particular phenotype (see Section IV,A,3). If selection is not a factor, as may well be the case with BL (although this is not certain), the tumor must have arisen from cells disseminated from a single focus. This could be by metastasis from a single primary tumor, but there might conceivably have been dissemination of cells that had taken some, but not all, of the steps toward transformation. 3. There are multiple pleoclonal tumors. Here the probability of transformation must be high. In congenital neurofibromatosis there is a genetic factor affecting many cells, but other factors may also be involved in the transformation. If so, the probability that each step will occur must be high. In the case of the pleoclonal B-cell lymphomas reported by Sklar et al. (1984), the question arises of why they consisted of just two clones. We shall consider this later when discussing biclonal animal tumors (Section VII,B,2). Many gaps in our knowledge are due to the fact that some tumors have been studied in only a few patients and others have not been studied at all. The previous discussion highlights three areas, which overlap to some extent, where further investigation might be especially rewarding. First, some, indeed according to Knudson et al. (1973) virtually all, human tumors occur in two forms: a nonheritable form, and a heritable form in which a predisposition to the tumor is inherited as a dominant character. Familiar examples are retinoblastoma, Wilms’ tumor, neurofibroma, and carcinoma of the colon. The heritable forms of retinoblastoma and Wilms’ tumor have been reported on the basis of a few cases to be monoclonal; congenital neurofibroma is pleoclonal. It would be of interest to compare the clonality of the two forms of these and other tumors. Second, some tumors occur as solitary tumors in some patients and as multiple tumors in others. In the case of neurofibroma, the nonhereditary form is typically solitary and the hereditary form multiple, and in breast cancer the hereditary factor seems to be stronger in patients with bilateral tumors than in those with a unilateral tumor. There are, however, exceptions to this rule; a patient may, for example, have just one wart or many, and there is no reason to suppose that the multiple ones are congenital. It would be of interest to study the clonality of individual solitary tumors and multiple tumors

TUMOR CLONALITY

223

and of the disease as a whole when there are multiple tumors. Some studies of this kind have been undertaken (e.g., with BL) but, as we have seen, these have not been entirely conclusive. Third, with some tumors, agents such as hormonal stimulation or an exogenous virus, which might be expected to act on a large number of target cells, appear to play an etiological role. A study of clonality might give some indication of whether, as appears to be the case with Epstein-Barr virus (EBV) in BL, other factors are also involved.

2. Animal Tumors It is possible in laboratory animals to study the effect of carcinogen dosage on tumor clonality, and to choose regimes which consistently yield a high proportion of pleoclonal tumors, almost certainly because the probability of transformation is high. As already mentioned (Section V,A,2), there is evidence which suggests that many of the MC-induced fibrosarcomas studied by Woodruff et al. (1986) were biclonal, and this appears to be true also of some human B-cell lymphomas (Section VII,B,l). There would seem to be two possible explanations. First, the probability of transformation, though too high for all the tumors to be monoclonal, was not sufficiently high to yield a significant number of tumors with more than two clones. Second, several, and perhaps many, cells are transformed in each mouse or patient, but many of the incipient neoplastic clones disappear as the result of clonal interaction. In putting forward this suggestion, Woodruff et al. postulated that when a second clone arises in the presence of an established clone a state of positive symbiosis may or may not develop. If it does, a third clone is unlikely to compete effectively with the established symbiotic pair; if it does not, one of the two clones is likely to disappear, after which the cycle may be repeated. The first explanation seems more likely in the case of the human lymphomas, the second for the murine fibrosarcomas. Other experimental tumors of great interest are the lymphomas that develop in transgenic mice bearing the c-myc oncogene coupled to the lymphoid-specific Ig heavy-chain enhancer. Adams et al. (1985) identified these as B-cell lymphomas and reported that, as judged by IgH-locus rearrangement patterns, most of them were monoclonal. In later experiments (Langdon et al., 1986) it was found that in young transgenic mice, before tumors developed, there was an expanded polyclonal population of B-cell precursors which were regarded as nonmalignant because they failed to grow upon transplantation, whereas cells of the monoclonal population that developed later were readily transplantable. In each case the assessment of clonality was based on the pattern of Ig-gene rearrangement. It is not clear why the members of the polyclonal population, even if they were premalignant rather than malignant, were not transplantable, and it would be of interest to repeat the transplantation tests at several cell dose levels. Assuming, however, that the distinction is valid, it would seem that progression to malignancy in these mice requires at least one further step, and the probability of this occurring is relatively low.

224


VIII. Conclusions and Perspectives If, as seems certain, carcinogenesis is a multistep process, a discussion of tumor clonality is meaningful only if we specify the category of cell that is to be taken as the founder of a neoplastic clone. The definition of clonality we have adopted specifies that this will be an initially normal cell that takes one or more of the steps on the road to transplantation before it divides, and this corresponds to clonality as determined with X-linked markers. It is, however, sometimes useful to consider clones with a different starting point, and to avoid confusion the terms N clone, T clone and P clone are proposed to designate clones which have arisen from a single normal, transformed, or part-way cell, respectively; and N-monoclonal, T-monoclonal, and P-monoclonal to denote tumors whose neoplastic cell population consists of a single N clone, T clone, or P clone. The terms monoclonal and pleoclonal without qualification stand for N-monoclonal and N-pleoclonal, respectively. A further problem of definition that will not be resolved until we can obtain reliable information about the distribution of subpopulations of different clonal origin in a single tumor mass, concerns the distinction between a pleoclonal tumor and a composite tumor formed by the coalescence of two or more monoclonal tumors. Only two human tumors, congenital neurofibroma and the rare congenital trichoepithelioma, have been shown to be regularly pleoclonal; a few others have been found to be so occasionally. Evidence of monoclonality has been reported on many occasions, but more data are required before we can draw up anything like a complete list of the kinds of human tumor that are nearly always monoclonal, and the proportion, if any, of each kind that are pleoclonal. The possibility of using X-linked RFLP as markers instead of the G d - P D alloenzymes should make it much easier to obtain this information. Mosaicism exists not only for X-linked gene products, but also for products of Ig genes, which are autosomally encoded (See Herzenberg et al., 1976), and this has been used to study the clonality of Ig-producing tumors (Section IV,C). If other instances of fixed inactivation at autosomal loci were found, it would open the way to studying in males the clonality of a wide variety of tumors which at present can only be studied in females. Whatever marker of clonality is used, we cannot rule out the possible existence of clonal subpopulations too small to be detected. There is evidence that such populations do exist in some apparently monoclonal experimental tumors, and may later expand sufficiently to be detectable or even predominant. If they exist also in some human tumors, do they, on occasion, give rise to metastases or recurrences? The only way to find an answer to this question is to undertake a systematic comparison of the clonal composition of metastatic (or recurrent) tumors and the primary tumors from which they have arisen.

TUMOR CLONALITY

225

If a tumor conforms to our definition of monoclonality, it may be concluded (Section VI1,A) that the probability of transformation was low, or that susceptible target cells were sparsely distributed, or that all clones except one have somehow been eliminated. Without other evidence, however, we cannot distinguish between these possibilities; nor can we say anything about the ancestry of the tumor cells (which we shall refer to as the pattern of carcinogenesis) beyond the fact that they have all developed from a single normal cell. Two possible but very different patterns are illustrated diagramatically in Fig. 1. In the first (Fig. 1A) the tumor, in addition to being N-monoclonal, is T-monoclonal, since it has developed from a single transformed cell; in the second (Fig. 1B) it is T-pleoclonal. The development of an N-monoclonal tumor in a field of altered cells is consistent with either pattern, although when this happens the second is perhaps the more likely. The paradox referred to in Section I is thus resolved. T o begin to distinguish between different patterns we must be able to distinguish between cells at different stages on the way to transformation. Morphology, and tumorigenicity on transplantation to a histocompatible or immunodeficient host, may be of some help, but more precise markers are urgently needed. T o take advantage of these as they are developed, however, we will also require sensitive methods for recognizing N-clonal, P-clonal, and T-clonal subpopulations at the cellular level. If a tumor is pleoclonal it is likely that none of the conditions of the preceding paragraph are fulfilled. It is, however, perhaps worth bearing in mind the theoretical possibility that normal or part-way cells of a different Xp or XM phenotype may have become transformed as a consequence, in part, of stimulation by a growth factor released by tumor cells, or have been transfected with tumor cell DNA. There does not seem to be any evidence to suggest that in vitro transfection in the laboratory has a natural in vivo counterpart, but who knows? It would be of interest to compare the numbers of N clones, P clones, and T clones at different stages during carcinogenesis in tumors of various kinds. A start could be made with retinoblastoma, Wilms’ tumor, and osteosarcoma, and as new markers are developed other tumors should become accessible to study. Tumors in which there is a clinically recognizable preneoplastic or preinvasive phase, including carcinoma of the colon in patients with familial polyposis coli, malignant hepatoma in patients with cirrhosis of the liver, and carcinoma of the cervix, would seem particularly appropriate for this type of investigation. The study of tumor clonality, like other areas of cancer research, has gained much from the use of appropriate animal models. Chemically induced murine fibrosarcomas have proved useful because the proportion of monoclonal tumors can be altered simply by altering the dose of carcinogen, and similar experiments could be set up to yield a range of tumors induced with a variety

226


A

0 @ 0

NORMAL CELL PART WAY CELL FULLY TRANSFORMED CELL

B

FIG. 1. Diagram illustrating two different patterns of carcinogenesis. In each case the tumor is assumed to develop in two steps from a single normal cell (open circle). Tumor arises (A) from one transformed cell or (B) from many. Hatched circles represent partly transformed cells; closed circles, fully transformed cells.

of different agents. Experiments with B-cell lymphomas in transgenic mice bearing a c-myc oncogene coupled to the lymphoid-specific Ig heavy-chain enhancer have extended the study of clonality to preneoplastic cells, and thus opened up a new field which invites further analysis. Already the study of tumor clonality has raised many questions of biological importance, and suggested some answers, but there are unsolved technical problems which limit progress in this field. There are also semantic and psychological obstacles to be surmounted. Monoclonality has been described as the cornerstone of current theories of carcinogenesis and, with some qualifications, so it is. But unless we start from unambiguous definitions and examine their implications rigorously, the cornerstone becomes a stumbling block.

ACKNOWLEDGMENTS The author thanks the Medical Research Council (U.K.) for a series of project grants, and Professor H . J. Evans for the privilege of working in his unit. He also thanks Professor Evans, and Drs. J. D. Ansell, N. D. Hastie, and H . S. Micklem for helpful suggestions and critical reading of this review, and Alan R. Liss Inc. for permission to quote from an article published in The International Journal .f Cancer (Woodruff el al., 1986).

TUMOR CLONALITY

227

REFERENCES Adams, J. M., Harris, A. W., Pinkert, C. A., Corcoran, L. M., Alexander, W. S., Cory, S., Palmiter, R. D., and Brinster, R . L. (1985). Nature (London) 318, 533-538. Alexander, P. (1985). Br. J. Cancer 51, 453-457. Ansell, J. D., and Micklem, H . S. (1986).In “Handbook of Experimental Immunology” (D. M. Weir, ed.), 4th. Ed., pp. 56.1-56.18.Blackwell, Oxford. Ansell, J. D., Hodson, B. A., and Woodruff, M. F. A. (1986).Br. J . Cancer 54, 853-855. Barnes, D. W. H., Carr, D. E. F., Evans, E. P., and Loutit, J. F. (1970).1nt.J. Radiat. Biol. 18, 53 1-537. Barnes, D. W. H . , Evans, E. P., and Loutit, J. F. (1971).Nature (London) 233, 267-268. Bucher, T . , Bender, W., Fundele, R., Hofner, H., and Linke, I. (1980). FEBS Lett. 115, 319-324. Canellos, G . P., and Whang-Peng, J. (1972). Lancet 2, 1227-1229. Cattenach, B. M., and Isaacson, J. H. (1965).Z. Vererbungsl. 96,313-323. Cavanee, W. K., Dryja, T. P., Phillips, R . A., Benedict, W. F., Godbout, R., Gallie, B. L., Murphree, A. L., Strong, L. C., and White, R. L. (1983). Nature (London) 305, 779-784. Charles, D. J., and Pretsch, W. (1984). M o w News Lett. 71, 37-38. Chen, S. H., Malcolm, L. A., Yoshida, A., and Giblett, E. R . (1971). Am. J . Hum. Gmet. 23, 87-91. Deamant, F. D., and Iannaccone, P. M. (1985).J.Natl. Cancer Znsf. 74, 145-150. Deamant, F. D., Vijh, M., Rossant, J., and Iannaccone, P. M . (1986). Int. J . Cancer 37, 283-286. DeMars, R., and Nance, W. E. (1984).In “Retention of Functional Differentiation in Cultured Cells” (V. Defendi, ed.), pp. 35-48. Wistar Institute, Philadelphia. Dryja, T . P., Rapaport, J. M., Epstein, J., Gorin, A. M., Weichselbaum, R . , Koupos, A., and Cavanee, W. K. (1986). Am. J . Hum. Genet. 38, 59-66. Evans, H . J. (1982). I n “Host Factors in Human Carcinogenesis” (B. Armstrong and H. Bartsch, eds.), pp. 35-36.IARC, Lyon. Evans, H. J. (1986). Zn “General Toxicology of Environmental Chemicals. Part A: Basic Principles and Mechanisms of Action” (C. Ramel, B. Lambert, and J. Magnusson, eds.), pp. 41-69. Liss, New York. Feinberg, A. P., and Vogelstein, B. (1983). Nature (London) 301, 89-92. Fialkow, P. J. (1972).Adu. Cancer Res. 15, 191-225. Fialkow, P. J. (1976). Biochim. Biophys. Acfa 458, 283-321. Fialkow, P. J., Gartler, S. M., and Yoshida, A. (1967). BOG. Natl. Acad. Sci. U.S.A. 58, 1468-1471. Fialkow, P. J., Klein, G., Gartler, S. M., and Clifford, P. (1970). Lancet 1, 384-386. Fialkow, P. J . , Sagebiel, R . W., Gartler, S. M., and Rimoin, D. L. (1971a).N. Engl. J. Med. 284, 298-300. Fialkow, P. J., Klein, G., Giblett, E. R . , Gothoscar, B., and Clifford, P. (1971b). Lancet 1, 883-886. Fialkow, P. J., Klein, E., Klein, G., Clifford, P., and Singh, S. (1973).J . Exp. Med. 138, 89-102. Fitzgerald, P. H., Pickering, A. F., and Eiby, J. (1971). Br. J . Haeniafd. 21, 473-480. Forrester, L. M., and Ansell, J. D. (1985). Gmef. Res. 45, 95-100. Friedman, J. M., and Fialkow, P. J. (1976). Transplant. Rev. 28, 17-33. Froland, S. S., and Natvig, J. B. (1972).J. Exp. Med. 136, 409-414.

228


Gahrton, G., Linston, J , , and Zech, L. (1974). Blood 43, 837-840. Gartler, S. M., Gandini, E., Hutchison, H . T . , Campbell, B., and Zechhi, G . (1971).Ann. Hum. Genet. 35, 1-7. Harnden, D. G., and Klinger, H . P., eds. (1985). “An International System for Human Cytogenetics,” p. 74. Karger, Basel. Herzenberg, L. A,, Herzenberg, L. A,, Black, S . J . , Loken, M. R., Okumura, K . , van der Loo, W., Osbourne, B. A,, Hewgill, D., Goding, J. W., Gutman, G . , and Warner, N. L. (1976).Cold Spring Harbor Qmp. Quant. Biol. 41, 33-45. Howell, S., Wareham, K. A., and Williams, E. D. (1985).Am. J. Pathol. 121, 426-432. Iannaccone, P. M., Gardner, R . L., and Harris, H . (1978).J. Cell Sci. 29, 249-269. Kamada, N., and Uchino. H. (1978).Blood 51, 843-850. Killman, s. (1972).Hacmatoiogica 57, 641-649. Knudson, A. G . (1973). Adv. Cancer Res. 17, 317-352. Knudson, A. G., Strong, L. C., and Anderson, D. E. (1973).B o g . Med. Genet. 9, 113-158. Krolick, K.A,, Isakson, P. C., Uhr, J . W . , and Vitetta, E. S. (1979).Immunol. Reu. 48,81-106. Langdon, W. Y.,Harris, A. W., Cory, S., and Adams, J . M. (1986).Cell 47, 11-18. Linder, D., and Gartler, S. M. (1965). Science 150, 67-69. Lyon, M. F. (1974). Roc. R. Soc. London Ser. B 187, 243-268. McGee, J . O’D., and Al Adani, M. S. (1976).I n “Scientific Foundations of Oncology” (T. Symington and R . L. Carter, eds.), pp. 45-52. Heinemann, London. McKillop, W. J., Ciampi, A,, Till, J. E., and Buick, R . N. (1983).J.Natl. Cancer Insf. 70, 9-16. McLaren, A. L. (1975).“Mammalian Chimeras.” Cambridge Univ. Press, London and New York. McMahon, A. (1983). Genet. Res. 42, 77-89. McMahon, A,, Fosten, M., and Monk, M . (1983).J. Embryol. Exp. Morphol. 74, 207-220. Micklem, H . S. (1986). Blood Cells 12, 119-126. Migeon B. R., Wolf, S. F., Axelman, J., Kaslow, D. C., and Schmidt, M . (1985).Roc. Natl. Acad. Sci. U.S.A. 82, 3390-3394. Murray, R . F., Hobbs, J., and Payne, B. (1971). Nafure (London) 232, 51-52. Nance, W. E. (1964).Cold Spring Harbor Symp. Quanf. Biol. 29, 415-425. Pedersen, B. (1973).Br. 1.Haemafol. 25, 141-145. Potter, M . (1972). Physiol. Reu. 52, 631-719. Raskind, W.H., and Fialkow, P. J. (1987). Adv. Cancer Rcs. 49, 127-167. Reddy, A. L., and Fialkow, P. J. (1979).J. Exp. Med. 150, 878-887. Reddy, A. L., and Fialkow, P. J. (1983). Nature (London) 304, 69-71. Rowley, J . D. (1981). In “Genes, Chromosomes and Neoplasia” (F. E. Arrighi, P. N. Ross, and E. Stubblefield, eds.), pp. 273-296. Raven, New York. Schnipper, L. E. (1986).N. Engl. J . Mcd. 314, 1423-1431. Silagi, S., Darlington, G., and Bruce, S. (1969).Roc. Nafl. A c Q ~Sci. . U.S.A. 62, 1085-1092. Sklar, J., Cleary, M . L., Thielemans, K., Gralow, J., Warnke, P., and Levy, R . (1984).N . Engl. J . Med. 311, 20-27. Solomon, E. (1984). Nature (London) 309, 111-112. Stevenson, G. T . , Elliott, E. V., and Stevenson, F. K. (1977). Fed. R o c . , Fed. Am. SOC.Exp. Biol. 36, 134. Takagi, N., and Susaki, M . (1975). Nafure (London) 256, 640-642. Tanooka, I. M., and Tanaka, K. (1982). Cancer Res. 42, 1856-1858. Tough, I. M., Court Brown, W. M., Baikie, A. G., Buckton, K. E., Harndon, D. G., Jacobs, P. A , , King, M . J., and McBride, J. A. (1961). Lancef 1, 411-417. Tough, I. M., Jacobs, P. A., Court Brown, W. M., Baikie, A. G . , and Williamson, E. R. D. (1963). Lancet 1, 844-846.

TUMOR CLONALITY

229

Vogelstein, B., Fearon, E. R . , Hamilton, S. R., and Feinberg, A. P. (1985). Science 227, 642-645. West, J. D. (1984). In “Chimeras in Developmental Biology” (N. Le Douarin and A. McLaren, eds.), pp. 39-63. Academic Press, New York. West, J . D., Frels, W. I., and Chapman, V. M . (1977). Cell 12, 873-882. Williams, E. D., Wareham, K. A., and Howell, S. (1983). Br. J . Cancer 47, 723-726. Willis, R . A. (1960). “Pathology of Tumors,” 3rd Ed. Butterworths, London. Woodruff, M. F. A. (1980). “The Interaction of Cancer and Host.” Grune & Stratton, New York. Woodruff, M. F. A. (1983). Br. J . Cancer 47, 589-594. Woodruff, M . F. A,, Ansell, J. D., Forbes, G. M., Gordon, J. C . , Burton, D. I., and Micklem, HS. (1982). Nature (London) 299, 822-824. Woodruff, M. F. A,, Ansell, J . D., Hodson, B. A,, and Micklem, H. S. (1984). Br. J . Cancer 49, 5-10. Woodruff, M. F. A,, Ansell, J. D., Hodson, B. A,, and Potts, R. B. (1986). Int. J . Cancer 38, 747-751. Yoshida, A , , Steinmann, L., and Herbert, P. (1967). Nature London 216, 275-276.


NEWBORN MACROSOMY AND CANCER Lev M. Berstein Petrov Research lnstltute of Oncology, Leningrad 186646, USSR

I. Introduc

'

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

................................................. id Optimum Birthweight: Ranges of Macrosomy . . . B. Macrosomy Incidence in Healthy Populations . . . . . . . . . . . . . . . . . . . C. A Large Fetus as a Marker of Potential Pathology: History of the Problem ................................ 111. Basic Evidence and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Incidence of Large-Baby Birth Reported in Cancer Patients Based on Case Histories ....................................... B. Anthropometric and Hormonal-Metabolic Characteristics of Cancer Patients in Whom Large-Infant Births Were Reported in Case Histories C . Prognosis quo ad Vitam in Cancer Patients with Large-Baby Births Reported in Case Histories D. Retrospective Data on Birt Subsequently Developed Cancer: Incidence of Large-Baby Birth E. The Relationship between Transplacental Carcinogenesis, Its Modification with Glucose, and the Large-Fetus Factor. . . . . . . . . . . . . . . . . F. Some Factors Promoting Large-Baby Birth . . . . . . . . . . . . . . . . . . . . . . . . . . . G. The Cancrophilia Syndrome, according to V. M. Dilman, and the Large-Fetus Problem H. the High-Birthweight Infant I. Acceleration of Physical Dev and Cancer . . .

231 235 235 236 236 239 239 241 245 250 254 255 261 263 266

J. 267

a High Infant Birthweight

K. High Infant Birthweight ................ Prophylaxis of Large-In Prevention of Cancer ......................... IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

269

L.

270 272 273

I. Introduction A considerable body of evidence shows that the internal environment of the organism contributes to a great extent to cancer induction, although the process of carcinogenesis is ultimately realized on the cellular level. The 231 ADVANCES IN CANCER RESEARCH, VOL. 50

Copyright 0 1988 by Academic Press, Inc. All rights ofreproduction in any form reserved

232

LEV M . BERSTEIN

organism-tumor relationship is a highly versatile one. One of its major aspects relates to the endocrine-metabolic disturbances observed in cancer patients and their role in the development and clinical course of cancer. The conclusion that these disturbances do play an important role is based first on the ability of hormones to induce tumors under certain conditions (Miihlbock and Boot, 1959; Lipshutz, 1970; Shabad, 1979) and to their modifying role resulting in intensified or inhibitory effects on neoplastic transformation (Furth, 1970; Berenblum, 1978; Bogovsky, 1978). On the other hand, the introduction of the concept of “endocrine-metabolic” disturbances implies that the analysis of this relationship cannot be restricted to hormonal influences alone. However, the importance of metabolic disturbances in creating conditions that promote cancer development has not been recognized for long. No due attention was given to metabolic disorders existing prior to and associated with cancer induction, while ever-increasing interest was given to studies of these disturbances in cancer patients, that is, in the presence of clinically manifested tumors (Kavetzki, 1962; Shapot, 1979; Barclay and Skipski, 1975). For almost three decades, Professor V. M. Dilman and his co-workers at the Petrov Research Institute of Oncology in Leningrad directed their efforts to exploring the role of age-associated disturbances of neuroendocrine regulation in the development of the cancrophilia syndrome, implying a complex of conditions facilitating the tumor growth. An important component of these conditions is the associated disorders of lipid and carbohydrate metabolism (decreased glucose tolerance, insulin hyposensitivity , hyperlipidemia, etc .). The occurrence of these disorders is based on the prevalent utilization of free fatty acids rather than glucose as an energy substrate by the peripheral tissues. Metabolic disturbances supposedly increase the risk for cancer development, particularly through intensified cell proliferation, decreased immune reactivity (metabolic immunodepression phenomenon), and reduced DNA repair (Dilman, 1958, 1968, 1978, 1981, 1983). It is therefore quite obvious that the understanding of the factors promoting the initiation of the endocrinemetabolic disturbances and the search for possible therapeutic and preventive measures to eliminate these disturbances are of significant theoretical and clinical importance. The diversity of forms and ways in which hormonalmetabolic lesions develop in cancer patients present certain difficulties in their systematization despite the practical value of such attempts. Any working classification relating to endocrine-metabolic disturbances seems to be uncertain, due chiefly to the differences in the underlying criteria. Nevertheless, disturbances of three types can be distinguished: (1) disturbances which exert a stimulatory effect on neoplastic transformation or associated with subsequent tumor growth, (2) disburbances which have no effect, and (3) disturbances which reduce the probability of tumor induction. It also seems possible to work out a classification of endocrine-metabolic disturbances on a

NEWBORN MACROSOMY AND CANCER

233

“hormone-substrate” basis which would distinguish the secretion of which hormones and the activity of which endocrine glands have changed. Examples illustrating a classification of this type could include data on the redistribution of excreted steroid (adrenal) hormone fractions in breast cancer patients (Bulbrook et al., 1971) and data on the decreased level of triiodothyronine and increased thyrotropin concentration in some types of neoplasms (Mittra and Hayward, 1974; Rose and Davis, 1979). Endocrine-metabolic disturbances in cancer patients can also be characterized by whether they are related to the presence of the tumor in the organism. The first group of disturbances includes, in particular, different forms of ectopic endocrine syndromes accompanied by hormone production in the tumor tissue (Rees and Ratcliffe, 1974). In addition, just in the tumor tissue an intensive transformation of hormones (primarily that of steroid hormones) takes place, an accumulation of hormone-binding proteins (transcortin, for example) may be observed, and the loss of hormone receptors due to dedifferentiation or, conversely, probably occurring by ectopic expression may be noted (Cikes, 1978). Of significance also may be the biotransformation of hormones in nontumor tissues of the tumor carrier (for example, the process of aromatization of androgens, namely, their conversion to estrogens in adipose tissue, the degree of which is associated with the fat content in the body or which can be independently increased in cancer patients) (Forney et al., 1981). A relatively large group of endocrine-metabolic disturbances can develop in cancer patients as side effects following cancer therapy (hypercholesterolemia and hot flashes after ovariectomy , frequent gain of body weight following unilateral mastectomy, steroid diabetes developed after application of some types of hormone therapy). The disturbances induced by therapy or related to further progression of the disease following previous radical surgery in cancer patients can be referred to as late disturbances. It seems reasonable to distinguish them from the concomitant endocrine-metabolic disturbances found in the course of tumor detection or the first clinical remission, as well as from previously existing (early) disturbances revealed in the medical history (MacMahon et al., 1973), or on the results of subsequent examinations (Bulbrook et al., 1971). It is clear that such a classification would be based chiefly on the determination of the time of detection of the disorders mentioned. The date when the cancer diagnosis was established is taken as a base point. From the preventive point of view, elaboration of measures aimed at the detection and control of early or previous hormone-metabolic disturbances that increase cancer risk is most justified. In this respect, special attention should be paid to the condition of pregnancy. Along with the onset of menarche and menopause, pregnancy is attributed to be the most important biological event in the life of a woman. Its possible

234

LEV M .BERSTEIN

influence on tumorigenesis has been explored for many years (Verhagen, 1974; Querlen et al., 1978; Napalkov et al., 1981). Experimental data on carcinogenesis, including transplacental carcinogenesis and clinical observations in regard to the coexistence of pregnancy with certain types of neoplasms, will not be considered here. It should be noted that in a great number of cancer epidemiological studies pregnancy is primarily regarded as the expression of a specific state of the reproductive function. In the course of pregnancy, however, changes occur not only in the reproductive system, but also in other homeostatic systems of the organism. In this respect two features are worth mentioning: the significant role of metabolic changes (fat and carbohydrate metabolism, in particular) in the deviations of homeostasis and the definite similarity of these changes to the metabolic disorders characteristic of aging and cancer. In the beginning of this century, Professor N. N. Petrov stressed that serological tests clearly showed that all of the same reactions were positive in both cancer patients and pregnant women. “In both cases similar products of cellular (lipid-protein) metabolism were released and similar defense enzymes were produced” (Petrov, 1914). According to Alvarez et al. (1967), pregnancy and cancer present two growth patterns (normal and abnormal, respectively) evoking a stress response with related metabolic features. Of equal importance also is that multiple pregnancies make the organism biologically more aged (Arvay, 1975) and, as assumed (Wexler, 1975), this is related to the progression of metabolic disorders. So far as the similarity of metabolic shifts in pregnancy, aging, and cancer, the contribution of metabolic changes in pregnancy to the development of a predisposition to cancer should be recognized. No due regard has been given to this fact previously. The complexity of this problem is enhanced by the long intervals, lasting sometimes for years, between single or multiple pregnancies and the clinical detection of tumors. In this respect the search for factors of cancer risk, including markers of metabolic disturbances in pregnancy accessible for retrospective analysis, is much needed. It is common knowledge that the status of the physical development of a newborn can be the first signal of apparent, or more frequently, occult pathology of the maternal organism (Navarette et al., 1970; Hytten and Leitch, 1971). The conclusion concerned is related to the subsequent morbidity rate of the child itself. Within the past 15-20 years data on the relationship of the excessive weight of a baby at birth (macrosomy) and cancer development have been collected. In this work the subject will be given fair consideration and treatment.


235

II. Definition

A. THEMEAN,NORMAL, AND O P T I M U M BIRTHWEIGHT: RANGES OF MACROSOMY Many factors affect the body weight of infants at birth. Among these, the sex, the birth order, and the length of gestation should be stressed (Hytten and Leitch (1971) and Meredith (1970). According to their data, collected from different sources, the lowest averages (2600-2900 g) were found in South Indians, Ceylonese, African pygmies, and Australian Aborigines. The next group (2900-3200 g) was represented by South and Central Americans, East and South East Asians, South Africans, Polynesians, and other Pacific Islanders. The highest averages were reported for the North American Indian tribes: from 3510 g for Navajo, 3720 g for Sioux, 3730 g for Commanche, and up to 3830 g for Cheyenne (Adams and Niswander, 1968). An intermediate position was occupied by Europeans (Caucasians). Most of their averages were between 3200 and 3500 g (Hytten and Leitch, 1971). Thus, when discussing the normal ranges of birthweight, ethnic variations should be given due consideration. The above mean values ordinarily represent live births (Meredith, 1970); this does not mean, however, that cases with some obstetric pathology were excluded in the birth series (Hytten and Leitch, 1971). The mean weight of babies born at term, namely, at 40 weeks, was about 80 g above the overall mean birthweight, although this difference may vary in both directions (McKeown and Gibson, 1951). The mean birthweight is slightly shorter than the median because a few more babies are born at the “light” rather than the “heavy” end of the range (Hytten and Leitch, 1971). The birthweight which is somewhat higher than the mean birthweight is regarded as the “optimum” birthweight. This conclusion is based on the fact that the perinatal mortality rate is in the lower range level of the higher values (plus 50-150 g) for this parameter and not in the mean birthweight ranges for this population (Cavalli-Sforza and Bodmer, 1971; Hytten and Leitch, 1971, Terrenato, 1983). An approach to the solution of this problem needs refinement, because in addition to the relation to the perinatal death rate, both “immediate” and “delayed” effects of the excess birthweight on the health status of subjects should be considered. From the practical point of view it is, however, justified to choose the birthweight of 3400 g at 40 weeks of gestation as representing the “optimum” birthweight for Caucasians (Hytten and Leitch, 1971), or 3500 g for males and 3400 g for females (Kogan, 1971). This value differs significantly from the present ranges of macrosomy, although the latter should not be regarded as completely estimated. There are

236

LEV M . BERSTEIN

variable values in the literature for the weight of a macrosomal fetus. According to different authors these values are as follows: > 3850 g (Ganguly et al., 1972), > 4000 g (Boyd et af.,1983), >4100 g (Horger and Miller, 1975),and >4500 g (Steel et al., 1979). For practical purposes and based on available recommendations, the birthweight of 4000 g for babies born as the result of a single pregnancy at 40 1 weeks of gestation will be regarded here as the lower limit of macrosomy.

*

B. MACROSOMY INCIDENCE IN HEALTHY POPULATIONS Reports on macrosomy incidence are not encountered as frequently as those on mean birthweight, but birthweight data by percentage of distribution of separate weight categories better characterizes the groups studies when compared with mean indices of physical development. It is the general impression that in Caucasians the incidence of birthweights of > 4000 g and > 4500 g is 9-15% and 1.2-2.5%, respectively. According to studies done in different geographical areas of the Soviet Union, macrosomy incidence (> 4000 g) is 8.9% in Kiev, 10.4% in Moscow, 11.9% in Vilnus, and 12.4% in Kalinin (Ignatieva, 1973). In Japan macrosomy incidence is reported to be 3-4% (Hirayama, 1980), and in American Indian tribes the incidence ranges from 13-14% in Hopi and Zuni to 38-41% in Cheyenne and Kiowa (Adams and Niswander, 1968). Among large newborns the males dominate (Boyd et al., 1983), and the average male birthweight is 140-150 g heavier than that for females (Hytten and Leitch, 1971). It should be noted that the length of gestation affects the incidence of macrosomy. According to Boyd et al., (1983),the incidence of birthweights of >4OOO g a t 36 weeks gestation is 1.7%, at 37 weeks 2.1 %, at 38 weeks 4.8%, at 39 weeks 8.2%, at 40 weeks 12.3%, at 41 weeks 15.8%, and at 42 weeks 21.0%. Of some importance also is the birth order (Hytten and Leitch, 1971; Ignatieva, 1973).

C. A LARGEFETUSAS A MARKER OF POTENTIAL PATHOLOGY: HISTORY OF THE PROBLEM The history of the subject is indirectly and surprisingly related to the general history of the measurement of the birthweight. Cone (1961)reported that the earliest records of birthweight date from the end of the seventeenth and the middle of the eighteenth centuries. These records had been collected by F. Mauriceau, T. Lobb, and W. Smellie, who claimed that the mean birthweight was 13-14 lb (5850-6300 g). According to present medical knowledge, such a high birthweight had to be accompanied by a high rate of pathological complications. By the end of the eighteenth century it became


237

obvious that such high mean-birthweight data were in error. Systematic studies of birthweight probably began in the first half of the nineteenth century, about 150 years ago. Significant contributions of L. Quetelet must be mentioned in this respect (Cone, 1961; Tanner, 1981). Pathological conditions related to newborn macrosomy are schematically presented in Fig. 1 . From the historical point of view it is natural that obstetric complications which affect the fate of a mother attract much attention. During the birth of a large baby, a “clinically contracted pelvis” frequently occurs when the size of the fetus’ head does not fit the dimensions of the maternal pelvis. Premature and early bursting of waters and uterine interia are also observed. A large fetus is frequently in breech or oblique position (Ignatieva, 1973; Pedersen, 1977). These complications, in addition to metabolic disturbances relating to macrosomy and probably to population-geneticregularities (CavalliSforza and Bodmer, 1971; Altukhov, 1984), give rise to both intrauterine and early-neonatal mortality rates of newborn babies (Boyd et al., 1983). Consequences of the birth of a large baby appear to affect both the parents and the progeny. Special emphasis in this respect was given to diabetes mellitus. In the early 1930s, H. Bix, E. Skipper, and E. Allen (see Pedersen, 1977) independently suggested that the birth of a large baby might be a precursor of maternal diabetes. Later, this suggestion was confirmed by a number of investigations (Kriss and Futcher, 1948; Kritzer, 1952; Horger and Miller, 1975; O’Sullivan and Mahan, 1980). The concept of prediabetes or the prediabetic state has been developed Uackson, 1954; Hagbard, 1958), and large-baby birth was attributed to be one of its significant markers, which is of independent prognostic importance in respect to the possible, subsequent development of diabetes mellitus (Pedersen, 1977). Among potential diabetics (diabetes mellitus in near relatives; a high perinatal death rate on previous births; glucose tolerance disorders observed in the course of pregnancy), the large-baby factor failed to account for a rise in the incidence of diabetes mellitus, but a 3-fold greater risk in developing its decompensated form was noted (O’Sullivan and Mahan, 1980). According to Pedersen (1977), there

A. immediate manifestations of pathology

B. distant manifestations of pathology

FIG. 1. Variants of pathological complications associated with high-birthweight infants.

238

LEV M. BERSTEIN

are two groups of females in whom a large-fetus birth preceded the development of diabetes mellitus: (1) young women of normal body weight who delivered large fetuses not long before the clinical detection of diabetes and (2) aged women with an excess gain of body weight who delivered large fetuses many years before the clinical detection of diabetes mellitus. This point of view indirectly corresponds to the concept that newborn macrosomy precedes the development of maternal diabetes of both the first type (insulin-dependent, juvenile-onset diabetes) and the second type (insulin-independent, maturityonset diabetes). The influence of the large-baby birth factor on the parental morbidity rate is hardly determinable. Several authors only (Babbott et al., 1958; Malins and Fitzgerald, 1965) succeeded in confirming previous observations of Jackson (1954), who claimed that the risk of developing diabetes mellitus was 2.5- to %fold higher in fathers who had babies weighing > 4100 g at birth. The studies showed that when diabetes mellitus was subsequently found in the father, the birthweight of his baby was 150 g greater than that in the control group, while a similar situation in women showed a 400-g increase (Nillsson, 1962). If the paternal prediabetic state is of any importance, its influence on baby birthweight appears to be less significant than the maternal prediabetic state (Pedersen, 1977). This is in agreement with the prevalent influence of maternal characteristics over paternal (Ounsted and Ounsted, 1966; Hytten and Leitch, 1971). Apart from the data reported relating to the subsequent development of diabetes mellitus, additional information on the morbidity rate in parents who have babies with large birthweights is almost nonexistent. Babies of high birthweight were characterized by clinical and metabolic adaptation disorders observed during the neonatal period and by decreased reactivity in the first year of their lives (Pedersen, 1977; Vereskova, 1979). They showed a high incidence of some infectious diseases (as do babies with low birthweights) (Altukhov, 1984), and the possibility of developing diabetes mellitus is increased (Baranov and Stroikova, 1980). Examination of these patients between ages of 13 and 30 years revealed high levels of reactive hyperinsulinemia when compared with subjects whose birthweights were < 4000 g (Motocu et a l . , 1976). Marked deviations of somatic and hormonal states were also observed in the macrosomal descendents of parents with diabetes mellitus (Radder and Terpstra, 1975; Amendt et a l . , 1976). Convincing experimental evidence is also available in this field (Bartelheimer and Kloos, 1952). Thus, it seems apparent that the excessive birthweight of an infant introduces a great number of problems for specialists in different medical disciplines. (The incidence of high birthweights among Caucasians mentioned above, is 10%.) What are the links between the above reported information and the main objective of this chapter? Following such a long, but essential

239


outline, which has provided a general background of the large-fetus factor, it should be stressed that the main purpose of the further presentation of this subject is to emphasize that studies of newborn macrosomy are not only a target of research for obstetricians, diabetologists, and pediatricians, but also a special problem for oncologists.

Ill. Basic Evidence and Discussion

A. INCIDENCEOF LARGE-BABY BIRTHREPORTED IN CANCER PATIENTSBASEDON CASEHISTORIES Our first attempts to explore this problem (1968-1969) showed that among breast cancer patients the number of females in whom case histories reported the birth of a large baby (> 4000 g or 4100 g) was 1.5 and 2 times that of the control group, respectively (Berstein and Hint, 1970). These encouraging findings stimulated our interest in conducting a similar analysis among patients with different neoplasms. The study was carried out in 1970-1971 (Berstein, 1973, 1981), and its main results are summarized in Tables 1-111 and Figs. 2-6. It should be noted that reports on single pregnancies completed at 40 1 weeks of gestation by the birth of live babies were taken into consideration. Table I shows the data on 1035 patients and 421 healthy women who gave birth to 1703 and 648 babies, respectively. The patients were subdivided into 14 nosological groups; each included not less than 20 patients. When compared with controls, significant increase in the number of women with large-baby births was observed for colorectal cancer, skin melanoma, cervical and endometrial cancer, malignant ovarian tumors, and gastric and breast cancer. This observation was more pronounced in patients > 50 years of age at the time the case histories were taken or at the time of clinical detection of the disease (Table I, Fig. 2). Calculations based on the Mantel-Haenszel formula (Mantel and Haenszel, 1959) showed that the risk of developing some types of cancer was 1.69- to 2 .O4-fold greater in women > 50 years of age who had previously delivered babies of high birthweight (Fig. 3). In this group of female patients with the neoplasms listed above, the mean birthweight of their babies was higher than in the corresponding control group, and again this observation was more pronounced in patients who developed cancer at over 50 years of age (Table 11).It should be stressed that the higher the average age of the cancer patients (i.e., the longer the period between the delivery and the clinical detection of cancer), the greater is the number of birthweights of >4000 g among their babies. The opposite observation is made in healthy women (Fig. 4), probably because of an acceleration phenomenon (see Section IIIJ). The “shift to the right,” namely, in the direction of higher birthweight babies in women who subsequently became cancer patients, was noted

*

240

LEV

M.BERSTEIN

TABLE I PERCENTAGE OF WOMENWITH LARGE BABIES (>4000 c)AT BIRTHAMONG CANCER PATIENTS AND HEALTHY WOMEN",^ Age at examination (years)

20-49

Type of cancer Cervical cancer Endometrial carcinoma Chorioepithelioma Ovarian cancer Ovarian benign tumors Lymphogranulomatosis Uterine myoma Gastric cancer Lung cancer Bone and soft tissue cancer Colorectal cancer Melanoblastoma Breast cancer Benign breast disease Healthy women

33.3 f 10.5 f 28.6 i 25.0 f 20.0 i 5.6 f 26.5 f 20.0 i 12.5 f 16.0 i 31.8 f 38.2 f 23.0 f 17.1 f 20.1 i

7.2 (42) 6.9 (19) 9.9 (21) 8.2 (28) 7.2 (30) 5.7 (18) 7.7 (34) 10.3 (15) 11.6 (8) 7.3 (25) 9.9 (22) 12.5 (15) 3.6 (139) 3.5 (117) 2.4 (273)

250 41.6 f 7.1 (48) 42.6 f 5.7 (73) (1) 46.7 f 9.1 (30) 25.0 f 12.5 (12) (3) 50.0 f 15.5 (8) 43.5 10.2 (23) 25.9 f 8.5 (27) 33.3 f 18.5 (6) 45.8 f 5.9 (72) 46.7 i 8.5 (34) 35.5 i 4.1 (138) 14.8 6.9 (27) 14.2 f 2.9(148)

*

All cases

37.8 i 35.9 f 31.8 36.2 f 21.4 f 9.5 f 31.0 f 34.2 f 22.8 i 19.4 i 42.5 f 40.8 f 29.2 f 16.7 i 18.1 i

*

5.1 (90) 5.0 (92) 9.9 (22) 6.3 (58) 6.3 (42) 6.5 (21) 7.1 (42) 7.7 (38) 7.2 (35) 7.0 (31) 5.1 (94) 7.0 (49) 2.7 (277) 3.1 (144) 1.8 (421)

"From Berstein (1981). bPercentagesare means f SE.

when the data were distributed by weight categories (Fig. 5). The occurrence of a greater number of large-baby births in cancer patients persists or is even intensified with progression of the birth order (Fig. 6). The trend is observed not only when the birthweight is > 4000 g, but also when > 4100 g and > 4500 g are the criteria of macrosomy. In the latter cases, the difference from the controls is found chiefly in male babies (Table 111). Thus, a large-baby birth is one of the early markers of a possible risk of cancer development long before its clinal detection.' This fact (see also Section III,C) allows early detection of those women who need observation and medical control. The fact that large-baby births are frequently found in case histories of cancer patients over 50 years of agez is additional proof of the necessity of classifying some forms of cancer by individual pathogenetic types (de Waard and Laive, 1960; de Waard, 1979; Dilman, 1968, 1974; Berndt and Landmann, 1969; Bokhman, 1972; Berstein, 1973; Semiglazov, 1980). 'According to our data an interval between the large fetus birth (birthweight of 4000 g and more) and cancer detection was on the average 25.5 i 0.7 years for breast cancer. 2The findings of Semiglazov (1980)and Umenushkina (1980)showed similar trends.

241

NEWBORN MACROSOMY AND CANCER >50 years

Colorec- Melano- Cervical Ovarian Endomet- Gastric Breast Cancer of Lung Benign Normals tal car- blastorna cancer carci- rial car- cancer cancer bone and cancer breast (healthy soft tissues disease women) cinorna norna cinorna

42.6.

43.5’

35.5.

33.3

25.9

14.8

14.2

23.0

16.0

12.5

17.1

20.1

26 Age of mother at first delivery (years)

FIG.9. Age at primary term and the “large-baby syndrome” in cancer patients. M, Pa, without large-baby syndrome. tients with large-baby syndrome; x - - - ~ patients

individual differences combined with specific risk factors may play an important role both in the predisposition to cancer development in certain sites and, conversely, in antitumor protection. If an attempt is made to classify cancer risk factors into irreversible, reversible, and potentially reversible types, as has been done by Bierman (1978) in respect to atherosclerosis, then the large-fetus syndrome (which is an irreversible anamnestic factor) might rather be attributable to reversible factors that might weaken the associated hormonal-metabolic disturbances (see Section I11,L).

K. THESO-CALLED HORMONE-INDEPENDENT CANCERS AND THE INCIDENCE OF HIGHINFANTBIRTHWEIGHT The present division of different neoplasms into hormone-dependent and hormone-independent types becomes more and more obscure. In fact it would be more correct to regard all tumors as well as normal tissues as being potentially hormone dependent (Kavetzki, 1977). However, the degree of hormone dependence may vary within one and the same nosological entity (Jensen, 1981). The high incidence of large-infant birth revealed in case histories of patients suffering from neoplasms which traditionally [with no due regard to the accumulated relevant data (see Murakami and Masui, 1980; Weiss et a/.,

270

LEV M . BERSTEIN

1981, Dilman, 1983)] were not considered hormone dependent (Table I, Fig.

2) is evidence of how incorrect this point of view is and of the general significance of the large-fetus factor in respect to oncological precaution.

L. PROPHYLAXIS OF LARGE-INFANT BIRTHAS A MEASURE OF METABOLIC PREVENTION OF CANCER Metabolic prevention of cancer development can be classified as primary and secondary. The latter is directed to the correction of endocrine-metabolic disturbances in cancer patients who have been subjected to radical therapy (surgery, in particular), and its ultimate objective is to prevent further progression of the tumor process (Dilman et d.,1975; Dilman, 1983). Primary prevention is oriented to the detection and elimination of early hormonalmetabolic disturbances contributing to the development of the cancrophilia syndrome (Section 111,G). Control of disturbances leading to neonatal macrosomy is also part of the primary metabolic prevention of cancer. Prevention and elimination of these disorders should involve efforts of oncologists as well as obstetricians, pediatricians, and endocrinologists. O n the one hand, we support the opinion of Crampton (1964) that older women who have had a large-infant birth need to be under constant medical observation, considering the possibility of their developing not only diabetes mellitus but also certain neoplasms. It seems justified to take measures for this “risk group” directed mainly to the correction of lipid-carbohydrate metabolism disorders. Consideration of a number of case history findings (onset of menarche, cancer-associated heredity), anthropometric findings (excess body weight, increased body surface area), and laboratory data (including also blood carcinoembryonic antigen level) enables the identification of those persons who need periodic medical examinations. O n the other hand, attention should also be given to the metabolic control of persons with excess body weight at birth, taking into account their tendency to develop during childhood and adolescence some types of cancer (Section III,D), diabetes mellitus (Section II,C), and retention of some endocrinemetabolic disorders until an older age (Motocu et al., 1976). Of significant importance is the elimination of conditions which could lead to a large-infant birth. Although, according to observations of ethnographers, primitive tribes restricted the diets of pregnant women (see Jeliffe, 1962), these measures exerted little influence on the birthweights of newborns (Page, 1969; Reynolds and Taper, 1982), and such extremely low caloric intake can result in ketosis and functional lesions of the central nervous system (Felig, 1977). Diabetes mellitus in pregnant women can be treated effectively with antidiabetic biguanides (Pedersen, 1977; Stowers and Sutherland, 1975). Our experimental data also showed that the administration of these drugs to rats


271

who had previously received alloxan resulted in decreased birthweights of their offspring; but a similar effect was not observed in alloxan-untreated normal pregnant rats (Table XV). On the basis of these findings, we concluded that the action of these drugs was associated with their normalizing influence on glucose tolerance and the blood insulin level. With the aim of preventing a large-infant birth and the associated hormonal-metabolic disturbances which are high cancer risk factors, biguanides and drugs of similar activity may be used in persons with signs of gestation diabetes, a family history of diabetes, previous large-infant births, and excess maternal body weight. Biguanides are widely used for the elimination of different manifestations of the cancrophilia syndrome (Dilman, 1981, 1983). Although biguanides fail to cause developmental pathology in progeny (Sterne, 1969; personal data), it appears justified to use them prior to the onset of pregnancy. For example, these drugs were administered in early-developing autoimmune lesions of the thyroid associated with decreased glucose tolerance, hyperinsulinemia, and subsequent birth of large infants (Section II,F,4). With biguanide, metabolic disturbances were eliminated and infant birthweights were decreased, although the interval between the administration of the drug and the delivery had been fairly long (Fig. 10). An increased occurrence of latent autoimmune thyroiditis was observed among children who outwardly appeared to be healthy (Inoue et al., 1975). The detection and timely treatment of this condition should be regarded as an important preventive measure of thyroid pathology and endocrine-metabolic disturbances relating to large-infant birth and other manifestations of the hormonal-metabolic complex which increase the risk of cancer development (Section 111,G).

TABLE XV EFFECT OF PHENETHYLBIGUANIDE (PHENFORMIN) ON WEIGHT OF FETUSES IN R A T S " ~ ~ Weight of fetuses (9) Administration of biguanide'

-

+

Normal pregnancy

Pregnancy following alloxan introduction

5.21 f 0.05 (114) 5.26 f 0.04(145)

5.40 f 0.05 (71)d 4.92 f 0.05 (76)d

"Data from Berstein and Alexandrov (1976); Berstein ct al. (1977a). *Number of fetuses given in parentheses. CBiguanide(5-10 mg/day) was administered orally from day 7 to 21 of pregnancy. $Difference is statistically significant (p < 0.01).

272

LEV M . BERSTEIN

5.50

p1.2 < 0.02

p1‘3

5.46*0.06(21)

< 0‘01

5.50

5.40*0.07(26) 5.40

5.40 5.37*0.04(43)

5.30

5.30

\

\

5.20

y.

5.20

14*0.06(26)

Q)

5.10

5.10

\

\

\ 5.00

\

4.90

4.96*0.08(35) 1

2

3

G12

13-19

\c

5.00 4.90

220

FIG. 10. The effect of phenethylbiguanide (phenformin) on the birthweight of fetuses of rats subjected to immunization with homologous thyroid antigen (Berstein ct al., 1981). Abscissa, birthweight (g); ordinate, interval between delivery and termination of immunization (in weeks). 0-0, hatched bars, immunization; x---x, dotted bars, immunization t phenformin (7.5 mg from day 1 of the 3-week immunization to 30 days after its termination); open bars, normal pregnant rats. In parentheses, number of fetuses.

IV. Conclusions The data reviewed can be briefly summarized as follows. The main conclusion is that large-infant birth is an early marker of conditions contributing to the development not only of diabetes but also of a number of neoplasms. The importance of the large-fetus syndrome in increased cancer risk is high for women aged 50 and older in whom large-infant births were revealed in case history. Oncologically , problems resulting from a large-infant birth involve both the mother’s health and that of her progeny. Hormonal-metabolic disorders resulting from newborn macrosomy can have delayed or immediate effects on the health of a mother and her fetus: “delayed” pattern (latent for many years after a large-infant birth; a typical complex of hormonal-metabolic disturbances); “immediate” pattern (direct effect on the target tissue facilitating under certain conditions the action of carcinogenic agents to become manifest in early and late periods of ontogenesis). In both, these factors play the role of “conditional influences” (Berenblum, 1978). The same factors are probably important in the prognosis of some tumors (Section II1,C). This, however, needs further study. The elimination of metabolic factors promoting large-infant birth and its unfavorable consequences may be regarded as a preventive measure in cancer control. The perspectives and


273

further analysis of the problem will require a more precise definition of the risk among persons born with high weights or of those who have large infants as revealed in case histories.

ACKNOWLEDGMENTS I express my sincere. gratitude to my teacher, Prof. V. M. Dilman, for constant attention to my work and my colleagues for assistance in conducting this study, especially to Drs. V. A. Alexandrov, E. K. Hint, and B. A. Kolygin.

REFERENCES Abe, R., Kumagai, N., and Kimura, M. (1976). TohokuJ . Exp. Mcd. 120, 351-359. Adams, M. S., and Niswander, J. D. (1968). Hum. Biol. 40, 226-233. Alexeev, N. A., and Vorontsov, I. M . (1979). “Leukemia in Childhood.” Medisina Publ., Leningrad (in Russian). Alexandrov, V. A. (1983). In “Modulators of Experimental Carcinogenesis” (V. Turusov and L. Montesano, eds.), pp. 65-78. Altukhov, Ju. P. (1984). Vcstn. Acad. Nauk. Mcd. N7, 16-21 (in Russian). Alvarez, R . R., Goodell, B. W., and Zighelboim, J. (1967). Am. J . Obstct. Cynccol. 97, 419-442. Amendt, P., Michaelis, D., and Hildmann, W. (1976). Endokrinologic 67, 351-361. Andrezen, V. P., and Nesmelova, N. M . (1973). “Methodological Recommendations on Evaluation of the Physical Development of Children of Early Age.” Leningrad. (in Russian). Andronescu, A. (1970). “Anatomy of a Child.” Bucuresti, (in Russian) Annegers, J. F., and Malkasian, G. D. (1981). Cancer 48, 856-859. L a y , A. (1975). In “Hypothalamus, Pituitary and Aging,” pp. 362-375. Thomas, Springfield, Ill. Babbott, D., Rubin, A., and Ginsburg, S.J. (1958). Diabctcs 7, 133-139. Baranov, V. G., and Stroikova, A. S. (1980). “Diabetes in Children” Medisina Publ., Leningrad (in Russian). Barclay, M., and Skipski, V. P. (1975). Bog. Biochtm. Phannacol. 10, 76-111. Bartelheimer, H., and Kloos, K. (1952). Z. Gcs. Exp. Mcd. 119, 246-255. Bastenie, P. A., Neve, P., Bonnyns, M., Vanhaelst, L., and Chailly, M. (1967). Lancct 2, 915-918. Bennett, P. H., and Miller, M . (1970). In “Diabetes” (R. R . Rodriguez and J. Vallance-Owen, eds.) Exc. Med. Int. Congr. Ser.231, 318-324. Knowler, W. C., Rushforth, and N. B. ct 01. (1979). In “Diabetes and Obesity” Bennett, P.H., Vague and Ph. Vague, eds.). Exc. Mcd. Int. Congr. Ser. 454, 117-126. Berenblum, I. (1978). J. Natl. Cancer Inst. 60, 723-726. Berndt, H., and Landmann, R . (1969). Arch. Gcschwulslforsch. 33, 157-163. Berstein, L. M . (1973). Vopr. Oncol. 3, 48-54 (in Russian). Berstein, L. M. (1975). In “Methodological Aspects of Studies of Breast Cancer Epidemiology,” pp. 69-75. Tallinn:Bit, (in Russian). Berstein, L. M. (1981). In “Malignant Tumors and Pregnancy” (N. P. Napalkov, Ja. V. Bokhman, and R. I. Wagner, eds.), pp. 47-54. Leningrad, (in Russian). Berstein, L. M . , and Alexandrov, V. A. (1976). Bull. Exp. Bid. Mea’. 7 , 825-827 (in Russian) Berstein, L. M., and Alexandrov, V. A. (1977). Bull. Exp. Biol. Med. 8 , 217-220, (in Russian). Berstein, L. M., and Alexandrov, V. A. (1984). Cancer Left. 25, 171-176. Berstein, L. M . , and Dilman, V. M. (1981). Vopr. Oncol. 6, 54-58 (in Russian). Berstein, L. M., and Hint, E. K. (1970). Akush. Ginekol. 6, 65-66 (in Russian).

u.

274

LEV M . BERSTEIN

Berstein, L. M . , and Hint, E. K. (1975). Zrauoohr. Sou. Esfon. 1, 19-23 (in Russian). Berstein, L . M . , Alexandrov, V. A., and Vasilieva, I. A. (1977a).Pathol. Physiol. 2, 60-63 (in Russian). Berstein, L. M . , Fedoreev, G. A , , Kolygin, B. A., and Dilman, V. M. (1977b).Pediatria 8, 63-66 (in Russian). Berstein, L. M., Levinson, L. L . , and Strukova, G . N. (1980a).Akush. Ginccof. 8, 44-46 (in Russian). Berstein, L. M . , Okulov, V. B., Alexandrov, V. A,, and Evtushenko, T. P . (1980b). Probl. Endokrinol. 2, 71-74 (in Russian). Berstein, L. M., Okulov, V. B., Alexandrov, V. A., Fedorova, L. V . , and Dilman, V . M . (1981).Bull. Ex). Biol. Mcd. 5, 616-617 (in Russian). Bierman, E. L. (1978).Fed Proc., 37, Fed. Am. SOC.Exp. Biol. 37, 2832-2836. Bodansky, 0. (1975). “Biochemistry of Human Cancer.” Academic Press, New York. Bogovsky, P. A. (1978).Ail-Union &m#. Leningrad 3-8 (in Russian). Bokhman, Ja. V. (1972). “Endometrial Cancer.” Kishinev:Shtinza (in Russian). Bokhman, Ja. V., Pryanishnikov, V. A., and Chepik, 0. F. (1979). “Complex Therapy of Endometrial Cancer. ” Medisina, Moscow. (in Russian). Boyd, M . E., Usher, R . , and McLean, F. H . (1983). Obstet. Gynccol. 61, 715-722. Breslow, N . E., and Langholz, B. (1983). Int. J. Cancer 32, 703-716. Brinton, L.A,, Williams, R . R., and Hoover, R . N. et al. (1979).J.Natl. Cancer Inst., 62, 37-44. Bulbrook, R . D., Hayward, J. L . , and Spicer, C . C . (1971). Lancet 2, 395-398. Butterstein, G. M . , and Garone, M . (1977). Biol. Reprod. 16, 479-485. Cavalli-Sforza, L. L., and Bodmer, W . F. (1971). “The Genetics of Human Population.” Freeman, San Francisco. Christakis, G., Severinghouse, E. L., and Maldonado, Z. el al. (1965).Amcr. J . Cardiol. 15, 320-332. Cikes, M . (1978). Bur. J . Cancer 14, 211-216. Cone, T.(1961).Pediatrics 28, 490-498. Cox, E. B.,Laszlo, J., and Freiman, A. (1979).J.A . M. Med. Assoc. 242, 2691-2695. Crampton, J. H . (1964).In “Diabetes” (P. Williams, ed.), pp. 513-521. Creagan, E. T.,and Fraumeni, J. F. (1972).J . Natl. Cancer Inst. 49, 959-967. Daling, J . R . , Starzyk, P., Olshan, A. F., and Weiss, N . S. (1984).J . Natl. Cancer Inst. 72, 1039-1041. de Waard, F. (1979).J . Natl. Cancn Inst. 6 3 , 549-552. de Waard, F., and Laive, J. (1960). Br. J . Cancer 14, 437-448. de Waard, F., Cornelis, J. P., Aoki, K.,and Yoshida, M . (1977).Cancer 40, 1269-1275. Dilman, V. M . (1958). Roc. Inst. Physiol. 7, 326-336 (in Russian). Dilman, V. M . (1968). “Aging, Climacteric and Cancer.” Medisina, Leningrad. (in Russian). Dilman, V . M . (1973). Vopr. Oncol. 5, 101-111. (in Russian). Dilman, V . M . (1974). “Endocrinological Oncology.” Medisina, Leningrad (in Russian). Dilman, V . M. (1975).Hum. Physiol. 2, 352-358 (in Russian). Dilman, V. M. (1978). Hum. Physiol. 4, 579-595 (in Russian). Dilman, V. M . (1981). “The Law of Deviation of Homeostasis and Diseases of Aging.” Wright, Boston. Dilman, V. M . (1983). “Endocrinological Oncology,” 2nd Ed. Medisina, Leningrad (in Russian). Dilman, V. M . , Berstein, L. M., Tsyrlina, and E. V. ct al. (1975). Vopr. Oncol. 11, 33-39 (in Russian). Doll, R . (1980). Cancer 45, 2475-2485. Donegan, W.L . , Hartz, A. J., and Rimm, A. A. (1978). Cancer 41, 1590-1594. Donovan, B. T., Van Der Werff Ten Bosch (1974). “Physiology of Sexual Development” (Russian trans].). Pedagogika, Moscow.


275

Dowsett, M., McGarrick, G. E., Harris, A. L. et al. (1983). Br. J . Cancer 47, 763-770. Elwood, J. M . , Cole, Ph., Rothman, K. J., and Kaplan, S. D. (1977).J.Not[. Cancer Znst. 59, 1055-1060. Enzi, G., Inelrnan, E. M . , and Caretta, F. (1980). Diabetologia 18, 135-140. Farewell, V. T. (1977). Canccr 40, 931-936. Fasal, E., Jackson, E. W., and Clauber, M. R . (1977).J. Nafl. Cancer Inst. 47, 501-510. Felig, P. (1977). Med, Clin. N . Am. 61,43-66. Forney, J . P., Milewich, L., and Chen, G. T. et d . (1981).J . Clin. Endocrinol. Metabol. 53, 192-199. Frisch, R . E. (1980).Fed. BOG., 39, Fed. Am. Soc. Ex#. Biol. 2395-2400. Frisch, R . E., and McArthur, J . W. (1974). Science 185, 949-951. Frisch, R . E., and Revelle, R. (1970). Science 169,397-400. Furth, J . (1970).Inf. Cancer Con-gr., 1Ofh 1, 288-299. Ganguly, M., Elwood, J . H., and McKenzie, G. (1972).Am. J . Epidemiol. 96, 161-167. Gold, E., Gordis, L., Tonascia, J . , and Szklo, M. (1979).Am. J . Epihmiol. 109,309-319. Grufferman, S.,Wang, H . H . , DeLong, E. R. et QI. (1982).J. Nafl. Cuncer Insf. 68, 107-113. Haenszel, W. M. (1964). Natl. Cancer Znst. Monogr. 15, 21-33. Hagbard, L. (1958). Acta Obsfef. Gynecol. &and. 37, 497-518. Harding, P. G.,Kinch, R. A. H., and Stevenson, J . A. F. (1962). Diabetes 11, 321-328. Hill, M. J. (1975). Cancer Res. 35, 3398-3403. Hirayama T. (1980). Zn “Recent Advances in Management of Children with Cancer” (M. Kobayashi, ed.), Children’s Cancer Association, Tokyo. pp. 27-43. Hoel, D. G., Wakabayashi, T . , and Pike, M. C. (1983).Am. J . Epidemiol. 118, 78-79. Horger, E. O.,and Miller, M. C. (1975). Obstet. Gynecol. 45, 150-154. Hytten, F. E., and Leitch, I. (1971). “The Physiology of Human Pregnancy.” Blackwell, Oxford. Ignatieva, R . K. (1973). “On the Statistics of Prematurity.” Medisina, Moscow (in Russian). Inoue, M., Taketani, N., Sato, T., and Nakajima, H . (1975).Endocrinol. Jpn., 22, 483-488. Itoh, K., and Maruchi, N. (1975). Lancet 2, 1119-1121. Iversen, T. (1966).Acfa Pcdiatr. Scund. Suppl. 167, 1-219. Jackson, E. W., Norris, F. D., and Klauber, M. R. (1969).Cancer 23, 913-920. Jellife, D. B. (1962). Am. J. Clin. Nutr. 10, 19-45. Jensen, E. V. (1981). Cancer 47, 2319-2326. Juret, P.,Couette, J.-E, Brune, D., and Vernhes, J.-C. (1974). Eur. J . Cancer 10,591-594. Juret, P., Couette, J.-E., Mandard, A. M . ef al. (1976). Eur. J . Cancer 12, 701-704. Kavetzki, R. E. (1962). “Tumor and Host.” State Med. Publ., Kiev (in Russian). Kavetzki, R. E. (1977). “Interrelation of Host and Tumor.” Naukova Dumka, Kiev (in Russian). Kennedy, G. C., and Mitra, J. (1963).J.Physiol. (London) 166, 408-418. Kholdin, S. A. (1962). Vopr. Oncol. 6,28-35 (in Russian). Kirchoff, H . (1967). Geburf Frauenheikd., 27, 565-571. Knopp, R. H., Saudek, Ch. D., Arky, R. A., and O’Sullivan, J. B. (1973). Endocrinofou 92, 984-988. Koqak, M., Hipkin, L. J., and Davis, J. C. (1981).Eur. J. Cancer 17, 123-124. Kogan, R. B. (1971). “The Health of Early Age Children in the Soviet Union.” Medisina, Moscow (in Russian) Konradi, L. I . , and Matveeva, 0. F. (1971). Probl. endocrinol. 6, 23-25 (in Russian). Kriss, J . P., and Futcher, P. H. (1948).J. Clin. Endocrinol. 8 , 380-389. Kritzer, M. D.(1952).Med. Clin. N. Am. 36, 1151-1155. Kunitz, S. J., and Levy, J . E. (1981). Zn “Ethnicity and Medical Care” (A. Harwood, ed.), pp. 337-196.Harvard Univ. Press, Cambridge, Mass.

276

LEV M . BERSTEIN

Kurbatova, 0. L., Botviniev, 0. K., Deschekina, M . F. ct d. (1982). Gcnetika 7, 1173-1182 (in Russian). Leboeuf, G., and Ducharme, J. R. (1966). Pcdiatr. Clin. N . Am. 13, 19-42. Lipschutz, A. (1970). Vopr. oncol. 61,94-104 (in Russian). Loffer, F. D. (1975).Am. J . Obstet. Gynecol. 123, 180-184. Loucks, A. B., Horvath, S. M., and Freedson, P. S. (1984). Hum. Biol. 56, 383-392. Lundholm, K.,Holm, G., and Schersten, T. (1978). CQW~Res. 38, 4665-4670. McKeown, T.,and Gibson, J. R. (1951). Br.1. SOC.Med. 5, 98-110. MacMahon, B., and Newill, V. A. (1962).J . Not/. Cancer Znst. 28, 231-244. MacMahon, B., Cole, P., Lin, T. M. et d . (1970). Bull. WHO 43, 209-221. MacMahon, B., Cole, P., and Brown, J. (1973).J . Nad. Cancer Znst. 50, 21-42. MacMahon, B., Purde, M., Cramer, D., and Hint, E. (1982). J. N d . Canccr Inst. 69,

1035-1038.

Malinas, R . R. (1979). Excrcisc Sport Sci. Reu. 6, 203-255. Malins, J. M., and Fitzgerald, M. G. (1965).Diabetes 14, 175-181. Mann, G.V., and Shafer, R. D. (1966).J . Am. Mcd. Assoc. 197, 1071-1073. Mantel, N., and Haenszel, W . (1959).J. Natl. Cancer Znst. 22, 719-748. Meredith, H. V. (1970). Hum. Bid. 42, 217-264. Miller, A. B., Barclay, T. H., and Choi, N. W. (198O).J. Chron. Dis. 33, 595-605. Mittra, I., and Hayward, J. L. (1974). Lancet 1, 885-888. Morse, E. H., and Clarke, R . P. (1975).Am. J. Clin. Nut?. 28, 1000-1013. Motocu, M., Duma, V . , Blaga, S. el al. (1976). Clujul. Mcd. (Romania) 49, 169-171. Miihlbock, O.,and Boot, L. (1959). CZBA Found. Symp. Carcinogen. Mech. Action 113-127. Murakarni, H . , and Masui, H . (1980).Froc. Natl. Acad. Sci. U.S.A. 7 7 , 3464-3468. Naeye, R. L., Sims, E. A. H., and Welsh, G. W. (1966).Arch. Pathol. 81, 552-560. Napalkov, N. P . , Bokhman, Ya. V., Wagner, R . I., eds. (1981). “Malignant Tumors and Pregnancy.” Leningrad, (in Russian). Navarette, V., Rojas, C., Alger, C., and Paniaqua, H . E. (1970).Lancef 2, 994-995. Newsholme, E., and Start, K. (1977). “Regulation of Metabolism.” Mir, Moscow (in Russian transl.). Nilsson, S. E. (1962). A d a Mcd. Scand. (Suppl.) 375, 1-101. Osborne, R . H., and De George, F. V. (1967).Canccr 20, 263-270. Osinski, S. P., Bubnovskaya, L. N., and Sergienko, T. K. (1983). Exp. Oncol. 3, 35-37 (in Russian). O’Sullivan, J. B., and Mahan, C . M . (1980).J . Chron. Dis. 33, 37-45. Ounsted, M., and Ounsted, C . (1966).Nature (London) 212, 995-997. Page, E. W. (1969).Am. J. Obstet. Gynecol. 104, 378-385. Peckharn, C. H . (1971).Am. J . Obstet. Gynecol. 111, 1-7. Pedersen, J. (1977). “The Pregnant Diabetic and Her Newborn,” 2nd Ed. Munksgaard, Copenhagen. Peto, R., Roe, F. J. C., Lee, P. N. el 01. (1975). Br. J , Cancer 32, 411-426. Petrov N. N. (1914). Cited in Serebrov, A. I., “N. N. Petrov” (1972). Medisina, Moscow (in Russian). ~ ~ QZnt. Congr. Scr. 238, 102-110. Prernachandra, B. N. (1971). E X C C TMed. Querlen, D . , Cappelaere, P., Crepin, G. ct 01. (1978).“Cancers et grossesse.” Masson, Paris. Radder, J. K. and Terpstra, J. (1975).Diabetologia 11, 135-138. Ramirez, V. D. (1973).Hand. Physiol. 2, 1-28. Randle, P. J , , Garland, P. B., Hales, C. N., and Newsholrne, E. A. (1963). Lancet 1, 785-789. Reaven, G. N., Olevsky, J., and Farquhar, J . W. (1972). Lancet 1, 1247-1249. Reddy, B. S.,Hedges, A. R., Laakso, K., and Wynder, E. L. (1978). Cancer 42, 2832-2838. Rees, L. H., and Ratcliffe, J. G. (1974). Clin. Endocrinol. 3, 263-302.

277 NEWBORN MACROSOMY AND CANCER Reynolds, L. K., and Taper, L. J. (1982). Fed. Boc., 41, Fed. Am. Soc. Ex. Bid. (Abstr.). Rhomberg, W. (1975).Dtsch. Med. Wochenschr. 100, 2422-2427. Rose, D. P., and Davis, T. E. (1979). Cancer 43, 1434-1438. Ryatsep, R. V., Sotnikova, E. N., and Buchny, A. E. (1981). Genefika 4, 740-746 (in Russian). Saez, S. (1971). Eur. J . Cancer 7, 281-389. Salonen, T.,and Saxen, L. (1975). Int. J . Cancer 15, 941-946. Schindler, A. E. (1978). In “Endometrial Cancer” (M. G. Brush, R . J. B. King, and R . W. Taylor, eds.), pp. 29-35. Bailliere Tindall, London. Semiglazov, V. F. (1980).Chirurgia 12, 27-31 (in Russian). Shabad, L. M . (1979). “Evolution of Theories of Carcinogenesis.” Medisina, Moscow (in Russian). Shapot, V. S. (1979).Adu. Cancer Res. 30, 89-150. Sherman, B., Wallace, R., Bean, J., and Schlabaugh, L. (1981).J . Clin. Endocrinol. Mcfab. 5 2 ,

488-494. Sievers, M. L. (1966). Publ. Health Rep. 81, 1075-1083. Smith, R . L., Salsbury, D. G., and Gilliam, A. G. (1956).J.Natl. Cancer Inst. 17, 77-89. Staszewski, J. (1971).J. Natl. Cancer Insf. 47, 935-947. Steel, J. M., Gray, R. S., and Clarke, B. F. (1979).Br. Med.J. 1, 1303-1305. Stein, J. H., West, K. M., and Robey, J. M . (1965).Arch. Intern. Mcd. 116, 842-845. Stenkvist, B., Bengtsson, E., Dahlqvist, B., and Nordin, B. (1982). Cancer 50,2884-2893. Sterne, J. (1969). In “Oral Hypoglycaemic Agents” (G. D. Campbell, ed.), pp. 193-245. Academic Press, London. Stowens, D., Cook, M. G., and Mallard, A. (1961).Mincrua Pediatr. 13, 865-890. Stowers, J. M., and Sutherland, H . W. (1975).In “Carbohydrate Metabolism in Pregnancy and Newborn” (H. W. Sutherland, ed.), pp. 205-220.Churchill Livingstone, London. Tannenbaum, A. (1959). In “Physiopathology of Cancer” (F. Homburger, ed.), pp. 517-562. Hoeber-Harper, Berlin. Tanner, J. M . (1973)Nature (London) 243, 95-96. Tanner, J. M . (1981). “A History of the Study of Human Growth.” Cambridge Univ. Press, London. Taylor, G. O., and Akande, E. 0. (1975). Er. J . Obstef. Gynaccol. 82, 297-302. Terrenato, L. (1983).Ann. Hum. Genet. 47, 67-71. Thomas, B. S.,Bulbrook, R. D., Hayward, J. L., and Millis, R. R. (1982).Eur. J. Cancer. Clin. Oncol. 18, 447-452. Till, M., Rapson, N., and Smith, P. G. (1979). Br. J. Cancer 40, 62-71. Umenushkina, L. N. (1980). In “Oncological Aspects of the Lack of Ovulation” Ua. V. Bokhman, ed.); pp. 105-112,Pskov (in Russian). Van-Duuren, B. L., Smith, A. C . , and Melchionne, S. M . (1978). Cancer Rcs. 38, 865-866. Verhagen, A. (1974).“Tumor and Graviditat.” Springer-Verlag, Berlin. Vereskova, N. E. (1979). Big baby: Group of the risk. Thesis, Moscow (in Russian). Villapando, S., Cisneros, I., Garsia-Bulnes, G. ct al. (1982). Acta Endocrinol. 99, 500-507. Vorontsov, I. M., Kuracheva, N. A,, and Mironovich, V. K. (1972). “Acute Leukemia in Children.” Medisina Publ., Leningrad (in Russian). Vorontsov, I. M., Zinevich, L. N., Andrezen, V. P., and Chanin, L. A. (1973).Pediatria 12, 40-45 (in Russian). Weiss, N. El, Daling, J. R., and Wong Ho Chow (1981).J. Natl. Cancer Inst. 67, 57-60. Wertelecki, W., and Mantel, N. (1973). Pediatr. Res. 7, 132-138. Wexler, B. C. (1975).In “Hypothalamus, Pituitary and Ageing” (A. Everitt, ed.), pp. 333-361. Thomas, Springfield, 111. White, P. (1965).Med. Clin. N. Amc. 49, 1015-1025. Widow, W., and Lehr, V. (1979). Arch. Ceschwulstfrsch. 49, 535-543.

278

LEV M. BERSTEIN

Will, L. C., and WacCay, C. M. (1948). Arch. Biochem. 2, 481-485. Winter, K. (1964). Aerzfl. Forfbild. 58, 1101-1114. Winter, R . J., and Green, 0. C. (1976).J. f‘ediafr. 89, 401-405. Wynder, E. L., and Hill, P. (1977). Lancef 2, 840-842. Wynder, E. L., and Reddy, B. S. (1974). Cancer 34, 801-806. Wynder, E. L . , Esher, G. C., and Mantel, N. (1966). Cancer 19, 489-497. Yamashita, N., Maruchi, N., and Mori, W. (1979). Cancer Left. 7, 9-13. Zeltzer, A. (1968). “Causes and Appearance of Accelerated Growth.” Moscow, Berlin. (in Russian transl.) Zumoff, B., Corzynski, J. G . , Katz, J. L. ef al. (1982). Anficancer Res. 2, 59-62. NOTEADDEDIN PROOF. Several additional published studies are of interest, although they present some controversial data. Children with low birth weight were shown to have a higher risk for testicular cancer (R. H . Depuecf a f . , 1983,J. Nafl. CancerInsf. 71, 1151-1155; L. M. Browncf d . , 1986, CancerRcs. 46, 4812-4816), a higher mortality rate from neuroblastoma (C. Cole Johnson and M . R. Spitz, 1985, J. Nod Cancer Insf. 74, 789-792), and some reticuloendothelial tumors (D. E. Eisenberg and T. Sorahan, 1987,J. Nafl. Cancer Insf. 78, 1095-1 100). However, some investigators failed to establish the said correlation (G. C. Wimdham ct d . , 1985, Am. J . Epidemiol. 121, 49-56; K. E. Malone and J. R . Daling, 1986,J. Nafl. Cancer Inst. 77, 829-830). Conversely, G . C . Windham ef a l . , showed the risk of renal cancer to rise with increased weight at birth. It should be also added that the frequency of large-baby birth is increased in women suffering from psoriasis (P.Propping ef d . , 1985, Hum. Gmef. 71,92). The risk of macrosomy in newborns was shown to be in direct correlation with mother’s weight at birth (M. A. Klebanoff ef d . , 1985 Am. J . Obsfef. Gynecol. 153, 253-256). Interestingly, macrosomic newborns (birth weight of 4100-5000 g) revealed lower blood T lymphocyte, B lymphocyte, and IgG levels on day 5 of life than normosomic ones. The reaction of blastogenic transformation to insulin was more pronounced in the former group (E. B. Kravetz el d . , 1984, B o b . Mofh. Child. Hcalfh Care 3 , 40-41, in Russian).

LOUVAIN RAT IMMUNOCYTOMAS

Hew6 Bazin,' Warren S. Pear,t and Janos Sumegii 'Experlmental Immunology Unlt, Faculty of Medlclne, Unlverslty of Louvaln, Brussels 1200, Belglum tDepartment of Tumor Blology, Karollnska Instltute, Stockholm 10401, Sweden

I. Introduction . . . . . . . 11. Animals . . . . . . . . . . . . . A . Louvain Rats . . . . B . Availability of Louvain Rats . . . . 111. Incidence of the Ileocecal Lymphoid T A. InRats . . . . . . . . .

,...........

IV. V. Biosynthesis of Immunoglobulins . . . . . . . . . . A. Rat Immunoglobulins B. Production of Monoclo VI . Zn Vivo Transplantation, in Vitro Cell Culture, and

B.

In Vitro Cell Culture . . .

VII.

C. Cytogenetic and Molecular Studies D. Attempts to Modify the Louvain IR E. Discussion on the Incidence of the L VIII. General Conclusions

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

279 280 281 281 282 282 282 283 287 287 288 292 292 293 294 294 294 294 295 305 305 305 307

I. introduction The synthesis and release of large quantities of immunoglobulins by tumors have always been considered highly interesting, the secreted products being a valuable marker of the tumor cell vitality and also a precious source of reagents. Human myelomas or rodent plasmacytomas have contributed greatly to the discovery of the various immunoglobulin isotypes and of their respective physiologies. In animals, except for those species which live very close to 279 ADVANCES IN CANCER RESEARCH, VOL. 50

Copyright 0 1988 by Academic Press, Inc. All rights ofmproduction in any form reserved.

280

H E R d BAZIN ET AL.

humans, medical care is rarely individual and the discovery of such tumors is rather rare. However, they have been described in many species as spontaneous growth: mouse (Dunn, 1954), cow (Pedini and Romanelli, 1955), dog (Bloom, 1946; Jennings, 1949; Groulade et al., 1959; Medwav et al., 1967; Osborne et al., 1968), hamster (Garcia et al., 1961), pig (Englert, 1955), horse (Cornelius, 1959; Dorrington and Rockey, 1964), rabbit (Pascal, 1961), cat (Farrow and Penny, 1971; Kehoe et al., 1972), ferret (Methiyapun et al., 1985). However, these tumors have rarely been utilized in research, since they generally cannot be transplanted in histocompatible animals or easily adapted to culture in vitro. The BALB/c induced plasmacytomas developed by Michael Potter (Potter and Robertson, 1960; Potter, 1972) are the most employed immunoglobulin-secreting models in laboratories. However, their use has been sometimes limited by the size of the mice, which, for example, are not well adapted for large production of monoclonal immunoglobulins, or by the absence of IgE-secreting tumors. The inbred Louvain strain of rat provided a completely new system. These rats have an exceptional tendency to develop spontaneous lymphoid tumors, predominantly arising in their ileocecal lymph nodes and generally secreting monoclonal immunoglobulins. This article aims to summarize the data obtained so far on this model.

II. Animals Ancestors of the Louvain rats were imported from an unknown place around 1937 (Maldague, 1967) or 1941 (Maisin, 1959), probably as a unique couple (Maisin, 1959). They were considered of Wistar origin, but without any background and certification. Since they have the same haplotype RT-1” of their major histocompatibility complex (MHC) as Wistar rats, this origin is likely. For a long time, they were bred as an outbred stock in the University of Louvain and generally called “L.” In the 19509, Maisin et al. (1955a,b, 1957) and Maldague et al. (1958) identified a special type of lymphoid tumor generally appearing in the ileocecal area of Louvain rats and called them “leucosarcomas” or “undifferentiated myelomas” (Maisin et al., 1957). Later they reported that some of these tumors were secreting “paraproteins” (Maisin et al., 1963; Deckers, 1964). In 1970, Bazin and Beckers started breeding rats obtained from various colonies kept in different places by the faculty of medicine of the University of Louvain which had been kindly given to them by J. and M. Maisin, R. de Meyer, and C . Deckers. They were all derived from the rat colony of the late Professor Joseph Maisin. Bazin and Beckers bred 28 different and distinct lines in which they observed the tumor incidence in the ileocecal area. The


281

model was called Louvain in honor of its native university, abbreviated to LOU to satisfy the international nomenclature rules (Altman and Katz, 1979). The line representing the highest incidence of lymphoid ileocecal tumor was chosen as the histocompatibility reference for the model and was called LOU/C. A line presenting the lowest incidence of ileocecal tumor and a histocompatibility identical to the LOU/C rats as judged by reciprocal skin grafts was called LOU/M. The LOU/M males of each generation are still checked for their acceptance of skin grafts from L O U R rats. WSL, which can be added to LOU/C or LOU/M, refers to the breeding colony of Bazin (Festing and Staats, 1973). WSL are the initials of WoluwC Saint Lambert, the suburb of Brussels where the faculty of medicine of the University of Louvain is located.

A. LOUVAIN RATS The LOU/C as well as the LOU/M rats are albino, fairly placid animals, easy to handle, and also to breed. The litter size of the LOU/C rats based on 183 gestations was found to be 7.67 at weaning (P. Billaut, Institut Pasteur de Lille, France, personal communication), which can be considered a good reproductive performance for an inbred strain of animals. The distribution pattern of the Louvain rats for 28 established polymorphisms have been published in Bender et al. (1984). Moreover, their immunoglobulin allotypes which can also be used as genetic markers are given in Table I (Bazin et al., 1974b;.Beckers et al., 1974; Beckers and Bazin, 1975; Gutman et al., 1983).

B. AVAILABILITY OF LOUVAIN RATS Louvain rats have always been freely distributed from our laboratory. Since 1975 LOU/M and since 1976 LOU/C can be obtained in the United States.’ They can also be obtained in France, in the Federal Republic of Germany, in Japan, and in many animal houses from universities or industries and from commercial breeders. *

‘Chief, Small Animal Section, Veterinary Resources Branch, Division of Research Services, National Institutes of Health, Building 14A, Room 103, Bethesda, Maryland 20205. *In France: Centre National de la Recherche Scientifique, Centre de Slection et d’Elevage des Animaux de Laboratoire 45045, Orleans-La-Source. In the FRG: Zentralinstitut f i r Verzuchstierzucht,Herman-Ehlers-Ale57, 3000 Hannover 91. In Japan: Central Institute for Experimental Animals, 1430 Nogawa, Kawasaki, Kanagawa 213.

HER^ BAZIN ET AL.

282

TABLE I NOMENCLATURE OF RATIMMUNOGLOBULINS Heavy chain IgG subclass Isotype‘ Allotypeb

Light chain

IgM

IgD

IgA

IgE

IgGl

IgG.

IgG

IgG,,

x

x

-

-

Igh-la Igh-lb

-

-

-

Igh-Pa Igh-2b

Igh-3a Igh-3b

Igk-la Igk-lb

-

-

-

“From Bazin el nl. (1974a). bFromGutman ct al. (1983).

Ill. Incidence of the lleocecal Lymphoid Tumors in Rats

A. IN RATS In the literature, descriptions of well-defined types of spontaneous rat tumors arising in the ileocecal area of rats have been remarkably concordant over the years. Since the beginning of the century this type of ileocecal lymphosarcoma or reticulum cell sarcoma has been cited (see Table 11.). Sporadic cases were mentioned in Woolley and Wherry (1911), Bullock and Curtis (1930), Curtis et ul. (1931), and McEuen (1938, 1939). The high and very special incidence of ileocecal lymphoid tumors in rats was particularly emphasized in a series of papers by Curtis and Dunning (1940) and Dunning and Curtis (1946) in the United States and also by Roussy and Guirin (1942) and Guirin (1954) in France. The description of most if not all these tumors was consistent with an identification of immunocytomas, although none of them were investigated for immumoglobulin-secreting properties. Moreover, the mean incidence of these ileocecal lymphosarcomas was estimated to be 0-3 % .

B. IN THE LOUVAIN RATS Two substrains of Louvain rats have been established: the LOU/C and the LOU/M substrains which, respectively, have a high and low spontaneous incidence of ileocecal malignant immunocytomas. This appellation is generally abbreviated in the laboratory to IR (for immunocytome de rut) or RIC (for rat immunocytomas). The largest group of LOU/C rats observed simultaneously has shown an IR incidence of 31.6% in the male (159 IR tumors in 503 animals) and 16.1% in the female (76 I R tumors in 472 animals). In different groups of rats kept under observation for the past 10 years, tumor incidences from 28.7% (Bazin et al., 1980) to 34.4% (Bazin and Beckers, 1976), and from 14.3% (Bazin et al., 1973) to 17% (Bazin and


283

Beckers, 1976) were found, respectively, in LOU/C male and female rats. These results do not reflect the real incidence in the colony, where the IR appearance was never homogeneous, being sometimes as much as 57% (in males) and 29% (in females) for some litters and very low or nil for others (Beckers and Bazin 1978). The ages of tumor development of LOU/C, LOU/M, and their F' hybrids are given in Fig. 1. No tumor appeared in rats < 7 months old. The maximum incidence was at 12 months and then decreased slowly with age.

IV. Macroscopic and Microscopic Description of the IR Tumors or RIC The site of origin of the IR tumors developed by Louvain rats is, in most cases, the ileocecal lymph node area. Ileocecal lymph nodes are a group of three to five nodes, of which one is generally larger than the others, which are located near the cecum with two to four smaller ones along the terminal ileum. They do not seem to have a special anatomy other than the other mesenteric lymph nodes. The IR tumors appear in the peritoneal cavity in the form of solid masses. They can be easily detected by palpation of the abdomen as they are mobile under the fingers. Ascitic fluid is sometimes present and can be of help in detecting the primitive tumors, because it is present in large volumes: 100 ml or even more in old male rats. The tumors grow fast and generally kill their bearers in a month after detection. It was possible to study the development of IgE-secreting tumors in LOU/C strain rats by titration of their IgE serum levels as soon as they were 8 months old. IgE serum level is always very low in normal animals. The majority of the IR tumors, at least those secreting myeloma IgE, killed their bearers in 1 or 2 months (Bazin, 1985). At necropsy, the IR tumors appear nearly always in one or more nodule(s) in the ileocecal area. Depending on how long the tumor has been progressing, metastasis can be found. The percentage of Louvain rats with primary tumors involving the ileocecal lymph nodes, cecum, spleen, mesenteric lymph nodes, liver, mediastinal lymph nodes, and epiploon were 98, 92, 62, 60, 55, and 35%, respectively. Few metastases were found elsewhere (Beckers and Bazin, 1978). The tumors were always highly vascularized and necrotic in places. The histological aspect of the I R tumors, at least as they appeared in Louvain rat ancestors, was described in Maldague et d.(1958). This description can still be considered perfectly valid. In most cases the cells cannot be described as plasma cells, but as poorly differentiated lymphoid cells. The secreting tumor exhibits a marked uniformity in size, a fine granular nucleus with relatively small nucleoli, and a rim of deeply basophilic and pyroninophilic cytoplasm (Bazin et al., 1972, 1973). In the electron microscope, the cells can display a well-developed ergastoplasmic reticulum, but, many cells, in the same secreting tumors may have their ribosomes clustered in rosettes of 5-10

ILEoCECAL

TABLE I1 TUMORS REPORTED IN RATS ~

~~

Number of Strain

Number of animalsexamined

a n i m a l s with

tumors

Number of animals with ileocecal lymphosarcoma or reticulum cell sarcoma

Incidence in total population

Reference

Wild rats (Rattu Amoeginc)

23,000

21

1

-

Wolley and Wherry (1911)

Fisher

31,868

-

Bullock and Curtis (1930)

110

41

August

92

5

Marshall

43

1

Zimmerman

31

10

Copenhagen

100

0

Thompson Hybrids between August and F d e r strains

“A hooded strain”

Fisher line 344

AxC line 9935 Experimental colony strain Wistar stock colony -

Colony of 2000-5000 animals observed for 2 % years

2

1

44

16

Curtis d d.(1931)

99

-

-

300

2

1 1

McEuen (1938, 1939)

1-1.5%

> 1-1.5% -

Curtis and Dunning (1940)

Ratdiffe (1940)

Stewart and Jones (1941)

Wistar albino rats Osborne-Mendd albino rats

21

83 (necxupsied) 323

Jenney (1941)

3

Nelson and Moms (1941)

-

Roussy and GuCrin (1942)

1%

Farris and Yeakel (1944)

Noninbred albino rats

4,@30

Wistar albinos

1 ,@30

Gray Norways

185

-

Copenhagen line 2331

4,596

605

and Fisher line 344

7,059

97

Hybrids between Copenhagen and Fisher strains

3.970

172

498

184

1

30

1

-

-

786

189

1,342

791

6

2.2 to 0%

Osborne-Mendel Long Evans strain Noninbred albino rats Rochester strain of Wistar rats Wistar strain

2.5%

Dunning and Curtis (1946)

-

Moon d al. (1950)

12

1%

Guirin (1954)

23

-3%

Crain (1958)

83

46

2

Noninbred Sprague-Dawley strain

125

52

1

Wistar rats

495

WistarFu Line

Wag rats Buffalo Marshall 520

Saxton d d. (1948)

Gilbert and Gillman (1958) Gilbert d d. (1958) Kim d al. (1960)

Possible primary in the mesenteric lymph node

Thompson d nl. (1961) Tuchmann-Duplessis and Mercier-Parot (1962) Snell (1968)

HERM BAZIN ET AL.

286 I60

A

I50 I40

I U

I30

58

110

B 3 P3

80

0

z

I20 I00 90

70 60 50 40

30

20 10

0 5

0

10

15

20

30

25

MONTHS

I

U 0

120

5B

110

L

80

100 90

0

70

I

P3

60 50 40

z

30

20 10

0 0

5

10

15

20

25

30

I60 1-50 140

-

I

130 -

0

110-

a

3B B 3 !3l

120 100

C

-

90 -

60 50 -

80 70

z

0

5

10

15

20

25

30 MONTHS

FIG. 1. Age of incidence of 1163 immunocytomas. (A) In male and female rats; (B) in male rats; (C) in female rats.


287

elements, without any ergastoplasmic membrane system being present. At the other extreme, the cell population becomes markedly polymorphic with regard to size, whereas the nucleus is grossly granular, the nucleoli of monstrous appearance, and the cytoplasm poorly stainable, vacuolated, and of indistinct boundaries. In these cases, the ergastoplasmic membrane system is poorly developed, even if the ribosomes are still in good number (Bazin et al., 1972). Burtonboy et al. (1978) studied 15 spontaneous IR tumors which originated in the ileocecal lymph nodes of LOU/C rats and noted the presence of intracisternal A particles but, despite close scrutiny, they did not find a typical image of a C-type particle.

V. Biosynthesis of Immunoglobulins A. RATIMMUNOGLOBULINS 1. Historic Background The first data concerning rat serum proteins were obtained by immunoelectrophoretic analysis (Grabar and Courcon, 1958; Escribano and Grabar, 1962). They distinguished two precipitating lines in the y-globulin area. Some years later, Arnason et al. (1963,1964) identified three classes of proteins with antibody activities in rat serum as IgM, IgX (later as IgA), and IgG. The IgM class was clearly characterized by its susceptibility to cysteine and its molecular weight. The IgG subclasses were somehow difficult to identify. The fastest IgG subclass, IgG1; was for a long time confused with the IgA class and called IgX and then IgA (Arnason et al., 1963,1964). This immunoglobulin was shown to be an IgG subclass by different authors (Banovitz and Ishizaka, 1967;Jones, 1969). Then the “IgG” subclass was divided in two by Binaghi and Sarando de Merlo (1966). The IgG,, subclass was the last to be identified with the help of LOU/C myeloma immunoglobulins (Bazin et al., 1973,1974a). The IgA isotype was demonstrated, using a cross-reaction between rat IgA and a rabbit anti-mouse IgA serum (Nash et al., 1969) and was later confirmed by a cross-reaction between human and rat IgA (Orlans and Feinstein, 1971). Reaginic antibodies have been shown to exist in rats (Mota, 1963, Binaghi and Benacerraf, 1964). Stechschulte and Austen (1970) and Jones and Edwards (1971) characterized the rat IgE with the help of antisera. Their results were fully confirmed with the Louvain rat IgE monoclonal proteins (Bazin et al., 1974~).The last rat immunoglobulin isotype, IgD, was identified by cross-reaction using a chicken anti-human IgD serum (Ruddick and Leslie, 1977) and by rat monoclonal IgD immunoglobulins (Bazin et al., 1978).

2. Nomenclature of the R a t Immunoglobulins On the one hand, rat IgM, IgD, IgA, and IgE seem to have biochemical and physiological properties quite analogous to their human homologs. O n the other hand, however, if rat IgG is identical to human or mouse IgG, the

288

HER-

BAZIN ET AL.

various IgG subclasses cannot be related to (sub)classes of human or mouse IgG. Nevertheless, mouse IgGSand rat IgG, seem to have common antigenic determinants as well as physiological properties (Der Balian et al., 1980;Moon d al., 1980). Mouse and rat IgG, also have common antigenic determinants, but the homocytotropic IgG subclass is the IgG, in the mouse and the IgG,, in the rat. For such reasons and without more data on the biochemistry of the rat IgG molecules, it is impossible to correlate the rat IgG subclasses with those of the human or the mouse. Figure 2 gives a schematic representation of immunoelectrophoretic analysis in agarose of normal rat serum, using an antiserum against the various immunoglobulin isotypes (Rousseaux and Bazin, 1979;Carter and Bazin, 1980).

B. PRODUCTION OF MONOCLONAL IMMUNOGLOBULINS BY THE IR TUMOR OR RIC 1. Class-Distribution of Monoclonal Immunoglobulin

Synthesized by IR Tumors Animals carrying an IR tumor were killed as late as possible and both their sera and urine were analyzed for monoclonal immunoglobulins. From 1163 IR tumours obtained to date in our rat colony of LOU/C, LOU/M, and Louvain F, hybrids, 20 tumors were not identified, as the rats in which these IR tumors were discovered were in poor condition; 379 were not secreting and 764 were secreting complete immunoglobulin molecules with or without BenceJones proteins (Table 111). The age distributions of the IR tumors in relation to their immunoglobulin-secreting properties and sex are given in Fig. 3. No significant difference was found between these groups of animals. Table IV gives the distribution of serum monoclonal immunoglobulins and Bence-Jones (BJ) proteins synthesized by 250 IR tumors which appeared consecutively in our Louvain rat colony. The class distribution of 758 secreting IR tumors is given in

-

ANTI RAT lg

FIG. 2. Schematic representation of combined immunoelectrophoreticpattern in agarose gel of the eight rat immunoglobulin classes and subclasses.

289

LOUVAIN RAT IMMUNOCYTOMAS TABLE 111 IMMUNOGLOBULIN-SECRETING PROPERTIES OF 1 163 IR TUMORS Total immunocytomas

Not determined

Nonsecreting tumor

Secreting tumor

1163 100

20 1.7

379 32.6

764 65.7

Number of tumors Percentage

Table V. This class distribution was found to be very similar for monoclonal immunoglobulins synthesized by the pure Louvain IR tumors or by IR tumors which appeared in various crosses of these rats with different inbred rat strains (Carter and Bazin, 1980). By comparison, the class distribution of the monoclonal immunoglobulins synthesized by the human myelomas and by the mouse plasmacytomas is given in Table VI. Clearly the two rodent models differ from the human one, the monoclonal immunoglobulins produced by the BALB/c mouse model being predominantly of the IgA isotype and those by the Louvain rat model of the IgE isotype. Table VII gives the class distribution of the IgG subclasses in the same three models. Once again, the three distributions differ significantly.

2. Synthesis of Monoclonal Immunoglobulins by the IR Tumors In rats bearing primitive IR tumors the serum levels of monoclonal immunoglobulin were found to be quite variable. An example is given in Bazin and Beckers (1976) concerning the IgE isotype: IgE serum levels were found from 1 to 37 mg/ml, the majority of the titers being around 10 mg/ml. Beckers and Bazin (1978) gave serum level titrations for the other immunoglobulin isotypes, showing that these levels could be very high in some cases. As usual in such immunoglobulin-secreting tumors, the serum levels of the normal polyclonal immunoglobulin were all lower than in the control animal (H. Bazin, unpublished results). TABLE IV DISTRIBUTION OF THE MONOCLONAL IMMUNOGLOBULINS SYNTHESIZED BY 250 CONSECUTIVE LOWAINIR TUMORS

Number of IR tumors Absolute percentage Percentage of secreting tumors

Secreting tumors

Monoclonal Ig with or without BJ proteins

BJ proteins only

185 74 100

159 64 86

26 10 14

290

HERVI?, BAZIN E T AL.

160 150 140

vI 130

a 120

8

110

3

100

90

L 80 70

1

w ip E

60

50 40 30

20 10

0 5

0

10

15

20

25

m MALES

160

30

MONTHS FEMALES

,

150

-

140

-

130

-

B

100 120 110

70

-

60

-

90

80

50 40

30

20 10

0 0

5

m MALES

10

20

15

K m

25

30

MONTHS

FEMALES

FIG. 3. Age of incidence of secreting (A) and nonsecreting (B)irnmunocytomas.

The properties of the Louvain IR tumors which secrete monoclonal immunoglobulins, when transplanted, are quite variable. Most of the IgM- or IgG-secreting tumors retain their properties for years. IgE and IgD tumors

291


CLASSDISTRIBUTION OF

THE

TABLE V MONOCLONAL IMMUNOGLOBULINS SYNTHESIZED BY 758 IR

TUMORS

Number oftumors Percentage

Secreting tumors

IgM

IgD

IgA

IgE

IgG,

IgG2.

IgG2b

IgGz,

758 100

24 3.2

6 0.8

34 4.5

342 45.1

261 34.4

50 6.6

4 0.5

37 4.9

TABLE VI CLASS DISTRIBUTION, IN PERCENTAGE, OF MONOCLONAL IMMUNOGLOBULINSSYNTHESIZED IN THE HUMAN, THE RAT, AND THE MOUSE Species

Reference

IgM

IgD

IgA

IgE

IgG

Human Rat Mouse

Oberdorfer ct al. (1973) This article Potter (1972)

6.9 3.2 0.5

0.9 0.8 Very low

18.8 4.5 73.7

Very low 45.1 0

73.3 46.4 25.8

TABLE VII SUBCLASS DISTRIBUTION, IN PERCENTAGE, OF IgG MONOCLONAL IMMUNOGLOBULINS I N THE HUMAN, THE RAT,AND THE MOUSE SYNTHESIZED Species

Reference

Human Subclasses Percentage

IgGi 73

IgGi 12

IgG4 7

Oberdorfer et al. (1973)

8

Rat Subclasses Percentage

IgGi 39.5

IgG2. 26.7

IgGZb 32.5

IgG2, 1.1

This article

Mouse Subclasses Percentage

IgC, 34.4

IgGib 0.5

IgG3 4.9

Potter (1972)

6.6

seem to be more prone to losing their secreting properties, but good producers have been obtained in all cases (Fig. 4).

3. Antigen-Binding Properties Two monoclonal immunoglobulins of the IgM (IR473) and of the IgA (IR22) isotypes, respectively, have been found to have rheumatoid factor properties for rat IgG, molecules. No other binding capacities have been identified, particularly for the common antigens (kindly given by M. Potter, National Institute of Health, Bethesda, Maryland) found capable of binding to BALB/c monoclonal proteins.

292

HER-

BAZIN ET AL.

FIG. 4. Agarose gel electrophoresis of sera from (top row) normal rat (NRS), IR202 (IgMsecreting tumor), IR731 (IgD-secreting tumor), IR1060 (IgA-secreting tumor), IR162 (IgEsecreting tumor), and (bottom row) normal rat (NRS), IR595 (IgC,-secreting tumor), IR418 (IgGz,-secreting tumor), IR863 (IgCzb-secretingtumor), and IR1148 (IgGz,-secreting tumor).

VI. In Vivo Transplantatlon, in Vltro Cell Culture, and Storage of the IR Tumors or RIC A. I N VIvO TRANSPLANTATION Between 90 and 95% of the primitive Louvain IR tumors can be transplanted in histocompatible animals. The tumor is removed under clean conditions and teased apart in a Petri dish. The tumor tissue is minced until it

293


can be aspirated through a 19-gauge needle into a syringe. Between 0.1 and 0.2 ml (%lo million cells) are injected subcutaneously into one side of the chest. After a first subcutaneous transplantation, the tumor is generally injected in the intraperitoneal cavity, using dissociated cells from the solid tumor. In nearly all cases, after a few passages, it is possible to obtain an ascitic tumor which can be transplanted by injecting 0.2-0.5 ml of ascitic fluid into the peritoneal cavity. The latent periods of transplanted Louvain IR tumors are generally shortened after two or three passages from 14-2 1 days to 7-10 days. No F, hybrid effects have been observed in inoculating small numbers of I R cells in LOU/M rats or in hybrids of first generation between this strain and rats of various other inbred strains (Table VIII).

B. IN VZTROCELLCULTURE Burtonboy et al. (1973) first established continuous cell lines from IR tumors, using the procedure established for long-term culture of hematopoietic cells of human origin (Moore et al., 1967), and found that ascitic fluid provided a more suitable starting material than cells from solid tumors. Later, Bennich et al. (1978) adapted the IR2 and IR162 IgE-secreting IR tumors to in vitro culture and Bazin (unpublished results) adapted a dozen IR tumors to in vitro culture. Two tumors have been particularly cultivated as a fusion line for obtaining of rat-rat hybridomas: the first one, the S210 cell line, a x Bence-Jones producer (Bazin et al., 1972), was adapted to in vitro culture by Burtonboy et al. (1973) and given to C. Milstein and then called Y3 Ag1,2,3 (GalfrO et al., 1979); the second one, IR983F, a nonsecreting cell line, was adapted to cell culture from the IR983 tumor by Bazin (1981,1982). In most of the cases, the IR tumors can easily be adapted to in vitro cell culture using conventional technology (Bazin, 1982; Bazin, 1986; De Clercq et al., 1986). TABLE VIII BETWEEN RATSOF LATENCY PERIOD AND SURVIVAL TIMEOF LOU/M RATSAND Fr HYBRIDS THESE STRAINS AND VARIOUS OTHERINBREDRATS'

Rats LOUlM (LOUIM x B N ) F , (LOU/M x AUG) F, (AxC x LOUIM) (DA x LOU/M)F, (KGH x LOU/M)F, (OKA x LOUIM) F,

Number of rats Rt-1 UIU

u/n u/c ah a/u g/u klu

Latency period (in days)

13.7 15.9 15.0 15.5 14.7 15.6 14.2

'Each rat was subcutaneously inoculated with 625 IR102 cells.

f

0.5 0.9

f

0

f

0.5 0.2 1.1 0.5

f

f

f

f

Survival time (in days)

28.0 f 33.3 f 33.7 f 33.0 f 33.7 f 31.2 f 30.6 x

1.4 0.9 0.4 0.8 1.8 1.4 1.2

HER^ BAZIN ET AL.

294 C.

STORAGE

Louvain IR tumors can easily be stored in liquid nitrogen as described by Bazin et al. (1972). Primitive tumors, kept for a continuous period of 14 years in liquid nitrogen, have recently been transplanted in histocompatible animals, and three of four were recovered. IR cells adapted to in vitro culture can also be frozen as described by Bazin (1986) for rat-rat hybridoma cells and kept in liquid nitrogen for years.

VII. Etiology of the IR Tumors or RIC The etiology of the IR tumors is still poorly understood; however, some data have been found which must be taken into account in any research concerning this problem.

A. INCIDENCEOF IR TUMORS IN RODENTS Monoclonal immunoglobulin-secreting tumors are not uncommon in rodents, particularly in the mouse species. Induced BALB/c plasmacytomas predominantly produced monoclonal immunoglobulins of the IgA isotype (Potter, 1972), whereas plasma cell leukemias of (CBA x DBA)F, or BALB/c mice (RaskNielsenetal., 1968)andreticulumcell sarcomaofSJL/J mice (Waneboetal., 1966; McIntire and Law, 1967) essentially synthesize IgG monoclonal proteins. However, all these tumors cannot be directly compared to the rat I R tumors, which all seem to appear in the same precise location. On the other hand, the plasma cell neoplasms described by Dunn (1957) seem to be similar if not identical to the IR rat tumors: the incidence of these tumors was estimated to be roughly 1 % of old C3H mice. They originate in alocalized areaofthe gastrointestinal tract, at the ileocecal junction. They show great histological variations, ranging from undifferentiated appearance to features characteristic of plasma cell. At least some of them synthesize monoclonal immunoglobulins (Potter d al., 1957). No etiological factors have been described for these C3H tumors.

B. GENETIC CONTROL OF THE IR TUMOR INCIDENCE Genetic studies of rat ileocecal lymphosarcomas were first conducted by Curtis d al. (193 1). They concluded that crosses and backcrosses with the August and the

Fisher strains suggest an inherited susceptibility to mesenteric lymph node sarcoma. Later, Curtis and Dunning (1940) and Dunning and Curtis (1946) inferred that “some influences other than those postulated by orthodox genetic hypotheses play a determining role in the incidence of these neoplasms, for tabulation of their gross presence or absence is an inadequate index of existence of the responsible genes.”


295

The first approach for studying the genetic control of the IR tumor, using the Louvain model, was the observation of the incidence of these tumors in various inbred strains of rats: LOU/C, LOU/M, AUG, AxC, and OKA. LOU/C had a high incidence whereas the LOU/M, AUG, and AxC rats had a low or a very low incidence and the OKA, a null incidence. (LOU/C x AUG)F, and (LOU/C x AxC)F, had an incidence intermediate between that of LOU/C and that of AUG or AxC rats (Beckers and Bazin, 1978). This incidence showed the susceptibility of both the AUG and the AxC strains and suggested a dominant factor in the LOU/C rats to induce IR tumors. On the other hand, (LOU/C x OKA)F, rats had a null incidence (Beckers and Bazin, 1978) or, at least, a very low incidence (Bazin et al.,1987), suggesting that OKA rats have one or more loci of resistance of susceptibility to IR tumors. Considering the two sublines of Louvain rats, the LOU/C and the LOU/M, which have a high and a low incidence of IR tumors, respectively, Bazin et al., 1980) studied the reciprocal hybrids of the first generation between both sublines (LOU/C x LOU/M)F, and (LOU/M x LOU/C)F, and showed that the transmission of the high tumor incidence from parents to offspring is determined by the female as well as by the male. Moreover, the mean latent periods of the IR tumors were not significantly different between the two hybrid groups. As the OKA inbred strain of rats is clearly resistant to IR tumor incidence, congenic strains with various loci of the OKA strain have been introduced in the LOU/C genetic background. Various hybrids or congenic rats between the LOU/C and the OKA strains were studied in order to localize the resistance locus(i) of the OKA strain (Table IX). The LOU/C.Rt-l'(0KA) rats having the MHC of the OKA strain in the LOU/C background showed no IR tumor incidence, indicating that the MHC of the rat or a gene closely linked to it determines resistance to that tumor development. Moreover, the very low incidence of the (LOU/C x OKA)F, hybrids demonstrated the dominance of the OKA resistance upon the LOU/C susceptibility. The congenic rats with LOU/C genetic background, having either the heavy or the x light-immunoglobulin chain loci from the OKA strain, have an IR tumor incidence identical to the LOU/C strain (Bazin et a l . , 1986). Thus, the resistance of the OKA strain to the appearance of IR tumor is not correlated with special properties of immunoglobulin heavy or x light-chain segments. C.

CYTOGENETIC AND MOLECULAR STUDIES 1. Chromosomal Translocation in the R I C Involves the Chromosomes

Bearing the c - m y c and IgH Chain Loci Studies on mineral oil-induced BALB/c plasmacytomas (MPC) have shown that two types of translocations, t( 12;15) and t(6; 15), are regularly associated with plasmacytogenesis (Ohno et al., 1979; Potter et al., 1984).

TABLE IX OF ILEOCECALIR TUMORS IN LOUIC, OKA, AND RELATED RATS INCIDENCE Allotype Strain

Sex

Rt-I

IgK-1

IgH-2

Number of IRInumber of rats under observation

Observed IR incidence

("/.I LOUIC

W

W

ulu ulu k/k

9

kk

W W

9 OKA (LOUIC x OKA) F, LOUIC x IgK-lb(0KA) LOUIC x IgH-2b(OKA)

W

LOUIC x Rt-l'(0KA)

W

u/k ulu ulu ulu ulu k/k

9

k/k

0 9

ala ala bh bh ah

bh

ala ala bh bh ah afa

bh

ala

ala ala

b/b bfb ala ala

ala

ala

431163 4/42 0168

0174 21185 9167 8148 371114 181115 0191 017 1

25.3 9.5 0 0 1.08 13.4 16.7 32.4 15.6 0 0


297

Wiener et al. (1982), by G-banding analysis of seven Ig-secreting Louvain IR tumors, showed a consistent translocation of the distal part of the q-arm of chromosome 7 to the telomeric end of chromosome 6 (Fig. 5). The break points were assigned to q3.3 on chromosome 7 and q3.2 on chromosome 6. G-Banding analysis has been performed on an additional 12 RIC and the t(6;7) translocation has been identified in every tumor (A. Szeles and F. Wiener, personal communication). As a result of the consistency of the translocation event and the banding homologies between the mouse and rat chromosomes involved in the translocation, we hypothesized that the RIC translocation would involve the same loci as in the 12;15 MPC translocation: the c-myc and IgH. By utilizing rat-mouse somatic cell hybrids (Szpirer et al, 1984), we have localized the c-myc oncogene to chromosome 7 (Sumegi et al., 1983) and the IgH cluster to chromosome 6 (Pear et al., 1986b). Cytogenetic studies of 19 RIC have not identified any tumors which carry a translocation between the chromosome 7 and x light-chain gene-bearing chromosome 4 (Perlmann d al., 1985) or the X light chain-carrying chromosome 11 (W. S. Pear and G. Wahlstrom, unpublished results). This finding suggests that variant translocations are either absent or occur at a low frequency in the RIC.

2. The Chromosomal Breakpoints Cluster at the 5’ End of the c-myc Gene Cleavage of rat genomic DNA with EcoRI generates a 17-kb c-myc-specific fragment which contains all three exons and 5‘ and 3’ flanking sequences (Sumegi et al., 1983; Steffen, 1984). Hybridization analysis has shown that an additional c-myc-specific fragment is detectable in 13 of 15 RIC (Table X). Mapping of the c-myc-associatedbreakpoints by genomic Southern hybridization with appropriate DNA fragments as probes has shown that, similar to Burkitt’s lymphomas (BL) and MPC, no breakpoints have been identified which interrupt the c-myc protein coding region (Pear et al., 1986a). In 10 of 12 tumors exhibiting c-myc rearrangement, the translocation breakpoints are clustered within a 1.5kb XbaI-Hind111 restriction fragment which contains the proximal c-myc promoter and 5’ flanking sequences (Fig. 6) (Pear et al., 1986~).Sequence analysis (W. S. Pear, unpublished) has shown that this region is approximately 75 % homologous to a similar region in the mouse (Corcoran d al., 1985) and 60% homologous to a similar region in the human (Siebenblist d d.,1984). Several subregions of this 1.5-kb fragment show >85 % homology between the three species ( W . S . Pear, unpublished). Significantly, it is in these regions that c-myc regulatory elements have been proposed to reside in the murine and human c-myc genes p a n g et d.,1985; Remmers d al., 1986; Bentley and Grodine, 1986). This region is also the site of proviral DNA

298

HER-

BAZIN ET AL.

FIG.5. Comparison of the murine chromosomes involved in the t(12:15) translocation with the rat chromosomes involved in the t(6;7) translocation.

insertion in murine T-cell lymphomas (Corcoran el al., 1984; Li et al., 1984; Selton et al., 1984; Wirschubsky et al., 1986) and in Moloney virus-induced rat thymomas (Steffen, 1984). The c-myc breakpoint in only 1 of 13 tumors occurs in the first intron (Fig. 6). This is in contrast to >90% of the BALB/c plasmacytomas (reviewed in Cory, 1986) and the majority of sporadic BL (Pelicci et al., 1986), in which the c-myc breakpoints occur in either the first exon or intron. In one tumor, IR221,the c-myc-associated breakpoint occurs approx ~ 5 0 0 base pairs 3’ of the third exon (Fig. 6). Although translocations involving the light-chain occur 3 ’ to the c-myc third exon, a similar translocation event has not been described in the translocations involving the heavy-chain loci. One case of human plasma cell leukemia has been described in which the c-myc is interrupted by repetitive sequences immediately 3 ’ of the third exon (Hollis et al., 1986). Recent evidence (reviewed in Piechaczyk et al., 1987) suggests that sequences 3 ’ to the third exon are important in determining c-myc mRNA stability.

299


A

221

outside: 9. 304

-1

kb

B 28s

+

FIG.6. (A) Restriction enzyme map of the 17-kb EcoRI c-my-containing restriction fragment. The three c-myc exons are shown by the crosshatched boxes. The dotted lines indicate the borders of the c-myc breakpoint regions. The names of the tumors are listed within appropriate breakpoint regions. (B) Northern blot showing c-myc expression in several RIC. The hybridization probe was a third-exon c-myc probe.

Two tumors, IR9 and IR304, do not show rearrangement within the 17-kb EcoRI fragment. Further restriction mapping has shown that these tumors are not rearranged 20 kilobases 5 ' of exon 1or 23 kilobases3 ' of exon 3 (Pear et al., 1986a). The DNA recombination event is accompanied by an elevated level of c-mycspecific RNA in the tumor cells (Fig. 6) (Pear et al., submitted). Except for the IR49 in which the translocation occurs in intron 1, the size of the mRNA is normal. It is interesting that the IR304, in which the c-myc gene is intact over at least a 40-kb region surrounding the exons, expresses a similar level of mRNA as those tumors in which the c-myc-associated breakpoints are within 1.5 kb of the promoters. Significantly, the IR221, in which the breakpoint is immediately 3' of the third exon, shows a lower level of c-myc transcription than the other RIC (Pear et al., submitted).

3. Translocation Targets in the IgH Locus Although there are exceptions, both MPC and BL frequently show a relationship between the IgH translocation target and the expressed isotype (reviewed in Cory, 1986). This is also true in the RIC in which genomic

300

HERV?? BAZIN ET AL. TABLE X SUMMARY OF IgH COMICRATION AND c-myc REARRANGEMENT STUDIES

Tumor IR9 IR27 IR33 IR49 IR50 IR72 IR74 IR75 IR88 IR89 IR209 IR221 IR222 IR223 IR241 IR304

Ig secreted

c-myc Target"

BJ

ND ND ND ND

YIlX

y2Jx

BJlx elx EIX

None EIX elx E

y2Jx y2Jx e

BJ YIlX y2.lx

S-E S-E S-E

s-P S-E

ND Line ND

c-myc Rearrangementb

-

+ + + +

+

+ +

+ + +

S-E C-e S-Yl

+ +

ND

-

+

"C, Within coding region; S, within switch region; ND, not determined. 'c-myc Rearrangement was determined by EcoRI digestion and hybridization to appropriate probes.

Ig-comigration experiments have shown a correlation between Ig production and the translocation targets (Table X). Of seven E-expresing RIC, six show recombination between the E locus and the c-myc (Pear et al., 1986~).As described below, cloning of the rearranged c-myc fragments from IR50, IR75, IR209, and IR223 has confirmed the genomic c-myc breakpoint and Ig comigration results. The analysis of the IR50 has shown that the c-myc is juxtaposed head-tohead with the switch E region (Fig. 7) (Pear el al., 1986a). Genomic hybridization studies of the other six tumors in which the c-myc recombines with the E locus show that the switch E is the target in five of these tumors (Table X). Head-to-head rearrangement of the c-myc with an Ig-switch region is similar to the findings in all molecularly analyzed MPC and sporadic BL (reviewed in Cory, 1986). This is consistent with the hypothesis that the recombination mechanism for normal IgH switch renders the chromosome temporarily susceptible to illegitimate recombination with other DNA sequences. Cloning and sequencing of the rearranged c-myc and its reciprocal from the IR223 (Fig. 8) has shown that, in contrast to the aforementioned results in RIC, MPC, and BL, a switch region is not involved in the recombination between c-myc and IgH sequences (Pear et al., 1986~).Instead, the IgH breakpoint occurs in the intron between exons I and I1 of the E domain. The involvement of this region in the translocation may indicate that the switch

30 1


MYC.900 0

P

a

I kb

FIG. 7. Restriction maps depicting the recombination in the IR50. MYC.900 is the 900-base pair BumHI fragment which contains the proximal c-myc promoter and 800 base pairs 5 ' flanking sequences (see Fig. 6). R50.5 is the rearranged c-myc BumHI fragment from the IR50. R-cp is the rat e locus. The arrowheads indicate the c-myc and switch e breakpoints. The thin arrows indicate the transcriptional orientations of the c-myc and e loci. Restriction sites: B, BamHI, P,B u I I , Bst, BstEII, H, HindIII, K, KpnI, S, SucI, Bg, BglII. Chl-CH4 indicates the e-coding domains, and S, is the switch e region.

recombination mechanism confers recombinatory potential on sequences outside of the Ig-switch regions. Consistent with this hypothesis are the findings by several groups which show that specific regions within the IgH constantregion locus may exert B-cell-specificenhancer or promoter activity (Ephrussi et al., 1985; Grosschedl and Baltimore, 1985; Luzker and Alt, personal communication). Although a switch region is not involved at the IR223 breakpoint, the c-myc and E loci are juxtaposed head-to-head. Unlike the other five t-producing tumors, rearrangement in the t-secreting IR75 does not involve the E locus (Pear et al., 1986a). Cloning of the 12-kb rearranged c-myc EcoRI fragment has shown that the c-myc is juxtaposed headto-head to the switch p region (Fig. 9). Unexpectedly, the switch p does not lead into the c-p region. Instead, a region derived from upstream of the switch y1 region has been joined in the opposite transcriptional orientation to the switch p . This particular configuration requires multiple chromosomal events, only one of which, the t(6:7) reciprocal translocation, is visible at the cytogenetic level. We have previously analyzed an exceptional MPC, ABPC45, where an inversion has occurred in the region juxtaposed to the rearranged c-myc fragment (Fahrlander et al., 1985). This case is very similar to the IR75, but with some important differences. In the ABPC45, the intact c-myc gene is juxtaposed head-to-head to the switch Q region. This is not followed by c,, as expected, but by another IgH-derived inverted region, containing switch p

HER^ BAZIN ET AL.

302 B

MYCSOO

A

A

B

P

A

I

223.24

n

A

R-EP

CHI

CHI

A L - _

*223.96

c14

I-

100 bp

- - _- _---__,-m y c

S

H

c 13

- _- _- _---_

BP

B

A

A

H

H

V

5 ' flank

B 1

SUP lkb

FIG.8. Restriction maps depicting the recombination in the IR223. MYC900 is the 900-base pair BamHI fragment which contains the proximal c-myc promoter and 800 base pairs of 5' flanking sequences (Fig. 6). 223.24 is the rearranged c-myc frangment which contains the c-myc coding region (exons 2 and 3). Only exon 1 is shown in the figure. R-EP is the rat e locus. 223.96 is the c-myc reciprocal product. The c-myc and e breakpoints are shown by the open arrowheads. The transcriptional orientations are indicated by the thin arrows. CHI-CH4 are the c-coding domains. S,, is the switch e region. Restriction sites: B, BamHI; A , AuaI; P , A u I I ; Bgl, BgfII; H, HindIII; S, SACI; K, KpnI.

sequences and the IgH enhancer. An important difference between the ABPC45 and IR75 is the demonstrated presence of the IgH enhancer in the rearranged c-myc-carryingstructure in the former. Hybridization experiments have shown that the IgH enhancer is not present in the 12-kb region upstream of the IR75 c-myc breakpoint (W.S. Pear, unpublished results). The IR209 is a +yZa producer whose c-myc mRNA size and level are similar to several other RIC (Fig. 6). In order to characterize the translocation in this tumor, a rearranged 12-kb EcoRI c-myc-specific fragment and an overlapping 8.5-kb BumHI fragment were cloned (Fig. 10). The c-myc breakpoint in this tumor occurs 850 base pairs upstream of the proximal promoter. Unlike our experience with the IR50, IR75, and IR223, hybridization experiments with probes derived from upstream of the c-myc breakpoint did not detect unique IgH-derived regions (Pear et al., submitted). Instead, hybridization to BumHI-, EcoRI-, or HindIII-digested rat DNA revealed a repetitive pattern consistent with the LINE family of moderately repetitive elements (D'Ambrosio d u l . , 1986).

1

303


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

I’ s2

0

EcoRl

0

Hindlll

v

Kpnl

er

i

Lb

-1

BamHl A

Xbal Fokl

‘ 4 ................................. ......................... bb

3’

cr

I’

sr

-1w

FIG.9. Restriction maps depicting the IR75 recombination. Rat c-myc is the 17-kb EcoRI c-myc-containing restriction fragment. The region containing the c-myc exons is bracketed with 5’-3’. IR75R Myc is the 12-kb rearranged c-myc fragment from the IR75. cy is the rat cyl locus. The dotted line under the cyl indicates the switch and coding regions of the y l locus. IgH, shows the rat switch p region and surrounding sequences. The thin arrows show the transcriptional orientations of the c-myt, y l , and switch p loci. The c-myc, p l , and switch p breakpoints are depicted. Symbols for restriction enzymes: 0 ,EcoRI; A , XbuI; V , KpnI; 0 , HindIII; 0 , BumHI; B, FokI.

The LINE family consists of long, interspersed repetitive elements whose longest members are > 7kb. In addition to rat, LINE family members have been described in primates (KpnI repeats). , canine, and homologs have been described in Drosophila (reviewed in Singer and Skowronski, 1985; Fawcett et a l . , 1986). In all of the above species, these elements contain open reading frames and are abundantly transcribed by RNA polymerase I1 (reviewed in Singer and Skowronski, 1985). These elements are thought to be capable of retroposition (Hattori et al., 1986), and the studies of LINE elements in Drosophila show that these particular elements are capable of transposition (Fawcett et al., 1986). Sequence and hybridization analysis of the IR209 c-myc-juxtaposed DNA has shown that LINE-specific DNA extends at least 8 kb upstream from the breakpoint (Fig. 10) (Pear etal., submitted). In their summary of LINE insertions, Furano et al. (1986) have noted that these insertions resemble transposable elements in several respects: (1) the target sites are AT rich, (2) there is a 7- to 15-base pair target site duplication which flanks the LINE family member,

304 RAT

H E R M BAZIN ET AL. LINE

IR209 C-MY C (R)

Ikb

FIG. 10. Comparison of the restriction maps of a typical rat LINE family member and the rearranged c-myc from the IR209. The typical LINE family member and the A, B, C, and D regions correspond to the pattern described by d’Ambrosio ct al. (1986). The arrowhead shows the c-myc breakpoint in the IR209. The entire region to the right of the arrowhead is homologous to LINE DNA. The B-like region is homologous to the LINE B region. Symbols for restriction enzymes: V, EumHI; E, EcoRI; 0 , HindIII; v , AuuI; BstEII.

.,

(3) there is an alternating purine-pyrimidine stretch in the target site, and (4) the LINE region inserts near a hexanucleotide which is also located near the A-rich right end. On the other hand, several reports have described LINE insertions which appear to occur independently of transposition (Soares et al., 1985). Although the IR209 contain a t(6;7) translocation, our present results do not allow us identify the exact mode of c-myc-LINE recombination. The question arises as to the origin of the LINE region and what accounts for c-myc activation in the IR209. One possibility is that the LINE has originated from the ZgH region, and it is the IgH chromatin which is influencing the c-myc expression. Another possibility is that it is the interruption of an important c-myc regulatory region which is affecting the aCtivation. Alternatively, the LINE region itself may be activating the c-myc. From this perspective, it is relevent that a LINE element may be involved in c-myc activation in canine venereal tumor (Arman et al., 1986). In order to study the potential of the LINE region to activate the c-myc, we have tested the ability of various LINE restriction fragments to substitute for the SV40 enhancer. At present, we have found one region which displays enhancer activity in fibroblasts (W.S. Pear, unpublished results).

LOUVAIN RAT IMMUNOCYTOMAS D.

305

ATTEMPTS TO MODIFY THE LOUVAIN IR TUMORAL INCIDENCE

Various manipulations have been employed to modify the incidence or the latent period of IR tumors in the LOU/C rats: monthly intraperitoneal injections of 1 ml of pristane (Janssen Pharmaceutica, Beerse-Belgium) or of complete Freund adjuvant (Difco, United States) from the age of 2.5 months; a single dose of 3 or 5 gray at monthly intervals on the whole body or localized on the ileocecal area; and administration of an acellular extract of I R tumors to newborn LOU/C rats (Bazin and Beckers, 1976). None of these attempts significantly modified the incidence or the latent period of the IR tumors in the L O U R rats used for these experiments. Moriamk et al. (1977), however, removed the ileocecal lymph nodes of 5- to 21-day-old baby rats and observed that the IR tumor incidence in these animals was significantly reduced in comparison to their controls. Castration of young male L O U K rats did not modify their IR tumor incidence (H. Bazin and A. Beckers, unpublished results).

E. DISCUSSION ON THE INCIDENCE OF THE LOUVAIN RATIR TUMORS Immunocytomas seem to be a common tumor, though relatively rare, in rats. Very similar neoplasms seem to exist in mice also. Cecal ulceration was first considered as important in the etiology of such tumors. Half of the rats of Bullock and Curtis (1930) and Curtis and Dunning (1960) showed ulcerated lesions in their cecum. Dunn (1954) also pointed out this ulceration in the etiology of ileocecal lymphosarcoma of the old C3H mice. Chronic antigenic stimulations have always been considered a possible etiological factor of B-cell neoplasia, at least for those secreting monoclonal immunoglobulins such as the human myelomas or the BALB/c induced plasmacytomas (Potter, 1972). The fact that the majority of the Louvain I R tumors secrete monoclonal IgE strongly suggests a parasitic implication (by helminths), which could be located in the cecum and which first stimulate the ileocecal lymph nodes. However, one can also consider that cancerogenic substances could be synthesized in the cecum, a special organ in which many bacteria and parasites can live. In this hypothesis, the reduction of IR tumor incidence by ileocecal lymphadenectomy could be interpreted as a means of diluting such antigenic cancerogenic substances before they reach the lymph nodes. Last, sex certainly influences the IR tumor incidence, however, by an unknown mechanism.

VIII. General Conclusions The Louvain rat model of immunoglobulin-secreting tumors has certainly been an interesting and valuable tool in immunology. It has provided many new possibilities for experimental research: readily available monoclonal

306

H E R a BAZIN E T AL.

immunoglobulins of eight different classes or subclasses from a highly utilized laboratory animal species, and especially the first IgE and IgD monoclonal immunoglobulins identified in an animal species. Finally, it allows the development of a rat-rat hybridoma technology which has many features distinctive from the mouse model, at least in its antibody repertoire. Without a doubt, etiological factors which can influence this tumor incidence and also mechanisms of cell transformation at the level of chromosomal constitution of the IR tumor have been found. Our cytogenetic and molecular studies of the IR tumors or RIC show that in three different tumors (BL, MPC, RIC) in three different species (human, mouse, rat), nearly identical genetic loci (c-my, Ig) are juxtaposed via chromosomal translocation. These three tumors represent at least two different stages of B-cell maturation, and the natural histories and modes of induction of the tumors are very different. The finding that c-myc-Ig juxtaposition occurs in these three tumors suggests that this configuration plays a central role in the genesis of these B-cell tumors. Our findings also indicate that sequences, in addition to Ig-switch sequences, may serve as translocation targets. The involvement of a LINE region in the IR209 raises the possibility that c-myc activation can occur without interposition into Ig-influenced chromatin. However, one must admit that these data alone cannot explain the tumor incidence. Other factors probably exist which are also important and possibly as important as the genomic ones. They must be determined in order to understand fully the reasons why tumors of the B lymphoid can develop in some organisms and not in others. All these factors are also poorly understood in the human myelomas or in the BALB/c plasmacytomas. But by comparing the various results obtained in these models, it may be possible in the near future to understand why B lymphocytes can evolve into either a normal short-lived plasma cell or a long-lived memory cell that is a highly aggressive tumor cell capable of killing its host in a very short period of time.

ACKNOWLEDGMENTS The authors acknowledge AndGe Beckers, Francoise Cormont, Martine Moriamt, Guy Burtonboy, Christian Deckers, Pierre Querinjean, and Jean Rousseaux for their generous contributions and many other colleagues for their help and advice, as well as Bernadette Platteau, Jenny Naze-Demets, FranCoise Nisol, Jean-Pierre Kints, Jean-Marie Malache, Rent Meykens, Pascal Palombi, and Alain De Cremer for their excellent technical assistance, and FranCoise Bolle and Liliane De Greef for their help in preparing the manuscript. This work was supported by the Fonds Canctrologique de la Caisse GnCrale d'Epargne et de Retraite (Belgique), contract no. 041/BDIIBC, by the Fonds de la Recherche Scientifique MGdicale (Belgique) contract no. 3.4518.76 and by contract no. Bio-c-358-81-B of the European Communities (publication no. 2308). This work was also supported by PHS grant number 3ROlCA14054 awarded by the


307

National Cancer Institute, DHHS, and by the Swedish Cancer Society. W.S.P. and J.S. are recipients of fellowships from the Cancer Research Institute and the Concern Foundation. W.S.P. has also been supported by the University of Rochester and the Karolinska Institute during various phases of this work. H.B. is staff member of the Commission of the European Communities.

REFERENCES Altman, P. L., and Katz, D. D. (1979).In “Inbred Genetically Defined Strains of Laboratory Animals. Part I. Mouse and Rats.” Biol. Books, Fed. Am. SOC.Exp. Bid. 3, 233-256. Arman, E., Katzir, N., Rechavi, G., and Givol, D. (1986). Cum Top. Microbiol. Immunol. 132,

90-97. Amason, B. G., de Vaux St. Cyr, C., and Grabar, P. (1963). Nature (London) 199, 1199-1200. Amason, B. G . , de Vaux St. Cyr, C., and Relweld, E. H . (1964).Int. Arch. Allngy. Appl. Zmmunol. 25, 206-224. Banovitz, J., and Ishizaka, K. (1967).Proc. Soc. Exp. Bid. Med. 125, 78-82. Bazin, H . (1981).Louuuin Mcd. 100, 3-8. Bazin, H. (1982).Protidcs Bid. Fluids 29, 615-618. Bazin, H . (1985). “Mechanisms of B Cell Neoplasia” (F. Melchers and M . Potter, eds.), pp. 208-216.Roche, Basel. Bazin, H . (1987).In “Hybridoma Formation” (A. H . Bartal and Y. Hirshaut, eds.)., pp. 337-378. Humana Press, Clifton, New Jersey. Bazin, H., and Beckers, A. (1976).Nobel Symp (33), 153-171. Bazin, H., Deckers, C., Beckers, A., and Heremans, J. F. (1972). Int. J. Cuncn 10, 568-580. Bazin, H., Beckers, A., Deckers, C., and Moriamk, M. (1973).J. Natl. Cuncer Znst. 51,

1359-1361. Bazin, H., Beckers, A,, and Querinjean, P. (1974a).Eur. J. Immunol. 4, 44-48. Bazin, H., Beckers, A., Vaerman, J. P., and Heremans, J. F. (1974b). J . Immunol. 112,

1035-1041. Bazin, H . , Querinjean, P., Beckers, A., Heremans, J. F., and Dessy, F. (1974~). Immunology 26,

713-723. Bazin, H., Beckers, A., Urbain-Vansanten, G., Pauwels, C . , Bruyns, C., Tilkin, A. F., Platteau, B., and Urbain, J. (1978).J. Immunol. 121, 2077-2082. Bazin, H., Rousseaux, J., Kints, J. P., and Herno, J. (1980). Cuncer Lett. 8, 353-357. Bazin, H . , Kints, J. P., and Rousseaux, J. (1986). Anticuncn Rcs. 6, 45-48. Beckers, A,, and Bazin, H . (1975). Immunochcmistry 12, 671-675. Beckers, A,, and Bazin, H. (1978).J. Nutl. Cunccr Insf. 60, 1505-1508. Beckers, A,, Querinjean, P., and Bazin, H . (1974). Immunochmistry 11, 605-609. Bender, K., Giinther, E., Adams, A., Baverstock, P.R., Den Bieman, M., Bissbort, S.,Brdicka, R., Butcher, G . W., Cramer, D. V., Von Deimling, O., Festing, M. F. W., Gutmann, R . D., Hedrich, H . J., Kendall, P. B., Kluge, R., Moutier, R . , Womack, J. E., Yamada, J . , and Van Zutphen, B. (1984).J. Zmmunogmct. 19, 257-266. Bennich, H., Karlsson, T., Bazin, H . , and Zeuthen, J. (1978).Protidcs Bid. Fluids 25, 559-562. Bentley, D. L., and Groudine, M. (1986).Mol. Cell. Biol. 6, 3481-3489. Binaghi, R . A,, and Benacerraf, B. (1964).J. Immunol. 92, 920-926. Binaghi, R. A., and Sarando de Merlo, E. (1966).Int. Arch. Allngy Appl. Zmmunol. 29, 105-111. Bloom, F. (1946). Cunccr Rcs. 6, 718-722. Bullock, F. D., and Curtis, M. R. (1930).J. Cancer Res. 14, 1-115. Burtonboy, G., Bazin, H . , Deckers, C., Lamy, M., and Heremans, J. F. (1973).Eur. J. Cancer 9, 259-262.

308

HERW?. BAZIN ET AL.

Burtonboy, G., Beckers, A., Rodhain, J . , Bazin, H . , and Lamy, M . E. (1978). J . Natl. Cancer Inst. 61, 477-484. Carter, P. B., and Bazin, H . (1980). In “The Laboratory Rat” (H. J. Baker, J. R . Lindsey, and S. H . Weisbroth, eds.), pp. 182-212. Academic Press, New York. Corcoran, L. M., Adams, J. M . , Dunn, A. R., and Cory, S. (1984). Cell 37, 113-122. Corcoran, L. M., Cory, S.,and Adams, J. (1985). Cell 40, 71-79. Cornelius, C. E. (1959). Cornell. Vet. 49, 478-493. Cory, S. (1986). Adu. Cancer RCS.47, 189-234. Crain, R. C. (1958). Am. J. Puthol. 34, 311-323. Curtis, M . R., and Dunning, W. F. (1940). Am. J. Cancer 40, 299-309. Curtis, M . R., Bullock, F. D., and Dunning, W. F. (1931). Am. J . Cuncer 15, 67-121. D’Ambrosio, E., Waitzkin, S. D., Witney, F. R., Salemme, A., and Furano, A. V . (1986). Mol. CcN. Biol. 6 , 411-424. Deckers, C . (1964). Rotidcs Biol. Fluids 2, 105-108. De Clercq, L., Cormont, F., and Bazin, H . (1986). In “Methods in Enzymology” Langone and H. Van Vunakis, eds.), Vol. 121, pp. 234-238. Academic Press, Orlando, Florida. Der Balian, G., Slack, J., Clevinger, B. L., Bazin, H., and Davie, J. M . (1980). J. Exp. Med. 152, 209-218. Dorrington, K. J . , and Rockey, J. H. (1964). J . Biol. Chnn. 243, 651 1-6513. Dunn, T . B. (1954). Roc. Am. Assoc. Cunccr Res. 1, 13-16. Dunn, T. B. (1957).J. Nutl. Cancer Inst. 19, 371-391. Dunning, W. F., and Curtis, M. R. (1946). Cunccr Res. 6, 61-81. Englert, H. K. (1955). Zentrulbl. Vetgninnmd. 2, 607-628. Ephrussi, A., Church, G. M., Tonegawa, S., and Gilbert, W. (1985). Science 277, 134-140. Escribano, M . J., and Grabar, P. (1962). C.R.Hcbd. Scunccs Acud. Sci. 255, 206-208. Fahrlander, P . , Sumegi, J., Yang, J., Wiener, F., Marcu, K., and Klein, G. (1985). Boc. Ndl. Acud. Sci. U.S.A. 82, 3746-3750. Farris, E. J., and Yeakel, E. H . (1944). Am. J. Puthol. 20, 733-781. Farrow, B. R. H., and Penny, R . (1971).J. Am. vet. Mcd. Assoc. 158, 606-611. Fawcett, D. H., Lister, C . K., Kellett, E.,and Finnegan, D. J. (1986). Cell 47, 1007-1015. Festing, M . , and Staats, J. (1973). Transplantdon 16, 221-245. Furano, A. V., Somerville, C . C . , Tsichlis, P. N., and D‘Ambrosio, E. (1986). Nuclcic Acids RCS.14, 3717-3727. GalfrC, G., Milstein, C., and Wright, B. (1979). Nuturc (London) 277, 131-133. Garcia, H . , Baroni, C., and Rappaport, H. (196l).J. Nutl. Cancer Inst. 27, 1323-1333. Gilbert, C., and Gillman, J. S. (1958). S.Af.. J . Med. Sci. 23, 257-272. Gilbert, C., Gillman, J., Loustalot, P., and Lutz, W. (1958). Br. J . Cancer 12, 565-593. Grabar, P., and Courcon, J. (1958). Bull. Soc. Chim. Biol. 40, 1993-2003. Grosschedl, R., and Baltimore, D. (1985). Ccll41, 885-897. Groulade, J., Morel, P., Greyssel, R., and Groulade, P. (1959). Bull. Acad. Vet. Fruncc 6 , 354-361. Gukrin, M . (1954). Legrand, Paris. Gutman, G . A., Bazin, H., Rockhlin, 0. V., and Nezlin, R. S. (1983). Trunsplunf R o c . 15, 1685-1 686. Hattori, M., Kuhara, S., Takenaka, O., and Sakaki, Y. (1986). Nature (London) 321, 625-628. Hollis, G. F., Gazdar, A. F., and Kirsch, I. R . (1986). Curr. Top. Microbiol. Zmmunol. 132, 225-226. Jenney, F. S. (1941). Cancer Res. 1, 407-409. Jennings, A. R . (1949).J. Comp. Puthol. Thcr. 59, 113-118. Jones, V. E. (1969). Immunology 16, 589-599.

u.


309

Jones, V. E., and Edwards, A. J. (1971). Immunology 21, 383-385. Kehoe, J. M., Hurvitz, A. L., and Capra, J. D. (1972).J . Zmmunol. 109,511-516. Kim, U.,Clifton, K. H., and Furth, J . (1960).J . Nall. Cancer Znst. 24, 1031-1055. Li, Y., Holland, C. A,, Hartley, J. W., and Hopkins, N. (1984).Proc. Nafl. Acad. Sci. U . S . A . 81, 6808-681 1. McEuen, C. S. (1938).Am. J . Cancer 34, 184-195. McEuen, C. S. (1939). Am. J. Cancer 36, 383-985. McIntire, R . R . , and Law, L. W. (1967).J.N a L Cancer Znsf. 39, 1197-1203. Maisin, H . (1959). In “Contribution h ]‘Etude du Syndrome Am. Vet. Med. Assoc. 150,386-395. Methiyapun, S.,Myers, R. K., and Pohlenz, J. F. L. (1985). Vet. Pafhol. 22, 517-519. Moon, H. D., Simpson, M. E., Li, C. H., and Evans, H. M. (1950). Cancer Res. 10,297-308. Moon, N., Der Balian, G., Venturini, D., Bazin, H., and Davie, J. M. (1980). Zmmunogenefics 11, 199-203. Moore, G. E., Garner, R . E., and Franklin, H. A. (1967).J.Am. Med. Assoc. 199, 87. Moriami, M., Beckers, A., and Bazin, H . (1977). CancerLeff.3, 139-143. Mota, I. (1963). Ann. N. Y. Acad. Sci. 103,264-277. Nash, D. R . , Vaerman, J. P., Bazin, H . , and Heremans, J . F. (1969).J. Zmmunol. 103,145-148. Nelson, A. A,, and Morris, H. J. (1941).Arch. Pafhol. 3, 578-584. Oberdorfer, A., Schnauffer, K., Lange, H . J., and Neiss, A. (1973).2.Klin. Chcm. Klin.Biochem. 11, 51-64. Ohno, S., Babonits, M., Wiener, F., Spira, J., Klein, G., and Potter, M . (1979). Cell 18, 1001 - 1007. Orlans, E., and Feinstein, A. (1971).Nafure (London) 233, 45-47. Osborne, C . A., Perman, V., Sautter, J . H . , Stevens, J. B., and Hanlon, G. F. (1968).J.Am. Vef.M d Assoc. 153, 1300-1319. Pascal, R . R . (1961). Carnell Vet. 51, 528-535. Pear, W. S.,Ingvarsson, S., Steffen, D., Munke, M., Francke, U., Bazin, H., Klein, G., and Sumegi, J. (1986a).Roc. Nafl. Acad. Sci. U . S . A . 83, 7376-7380. Pear, W. S.,Wahlstrom, G., Szpirer, J., Levan, G., Klein, G., and Sumegi, J . (1986b). Zmmunogenetics 23, 393-395. Pear, W. S.,Wahlstrom, G., Nelson, S. F., Ingvarsson, S., Bazin, H., Klein, G., and Sumegi, J . (1986~).Curr. Top. Microbiol. Zmmunol. 132, 169-174. Pedini, B., and Romanelli, V. (1955). Arch. Vet. Zfal. 6 , 193-214. Pelicci, P., Knowles, D., Magrath, I., and Dalla-Favera, R . (1986).Proc. Nafl. Acad. Sci. U . S . A . 83,2984-2988. Perlrnann, C., Sumegi, J., Szpirer, J.. Levan, G., and Klein, G. (1985).Zmmunogmfics22,97-100. Piechaczyk, J., Blanchard, J. M., and Jeanteur, P. (1987).Trends Gen. 3, 47-51. Potter, M. (1972). Phys. Reu. 52, 631-719. Potter, M., and Robertson, C. L. (1960).J. Natl. Cancer Znsf. 25, 847-861. Potter, M., Fahey, J . L., and Pilfrim, H. J. (1957). Proc. Soc. Exp. Biol. 94, 327-333. Potter, M . , Wiener, F., and Mushinski, J. F. (1984). Adu. Viral Oncol. 4, 139-162. Rask-Nielsen, R., McIntire, K. R . , and Ebbesen, P. (1968).J. Nafl. Cancer Znsf. 41, 495-504. Ratcliffe, H . L. (1940). Am. J. Pathol. 16, 237-254. Remmers, E. F., Yang, J. Q., and Marcu, K. B. (1986).EMBOJ. 5, 895-904. Rousseaux, J . , and Bazin, H. (1979). Vef.Zmmunol. Zmmunopafhol. 1, 61-78. Roussy, G.,and Guerin, P. (1942). Bull. Assoc Fr. E f . Cancer 30, 17-28. Ruddick, J. H., and Leslie, G. A. (1977).J . Zmmunol. 118, 1025-1031. Saxton, J. A., Sperling, G . A., Barnes, L. L., and McCay, C. M. (1948).Acfa Unio Znf. Cancerum 6,423-431.

310

HER*

BAZIN E T AL.

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

3215-3222. Siebenlist, U., Hennighaussen, L., Battey, J., and Leder, P. (1984).Cell 37, 381-391. Singer, M.F.,and Skowronski, J. (1985).Trcndc Biochem. Sci. 10, 119-122. Soares, M.B., Schon, E., and Efstratiadis, A. (1985).J. Mol. Evol. 2 2 , 117-133. Snell, K.C . (1964).In “Pathology of Laboratory Animals” (E. Ribelin and J. R . McCoy, eds.), pp. 1-60. Thomas, Springfield, Illinois. Stechschulte, D. J., and Austen, K. F. (1970).J. Immunol. 1 0 4 , 1052-1062. Steffen, D.(1984). Proc. Natl. Acad. Sci. U . S . A . 81, 2097-2101. Stewart, H . L., and Jones, B. F. (1941). Arch. Pathol. 31, 37-54. Sumegi, J. (1985).In “Mechanisms of B Cell Neoplasia” (F. Melchers and M . Potter, eds.), pp.

244-247. Sumegi, J., Spira, J . , Bazin, H . , Szpirer, J., Levan, G., and Klein, G. (1983).Nature (London)

306, 497-498. Szpirer, J . , Levan, G., Thorn, M . , and Szpirer, C . (1984). Cytogenet. Cell &net. 38, 142-149. Thompson, S. W., Huseby, B. A., Fox, M . A , , Davis, C . L., and Hunt, R . D. (1961).J.Natl. Cancer Inst. 27, 1037-1057. Tuchmann-Duplessis, H., and Mercier-Parot, L. (1962). C . R . Acad. Sci. 2 5 4 , 1535-1537. Wanebo, H . J., Gallmeier, W . M., and Boyse, E. A. (1966). Science 1 5 4 , 901-903. Wiener, F.,Babonits, M . , Spira, J., Klein, G., and Potter, M . (1980).Som. Cell. Genet. 6 , 731 -738. Wiener, F., Babonits, M., Spira, J., Klein, G . , and Bazin, H. (1982).1nt.J. Cancer29, 431-437. Wirschubsky, Z . , Tshichlas, P., Klein, G., and Sumegi, J. (1986). Int. J. Cancer 38, 739-746. Woolley, P. G . , and Wherry, W . B. (1911).J.Med. Res. 2 5 , 205-215. Yang, J. Q.,Bauer, S. R . , Mushinski, J. F., and Marcu, K. B. (1985). EMBO J . 4 , 1441-1 447.b.

A

human tumors, 217 pleoclonal tumors, 212, 214, 215 Allotype Louvain rat immunocytomas and, 281, 282 tumor clonality and, 210 Alloxan, newborn macrosomy and, 271 Amines, chemical carcinogenesis and experimental models, 27, 29 mechanisms, 39, 41, 42 Amino acids chemical carcinogensis and, 46 Epstein-Barr virus proteins and transformed cells, 110. 114, 118, 127, 130, 134 virus-produced cells, 145 stroma and, 161 tumor clonality and, 215 Aneuploidy, chemical carcinogenesis and, 33 Antibodies chemical carcinogenesis and, 54, 55 Epstein-Barr virus proteins and diseases, 97-100 transformed cells, 105, 107, 113, 114, 119, 121, 124, 126-128, 133, 134, 136 virus-produced cells, 140, 142, 147, 148 Louvain rat immunocytomas and, 287, 306 newborn macrosomy and, 259 oncogenes and, 84 tumor clonality and, 215 Anticomplement immunofluorescence, Epstein-Barr virus proteins and, 104, 105, 107, 109, 110, 112-114, 121 Antigens Epstein-Barr virus proteins and genome, 103 latent infection membrane protein, 133, 134, 137 transformed cells, 104-131 virus-produced cells, 138-140, 142, 147 gastrointestinal diseases and, 14 Louvain rat immunocytomas and, 288, 291, 305

Abelson murine leukemia virus, oncogenes and, 82 Acetylation, chemical carcinogenesis and, 41 Actin gastrointestinal diseases and, 18, 19 stroma and, 170, 177 Actinomycin D, Epstein-Barr virus proteins and, 141 Acute mononucleosis, Epstein-Barr virus proteins and, 113, 135 Acyclovir, Epstein-Barr virus proteins and, 99 Adenocarcinomas chemical carcinogenesis and, 29, 32 gastrointestinal diseases and, 7, 10, 16, 18 oncogenes and, 89 stroma and basement membranes, 176 disorden, 180-182 epithelium, 166 Adenomas chemical carcinogenesis and, 50 gastrointestinal diseases and, 11, 12, 14, 22 Adenomatosis. stroma and, 177 Adenovirus, Epstein-Barr virus proteins and, 102 Adhesion, stroma and, 186 epithelium, 168 glycoproteins, 170-172 Adipocytes chemical carcinogenesis and, 49 newborn macrosomy and, 255 Aflatoxins, chemical carcinogenesis and, 39, 41, 57 Aging, newborn macrosomy and, 261, 265 Alcoholism, newborn macrosomy and, 264 Alkaline exonuclease, Epstein-Barr virus proteins and, 145, 146 Allelic loss, tumor clonality and, 209, 210 Alloenzymes, tumor clonality and, 224 animal tumors, 218, 219 distinction, 202-205, 211 311

312

INDEX

Antigens ( c o n t . ) newborn macrosomy and, 261, 262 oncogenes and, 72-74, 87-89 malignant genotype, 84, 86, 87 malignant phenotype, 74, 79 maturation arrest, 92 tumor clonality and, 210, 211 Antithyroid antibodies, newborn macrosony and, 259, 261 Arachidonic acid, chemical carcinogenesis and, 52 Arginine, Epstein-Barr virus proteins and, 112, 114, 127, 141

Asbestos chemical carcinogenesis and, 28 stroma and, 181 Ascites tumors, tumor clonality and, 199 Atherosclerosis, newborn macrosomy and, 263, 265, 269

Atrophic gastritis, gastrointestinal diseases and, 5, 7 Autoimmune thyroiditis, newborn macrosomy and, 259, 261 Autoradiography, tumor clonality and, 204 Avian myeloblastosis virus, oncogenes and, 83

B lymphoid tumors, Louvain rat immunocytomas and, 306 Bacteria Epstein-Barr virus proteins and, 108, 114, 146

Louvain rat immunocytomas and, 305 stroma and, 162 Barret'a epithelium, gastrointestinal diseases and, 2 Basal cell carcinomas, tumor clonality and, 217

Basement membranes oncogenes and, 77 stroma and, 186 alterations, 184 epithelium, 161, 166 glycoproteins, 172 invasiveness, 173-176 Benzene, stroma and, 188 Biguanides, newborn macrosomy and, 270, 271

Bile acids, gastrointestinal diseases and, 11 Biomonitoring, chemical carcinogenesis and, 54-58

Birth order, newborn macrosomy and, 235, 236

Avian myelocytomatosisvirus, oncogenes and, 83 Azaserine, chemical carcinogenesis and, 29

B B cell growth factor, Epstein-Barr virus proteins and, 96, 137 B cell lymphoma Epstein-Barr virus proteins and, 100 tumor clonality and, 212. 222, 223, 225 B cells, Epstein-Barr virus proteins and, 149, 150 diseases, 99, 100 transformed cells, 120, 133, 135, 136 virus-produced cells, 138

B lymphocytes Epstein-Barr virus proteins and, 149 biology, 96 diseases, 98, 100 transformed cells, 104, 120, 132 Louvain rat immunocytomas and, 306 tumor clonality and, 210

Bladder chemical carcinogenesis and, 29, 39, 57 stroma and alterations, 184, 185 disorders, 181 epithelium, 162 Bone marrow Epstein-Barr virus proteins and, 100 gastrointestinal diseases and, 19 oncogenes and, 82 stroma and, 188 tumor clonality and, 199, 208 Bowel, gastrointestinal diseases and, 18 Brain tumor, chemical carcinogenesis and, 30 Breast cancer newborn macrosomy and, 233 accelerated development, 267 case histories, 239 characteristics, 241, 242, 245 factors, 258, 259 prognosis, 247, 249, 250 risk factors, 267, 268 stroma and

INDEX

disorders, 181 epithelium, 165-169 fibroblasts, 178 glycoproteins, 171 Brunner's glands, gastrointestinal diseases and, 16 Burkitt's lymphoma Epstein-Barr virus proteins and biology, 96 diseases, 96-98 transformed cells, 105, 108, 109, 128, 133, 135, 136 virus-produced cells, 138 Louvain rat immunocytomas and, 297-300 tumor clonality and, 208, 210, 216, 223

C Cancrophilia syndrome, newborn macrosomy and, 232, 261-269 accelerated development, 266 epidemiology, 264 factors, 261 prophylaxis, 270, 271 Carbohydrate gastrointestinal diseases and, 18 newborn macrosomy and, 232 cancrophilia syndrome, 263 case histories, 241, 245 factors, 255, 258, 259, 261 prognosis, 250 prophylaxis, 270 retrospective data, 254 transplacental carcinogenesis, 254, 255 stroma and, 168, 169 Catalysis, Epstein-Barr virus proteins and, 146 Cathespins, stroma and. 162 cDNA, Epstein-Barr virus proteins and genome, 102, 103 transformed cells, 124, 126, 127, 129, 130, 136, 137 Cecum, Louvain rat immunocytomas and, 283, 305 Cell culture, Louvain rat immunocytomas and, 293 Cell surface glycoconjugates, gastrointestinal diseases and, 17

313

Cervix chemical carcinogenesis and, 29 newborn macrosomy and, 239, 267, 268 stmma and, 177 tumor clonality and, 217, 225 Chediak-Higashi syndrome, Epstein-Barr virus proteins and, 100 Chemical carcinogenesis, 25, 26, 59, 60 biological concepts genetic changes, 36-38 multistage tumor development, 33-35 tumor clonality, 35, 36 tumor promotion, 38. 39 biomonitoring, 54-58 experimental models animal, 26-30 cell culture, 30-33 mechanisms cellular basis for tumor promotion, 49-51 DNA interactions, 41-45 metabolism, 39-41 oncogene activation, 45-48 phenotype of initiated cells, 48, 49 phorbol ester tumor promotion, 51-54 transgenic mice, 58. 59 Chemotherapy, chemical carcinogenesis and, 45, 55 Chimeras, tumor clonality and animal tumors, 217-219 distinction, 202, 207, 211 origin, 199 Cholesterol, newborn macrosomy and, 242 Chondroitin sulfate, stroma and, 168 Chromatids, chemical carcinogenesis and, 53 Chromatin Epstein-Barr virus proteins and, 104 Louvain rat immunocytomas and, 304, 306 Chromosomes chemical carcinogenesis and, 33, 38, 53 Epstein-Barr virus proteins and diseases, 98 transformed cells, 104, 109, 110, 112, 113, 138 Louvain rat immunocytomas and, 295-299, 301, 306 tumor clonality and animal tumors, 219 distinction, 202, 203, 205-210 origin, 199 pleoclonal tumors, 215, 216

314

INDEX

Chronic atrophic gastritis, gastrointestinal diseases and, 6, 19 Chronic lymphatic leukemia, tumor clonalitg and, 210 Chronic mononucleosis, Epstein-Barr virus proteins and, 99 Chronic myeloid leukemia, tumor clonality and, 208, 209, 216, 217 Chronic ulcerative colitis, gastrointestinal diseases and, 9, 16 Chymotrypsin, Epstein-Barr virus proteins and, 131 Cisplatin, chemical carcinogenesis and, 45, 58 Cleavage, Epstein-Barr virus proteins and, 112. 131 Clonality, tumor, see Tumor clonality Clones chemical carcinogenesis and, 60 biological concepts, 35-38 experimental models, 31, 32 mechanisms, 48, 49, 51 transgenic mice, 59 Epstein-Barr virus proteins and biology, 96 genome, 102, 103 transformed cells, 104, 109, 117, 124, 126, 127, 129, 130, 133, 136, 137 gastrointestinal diseases and, 17 Louvain rat immunocytomas and, 302 oncogenes and, 90 stroma and, 166 Collagen, stroma and alterations. 183-185 basement membranes, 174, 175 disorders, 179-182 epithelium, 160-167, 169, 170 glycoproteins, 171, 172 Collagenases, stroma and alterations, 183 basement membranes, 173, 175 epithelium, 161-164 Collagenolysis, stroma and, 186 alterations, 183, 184 basement membranes, 174 epithelium, 162-165 Colon chemical carcinogenesis and, 29, 50 gastrointestinal diseases and, 9-13 modulators, 13-15 normal, 7-9

stomach, 4, 6 newborn macrosomy and, 264 stroma and, 168, 182 tumor clonality and, 222, 225 Colonic mucoprotein antigen, gastrointestinal diseases and, 16 Colon-specific antigens, gastrointestinal diseases and, 16 Colorectal cancer, newborn macrosomy and, 239, 258 Connectin, stroma and, 172 Connective tissue stroma and, 159, 160, 186 alterations, 184 disorders, 178, 180, 182 epithelium, 168 glycoproteins, 171 tumor clonality and, 198 Cornea, stroma and, 163 Cyclamate, chemical carcinogenesis and, 29 Cyclic AMP, gastrointestinal diseases and, 8 Cycloheximide, Epstein-Barr virus proteins and, 141, 142, 144 Cysteine, Louvain rat immunocytomas and, 287 Cytoplasm Epstein-Barr virus proteins and, 102, 109 Louvain rat immunocytomas and, 283, 287 stroma and, 177 Cytosine, tumor clonality and, 205 Cytotoxicity chemical carcinogenesis and, 60 experimental models, 30, 31 mechanisms, 43, 49, 50, 53 Epstein-Barr virus proteins and, 98, 133, 134 gastrointestinal diseases and, 18

D Debrisoquine, chemical carcinogenesis and, 41 Deficits, oncogenes and, 88 cellular heterogeneity, 91 malignant phenotype, 76, 79 maturation arrest, 92 Degradation, stroma and, 186 alterations, 184 basement membranes, 173, 175

INDEX epithelium, 160, 162-164, 169. 170 Deletion, tumor clonality and, 208, 209 Deoxyadenosine, chemical carcinogenesis and, 46 Deoxycholic acid, gastrointestinal diseases and, 14, 15 Deoxyguanosine, chemical carcinogenesis and, 42, 44 DES, chemical carcinogenesis and, 30 Desmoplasia, stroma and disorders, 180-182 epithelium, 160, 165-170 glycoproteins, 171 Diabetes, newborn macrosomy and, 237-239, 272 case histories, 241, 245 epidemiology, 264 factors, 255, 257, 259 prognosis, 248 prophylaxis, 270, 271 Diacylglycerol, chemical carcinogenesisand, 51 Differentiation chemical carcinogenesis and, 26, 60 mechanisms, 48-50, 52 Epstein-Barr virus proteins and, 97, 149, 150 gastrointestinal diseases and antigenic determinants, 15-22 esophagus, 2 , 3 large intestine, 7-15 stomach, 3-7 Louvain rat immunocytomas and, 283 oncogenes and, 72, 73, 87, 88 cellular heterogeneity, 89, 91 malignant genotype, 80-87 malignant phenotype, 76-79 maturation arrest, 92 stroma and, 188 basement membranes, 173, 175, 176 glycoproteins, 170, 172 tumor clonality and, 199 Differentiation antigens, oncogenes and, 72-74, 87-89 malignant genotype, 84, 86, 87 malignant phenotype, 74, 76 Dimethylhydrazine. gastrointestinal diseases and, 10, 11, 18 Dimethylsulfoxide, gastrointestinal diseases and, 21, 22 DNA chemical carcinogenesis and

315

biomonitoring, 54-58 experimental models, 31 mechanisms, 40-46, 48, 49, 53 Epstein-Barr virus proteins and, 150 diseases, 98 genome, 100-102 transformed cells, 104, 109, 112, 114, 118, 120, 121, 126, 127, 137 virus-produced cells, 140, 143, 146, 147 gastrointestinal diseases and esophagus, 2 large intestine, 8, 10, IS, 14 stomach, 5-7 Louvain rat immunocytomas and, 297, 300, 302, 303 newborn macrosomy and, 232, 263 oncogenes and, 84, 87 tumor clonality and, 205, 207, 209, 210, 225 DNA polymerase, Epstein-Barr virus proteins and, 142, 144-146 Drosophilia Louvain rat immunocytomas and, 303 oncogenes and, 75, 82, 85 Duodenum, gastrointestinal diseases and, 6 Dysgammaglobulinemia, Epstein-Barr virus proteins and, 100

Early antigen, Epstein-Barr virus proteins and diseases, 97 transformed cells, 120 virus-produced cells, 139, 144, 147 Elastase, stroma and, 162 Elastin, stroma and, 186 alterations, 183 epithelium, 160, 165, 168, 169 Elastogenesis, stroma and, 168 Elastolysis, stroma and, 168 Elastosis, stroma and, 165, 168 Electron microscopy Epstein-Barr virus proteins and, 113 gastrointestinal diseases and, 14 Louvain rat immunocytomas and, 283 stroma and, 165, 175, 183 Electrophoresis, tumor clonality and animal tumors, 218 distinction, 203-206, 211

316

INDEX

ELISA chemical carcinogenesis and, 55 Epstein-Barr virus proteins and, 107 Embryogenesis stroma and, 176, 186 tumor clonality and, 203 Endocrine cells, gastrointestinal diseases and, 3, 4, 9

Endocrine disturbances, newborn macrosomy and, 252, 233 cancrophilia syndrome, 263 prophylaxis, 270, 271 risk factors, 267 Endocytosis, stroma and, 162 Endometrial cancer, newborn macrosomy and, 259, 250, 258, 267 Endothelial cells gastrointestinal diseases and, 19 tumor clonality and, 198 Enzymes chemical carcinogenesis and biomonitoring, 55 experimental models, 28 mechanisms, 40, 45, 46, 51, 52 Epstein-Barr virus proteins and transformed cells, 104, 105 virus-produced cells, 141, 144-148 gastrointestinal diseases and, 13, 17 newborn macrosomy and, 234 stroma and, 186 basement membranes, 173. 174 epithelium, 160-162, 164, 168, 169 tumor clonality and animal tumors, 218 distinction, 204-206, 211 pleoclonal tumors, 215, 216 Epidermal cells, oncogenes and, 77, 78, 90 Epidermal growth factor gastrointestinal diseases and, 14 oncogenes and, 82 Epidermal proliferative unit, chemical carcinogenesis and, 45 Epidermis stroma and alterations, 184 disorders, 179, 180 epithelium, 163 tumor clonality and, 211 Episomes, Epstein-Barr virus proteins and, 100, 113, 137

Epitheliomas

oncogenes and, 89 stroma and, 179 Epithelium chemical carcinogenesis and, 60 biological concepts, 33, 35 experimental models, 28, 50-32 mechanisms, 41, 49, 50 Epstein-Barr virus proteins and, 150 biology, 95, 96 diseases, 98 virus-produced cells, 158 gastrointestinal diseases and antigenic determinants, 16, 17, 19, 22 esophagus. 2, 3 large intestine, 7-10, 12-15 stomach, 3-6 oncogenes and, 77 stroma and, 160, 186, 188 alterations, 183-185 basement membranes, 173-176 collagen, 160, 167 collagenases, 161 collagenolysis, 163-165 collagenolytic enzymes, 162 desmoplasia, 165 - 167 disorders, 180-182 elastogenesis, 168 fibroblasts, 177 glycoproteins, 171, 172 plasminogen activator, 162, 163 productive changes, 165 proteoglycans, 168, 169 reactivity, 169, 170 Epitopes Epstein-Barr virus proteins and, 121, 125, 126

gastrointestinal diseases and, 17 Epstein-Barr virus chemical carcinogenesis and, 53 tumor clonality and, 223 Epstein-Barr virus proteins, 149, 150 biology lymphoblastoid cell line, 96 viral tropism, 95, 96 diseases Burkitt’s lymphoma, 96-98 infectious mononucleosis, 98-100 lymphoproliferative disorder, 100 nasopharyngeal carcinoma, 98 genome DNA structure, 100-102

317

INDEX transcription, 102, 103 transformed cells, 137, 138 differential expression, 134-136 EBNA, 104 EBNA-1, 104-113 EBNA-2, 113-121 EBNA-3, 121-124 EBNA-4, 124-127 EBNA-5, 127-131 identification, 136, 137 latent infection membrane protein, 131-134

virus-produced cells, 138, 139 early genes, 141-147 gene map, 140, 141 productive cycle, 139, 140 virus particle, 147-149 Equilibrium, stroma and, 169, 170 Ergastoplasmic reticulum, Louvain rat immuncytomas and, 283, 287 Escherichia coli. Epstein-Barr virus proteins and, 146, 147 Esophagus chemical carcinogenesis and, 30, 49 gastrointestinal diseases and, 1-3, 13 Estradiol. gastrointestinal diseases and, 9 Estrogen gastrointestinal diseases and, 8 newborn macrosomy and, 250 Ethionine, chemical carcinogenesis and, 31

generalized changes, 177, 178 glycoproteins, 171 local changes. 177 tumor clonality and, 198, 199, 206 Fibronectin, stroma and basement membranes, 173 epithelium, 160 glycoproteins, 170-172 Fibrosarcomas gastrointestinal diseases and, 19 stroma and, 161 tumor clonality and, 225 animal tumors, 217, 219, 223 distinction, 204, 206, 211 pleoclonal tumors, 212, 214 Fibrosis, stroma and, 180-183, 186 Filaments, Epstein-Barr virus proteins and, 147

Fixation chemical carcinogenesis and, 42 Epstein-Barr virus proteins and, 139 Fluorescence chemical carcinogenesis and, 57 Epstein-Barr virus proteins and, 110, 139, 140

oncogenes and, 84 tumor clonality and, 204 Flutamide. gastrointestinal diseases and, 8 Fractionation, Epstein-Barr virus proteins and, 113, 124 Friend erythroleukemia cells, gastrointestinal diseases and, 19, 21 Fujinami sarcoma virus, oncogenes and, 82 Fusion, tumor clonality and, 198, 217

F-actin, gastrointestinal diseases and, 18 Fat, newborn macrosomy and, 250 Fatty acids, newborn macrosomy and, 232,

G

263, 264

Feedback, stroma and, 170 Fibrils, stroma and, 162, 165, 183 Fibroadenomatosis, newborn macrosomy and, 242 Fibroblasts chemical carcinogenesis and, 33, 43, 50 Epstein-Barr virus proteins and, 105, 120, 132, 137, 150

gastrointestinal diseases and, 9, 14 Louvain rat immunocytomas and, 304 stroma and, 186 alterations, 183 epithelium, 163-169

fi-Galactosidase, Epstein-Barr virus proteins and, 105, 109, 110, 124, 125, 130, 131, 134

Gardner syndrome, gastrointestinal diseases and, 12 Gastrin, gastrointestinal diseases and, 4, 5 Gastrinomas, gastrointestinal diseases and, 4 Gastritis, gastrointestinal diseases and, 6 Gastrointestinal diseases, 1, 2 antigenic determinants, 15-22 esophagus, 2, 3 large intestine, 7-13 modulators, 13-15

318

INDEX

Gastrointestinal diseases (cont. ) stomach, 3-7 Genotoxicity, chemical carcinogenesis and, 34, 35, 53, 60 Genotype, malignancy and, 74, 79-81, 86-88 dominant oncogenes, 81-85 recessive oncogenes, 85, 86 Glucose, newborn macrosomy and, 232 cancrophilia syndrome, 263 epidemiology, 265 factors, 255, 257, 261 prophylaxis, 271 retrospective data, 254 transplacental carcinogenesis, 254, 255 Glucose phosphate isomerase. tumor clonality and, 211, 218 Glucose-6-phosphate dehydrogenase, tumor clonality and, 224 distinction, 203-207, 209 human tumors, 216, 217 pleoclonal tumors, 212, 215, 216 Glycine. Epstein-Barr virus proteins and, 127 Glycolipid, gastrointestinal diseases and, 18 Glycoprotein Epstein-Barr virus proteins and, 146, 148 stroma and, 186 adhesion, 170-172 basement membranes, 173, 176 epithelium, 160, 169 Glycosaminoglycans. stroma an d , 186 alterations, 183 basement membranes, 175 epithelium, 168, 169 Glycosidases, stroma and, 169 Glycosylases, chemical carcinogenesis and, 44 Glycosylation. Epstein-Barr virus proteins and, 131, 137, 148 Goblet cell antigen, gastrointestinal diseases and, 16 Granulocyte-macrophage colony-stimulating factor, oncogenes and, 82 Granulocytes, tumor clonality and, 208, 216 GTP, chemical carcinogenesis and, 45 Guillain-BarrC syndrome, Epstein-Barr virus proteins and, 100

H Haplotype, Louvain rat immunocytomas and, 280 Harvey murine sarcoma virus, oncogenes and, 82, 83

Hematopoiesis, tumor clonality and, 200 Hematopoietic cells chemical carcinogenesis and, 49 Louvain rat immunocytomas and, 293 oncogenes and, 82, 83 stroma and, 188 Hematopoietic system, newborn macrosomy and, 255 Heparan sulfate, stroma and, 169, 172, 173 Hepatocytes chemical carcinogenesis and, 49 tumor clonality and, 205, 216 Hepatomas, tumor clonality and, 216, 220 Herpes simplex virus, Epstein-Barr virus proteins and, 141, 142, 144, 146, 147, 149 Heterogeneity chemical carcinogenesis and, 26, 31 Epstein-Barr virus proteins and, transformed cells, 99, 128, 142 gastrointestinal diseases and, 16, 17 oncogenes and, 72, 73, 87 cellular, 89-91 maturation arrest. 92 tumor clonality and, 199, 200 High-pressure liquid chromatography, chemical carcinogenesis and, 55 Histamine, gastrointestinal diseases and, 4 Histocompatibility, Louvain rat immunocytomas and, 280. 281, 292, 294 Hodgkin's disease, newborn macrosomy and, 251 Homeostasis newborn macrosomy and, 234, 261 stroma an d , 173, 187 Homogeneity Epstein-Barr virus proteins and, 112, 145 Louvain rat immunocytomas and, 283 HomoIogy chemical carcinogenesis and, 26, 45 Epstein-Barr virus proteins and transformed cells, 109, 110, 118, 120, 129, 133 virus-produced cells, 141, 144-147 gastrointestinal diseases and, 14 Louvain rat immunocytomas and, 287, 294 oncogenes an d , 71, 81 stroma and, 164 tumor clonality and, 209 Hormones chemical carcinogenesis and experimental models, 28, 32 mechanisms, 49, 52

319

INDEX

gastrointestinal diseases and, 4, 8, 9, 14 newborn macrosomy and, 232, 233, 272 accelerated development, 266 cancrophilia syndrome, 261, 263 case histories, 241 characteristics, 241-245 definition, 238 dependence, 269, 270 epidemiology, 266 factors, 261 prognosis, 246, 250 prophylaxis, 270, 271 retrospective data, 254 risk factors, 267, 269 stroma and, 169, 177 tumor clonality and, 198, 223 Hyaluronic acid, stroma and, 168, 169 Hybridization Epstein-Barr virus proteins and genome, 103 transformed cells, 110, 118, 121 virus-produced cells, 142, 145 Louvain rat immuncytomas and etiology, 295, 297, 300, 302, 303 immunoglobulins, 288 incidence, 283 in vitro cell culture, 293 in vivo transplantation, 293 stroma and, 173 tumor clonality and distinction, 204, 205 origin, 198, I99 pleoclonal tumors, 214 Hybridomas Epstein-Barr virus proteins and, 116 gastrointestinal diseases and, 17 Louvain rat immunocytomas and, 293, 294, 306 Hydrazine, chemical carcinogenesis and, 29 Hydrolysis chemical carcinogenesis and, 55, 57 stroma and, 169 Hyperglycemia, newborn macrosomy and, 255, 257 Hyperinsulinemia, newborn macrosomy and cancrophilia syndrome, 263 characteristics, 245 definition, 238 factors, 255, 257, 259, 261 prophylaxis, 271 transplacental carcinogenesis, 255 Hyperlipidemia, newborn macrosomy and, 263

Hyperplasia chemical carcinogenesis and experimental models, 28, 29, 32 mechanisms, 45, 49 gastrointestinal diseases and, 4, 11 newborn macrosomy and, 255 stroma and, 179, 180 Hyperproliferation, gastrointestinal diseases and, 6, 19 Hypertriglyceridemia, newborn macrosomy and, 245 Hypothalamus, newborn macrosomy and, 261, 262

I Ileocecal lymph nodes, Louvain rat immunocytomas and, 280, 283, 287, 305 Ileocecal tumors, Louvain rat immunocytomas and animals, 280, 281 etiology, 294, 296, 305 immunoglobulins, 282-285 Ileum, Louvain rat imrnunocytomas and, 283 Immune system, oncogenes and, 76 Imrnunoassay, chemical carcinogenesis and, 57 Immunocytomas, Louvain rat, 279, 280, 305, 306 animals,. 280, 281 description, 283. 287 etiology, 294 chromosomal breakpoints, 297-299 chromosomal translocation, 295-297 incidence, 294, 295, 305 translocation targets, 299-304 immunoglobulins history, 287 nomenclature, 282, 287, 288 production, 288-291 incidence, 282-286 storage, 294 in vitro cell culture, 293 in who transplantation, 292, 293 Immunoelectrophoresis, Louvain rat immunocytomas and, 287, 288 Immunofluorescence Epstein-Barr virus proteins and, 124, 132 gastrointestinal diseases and, 19

320

INDEX

Immunofluorescence (cont.) stroma and, 182 Immunoglobulins Louvain rat immunocytomas and, 279, 280, 305, 306

animals, 281 description, 283 etiology, 294, 295, 297, 305 history, 287 incidence, 282 nomenclature, 282, 287, 288 production, 288-291 translocation targets, 299-304 tumor clonality and, 224, 226 animal tumors, 223 distinction, 202, 210 human tumors, 216 pleoclonal tumors, 212 Immunotherapy, gastrointestinal diseases and, 18 Infectious mononucleosis, Epstein-Barr virus proteins and diseases, 98-100 transformed cells, 121, 133, 134 Inflammation gastrointestinal diseases and, 5 stroma and, 184 Inhalation, chemical carcinogenesis and, 28 Initiation chemical carcinogenesis and, 26, 59, 60 biological concepts, 33-36, 39 biomonitoring, 55 mechanisms, 42, 48-51, 53 Epstein-Barr virus proteins and genome, 102 transformed cells, 104, 110, 119, 136 stroma and, 187 tumor clonality and, 198 Insulin, newborn macrosomy and cancrophilia syndrome, 263 characteristics, 242 factors. 257 prophylaxis, 271 transplacental carcinogenesis, 255 Internal repeat, see also Third internal repeat array Epstein-Ban virus proteins and, 100, 102, 128 Internalization. Epstein-Barr virus proteins and, 148 Intestinal mucosal-specific glycoprotein, gastrointestinal diseases and, 16

Intracisternal A particles, Louvain rat immunocytomas and, 287 Invasion, oncogenes and, 72, 73, 87 malignant genotype, 81 malignant phenotype, 74-76, 79

J Jaws, Epstein-Barr virus proteins and, 97

K Karyotype, oncogenes and, 89, 90, 92 Karyotypic markers, tumor clonality and, 202

Keratinocytes, stroma and, 174 Ketosis, newborn macrosomy and, 270 Kidney chemical carcinogenesis and, 30 newborn macrosomy and, 251, 255

L Lactation chemical carcinogenesis and, 28 newborn macrosomy and, 265 Laminin, stroma and alterations, 184 basement membranes, 173, 174, 176 epithelium, 160 glycoproteins, 170, 172 Large intestine, gastrointestinal diseases and, 1, 2, 9-13

modulators, 13-15 normal, 7-9 Latent infection membrane protein genome, 103 transformed cells, 131-137 virus-produced cells, 149, 150 Leiomyomas, tumor clonality and, 221 Leucosarcomas, Louvain rat immunocytomas and, 280 Leukemia Louvain rat immunocytomas and, 298 newborn macrosomy and, 251, 259 oncogenes and, 92 stroma and, 188 tumor clonality and, 199, 208, 209

321

INDEX Leukemic blast cells, oncogenes and, 92 Leukocyte migration inhibition, Epstein-Barr virus proteins and, 131 Leukocytes Epstein-Barr virus proteins and, 131 stroma and, 162, 168 tumor clonality and, 198, 199, 206 Light microscopy, stroma and, 174, 175, 183 Lipidemia. newborn macrosomy and, 265 Lipids, newborn macrosomy and, 232 cancrophilia syndrome, 263 epidemiology, 265 factors, 259 prophylaxis, 270 Liver chemical carcinogenesis and biological concepts, 39 experimental models, 27, 31 mechanisms, 41, 42, 49, 50 Louvain rat immunocytomas and, 283 stroma and, 167, 180 tumor clonality and, 205, 225 Louvain rat immunocytomas. see Immunocytomas, Louvain rat Lung cancer chemical carcinogenesis and, 28, 41, 49, 55 newborn macrosomy and, 259 stroma and, 169, 178, 179, 182 tumor clonality and, 208 Lymph nodes Louvain rat immunocytomas and, 280, 283, 294, 305 newborn macrosomy and, 249 Lymphoblastoid cell lines, Epstein-Barr virus proteins and biology, 96 transformed cells, 119, 120, 128, 133-136 Lymphocyte-detected membrane antigen, Epstein-Barr virus proteins and, 133, 134 Lymphocytes chemical carcinogenesis and, 59 Epstein-Barr virus proteins and, 100, 120 stroma and, 183 Lymphoid cells, Epstein-Barr virus proteins and, 130 Lymphoid tissues newborn macrosomy and, 251 oncogenes and, 80 Lymphoid tumors, Louvain rat immunocytomas and, 280, 281

Lymphokines, Epstein-Barr virus proteins and, 131 Lymphomas chemical carcinogenesis and, 59 Epstein-Barr virus proteins and, 96, 100, 108 Louvain rat immunocytomas and, 298 tumor clonality and, 208, 210, 225 Lymphoproliferative diseases, Epstein-Barr virus proteins and, 99, 100 Lymphosarcomas Louvain rat immunocytomas and, 282, 294, 305 newborn macrosomy and, 251 Lysis Epstein.Barr virus proteins and, 133, 134, 137 stroma and, 186-188 basement membranes, 173-175 epithelium, 160, 164, 169

M crophages newborn macrosomy and, 263 oncogenes and, 83 stroma and, 164, 168, 183 tumor clonality and, 198, 199, 206 Macrosomy, newborn, 231-234, 272, 273 accelerated development, 266, 267 cancrophilia syndrome, 261-263 case histories, 239-241 characteristics, 241-245 epidemiology, 263-266 factors, 255, 257-261 healthy populations, 236 hormones, 269, 270 pathology, 236-239 prognosis, 245-250 prophylaxis, 270, 271 ranges, 235, 236 retrospective data, 250-254 risk factors, 267-269 transplacental carcinogenesis, 254-256 Major histocompatibility complex, Louvain rat immunocytomas and, 280, 295 Malaria, Epstein-Barr virus proteins and, 97, 136 Malignancy, oncogenes and, 71-74, 87-89 cellular heterogeneity, 89-91

322 Malignancy, ocogenes and ( c o n t . ) genotype, 79-81, 86, 87 dominant, 81-85 recessive, 85, 86 maturation arrest, 92 phenotype, 74, 79 growth control, lack of, 76-79 positional control, lack of, 74-76 Mammary gland newborn macrosomy and, 255 stroma and alterations, 185 basement membranes, 176 epithelium, 163, 166, 168 Mammary tumors, chemical carcinogenesis and, 46, 59 Mast cells, stroma and, 164 Melanoma chemical carcinogenesis and, 27 newborn macrosomy and, 239, 267 stroma and basement membranes, 175 epithelium, 162, 163, 169 fibroblasts, 178 glycoproteins, 171 Membrane antigen, Epstein-Barr virus proteins and, 97, 139 Menarche. newborn macrosomy and, 233 accelerated development, 266 factors, 257-260 risk factors, 267 Menopause, newborn macrosomy and, 233, 259, 266 Mesenchymal cells chemical carcinogenesis and, 30, 35 stroma and alterations, 185 basement membranes, 176 disorders, 180 epithelium, 160, 164, 169 fibroblasts, 178 Metachromasia. stroma and, 183 Metalloproteinases, stroma and, 161 Metaphase, Epstein-Barr virus proteins an d , 104 Metaplasia. gastrointestinal diseases and, 7 Methylation, tumor clonality and, 205 Methylcholanthrene, tumor clonality and, 219, 223 MFO system, chemical carcinogenesis and, 39, 40

IN1DEX Microautoradiography, gastrointestinal diseases and, 4, 5, 10 Microfilaments, gastrointestinal diseases and, 18, 19 Migration, oncogenes and, 74-78, 89 Mitochondria, Epstein-Barr virus proteins and, 132 Mitogen chemical carcinogenesis and, 49, 50 Epstein-Barr virus proteins and, 133 gastrointestinal diseases and, 5 Mitosis Epstein-Barr virus proteins and, 104, 112 gastrointestinal diseases and, 2, 6 , 7 oncogenes and, 72. 87, 89 malignant phenotype, 77-79 stroma and, 179 tumor clonality and, 208, 210 Moloney virus, Louvain rat immunocytomas and, 298 Monoclonal antibodies chemical carcinogenesis and, 55 Epstein-Barr virus proteins and transformed cells, 109, 116, 131 virus-produced cells, 141, 142, 146 gastrointestinal diseases and, 17, 18 oncogenes and, 76 stroma and, 176 tumor clonality and, 215 Monoclonal immunoglobulins, Louvain rat immunocytomas and, 280, 288-291, 294, 305, 306 Monoclonality, 198, 224-226 animal tumors, 218-220 definition, 201 distinction, 202-211 factors, 220-223 human tumors, 216. 217 number, 215 Morphology Epstein-Barr virus proteins an d , 122 gastrointestinal diseases and, 2, 18 newborn macrosomy and, 245, 261 stroma and, 170, 172, 173, 176 tumor clonality an d , 217, 219, 225 mRNA Epstein-Barr virus proteins and genome, 102, 103 transformed cells, 110, 129, 133, 136, 137, 139 virus-produced cells, 140, 142

INDEX Louvain rat immunocytomas and, 298, 302 Mucin, gastrointestinal diseases and, 17 MW cav~olz,tumor clonality and, 204, 206, 211 Mus mwculw, tumor clonality and, 204, 205, 211. 215 Mutagenesis chemical carcinogenesis and experimental models, 30 mechanisms, 40, 41, 43, 46, 53 gastrointestinal diseases and, 5 Mutation chemical carcinogenesis and, 60 biological concepts, 36, 39 experimental models, 30 mechanisms, 42, 43, 46, 48, 54 Epstein-Barr virus proteins and. 141 oncogenes and malignant genotype, 82, 83, 85, 86 maturation arrest, 90 tumor clonality and, 199, 209, 221 Myelomas, Louvain rat immunocytomas and, 279, 306 animals, 280 etiology, 305 immunoglobulins, 289 Myofibroblasts, stroma and, 166

N %-Napthylamine,chemical carcinogenesis and, 29 Nasopharyngeal carcinoma, Epstein-Barr virus proteins and, 98, 108, 146, 150 Nasopharynx. Epstein-Barr virus proteins and, 96 Necropsy, Louvain rat immunocytomas and, 283 Neoantigens. oncogenes and, 74, 79 Neoplasia, gastrointestinal diseases and, 9-11 Neoplasms chemical carcinogenesis and, 26 Epstein-Barr virus proteins and, 97 gastrointestinal diseases and, 18 Louvain rat immunocytomas and, 294, 305 stroma and alterations, 184, 185 basement membranes, 175

323

disorders, 182 epithelium, 167 fibroblasts, 178 Neoplastic cells chemical carcinogenesis and biological concepts, 34-36, 38 experimental models, 31 mechanisms, 42, 50 tumor clonality and, 224 animal tumors, 219, 223 definition, 200, 201 distinction, 202, 205, 207, 209, 211 human tumors, 216, 217 origin, 199 pleoclonal tumors, 212 Neoplastic transformation, newborn macrosomy and, 232 Neural crest, gastrointestinal diseases and, 4 Neuroblastomas, newborn macrosomy and, 251 Neurofibromas, tumor clonality and, 224 factors, 221, 222 human tumors, 216 pleoclonal tumors, 212, 213 Newborn macrosomy, see Macrosomy, newborn Nit rosamines chemical carcinogenesis and, 57 gastrointestinal diseases and, 5 N-Nitrosamines. chemical carcinogenesis and, 29, 39, 55 N-Nitroso compounds, chemical carcinogenesis and, 28, 29 N-Nitrosodiethylamine, tumor clonality and, 220 Nitrosomethylurea, newborn macrosomy and, 254 N-Nitrosomethylurea, newborn macrosomy and, 254-256 Nonesterified fatty acids, newborn macrosomy and, 242 Nuclear antigens, Epstein-Barr virus proteins and, 120-122, 126, 149 Nucleosides, chemical carcinogenesis and, 55, 59 Nucleotides chemical carcinogenesis and, 32, 44, 46, 55, 57 Epstein-Barr virus proteins and, 102, 145 Nutrition, gastrointestinal diseases and, 5, 11

324

lNDEX

0 Obesity, newborn macrosomy and, 259 Oncogenes chemical carcinogenesis and biological concepts, 36, 38 mechanisms, 45-48, 53, 54 transgenic mice, 58, 59 Epstein-Barr virus proteins and, 98, 109, 120

gastrointestinal diseases and, 18, 19 Louvain rat immunocytomas and, 297 stroma and, 164, 187, 188 tumor clonality and, 220, 223, 225 Oncogenes, malignancy and, 71-74, 87-89 cellular heterogeneity, 89-91 genotype, 79-81, 86, 87 dominant, 81-85 recessive, 85, 86 maturation arrest, 92 phenotype, 74, 79 growth control, lack of, 76-79 positional control, lack of, 74-76 Oophorectomy, gastrointestinal diseases and, 8, 9

Organogenesis, stroma and, 186 Ornithine carbamoyltransferase, tumor clonality and, 205, 216, 220 Ornithine decarboxylase, gastrointestinal diseases and, 8, 11 Osteosarcoma newborn macrosomy and, 251 stroma and, 167, 178 tumor clonality and, 208, 209, 225 Ovary chemical carcinogenesis and, 28 newborn macrosomy and, 239, 255, 267 Oxygen, chemical carcinogenesis and, 53

P Pancreas chemical carcinogenesis and, 29, 59 newborn macrosomy and, 261 Papilloma viruses, Epstein-Barr virus proteins and, 150 Papillomas chemical carcinogenesis and experimental models, 27 mechanisms, 36, 46, 48, 53

tumor clonality and, 217 paraproteins, Louvain rat immunocytomas and, 280 Parasites, Louvain rat immunocytomas and, 305

Pentagastrin. gastrointestinal diseases and, 5 Pepsinogen, gastrointestinal diseases and, 3 Peptic ulcer, gastrointestinal diseases and, 5, 6 Peptides Epstein-Barr virus proteins and, 105, 107-110, 114, 116, 119, 133, 134

stroma and, 161 Phagocytosis, stroma and, 161, 183 Phenethylbiguanide, newborn macrosomy and, 271, 272 Phenobarbitone, tumor clonality and, 220 Phenoclones. oncogenes and, 90 Phenotype chemical carcinogenesis and, 26, 60 biological concepts, 34, 36 experimental models, 32 mechanisms, 41, 43, 45, 48, 49, 51, 53, 54

Epstein-Barr virus proteins and diseases, 96-98 transformed cells, 120, 136, 138 malignancy and, 71, 72, 74, 79, 87, 88 cellular heterogeneity, 89-91 genotype, 79, 81, 83, 84 growth control, lack of, 76-79 maturation arrest, 92 positional control, lack of, 74-76 stroma and, 187 alterations. 185 basement membranes, 174 epithelium, 167 fibroblasts, 177 tumor clonality and, 225 animal tumors, 218 distinction, 203, 206, 207, 211 human tumors, 216, 217, 222 origin, 199 pleoclonal tumors, 212 Philadelphia chromosome, tumor clonality and, 208, 209 Phorbol esters chemical carcinogenesis and, 31, 50-54, 60 stroma and, 183 tumor clonality and, 218

INDEX Phosphoglycerate kinase-1, tumor clonality and animal tumors, 218, 219 distinction, 204, 206, 207, 211 pleoclonal tumors, 212-215 Phosphorylation chemical carcinogenesis and, 52, 55 Epstein-Barr virus proteins and, 127, 146 stroma and, 170 Pituitary, newborn macrosomy and, 255 Plasma cells, Louvain rat immunocytomas and, 283, 294, 306 Plasma membrane Epstein-Barr virus proteins and. 131-133 stroma and, 187 Plasmacytomas Louvain rat immunocytomas and, 279, 280, 306 etiology, 294, 295, 298-300, 305 immunoglobulins, 289 tumor clonality and, 210 Plasmacytosis, Epstein-Barr virus proteins and, 100 Plasmids chemical carcinogenesis and, 42, 46 Epstein-Bart virus proteins and, 112, 137 Plasmin, stroma and, 162, 163 Plasminogen activator gastrointestinal diseases and, 15 stroma and, 161, 162 Plasmodium falcifiarum. Epstein-Barr virus proteins and, 97, 112 Platelet-derived growth factor, oncogenes and, 81 Pleoclonal tumors, 198, 224 animal tumors, 218, 219 definition, 201 distinction, 202-211 factors, 221, 222 human tumors, 216 number, 212-215 origin, 199, 200 spatial distribution, 215, 216 Polycyclic aromatic hydrocarbons, chemical carcinogenesis and, 29 Polymerization, stroma and, 168, 170, 171 Polymorphisms, Louvain rat immunocytomas and, 281, 287 Polypeptides Epstein.Barr virus proteins and genome. 103

325

transformed cells, 105, 113, 114, 119, 121-125, 127, 129, 137 virus-produced cells, 140-142, 145, 147, 148 gastrointestinal diseases and, 19 stroma and, 168, 171 Polyposis chemical carcinogenesis and, 50 gastrointestinal diseases and, 5, 12 Polyposis coli stroma and, 178 tumor clonality and, 225 Polyps chemical carcinygenesis and, 29 gastrointestinal diseases and, 6, 7 Pregnancy chemical carcinogenesis and, 28, 59 newborn macrosomy and, 233, 234 case histories, 239 definition, 237 epidemiology, 263-266 factors, 257, 258 prophylaxis, 270, 271 risk factors, 268 transplacental carcinogenesis, 255 Priming, oncogenes and, 74 Procollagenases, stroma and, 164 Progesterone, gastrointestinal diseases and, 9 Prolactin, chemical carcinogenesis and, 28 Proliferation chemical carcinogenesis and biological concepts, 38 experimental models, 27-30 mechanisms, 48, 52, 53 Epstein-Barr virus proteins and, 149 diseases, 98-100 transformed cells, 119, 135 gastrointestinal diseases and, 1, 2 antigenic determinants, 15-22 esophagus, 2, 3 large intestine, 7-15 stomach, 3-7 newborn macrosomy and, 232 oncogenes and, 72, 73, 87-89 cellular heterogeneity, 89 malignant genotype, 82, 83, 85 malignant phenotype, 74, 76, 78 stroma and, 188 alterations, 184, 185 disorders, 180 epithelium, 168

326

INDEX

Proliferation (cont.) tumor clonality and, 199, 201, 217 Proline, Epstein-Barr virus proteins and, 114, 127 Promotion chemical carcinogenesis an d , 26, 59, 60 biological concepts, 36, 38 mechanisms, 49-54 Epstein-Barr virus proteins and, 102, 144 stroma and, 187 Promyelocytic leukemia cells, chemical carcinogenesis and, 52 Prostaglandins, chemical carcinogenesis and, 32 Proteases Epstein-Barr virus proteins and, 112 gastrointestinal diseases and, 15 oncogenes a nd, 74 stroma and, 161, 162, 164, 169 Protein chemical carcinogenesis an d biomonitoring, 54-57 mechanisms, 40, 45, 46, 51 Epstein-Barr virus proteins and, see Epstein-Barr virus proteins gastrointestinal diseases an d , 5, 8, 14, 18, 19 Louvain rat immunocytomas and, 288, 291, 294, 297 newborn macrosomy and, 233 oncogenes and, 81. 86, 87 stroma and, 187 epithelium, 162-164, 170 glycoproteins, 170, 171 Protein kinase chemical carcinogenesis and, 52 Epstein-Barr virus proteins and, 104 Protein kinase C chemical carcinogenesis an d , 51, 52, 54, 60 stroma a nd, 170 Proteoglycans, stroma and basement membranes, 173, 175 epithelium, 160, 165, 168, 169 Proteolysis Epstein-Barr virus proteins and, 112 stroma and, 161, 164 Protooncogenes chemical carcinogenesis an d , 43, 53 stroma and, 187

Pseudosarcoma. stroma and, 165, 166 Purification, Epstein-Barr virus proteins and, 104, 107, 109, 112 Pyloroplasty, gastrointestinal diseases and, 4

R Radiation, tumor clonality and, 198, 208 Radioimmunoassay chemical carcinogenesis and, 55 Epstein-Barr virus proteins and, 134 Radioimmunoelectrophoresis, Epstein-Barr virus proteins and, 104 Raji cell, Epstein-Barr virus proteins and transformed cells, 116 virus-produced cells, 138, 143, 144, 147 Rectal cancer newborn macrosomy and, 264 stroma and, 182 Reflex esophagitis, gastrointestinal diseases and, 2 Restriction endonuclease, Epstein-Barr virus proteins and, 102 Restriction fragment length polymorphism, tumor clonality and,224 distinction, 202, 203, 205 human tumors, 217 Reticulum cell sarcoma, Louvain rat immunocytomas and, 282. 294 Retinoblastoma stroma an d , 178 tumor clonality and, 208, 209, 222, 225 Retinoic acid, gastrointestinal diseases and, 21, 22 Retrovirus chemical carcinogenesis and, 45, 53 gastrointestinal diseases and, 19 Ribonucleotide reductase, Epstein-Barr virus proteins and, 144, 146 Ribosomes, Louvain rat imrnunocytomas and, 283, 287 RNA chemical carcinogenesis and, 52 Epstein-Barr virus proteins and, 102, 142, 149 gastrointestinal diseases and, 5, 8 Louvain rat immunocytomas and, 299 RNA polymerase I1 Epstein-Barr virus proteins and, 112

327

INDEX

Louvain rat immunocytomas and, 303 RNA polymerase 111. Epstein-Barr virus proteins and, 102 Rous sarcoma virus, stroma and, 180

S Saccharin, chemical carcinogenesis and, 29 Saliva, Epstein-Barr virus proteins and, 95, 99 Scar cancer, stroma and disorders, 180-183 epithelium, 167 Scar formation, stroma and, 179 Scleroderma, stroma and, 181 Serotonin, gastrointestinal diseases and, 4 Simian sarcoma virus, oncogenes and, 81 Skin cancer, stroma and, 178 Skin grafts, Louvain rat immunocytomas and, 281 Small intestine gastrointestinal diseases and, 2. 4, 6, 7 oncogenes and, 77 Smoking, chemical carcinogenesis and, 55, 57 Somatostatin, gastrointestinal diseases and, 5 Spleen, h u v a i n rat immunocytomas and, 283 Squamous cells, stroma and, 165, 166, 175, 184 Stem cells gastrointestinal diseases and, 3, 15 oncogenes and, 73, 74 cellular heterogeneity, 89, 90 malignant genotype, 82 malignant phenotype, 77, 78 maturation arrest, 92 tumor clonality and, 199 Steroids newborn macrosomy and, 233, 264 stroma and, 168 Stomach gastrointestinal diseases and, 1, 2, 4-7 antigenic determinants, 19 normal, 3, 4 stroma and, 165, 182 Stroma gastrointestinal diseases and, 7 tumor clonality and, 198, 199, 218

Stroma, malignant growth and, 159, 160, 186-188 alterations, 183-185 basement membranes, 173-176 disorders, 178, 179 scar cancer, 180-183 wounds, 179, 180 epithelium, 160 collagen, 160, 167 collagenases, 161 collagenolysis, 163-165 collagenolytic enzymes, 162 desmoplasia, 165-167 elastogenesis, 168 plasminogen activator, 162, 163 productive changes, 165 proteoglycans, 168, 169 reactivity, 169, 170 fibroblasts, 177, 178 glycoproteins, 170 fibronectins, 170-172 laminin, 172 Subclones, tumor clonality and, 200 Sugar, Epstein-Barr virus proteins and, 148 Sulfated glycopeptide antigen, gastrointestinal diseases and, 16 Superinfection, Epstein-Barr virus proteins and, 138 Symbiosis, tumor cIonality and, 223

T T cells, Epstein-Barr virus proteins and diseases, 98, 100 transformed cells, 133-135 T lymphocytes, Epstein-Barr virus proteins and, 149 Temperature, oncogenes and, 82, 83, 85 Testosterone chemical carcinogenesis and, 29 gastrointestinal diseases and, 8 Tetracycline, chemical carcinogenesis and, 42, 43 Third internal repeat array, Epstein-Barr virus proteins and, 108-110 Thymidine kinase, Epstein-Barr virus proteins and, 144, 146 Thymomas, Louvain rat immunocytomas and, 298

328

INDEX

Thyroglobulin, newborn macrosomy and, 259 Thyroid newborn macrosomy and, 259, 261, 262, 271

stroma and, 177 Thyrotropin, newborn macrosomy and, 233 Tissue type plasminogen activator, stroma and, 162 Transcription Epstein-Barr virus proteins and, 102, 103, 141, 148

Louvain rat immunocytomas and, 299, 301, 303

Transfect ion chemical carcinogenesis and, 38, 45, 48 Epstein-Barr virus proteins and transformed cells, 104, 105, 120, 121, 126, 132

virus-produced cells, 144 tumor clonality and, 225 Transformation newborn macrosomy and, 232 tumor clonality and, 198, 225 animal tumors. 218 definition, 200, 201 distinction, 206, 207, 210 factors, 220-223 human tumors, 217 origin, 198, 199 pleoclonal tumors, 212, 213 Transgenic mice, chemical carcinogenesis and, 58-60 Translation, Epstein.Barr virus proteins and, 109, 142, 148

Translocation Epstein-Barr virus proteins and, 98 Louvain rat immunocytomas and, 306 breakpoints, 297-299 chromosome, 295-297 targets, 299-304 tumor clonality and, 208-210, 218 Transplacental carcinogenesis. newborn macrosomy and, 254-256 Transplantation Louvain rat immunocytomas and, 290, 292, 293

tumor clonality and, 199. 201, 217, 223 Transplantation antigens, tumor clonality and, 211, 215 Transposition, Louvain rat immunocytomas and, 304

Triated thymidine, gastrointestinal diseases and antigenic determinants, 19 esophagus, 2 large intestine, 9, 11, 12, 14, 16 stomach, 5 Trichoepithelioma. tumor clonality and, 216, 224

Triglycerides, newborn macrosomy and, 242 Triiodothyronine, newborn macrosomy and, 233

Trophoblasts, tumor clonality and, 206 Tropomysin, gastrointestinal diseases and, 19 Trypsin stroma and, 163 tumor clonality and, 206 Tryptophan, chemical carcinogenesis and, 29 Tuberculosis, newborn macrosomy and, 264 Tumor clonality, 197, 198. 224-226 animal tumors, 217-220 definition, 200, 201 distinction, 202, 203 cell surface antigens, 211 chimerism. 211 chromosomal markers, 208-210 immunoglobulins, 210 X-linked markers, 203-207 factors, 220-223 human tumors, 216, 217 origin, 198-200 pleoclonal tumors number, 212-215 spatial distribution, 215, 216 Tumor promoting agent chemical carcinogenesis and, 51, 54 Epstein-Barr virus proteins and, 149 gastrointestinal diseases and, 11, 14, 15 Tumorigenesis chemical carcinogenesis and biological concepts, 33 experimental models, 32 mechanisms, 42, 43, 53 Epstein-Barr virus proteins and, 104 Tumors chemical carcinogenesis and, 26, 60 biological concepts, 33-39 experimental models, 26-28, 31 mechanisms, 41, 42, 49-51 phorbol ester, 51-54 transgenic mice, 58. 59 Epstein-Barr virus proteins and, 150

329

INDEX diseases, 98 transformed cells, 116, 132 virus-produced cells, 138, 144 gastrointestinal diseases and antigenic determinants, 15, 17, 18, 22 large intestine, 8-10, 14, 15 stomach, 7 Louvain rat immunocytornas and, 279, 280, 305, 306 animals, 280 description, 283 etiology, 295, 297-302, 304, 305 immunoglobulins, 290, 291 incidence, 282, 283 storage, 294 in m l m cell culture, 293 in viuo transplantation, 292 newborn macrosomy and, 232, 233, 272 cancrophilia syndrome, 261, 263 epidemiology, 264 prognosis, 245, 250 retrospective data, 254, 255 oncogenes and, 73, 74, 89, 91 stroma and alterations, 183-185 basement membranes, 173-176 disorders, 179, 180, 182 epithelium, 160-166, 168 fibroblasts, 177, 178 glycoproteins, 170-172

U Ultraviolet light chemical carcinogenesis and, 38, 43 Epstein-Barr virus proteins and, 149 Urogastrone, gastrointestinal diseases and, 14 Urokinase plasminogen activator, stroma and, 162, 163

Uterine leiomyomas, tumor clonality and, 197, 216, 222 Uterus, newborn macrosomy and, 237

V Vaginal cancer, chemical carcinogenesisand, 30 Vagotomy, gastrointestinal diseases and, 4 Viral capsid antigen, Epstein-Barr virus proteins and diseases, 97- 99 transformed cells, 120 virus-produced cells, 139, 140, 147 Viruses, stroma and, 167, 185

W Warts, tumor clonality and, 221, 222 Wilms’ tumor newborn macrosomy and, 251 tumor clonality and, 208-210, 222, 225 Wound healing, stroma and, 186 disorders,n 179, 180, 182 glycoproteins, 170 Wounding, stroma and, 178-180, 182, 183

X Xeroderma pigmentosum, chemical carcinogenesis and, 43 X-linked lymphoproliferative syndrome, Epstein-Barr virus proteins and, 100, 136 X-linked markers, tumor clonality and, 224 alloenzymes, 203-205 assays, 205-207 distinction, 202, 203 pleoclonal tumors, 213 RFLP, 205


This page intentionally left blank

Advances in Cancer Research Volume 50

Advances in Botanical Research, Volume 50

Advances in Cancer Research

Advances in Cancer Research Volume 51

Advances in Cancer Research Volume 43

Advances in Cancer Research, Volume 91

Advances in Cancer Research Volume 42

Advances in Cancer Research Volume 41

Advances in Cancer Research Volume 78

Advances in Cancer Research, Volume 106

Advances in Cancer Research, Volume 84

Advances in Cancer Research, Volume 20

Advances in Cancer Research Volume 76

Advances in Cancer Research Volume 65

Advances in Cancer Research Volume 40

Advances in Cancer Research Volume 63

Advances in Cancer Research Volume 35

Advances in Cancer Research Volume 75

Advances in Cancer Research Volume 49

Advances in Cancer Research Volume 32

Advances in Cancer Research Volume 82

Advances in Cancer Research, Volume 57