Advances in Cancer Research Volume 48

ADVANCESINCANCERRESEARCH VOLUME 48

This Page Intentionally Left Blank

ADVANCES IN CANCERRESEARCH €dited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

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

Volume 48- 1987

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

Orlando San Diego New York Austin Boston London Sydney Tokyo Toronto

COPYRIGHT 0 1987 BY ACADEMIC PRESS. 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. Orlando, Florida 32887

United Kingdom Edition published by

ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road, London NWI 7DX

LIBRARY OF CONGRESS CATALOG CARD N U M B E R : 52-1 3360 ISBN 0-12-006648-3

(alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA

8 7 8 8 8 9 9 0

9 8 7 6 5 4 3 2 1

CONTENTS

Oncotrophoblast Gene Expression: Placental Alkaline Phosphatase WILLIAM

H.

FISHMAN

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

11. Oncotrophoblast Genes . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Induction of Alkaline Phosphatase in Cultured Cell

IV. Possible Mechanisms of Induction

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

VI. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 11 18 19 21 28 29

Cellular Events during Hepatocarcinogenesis in Rats and the Question of Premalignancy S. SELL,J. M. HUNT, B. J . KNOLL, A N D H. A. DUNSFORLI Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activation of Carcinogens and Initiation and Promotion in the Liver . . . . Markers for Cellular Lineage during Hepatocarcinogenesis . . Monoclonal Antibodies in Chemical Carcinogenesis . . . . . . . . . . . . . . . . . . Analysis of Phenotype of Carcinogen-Altered Cells by in Vioo Transplantation and in Vitro Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................. VI. Gen e Expression in Liver Carcinogenesi VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ......................

I. 11. 111. IV. V.

36 38 46 57 70 86 101 102

Human Papillomaviruses and Genital Cancer HERBERTPFISTEH

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Biology of Papillomaviruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. Human Papillomaviruses from Genital Tumors. . . . . . . . . . . . . . . . . . . . . . . IV. Characteristics of HPV-Induced Genital Lesions . . . . . . . . . . . . . . . . . . . .

.

V

113 114 122 124

vi

CONTENTS

V. Human Papillomaviruses in Cervical Cancer. ........................ VI. Speculations on an Etiologic Role in Carcinogenesis . . . . . . . . . . . . . . . . . . VII. Concluding Remarks., ........................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

131 135 141 142

Herpes Simplex Type 2 Virus and Cervical Neoplasia VLADIM~R VONKA, JIRf

KAfiKA AND

ZDENEK ROTH

I. Introduction. . . . . . . . . . . .................................. 149 11. Criteria for a Causal Rela between a Particular Virus and a Particular Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Nature of Association of HSV-2 with Cervical Neoplasia. . . . . . . . . . . 151 VI. Prague Prospective Study. . . . . . . . . VII. Houston Prospective Study .................................. VIII. General Discussion. . . . . . . . . . . . . . . . . . . .

182

Transforming Genes and Target Cells of Murine Spleen Focus-Forming Viruses WOLFRAM OSTERTAG, CABOLSTOCKING, GREGORY R. JOHNSON, NORBERT KLUGE, RECINEKOLLEK, THOMAS FRANZ,A N D NORBERT HESS 1. 11. 111. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Normal Hematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Etiology of Nonviral Myeloid Neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Biology of Murine Spleen Focus-Forming Viruses.. . . . . . . . . . Target Cells for Transformation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

193 194 218 243 304 323 329

357

ONCOTROPHOBLAST GENE EXPRESSION: PLACENTAL ALKALI N E PH0s PHATASE William H. Fishman Cancer Research Center. La Jolla Cancer Research Foundation, La Jolla. California 92037

1. Introduction

The discovery of the “Regan isoenzyme,” with the properties of placental alkaline phosphatase, in a patient (Regan) with terminal bronchogenic cancer (Fishman et al., 1968a) initiated my interest in oncotrophoblast gene expression. The finding was made possible then by an understanding of the biochemical, histochemical, and electrophoretic means of distinguishing alkaline phosphatases present in placenta, intestine, liver, and bone. Briefly, the patient’s primary and metastatic tumor tissues were enriched by an alkaline phosphatase which was heat stable, L-phenylalanine sensitive, and hydrolyzed by neuraminidase. Also, its electrophoretic mobility was identical to placental alkaline phosphatase (PLAP). The L-phenylalanine-sensitive enzyme was demonstrated histochemically in tumor cells of both the primary lesion and its metastases. In addition to lung cancer, the Regan isoenzyme has been identified in neoplasms of the testis, ovary, pancreas, breast, colon, lymph tissue, kidney, stomach, and bladder (Stolbach et al., 1969,1972; Belliveau et al., 1973; Cadeau et al., 1974; Fishman et al., 1975; Higashino et al., 1972; Jeppsson et al., 1984; Nathanson and Fishman, 1971; Usategui-Gomez et al., 1973; Uchida et al., 1981a). Two years later, the Nagao isoenzyme (Nakayama et al., 1970) was found in another lung cancer patient. It differed from PLAP by its slower migration on starch gel and its much greater inhibition by Lleucine. This isoenzyme is most frequently expressed in germ cell tumors and in ovarian cancer and serves as a useful tumor marker in patients with those tumors (Inglis et al., 1973; Lange et al., 1982; Epenetos et al., 1985). The first widely accepted instance of oncotrophoblast gene expression was the production of human chorionic gonadotropin (hCG) in patients with choriocarcinoma. In this tumor, the cancer cells overpro1 ADVANCES IN CANCER RESEARCH, VOL. 48

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

WILLIAM H . FISHMAN

duce hCG, the product of a gene active in trophoblast cells which are progenitors of choriocarcinoma. The amount of this trophoblast gene product in the urine of choriocarcinoma patients has served as an invaluable tumor marker to the medical oncologist in the diagnosis and management of the disease. In fact, the dramatic success of methotrexate as a chemotherapeutic agent in choriocarcinoma (Li et al., 1956) could not have been achieved without the use of the hCG marker. More recently, the placental protein SP1 (Tatarinov et al., 1974) was found elevated in the serum of patients with gestational trophoblastic disease. There is often a parallelism of SP1 with hCG in such patients, but discordant behavior has been observed (Seppala and Rutanen, 1982). Placental proteins as tumor markers have been reviewed by Stigbrand and Engvall (1982). Histaminase is another example of an oncotrophoblast enzyme. Its levels are elevated most frequently in effusions collected from ovarian cancer patients (87%) according to Lin et al. (1979). This has been followed by systematic studies aimed at isolating other oncotrophoblast proteins. At least five hitherto unrecognized placental proteins have been isolated, characterized, and assayed in a variety of clinical conditions (Bohn, 1983).A number show a positive correlation with cancer. A tumor calcium-binding protein, oncomodulin, has been reported to be synthesized b y placenta and parietal yolk sac and a wide variety of tumors, but not by normal fetal and adult tissues (Brewer and MacManus, 1985; MacManus et al., 1982). Accordingly, the phenomenon of oncotrophoblast gene expression in humans is well established. This system is attractive because oncotrophoblast expression can be interpreted in terms of the trophoblast nature of these particular gene products, particularly relevant being their migratory properties and replication propensity. Moreover, the availability of a number of human cancer cell lines expressing one or other trophoblast genes makes feasible interesting experiments on the significance of their expression. In this article, our purpose will be to provide a critical account of the past and current work in this field at the beginning of the gene cloning and sequencing era of PLAP and its related proteins. In particular, the discussion will focus on the place of oncotrophoblast gene expression in oncodevelopmental biology, the significance of PLAPlike enzyme in testis, seminoma, and other tumors, and the inferences which may be drawn from the PLAP-type enzymes regarding gene structure and regulation.

PLACENTAL ALKALINE PHOSPHATASE

3

II. Oncotrophoblast Genes

Oncotrophoblast genes represent a category of oncodevelopmental genes. The latter are defined as characteristic of certain defined stages in embryonic development, usually not appreciably expressed in adult tissue, but expressed inappropriately in cancer cells. Inappropriate expression includes both ectopic and eutopic types, terms introduced to distinguish between unexpected and expected expression. This distinction is becoming less and less real as ultrasensitive analytical methodology demonstrates low but significant expression of oncodevelopmental genes in a number of normal tissues. Oncodevelopmental gene products can also be identified with extraembryonic membranes, such as a-fetoprotein (AFP) in the yolk sac, or with structures of the postembryonic fetus, such as carcinoembryonic antigen (CEA) in intestine and AFP in fetal liver. Assuming that gene products which are located on the cell surface do control the destiny of the cell, it is clear that the repertoire of genes which gives the cancer cell the ability to divide rapidly and to migrate to new tissue sites and prosper there is the very same collection of genes which operate under regulatory and differentiation controls of the conceptus. Cell transformation can therefore be regarded as the inappropriate unregulated expression of certain embryonic, extraembryonic, and other genes. Some of these genes may have undergone point mutation (Santos et al. 1984). In this regard, some oncodevelopmental genes include the socalled c-oncogenes or protooncogenes. For example, c-fos is expressed in extraembryonic membranes (especially placenta) of the mouse embryo and in certain tumors, according to Adamson et al. (1983). Also, using polyclonal and monoclonal antibodies prepared against synthetic polypeptides representing highly conserved regions of the protein products of sis, ras, and fes oncogenes, Niman et al. (1985) detected oncogene-related proteins in the urine of cancer patients and pregnant women in greater than normal amounts. Examples such as these draw attention to the importance of defining the role of protooncogenes in normal development in order to understand what truly may be their role as oncogenes in transformation. Accordingly, oncotrophoblast genes may be found to share properties of oncogenes, since trophoblast cells are responsible for performing the implantation of conceptus in the uterine endometrium, involving a process of cell migration, angiogenesis, and replication. In later pregnancy, one frequently observes trophoblast cells disseminated

4

WILLIAM H. FISHMAN

through the body, especially in the lung, which fortunately usually disappear after parturition (Douglas et al., 1959; Attwood and Park, 1961).Also relevant is the report of Log et al. (1981) that carcinomas in several strains of mice were induced by inoculating mouse trophoblast cells that had been obtained from successful culturing of trophoblast cell lines. Clearly, there is reason to look for the explanation of tumor behavior in the expression of trophoblast genes. A. EVOLUTION OF PLAP

The hypothetical genealogy of PLAP is unique, and so far no counterpart has been described for other mammalian enzymes. As illustrated in Fig. 1, three gene loci have been recognized which have a degree of organ specificity: placental, intestinal, and tissue-unspecific type. Thus, it is thought that through the process of gene duplication and mutation, the term-trophoblast alkaline phosphatase (AP) genes derives from an intermediate intestinal gene which, in turn, originated from a tissue-unspecific AP gene. The term-trophoblast AP genes may give rise to two separate genes: the PLAP gene with its known multialleles, and the PLAP-like gene with its presumed variants as suggested from immunological studies (Millan and Stigbrand, 1983). The neoplastic counterpart to intestinal alkaline phosphatase has been reported as “variant” by Warnock and Reisman (1969) and as the “Kasahara” isoenzyme by Higashino et al. (1972). During the evoluANCESTRAL GENE

I

7 TISSUE-UNSPECIFIC PRECURSOR GENE

INTERMEDIATE INTESTINAL AP GENE

I

TERM-TROPHOBLAST AP GENES

1 3

TISSUE-UNSPECIFIC

INTESTINAL

AP GENE

1

AP GENE

TISSUE.UNSPECIFIC AP (LIVER. BONE. EARLY PLACENTA) AP

1

INTESTINAL AP IAP

PLAP

PLAP-LIKE

GTE “5“‘

ALLELIC VARIANTS OF PLACENTAL AP PLAP

FIG.1. Hypothetical genealogy of PLAP.

VARIANTS OF PLAP-LIKE AP PLAP-LIKE AP

PLACENTAL ALKALINE PHOSPHATASE

5

tion of these isoenzymes, the catalytic site domain has been conserved, as shown by the work of Whitaker and Moss (1979). They found identical amino acid sequences at the active site both for bacterial and mammalian AP upon analysis of radiochemically labeled peptides derived from proteolysis of the native proteins. The noncatalytic site domains, however, do show variability, particularly at the PLAP locus which the multiple allelic forms (Beckman and Beckman, 1969; Robson and Harris, 1965) suggest. The PLAP gene is unique in the fact that it produces at least 18 alleles which represent 2.5% of the total gene product, the rest being accounted for by six common phenotypes. No other enzyme has yet been described with this degree of phenotypic diversity. Other cases may be found through studies of nonprimate, primate, and human species. There is agreement that term PLAP is a product of a gene which appeared late in evolution and that the tissue-unspecific gene has been operating from earliest evolutionary times to the present. Manning et al. (1970) demonstrated that animal species lower than the human lacked PLAP in their placentas, but the latter tissues were endowed with tissue-unspecific enzyme. This finding was true for the lower apes, the African green monkey, rhesus monkey, and baboon (Manning et al., 1969). However, in the chimpanzee and orangutan placentas, but not in the spider monkey or the lowland gorilla (Doellgast and Benirschke, 1979; Goldstein and Harris, 1979), the placental alkaline phosphatase shared immunological determinants and biochemical properties with PLAP. This similarity was observed also in baboon lung and several Old World monkeys (Chang et al., 1979; Harris, 1982). The biological rule which states that “ontogeny repeats phylogeny” would appear to apply when one studies PLAP in relation to normal development. During the first trimester, the placenta essentially expresses the tissue-unspecific form of AP and not PLAP, but in the second and third trimesters, PLAP predominates. This demonstration of developmental phase-specific expression by L. Fishman et al. (1976),confirmed by Sakiyama et al. (1979,1980),does provide a basis for interpreting oncotrophoblast expression (see below).

B. THEPLAP GENE According to Millan (1986), the PLAP gene has been cloned and sequenced. This accomplishment was achieved by screening a bacteriophage hgtll human S-phenotypic placental cDNA library with poly-

6

WILLIAM H. FISHMAN

clonal antibodies against CNBr fragments of the PLAP protein. The complete amino acid sequence was inferred from the cDNA, and it is 513 amino acids long. The precursor protein exhibits a 21 amino acid hydrophobic signal peptide preceding the NH2-terminal amino acid of mature PLAP. The carboxy terminus of the protein represents a highly hydrophobic membrane anchoring domain ending at the bromelain cleavage site. The presumed glycosylation sites are identified at Asn128 and Asn-262. The amino acid sequence is consistent with prior observations on PLAP protein listed below. The sequence of the first 40 NH2-terminal amino acids of PLAP has been established (Sussman, 1984; Ezra et al., 1983). They can be split off in a 10,000 kDa fragment by trypsin (Jemmerson et al., 1984a). The C-terminal sequence, on the other hand, is released in a 2000 Da fragment by bromelain proteolysis (Kottel and Hanford, 1980; Neuwald and Brooks, 1981) and by subtilisin (Abu-Hasan and Sutcliffe, 1984). After trypsin and bromelain treatment, the major 55 kDa component is resistant to further proteolysis unless it is denatured beforehand (Jemmerson et al., 1984a). It contains the catalytic site. Thus, the two ends of the PLAP molecule can be recovered after specific proteolysis. AND ITS MEASUREMENT C. THEGENEPRODUCT

The cell surface membrane of cancer cells has been found to be the location of PLAP enzyme in the electron microscope studies of many workers (Sasaki and Fishman, 1973; Lin et al., 1976; Miyayama et al., 1976, 1983; Jemmerson et al., 198513).More recently, in addition to the cell surface membrane, enzyme-positive cytoplasmic sites such as endoplasmic reticulum, Golgi apparatus, mitochondria1 membranes, and vesicles have been demonstrated with the introduction of saponin into the reaction medium and b y prior blocking (Tokumitsu et al., 1981a) of the plasma membrane PLAP with specific antibody. The membrane location of PLAP was expected from the observation that organic solvents (butanol) or detergents were necessary to free the enzyme. The conformation of the PLAP molecule has been visualized (Fig. 2) at the electron microscope level by the use of rotary shadowing and negative staining techniques (Takeya e t al., 1984). One can clearly recognize a rectangularly shaped molecule with a central space, conceivably separating the monomers. Its dimensions are 7.5 x 5.5 nm. Also, using gold-labeled antibody technique, clusters of PLAP can be seen (Jemmerson et al., 1985a) on the surface of microvilli of tumor cells and syncytiotrophoblast cells (Fig. 3).

PLACENTAL ALKALINE PHOSPHATASE

7

FIG.2. Appearance of human PLAP as visualized by rotary-shadowing technique (left) and by a negative staining procedure (right). Reproduced with permission from Takeya et u1. (1984).

FIG.3. Clustering of PLAP on villi of human syncytiotrophoblast (left) and cancer cells (A-431) (right). Reproduced with permission from Jemmerson et al. (1985a).

8

WILLIAM H. FISHMAN

The spatial arrangements of the antigenic sites can be approached by a combination of monoclonal antibody binding and proteolysis. Thus, each of two different sets of monoclonal antibodies, which recognizes different epitopes, blocks proteolysis by trypsin and bromelain (Jemmerson et al., 1984a, 1985a).Two of the monoclonal antibodies exhibit overlapping proteolysis-blocking effects. These results would fit a picture of the amino and the carboxyl ends of the polypeptide chains being positioned close to each other at the cell surface. Such a view has been proposed for Escherichia coli alkaline phosphatase by Wyckoff et al. (1983). Finally, Takeya et al. (1986) have proposed from their immunoelectron microscope studies on the binding of two common alleles of PLAP with monoclonal antibodies with allelic specificity that the two subunits of PLAP are arranged countersymmetrically. Placental alkaline phosphatases represent the dimeric glycoprotein products of multiple alleles of one of three AP genes, the others being intestinal and tissue-unspecific types (see Section 11,A). Greene and Sussman (1973) found no differences between PLAP and the Regan isoenzyme in their NHz-terminal amino acid sequences, subunit molecular weights, and two-dimensional peptide maps. Sussman (1984) has recently summarized a structural analysis of human alkaline phosphatases, while Jemmerson et al. (1984a) reported on the functional organization of the PLAP polypeptide chain. Finally, progress has been made in the methodology of measuring PLAP and PLAP-like alkaline phosphatase. Assays can be grouped under four categories: catalytic assays (Fishman and Green, 1967; Fishman et al., 1968b; Green et al., 1971; Anstiss et al., 1971; Haije et al., 1979), electrophoretic separations (Inglis et al., 1971; Forman et al., 1976), immunoassays (Sussman et al., 1968; Usategui-Gomez et al., 1973; Lehmann, 1975; Doellgast, 1977),and radioimmunoassays (Iino et al., 1972; Jacoby and Bagshawe, 1972; Chang et al., 1975; Holmgren et al., 1978). Currently, sandwich ELISA (Millan and Stigbrand, 1981) techniques have proved useful in longitudinal studies of patients with seminoma (Lange et al., 1982). Similarly, a sensitive radioimmunoassay introduced by Nustad et al. (1984) has been applied to the evaluation of PLAP in pre- and postoperative sera from the Danish testicular cancer study. In recent years, advantage has been taken of the exquisite specificity of monoclonal antibodies to PLAP (Slaughter et al., 1981; Millan et al., 1982a; Millan and Stigbrand, 1983; Jemmerson et al., 198513) and to PLAP and PLAP-like isozymes (Millan et al., 1985) to fashion methodologies. These are being applied to studies mainly of testicular

9

PLACENTAL ALKALINE PHOSPHATASE

and ovarian cancer by McLaughlin et al. (1983), DeGroote et al. (1983), Horwich et al. (1985), Tucker et al. (1985), Epenetos et al. (1985), Eerdekens et al. (1985), and Van de Voorde et al. (1985). OF AP ISOZYMES D. PROPERTIES

The properties of the accepted forms of alkaline phosphatase in various cancer cell lines are summarized in Tables I and 11. These have been reviewed by Fishman (1974), Stigbrand et al. (1982), and Nozawa and Fishman (1982). Briefly, tissue-unspecific AP (which in tumors was termed non-Regan) is completely distinct from intestinal alkaline phosphatase (IAP), PLAP, and PLAP-like AP because of its heat lability, L-homoarginine sensitivity, and its specific reaction with polyclonal antisera to liver AP. On the other hand, both PLAP and PLAP-like isozymes have great heat stability and react preferentially with antisera to placental alkaline phosphatase. Although fetal intestine (Kasahara isozyme), TABLE I CLASSES AND PROPERTIES OF TUMOR-ASSOCIATED ALKALINEPHOSPHATASES Isozyme/developmental counterpart

Properties Molecular weight (subunit) N-Terminal sequence pH optima Heat stability 5WC, 30 min 65"C, 5 m i n Amino acid sensitivity L-phenylalanine (5mM) L-Homoarginine (5 mM) L-Leucine (5 mM) Electrophoretic migration Neuraminidase sensitivity Reaction with antisera to Liver AP Intestinal AP Placental AP ~~

ND, Not determined.

Tissueunspecific AP (non-Regan)/ early fetal placenta

Intestinallike AP (&Sahara)/ fetal intestine

Placental AP (Regan)/term placenta

Placentallike AP (Nagao)/term placenta, testis

ND" ND 10.1

ND ND 10.1

64,000 Ile-Ile-Pro 10.6

65,000 ND 10.6

-

+

-

-

+++ +++

+++ +++

+ +++ + Fast +

+++ 5 ++ Fast +

+++ 2 + Intermediate +

+++ * +++ Intermediate +

-

-

-

++

+ +++

+ +++

+ -

+

10

WILLIAM H. FISHMAN

TABLE I1 N H z - T SEQUENCE ~ ~ ~ AND ~ ~MOLECULAR ~ PROPERTIES OF SEVERAL ENZYME TYPES' Enzyme source (reference) Liver (Badger and Sussman, 1976) Intestine (Kamoda et al., 1981) Placenta (Badger and Sussman, 1976; Behrens et al., 1983) Lung tumor (Behrens et al., 1983) KB cell heteromer (Luduena and Sussman, 1976)

NHz-terminal sequence

M , subunit

Leu-Val-Phe Phe-Ile-Pro Ile-Ile-Pro-Val

69,000 68,000 64,000

Ile-Ile-Pro-Val Ile-Ile-Pro (PLAP) Phe-Ile-Pro (fetal IAP)

64,000 64,000 72,000

From Sussman (1984).

PLAP, and PLAP-like enzyme are equally inhibited by L-phenylalanine, fetal intestinal AP is completely heat labile (5 min at 65°C) and is preferentially bound by antisera to intestinal AP. It should be noted that adult intestine alkaline phosphatase is not cleaved by neuraminidase treatment, unlike the other organ-specific and -unspecific AP isozymes.

E. BIOSYNTHESIS OF PLAP Ito and Chou (1983) demonstrated three consecutive stages in the biosynthesis of PLAP, represented by a 60,000 Da preprotein, a 61,500 Da glycopolypeptide, and a 64,500 Da PLAP monomer, by pulse-chase labeling experiments with intact JEG-3 choriocarcinoma cells. ~ - [ ~ ~ S ] m e t h i o n iwas n e the label for the polypeptide, and [3H]glucosamine and [3H]mannose were the labels for the carbohydrate moiety of the glycopeptides. Interestingly, in the longer period of labeling (1 hr) leading to the 64,500 Da product, it was mainly the incorporation of [3H]glucosamine both as glucosamine and as N-acetylneuraminic acid which accounted for the increase in molecular weight. The 64,500 Da product, but not the 61,500 one, was cleaved by neuraminidase. In the presence of the protein glycosylation inhibitor tunicamycin, choriocarcinoma cells produce the PLAP monomer without carbohydrate units, a 58,000 Da polypeptide. However, the product synthesized by cell-free extracts was a 60,000 Da polypeptide believed by Ito and Chou (1983) to be a preprotein with a 2000 Da signal peptide. This PLAP preprotein is incorporated into microsomal vesicles with the subsequent events of signal peptide cleavage and core glycosylation taking place.

PLACENTAL ALKALINE PHOSPHATASE

11

Some experimental evidence has provided an insight into the dynamics of PLAP transport from its biogenesis in the endoplasmic reticulum to its terminal in the plasma membrane. Thus, using HeLa TCRC-1 cells, Hanford and Fishman (1983) demonstrated that the transit time of a molecule of PLAP from its first [35S]methionine labeling in the endoplasmic reticulum until its incorporation into the cellsurface membrane is 60 min. Also, Tokumitsu and Fishman (1983), by employing the electron microscope to study the labeling as a function of time, demonstrated that cycloheximide arrested the entire process of transport in 10 min. For example, at 10 min, activity disappeared completely from the endoplasmic reticulum and was less intense in the Golgi apparatus. Ill. Induction of Alkaline Phosphatase in Cultured Cells

Much of our present knowledge of gene regulation and gene expression originated from Jacob and Monod’s pioneer studies of enzyme induction in microorganisms. The hope has been expressed that similar advances will come from work on enzyme induction in eukaryotes. One such system of current interest is the induction of alkaline phosphatase in cultured cells. As early as 1961, it was known that corticosteroids added to the medium of HeLa cells increased alkaline phosphatase activity (Cox and McLeod, 1961).RNA and protein synthesis were required (Griffin and Cox, 1966), but apparently not that of the alkaline phosphatase protein. The mechanism envisioned then was that of a key substance which improved substrate binding to Zn, leading to increased catalytic activity. Later, the degree of phosphorylation of the enzyme protein was believed to control the binding of Zn at the active site (Cox et aZ., 1975). This view was accepted without contest until 1974 when several immunological methods (Singer and Fishman, 1975; Hamilton and Sussman, 1981; Hanford et aZ., 1981; Ito and Chou, 1983) demonstrated unequivocally that the enhanced enzyme activity was due to the de nouo biosynthesis of alkaline phosphatase protein.

A. RECOGNITION OF ISOENZYMES AS SICNI~~CANT COMPONENTS IN THE AP INDUCTION Nitowsky and Herz (1963) distinguished between heat-stable and heat-labile forms of alkaline phosphatase in their studies of hormonal regulation of alkaline phosphatase in cultured cells. However, that

12

WILLIAM H. FISHMAN

this distinction resided in two different isozymes became clear when it was found that cultured cell lines produced more than one isozyme of alkaline phosphatase. Thus, Singer and Fishman (1974) characterized two monophenotypic sublines of HeLa, one producing PLAP (Regan isoenzyme) and the other tissue-unspecific enzyme (non-Regan). These differences were manifested in the degree of sensitivity to L-phenylalanine, L-homoarginine, and heat as demonstrated on isozyme bands separated by gel electrophoresis. Of great value was the specificity of interaction with polyclonal antibodies as viewed by antigen retardation on polyacrylamide gel electrophoresis. Although the intestinal enzyme is inhibited by L-phenylalanine, its thermolability, slow migration in gel electrophoresis, and resistance to neuraminidase action were the first set of conditions (Fishman et al., 1968b) for recognizing intestinal alkaline phosphatase. As will be described, the ability to measure each AP isozyme in the presence of the others has made possible an accurate evaluation of the isozyme component(s) produced by cells in culture.

B. DEPENDENCE OF PLAP INDUCTION ON CELLDENSITY AND CELLCYCLE PLAP in cancer cell lines exhibits a growth-dependent induction which is maximal after 6-10 days of culture when cells have reached the stationary phase. This phenomenon is not restricted to PLAPproducing cancer cells, but is seen in normal fibroblasts and is absent from the intestinal isozyme-producing cell line, HT-20. That enzyme protein synthesis takes place in autoinduced fibroblasts is suggested by cycloheximide-blocking studies of Maziere et al. (1977). The autoinduction could be due to cell :cell interaction, but not to enzyme leakage, changes in morphology, or the technique of subculture, according to Miedema (1968). Singer and Fishman (1976) explained the divergent results of Griffin and Ber (1969) (induction is restricted to S phase) and of Melnykovych et al. (1967) (induction is related to early GI) on the basis of a two-step mechanism. The first step of induction is dependent on DNA synthesis, whereas the second step is a function of the sequence of transcription and translation occurring in the G1 period. Thus, cell lines of low levels of constitutive alkaline phosphatase such as those employed by Griffin and Ber undergo both steps, whereas cell lines possessing high constitutive levels such as HeLa TCRC-1 experience only the second step. The results of Singer and Fishman (1976) provide direct evidence for an arrest in G1 by prednisolone thus explain-

PLACENTAL ALKALINE PHOSPHATASE

13

ing the elongation of GI of Kollmorgan and Griffin (1969). Xue and Rao (1981) also reported that sodium butyrate blocks PLAP induction in HeLa cells preferentially in early GI phase of the HeLa cell cycle. A parallel exists between oncotrophoblast and oncofetal gene expression and the events of the cell cycle. Thus, Tsukada and Hirai (1975), using the AH66 hepatoma cell line, and Sell et al. (1975), employing fetal rat hepatocytes, reported that a-fetoprotein production is initiated in late GI. Also, prednisolone was found to stimulate a-fetoprotein synthesis in hepatocytes in GI, according to Belanger et al. (1975). C. GLUCOCORTICOID INDUCTION OF ALKALINEPHOSPHATASE From 1961 on, HeLa cell lines have been continually studied from the point of view of glucocorticoid induction (Cox and MacLeod, 1961; Melnykovych, 1962; Nitowsky and Herz, 1963; Fishman, 1969; Ghosh et al., 1972; Singer and Fishman, 1974; Lalitha and Nagarajan, 1977). All HeLa cell lines are not equivalent in their hormone response, as demonstrated by the monophenotypic expression of HeLa TCRC-1 (Regan isoenzyme) and HeLa TCRC-2 (tissue-unspecific isoenzyme) in the early studies of Kottel and Fishman (1978). It now appears that the isozyme species induced by glucocorticoid is not predictable. Thus, although many reports (reviewed by Nozawa and Fishman, 1982) have indicated that in most cell lines glucocorticoid steroids induce PLAP, a number of cell lines monophenotypic for tissue-unspecific alkaline phosphatase-HeLa TCRC-2 (Singer and Fishman, 1974), KMK-2 (Tokumitsu et al., 1979), SW-620 (Herz and Halwer, 1983), and intestinal alkaline phosphatase, HT-29 (Herz et al., 1981)-were not induced by prednisolone. On the other hand, the tissue-unspecific alkaline phosphatase of newly established cultures of human brain tumors increased markedly in the presence of prednisolone in the medium (Takahara et al., 1982). The lack of PLAP response to glucocorticoid induction may be explained. For example, the failure of the PLAP-like enzyme of BeWo cells to be induced by steroid was due to the absence of corticosteroid receptors on the cell surface, according to Speeg and Harrison (1979). Singer and Fishman (1975) observed that during the prednisolone induction of PLAP, there was a simultaneous reduction in the expression of intestinal-type AP. This was true of F1-amnion and Hep-2 cell lines; both of which express fetal intestine (oncoamnion) AP (Honda et al., 1973; Fishman and Singer, 1976; Higashino et al., 1975). In addition, there was evidence of a distinct electrophoretic band which

14

WILLIAM H. FISHMAN

shared both PLAP and intestinal AP properties (antigenic and biochemical). On the other hand, HeLa D98AHz which expresses only the intestinal AP, continues to do so to a lesser extent in the presence of prednisolone, but surprisingly generates PLAP as well, according to Kottel and Fishman (1978) and Hanford et al. (1951). Finally, additional evidence for the existence of interlocus heteromeric intestinal and placental isozymes has been reported by Wray and Harris (1982). Using the electrophoretic technique of enzyme-antibody complex retardation, they found that in the Hep2/5 cell line, two out of the four isozymes separated by polyacrylamide gel electrophoresis were retarded by both PLAP and intestinal AP-specific monoclonal antibodies. As one now accepts the view that separate genes code for placental and intestinal AP, it seems possible from their inverse responses to prednisolone that the two genes may be located close to each other. An analogous situation exists in the case of the albumin and AFP genes which bear a tandem relationship. Early development causes a turning off of the dominant AFP gene in fetal liver, while the albumin gene is amplified later in postfetal life (Koga and Tamaoki, 1974). The difference is that albumin and AFP are immunologically distinct, although exhibiting significant homology, whereas placental and intestinal isozymes share several antigenic determinants. With regard to the specificity of the steroid hormone itself, it was established earlier by Melnykovych (1962) that prednisolone and dexamethasone were the most effective of the hydrocortisone series, while androgens, estrogens, and estradiol were altogether ineffective. However, other steroids such as progesterone act as antiinducers, blocking cortisol induction (Cox, 1971). D. BUTYRATEINDUCTION Differences have been observed in the enzyme histochemical appearance of prednisolone and butyrate-induced cells; the prednisolone-treated cells show rather uniform staining, whereas in the case of butyrate-induced cells, only a minor proportion exhibit intense staining (Tokumitsu, 1984). Griffin et al. (1974) reported butyrate as an inducer of PLAP in HeLa cells, and a number of other workers have since expanded knowledge of this phenomenon to other cell lines, including Deutsch et al. (1977), Chou (1979), Littlefield et al. (1980), Cox (1981), Herz et al. (1981), and Hanford and Fishman (1983). Unlike those of prednisolone, butyrate effects can be variable and even unpredictable, yet, in many instances, the two inducers modulate the same isoenzymes in HeLa cells. In two non-HeLa cervical

PLACENTAL ALKALINE PHOSPHATASE

15

cancer cell lines (C41 and DOT),butyrate is ineffective with regard to PLAP induction (Brahmacupta and Melnykovych, 1980; Herz et al., 1981; Kottel and Fishman, 1981).Again, unlike prednisolone, butyrate induces the intestinal isozyme of the HT-29 cell line derived from colon carcinoma (Herz et al., 1981). In the HRT-18 rectal cancer cell line, Morita et al. (1982) report that butyrate causes an enhancement of PLAP with a simultaneous fall in the tissue-unspecific AP. Also, the latter isozyme is induced 15-fold by butyrate, while PLAP increases twofold in the uterine cervical cancer cell line SKG-IIIa, according to Nozawa et al. (1983).Often, the butyrate will induce tissue-unspecific isozyme as well as PLAP. Some characteristics of the butyrate induction include its reversibility (Herz et al., 1981) and the variability of the induction period (Deutsch et al., 1977; Chou, 1979; Littlefield et al., 1980; Tokumitsu et al., 1981a). Also, like prednisolone and hyperosmolarity, butyrate arrests cells in the GI phase of the cell cyle (Xue and Rao, 1981). It is believed that the butyrate phenomenon is manifested by a combination of inhibition of phosphorylation and hyperacetylation of histones (Riggs et al., 1977; Candido et al., 1978) and alterations in chromatin structure (Prasad, 1980; Fallon and Cox, 1981). Xue and Rao (1981) propose that the two mechanisms are not necessarily dependent on each other and report a butyrate-enhanced protein which may regulate the transition of early GI cells to the next phases of the cell cycle.

E. INDUCTION BY HYPEROSMOLAR GROWTHCONDITIONS Nitowsky and Herz (1963)first observed that supplementing growing control HeLa S3 cells in culture with sodium sulfate resulted in an increase in the specific activity of alkaline phosphatase. It was then demonstrated that this effect was due to an osmotic rather than an ionic mechanism. The isozyme specificity of hyperosmolar induction is interesting. For most human cancer cell lines, it is PLAP which is induced while the tissue-unspecific isozyme does not undergo induction. Moreover, different mechanisms appear to account for corticosteroid versus hyperosmolar induction. These considerations have been reviewed in detail by Herz (1984).

F. INDIVIDUALITY OF INDUCTION MECHANISMS As Herz has pointed out, each inducer exerts an independent effect on the expression of individual isozymes, and these effects can be additive or synergistic, depending on the circumstances. Thus, the

16

WILLIAM H. FISHMAN

three inducers (glucocorticoids, hyperosmolarity, butyrate) produce additive effects in HeLa S3 cells (Herz et al., 1981), whereas butyrate and hyperosmolarity together increase specific activity 1000-fold or more in HT-29 cells. With regard to glucocorticoids and hyperosmolarity, an additive result is evident in HeLa cervical cancer cells (Nitowsky and Herz, 1963), whereas in bladder tumor cells, a synergistic effect is observed (Herz and KOSS,1979). G. CYCLICAMP INDUCTION OF ALKALINEPHOSPHATASE The best documented case of cyclic adenosine monophosphate (CAMP) induction was observed by Chou and Robinson (1977) with BeWo choriocarcinoma cells; PLAP but not tissue-unspecific enzyme was overproduced, according to Hamilton et al. (1979). Previous studies on a hybrid hamster-mouse cell line did not permit a conclusion as to whether the cAMP or the butyrate released from the dibutyryl derivative was the effective inducing agent (Koyama et al., 1972). Firestone and Heath (1981) demonstrated the production of alkaline phosphatase-specific mRNA by cAMP and that the expression of enzyme activity and normal intercalation into the plasma membrane was dependent on enzyme protein glycosylation.

H. HALOGENATED NUCLEOSIDE AND DNA SYNTHESIS INHIBITOR INDUCTION Koyama and Ono (1971) first observed that bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR) increased alkaline phosphatase activity in a mouse-Chinese hamster hybrid cell line. Similar results were reported in choriocarcinoma and HeLa cell lines by Goz (1974), Edlow et al. (1975), Bulmer et al. (1976), Chou and Robinson (1977), Speeg et al. (1978), and Hamilton et al. (1979). Both halogenated nucleosides are not equivalent in the effects they produce in choriocarcinoma cells. Thus, IUdR induces both PLAP and tissue-unspecific isozyme (Speeg et al., 1978), whereas BUdR induces PLAP only (Hamilton et al., 1979). PLAP in HeLa and choriocarcinoma cells can be increased by a number of DNA synthesis inhibitors such as mitomycin C, methotrexate, lp-D-arabinofuranosylcytosine, phleomycin, and hydroxyurea (Bulmer et al., 1976; Chou and Robinson, 1977; Chou, 1979; Speeg et al., 1977; Deutsch et al., 1977). Since other agents which counter cell proliferation do not increase activity, Chou and Robinson (1977) and Herz (1984) suggest that these DNA biosynthesis inhibitors alter chromatin structure rather than stopping DNA synthesis.

PLACENTAL ALKALINE PHOSPHATASE

I.

INDUCTION OF

17

“FIRST TRIMESTER” PLACENTAL

ALKALINE PHOSPHATASE

The developmental counterpart to a heat-sensitive, L-phenylalanine-insensitive alkaline phosphatase occurring in lung cancer, meningioma, craniopharyngiomas, and pancreatic cancer was a mystery for many years after the discovery of the Regan isoenzyme (Fishman, 1974). L. Fishman et al. (1976) reported the existence of an alkaline phosphatase with these properties which predominated in chorionic tissue from 8- to 12-week gestation. On the other hand, the termplacental alkaline phosphatase became increasingly evident in placentas from 12- to 40-week gestation. This developmentally controlled switch in two isozyme forms has its counterpart in the expression of one or both tissue-unspecific and PLAP isozymes in cancer cells, as described by Fishman et al. (1975). The first trimester isozyme was purified by Sakiyama et al. (1979) and was shown to resemble human liver AP in immunological and amino acid inhibition properties, but exhibited a different electrophoretic mobility on nondenaturing polyacrylamide gels. Chou (1978) introduced TsA mutants of SV40 into the genome of normal placental cells and reported these transformed cells were temperature sensitive for growth and differentiation. At 40°C, the TsAtransformed cells assume a normal phenotype and express significant amounts of first trimester isozyme and hCG, whereas at 33”C, the cells show the transformed phenotype and low activity. Apparently, the incorporation of the virus into the genome interferes with the expression of the PLAP gene, which is apparently turned off completely, or perhaps only the first trimester placental cells surviving in the mature placenta integrated the virus and then overgrew the original termplacental cells. The properties of the enzyme purified from the TPA30-1 cell line were found identical to first trimester AP by Sakiyama et al. (1980). Most interesting is the fact that this cell line undergoes induction of its first trimester AP by dexamethasone, whereas choriocarcinoma PLAP was not affected by the glucocorticoid (Chou and Ito, 1984; Speeg and Harrison, 1979). J. INDUCTION BEHAVIOR OF GASTROINTESTINAL CANCER CELLLINES A number of alkaline phosphatase isozyme-producing human gastrointestinal and pancreatic cell lines are known. Thus, PLAP was detected in a human ileocecal carcinoma cell line (HCT-8) by Singer et aZ. (1976). Later, Tsao et al. (1982) found a rectal adenocarcinoma cell line (HRT-18) with a similar PLAP isoenzyme, but exhibiting an

18

WILLIAM H. FISHMAN

unusual heat-labile isoenzyme. The latter contains antigenic determinants which are absent from PLAP and tissue-unspecific isoenzymes. Of the cell lines studied by Benham et al. (1981), HT-29 expressed 25% of its total AP as PLAP, a figure higher than one reported by Herz et al. (1981). Also, a Kasahara isoenzyme was found to be produced by a salivary gland tumor cell line (OKK) by Tanaka et al. (1983).A cell line which expresses only tissue-unspecific AP is the KMK-2 gastric carcinoma cell line of Tokumitsu et al. (1979). It should be noted that unlike the PLAP gene, intestinal AP gene does not produce allelic variants. Amnion cells produce an AP which corresponds to fetal intestine (Honda et al., 1973; Singer and Fishman, 1976; Higashino et al., 1975). The experiments (Singer, 1976) which manipulate the HeLa cell expression of this enzyme in immunosuppressed rats have a more plausible interpretation if the isoenzyme produced is viewed as an amnion rather than an intestinal gene product. In these cell lines, one is dealing often with the intestinal alkaline phosphatase, PLAP, and tissue-unspecific AP in respone to induction (refer also to Kam et al., 1984). Of interest is the fact that intestinaltype alkaline phosphatase (Kasahara isozyme) was produced by human hepatoblastoma cell line HVH-6 clone 5, according to Yamamoto et al., 1984. IV. Possible Mechanisms of Induction

The evidence to date suggests that in addition to the effects on cell cycle discussed previously, chromatin alterations per se may be a relevant mechanism. Thus, Fallon and Cox (1981), using the techniques of premature chromosome condensation and quinacrine dihydrochloride fluorescence, demonstrated that the time course of butyrate-mediated hCG synthesis correlates well with the extent of chromatin decondensation in synchronized HeLa cells. That chromatin structures and functions may be altered by butyrate is an opinion also expressed by Ito and Chou (1983).Their studies demonstrate that butyrate operates at the transcriptional level in inducing the production of PLAP in choriocarcinoma cell lines. Whatever the mechanism of butyrate induction, it has a number of phenomena to explain. Thus, morphological changes in butyrate-induced HeLa cells have been reported by Deutsch et al. (1976) such as increased number of desmosomes and tonofilament bundles. These alterations are accompanied by increased biosynthesis of hCG (Ghosh et al., 1977), free a chains for glycopeptide hormones (Lieblich et al.,

PLACENTAL ALKALINE PHOSPHATASE

19

1977), and FSH (Ghosh and Cox, 1977). Also, in addition to the Regan isoenzyme, PLAP and 5’-nucleotidase (Deutsch et al., 1976) and sialyltransferase 1 (P. H. Fishman et al., 1974) are other cell membraneassociated enzymes induced by butyrate. V. Oncotrophoblast Gene Expression in Normal Nontrophoblast Tissues

A. TESTIS

The sensitive technique of microzone cellulose acetate electrophoresis coupled with the use of fluorescent substrates and tissue-specific alkaline phosphatase antibodies demonstrated a heat-stable, L-phenylalanine-sensitive isozyme resembling PLAP in normal human testis (Fishman and Singer, 1976).The suggestion was made then that testicular teratocarcinoma may be expressing testicular AP genes, and these may be operating in placenta. Later, Chang et al. (1978, 1980) concluded that the testis heat-stable enzyme shared the L-leucine inhibition property with the rare placental D variant and the Nagao isoenzyme. Confirmatory studies were published by others (Goldstein et al., 1982; Millan et al., 1982a). The testicular tumor with the greatest probability of expression of PLAP-like enzyme is seminoma, according to Fishman et al. (1979), Wahren et al. (1979), Uchida et al. (1981b), Lange et al. (1982), and Jeppsson et al. (1984). In addition to PLAP, the testis produces at least four other trophoblast proteins, hCG, SP1, hPL, and PP5, according to Chard (1982). B.

OVAHY

The presence of PLAP in normal ovary was detected by heat stability and L-phenylalanine sensitivity measurements (Benham et al., 1978; Doellgast and Homesley, 1984) and with monoclonal antibodies (McLaughlin and Johnson, 1984; Nouwen et al., 1985). The Nouwen et al. study demonstrated PLAP in germinal inclusion cysts (Fig. 4) of normal ovary, and these authors proposed that such cysts may represent the origin of serous ovarian tumors and of PLAP-positive endometrioid carcinoma. The observations of Sasaki and Fishman (1973) and Fishman et al. (1975) demonstrated the enrichment of the ovarian cancer cells and the ascitic fluids in ovarian cancer patients with PLAP enzyme. In fact, this tissue enrichment was seen most frequently in ovarian cancer and testicular cancer. Subsequent studies have amply confirmed these observations [Nathanson and Fishman

20

WILLIAM H . FISHMAN

FIG.4. Germinal inclusion cyst in normal ovary exhibiting location of PLAP in the . from Nouwen et al. (1985) germinal epithelium. Final magnification ~ 2 4 0Reprinted with permission.

(1971), Cadeau et al. (1974), Benham et al. (1978), Haije e t al. (1979), McLaughlin et al. (1983), and Nouwen et al. (1985)l. C. CERVIX Three different laboratories found PLAP in normal human uterine cervix. Malkin et aZ. (1979) employed heat stability, sensitivity to Lphenylalanine, and inactivation by a polyclonal antibody anti-PLAP antibody. Nozawa et al. (1980) reported the presence of PLAP cytochemically in the reserve cell population of uterine cervical epithelium. Biochemical evidence demonstrating PLAP in the nonmalignant human cervix was published by Goldstein et al. (1980), and a detailed comparison of the characteristics of the enzyme in cervix and other tissues was completed by Goldstein et al. in 1982.

D. THYMUS Goldstein et al. (1982) found human thymus to express a measurable amount of PLAP-like enzyme.

PLACENTAL ALKALINE PHOSPHATASE

21

E. LUNG On the basis of the inhibition profiles of heat-stable AP of human lung, Goldstein and Harris (1979) classified this isozyme as PLAP. It is of interest that Chang et al. (1979) and Harris (1982) found heat-stable AP indistinguishable from PLAP in baboon lung and several Old World monkeys. The increased serum heat-stable PLAP found in smokers is reasonably attributed to the lung by Maslow et al. (1983). There is a consensus of opinion that the enzyme in lung and cervix is PLAP and that in testis and thymus it is PLAP-like, based on amino acid inhibition and electrophoretic evidence (Chang et al., 1978, 1980; Goldstein e t al, 1982) and on immunological studies with polyclonal (Wei and Doellgast, 1980, 1981) and monoclonal antibodies (Millan and Stigbrand, 1983; McLaughlin and Johnson, 1984; Travers and Bodmer, 1984). Especially definitive is the evidence of great similarity in the panel of five monoclonal antibody reactivities of normal testis and seminoma identifying PLAP-like protein in testis and seminoma (Millan and Stigbrand, 1983; Millan, 1983; Jeppsson et al., 1984).An opportunity to investigate human testicular teratocarcinoma is now also provided by eight cell lines (Andrews et al., 1980) with differing expression of alkaline phosphatase isozymes. Quantitatively, Goldstein e t al. (1982) report PLAP and PLAP-like enzyme in these nontrophoblastic tissues having activities in the range of 0.010.63 IU/g.

VI. Discussion

A. ONCOTROPHOBLAST GENEEXPRESSION AND ONCODEVELOPMENTAL BIOLOGY

Fishman ( 1976) has emphasized the importance of understanding the chronological sequence of normal development in the human and relating to it information coming from cancer cell oncodevelopmental gene expression to provide a framework for the interpretation of apparently unrelated expression of genes. This framework has proved useful in evaluating the possible fourth gene locus in testis of PLAPlike isozyme. Thus, on a developmental basis, the PLAP-like gene is expressed earliest in the gamete and makes only a minor appearance in the term-placental stage. This view, along with the PLAP-like expression in the nonhuman and human primate placenta, would favor

22

WILLIAM H. FISHMAN

regarding the PLAP-like gene as the evolutionary precursor of the PLAP gene. This hypothesis can be tested most directly by cloning and sequencing the relevant genes. Another rationale for using the developmental framework as we have derives from the pathologist’s nomenclature of tumors, which is based on the three germ layers (Pitot, 1981) and their combinations. Thus, carcinoma denotes a tumor of embryonic ectodermal or endodermal tissue, sarcoma derives from mesodermal tissue, and teratocarcinomas from all three germ layers. A list of neoplasms of counterpart developmental tissues and their marker proteins has been published (Fishman, 1983). A picture of the chronology of preembryonic development is seen in Fig. 5. Beginning with the gametes fusing with each other to form the zygote, the fertilized egg progresses through the morula and blastoGAMETES

DAYS GESTATION

I

ZYGOTE

1

I

2

MORULA

3 4

I

5

BLASTOCYST

6

7

[Implantation] TROPHOBLAST

8 9

AMNION PRIMITIVE YOLK SAC

EMBRYOBLAST ENDODERM & ECTODERM

10

SPECIALIZED EMBRYONIC

11

12 13

CHORION

14-21

YOLK SAC

I

I

MESODERM

I

Neural [NEURAL CREST CELLS Crest [c-CELLS MELANOCY TES

21-28

Endoderm [GUT HORMONE CELLS [PANCREATIC ISLET CELLS Yolk Sac/ [GERM CELLS Chorlon [ HEMATOPOIETIC STEM 1 CELLS 28-56

I

I

ORGANOGENESIS THE EMBRYO

I

FIG.5. Chronology of preembryonic development in the human.

PLACENTAL ALKALINE PHOSPHATASE

23

cyst, with the latter implanting itself in the uterine endometrium by the seventh day. In the following few days, one can observe the outer layer of trophectoderm which progresses through the chorion, and by the twelfth week, the placenta is formed by fusion of amnion and chorion. The inner cell mass of the blastocyst is the progenitor of the fetus, developing first after 4 weeks gestation into an embryo and, upon completion of the rudiments of the organ systems, becoming the fetus. Coincident with the recognition of the embryo, one can identify the extraembryonic membranes, such as the yolk sac, amnion, and the neural streak, which is the residence of neural crest cells. It is to be noted that two widely known oncodevelopmental proteins, AFP and hCG, are products of preembryonic genes.

B. DESCENDANTS OF SPECIALIZED MIGRATORY EMBRYONIC CELLS RESIDENTIN THE ADULT In the preembryonic period, one can also recognize a variety of specialized migratory embryonic cells, among which are the germ cells. These are the progenitors of the testis which, in the adult, produces PLAP-like isozyme. This subject merits discussion, as the role of specialized migratory embryonic cells in development and neoplasia is not widely appreciated. The category of specialized migratory embryonic cells is of more recent interest, having emerged, so to speak, from their cryptic residence in normal tissues to occupy center stage in contemporary clinical oncology. A case in point is medullary thyroid cancer which arises from the so-called calcitonin-producing cells (C cells) resident in normal thyroid. They are descendants of neural crest cells first recognizable in early fetal development and which are disseminated throughout the body where they were identified by the polypeptide hormones their neoplastic counterparts produced. In the case of medullary thyroid cancer, the earliest recognition of the neoplastic process is a greater than normal production of calcitonin by hypertrophied C cells (DeLellis et al., 1977). Once the cells have become frankly malignant, they often express CEA (DeLellis et al., 1978), an oncodevelopmental protein associated with fetal intestine expression. Polypeptide hormone production has also identified the neural crest origin of gastrointestinal tract endocrine tumors (O’Briain and Dayal, 1981). A current view, however, is that these tumors are of endoderm origin. Germ cell neoplasms are now diagnosable in genital and extragenital sites (deposited in fetal life) by measurements of PLAP, hCG, and AFP (Wahren et al., 1979).

24

WILLIAM H. FISHMAN

There are also the hematopoietic cell neoplasms which represent arrest at different stages of normal differentiation progression in the bone marrow. Since this process of hematopoietic cell generation is first recognized in the blood islands in the preembryonic period and operates in the same way in its colonies in the bone marrow, spleen, and lymph nodes of the adult, this is an example of an embryonic cellgenerating mechanism operating in tissues of the adult. It may be well to remember that all tissue regeneration phenomena in the adult represent a recapitulation of the fetal tissue generation process, be it regeneration of liver or skin or lining of the intestine. Consequently, it is to be expected that certain developmental markers may be expressed in adult tissues by virtue of such ongoing tissue renewal. Thus, it is necessary to recognize that a degree of differentiation may be proceeding in specialized cells which established their particular residence in one or more adult tissues as a consequence of antecedent cell migration in the embryo. That such cells may undergo transformation in the adult with the overproduction of cell-specific peptide hormones is a significant departure from the view that all cells in a particular organ have “adult” characteristics and that all cells are involved in the malignant transformation of a given tissue rather than a subpopulation. What is the relevance of these considerations to Cohnheim’s embryonal rest theory of cancer (Cohnheim, 1889)? That theory proposed that surplus undifferentiated embryonic cells, as in a nevus or teratoma, were prone to undergo neoplastic transformation. In the present context, however, it is the descendants of specialized embryonic cells performing normal physiological functions which progress from hyperplasia to neoplasia. OF ONCOTROPHOBLAST GENEEXPRESSION C. MECHANISMS

The opportunity to investigate the mechanisms of oncotrophoblast gene expression experimentally is provided by the various cancer cell lines referred to in this article. Clearly, there is evidence of a whole spectrum of expression, from monophenotypic to polyphenotypic cell lines with differing levels and specificity of response to enzyme-inducing agents in the culture medium of cells. There is strong evidence that the levels of enzyme activity are direct reflections of the amount of enzyme protein, and the phenomenon of induction is explainable as a phenomenon of increased transcription of the gene. What evidence supports concordance of trophoblast gene expression? For the most part, comparisons are most complete for hCG and

PLACENTAL ALKALINE PHOSPHATASE

25

PLAP. In studies of 22 ovarian cancer patients, 59% showed Regan isoenzyme and 68% hCG in the ascitic fluid. Progressively increasing levels of each marker generally correlated with the extent of disease, although in a few cases only one marker correlated. These longitudinal studies in patients with ovarian cancer (Fishman et al., 1974) have demonstrated trophoblast gene expressions which can be interpreted as development-phase specific. For example, the early placental form, non-Regan isoenzyme, is often correlated with hCG first trimester expression, while PLAP-dominated enzyme production (a second and third trimester phenomenon) was not accompanied by similar hCG levels. In seminoma, there is also good concordance in the expression of both oncotrophoblast markers (Lange et al., 1982). Next, in a uterine cervical cancer cell line (SKG-IIIa). Nozawa et al. (1983)reported that sodium butyrate induced concordant expression of “early placenta” AP, pregnancy-specific PI-glycoprotein, and hCG a subunit. Finally, there were two earlier reports in cancer patients of concordance; Charles et d.(1973)measured three placental proteins, hCG, human chorionic somatomammotropin, and PLAP in lung carcinoma, and Belliveau et al. (1973)showed that a primary mediastinal choriocarcinoma overproduced Regan isoenzyme, hCG, and CEA. Another recent example is the finding by Uchida et al. (1981b) of gastric cancer metastatic to bone, which elaborated hCG and both PLAP and intestinal alkaline phosphatase.

D. PLAP

AND

PLAP-LIKEISOZYMES

The current use of these terms was defined by Stigbrand (1984a,b) and the UmeH satellite symposium of the 11th Annual Meeting of the International Society of Oncodevelopmental Biology and Medicine in Stockholm. Thus, PLAP is equivalent to the Regan isoenzyme in tumors and the heat-stable alkaline phosphatase of term placenta, whereas PLAP-like includes all L-leucine-sensitive, heat-stable alkaline phosphatases both from tumors, as in the Nagao isoenzyme, and in tissues such as testis and thumus. Also, PLAP and PLAP-like enzymes can be distinguished from each other by the use of monoclonal antibodies (Millan and Stigbrand, 1983; McLaughlin et al., 1984; Travers and Bodmer, 1984; Jeppsson et al., 1984). Accordingly, the evidence is mounting that PLAP-like AP could represent a fourth locus in addition to the genes for tissue-unspecific AP, intestinal AP, and PLAP.

26

WILLIAM H. FISHMAN

E. PHENOMENA TO BE EXPLAINED BY GENESTRUCTURE AND GENEREGULATION It is widely agreed that further understanding of oncotrophoblast genes and their regulation must await cloning and sequencing of the relevant genes, One such PLAP gene has recently been cloned and sequenced (Millan, 1986). When one examines the current information on human PLAP alkaline phosphatases, there are clearly a number of significant domains. Thus, the membrane insertion site is -2000 Da and is cleaved by the proteolytic enzyme bromelain (Kottel and Hanford, 1980; Neuwald and Brooks, 1981). The 10,000 Da segment can be split by trypsin from the end of the molecule farthest from the membrane (Jemmerson et al., 1984).The intervening segment of 55,000 Da binds most of the monoclonal antibodies to PLAP and also is the site of catalytic activity. Polyclonal and monoclonal antibodies appear to recognize conformational determinants. How will these features be explained when all the PLAP genes are cloned? Will they differ? To what extent? The overlapping PLAP and intestinal AP antibody reactions suggest that there is a degree of homology between the two genes. Where is this homology to be seen in these two genes and their products? Evidence also exists that the placental and intestinal genes are closely linked in the genome. Thus, interlocus heteromers were demonstrated by Singer and Fishman (1975) in F1-amnion and Hep-2 cells using polyclonal antibodies to PLAP and intestinal AP, and by Wray and Harris (1982) employing specific monoclonal antibodies in Hep 2/ 5 HeLa cells. Also awaiting explanation from the amino acid sequence of the PLAP and PLAP-like proteins is the basis for the exquisite specificity of the L-phenylalanine, L-leucine, and L-Phe-Gly-Glyamino acid inhibitions. What is the architecture of the active site and its environs which explains these specificities? What are the structural features which favor the “uncompetitive” nature of these inhibitions? Finally, are there enhancing nucleotide sequences flanking the PLAP gene, the modulation of which may account for the induction phenomena? Can the specificity of PLAP induction be explained by the absence of enhancers flanking the IAP and AP genes? By what mechanism does one explain the inverse simultaneous modulation of intestinal and placental isoenzymes in HeLa D98AH2 cells? How can one explain the allelic polymorphism of PLAP and its lack in IAP and tissue-unspecific AP? One may be confident that the answers to these questions will be forthcoming in the near future.

PLACENTAL ALKALINE PHOSPHATASE

F. FEASIBILITY OF PLAP AS AND

THE

27

TARGET FOR IMMUNOLOCALIZATION

IMMUNOTHERAPY

The current era of radioimmunolocalization of tumors was ushered in by the radioactive iodine-labeled CEA antibody techniques of Goldenberg et al. (1978) and Mach et al. (1980).In their critical review of theoretical and practical aspects of radioimmunolocalization, Begent and Bagshawe (1983) point out the great desirability of antigenic targets which are integrated into the plasma membrane and refer to phenomena which affect the interpretation of results such as variation in, antibody distribution, antigen-antibody complexes, and nonspecific accumulation of radioisotope in normal organs. Two laboratories have investigated the immunolocalization of human tumor xenografts in nude athymic mice. Jemmerson et al. (1984) injected A431 cells which produce PLAP subcutaneously on one side of the neck and SNG cells which do not produce PLAP on the other side of the neck. After the tumors became visible, the mice were injected intraperitoneally with 1251-labeledF(ab’)2 fragment of the F11 monoclonal antibody (Millan et al., 198213). Four days later, the concentration ratio in the PLAP-positive A431 tumor was 10-fold greater than was observed in the PLAP-negative SNG tumor. Similarly, Jeppsson et al. (1984) reported a 6.5-fold enrichment of radioactivity in HeLa Hep-2 xenografted tumors using monoclonal antibody H7 (Millan and Stigbrand, 1983). Undoubtedly, the fact that PLAP is most accessible by virtue of its outer cell membrane location has contributed to these favorable ratios. Both immunotherapeutic and immunodetection targeting are based on the principle of the specific uptake of the antibodies by the target tumors. Aside from highly radioactive isotopes incorporated into the antibody, highly toxic proteins or cytotoxic drugs can be coupled to the specific antibody. An objective demonstration of the advantage of an integral cell membrane protein versus a cytoplasmic soluble one is a study by Tsukazaki e t al. (1985).When tested under similar conditions, they reported that ricin A conjugates to a monoclonal antibody for PLAP in a tumor cell line kill PLAP-producing tumor cells. On the other hand, anti-AFP ricin A conjugate was not cytotoxic to either PLAP-producing or AFP-producing tumor cells. It may be reasonable to suggest that PLAP-inducing agents administered prior to detection and therapeutic procedures could increase the number of target molecules on the cell surface of tumor cells. Such an event would help significantly the effectiveness of detection and therapy.

28

WILLIAM H. FISHMAN

VII. Conclusions

Oncotrophoblast gene expression is reviewed within the context of the interrelationship of neoplasia with developmental biology. It has been possible to develop models of cell lines in culture which appear to reflect the inappropriately great expression of PLAP, PLAP-like, IAP, and tissue-unspecific AP observed in neoplasms of individual patients. These expressions can be restricted to individual isozymes or can include two or more isozymes in conjunction with other trophoblast gene products. In some cases, induction of one isozyme occurs apparently at the expense of another. The inducing agents most commonly used in the laboratory are prednisolone, butyrate, and hyperosmolarity, and these have been employed individually and in combination. Depending on the cell line and the inducer chosen, one can manipulate the expression of the individual AP genes at will. This expression takes place with transcription of the gene, correlates with chromatin condensation, and is a function of the cell cycle at GI. Of interest is the possibility that a fourth gene locus (PLAP-like) may exist. The gene product has been identified in testis, ovary, and thymus, and in the majority of cancer patients harboring seminoma or ovarian carcinoma. The progenitor cells in these situations are the germ cells. This fact has stimulated a discussion of neoplasms originating from descendants of specialized migratory embryonic cells resident in the adult. Thus, attention has been focused anew on these latest reflections of the interrelationship of cancer and developmental biology. Included also is a discussion of the current views of the evolutionary history of PLAP and the other isozymes. The phenomena which cloning and sequencing of the gene for PLAP and the other AP genes can be expected to explain include the following: the precise homology in the structures of PLAP from tumors and placenta, the differences in structure which have resulted in so many allelic forms of PLAP, the distinguishing feature of the gene for PLAP, the evolutionary history of AP, precise explanation of the uncompetitive inhibition, the overlapping homology between PLAP and intestinal alkaline phosphatase, and the definition of the nucleotide sequences which regulate the expression of PLAP and other alkaline phosphatase genes. Clearly, the field is on the threshold of making a major step forward.

PLACENTAL ALKALINE PHOSPHATASE

29

ACKNOWLEDGMENTS The research conducted in this laboratory has been supported in part by Cancer Center Support Grant P30 CA 30199-04, and Grants R01 CA 21967-08 and R01 CA 31378-03 from the National Cancer Institute, NIH, Bethesda, Maryland. I am grateful to Dr. E. Ruoslahti for reading the manuscript and offering his valuable comments. My sincere thanks to Diane Lowe for typing the manuscript.

REFERENCES Abu-Hasan, N. S., and Sutcliffe, R. G. (1984).In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 117-126. Liss, New York. Adamson, E. D., Muller, R., and Verma, I. (1983). Cell Biol. Int. Rep. 7, 557-558. Andrews, P. W., Bronson, D. L., Benham, F., Strickland, S., and Knowles, B. B. (1980). Int. J. Cancer 26,269-280. Anstiss, C. L., Green, S., and Fishman, W. H. (1971). Clin.Chim. Actu 33, 279-286. Attwood, H. D., and Park, W. W. (1961).J.Obstet, Gynaecol. Br. Commonw. 68, 611617. Badger, K., and Sussman, H. H. (1976).Proc. Natl. Acad. Sci. U.S.A. 73, 2201-2205. Beckman, G., and Beckman, L. (1969). Hum. Hered. 19,524-529. Begent, J. H., and Bagshawe, K. D. (1983).In “Oncodevelopmental Markers” (W. H. Fishman, ed.), pp. 167-188. Academic Press, New York. Behrens, C. M., Enns, L. A., and Sussman, H. H. (1983). l3iochern.J.211,553. Belanger, L., Hamel, D., Lachance, L., Dufour, D., Tremblay, M., and Gagnon, P. M. (1975). Nature (London)256,657-659. Belliveau, R. E., Wiernik, P. H., and Sickles, E. A. (1973). Lancet 1,22-24. Benham, P. J., Povey, M. S., and Harris, H. (1978). Clin. Chim. Acta 86, 201-215. Benham, P. J., Fogh, J., and Harris, H. (1981). Int. J. Cancer 27, 637-644. Bohn, H. (1983). In “Oncodevelopmental Markers” (W. H. Fishman, ed.), pp. 69-84. Academic Press, New York. Brahmacupta, P., and Melnykovych, G. (1980).J. Cell. Physiol. 105, 227-233. Braunstein, G. D., Vaitukaitis, J. L., Carbone, P. P., and Ross, G. T. (1973).Ann. Intern. Med. 78,39-45. Brewer, L. M., and MacManus, J. P. (1985).Det.. Biol., 112, 49-58. Bulmer, D., Stocco, D., and Morrow, J. (1976).J . Cell. Physiol. 87, 357-365. Cadeau, J., Blackstein, M. E., and Malkin, A. (1974). Cancer Res. 34, 729-732. Candido, E. P. M., Reeves, R., and Davie, J. R. (1978). Cell 14, 105-113. Chang, C. H., Raam, S., Angellis, D., Doellgast, G. F., and Fishman, W. H. (1975). Cancer Res. 35, 1706-1712. Chang, C. -H., Angellis, D., and Fishman, W. H. (1978).Scand.1. Immunol. 8,543-546. Chang, C., Anstiss, C. L., Angellis, D., and Fishman, W. H. (1979). Immunol. Commun. 8,563-579. Chang, C. -H., Angellis, D., and Fishman, W. H. (1980). Cancer Res. 40, 1506-1510. Chard, T. (1982). In “Pregnancy Proteins, Biology, Chemistry, and Clinical Application” (J. G. Grudzinskas, B. Teisner, and M. Seppala, eds.), pp. 3-24. Academic Press, New York. Charles, M. A., Claypool, R., Schaaf, M., Rosen, W. E., and Weintraub, W. E. (1973). Arch. Intern. Med. 132,427-431. Chou, J. Y. (1978). Proc. Notl. Acad. Sci. U.S.A. 75, 1409-1413. Chou, J. Y. (1979). In Vitro 15, 789-795.

30

WILLIAM H. FISHMAN

Chou, J. Y., and Ito, F. (1984). In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 167-186. Liss, New York. Chou, J. Y., and Robinson, J. C. (1977). In Vitro 13,450-460. Cohnheim, J. (1889). “Lectures on General Pathology.” New Sydenham Society, London. Cox, G. S. (1981). Biochemistry 20, 4893-4900. Cox, R. P. (1971). Ann. N . Y. Acad. Sci. 179, 596-610. Cox, R. P., and Griffin, M. J. (1967). Arch. Biochem. Biophys. 122,552-562. Cox, R. P., and MacLeod, C. M. (1961). Nature (London)190,85-87. Cox, R. P., Elson, N. A., Tu, S., and Griffin, M. J. (1971).J. Mol. Biol. 58, 197-215. Cox, R. P., Ghosh, N. K., Bazzell, K., and Griffin, M. J. (1975). In “Isozymes. I. Molecular Structure” (C. L. Markert, ed.), pp. 343-365. Academic Press, New York. DeGroote, G., De Woeb, P., Van de Voorde, A,, De Broe, M., and Fiers, W. (1983).Clin. Chem. 29, 115-119. DeLellis, R. A., Nunnemacher, G., and Wolfe, H. J. (1977). Lab. Invest. 36, 237-248. DeLellis, R. A., Wolfe, H. J., Rule, A. A., Reichlin, S., and Tashijian, A. H., Jr. (1978).N. Engl. J . Med. 299, 1082. Deutsch, S. I., Silvers, D. N., Cox, R. P., Griffin, M. J., and Ghosh, N. K. (1976).J.Cell Sci. 21, 391-406. Deutsch, S. I., Ghosh, N. K., and Cox, R. P. (1977). Biochim. Biophys. Acta 499,382391. Doellgast, G . J. (1977).Anal. Biochem. 82,278-288. Doellgast, G. J., and Benirshke, K. (1979). Nature (London)280, 601-602. Doellgast, G. J., and Holmesley, H. D. (1984). Obstet. Gynecol. 63, 324-329. Douglas, G. W., Thomas, L., Carr, M., Cullen, N. M., and Morris, R. (1959). Am. J. Obstet. Gynecol. 78, 960-973. Eerdekens, M. W., Nouwen, E. J., Pollet, D. E., Briers, T. W., and De Broe, M. E. (1985). Clin. Chem. 31,687-690. Edlow, J. B., Ota, T., Relacion, J., Kohler, P. O., and Robinson, J. C. (1975). Am. J. Obstet. Gynecol. 121, 674-681. Epenetos, A. A., Munro, A. J., Tucker, D. F., Gregory, W., Duncan, W., MacDougall, R. H., Faux, M., Travers, P., and Bodmer, W. F. (1985). B r . ] . Cancer 51,641-644. Ezra, E., Blacker, R., and Udenfriend, S. (1983). Biochem. Biophys. Res. Commun. 116, 1076-1083. Fallon, R. J., and Cox, R. P. (1981). Somatic Cell Genet. 7 , 193-204. Firestone, G. L., and Heath, E. C. (1981).J. Biol. Chem. 256, 1404-1411. Fishman, L. Miyayama, H., Driscoll, S. G., and Fishman, W. H. (1976). Cancer Res. 36, 2268-2273. Fishman, P. H., Simmons, J. L., Brady, R. O., and Freeze, E. (1974). Biochem. Biophys. Res. Commun. 59,292-299. Fishman, W. H. (1969). Ann. N.Y. Acad. Sci. 166, 745-759. Fishman, W. H. (1974). Am. J . Med. 56,617-650. Fishman, W. H. (1976). Cpncer Res. 36,3423-3428. Fishman, W. H. (1983). In “Oncodevelopmental Markers” (W. H. Fishman, ed.), pp. 119. Academic Press, New York. Fishman, W. H., and Green, S. (1967). Enzymologia 31, 89-99. Fishman, W. H., and Singer, R. M. (1976). Cancer Res. 36,4256-4261. Fishman, W. H., Ghosh, N. K., Inglis, N. R., and Green, S. (1968a). Enzymologia 34, 3 17-32 1. Fishman, W. H., Inglis, N. R., and Ghosh, N. K. (1968b). Clin. Chim. Acta 19, 71-79.

PLACENTAL ALKALINE PHOSPHATASE

31

Fishman, W. H., Inglis, N. R., Stolbach, L. L., and Krant, M. J. (1968~). Cancer Res. 28, 150- 154. Fishman, W. H., Inglis, N. R., Vaitukaitis, J., and Stolbach, L. L. (1975). Natl. Cancer lnst. Monogr. 42,63-73. Fishman, W. H., Krishnaswamy, P. R., Fishman, L., Millan, J. L., and McIntire, K. R. (1979). In “Carcino-embryonic Proteins” (F. G. Lehmann, ed.), Vol. 2, pp. 699-708. Elsevier, Amsterdam. Forman, D. T., Moss, D. W., and Whitaker, K. B. (1976).Clin. Chim. Acta 66,287-290. Ghosh, N. K., and Cox, R. P. (1977). Nature (London)267,435. Ghosh, N. K., Ruckenstein, A., Baltimore, R., and Cox, R. P. (1972). Biochim. Biophys. Acta 286, 175-185. Ghosh, N. K., Rukenstein, A., and Cox, R. P. (1977). Biochern.1. 166,265-274. Goldenberg, D. M., Deland, F. H., Kim, E. E., Bennett, S., Primus, F. J., van Nagell, J. R., Estes, N., DeSimone, P., and Rayburn, P. (1978). N . Engl. J . Med. 298, 13841388. Goldstein, D. J., and Harris, H. (1979). Nature (London)280, 602-605. Goldstein, D. J., Blasco, L., and Harris, H. (1980).Proc. Natl. Acad. Sci. U.S.A.77,42264228. Goldstein, D. J., Rogers, C., and Harris, H. (1982). Clin. Chim. Acta 125, 63-75. Goz, B. (1974).Cancer Res. 34,2393-2398. Green, S., Anstiss, C. L., and Fishman, W. H. (1971). Enzymologia 41, 9-26. Greene, P. J., and Sussman, H. H. (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 2936-2940. Griffin, M. J . , and Bern, R. (1969).J . Cell Biol. 40, 297-304. Griffin, M. J., and Cox, R. P. (1966).J. Cell Biol. 29, 1-9. Griffin, M . J., Price, G. H., Bazzell, K. L., Cox, R. P., and Ghosh, N. K. (1974).Arch. Biochem. Biophys. 164,619-623. Haije, W. G., Meenvaldt, J. H., Talerman, A., Kuipers, Tj., Baggerman, L., Tecuso, A. H., Van Der Pompe, W. B., and Van Driel, J. (1979). Int. J . Cancer 24,288-293. Hamilton, T. A., and Sussman, H. H. (1981). Bi0chem.J. 198,29-35. Hamilton, T. A,, Tin, A. W., and Sussman, H. H. (1979). Proc. Natl. Acad. Sci. U.S.A.76, 323-327. Hanford, W., and Fishman, W. H. (1983). Anal. Biochem. 129, 176-183. Hanford, W. C., Kottel, R. H., and Fishman, W. H. (1981). Bi0chem.J. 200,461-464. Harris, H. (1982).Homey Lect. Ser. 76, 95-124. Herz, F. (1984).In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 139-166. Liss, New York. Herz, F., and Halwer, M. (1983).Biochim. Biophys. Acta 762, 289-293. Herz, F., and Koss, L. G. (1979). Arch. Biochem. Biophys. 194,30-36. Herz, F., Lewis, J., Jr., and Lipsett, M. B. (1961). Am. J . Obstet. Gynecol. 82, 631640. Herz, F., Miller, 0.J., Miller, D. A., Auersberg, N., and Koss, L. G. (1977). Cancer Res. 37,3209-3213. Herz, F., Schermer, A., Halver, M., and Bogart, L. H. (1981). Arch. Biochem. Biophys. 210,581-591. Higashino, K., Hashinotsume, K., Kang., Y., Takaheshi, Y., and Yamamura, Y., (1972). Clin. Chim. Acta 24, 67-81. Higashino, K., Kudo, S., Ohtani, R., Yamasura, Y., Honda, T., and Sakurai, J. (1975).Ann. N . Y . Acad. Sci. 259,337-346. Holmgren, P. A., Stigbrand, T., Damber, M. B., Von Schoultz, B., and Wahren, B. (1978). Scand. J . Zmmunol. 8, (Suppl. 8), 515-518.

32

WILLIAM H. FISHMAN

Honda, T., Kurabori, T., Ishigami, S., and Sakurai, J. (1973). Igaku-no-Azumi 86,313314. Horwich, A., Tucker, D. F., and Peckham, M. J. (1985). Br.J. Cancer 51,631-640. Hugon, J., and Borgers, M. (1966).J. Histochem. Cytochem. 14, 629. Iino, S., Abe, K., Oda, T., Suzuki, H., and Sugiura, M. (1972). Clin. Chim. Acta 42, 161165. Inglis, N. R.,Guzek, D. T., Kirley, S., Green, S., and Fishman, W. H. (1971).Clin. Chim. Acta 33,287-292. Inglis, N. R.,Kirby, S., Stolbach, L. L., and Fishman, W. H. (1973). Cancer Res. 33, 1657-1661. Ito, F., and Chou, J. Y. (1983). Biochem. Biophys. Res. Commun. 111,611-618. Jacoby, B., and Bagshawe, K. D. (1972). Cancer Res. 32,2413-2420. Javadpour, N. (1983). Cancer 52,887-889. Jemmerson, R.,and Stigbrand, T. (1984).FEES Lett. 173,357-359. Jemmerson, R., Shah, N., Takeya, M., and Fishman, W. H. (1984a).In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 105-115. Liss, New York. Jemmerson, R., Takeya, M., Shah, N., and Fishman, W. H. (19i84b).In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 245-256. Liss, New York. Jemmerson, R., Klier, F. G., Shah, N., Takeya, M., and Fishman, W. H. (1985a). J. Histochem. Cytochem. 33, 1227-1234. Jemmerson, R.,Shah, N., and Fishman, W. H. (1985b). Cancer Res. 45,3268-3273. Jemmerson, R., Millan, J. L., Klier, F. G., and Fishman, W. H. (1985~). FEES Lett. 179, 316-320. Jeppsson, A., Wahren, B., Brehmer-Anderson, E., Silfversward, C., Stigbrand, T., and Millan, J. L. (1984). 1nt.J. Cancer 34, 757-761. Kam, W. K., Bresalier, R. S., and Kim, Y. S. (1984). In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 207-222. Liss, New York. Kamoda, T., Sakagichi, Y., and Sukine, T. (1981). Clin. Chim. Acta 117, 167. Kellen, J. A., Bush, R. S . , and Malkin, A. (1976). Cancer Res. 36,269-271. Koga, K., and Tamaoki, T. (1974). Biochemistry 13,3024-3028. Kollmorgan, G. M., and Griffin, M. J. (1969). Cell Tissue Kinet. 2, 111-122. Konsoda, T., Sakagishi, Y., and Sekine, T. (1981). Clin. Chim. Acta 117, 167. Kottel, R. H., and Fishman, W. H. (1978). Scand. J. Immunol. 8, (Suppl. 8), 571-574. Kottel, R. H., and Fishman, W. H. (1981).Biochem. J. 200,679-684. Kottel, R. H., and Hanford, W. C. (1980).J. Biochem. Biophys. Methods 2,325-330. Koyama, H., and Ono, T. (1971). Exp. Cell Res. 69,468. Koyama, H., Kato, R., and Ono, T. (1972). Biochem. Biophys. Res. Commun. 46,305311. Lalitha, N., and Nagarajan, B. (1977). Indian J. Biochem. Biophys. 14,247-250. Lange, P. T., Millan, J. L., Stigbrand, T., Vessella, R. L., Ruoslahti, E., and Fishman, W. H. (1982). Cancer Res. 42,3244-3247. Lehmann, F. G. (1975). Clin. Chim. Acta 65,271-282. Li, M. C., Hertz, R.,and Spencer, B. (1956). Proc. Soc. E x p . B i d . Med. 93,361-366. Lieblich, J. M., Weintraub, B. D., Rosen, S. W., Ghosh, N. K., and COX,R. P. (1977). Nature (London)265, 746. Lin, C. W., Singer, R. M., Sasaki, M., Truett, M. L., and Fishman, W. H. (1976). J. Histochem. Cytochem. 24,659-667. Lin, C. W., Inglis, N. R., Rule, A. H., Turksoy, R. N., Chapman, C. M., Kirby, S. P., and Stolbach, L. L. (1979). Cancer Res. 39,4894-????

PLACENTAL ALKALINE PHOSPHATASE

33

Littlefield, B. A,, Cidlowski, N. B., and Cidlowski, J. A. (1980).Arch. Biochem. Biophys. 201, 174-184. Log, T., Chang, K. S. S., and Hsu, Y. C. (1981).Int. J. Cancer 27,365-372. Luduena, M. A,, and Sussnian, H. H. (1976).J.B i d . Chem. 251,2620-2628. Mach, J.-P., Carrel, S., Forni, M., Ritschard, J., Donath, A., and Alberto, P. (1980).N. Eng1.J. Med. 303, 5-10. McLaughlin, P. J., and Johnson, P. M. (1984).In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 67-75. Liss, New York. McLaughlin, P. J., Gee, H., and Johnson, P. M. (1983).Clin. Chim.Acta 130, 199-209. McLaughlin, P. J., Travers, P. J., McDicken, I. W., and Johnson, P. M. (1984). Clin. Chim. Acta 137, 341-348. MacManus, J. P., Whitfield, J. I., Boynton, A. L., Durkin, J. P., and Swierenga, S. H. H. (1982). Oncodev. Biol. Med. 3, 79-90. Malkin, A., Kellen, J. A,, and Caplan, B. (1979). In “Carcino-Embryonic Proteins” (F. G. Lehman, ed.), Vol. 2, pp. 679-684. Elsevier, Amsterdam. Manning, J. P., Inglis, N. R., Green, S., and Fishman, W. H. (1969). Enzymologia 37, 25 1-26 1. Manning, J. P., Inglis, N. R., Green, S., and Fishman, W. H. (1970). Enzymologia 39, 307-318. Maslow, C. W., Muensch, H., Azama, F., and Bertrand, M. (1983). Clin. Chem. 29,260263. Maziere, J. C., MaziBre, C., and Polonovsky, J. (1977). Biochimie 59, 221. Melnykovych, G. (1962). Biochem. Biophys. Res. Commun. 8, 81-86. Melnykovych, G., Bishop, C., and Swayze, M. A. B. (1972).J. Cell. Physiol. 70, 231. Miedema, E. (1968). Erp. Cell Res. 53, 488. Millan, J. L. (1983). Umea University Medical Dissertations. No. 107. Millan, J. L. (1986).J. B i d . Chem., in press. Millan, J. L., and Stigbrand, T. (1981). Clin. Chem. 27, 2014-2018. Millan, J. L., and Stigbrand, T. (1983). Eur. J. Biochem. 136, 1-7. Millan, J. L., Eriksson, A., and Stigbrand, T. (1982a). Hum. Genet. 62, 293-295. Millan, J. L., Stigbrand, T., Ruoslahti, E., and Fishman, W. H. (1982b). Cancer Res. 42, 2444-2449. Millan, J. L., Nustad, K., and NZrgaard-Pedersen, B. (1985). Clin. Chem. 31, 54-59. Miyayama, H., Doellgast, G. J., Memoli, V., Gandbhir, L., and Fishman, W. H. (1976). Cancer 38, 195-204. Miyayama, H., Tokumitsu, S. I., Nomura, H., and Takeya, M. (1983). Acta Histochem. Cytochem. 16,415-430. Morita, A., Tsao, D., and Kim, Y. S. (1982). Cancer Res. 42,4540-4545. Nakayama, T., Yoshida, M., and Kitamura, M. (1970). Clin. Chim. Acta 30,546-548. Nathanson, L., and Fishman, W. H. (1971). Cancer 27, 1388-1397. Niman, H. L., Andrew, M. H., Thompson, A. Y., Markman, M., Willems, J. J., Hernig, K. R., Habib, N. A., Wood, C. B., Houghten, R. A,, and Lerner, R. A. (1985).Proc. Natl. Acad. Sci. U S A . 82, 7924-7928. Nitowsky, H. M., and Herz, F. (1963). Biochem. Biophys. Res. Commun. 11, 261266. Neuwald, P. D., and Brooks, M. (1981). Cancer Res. 41, 1682-1689. Nouwen, E. J., Pollet, D. E., Schelstraete, J. B., Eerdekens, M. W., Hansch, C., Van de Voode, A., and D e Broe, M. E. (1985). Cancer Res. 45,892-902. Nozawa, S., and Fishman, W. H. (1982). In “Pregnancy Proteins: Biology, Chemistry, and Clinical Application” (J. G. Grudzinskas, B. Teisner, and M. Seppala, eds.), pp. 121-153. Academic Press, New York.

34

WILLIAM H. FISHMAN

Nozawa, S., Ohta, H., Izumi, S., Hayashi, S., Tsutsui, F., Kurihara, S., and Watanabe, K. (1980).Acta Histochem. Cytochem. 13, 521-530. Nozawa, S., Engvall, E., Kano, S., Kurihara, S., and Fishman, W. H. (1983). Int. J . Cancer 32,267-272. Nustad, K., Monrad-Hanson, H. P., Paus, E., Millan, J. L., Norgaard-Pederson, B., and the DATECA Study Group (1984). In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 337-348. Liss, New York. O’Briain, D. S., and Dayal, Y. (1981). In “Diagnostic Immunohistochemishy” (R. A. DeLellis, ed.), pp. 75-109. Masson, New York. Paiva, J., Damjanov, I., Lange, P. H., and Harris, H. (1983).Am. J. Pathol. 111,156-165. Pitot, k. C. (1981). In “Fundamentals of Oncology,” pp. 18-28. Dekker, New York. Pollet, D. E., Nouwen, E. J., Schelstraete, J. B., Renard, J., Van de Voorde, A., and De Broe, E. (1985). Clin. Chem. 31,41-45. Prasad, K. N. (1980). Life Sci. 27, 1351-1358. Riggs, M. G., Whitaker, R. G., Neumann, J. R., and Ingram, V. M. (1977). Nature (London)268,462-464. Robson, E. B., and Harris, H. (1965). Nature (London) 207, 1257-1259. Sakiyama, T., Robinson, J. C., and Chou, J. Y. (1979).J . Biol. Chem. 254,935-938. Sakiyama, T., Mano, T., and Chou, J. Y. (1980).J . Biol. Chem. 255,9399-9403. Santos, E., Martin-Zanea, D., Reddy, E. P., Pierotti, M. A., Della Porta, G., and Barbacid, M. (1984). Science 223, 661-664. Sasaki, M., and Fishman, W. H. (1973). Cancer Res. 33,3008-3018. Sell, S., Skelly, H., Leffert, H. L., Mueller-Eberhard, W., and Kila, S. (1975).Ann. N . Y. Acad. Sci. 259,45-59. Seppala, M., and Rutanen, E.-M. (1982). In “Pregnancy Proteins” (J. G. Geudzinskas, B. Teisner, and M. Sappala, eds.), pp. 235-240. Academic Press, New York. Seppala, M., Wahlstrom, T., and Bohn, H. (1979). Znt. J . Cancer 24, 6-10. Singer, R. M. (1976). Cancer Res. 36, 4262-4265. Singer, R. M., and Fishman, W. H. (1974).J.Cell B i d . 60,777-780. Singer, R. M., and Fishman, W. H. (1975).In “Isozymes. 111. Developmental Biology” (C. L. Markert, ed.), pp. 753-774. Academic Press, New York. Singer, R. M., and Fishman, W. H. (1976).Differentiation 5, 127-132. Singer, R. M., Tompkins, W. A. F., White, L. J., and Perry, J. E. (1976).J. Natl. Cancer Inst. 56, 175-178. Slaughter, C. A., Coseo, M. C., Cancro, M. P., and Harris? H. (1981). Proc. Natl. Acad. Sci. U S A . 78, 1124-1128. Slor, H., and Bustan, H. (1973). Experientia 29, 1214-1215. Speeg, K. V., Jr., and Harrison, R. W. (1979). Endocrinology 104, 1364-1368. Speeg, K. V., Jr., Azizskan, J. C., and Stromberg, K. (1978).Scand.J.Immunol. 8, (Suppl. 8). 527-532. Stigbrand, T. (1984a). In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. xix-xxiii. Liss, New York. Stigbrand, T. (1984b). In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 6-14. Liss, New York. Stigbrand, T., and Engvall, E. (1982). In “Human Cancer Markers” (S. Sell and B. Wahren, eds.), pp. 275-301. Humama Press, Clifton, New Jersey. Stigbrand, T., Millan, J. L., and Fishman, W. H. (1982). In “The Genetic Basis of Alkaline Phosphatase Isozyme Expression in Isozymes” (M. C. Rattazzi, J. G. Scandalios, and G. S. Whitt, eds.), Vol. 6, pp. 93-110. Liss, New York. Stolbach, L. L., Fishman, W. H., and Krant, M. J. (1969).N . Engl.]. Med. 281,757-762.

PLACENTAL ALKALINE PHOSPHATASE

35

Stolbach, L. L., Skillman, J., and Goodman, R. (1972).Arch. Surg. 105,491-493. Sussman, H. H. (1984). In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 87-103. Liss, New York. Sussman, H. H., Small, P. A., and Cotlove, E. (1968).J . Biol. Chem. 243, 160-166. Takahara, N., Herz, F., and Hirano, A. (1982).Acta Neuroputhol. 57,45-50. Takeya, M., Jemmerson, R., Shah, N., and Fishman, W. H. (1985). In press. Takeya, M., Klier, F. G., and Fishman, W. H. (1984).J . Mol. B i d . 173,253-264. Tanaka, M., Kudo, S., Higashino, K., and Kishimoto, S. (1983). Oncodeu. Biol. Med. 4, 245-252. Tatarinov, Y. S., Mesnyankina, N. V., Nikoulina, D. M., Novikova, L. A., Toloknov, B. O., and Falaleyeva, D. M. (1974). Int. J. Cancer 14, 548-554. Tokumitsu, S. I., and Fishman, W. H. (1983).J. Histochem. Cytochem. 31, 647-655. Tokumitsu, S. I., Tokumitsu, K., Kohnoe, K., and Takeuchi, T. (1979). Cancer Res. 39, 4732-4738. Tokumitsu, S . I., Tokumitsu, K., and Fishman, W. H. (1981a).Histochemistry 73, 1-13. Tokumitsu, S. I., Tokumitsu, K., and Fishman, W. H. (1981b).J.Histochem. Cytochem. 29, 1080-1087. Tokumitsu, S. I. (1984). In “Human Alkaline Phosphatases” (T. Stigbrand and W. H. Fishman, eds.), pp. 187-206. Liss, New York. Travers, P., and Bodmer, W. (1984).I n t . J . Cancer 33,633-641. Tsao, D., Morita, A., Bella, A., Jr., Luu, P., and Kim, Y. S. (1982).Cancer Res. 42, 10521958. Tsukada, Y., and Hirai, H. (1975). Ann. N . Y. Acud. Sci. 259, 37-44. Tsukazaki, K., Hayman, E. G., and Ruoslahti, E. (1985).Cancer Res. 45, 1834-1838. Tucker, D. F., Oliver, R. T. D., Travers, P., and Bodmer, W. F. (1985). Br. J. Cancer 51, 631-640. Uchida, T., Shikata, T., Shimizu, S. -I., Takimoto, Y., Iino, S., Suzuki, H., Oda, T., Hirano, K., and Suguira, M. (1981a). Cancer 48, 140-150. Uchida, T., Shimoda, T., Miyata, H., Shikata, T., Iino, S., Suzuki, H., Oda, T., Hirano, K., and Suguira, M. (1981b). Cancer 48, 1455-1462. Usategui-Gomez, M., Yeager, F. M., and Fernando d e Castro, A. (1973). Cancer Res. 33, 1574-1577. Van d e Voorde, A,, De Groote, G., De Waele, P., De Broe, M.E., Pollet, D., De Boever, J., Vanderkerckhove, D., and Fiers, W. (1985). Eur.J. Cancer Clin.Oncol. 21,6571. Wahren, B., Holmgren, P. A., and Stigbrand, T. (1979). I n t . J . Cancer 24, 749-753. Warnock, M. L., and Reisman, R. (1969). Clin. Chim. Actu 24, 5-11. Wei, S. C., and Doellgast, G. J. (1980).Biochem. Genet. 18, 1097-1107. Wei, S. C., and Doellgast, G. J. (1981). Eur. J. Biochem. 118, 39-45. Whitaker, K. B., and Moss, D. W. (1979).Biochem. J. 183, 189-192. Whitaker, R. B., Byfield, P. G. H., and Moss, D. W. (1976). Clin. Chim. Actu 71, 285291. Wray, L., and Harris, H. (1982).J.Immunol. Methods 55, 13-18. Wyckoff, H. W., Handschumacher, M., Krishna, M., Murray, H. M., and Sawadski, J. M. (1983). In “Advances in Enzymology” (A. Meister, ed.), pp. 453-480. Wiley, New York. Yamamoto, H., Tanaka, M., Nakabayashi, H., Sato, J., Okodi, T., and Kishimoto, S . (1984).Cancer Res. 44, 339-344. Xue, S., and Rao, P. N. (198l).J.Cell Sci. 51, 163-171.

This Page Intentionally Left Blank

CELLULAR EVENTS DURING HEPATOCARCINOGENESIS IN RATS AND THE QUESTION OF PREMALIGNANCY S. Sell, J. M. Hunt, B. J. Knoll, and H. A. Dunsford Department of Pathology and Laboratory Medicine. The University of Texas Health Science Center at Houston, Medical School, Houston, Texas 77225

I . Introduction

The association of the use of snuff with an increased incidence of cancers of the nasal cavity by John Hill (1761) and the classic observations on the appearance at an early age and high incidence of cancer of the scrotum in chimney sweeps in England by Percival Pott (1775)are most likely the earliest documentations of chemical carcinogenesis in the Western world. (A short history of chemical carcinogenesis is presented in Table I.) Pott recognized several critical features of the process of chemical carcinogenesis: (1) A long period was required between the time of first exposure and the appearance of tumors; (2) reversible “premalignant” epidermal lesions appeared prior to the development of cancer (soot warts); and (3) removal of soot by frequent bathing could prevent development of cancer. Later, others noted increased cancer incidence in people exposed to polycyclic hydrocarbons, particularly among workers in the German dye industry.In 1930, the first synthetic carcinogenic compound was identified (1,2 :5,6-dibenzanthracene), and a number of polycyclic hydrocarbons were found to be carcinogenic. Early studies used the development of epidermal cancers on the skin of exposed mice as the major test for carcinogenicity (see review by Heidelberger, 1975). The experimental induction of cancer of the liver by chemicals was first reported by Sasaki and Yoshida (1935), who noted liver cancer in rats treated with o-aminoazotoluene. Since then, a growing number of investigators have used various hepatocarcinogens to study the process of chemical induction of cancer of the liver (see reviews by Farber, 1963, 1980, 1981, 1982a,b; Pitot and Sirica, 1980; Becker, 1981; Williams, 1980; Sell and Leffert, 1982; Rabes, 1983). Sasaki and Yoshida (1935) first described a sequential series of cellular changes that have become the focus of most of the pathological analysis of the 37 ADVANCES IN CANCER RESEARCH, VOL. 48

Copyright 0 1987 by Academic Press, Inc.

All rights of reproduction in any form resewed.

38

S. SELL ET AL.

TABLE I A BRIEFHISTORY OF CHEMICAL CARCINOGENESIS Date

Investigators

1761

John Hill

1775

Percival Pott

1875

Von Volkmann

1915

Yamagiwa and Ichikawa Kennaway

1930s 1940s

Mottram, Row, Berenblum, and Shubik

1956

Doll

Observations Cautions against the immoderate use of snuff Scrota1 carcinoma in chimney sweeps Skin cancer in oil and tar workers in Germany Induced tumors on ears of rabbits with coal tar Isolated carcinogenic substances in coal tar benzo[a]pyrene Concept of initiation and promotion

Epidemiologic proof of association of lung cancer with smoking

early cellular events preceding liver cancer. These changes included foci of cellular change and nodular proliferation which appeared before liver cancer. I n the past few years, it has been recognized that the cellular changes induced in the liver of experimental animals by carcinogens are much more complex than previously recognized (Sell and Leffert, 1982). The purpose of this review is to consider in some detail these early carcinogen-induced changes and their significance. We will begin with a brief review of the activation of carcinogens by host metabolism and the concepts of initiation and promotion. These subjects have been reviewed in more detail elsewhere (see below) and are presented here as a brief orientation to the main subject of this review: early cellular and biochemical changes in the liver of rats exposed to chemical hepatocarcinogens. II. Activation of Carcinogens and Initiation and Promotion in the Liver

There are a large number of inorganic and organic chemicals that can cause cancer of the liver. In this review, we will consider only a few examples of those chemicals that cause cancer of the hepatic parenchymal cells and will not review cancers of stromal or ductular

HEPATOCARCINOGENESIS AND PREMALIGNANCY

39

cells. Most hepatocarcinogens are not active until they are metabolized (Heidelberger, 1975; Miller, 1970, 1978).Activated carcinogens are short-lived electrophilic forms that bind to tissue components, not only DNA, but also RNA and protein molecules. Most hepatocarcinogens are specific for the liver because the parent carcinogenic compound is selectively metabolized by enzymes in liver cells. These enzymes belong to the mixed function oxidase system, e.g., P-450 and P-448. The levels of these enzymes in liver are relatively high compared to other tissues, thus apparently explaining why these compounds selectively induce cancer of the liver. Even so, the major metabolic pathway for carcinogens in the liver results in inactivation of the parent compound rather than activation. Thus, most of the carcinogen metabolites are detoxified by aromatic ring hydroxylations, conjugations, glucuronidations, and hydrations to water-soluble substances that are excreted from the body in urine or bile. A relatively small fraction of the parent carcinogen is “activated” to an electrophilic form which starts the carcinogenic process by binding to cellular macromolecules. Cells that have been altered by carcinogen exposure are said to be initiated. Initiation means that the cell has acquired characteristics which may be expressed by cancer. The relationship of carcinogen binding to “initiation” is not well understood (Smuckler, 1983a,b). It is thought that cells which have acquired carcinogen adducts are initiated; however, it is not clear which carcinogen adducts are critical. The molecular events following initiation are also not well understood. This situation has inspired the “black box” analogy; i.e., the carcinogen is activated and binds to cellular macromolecules; then there is a black box where something happens that we do not understand, and, usually much later, cancer appears. The key to opening the black box is a clear understanding of the cellular events which follow hepatocarcinogen exposure. Once a cell has been initiated, it is believed to be altered in such a way that further events will result in expression of the cancer phenotype. Events permitting or stimulating expression of the cancer phenotype are generally growth-promoting events, and the process whereby the cancer phenotype is called forth is termed promotion. Recent evidence suggests that initiated cells may respond to cell membrane active growth factors and eventually become autonomous, either by producing their own growth factor (autocrines) or by devel: oping metabolic capabilities that bypass the requirement for growth factors (Goustin et al., 1984).

40

S. SELL ET AL.

A. MODELSOF HEPATOCARCINOGENESIS Different hepatocarcinogens and different carcinogenic regimens produce markedly different cellular reactions in the liver (Table 11) (Sell and Leffert, 1982; Sell et al., 1983). Earlier studies by Farber emphasized the similarity of morphological changes induced by different carcinogens in the liver prior to the development of cancer (Farber, 1956, 1973). Essentially those changes first seen by Sasaki and Yoshida (1935) were highlighted. In the classic model, small collections of altered hepatocytes arise which stain more basophilic than normal liver cells. This is followed by “nodules” of altered hepatocytes that compress the adjacent normal-appearing liver cells. These nodules increase in size during the carcinogenic process, and their morphological appearance suggests that they are the precursors of hepatocellular carcinomas. Because of this, they became known as “premalignant nodules.” The term premalignant should currently be restricted to describe a condition in which carcinogen exposure has resulted in initiation rather than to identify a particular cell population, since the precursor lesions to cancer have not been unequivocally identified. Cell populations believed to be at higher risk for cancer development are called putative premalignant cells. In the past few years a number of different regimens for exposing rats to chemical hepatocarcinogens as well as different chemicals have been developed in order to study different aspects of carcinogenesis. A limited description of some carcinogenic regimens will now be presented: the classic method of Teebor and Becker (1971) to produce nodules, several models that we have used to delineate different kinetics of a-fetoprotein production as related to putative premalignant cellular changes (Sell et al., 1983), and finally, some complex protocols designed to analyze the selective effects of promoting agents.

1 . Cyclic Feeding of 2-AcetylarninofEuorene (AAF) AAF has been used extensively as a hepatocarcinogen to study the early cellular events in the liver which precede hepatocellular carcinoma. In 1971, Teebor and Becker introduced a novel method of cyclic feeding of AAF that brought out the morphological sequence of foci to nodules to cancer. Since AAF is toxic and will kill the rats if administered at high carcinogenic doses of 0.06% continuously, Teebor and Becker fed 3-week “cycles” of a diet containing 0.06% AAF following a week of normal diet. With use of this regimen, increasing histological changes were noted at the end of each feeding cycle of 3 weeks on, 1 week off, of an AAF-containing diet. After the

TABLE I1 AFP PRODUCTION AND MORPHOLOGICAL CHANGES AFTER EXPOSURE OF RATS TO CHEMICAL HEPATOCARCINOGENSO AFP production Liver morphology Early

Late

(1-4 weeks)

Early

Late (10-18 weeks)

AAF Ethionine DAB

Foci, OC Foci, OC Necrosis, proliferation of

Nodules, OC, AH ducts Nodules, OC, AH ducts Nodules, OC, AH

++ ++ ++

+ + +++

DEN WY 14643 DEN, AAF, PH CD CD + carcinogen (AAF, ETH, DEN)

Foci, minicarcinomas Hepatocyte proliferation Foci, OC nodules Hepatocyte proliferation Massive oval cell proliferation

Nodules, AH, few OC Nodules, AH Nodules, OC, AH 0 Nodules, OC

0

++

+++ ++++

?

0 ? 0

0 ? 0

Carcinogen

oc

+ +

+++

++

Hepatomas ++++too +++too

+++

Abbreviations: OC, oval cells; AH, atypical hyperplasia; PH, partial hepatectomy; CD, Choline deficiency. (Modified from Sell et al., 1983.)

42

S . SELL ET AL.

first cycle, small foci of liver cells with altered staining characteristics, usually more basophilic than normal liver cells, were seen. After the second cycle, “micronodules” of larger foci that compressed adjacent liver tissue slightly were found. After three cycles, many larger nodules up to 1 cm in diameter distorted the liver grossly. If no more carcinogen was administered, all these changes were reversible. After four cycles, even larger nodules distorted the liver. Approximately 20 weeks following the fourth cycle, almost all animals developed liver cancer, even though no more carcinogen was given. During this 20week period, most of the hundreds of nodules present after the fourth AAF feeding cycle “involuted,” and usually only one or sometimes two cancers per liver resulted. This model has been used to produce foci and nodules for study, but, as we shall see below, other changes must also be evaluated. Clearly, in addition to foci and nodules, oval cell proliferation and duct formation as well as zones of atypical proliferation are noted with this regimen (Sell, 1978).

2 . Ethionine In 1953, Popper et al. first reported on the carcinogenic effects of ethionine, and Farber and his co-workers (Farber, 1963, 1981, 1982a,b) have used this model extensively to analyze the relationship of foci and nodule formation to the development of cancer. Ethionine administration produces a sequence of morphological events similar to those induced using AAF (Farber, 1956,1963). An often used protocol is to feed ethionine in increasing concentrations from 0.25 to 0.8% for a period of 16 weeks. From 4 to 8 months after this feeding is discontinued, hepatocellular carcinomas will develop. Early after the 16 weeks of feeding, livers of rats will have foci, oval cells, and later nodules prior to the appearance of cancer.

3. 3’-Methyl-4-dimethylaminoazobenzene(3I-Me-DAB) The early effects of 3I-Me-DAB are similar to, but different frcm, those seen with ethionine and AAF (Kinosita, 1937; Opie, 1944). The carcinogen is fed at a concentration of 0.05%. Early changes also include extensive proliferation of bile duct cells, much more so than with AAF or ethionine (Kinosita, 1940). In addition, a high incidence of cholangiocarcinomas is also found after 3I-Me-DAB feeding (Reuber et al., 1972), in contrast to a low incidence of this carcinoma observed with the other hepatocarcinogens.

HEPATOCARCINOGENESIS AND PREMALIGNANCY

43

4 . Diethylnitrosamine (DEN)

The early cellular changes following continuous administration of DEN are more subtle than those seen after AAF or ethionine (Magee and Barnes, 1967;Becker and Sell, 1979).In fact, there is very little early change noted. Some disruption in the normal orientation of the hepatocellular cords with formation of small islands of cells distinguishable morphologically and by altered histological staining may be seen. After a certain period, small collections of cells with morphological characteristics of “minicarcinomas” may be observed. Once these changes take place, overt and rapidly expanding hepatocellular carcinomas become obvious. It appears that carcinomas may arise in the livers of DEN-treated rats without clear-cut premalignant lesions (Dunsford and Sell, unpublished data).

5. Peroxisome Proliferators A number of drugs which have been proposed to lower blood lipids of human patients have been shown in long-term experiments to induce liver cancer in rats (Reddy and Lalwani, 1984).WY 14643 has been chosen as one of our examples because of its effects on serum AFP concentrations (Reddy et al., 1979).WY 14643 is administered by feeding at a concentration of 0.1% for periods up to 16 months. After this time, essentially all treated animals will develop hepatocellular carcinomas. Within 1 week, there is a marked increase in the number of dividing cells in the liver. After this time, there appears to be little or no alteration in the liver structure for some months, but neoplastic nodules are seen after prolonged exposure. WY 14643 also serves as a promoter for liver carcinogenesis (Reddy and Rao, 1978).Ciprofibrate and dl-(2-ethylhexyl)phthalate are examples of peroxisome proliferators now being studied (Reddy et al., 1984);Wy-14643is no longer commercially available.

B. PROMOTERS OF LIVERCARCINOGENESIS Promotion has been most extensively studied in the induction of epithelial cancer of the skin (Mottram, 1944;Berenblum and Shubik, 1947,1949;Boutwell, 1978;Pitot and Sirica, 1980;Van Duuran, 1969). In this system, croton oil, by itself not a carcinogen, will stimulate cancer formation when applied to an area of the skin previously exposed to a carcinogen such as 7,12-dimethylbenz[a]anthracene

44

S. SELL ET AL.

(DMBA) at a subcarcinogenic dose. Multiple applications of DMBA are required in order to stimulate cancer of the skin, but one application of DMBA followed by multiple applications of croton oil will be effective. Associated with carcinogen exposure in the skin is reversible focal proliferations of epithelial cells called papillomas. Promoting agents may act by enhancing the proliferation of cells in the papilloma so that ultimately a clone of cells is produced which has undergone malignant transformation. However, it is not clear that cells in the papilloma are premalignant, and it is possible that carcinomas may arise from dividing “stem” cells not in the papilloma itself (Smuckler, 1983a). Several noncarcinogenic substances promote the induction of liver cancer by hepatocarcinogens. Liver cancer promoters include phenobarbital, carbon tetrachloride, dichlorodiphenyltrichloroethane,butylated hydroxytoluene, and estrogenic steroids (Schulte-Hermann et al., 1982). Peraino et al. (1973)were the first to demonstrate two stages in hepatocarcinogenesis analogous to initiation and promotion described in dermatocarcinogenesis. They found that a diet containing 0.05% phenobarbital increased significantly the incidence of liver cancer in weanling Sprague-Dawley rats previously fed 0.02% AAF for 18 days and reduced the time at which liver carcinomas first appeared. Partial hepatectomy following carcinogen exposure (Craddock, 1972, 1974; Ishikawa et al., 1980; Hilpert, 1983) and feeding a choline-deficient diet during exposure to a carcinogen also appear to have promoting effects (Rogers, 1975). Testosterone or pituitary hormone administration to weanling male rats increases the carcinogenic effects of AAF feeding (Weisburger et al., 1964; Reuber, 1975), probably by altering the metabolism of AAF so that a lower dose is more effective (Lotlikar et al., 1964). A common feature of these promoting agents or events is the stimulation of proliferation of liver cells. However, the mechanism of action of these promoters is still unresolved. Promotion may involve selective pressure for growth or survival of initiated cells, altered metabolism of the carcinogen, or unknown changes in the effects of bound carcinogen on cellular macromolecules. Using the parallel with the skin model, Farber has proposed that nodules are essentially equivalent to skin papillomas and that prolonged exposure to a chemical hepatocarcinogen or promoters results in a differential selective advantage of initiated cells to proliferative stimuli (Farber, 1982a,b). The question remains: Which are the initiated cells?

HEPATOCARCINOGENESIS AND PREMALIGNANCY

45

C. COMPLEX REGIMENS

1 . The Solt-Farber Model A rapid model for the development of foci and nodules using two carcinogens and partial hepatectomy was developed by Solt et al. (1977) in order to permit more detailed and convenient study of these lesions. For this protocol, animals are first given an injection of 200 mgkg of DEN.The animals are allowed to recover for 2 weeks from the necrosis that follows this injection. They are then placed on a diet containing 0.02% AAF for 2 weeks. After 1 week on the AAF diet, a two-thirds partial hepatectomy is performed. Following this, the animals rapidly develop nodular liver lesions. The authors explain this process as follows: The DEN serves as the initiator, the AAF inhibits proliferation of noninitiated hepatocytes, and the partial hepatectomy stimulates the proliferation of the initiated hepatocytes. Regardless of whether this explanation is correct, the model does provide a convenient system for the production and study of nodules.

2. Choline Deficiency Rogers (1975) reported that rats fed a lipotrope-deficient, high fat diet along with AAF developed hepatocellular carcinomas more rapidly and in higher incidence than did animals fed AAF in a normal diet. More recently, this regimen has been modified to develop a system to produce rapid proliferation of cells after carcinogen exposure (Shinozuka et al., 1978a,b, 1979). It is not clear how a cholinedeficient diet enhances the effects of a hepatocarcinogen. It is thought that the choline-deficient diet may modify the metabolism of the carcinogen. Since choline deficiency alone may stimulate proliferation of liver cells (Sell et al., 1981a), it is also possible that choline deficiency somehow acts like a promoter in providing a selective proliferation stimulus to initiated cells. The choline deficiency AAF feeding regimen results in rapid proliferation of bile duct-like cells preceding nodule formation and produces cancers more rapidly and in higher frequency than with AAF fed in a normal diet. The nature of the proliferating cells will be discussed further below. In addition, administration of DEN to rats fed a choline-deficient diet results in an early proliferation of oval cells not seen with DEN alone (Sell et al., 1983).

46

S. SELL ET AL.

D. BILE DUCTPROLIFERATION Proliferation of bile ducts in the apparent absence of proliferation of hepatocellular elements may also be chemically induced. Two chemicals, 4,4'-diaminodiphenylmethane (DDPM) (Fukushima et al., 1979) and l-naphthylisothiocyanate (ANIT) (Lopez and Mazzanti, 1955; Goldfarb et al., 1962), have been used for this effect. The mechanism of action of these chemicals is not known. The relationship of bile duct proliferation to the process of chemical hepatocarcinogenesis will be considered later in this chapter. Ill. Markers for Cellular Lineage during Hepatocarcinogenesis

The identification of which cellular changes are precursors of cancer has been elusive because no clear phenotypic marker for putative premalignant cells has been identified. Most hypotheses have developed from morphological studies of livers of rats after exposure to hepatocarcinogens. Two phenotypic markers have been studied extensively, the enzyme y-glutamyltranspeptidase (GGT)and the serum protein a-fetoprotein (AFP). Because of our interest in AFP, this marker will be covered more extensively in this review. The relationship of GGT activity to hepatocarcinogenesis has recently been reviewed in detail by Hanigan and Pitot (1985b). A. SERUM AFP CONCENTRATIONS AND HEPATOCARCINOGENESIS Our insight into the question of whether different hepatocarcinogens might produce different effects was influenced greatly when the kinetics of AFP elevation in the serum of rats exposed to different carcinogens were compared (Sell and Leffert, 1982; Sell et al., 1979a; Sell, 1980).AFP is a serum protein with properties similar to those of albumin. It is produced in large amounts in the fetal liver and yolk sac so that the serum level of AFP at birth in the rat is 5000 pg/ml (Sell et al., 1974). This level is maintained until 4-5 weeks of age when liver cell proliferation ceases. The serum concentration then quickly falls to an adult level of less than 0.06 pg/ml. Garri I. Abelev (Abelev et al., 1963) first observed that elevations well above the adult normal level occurred in adult mice who bore hepatocellular carcinomas (see Abelev, 1971). It was quickly confirmed that high levels of serum AFP were found in every species examined when hepatocellular carcinoma was present and that the AFP was synthesized by the tumors. However, not all hepatocellular carcinomas make AFP, but most do

47

HEPATOCARCINOGENESIS AND PREMALIGNANCY

(Sell and Becker, 1978). In the human, between 80 and 90% of patients with hepatocellular carcinoma will have an elevated serum AFP during the course of the growth of the tumor (Sell, 1980). In the rat, serum concentrations of AFP over 10,000 pg/ml are found with some transplantable hepatocellular carcinomas, but the serum concentration produced by different hepatomas varies greatly (Sell and Morris, 1974). The kinetics of serum AFP elevations after exposure to three selected hepatocarcinogens as examples of different effects is illustrated in Fig. 1. Following feeding of AAF, there is a rapid elevation of AFP to about 10 times the normal level (Becker and Sell, 1974). This elevation is maintained for 16 weeks after exposure before gradually dropping back to almost normal. In 80% of the rats that develop hepatocellular carcinoma, reelevations of AFP are seen. 3’-Me-DAB produces a low early elevation followed by a rapid rise to a second plateau and then a final increase associated with the appearance of hepatocellular carcinomas. After exposure to DEN, little or no elevation of AFP is observed for several weeks (Becker and Sell, 1979). Then a sudden rapid elevation occurs when hepatocellular carcinomas appear. Essentially all DEN-induced hepatocellular carcinomas produce AFP

I

?2 v)

P t

F - O -

0.01 WEEKS

FIG.1. Serum concentration of AFP in rats exposed to different carcinogens (see text). DEN, Diethylnitrosamine; DAB, dimethylaminodiazobenzene; FAA,N-2-acetylaminofluorene; WY 14643,hypolipidemic agent; AFP, 0-fetoprotein.

48

S. SELL ET AL.

elevations. The time between exposure to DEN and the onset of the elevation of serum AFP is directly related to the dose of DEN; i.e., the higher the dose of DEN, the shorter the time until AFP elevations are seen. WY 14643, a hypolipidemic agent, induces an early rise in AFP that correlates with induction of hepatocyte proliferation. With continued exposure to WY 14643, the serum AFP falls to normal (Reddy et al., 1979). After many weeks, the tumors that arise do not produce serum AFP elevations. These observations suggested to us that different cellular events might be associated with the different effects of the hepatocarcinogens (Sell et al., 1979a; Sell, 1980). Elevations of serum AFP in the adult may be associated with noncarcinogenic as well as carcinogenic events. Noncarcinogenic hepatotoxic agents such as thioacetamide and CC14 will cause a temporary elevation of serum AFP related to the “step-down” phase of liver regulation following liver cell necrosis (Smuckler et al., 1976a). This pattern is similar to that seen following restituitive liver proliferation after partial hepatectomy (Sell et al., 1974).In some situations, such as after administration of phenobarbital (Smuckler et al., 1976b), serum elevations of AFP may occur prior to liver cell proliferation. Thus, an elevation of serum AFP in an adult does not necessarily indicate carcinogen exposure. Carcinogens usually produce prolonged elevations over weeks, whereas those seen with liver cell proliferation last only 5 or 6 days.

B. CELLULAR AFP AS A MARKERFOR HEPATOCARCINOGENIC EVENTS Further insight into the cellular events occurring early after hepatocarcinogen exposure in rats is obtained by analyzing the production of AFP at the cellular level (Sell and Leffert, 1982). In our original experiment, the serum AFP concentrations were determined using a sensitive radioimmunoassay during the first cycle of AAF feeding (Becker and Sell, 1974). As mentioned, the serum AFP concentration unexpectedly became elevated within the first few days and remained elevated for more than 16 weeks, 13 weeks longer than the 3-week exposure to AAF. During this time, very little cellular change was seen in the livers of the exposed rats. In the next experiment, the full four cycles of AAF were fed. Again, the serum AFP concentrations rose within a few days after feeding AAF. However, to our surprise the serum concentrations fell during the subsequent cycles so that at the time of maximum nodule formation the serum concentrations had returned to almost normal (Sell et al., 1983). The negative correlation of elevated serum AFP to the progressive development of nodules raised

HEPATOCARCINOCENESIS AND PREMALIGNANCY

49

the question of which cells in the liver were producing AFP after carcinogen exposure. Since most hepatocellular carcinomas induced by this regimen did produce AFP, the presumption was that the cells that the tumors came from would also produce AFP. In an immunofluorescent study, it had been reported previously that AFP was found in nodular cells (Okita et al., 1974). In the above experiment, we were unable to confirm this and became suspicious that AFP might actually be produced by cells other than nodular cells. In another series of experiments using Fischer rats instead of the Sprague-Dawley rats used previously, serum AFP concentrations continued to rise during four cycles of AAF feeding to much higher levels than seen in the Sprague-Dawley rats (Sell, 1978). In this experiment, serum AFP concentrations remained high when maximum nodule formation was present. It was felt that this situation now provided a high likelihood of determining which cells contained AFP. A systematic immunofluorescent examination of multiple sections from livers from rats with massive nodule formation and high serum AFP concentrations failed to identify AFP in nodular cells, even though albumin was easily detectable in most nodules (Sell, 1978). The observation that AFP was not found in nodular cells was reported by three laboratories independently at about the same time (Tschipysheva et al., 1977; Sell, 1978; Kuhlmann, 1978). After careful searching, AFP was found in nonnodular cell populations. These included small rounded oval cells, larger cells in a glandular-like organization (atypical hyperplasia), and occasionally in duct-like structures. Several Japanese pathologists had previously described the localization of AFP in oval cells (Dempo et al., 1975; Onoe et al., 1975; Fujita et al., 1975; Onda, 1976), but their observations had not received much attention because of the focus of the major hepatocarcinogenesis laboratories on foci and nodules as the putative premalignant cells. Our attention now turned to these other cell types, in particular oval cells. A short history of oval cells is presented in Table 111. In point of fact, small oval cells were among the first new cell types seen early after carcinogen exposure (Opie, 1944). The term oval cell was coined by Farber (1956), and Popper et al. (1957) concluded that these cells belonged to bile duct lineage because of the associated appearance of duct-like structures with oval cell proliferation. Since the cancers that develop after hepatocarcinogen exposure are primarily hepatocellular carcinomas and not ductular cancers, it is understandable that most attention was given to nodules. In most regimens where nodules

50

S. SELL ET AL.

TABLE 111 A CHRONOLOGY OF FINDINGS ON OVALCELLS

1944 1954 1956 1961 1964 1975 1981 1985

Opie describes appearance of small cells in the liver after exposure to butter yellow Price et al. note similar cells after DAB exposure Farber coins the term oval cells Popper concludes oval cells arise from bile duct proliferation Grisham and Porta note two distinct cell types, “mesenchymal” and “ductal,” during oval cell proliferation Dempo, Onoe, and Fujita demonstrate the localization of AFP in oval cells Sell et al. identify a periportal “stem” cell that participates in oval cell proliferation Yaswen et al. note increased expression of protooncogenes (c-myc and c-Kras) in oval cells when compared to normal liver

are produced, extensive oval cell proliferation also is seen. Thus, one must be careful in making conclusions regarding cells taken from “nodular livers,” as multiple cell types are present. C. OVALCELLPROLIFERATION Grisham and Porta (1964), on the basis of an autoradiographic study 1 month after ethionine feeding, concluded that proliferation of duct cells accounted for the oval cell types and proliferation of hepatocytes for the larger cells seen in nodules. In order to determine the origin of the oval cell population in our studies, we performed autoradiographic analysis of the cells that proliferate rapidly after the feeding of AAF in a choline-deficient diet (Sell et al., 1981b). In this protocol, Fischer male rats are fed a choline-deficient diet containing 0.05% AAF for 10-12 days followed by normal diet without carcinogen. This results in moderate proliferation of oval cells for the first 10-12 days, a much more rapid proliferation from days 12 to 22, and then a gradual involution or loss of oval cells after that. Nodules are not seen in this model until 28 days. Tritiated thymidine was injected on days 0, 1,2, 3, 7, 10, 14, 18, 21, 24, 28, 32, and 34 to different individual animals (see Fig. 2). Each animal was sacrified 24 hr after tritiated thymidine injection, and autoradiography of sections of the liver was performed after immunofluorescent labeling for AFP and albumin. The results indicated that the proliferating oval cells initially arise from a few small cells that are located near the bile ducts and have little cytoplasmic organelles and no distinguishing morphological features that identify a particular cell type. From day 1 to day 3 these cells consti-

HEPATOCARCINOGENESIS AND PREMALIGNANCY

51

v)

J J

W

0

1

a

W

>

3

8

0

10

I

1

1

20

30

38

CD-AAF DIET] PURINA LAB CHOW DIET 7 0

14 18 21 2 4 2 8 3 2 3 4 8 !

cbHdT

DAYS

FIG.2. Estimated increase in oval cells in livers of Fischer rats fed 0.06% N-2acetylaminofluorene in a choline-deficient diet for 12 days. The numbers at the bottom of the figure indicate the days that treated thymidine was injected 24 hr before sacrifice for autoradiography.

tute most of the proliferating cells. After day 3, proliferation of bile duct cells also becomes prominent. Electron microscopic autoradiography confirms that the first cells to proliferate are nondescript periportal small oval cells. At the height of oval cell proliferation many new duct-like structures appear in the midzone of the liver (Sell and Salman, 1984). These duct-like structures are usually heavily labeled when they first appear, indicating a rapid appearance. They also usually contain AFP and albumin, markers of liver cells rather than duct cells. Proliferation of oval cells induced by the choline-deficient AAF diet is associated with an exponential rise of AFP in the serum that correlates with the number of proliferating oval cells (Sell et aZ., 1981a). Many of the rapidly proliferating oval cells also contain AFP and albumin (Sell, 1983).To determine if the duct-like structures are related to true bile ducts, the bile ducts of carcinogen-fed animals were injected with a pigmented barium gelatin medium, and it was possible to demonstrate clearly that these newly formed ducts are connected to the true bile ducts (Dunsford et al., 1985). Bile duct proliferation induced by DDPM or ANIT is not associated with elevation of serum AFP, but the newly formed ducts are connected to the true bile ducts and the proliferating cells do not contain AFP or al-

52

S . SELL ET AL.

bumin. We have concluded that oval cell proliferation following carcinogen feeding involves two cell populations: stem cell-like periductular cells and bile duct cells. This raises the possibility that stem cells might be the earliest cellular precursors in the lineage to hepatocellular carcinoma, although the relationship of either oval cell or duct cell proliferation to hepatocellular carcinoma remains unknown.

D. CARCINOGEN-INDUCED LIVERLESIONS Additional tissue alterations associated with exposure to hepatocarcinogens must also be considered (Figs. 3 and 4).When analyzing the livers of rats exposed to four cycles of AAF feeding, islands of atypical hepatocytes were observed among the large numbers of oval cells and residual hepatocytes located between the nodules (Sell, 1978). Although these were relatively few in number 10 per liver) compared to true nodules (over 1000 per liver), many of these “atypical hyperplastic” zones contained AFP (Sell, 1978; Kuhlmann, 1981). In contrast, as stated above, none of the nodules contained AFP. Since many of the hepatocellular carcinomas which arise from these regimens also contain AFP, it is possible that these atypical hyperplastic foci may represent either precursors of the eventual cancers or might actually be microcarcinomas. The argument has been made that the occasional finding of a nodule within a nodule or atypical foci or carcinoma-like cells within a nodule is proof that all cancers arise within nodules (Farber and Cameron, 1980; Solt et al., 1977). Recently, we have examined some tissues sent to us by Dr. Cameron that contained large numbers of nodules. One of these nodules contained a nodule within a nodule that was strongly AFP positive. Does this represent a “malignant degeneration” within a nodule? Can such malignant change occur outside of a nodule, perhaps with even higher frequency than within the nodule? Do nodules represent an adaptive change to the toxic effects of carcinogens, and could nodules actually represent sites that are protected against malignant transformation? This last possibility may be considered heterodoxy, but is at least partially supported by the fact that hundreds of nodules are produced by some of the carcinogenic regimens, yet only one or a very few hepatocellular carcinomas develop (Teebor and Becker, 1971; Epstein et al., 1967; Enomoto and Farber, 1982; Kitagawa, 1976; Tatematsu et al., 1983b). On the other hand, only a relatively few zones of atypical hyperplasia are seen, numerically more consistent with the number of cancers that appear. The role of atypical hyperplastic zones is further explored in the

FIG.3. Examples of cellular changes induced in the liver of rats by chemical hepatocarcinogens. (A) AFP-positive atypical hepatocytes (atypical hyperplasia). Immunoperoxidase. x400. (B) Neoplastic nodules. ~ 4 0(C) . Oval cells. x250. (D) AFP-positive oval cell duct structures. Immunoperoxidase. x250.

FIG.4. (A) Early AFP-positive hepatoma induced by 11 weeks of continuous feeding of DEN. Immunoperoxidase. X 100. (B) Hepatocellular carcinoma 22 months after 5 cycles of AAF. ~ 2 0 0(C) . Cholangiofibroma,end of fifth cycle ofAAF feeding. X250. (D) Cholangiocarcinoma, 22 months after 5 cycles of AAF. x250.

HEPATOCARCINOGENESIS AND PREMALIGNANCY

55

DEN model of carcinogenesis in the rat (Becker and Sell, 1979) and in the identification of early AFP-positive lesions in the mouse (Koen et al., 1983).In an earlier study, we had determined that the kinetics of AFP elevation and early cellular changes in the livers of rats exposed to DEN were substantially different than with AAF. Very little if any oval cell proliferation occurs, and serum AFP elevations do not appear until after 6-20 weeks, depending upon the dose of DEN.In examining the livers of rats at the time of the earliest AFP elevations, small islands of atypical hepatocytes were found to be AFP positive (Dunsford and Sell, unpublished data). Further study of the fate of these islands and their relationship to hepatocellular carcinoma is under way. In the mouse, small hyperplastic lesions which contain AFP are seen early after carcinogen exposure and compress hepatic veins even when small (Koen et al., 1983). No serum AFP elevations are seen in mice which develop hepatocarcinomas spontaneously until actual cancers develop (Becker et al., 1977). The early AFP-positive lesions of Koen et al. (1983) could represent microcancers or premalignant lesions similar to those seen in the rat, but having a different morphological appearance. E. OTHER“MARKERS” OF “PHENOPLASIA” IN THE LIVER A large number of phenotypic markers have been studied in an attempt to identify a marker that would clearly demonstrate a precursor-product relationship between one of the many presumptive premalignant liver lesions discussed above and hepatocellular cancer. To date, such markers have provided circumstantial evidence, but not proof, of lineage (Farber, 1984c; Sell et al., 1983; Tatematsu et al., 1983b; Tsao et al., 1984b; Germain et al., 1985). A partial listing of phenotypic markers in normal hepatocytes, oval cells, duct cells, nodular cells, and hepatocellular carcinomas is given in Table IV. The most extensively studied markers are various enzymes and isozymes (Pitot and Sirica, 1980; Goldfarb and Pitot, 1976; Shapira, 1973). On the basis of different enzyme and isozyme content of nodules as determined histochemically and compared to the enzyme and isozyme content of hepatocellular carcinomas (Knox, 1976), strong circumstantial evidence for a relationship between nodules and cancer was obtained. However, conclusions regarding lineage depend on which markers one uses. We have already presented in detail conclusions based on AFP expression in various cell types. From a brief examination of Table IV, one can conclude that it is not possible to make a firm choice regarding the lineage of cancer from among the various possible pre-

56

S. SELL ET AL.

TABLE IV MARKERSOF MATURECELLTYPES IN LIVERUNDERGOING CHEMICAL CARCINOGENESIS”

Albumin AFP Keratin Enzymes ATPase p-Glucuronidase DT-diaphorase Epoxide hydrolase GGT Glucose 6-phosphataseC Isozymes Aldolase Pyruvate kinase

Hepatocytes

Bile ducts

Oval cellsb

Nodules

Carcinomas

++

0 0

++ ++ +

++

+ or0 ++, + or 0 +

0

0 0 0 0

0 0

0 0

+ 0 0 0

++

B L

++

0 0

+ + +

+ or0

+ or0

+ or0

A

A, B, and C K, K-L

0

K

0

+ or0 + + + 0

A, B, and C

Modifed from (Tatematsu et al., 1983a; Sell et al., 1983; Tsao et al., 1984a). Data for zones of atypical hyperplasia are insufficient for inclusion. Evaluation of markers for oval cells is complicated by the possibility of more than one cell type, i.e., stem cells and duct cells, being indistinguishable histologically. Conflicting data exist regarding the presence of glucose 6-phosphatase in bile duct and oval and nodular cells. (See Tatematsu et al., 1983a; Tsao et al., 1984a.) (I

malignant cell populations. Such a conservative conclusion leaves open the possibility of different pathways to liver cancers. The crux of the problem is that the phenotype of each hepatocellular carcinoma appears to be different from that of other hepatocellular carcinomas in some way. This is not surprising in view of the tremendous variation in chromosome composition found in different hepatocellular carcinomas (Nowell et al., 1967; Wolman et al., 1972). A marker that reflects the malignant and premalignant phenotype is clearly needed. Elevated expression of the placental isozyme of glutathione S-transferase (GST-P)has recently been described as a new marker phenotype associated with premalignant rat liver lesions (Sato et al., 1984). The association of the two glutathione-related enzymes, GST-P and GGT, as well as other detoxifying enzymes, such as epoxide hydrolase, with such lesions is consistent with in v i t r o analogs of the Haddow (1938) hypothesis discussed below. As useful as these markers are, however, they cannot as yet reliably indicate lineage relationships. We have also examined the extracellular components, fibronectin and laminin, in hepatocellular carcinomas and in the livers of carcino-

HEPATOCARCINOGENESIS AND PREMALIGNANCY

57

gen-treated rats (Sell and Ruoslahti, 1982; Sell, 1983). Fibronectin is found diffusely in normal liver lining the sinusoids and in connective tissue around vessels and ducts; laminin is found only in the basement membranes of vessels and ducts. In transplantable hepatocellular carcinomas, the degree of fibronectin staining in the connective tissue varied, and positive laminin staining was limited to vascular structures within the tumors. Neoplastic nodules contained very little fibronectin and no laminin. Large deposits of fibronectin were seen in areas of oval cell proliferation. Laminin was found around the newly formed duct-like structures seen after carcinogen treatment. Though these studies are of some interest, they do not provide data that can be applied to answer the lineage question other than to support the negative association between cells with bile duct properties and hepatocellular carcinomas. It should be stressed here that not all oval cells have bile duct properties and that the potential for minor cell types to serve as precursors for hepatocellular carcinoma must be kept in mind.

F. SUMMARY On the basis of the morphological changes seen in the liver, we have proposed different possible cellular lineages following exposure to hepatocarcinogens culminating in cancer (Sell and Leffert, 1982; Fig. 5). More recent observations described above suggest even more interconnecting pathways. These are illustrated in Fig. 6.

IV. Monoclonal Antibodies in Chemical Carcinogenesis

The study of tumor antigens in both humans and in experimental systems has been greatly enhanced by the introduction of hybridoma methodology by Kohler et al. in 1975. In contrast to conventional antisera, monoclonal antibodies react with individual antigenic determinants (epitopes) and theoretically permit identification of unique epitopes on cancer cells. Conventional antisera are composed of a mixture of antibodies of different specificities from which unique or tumor-associated specificities may not be distinguished. During the past 10 years, monoclonal antibodies have largely replaced most conventional antisera used in the study of tumor antigens (Sevier et al., 1981). The majority of the literature on monoclonal antibodies and cancer is concerned with the diagnosis and treatment of human cancer using new approaches for imaging and treatment via monoclonal anti-

58

S. SELL ET AL.

CANCER

t

\

PORTAL

FIG.5. Possible cellular lineages in experimental hepatocarcinogenesis in rats. Possible relationships between putative premalignant cellular changes in the livers of rats exposed to chemical carcinogens and the malignant tumor that eventually appears are depicted. Hepatocellular carcinomas may arise from altered hepatocytes (foci) that progress to nodules and then to cancer. Another putative premalignant cell population is the oval cell, which may progress directly to cancer or be a precursor lesion to nodules or areas of atypical hyperplasia that are the ultimate premalignant lesions. It is possible that neither nodules nor oval cells are premalignant and that carcinomas arise from altered hepatocytes either directly or from areas of atypical hyperplasia. (From Sell and Leffert, 1982.)

bodies directed at tumors (Sell and Reisfeld, 1985; Reisfeld and Sell, 1985). The application of monoclonal antibodies to the study of chemical carcinogenesis has been far more limited, possibly due to the fact that chemically induced tumors are at best weakly antigenic compared to virally induced tumors, and most chemically induced tumors show marked antigen heterogeneity with little cross-reactivity. In this section, we will review monoclonal antibodies applied to (1)chemically induced tumors other than hepatocellular cancers, (2)normal liver, (3) carcinogen metabolizing enzymes, (4) carcinogen adducts in the liver, and (5) preneoplastic cell populations in the liver.

A. MONOCLONAL ANTIBODIES TO MURINETUMORS Table V summarizes some of the reports of monoclonal antibodies to chemically induced tumors other than hepatomas in mice and rats.

HEPATOCARCINOGENESIS AND PREMALIGNANCY

59

CE

FIG.6. Some possible cellular lineages during hepatocarcinogenesis (updated). The classic pathway is through development of altered foci to nodules to cancer. Hepatocellular cancer may also arise from “stem” cells located in the portal area that are stimulated by carcinogens to proliferate and form oval cells. Oval cells may serve as precursors for nodules, atypical hyperplastic zones, or may progress to cancer without an intermediate form. Atypical hyperplastic zones may arise from oval cells or directly from initiated hepatocytes. It is also possible that none of the so-called premalignant lesions is in the direct lineage to cancer; cancer may arise directly from initiated hepatocytes without a morphological intermediate. Atypical hyperplastic zones may, in some instances, actually be microcarcinomas. The part played by bile duct proliferation and the duct-like structures induced by carcinogen exposure is not clear. These structures may have the capacity to differentiate into “normal” hepatocytes or evolve into liver cancers, atypical hyperplastic zones or biliary cancer. Regardless of the potential of each of these “preneoplastic” lesions to develop into cancer, it appears that each cell type can differentiate into a “normal” hepatocyte. Thus, most of the tissue alterations seen will “involute” or “remodel” with time after exposure to the carcinogen, but a few cells will develop malignant transformation. These cells may be in a preneoplastic lesion or may be unrecognizable at present from normal hepatocytes.

The unique tumor epitopes identified have not been sufficiently characterized to determine their significance. Other epitopes are on other previously identified cellular molecules. Gunn et al. (1980)used a cell line derived from a mammary adenocarcinoma, which occurred spontaneously in a rat to immunize syngeneic rats, and fused the immunized rat spleen cells with a mouse

S. SELL ET AL.

60

TABLE V MONOCLONAL ANTIBODIESTO CHEMICALLY INDUCED TUMORS Monoclonal antibody to: Murine mammary adenocarcinoma, spontaneous Murine adenocarcinoma, N-eth yl-N-nitrosourea Transitional cell carcinoma, mouse, 3methylcholanthrene Mouse fibrosarcoma, carcinogen not specified Adenocarcinoma of colon, rat, 2-azoxymethane Rhabdomyosarcoma, rat, nickel sulfide

Comment

Reference

Specific for tumor

Gunn et al. (1980)

Identifies I-A(%)antigen One to tumor only, one to tumor and liver Multiple to MULV(l), 3 to unique tumor antigens Small intestine type alkaline phosphatase Specific for desmin

Giedlin et al. (1983) Hellstrom et al. (1982) Klein (1981) Owens and Hartman (1984) Altmannsberger et al. (1985)

myeloma. Screening of the resulting hybridomas produced one monoclonal antibody that was specific for the cell line, but it did not stain other mammary adenocarcinomas and therefore was not useful as a general tumor-specific marker. Gunn et al. felt that the immunization of the rat would eliminate screening the many irrelevant hybridomas which would have been produced by immunizing a mouse; however, the number of tumor-specific hybridomas does not appear to differ from other investigators and our own work (see below) with xenogeneic immunization of the mouse. Hellstrom et al. (1982) used a tumor line derived from a chemically induced mouse bladder carcinoma to immunize rats. Fusion with mouse myeloma cells produced two monoclonal antibodies specific to this cell line. One of the monoclonal antibodies also reacted with hepatocytes. Klein (1981) demonstrated that chemically induced tumors in mice contain predominantly antigens associated with mouse leukemia virus (MULV), although they were able to produce three monoclonal antibodies to antigens specific to three separate tumors. Giedlin et aE. (1983) used a monoclonal antibody to the I-A(k) region of the major histocompatibility complex present on a mouse adenocarcinoma line to study this antigen in vivo and in vitro. They found that the antigen was present in vivo, but disappeared with even very brief growth in vitro, and would reappear when passed in vivo. This demonstrates the usefulness of monoclonal

HEPATOCARCINOGENESIS AND PREMALIGNANCY

61

antibodies in following an epitope over time. Owens and Hartman (1984)have used a monoclonal activity that reacts with the small intestine isotype of alkaline phosphatase in the rat to characterize the alkaline phosphatase produced by two chemically induced colon carcinomas, which appeared to be of the small intestine type. This monoclonal antibody was capable of immunoprecipitating the antigen without inhibiting its enzyme activity, which allowed the study of antibody binding on the same gels used for isotyping the alkaline phosphatase. Although somewhat peripheral to the study of hepatocarcinogenesis, these studies demonstrate several features of the use of monoclonal antibodies in the study of chemically induced tumors: (1)Chemically induced tumors do have specific antigens, although to date most appear to be largely specific for individual tumors so that they may not be useful in distinguishing other malignant cells from the normal or premalignant parenchymal cells; (2) epitopes detected by monoclonal antibodies can disappear and reappear when studied over time; and (3)careful selection of monoclonal antibodies can produce reagents which will aid in the further study and characterization of tumor-associated antigenic markers.

B. MONOCLONAL ANTIBODIES TO LIVERCELLS Monoclonal antibodies to liver cells have been used in the study of hepatocellular proliferation and chemical hepatocarcinogenesis. Table VI summarizes the reported monoclonal antibodies to rat liver and gives the structures identified. Behrens and Paronetto (1978)prepared polyclonal antisera to mouse liver plasma membranes. The initial antiserum was membrane specific, but not organ specific. Repeated absorptions were required to produce a liver-specific antisera which reacted with the entire cell surface. In contrast, careful screening of hybridomas prepared to similar antigens has resulted in a large number of liver-specific monoclonal antibodies to a variety of cellular and subcellular structures. Most of these monoclonal antibodies are in an early stage of characterization, and their potential to define better the cellular events in carcinogenesis has not yet been realized. Holmes et al. ( 1984) produced four monoclonal antibodies by immunizing mice with normal rat hepatocytes. Each monoclonal antibody recognizes a different epitope on the hepatocyte, as indicated by the specific sequence of the appearance of the epitope in developing fetal liver and the variable staining of 32 primary 3’-Me-DAB-induced hepatocellular carcinoma. Using the four monoclonal antibodies to stain the 32 primary tumors, they identified four phenotypes which differ from the

62

S. SELL ET AL.

TABLE VI MONOCLONAL ANTIBODIESTO HEPATOCYTES Structure identified

Antigen MW

Cytoplasm, hepatocyte

-

Plasma membrane, canaliculi and biomatrix Plasma membrane, canaliculi Plasma membrane, excluding canaliculi Liver cell membrane Rat liver glucocorticoid receptor ATPase-dependent sodium pump on plasma membrane Rat liver mitochondria, inner membrane Cytokeratin, bile ducts, canaliculi

105,000 105,000- 140,000

Comment 4 monoclonal antibod-

ies, hepatocyte specific variable expression, fetal hepatocytes and tumor Glycoprotein

Hixson et al. (1984) Cook et al. (1983) Poralla et al. (1984)

Prepared to purified glycoprotein

Rat hepatocytes and neurons

Fukamoto et al. (1984) Okret et al. (1984) Schenk et al. (1984); Leffert et al. (1985) Billet et al. (1984)

Similar to human, M , 40,000

Schmidt et al. (1984a)

94,000 Inhibits Na+, K+ATPase, a subunit

39,000

Holmes et al. (1984)

Glycoprotein

45,000-50,000

Reference

phenotypes determined by GGT staining or morphology. This is the first study to use monoclonal antibodies to attempt to define phenotypes of chemically induced tumors in the rat. Hixson et a2. (1984)' used a monoclonal antibody prepared to purified rat liver biomatrix to demonstrate that some components of the liver plasma membrane share epitopes with the liver biomatrix. Their monoclonal antibody (HEP 105)may define an epitope that plays a role in the interactions between hepatocyte and extracellular matrix. Glycoprotein antigens on the plasma membrane have been studied by several investigators interested in using monoclonal antibodies to define liver-specific antigens which play a role in the differentiation of cells in development and carcinogenesis. Cook et a2. (1983) produced three monoclonal antibodies to three polypeptide antigens synthesized by cultured he-

HEPATOCARCINOGENESIS AND PREMALIGNANCY

63

patocytes and present on the plasma membrane of cultured hepatocytes, which in vitro are localized only on the bile canaliculi. Poralla et al. (1984) produced a monoclonal antibody which stained the entire plasma membrane except for the canaliculi. Fukumoto et aZ. (1984) prepared monoclonal antibodies to a purified glycoprotein antigen extracted from isolated rat liver plasma membranes. By fluorescenceactivated cell sorting, one monoclonal antibody stained liver cells and hepatoma cells. These studies demonstrate that monoclonal antibodies can be produced which define specific regions of the hepatocyte plasma membrane, even when using crude cellular fractions to immunize mice. They also demonstrate that caution must be used when comparing immunohistochemistry with other detection methods and in comparing cultured cells or isolated cells with tissue sections from whole liver. For example, Hixson’s monoclonal antibody (Hixson et al., 1984) stained the entire plasma membrane of isolated hepatocytes, but only the canaliculi of hepatocytes in frozen sections. This implies that the staining pattern may be dependent on the conformation of the cells in section and that although the antigen may be present on the whole membrane, it is only available for binding at the canaliculi in frozen sections. Studies done in collaboration with Leffert (Schenk et al., 1984; Leffert et aZ., 1983) have demonstrated that two monoclonal antibodies which inhibit the action of the ATPase-dependent sodium pump give different staining patterns in frozen sections of liver monoclonal antibody. 9B1 stains only the canaliculi, whereas 9A5 stains canaliculi and the sinusoidal plasma membrane (Dunsford, Leffert, and Sell, unpublished data). It remains to be seen whether this represents a different distribution of the epitopes bound by these monoclonal antibodies or if this is an artifact resulting from altered conformation of the hepatocytes in frozen tissue sections or some other factor. Okret et al. (1983)have developed a series of monoclonal antibodies to the glucocorticoid receptor of rat hepatocytes. These monoclonal antibodies have made possible the study of this receptor in hepatocytes even when it is in its inactive form, thus overcoming limitations hindering the study of this receptor by conventional means based on receptor activity. A monoclonal antibody which may be useful in the study of carcinogenesis is one specific for rat and human liver mitochondria (Billet et al., 1984). The use of antibodies to cytoskeletal proteins has recently been applied to chemical hepatocarcinogenesis in the rat (Schmidt et al., 1984a). These investigators had initially characterized a cytoskeletal

64

S. SELL ET AL.

protein (p39) found by conventional antisera in the Novikoff hepatoma. Although it was initially thought that the p39 protein was specific for the hepatoma (Schmidt et d.,1981), subsequent characterization of monoclonal antibodies made to the purified protein demonstrated that the monoclonal antibodies stained bile ducts, but not hepatocytes. These authors recently have studied the localization of this protein during azo dye carcinogenesis (Schmidt et al., 1985). Immunofluorescent staining of sections of liver from a series of animals treated with 3’-MDAB demonstrated positive staining of oval cells and bile ducts and atypical bile ducts during the early stages, with no positivity of hepatocytes. By 9 weeks, rare nodules contained clusters of positive hepatocytes, suggesting that some nodular hepatocytes can express this antigen. By 19-22 weeks, hepatomas were all positively stained. Collaborative studies in our laboratory have shown that p39 also stains oval cells in Fischer rats fed CDAAF diet and does not stain Morris hepatoma 7777 (Dunsford and Sell, unpublished data). Although these data are preliminary, they suggest that the original Novikoff hepatoma may have been a cholangiocarcinoma and not a hepatoma.

ANTIBODIES TO CARCINOGENIC C. MONOCLONAL METABOLIZING ENZYMES

Monoclonal antibodies have been applied to the study of the metabolism of carcinogens by the cytochrome P-450 monooxygenases in the liver. Table VII summarizes some of the reported studies using monoclonal antibodies to P-450 induced by 3-methylcholanthrene (MC) or phenobarbital (PB) in rats and monoclonal antibodies to specific subtypes of P-450. Thorgeirsson et al. (1983) have demonstrated that a monoclonal antibody to MCP-450 is able to block its function and prevent N and C hydroxylation of AAF. Friedman et al. (1983) characterized the antigen precipitated by monoclonal antibodies to MCP450 and PBP-450 as 56,000 and 54,000 Da, respectively. Either or both of these P-450 activities are induced by many carcinogens and are being used in in vitro test systems for the study of potential carcinogens in the environment. Monoclonal antibodies have been used for radioimmunoassay to detect elevations of MCP-450 (Song et al., 1983) and as probes in Western blots (Thomas et al., 1984). Wiebel et al. (1984)has demonstrated that two hepatoma cell lines are inducible for both MCP-450 and PB450 on exposure to carcinogens and may therefore be useful in in vitro testing.

65

HEPATOCARCINOGENESIS AND PREMALIGNANCY

TABLE VII MONOCLONAL ANTIBODIESTO P-450" Monoclonal antibody to:

Characteristic antigen

MCP-450

-

MCP-450, PB450 MCP-450 P-450~

56,000 54,000

MCP-450, PB450

-

-

Comment

Reference

Inhibit N and C hydroxylation of AAF

Thorgeirson et a2. (1983)

Radioimmunoassay Probe on Western blot Demonstrate induction by 2 carcinogens in a hepatoma cell line

Friedman et al. (1983) Song et al. (1983) Thomas et al. (1984) Wiebel et al. (1984)

Abbreviations: MCP-450, P-450 induced by methylcholanthrene; PB450, P-450 induced by phenobarbital; P-450c, a specific isotype of P-450 associated with carcinogen exposure.

D. MONOCLONAL ANTIBODIESTO CARCINOGEN ADDUCTS Another area of interest is the ability of monoclonal antibodies to detect carcinogens bound to DNA and specific nuclear antigens found in carcinogen-treated liver. Table VIII summarizes studies on monoclonal antibodies to carcinogen adducts. These reagents could be useful in detecting the exposure of animals or humans to carcinogens, assaying for carcinogens in the environment, and in studying cellular events during chemical carcinogenesis. Schmidt et al. (198413)studied a unique nuclear antigen which appears during azo dye carcinogenesis and is present in all azo dye-induced hepatomas examined, but is not present in normal hepatocytes or in hepatocytes exposed to certain hepatotoxins. The antigen appears to be a 55,000 Da protein consisting of part of the nuclear matrix. The remaining papers cited in Table VIII deal with the study of DNA-carcinogen adducts. Monoclonal antibodies allow the detection of adducts in the livers of exposed animals of very low concentrations. For example, it is possible to detect one aflatoxin B (1) residue per 1,355,000 nucleotides (Groopman et al., 1982).Affinity chromatography with these monoclonal antibodies can be used to purify the adducted nucleotides and enhance

66

S. SELL ET AL.

TABLE VIII MONOCLONAL ANTIBODIESTO NUCLEI,DNA ADDUCTS, OR CARCINOGENS Monoclonal antibody to: 3’-Me-DAB induced specific antigen in nuclear matrix DNA modified by BPDE-1 Aflatoxin Bl DNA complex Aflatoxin BI Aflatoxin B1-modified DNA O(6)-ethylguanosine

Comment

Reference

One 55,000, one all polypeptides from nuclear matrix Immunoprecipitate BPDE adducts by affinity chromatography Specific for two adducts of BI Detects B1, Bz, MI, and DNA adducts by radioimmunoassay Detects BI-modified DNA by ELISA Radioimmunoassay and ELISA, DNA bind in oioo and in oitro with ENU

Schmidt et ~ l . (1984b) Santella et LIZ. (1984) Groopman et al. (1982) Groopman et d . (1984) Hertzog et LIZ. (1983) Wani et d . (1984)

OAbbreviations: 3’-Me-DAB, N,N-dimethyl-(p-rn-toly1azo)aniline; BPDE-1, ENU, eth7&8a-dihydroxy-Sa, 10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; y lnitrosourea.

the sensitivity of tests for these adducts in body fluids, cells, and the environment (Groopman et al., 1984). Monoclonal antibodies to carcinogen-modified nucleotide DNA have proved more useful than monoclonal antibodies to the purified adduct (Wani et al., 1984). Further enhancement of the affinity of the monoclonal antibody to the modified DNA was achieved by using guanine-imidazole ring opened aflatoxin B (1) modified DNA (Hertzog et al., 1983).

E. MONOCLONAL ANTIBODIES TO PUTATIVE PREMALIGNANT CELLS Our laboratory has attempted to identify epitopes on new populations of cells found in the livers of rats during chemical hepatocarcinogenesis which will be useful in their identification during the different stages of evolution of hepatomas. These monoclonal antibodies are being used to isolate purified populations of preneoplastic cells by fluorescence-activated cell sorting to test their tumorigenicity by transplantation into syngeneic rats. Standard hybridoma techniques have been used following immunization of young female BALB/c

HEPATOCARCINOGENESIS AND PREMALIGNANCY

67

mice with intact cells without adjuvant. To date, we have immunized mice with partially purified fractions of oval cells from the livers of F344 rats fed AAF in a choline-deficient diet; hepatocytes isolated from the livers of F344 rats containing GGT-positive hepatocytes induced using the Solt-Farber regimen (Solt and Farber, 1976); finely minced Morris hepatomas 7777 and 5123 passaged in Buffalo rats; and finely minced neoplastic nodules taken by sharp dissection from the livers of F344 rats following the administration of a carcinogenic dose of AAF in the diet. With the exception of the first monoclonal antibodies produced (OV-1 and T-1) which were screened by an ELISA assay using immobilized oval cells and hepatocytes in microtiter plates, all hybridomas were screened by indirect immunofluorescence on airdried acetone-fixed cryostat sections containing fetal, adult, and DENinduced nodular liver and Morris hepatoma 7777. Screening by indirect immunofluorescence on crystat sections greatly enhanced the yield of potentially useful monoclonal antibodies reactive with liver cell subpopulations from each fusion, as compared to the ELISA screening method. Table IX summarizes the monoclonal antibodies currently being characterized in our laboratory (Dunsford and Sell,

1987). The monoclonal antibodies that recognize oval cells (OV-1 to OV-5) all stain bile ducts, and none stains hepatocytes. Some also stain gastric mucosa and others stain connective tissue and smooth muscle. The significance of these apparent cross-reactions remains unclear. OV-1 has been used successfully to purify fractions of oval cells isolated from F344 rats fed a CDAAF diet and a C D ethionine diet by fluorescence-activated cell sorting. Transplantation experiments using such isolated cells are in progress. Monoclonal antibodies to hepatocytes (H-2 to H-5) stain the cytoplasm of hepatocytes, but not bile ducts. H-6 stains the plasma membrane of adult hepatocytes. H-1 stains only nodular cells. H-3 and H-4 stain only hepatocytes. CN-1 stains bile canaliculi of hepatocytes and the luminal surface of bile ducts and oval cell duct-like structures. Other tissues with shared epitopes include kidney tubules, testicular interstitial cells, lung, and connective tissue. The last group of monoclonal antibodies (T-1 to T-7) all stain the cytoplasm of Morris hepatoma 7777. Most also stain to some degree adult or nodular hepatocytes. Tissues with shared epitopes include gastric mucosa and renal and testicular tubules. T-6 is the only monoclonal antibody to stain only hepatomas. Table X summarizes the variable staining of some of these monoclonals on four hepatomas. These monoclonal antibodies and others being developed are being

TABLE IX CHARACTERIZATION OF MONOCLONAL ANTIBODIESBY INDIRECT IMMUNO~~UORESCENT STAINING OF CRYOSTAT SECTIONS OF FETAL, ADULT, AND NODULAR LIVERAND TRANSPLANTABLE M o m s HEPATOMA 7777" Monoclonal antibody

Fetal Nonhepatoma

Fetal Hepatoma

Adult Hepatoma

+ +

-

-

Bile ducts

Oval cells

Nodules

Hepatoma

+ + + +

-

-

+

+ + + + +

-

-

-

-

-

-

~~

ov-1 ov-2 OV-3

ov-4 OV-5 H-1 H-2 H-3 H-4 H-5 H-6 CN-1 T- 1 T-2 T-3 T-4 T-5 T-6 T-7

+ + +

-

-

+

+I-

+ -

-

+

-

-

+WK

+

-

+ + + + + + -

-

-

+ + + + + + + +

+/-

-

+wK

-

+ -

-

-

+

-

-

-

+3 +4

+ + +5 +

+6 +6 +6 + rare +/-

+ rare -

+WK

+1 -2 -

+ + + + + + + +

a 1, Tumor vessels +, cytoplasm of DEN-derived tumor ++; 2, tumor vessels ++; 3, stains predominantly hepatocytes in neoplastic nodules; 4, stains hepatocytes around nodules; 5, stains some nodules brightly, and only portions of other nodules; 6, stains variably, some nodules have decreased staining; WK, weak staining.

HEPATOCARCINOGENESIS AND PREMALIGNANCY

69

TABLE X VARIABLE STAININGOF DIFFERENT TUMORS BY MONOCLONAL ANTIBODIESTO HE PAT OM AS^ Monoclonal antibody T-1 T-3

T-4 T-5 T-6 T-7

7777

5123

+++ +++ ++ + +++ ++++

+/-

+ ++++ +++ ++ ++++

9098

DEN 308T

-

-

+++ +++ + + +++

+ + + ++ -

7777, Morris hepatoma passed intramuscularly in Buffalo rats; 5123, Morris hepatoma passed intramuscularly in Buffalo rats; 9098, Morris hepatoma passed intramuscularly in AC1 rats; DEN 308T, DEN-derived tumor passed intramuscularly in ACl rats.

used to study the cell populations which appear during chemical hepatocarcinogenesis. The appearance and disappearance of cell populations bearing the epitopes detected are being identified at selected intervals during the administration of chemical carcinogens. For example, preliminary studies indicate that oval cells are present within neoplastic nodules produced by AAF in Fischer rats. Two other laboratories are using monoclonal antibodies to study new cell populations seen during the early stages of chemical carcinogenesis. Faris et al. (1985)isolated oval cells from rats fed a CDAAF diet, transplanted them into the livers of allogeneic rats immediately following partial hepatectomy, and fed the transplanted rats a cholinedeficient diet for 12 weeks. At 12 weeks, there were GGT-positive foci in the recipient rat livers, which were donor derived as determined by assay with alloantiserum. Immunohistochemical staining of these foci with monoclonal antibodies that react with hepatocytes or with duct cells indicated that these foci were derived from hepatocytes and not duct cells. Unfortunately, the isolated oval cell suspensions used for transplantation were not completedly purified and contained hepatocytes. Germain et al. (1985) have used monoclonal antibodies and polyclonal antisera to different rat cytokeratins to study cells isolated from the livers of rats fed an azo dye for 4 weeks at a time corresponding to peak AFP production. The cells were sorted into four fractions by cell size and ploidy, with fractions 1-111containing tetraploid hepatocytes, with albumin production, and fraction IV containing diploid cells with AFP production. Double immunofluorescence microscopy

70

S. SELL ET AL.

on the cells of fraction IV with antibodies specific for M,52,000 cytokeratin (duct cells only) and for AFP revealed three populations, two expressing either one of the markers and a third expressing both. These authors interpret these results as supporting the origin of oval cells from bile ducts and suggestive of transition from the oval cell into an immature hepatocyte. This interpretation must at present by cautiously viewed, since their original sorting of cell types was dependent on methods that provide enrichment and only partial purification of cell populations.

F. SUMMARY The application of monoclonal antibodies to the study of chemical carcinogenesis has been limited. Monoclonal antibodies to chemically induced tumors in mice and rats have been used to demonstrate that chemically induced tumors do have specific epitopes, that epitopes can be followed over time, and that some monoclonal antibodies can aid in the study and characterization of tumor-associated markers. Monoclonal antibodies to DNA adducts and carcinogen metabolizing enzymes have been useful in the study of the metabolism of carcinogens and in the development of sensitive tests for the presence of carcinogens in the host or the environment. Monoclonal antibodies have been prepared to a variety of known and unknown hepatocellular and neoplastic cellular epitopes. Their utility in studying the new cell populations which arise during chemical hepatocarcinogenesis may make a substantial contribution to an understanding of the cellular lineage of hepatocellular carcinogenesis. V. Analysis of Phenotype of Carcinogen-Altered Cells by in Vivo Transplantation and in Vitro Culture

Transplantation and in vitro culture of cells represent experimental biological endpoints for cells which are precursors of tumors. Both transplantation in vivo and culture in vitro reflect “relative autonomy of growth” of the cells surviving the experimental regimens, an important phenotype associated with cancer. From an analytical standpoint, the ability of small numbers of purified premalignant cells to grow eventually like a cancer may be the only definite indication of which cells become cancer. In addition, perturbations and manipulations of the recipient of transplanted cells or of cultured cells may lead to elucidation of the essential factor(s) which promotes the progressive growth of initiated cells into cancer.

HEPATOCARCINOGENESIS AND PREMALIGNANCY

71

A. CONSIDERATIONS IN PLANNING AND INTERPRETING TRANSPLANTATION AND in Vitro CULTURE STUDIES Before discussing and criticizing liver cell transplantation experiments, some overall difficulties inherent in transplantation and culture studies should be mentioned. No experimental systems to date are totally free of some of the complications, but it is anticipated that some pitfalls may be avoided as new techniques are developed. The following is a partial listing of the major problems.

1. Tumors and premalignant liver lesions are heterogeneous at the cellular level. Transplantation of fragments or enzymatically dissociated cell suspensions will theoretically mean transplanting mixtures of cell populations unless some fractionations or enrichments can be performed. 2. Not all the transplanted cells may be viable. This may result in misinterpretation of which cells have actually been transplanted. In fact, enhancement of transplantation may occur if a favorable milieu is provided to the viable cells by dead cells in the transplanted mixture, the “Revesz effect” (Revesz, 1958). Reconstruction experiments in which cell mixtures are used may be useful for determining the extent to which this enhancement occurs in a particular experimental system. 3. Poorly defined selection pressures are operative in the in uiuo transplant site and in cell culture. In transplantation and cell culture, we can only study the cells which survive and are detectable. Selection is probably occurring, but we know neither what the selection pressures are nor whether the pressures are “trivial” or nontrivial. Thus, loss of cells may be due to mechanical damage or dehydration or may reflect nontrivial important biological characteristics of the dying cells. 4.Evolution of cell populations, or “progression,” occurs prior to as well as after transplantation or culture. Transplantation or culturing of cells is analogous to jumping from one moving train onto another. The more that is known about the evolution of liver cell populations prior to and after transplantation or in vitro culture, the greater will be the success in turning a clever technique into an analytical tool for probing cancer development at the single-cell level. 5. The results of transplantation and in vitro culture depend upon nonphysiological techniques, such as the choice of the transplant site or the culture conditions. 6. Criteria for “success” of transplantation or culture of presumptive premalignant liver cells must be based on the specific hypothesis

72

S. SELL ET AL.

TABLE XI EXAMPLES OF BIOLOGICAL BEHAVIORCRITERIA FOR TRANSPLANTED OR CULTURED LIVERCELLS Criteria Progression to carcinoma Persistence of cells DNA synthesis Gene expression Mitosis Histogenesis Angiogenesis Transformtion phenotypes Altered cell morphology Anchorage-independent growth 0

In transplant siten (Spleen) Lee et al. (1983) (Spleen) Mito et al. (1979a) (Liver) Laishes and Rolfe (1980) (Fat pad) Reddy et al. (1984) (Spleen) Lee et al. (1982)

In culture NAb Enat et al. (1984) Rabes et al. (1972)

Enat et al. (1984)

(Spleen) Lee et al. (1985) NTd

Williams et al. (1973)’ NA NA

NA NA

Heine et al. (1984) Shimada et al. (1983)

Transplant sites are indicated in parentheses. NA, Not applicable. Mitosis inferred from serial culture. NT, Not tested.

being tested by the investigator. Examples of criteria which have been applied to premalignant rodent liver cells are presented in Table XI. I n determination of malignancy, the ultimate criteria must be autonomous growth, long-term survival, and/or the ability to metastasize in uiuo. Alterations in other phenotypes are useful as markers, but not definitive.

B. In Viuo TRANSPLANTATION STUDIES IN RODENT HEPATOCARCINOGENESIS

I. Hepatomas The capacity for hepatomas to be serially transplanted in histocompatible or immunoincompetent host animals has become dogmatically established as a reference phenotype with which to compare the transplantability of various types of liver lesions that temporally precede the development of hepatomas (Reuber, 1975).Transplantation of neoplasms of the liver has been employed widely by investigators interested in particular growth, biochemical, or immunological char-

HEPATOCARCINOGENESIS AND PREMALIGNANCY

73

acteristics of a particular liver tumor. The older literature on subcutaneous transplantation of mouse hepatomas has been reviewed by Andervont and Dunn (1952, 1955). A common finding of investigators attempting to transplant primary liver tumors is that most, but not all, hepatocellular carcinomas defined histologically can be successfully transplanted (Andervont and Dunn, 1952, 1955; Hunt et al., 1985; Becker et al., 1973; Odashima and Morris, 1966; Reuber, 1975). Studies on transplantable tumors are, of course, limited to tumors which do, for reasons not entirely understood, grow and passage repeatedly (Reuber, 1966, 1975; Baldwin, 1973; Churchill et al., 1968; Novikoff, 1957). After repeated selection through transplantation, hepatomas can be adapted to grow aggresively in virtually any tissue site, as evident from the experiments leading to establishment of ascites forms of transplantable hepatomas (Odashima and Morris, 1966). On intravenous transplantation, an ascites form hepatoma will colonize the liver or lung, depending on the route of inoculation (Hornberger et al., 1981). Metastasis of intrasplenically transplanted mouse hepatomas to the liver was reported by Leduc (1959). Leduc and Wilson (1963) suggested that it was the blood flow of the spleen which routed implanted cells to the liver. This possibility must be considered in evaluating results using recent intrasplenic transplantation systems (Cameron et al., 1984; Lee et al., 1983), particularly in view of the classic descriptions of the ability of mechanical manipulation of organs to alter the biological behavior of intravenously transplanted tumor cells (Fisher and Fisher, 1962). The majority of currently used nonhuman transplantable and cultured hepatomas are derived from rats. These hepatomas, i.e., transplantable tumors arising in the liver, are generally assumed to be derived from hepatocellular carcinomas. This assumption may not be true for all tumors and is worth evaluating by histopathological examination of transplanted tumors, as was done for the Morris hepatomas (Morris and Meranze, 1974). Caution is warranted on account of the number of different cell types present in the liver in addition to the hepatocytes and in view of the as yet unknown target cell in carcinogenesis which gives rise to progenitor cells of liver tumors. Thus, the ambiguous term hepatoma does provide a degree of caution in classifying neoplasms of the liver. There are two additional ramifications of this ambiguity worthy of mention. First, considerable question exists as to the homology or similarity of chemically induced rodent liver hepatomas to liver neoplasms of humans (Popper et al., 1977). Adult human liver tumors thought to be chemically induced include hemangiosarcomas, hepato-

74

S. SELL ET AL.

cellular carcinomas, and hepatocellular adenomas. Popper et al. (1977) suggested that only the adenomas may have a histopathological counterpart in rats. A second ramification of the hepatoma ambiguity is the general lack of agreement on the classification of premalignant liver lesions in the mouse (Andervont and Dunn, 1952; Newberne, 1982; Becker; 1982; Koen et al., 1983).This lack of agreement occurs in addition to the high and variable spontaneous hepatoma incidence in certain mouse strains (Everett, 1984; Newberne, 1982; Tarone et al., 1981). These factors may account for the preponderance of work over the past 20 years using transplantable rat hepatomas (Potter, 1961). The possibility for viral etiology in mouse and rat hepatomas, though undocumented, must be considered in view of the association of HBV with human hepatocellular carcinoma (Blumberg and London, 1982) and the induction of hepatocellular carcinomas in turkey poults by MC29 virus (Beard, 1980).Thus, if a viral infection of certain target liver cells were important in hepatoma induction, transplants to a viremic recipient animal would be susceptible to viral infection, and transfer of virus could produce cancer in cells of recipient animals. The transplantation immunology of hepatomas has been studied extensively only for a limited number of hepatomas, namely, the azo dye-induced rat hepatomas of Baldwin and co-workers (Bowen and Baldwin, 1979; Lando et al., 1982), and the DEN-induced hepatomas of inbred guinea pig lines (Churchill et al., 1968; Bernhard et al., 1983; Dvorak et al., 1984; Zbar et al., 1969). Biochemical characterization of potentially immunogenic hepatoma cell surface molecules is progressing for other hepatoma cell lines (Glenney et al., 1980; Vischer and Reutter, 1978; Holmes and Hakomori, 1982; Chu and Doyle, 1985). The lack of a broad base of data on immunity to transplanted hepatomas (Baldwin, 1973; Shu et al., 1983)combined with the failure of some primary hepatomas to grow on transplantation does mean, however, that caution be observed in interpreting the transplantation experiments in which negative results are obtained. 2 . Putative Premalignant Liver Lesions Early transplantation studies to determine the growth potential of putative premalignant cell populations in the liver of rats or mice focused on correlating lesion histopathology with transplantability. Reuber (1971) studied spontaneous hyperplastic liver lesions in (C3H x Y)FI mice and concluded that hepatocellular carcinomas (271 30) were successfully transplanted subcutaneously in mice of this F1 strain, but areas (0/78) and nodules (0/56) of hyperplasia were not

HEPATOCARCINOGENESIS AND PREMALIGNANCY

75

(numerators = number of successful transplants; denominators = number of transplants attempted). A similar conclusion had been reached in earlier studies in which ACI strain rats received up to four 4-week feeding cycles of 0.025% N-2-fluorenyldiacetamide (Reuber and Firminger, 1963). The dietary carcinogen regimen used by these authors induced early-appearing hyperplastic areas and nodules as well as hepatomas and cholangiocarcinomas. Of 142 hyperplastic areas transplanted, none grew subcutaneously in ACI rats, and only 1 of 47 nodules grew on transplantation. In contrast, using ACT rats as transplant recipients, the fractions of liver carcinomas successfully transplanted were as follows: 2/7 for highly differentiated hepatomas; 31/37 for well-differentiated hepatomas; 9/9 for poorly differentiated and undifferentiated hepatomas; and 1/1 for a cholangiocarcinoma. The extent and reproducibility of these authors’ work is indicated by the overall fraction of recipient ACI rats successfully transplanted with these four histological types of carcinomas, namely, 293 successful of 385 attempted transplants. Nonetheless, the authors cautiously state, “The microscopic morphologic differentiation between hyperplastic nodules, small, presumably early hepatomas, and highly differentiated hepatocellular carcinomas is difficult and not without subjective factors.” In further experiments, hyperplastic areas and nodules induced in livers of Buffalo (BUF) and Wistar male rats by four 4-week feeding cycles of 0.025% N-2-fluorenyldiacetamide were transplanted into intrahepatic (beneath the liver capsule), intramuscular, intrasplenic, and intraperitoneal sites (Reuber and Odashima, 1967). Areas of hyperplasia (2-4 mm in diameter) transplanted syngeneically 14-20 weeks after the beginning of the experiment persisted in the intrahepatic site of male, but not of female recipients for BUF (4/12 successful) or Wistar (2/5 successful) strains. Hormonal or H-Y antigenicity effects may account for this sex difference. Fractions of successful intrahepatic transplant persistence for nodules of hyperplasia (4-6 mm diameter) transplanted 26 weeks after the beginning of the experiment were 6/9 and 3/9 for BUF male and female recipients, respectively, and 6/8 and 3/8 for Wistar male and female recipients, respectively. Neither areas nor nodules persisted at any of the other heterotopic transplantation sites tested. The nodules persisting in the intrahepatic sites increased 2-fold in diameter over the 40-52 weeks of observation, and cells of the nodules showed hydropic or fatty change histologically, but no evidence of progression to a more advanced stage. These results with transplanted areas and nodules contrast sharply with the relative ease of transplantation of large carcino-

76

S. SELL ET AL.

mas arising in animals from the same study. Reuber and Finninger (1963)and Reuber and Odashima (1967) transplanted tissue both autologously-from a biopsied lesion into a site in the same rat-and syngeneically into an untreated inbred recipient rat of the same strain. In the discussion above, results of both types of transplants have been pooled and called “syngeneic.” However, the wisdom of these investigators in using both types of transplant recipients is apparent in light of the ability of certain highly antigenic tumors-albeit UV and not chemically induced (Rusch and Baumann, 1939; Kripke, 1981-to grow only in immunosuppressed recipient animals. Reuber and Firminger (1963) observed consistent failure of 43 ACI rat strain-derived well-differentiated hepatomas to grow upon transplantation into either allogeneic SD strain rats or xenogeneic Hauschka mice. No growth of transplanted normal (Oh45 syngeneic transplants) or cirrhotic (0/87 syngeneic transplants) liver tissue was obtained. In the studies just described, conclusions regarding the possible premalignant potential of hyperplastic areas of nodules are limited by the possibility that the areas and nodules of hyperplasia fail to do more than persist even in the intrahepatic transplant site because of an inadequate milieu necessary for their continued growth and development into carcinomas if this is, in fact, the biological fate of cells within the transplanted tissues. Williams, Farber, and co-workers (Williams e t al., 1977, 1980; Ohmori et al., 1980) attempted to overcome this limitation by utilizing inguinal mammary fat pads as transplant sites in light of the successful use of this site for development of mammary tumors from transplanted premalignant rat mammary tissue (De Ome et al., 1959). Williams et al. (1977) generated hyperplastic liver nodules in C D F (Fischer) rats by feeding 0.05%N-2-fluorenyldiacetamide or 0.25-0.8% ethionine and transplanted these nodules autologously into mammary fat pads of the carcinogen-treated rats or into untreated syngeneic C D F rats. In autologous transplantations, liver-derived cells were detected in fat pads of 7/19 recipient rats examined over a 3- to 19-week period. In syngeneic transplantations, liver-derived cells were detected in fat pads of 13/19 recipient rats examined 10-38 weeks posttransplantation. These transplant persistence results are not significantly different from persistence results for mammary fat pad transplantation of normal liver fragments reported in the same study: Liver-derived cells were observed in 32/59 autologous transplants and 30/42 syngeneic transplants of normal liver tissue fragments. In a later study, Ohmori et al. (1980) transplanted 219 hyperplastic nodules into fat pads, but only recovered 3 transplants, one of which appeared to be an adenocarcinoma which may have

HEPATOCARCINOGENESIS AND PREMALIGNANCY

77

developed from a small number of carcinoma cells entrained in the fragment of transplanted nodule. Taken together, the tissue fragment transplantation results strongly indicate that cells of transplanted hyperplastic liver nodules can persist in intrahepatic or mammary fat pad sites, but do not progressively grow to produce lesions more advanced toward carcinomas. Considering the possibility of entrainment or entrapment of diverse cell populations in the transplanted fragments (Ohmori et al., 1980), it is noteworthy that the fragment transplantation studies have yielded such clear-cut negative results. Are the nodule fragments not transplantable because they are “false negative” due to crude technology coupled with inappropriate milieu in transplant site for growth and further development of nodule cells to carcinomas? Or, are nodule cells not precursors of carcinomas, contrary to much circumstantial evidence amassed over the past 30 years (Farber, 1984a,b)? Recent experiments have shown that survival of transplanted fragments of socalled persistent nodules (Farber, 1984a,b), explanted at 12 weeks from rats receiving the Solt-Farber regimen, in spleens of syngeneic recipient rats can be modulated by administration of liver tumor promotors to recipient rats (Hayes et d . ,1985). The prolonging effect of phenobarbital on transplant survival contrasted with an apparent reduction in the survival of transplants in recipient rats treated with Aroclor 1254.

3. Enzymatically Dissociated Premulignant Liver Cells A major difficulty in interpreting experiments involving transplanting excised fragments of liver lesions lies in the unknown cellular heterogeneity of the fragments transplanted. For example, the transplants of well-differentiated hepatomas of Reuber and Finninger (1963) contained many littoral cells. A logical progression of transplantation technology occurred from transplantation of liver lesion fragments to transplantation of suspensions of enzymatically dissociated (Seglen, 1976) liver cells subjected to different types of enrichment for interesting cell types. This approach reflects the change in focus of transplantation studies from correlating liver lesion histopathology with biological behavior in a transplant site, to identifying the precursor cell(s)of liver carcinomas. This latter, current focus is analogous to one of Koch’s postulates for infectious disease, although as Smuckler (19834 has discussed, Koch’s postulates likely cannot be directly applied to cancer. The first successful transplantation regimen in which premalignant liver-derived cells were shown to have convincing biological behav-

78

S. SELL E T AL.

ior, namely, proliferation, and colony formation in the transplant site, was devised by Laishes and Farber (1978). The Laishes-Farber transplantation procedure used donor premalignant liver cells dissociated by two-step collagenase perfusion of livers of F344 rats receiving the Solt-Farber regimen, i.e., DEN (200 mg/kg, ip) followed by selection with dietary AAF and two-thirds partial hepatectomy (PH) (Solt and Farber, 1976). This 5-week DEN/AAF/PH donor regimen very reproducibly yields livers containing relatively synchronously appearing, focally proliferating altered hepatocytes easily detected by histochemical staining of liver cryostat sections or isolated cell suspensions for the marker enzyme GGT. The cell suspensions prepared from such livers contain cells capable of colonizing livers of syngeneic recipient rats, but only if the recipient rats receive the same AAF/PH selection regimen used with the donor rats (Laishes and Farber, 1978; Laishes and Rolfe, 1980). Transplantation is achieved by intravenous injection of donor liver cell suspensions into mesenteric vein tributaries of the hepatic portal system immediately following surgical twothirds PH. The recipient rats develop hepatocellular carcinomas at about 80% incidence (Laishes and Rolfe, 1980). Hunt and co-workers developed a major histocompatibility complex alloantigen marker system based on the Laishes-Farber procedure to demonstrate, first, that 97% of colonies produced in the recipient rat livers were of donor origin (Hunt et al., 1982),and second, that at least 5/6 transplantable hepatocellular carcinomas developing after 17 months in the recipient rat livers were of donor origin (Hunt et al., 1985). This system for liver carcinoma production offers another element of technical flexibility, namely, the opportunity to utilize a variety of cell selection and purification strategies on the heterogeneous donor liver cell population prior to transplantation. The marker system generates genotypic mosaic livers, since parental strain donor rat liver cells are transplanted into F1 hybrid rats such that appropriate alloantisera can be employed to distinguish donor from host liver cells. The Laishes-Farber liver cell transplantation system has been criticized for the complexity of the donor rat regimen (Potter, 1984) and for the necessity of employing a carcinogen, AAF, in the recipient rat selection regimen. Thus, it is possible that the AAF treatment of the recipient rats additionally alters-e.g., by inducing additional mutations-the transplanted donor cells such that the developmental fate of these donor origin cells may not be analogous to that of carcinoma progenitor cells in rats given a more traditional long-term carcinogenic regimen of 16 weeks of dietary AAF (Reuber and Firminger,

HEPATOCARCINOGENESIS AND PREMALIGNANCY

79

1963). These criticisms must be carefully weighed as the genotypic mosaic liver transplantation model (Hunt et al., 1985) is exploited for the isolation of donor-origin premalignant liver cells during carcinoma development in the recipient rat livers. Since the original studies of Laishes and Farber (1978), numerous other investigators have transplanted suspensions of liver-derived cells from rats undergoing chemically induced hepatocarcinogenesis. The Pretlow group used Laishes-Farber AAF/PH recipient F344 rats for transplantation of collagenase-dissociated liver cells from syngeneic rats which had received 90 ppm DEN in drinking water for 5 weeks (Miller et al., 1982). These authors demonstrated that GGTpositive liver cell foci were produced in host rats receiving donor cells purified to 97% purity for hepatocytes using density gradient sedimentation in a zonal rotor. Although no quantitation of number of foci was reported and no other cell populations from the gradient were reported transplanted, the authors concluded that foci could arise from transplanted hepatocytes. Thus, their data do not permit conclusions to be drawn regarding the fate of transplanted oval cells which are generated in premalignant rat livers by many, though not all, carcinogenic regimens (Farber, 1956; Solt et d., 1977). As a more direct test of the ability of oval cells to produce donor-origin GGT-positive foci in livers of transplant recipients, Faris et al. (1985) intravenously transplanted oval cell-containing suspensions of liver cells from rats receiving 3 weeks of 0.05% AAF in choline-deficient diet into livers of recipient rats receiving choline-,deficient diet and PH prior to transplantation. These authors observed donor origin foci in the host rats, but because their transplanted cell suspension contained about 15% hepatocytes, no firm conclusion as to the nature of the cells producing the foci was possible. It is still significant that using a far less toxic selection regimen than that of Laishes and Farber (1978), transplanted cells did colonize the recipient rat livers. Hanigan and Pitot (1985a) were able to produce GGT-positive liver colonies in recipient F344 rats treated with dietary phenobarbital and PH prior to transplantation of donor liver cell suspensions isolated from syngeneic rats treated with DEN, PH, and phenobarbital (Pitot et al., 1978). Thus, additional work may well yield less toxic selection regimens for recipients of intravenously transplanted donor liver cell suspensions. It should be noted, however, that the burden is upon the investigator to use phenotype-independent marker systems in liver cell transplantations into livers of recipient rats to demonstrate donor cell origin of the liver lesions scored as endpoints. Genetically determined marker systems adaptable to transplantation studies include Class I major

80

S. SELL ET AL.

histocompatibility complex alloantigens (Hunt et al., 1982, 1985; Weinberg et al., 1985), enzyme isozymes (Rabes et al., 1982; Rabes, 1983), or other suitable strain-specific chemotypes (Condamine et al., 1971). Transplantation of premalignant liver cells and premalignant lesion fragments into heterotopic sites such as spleen or fat pad is technically attractive because it obviates the need for a complex marker system to distinguish donor liver tissue from nonhepatic recipient tissue. For example, the anterior eye chamber with its continuous outflow of intraocular fluid may provide a useful environment for analysis of secreted products of transplanted premalignant and malignant liver cells (Evarts et al., 1984). Interscapular, inguinal, axillary, and mammary fat pads of rats have been used successfully as transplantation sites for normal syngeneic hepatocytes (Jirtle et al., 1981).The fat pad sites are well suited for quantitating numbers of transplanted liver cells required to produce a single liver cell colony in recipient rats subjected to different experimental regimens (Jirtle and Michalopoulos, 1985; Bone et al., 1985). The colonies produced from transplanted normal hepatocytes grow in a flattened plaque-like manner, whereas liver cells from Solt-Farber DEN/AAF/PH donor rats produce colonies having a more spherical morphology in fat pads (R. Jirtle, personal communication). Normal hepatocytes transplanted into fat pads retain their responsiveness to the peroxisome proliferators ciprofibrate and di-(2-ethylhexyl)phthalate (Reddy et al., 1984), and the efficiency of colony formation can be altered by PH and by administration of the liver tumor promoter phenobarbital to the recipient rats (Jirtle and Michalopoulos, 1985).Thus, the apparent accessibility of the fat pad environment to physiological growth modulators makes it attractive for future studies with premalignant liver cells. Interest in the use of the spleen as a transplant site for enzymedissociated liver cells stems from extensive work of Mito and co-workers (Kusano and Mito, 1982; Mito et al., 1979a,b). At 16 months after transplantation, Kusano and Mito (1982) demonstrated hepatocytes, Ito cells, and endothelial cells, but not Kupffer cells in the spleens of rats receiving an unfractionated liver cell suspension containing 5 x lo6 hepatocytes. The spleen has been shown to support the growth and progression over 14 months to carcinoma of transplanted liver cells isolated from carcinogen-treated syngeneic donor rats having hyperplastic liver nodules (Lee et al., 1982, 1983).Finkelstein et al. (1983) transplanted 2 X lo6liver cells isolated at 4 months from livers of Solt-Farber (Solt and Farber, 1976) donor rats into spleens of recipient rats and scored the area of spleen occupied by hepatocytes 1 week later. Over this ex-

HEPATOCARCINOCENESIS AND PREMALIGNANCY

81

tremely short period, the application of the AAF/PH regimen used in earlier intrahepatic transplants (Laishes and Farber, 1978) to these recipients increased the spleen area occupied 10-fold over levels in rats receiving PH alone, AAF alone, or no treatment. Thus, the as yet ill-understood modulation of premalignant hepatocyte growth by AAF/PH selection can act on liver cells implanted in the spleen as well as in the liver of recipient rats. Cameron et al. (1984) compared spleen and liver transplant sites of AAF/PH-selected recipient rats and found equivalent growth in either site of transplanted hepatoma cells as well as of normal liver cells treated in vitro with methylnitrosourea (MNU). The treatment of liver cells in vitro prior to transplantation requires further evaluation as a useful technique for eventually identifying carcinogen-induced cellular damage to hepatocytes, leading to heritable malignant growth characteristics. Lee et al. (1985), Cameron et al. (1984), and Yoshimura et al. (1983) have achieved the first positive results with the so-called in vivo-in vitro-in uivo approach (Laishes et al., 1980). Lee, Cameron, and co-workers treated isolated donor liver cells in vitro with a carcinogen, MNU, prior to transplantation. Yoshimura et al. (1983)serially passaged oval cell-enriched nonparenchymal liver epithelial cells from rats fed ethionine in choline-deficient diet prior to intraperitoneal or subcutaneous transplantation of the cell lines and obtained highly anaplastic carcinomas in syngeneic rats as well as in nude mice. The lesions obtained in transplantation experiments following in vitro treatment or long-term culture of donor liver cells need to be cautiously evaluated until more is known about the effects on liver cells of in vitro treatments and serial cultivation. C. In Vitro CULTURE OF PUTATIVE PREMALIGNANT LIVERCELLS Persistence and limited capacity for serial subculturing in vitro was described by Slifiin et al. (1970)for cells of hyperplastic liver nodules (HLN) explanted from rats treated with AAF or aflatoxin B1. The HLN cultures survived up to 4 months and a maximum of 6 passages, whereas normal liver cells and cells from non-nodular regions of carcinogen-treated rat livers degenerated after 48 hr in culture. Rabes et al. (1972) explanted fragments of enzyme-altered (ATPasedeficient) hyperplastic liver nodules from rats receiving continuous DEN administration and showed in [3H]thymidine labeling index studies that in vitro cultures mirrored the in uivo lesions in their labeling index heterogeneity. These authors postulated that proliferative heterogeneity of cells of premalignant liver lesions may occur at a

82

S. SELL ET AL.

critical point during liver carcinogenesis, perhaps when irreversible commitment of certain cells to lineages leading to carcinomas occurs (Rabes, 1983). In vitro culture may be a means of selecting for such committed cells. In vitro analogs of the Haddow hypothesis, i.e., selective proliferation of cancer cells under conditions which inhibit growth of normal cells (Haddow, 1938), have been demonstrated by many groups of investigators (Grisham, 1983; Judah et al., 1977; Laishes et al., 1978; Diamond, 1969).Farber et al. (1979) quantitated greater resistance to cytotoxic carcinogens in vitro for hyperplastic liver nodule cells than for nonnodular liver cells. These experimental results still do not answer questions regarding cellular origins of carcinomas since first, the “microenvironment” becomes completely disrupted in vitro,and second, it is not known at present what in vitro biological behavior is strongly correlated with, much less causally related to, cellular lineage commitments toward a cancer endpoint. The development of novel cell culture methodology (Guguen-Guillouzo and Guillouzo, 1983; Enat et al., 1984), immortalization of liver cells by viral transformation (Isom et al., 1981; Chou and Schlegel-Haueter, 1981; Woodworth et al., 1984; Lafarge-Frayssinet et al., 1984), and improved single-cell resolving immunocytological techniques and nucleic acid sequence probes reviewed in this article should lead to more fruitful studies. Appropriate application of these techniques may lead to identification of properties of in vitro cultures of premalignant liver cells which are authentic indicators of irreversible, heritable commitment of certain cells to lineages culminating in liver cancers. A number of cell lines propagable in vitro have been derived from adult rat liver (reviewed by Grisham, 1983). Such cell lines have been tested for their responsiveness to chemical carcinogens and tumor promoters as well as for their capacity to induce tumors upon inoculation into rats. Williams (1976)has reviewed some of the earlier literature on use of liver epithelial cells for in vitro carcinogenesis and has discussed the need to evaluate carefully the variable histology of tumors produced upon in vivo inoculation of tumorigenic cell lines. Table XI1 summarizes results of investigators in this area over a 12year period. The identity of such cell lines is not clear on account of the tendency of hepatocytes to senesce rapidly in primary culture and the multiple cell types present in enzymatically dissociated adult rat liver cell preparations (Nagelkerke et al., 1983; Knook et al., 1982; Grisham, 1983).Another major problem in interpreting results of experiments using such liver cell lines is our present lack of understanding of the relationship between in vivo carcinogenesis and some of the biological and biochemical properties exhibited by the liver cell lines

83

HEPATOCARCINOGENESIS AND PREMALIGNANCY

TABLE XI1 EXAMPLES OF USE OF RAT LIVER-DERIVED EPITHELIAL CELLCULTURES IN CHEMICAL CARCINOGENESIS STUDIES" Authors

Cells

Williams et al. (1973)

TRL 2

Borenfreund et al. (1975) Montesano et al. (1975, 1977)

Adult Gunn rat

Treatmentb AFBI, NMU, N-OH-AAF, DMBA MAM, BP

Observations turn+ txd. cell linesc

aig+ txd. cell lines

IAR20 and IAR20-PC 1 IAR20-PC1

Serial passage

IAR 6

Serial passage

IAR 6

MNNG

IAR 6

DMN

IAR 2

Serial passage

IAR 2

MNNG

Leffert et al. (1977)

Primary adult F344d

None

Schaeffer and Heintz (1978) San et al. (1979)

RL-PR-C

AFBL

Virtually turn- and aig- at 38 weeks IAR20-PC1-3 22-25 weeks: aig-, tum30-46 weeks: aig+ turn+ 20 weeks: aigturn36-52 weeks: aig+ turn+ IAR6-10, turn- at 38 weeks IAR6-1, turn+ at 35 weeks, aig+ at 52-63 weeks IAR2-25, turn+ at 29 weeks IAR2-28, turn+ aig+ at 48-103 weeks Hormone reqts. for growth; albumin synthesis; AAF metabolism turn+, aig- txd. cells

T51 and ARL 11, 12, 14, 15, 16, 18 ARL 6, 17 Liver foci from AAF-fed rat

None

aig-, turn- cells

None PB in oitro

RL-PR-C Clone 12

DDBP None

aig+, turn+ cells PB-enhanced persistence of turn+ aig+ cells in cultures aig- turn+ txd. cells Cytoskeletal proteins differ from those of hepatoma cells

Kitagawa et al. (1980) Heintz et al. (1980) Franke et al. ( 1981)

MNNG

(continued)

84

S. SELL ET AL.

TABLE XI1 (Continued) Authors Lafarge-Frayssinet et al. (1981) Manson et al. (1981)

Cells

Treatment6

F 11

Serial passage !

BLBL

None

BL8L

McMahon et al. (1982)

FNRL

Serial passage

FNRL

HPI

NRL ST

HPI

TRL 12-13

NMU

NMU-3

None

Leffert et al. (1983) Brown et al. (1983)

Primary adult F344d TRL 1215

AAF

Shimada et al. (1983)

ARL 15 (C1.l)

Kaplan et al. (1982)

Wahid (1983) Heine et al. (1984)e

Primary adult Donyrud TRL 1215

Ethionine or AdoEt MNNG, MMS, AAF, AFBI, DMN, NP, DAB Fluorene, AFGZ, DMF, DASA, BP, pyrene 3’-Me-DAB Ethionine

Observations Cells aig+ by pass 29 Contact-inhibits hepatoma line JB1; low % GGT + cells Cells 1 0 0 more ~ sensitive to cytotoxicity than hepatoma JBl Spontaneously txd. to line ‘NRL ST’ big+) Growth inhibition in aig assay No growth inhibition in aig assay Txd. to line NMU-3 Decreased mitochondria, Golgi, RER, vesicles; increased lactic acid output AAF-DNA adduct detection Txd. to aig+, tum+ cells aig+ cells

aig- cells Tum- txd. cell lines Txn. to cells with altered morphology, multilaminar growth, decreased freq. of 1-ciliated cells and of IJs, increased nuclear area (continued)

HEPATOCARCINOGENESIS AND PREMALIGNANCY

85

TABLE XI1 (Continued) Authors

Cells

Tsao et al. (1984a)

WB-F344

Morel-Chany et al. (1985)

F

Kaufmann et al. (1986)

Liver cells from DMN-Actreated rat

Treatmentb TPA, PB, RA, and 5-Ac Serial passage

PB

Observations Modulation of enzyme activities aig- at pass 13-30; aig+ at pass 35180 Proliferative hepatocyte colonies

All cell lines used, unless otherwise indicated, are epithelial appearing, liver-derived cells from adult rats (see Grisham, 1983). At early passages cell lines were tumand aig-. Treatments are in uitro on cultured cells unless otherwise noted. Abbreviations used: txd., transformed; reqts., requirements; AFBI, aflatoxin B,; tum, tumorigenic in transplant recipient animals (+ or -); aig, anchorage-independent growth in soft agar (+ or -); DMN, dimethylnitrosamine; MNNG, N-methyl-N’-nitro-Nnitrosoguanidine; HPI, hepatic proliferation inhibitor; NMU, nitrosomethylurea; RER, rough endoplasmic reticulum; AAF, 2-acetylaminofluorene; AdoET, S-adenosylethionine; MAM, methylazoxymethanol acetate; BP, benzo[a]pyrene; MMS, methylmethane sulfonate; DAB, dimethyl-4-aminoazobenzene; NP, nitrosopyrrolidine; DMF, dimethylformamide; DASA, dimethylamino-4-azobenzene-4-sulfonicacid; DMBA, 7,12-dimethylbenzanthracene;N-OH-AAF, N-hydroxy-2-acetylaminofluorene; TPA, 12-0-tetradecanoylphorbol-13-acetate; RA, retinoic acid; 5-AC, azacytidine; 3’Me-DAB, 3’-methyl-4-dimethylaminoazobenzene; DDBP, dihydro-7,8-dihydroxybenzo[a]pyrene; PB, phenobarbital; AFG2, aflatoxin Gz; IJ, intermediate junction; DMNAc, methyl(acetoxymethy1) nitrosamine. d Primary rat liver cell cultures were used without serial passage. The karyotype of the nonethionine-treated cells changed with passage, and the same passage number cultures of nontransformed cells were used as parallel controls in these studies.

in vitro. For example, colony formation in soft agar (Montesano et al., 1977; Brown et al., 1983; Shimada et al., 1983), ultrastructural changes (Heine et al., 1984), and modulation of premalignant liver lesion “marker enzyme” activities (Tsao et al., 1984a) have been reported following treatment of liver cell lines with carcinogens or tumor promoters. The tumorigenic capacity of some of the liver cell lines developed by Montesano et al. (1975, 1977) varied depending on the passage number at the time of assay by transplantation. Some lines showed heterogeneity for GGT enzyme phenotype (Huberman et al., 1979). Culturing the well-differentiated rat hepatoma cell line H4-11EC-3 in cell aggregates induced rapid appearance of dedifferentiated variant cells in the cultures (Deschatrette, 1980). The culture variables operating in the complex “in uitro promotion” regimen of Kita-

86

S. SELL ET AL.

gawa et al. (1980) are at present unknown. Many observations and phenomena utilizing liver-derived cell cultures have now been described. The future of in vitro liver cell culture will depend on the ability of the investigators to formulate testable hypotheses relevant to in vivo carcinogenesis using these culture systems.

D. SUMMARY Transplantation and in vitro studies have largely focused on the hypothesis that some cells of hyperplastic liver nodules are progenitors of carcinomas. The more recent intrasplenic transplantation results of Lee, Farber, and co-workers support the hypothesis, in contrast to earlier extensive work of Reuber and co-workers using a different carcinogenic regimen and different transplant sites. Yet, the question of the cellular origin of rodent liver carcinomas is unanswered (Sell and Leffert, 1982; Peraino et al., 1984). Until technology is developed to follow the lineage of single cells within an intact organism, transplantation studies may be a fruitful way to determine experimentally the biological fate of particular types of liver cells arising during liver carcinogenesis. The immunology of transplantation of premalignant liver cells and of hepatomas is poorly understood for most current experimental transplantation systems. A better understanding of this transplant immunology may be an important point of attack on hepatocarcinogenesis, not only as an approach to following cell lineages in this well-studied rodent cancer model, but also for eventually designing targeted therapy regimens suitable for the virtually inoperable liver tumors.

VI. Gene Expression in Liver Carcinogenesis

A. CARCINOGENESIS AND PROTOONCOGENE MUTATIONS One of the principal characteristics of a cancer is the ability of the phenotype of the cancer cells to be expressed in subsequent generations (although notable exceptions exist). Thus, cancer grows as a population of cells with heritable characteris tics. This implies a critical heritable alteration in the DNA of the cancer cell, the nature of which is unknown. In this section, we will cover recent studies done to detect transforming genes activated by chemical carcinogens. In order to study this, genomic DNA is extracted from primary tumors or from intermediate-stage tissues undergoing carcinogenesis. The DNA is then transferred into NIH 3T3 cells, which are cultured until trans-

HEPATOCARCINOGENESIS AND PREMALICNANCY

87

formed foci appear. NIH 3T3 cells normally grow as a monolayer; tranformation refers to a change in the growth pattern of the NIH 3T3 cells so that foci of cells grow in multilayered colonies above the monolayer. This is believed to be a phenotypic characteristic of cancer cells. This assay is specific for dominant mutations in cellular genes (protooncogenes) which can transform NIH 3T3 cells: Mutations which are recessive or which affect genes that are not functional in NIH 3T3 transformation will be missed. Nonetheless, the mutations which have so far been detected affect genes that are related to the transforming genes (oncogenes) of several tumor retroviruses and, as such, could be involved in tumorigenesis (Varmus, 1984). These experiments should be considered in the context of three general hypotheses for the role of mutations in chemical carcinogenesis:

1. Any mutations which may be discovered, regardless of which gene is assayed, may be incidental to the process of carcinogenesis. Tumors change properties with growth (progression) and are sometimes quite heterogenous. These traits may be due to a random mutagenic process that could affect genes having nothing to do with tumorigenesis. 2. Carcinogenic treatments create conditions which favor the proliferation of cells containing mutations in one or several specific genes. This would imply a selective process for randomly occurring mutations. The mutations might occur at a normal rate or at an enhanced rate brought about by carcinogen-mediated induction of error-prone DNA repair processes. 3. Carcinogens may create mutations directly by chemical interactions with the DNA. Mutations of one or several specific genes might then play a role in the initiation or development of tumors. In each of these hypotheses, mutations do not necessarily initiate neoplastic transformation. The initiation event may take place by an entirely different mechanism, with mutations being required for further tumor development. Mutant protooncogenes have not been detected in rat liver tumors (Farber, 1984b; Knoll et al., unpublished). Thus, we will discuss results obtained in some other sytems. Alterations in the DNA of chemically induced tumors have been described in several systems. Sukumar et al. (1983)induced mammary carcinomas in Buf/N rats by single injections with N-nitroso-Nmethlyurea (MNU). Genomic DNA from 9/9 primary induced tumors were able to transform NIH 3T3 cells. DNA from the breasts of untreated animals were negative in the assay. Normal breast or other tissues from the treated animals were not tested. The transforming

88

S. SELL ET AL.

DNA from breast tumors was cloned and found to be a cellular gene related to the transforming gene of Harvey murine sarcoma virus (the Ha-rus-1 gene): The new transforming gene was called NMU-H-rus. DNA sequence analysis of one of these genes showed a single point mutation alterating amino acid 12 within exon 1, changing a glycine to a glutamic acid. Alteration of amino acid 12 had previously been shown in transforming versions of Ha-ras-1 from human and viral sources (Tabin et al., 1982; Reddy et ul., 1982). Another possible site of mutation, observed in some human tumors, is amino acid 61 (Yuasa et ul., 1983);this codon was not altered in the transforming gene that was analyzed. In the remaining 8 tumors, a change in codon 12 of the Ha-rus-1 gene was detected by virtue of a restriction enzyme site change that could be detected in Southern blots of total genomic DNA: No cloning or sequencing data were reported for these other tumors. In further studies (Zarbl et ul., 1985), 48 of 58 mammary carcinomas generated by NMU in several strains of rat were able to transform NIH 3T3 cells. Further, each of these tumors contained the very same G + A transition at position 35 (codon 12) of the Ha-rus-1 protooncogene, as described by Sukumar et ul. (1983). This mutation corresponds to the mutagenic specificity of NMU (Singer and Kusmierek, 1982), therefore arguing in favor of a direct mutagenic action of NMU rather than an indirect selection by NMU for random mutations at codon 12 of Ha-rus-1. Further, the physiological half-life of NMU is very short (several hours) (Druckrey et al., 1967), which suggests that the mutation in Ha-rus-1 was an early event in tumorigenesis. These studies suggest that most mammary tumors generated in the rat by NMU had undergone mutation of their Ha-rus-1 protooncogenes early in their development. However, this mutation is not necessary for the development of mammary carcinomas, since 18% of the NMU-induced tumors do not contain the mutant oncogene. Perhaps some other protooncogene, not detectable in the NIH 3T3 assay, is mutated in these tumors. Transforming Ha-rus genes were also detected in mouse skin carcinomas induced by DMBA with promotion by 12-tetradecanoylphorbol-13-acetate (TPA), and then passaged subcutaneously in syngeneic mice. The genomic DNAs from 3/3 transplanted carcinomas were able to transform NIH 3T3 cells. Primary foci of transformed NIH 3T3 cells were shown to contain several additional copies of the Ha-rus-1 gene, presumably acquired from the carcinomas. It is not known whether the Ha-rus-1 genes of mouse skin carcinomas contained mutations; however, DNA from several normal tissues did not transform NIH 3T3 cells (Balmain and Pragnell, 1983).

HEPATOCARCINOGENESIS AND PREMALIGNANCY

89

The mouse skin carcinoma system was further exploited to determine whether premalignant lesions contain transforming sequences (Balmain et ul., 1984). Sencar mice (specially bred for carcinogen sensitivity) were treated with DMBA and TPA to induce primary papillomas, which precede carcinomas. The genomic DNA from 4/5 papillomas and from 2/3 carcinomas (not transplanted) induced foci in NIH 3T3 cells. Normal tissue or epidermis induced to proliferate by TPA were negative in the transformation assay. NIH 3T3 cells transformed by these genomic DNAs contained additional copies of the Ha-rus-1 gene. In papillomas, carcinomas, and in NIH 3T3 cells transformed by DNA from papillomas and carcinomas, the amounts of Ha-rus-1 RNA were considerably elevated over normal epidermis and normal NIH 3T3 cells. The basic finding was that papillomas and carcinomas are essentially equivalent with respect to the transforming activities of their genomic DNAs and the amounts of Ha-rus-1 transcripts RNA that they contain. However, since only 5-7% of papillomas progress to carcinomas, events other than Ha-rus-1 activation must also occur during carcinogenesis. In addition to Ha-rus, other members of the M S family of oncogenes may be altered in tumors generated by carcinogenic treatments. Eva and Aaronson (1983) found that two of four methylcholanthrene-induced fibrosarcomas in the mouse contained transforming sequences related to the Kirsten sarcoma virus (Ki-rus). Guerrero et ul. (1984a) created thymomas in AKR/RF mice with either y irradiation or with NMU. Genomic DNA from both types of tumors (516 NMU tumors and 4/7 radiation-induced tumors) caused focus formation in NIH 3T3 cells. Foci generated by DNA from NMU tumors contained several additional copies of the mouse N-rus gene and increased amount of Nrus mRNA and protein. In contrast, foci generated by DNA of the radiation-induced tumors contained additional copies of the Ki-rus gene rather than the N-rus gene. Increased levels of Ki-rus mRNA and protein were detected in these transformed NIH 3T3 cells. In further studies (Guerrero et ul., 1984b),the transforming Ki-rus gene from one y radiation-induced tumor was sequenced and found to contain a single G --j A transition in codon 12. No further data were reported for the NMU-induced tumors; however, the apparent specificity of carcinogens for different protooncogenes is difficult to reconcile with the idea of random mutagenesis by carcinogens as a mechanism for generating activated oncogenes. To further clarify this point, Marshall et ul. (1984)treated the cloned human Ha-rus protooncogene (i.e., not mutant) in uitro with the carcinogenic metabolite of benzo[u]pyrene-7,8-diol 9,lO-epoxide. The chemically modified DNA efficiently induced foci in NIH 3T3 cells,

90

S. SELL ET AL.

while the unmodified DNA did not. Of 17 foci that were analyzed, all contained the human Ha-rus gene, and four were shown to have a mutation at codons 11or 12 (using a restriction enzyme site variation). Thus, it is clear that simple in vitro mutagenesis of a protooncogene can create a gene capable of transforming NIH 3T3 cells. In fact, this has been done in a systematic way by Seeburg et al. (1984) with the same human protooncogene, c-Ha-rus-1. Alteration of codon 12 from its normal glycine to any other amino acid (except proline) creates a gene able to transform NIH 3T3 cells. To summarize, these studies show a strong correlation between mutation of rus-type protooncogenes and carcinogenesis in several animal and in vitro systems. Further work is necessary to explain those tumors that do not contain detectable mutant protooncogenes and to determine the mechanism by which alteration of the rus protein might cause neoplastic transformation. In some other animal systems there appears to be no mutation of protooncogenes detectable by the NIH 3T3 transformation assay (Duesberg, 1985).Other assays may be needed to detect mutant protooncogenes in these tumor systems.

B. RAT LIVERCARCINOGENESIS AND PROTOONCOGENE EXPRESSION Because transforming oncogenes have not been detected in rat liver tumors, (Farber, 1984; however, see Zurlo and Yager, 1985), work has focused on the possibility that the expression of different protooncogenes may be altered in the rat liver during carcinogenesis. Thus, carcinogenesis in this sytem may involve perturbations in the control of protooncogene expression rather than in mutations of the protooncogenes themselves. In principle, an analysis of this sort is no different from examining the expression of AFP, GGT, etc. as a function of carcinogenesis except that protooncogenes are related to the transforming genes of retroviruses and may be more likely to be involved in tumorigenesis than the proteins or enzymes that have been traditionally studied. The results of several recent studies are summarized in Table XIII. The expression of the c-myc gene in Morris hepatomas (chemically induced, transplantable) was analyzed by Hayashi et ul. (1984). Each hepatoma (7794A, 7316A, and 5123D) produced more c-myc mRNA than normal liver (by a factor of 5-10). In addition, one hepatoma (7794A) showed a 5- to 10-fold amplification of c-myc DNA sequences compared to normal liver or to hepatomas 7316A, 5123D, and 3924. This finding is consistent with other experimental systems in which increased expression of c-myc accompanies cell division, such as liver

91

HEPATOCARCINOGENESIS AND PREMALIGNANCY

TABLE XI11 PROTOONCOGENE TRANSCRIPTS IN RAT HEPATOMAS AND

IN

PREMALICNANT LIVER

Protooncogene Tissues or cell Hepatocytes Normal liver Carcinogen treated Bile ducts' Oval cellso Nodulesf Carcinomas Regenerating liver Fetal liver

K-ras

H-ras

myc

N-ras

+" +

+ +

+o.c

+ +++ + +++a

++b +a.h

srca

+

+++

NTR NT

++

+ +J

+/O

+a.b.c.d

++++e

NT

+

+J

+

+ + NT + f

0

mos"

abla

0 0

0 0

0

0 0 NT 0

0 NT 0 0 0

0

Yaswen et al. (1985). Goyette et al. (1983, 1984). Makino et al. (1984a). Hayashi et al. (1984). Makino et al. (1984b). f Corcos et al. (1984); the term nodules refers to premalignant, hyperplastic growths (Farber, 1984a). R NT, Not tested. ++ on day 17,O on day 20. i ++ on day 17, + on day 20.

regeneration (Makino et al., 1984a) or treatment of cell cultures with mitogenic stimuli (Kelly et al., 1983; Campisi et ul., 1984). However, caution must be observed regarding intrepretation of these results, since increased oncogene expression has also been seen after partial hepatectomy (H. L. Leffert, personal communication). Protooncogene expression has also been extensively studied in rat livers during carcinogenesis. Makino et al. (198413) examined the levels of c-myc and c-Ha-rus mRNA in rat livers during treatment with the azo dye 3'-Me-DAB. c-Ha-rus mRNA was elevated about 2-fold in both liver tumor and in adjacent nonmalignant tissue. In contrast, crnyc mRNA was elevated (about 3- to 5-fold) only in the malignant tissue and not in the surrounding tissue. The increase in c-Ha-rus mRNA was observed by 5 days after the beginning of the carcinogenic diet and remained elevated throughout treatment. In a similar study, Corcos et ul. (1984) observed increased c-Ha-ras expression in both normal-appearing hepatocytes and in "nodules" isolated from rat livers 70 weeks after DEN administration. Expres-

92

S. SELL ET AL.

sion of Ki-rus and N-ras was also observed in both hepatocytes and in nodules, but with much greater variability. To explore further oncogene expression in various liver cell types during carcinogenesis, Yaswen et ul. (1985) used cell fractionation techniques to separate normal hepatocytes from oval cells. The expression of c-mos and c-abl was not detected in any cell fraction, while c-src was unchanged. Three other protooncogenes varied in their expression with respect to cell type and time of carcinogenesis (0.1% ethionine in a choline-deficient diet): c-Ha-rus, c-Ki-rus, and cmyc. Poly(A)+RNA from total liver contained elevated levels of all three transcripts within 2 weeks after the start of carcinogen administration: c-Ha-ras by a factor of 2-3, and c-myc and c-Ki-rus by factors of about 10. These increases persisted in tumors arising at 35 weeks. Kiras transcripts appeared early in hepatocytes (by 2 weeks), but were not abundant in oval cells until 9 weeks. c-myc expression followed a similar pattern. In contrast, the increased c-Ha-rus expression was seen in hepatocytes, but not in oval cells. In conclusion, specific patterns of oncogene expression can be observed in different cell types of the rat liver during chemical carcinogenesis. In future studies, more refined methods of analysis, such as in situ hybridization of oncogene probes to tissue sections or immunohistochemical analysis with antibodies against oncogene products will define more precisely the cellular pattern of oncogene expression in the preneoplastic liver. However, these studies may be limited by two factors: (1)The functions of most oncogenes are not known (for review, see Varmus, 1984),and (2) some oncogenes being studied (Kiras, Ha-rus, c-myc, and c-fos) are expressed at higher levels during regeneration of the liver following a partial hepatectomy (Makino et al., 1984a; Fausto and Shank, 1983; Fausto, 1984; Goyette et ul., 1983, 1984; Makino et ul., 1984b). These studies are complicated by the fact that sham hepatectomy can induce at least one oncogene (c-fos), so further controls will be needed (H. Leffert, personal communication). It will thus be difficult to distinguish oncogene expression occurring as a result of liver repair following toxic injury by the carcinogen from oncogene expression taking place as a part of neoplastic transfonnation.

C. CARCINOGENESIS AND TUMOR MRNA COMPLEXITY

If changes take place in the pattern of gene expression when normal tissue undergoes neoplastic development, then one should be able to observe differences in total mRNA sequence between tumor and nor-

HEPATOCARCINOGENESIS AND PREMALIGNANCY

93

ma1 tissue. Further, one might be able to detect such changes in preneoplastic tissue as a prelude to overt morphological changes. These hypotheses have driven large numbers of molecular hybridization studies in a diverse array of tumor systems. The results of these studies have been mixed, mainly because of methodological differences among different investigators. In general, the results show that the mRNA populations of hepatomas and normal liver are almost identical. A small number of mRNA sequences may be more or less abundant in the tumor compared to normal, but it is unlikely that genes which are turned off in the the normal tissue are turned on in the tumor. Changes in the abundance of some mRNA sequences can be observed as a function of carcinogenesis, but these experiments have been done with whole preneoplastic liver, thus limiting their sensitivity.

1 . Saturation Hybridization: Number of Genes Expressed In experiments of this type, labeled single-copy genomic DNA is hybridized with increasing amounts of RNA until a plateau value of hybridized DNA is reached. Provided the RNA is in sufficient excess, the plateau indicates the fraction of the genome which is transcribed. The value obtained from a mixture of two RNA populations can be compared with the value obtained with either population alone: additivity indicates nonidentical populations. In the most often cited study of this type, Groudine and Weintraub (1980) compared nuclear RNA from chick embryo fibroblasts (CEF) and from C E F transformed with Rous sarcoma virus (RSV). They found that 10-11% of the DNA hybridized to normal C E F RNA, while 13-15% hybridized to RSV C E F RNA. This difference in saturation corresponds to the activation of about 1000 new transcription units in RSV C E F cells, assuming that the RNA excess used was sufficient to allow hybridization by very scarce RNA molecules. This kind of analysis was subsequently applied to rat liver. Jacobs and Birnie (1980) found that polysomal mRNA from both normal liver and from the HTC hepatoma cell line hybridized to 1.5-1.6% of single-copy rat genomic DNA. When both RNAs were hybridized at the same time, no additivity was observed, indicating that identical polysomal mRNA sequences were present in both cell types. Thus, while Groudine and Weintraub (1980) found clear evidence of a qualitative difference between nuclear RNA from normal and transformed cells, no such difference was observed comparing rat liver with hepatoma cell line. The sensitivity of these experiments was not great enough to rule out the possibility one or several mRNA sequences might be unique to hepa-

94

S. SELL ET AL.

toma. Further, experiments of this type cannot detect extreme quantitative differences in the level of an mRNA transcript.

2 . Competitive Hybridization: Repetitive Sequences In the rat liver, many studies used competition hybridization to compare normal liver with transplantable hepatomas. In these experiments, one labeled RNA is hybridized with a slight excess of homologous genomic DNA in the presence of increasing amounts of competitor RNA, and hybridization is plotted as a function of competitor added. This kind of experiment generally detects only repetitive nucleotide sequences, particularly if the DNA is bound to a nitrocellulose filter. Drews et al. (1968)noted that excess nuclear RNA from the hepatomas 9121, 3924A, and H35TC1 competed less efficiently than liver nuclear RNA in hybridization of labeled liver nuclear RNA to rat DNA. On the other hand, liver nuclear RNA competed efficiently with labeled nuclear RNA from all three hepatomas for hybridization to rat DNA. Thus, the hepatomas were lacking some nuclear RNA sequences found in liver, but had no sequences unique to themselves. Similar findings were made by Mendecki et al. (1969) for the tumor 5123 and by Chiarugi (1969) for the tumor 5123 and the Yoshida hepatoma AH130. Because these studies assayed repetitive sequences present in RNA sequences, it is not possible to draw conclusions about the numbers of single-copy genes being expressed in tumors and normal liver or the abundance of their transcripts.

3. Kinetic Hybridization: Transcript Abundance Although the saturation hybridization experiments show no qualitative differences between liver and hepatoma, many studies have attempted to find quantitative differences in mRNA sequences. In these experiments, one of the poly(A)+ mRNAs is made into cDNA using reverse transcriptase in the presence of radioactive precursors. This permits the mRNA sequences from one tissue to be hybridized with the mRNA of another and for the rate of the reaction to b e followed by measuring radioactivity being driven into hybrid form. This is a kinetic method whose sensitivity is limited to abundant and moderately abundant mRNA sequences. Polysomal mRNA from Novikoff hepatoma is deficient in about 30% of the polysomal mRNA found in normal liver (comprising about 3-4 sequences of the most highly abundant mRNAs). Inversely, 8-10% of the polysomal mRNA from hepatoma failed to hybridize with normal liver mRNA, indicating that some polysomal mRNA from hepatoma was reduced in abundance in normal liver. Similar results were found

HEPATOCARCINOGENESIS AND PREMALIGNANCY

95

with nuclear RNA (Capetanaki and Alonso, 1979). Knochel et al. (1980), examining polysomal mRNA from Chang’s hepatoma, also found depletion of some mRNA sequences. Chiu et al. (1983) found that about 8% of the polysomal mRNA of the hepatoma 7777 failed to hybridize with normal liver polysomal mRNA. In all cases where evidence was found for hepatoma-abundant sequences, they comprised part of the least abundant class of mRNA sequences. To summarize, studies with established rat hepatomas suggested extensive depletion of mRNA sequences accompanied by the appearance of some mRNA sequences not present (or present at a much lower abundance) in normal liver.

4 . Sequence Complexity in Primary Tumors and Preneoplastic Liver Can these differences also be demonstrated for primary tumors and preneoplastic versus normal liver? In kinetic hybridizations, Knochel et al. (1980) found that polysomal mRNA from the livers of rats fed 3’Me-DAB for 17 weeks was identical in sequence content to normal liver polysomal mRNA. However, rats fed carcinogen for only 10 weeks showed some shift in abundances of liver polysomal mRNA: Some members of the abundant class became more abundant, while some scarce mRNAs became more scarce. Atryzek et al. (1980),using kinetic hybridization, examined polysomal mRNA from the livers of rats fed 0.05% ethionine in a choline-deficient diet for 8 weeks (preneoplastic liver) and found no difference from normal liver. Neoplastic livers (22-25 weeks of feeding) were also very similar, except that minor shifts in the abundance of some mRNA sequences might have occurred. Thus, no great differences seem to exist among the polysoma1 mRNA populations of normal, preneoplastic, and neoplastic liver. What about nuclear RNA? Fausto et al. (1982) compared nuclear RNA from primary liver tumors (generated by 0.05% ethionine in a choline-deficient diet) with normal liver and found that hybridization between labeled nuclear cDNA from tumor hybridized more slowly (in the low-abundance region) to normal nuclear RNA and with a lower saturation value (by several percent) compared with the homologous hybridization. These results suggested that some low-abundance mRNA sequences might be elevated in amounts in primary tumors. Nuclear RNA from AAF-induced primary tumors contained no tumor-specific sequences, as assayed by saturation hybridization (Austin et al., 1982). Several studies have examined nuclear RNA by competition hybridization. Aka0 and Kuroda (1981) analyzed nuclear RNA from primary

96

S. SELL ET AL.

hepatoma and from livers of rats fed 3'-Me-DAB for 7 weeks (preneoplastic). No changes were observed in the preneoplastic liver, while a depletion of about 15% of the nuclear RNA sequence from primary hepatoma took place. Shearer and Smuckler (1971) found no tumorspecific sequences among nuclear RNA from primary tumors induced with 3'-Me-DAB. As discussed above, this assay measures repetitive RNA sequences. In this brief survey, we have attempted to summarize what is known about changes in gene expression in the rat liver during carcinogenesis. A large number of studies have compared total mRNA sequences of normal liver, preneoplastic liver, and hepatoma. In general, shifts in the abundance of some mRNA sequences can be observed, particularly when transplantable hepatomas are analyzed, but these experiments have provided no evidence for the expression of tumorspecific genes. Thus, there appears to be no qualitative difference between normal liver and hepatoma, but quantitative differences may exist.

5. Analysis of Speci&c Transcripts The molecular hybridization methods used in the above studies suggest that the mRNA sequences of normal, preneoplastic, and neoplastic liver are almost identical. If mRNA sequences are unique to neoplastic liver, they must be few in number and/or very scarce in abundance. If such mRNA sequences can b e shown to exist, it would imply that transformation involves changes in the expression of a relatively small number of genes. Using recently developed recombinant DNA techniques, several groups have now demonstrated that transformed cells contain mRNA sequences which are less abundant in normal cells. Yamamoto et al. (1983),using differential colony hybridization (see Williams, 1981), isolated cDNA clones corresponding to a 0.6 kb mRNA, a 1.5 kb mRNA, and a cDNA clone encoding a repetitive sequence DNA element present in a large number of mRNA molecules in hepatoma, but not in normal or regenerating liver. It is not yet known what these mRNA sequences encode, and their expression as a function of carcinogenesis still needs to be investigated. Differentially expressed mRNA sequences have also been found in SV40transformed 3T3 cells compared to normal 3T3 cells (Schutzbank et al., 1982; Scott et al., 1983).One of these mRNA sequences was found in every mouse tumor examined (Scott et al., 1983). Thus, it is now possible to clone cDNAs which correspond to mRNAs differentially expressed in transformed cells. Although these mRNA sequences may eventually prove to be useful

HEPATOCARCINOGENESIS AND PREMALIGNANCY

97

as markers of carcinogenesis, it is still uncertain that the proteins they encode have any relevance to the development of tumors. For example, when Groudine and Weintraub (1980) demonstrated the expression of transformation-specific RNA in RSV-transformed CEF, one of these mRNA sequences encoded globin. In the rat liver, the expression of the AFP gene increases at least 30-fold during carcinogenesis (from a very low level in the normal adult liver) (Petropoulos et al., 1983), and transplantable hepatomas contain variable but much higher than normal levels of AFP mRNA (Sell et al., 1979b, 1979c; Sala-Trepat e t al., 1979). It is hard to imagine how either globin or AFP is relevant to carcinogenesis, although we simply may not understand their significance. Claims for tumor-specific gene products must always be viewed with caution.

D. CHANGES IN LIVERPROTEINS DURING CARCINOGENESIS When a normal tissue becomes malignant, one might expect changes to occur in the types and/or abundances of proteins present in the cells. Such changes might occur if the transcription of genes were turned on or off or if mRNA molecules were translated more or less efficiently or if posttranslational modification of proteins occurred differently. In many cases, investigators have measured changes in the amounts of specific proteins or enzymes and found large variations in activity (or amount) as a function of carcinogenesis. This approach suffers from being limited to only one or a few different proteins. It is possible that interesting proteins for which no assay exists may appear (or disappear) during carcinogenesis. To detect these, several groups have used gel electrophoresis to compare total proteins of tumors with their normal counterparts. Using two-dimensional gel electrophoresis (O’Farrell, 1975), one can generate “maps” (consisting of hundreds of proteins spots) of both normal and malignant tissue, then compare the two and look for differences. The kinds of differences reported are increases or decreases in the amounts of specific protein spots. Occasional claims are made for the appearance of “new” proteins during carcinogenesis; however, it is usually impossible to prove the absence of a new protein from the normal tissue. It must also be remembered that changes in protein function (rather than amount) may be important in carcinogenesis: These will be missed by two-dimensional gel analysis unless the altered function is acocmpanied by changes in protein size or charge. Finally, posttranslational modifications of proteins during carcinogenesis may change their migration and complicate the analysis of twodimensional gel patterns.

98

S. SELL E T AL.

Some studies have compared liver with hepatoma, with whole liver from carcinogen-fed rats, or with dissected hyperplastic nodules to determine whether nodules (or carcinogen-treated liver) have any relation to hepatoma in terms of protein constitution. In many cases, liver is compared with regenerating liver to identify proteins which may be present in higher amounts as a result of cell proliferation. Fetal liver may also be analyzed to identify proteins which are produced as a result of activation of fetal genes. The idea behind these cross comparisons is to identify a specific set of proteins which are overproduced (or underproduced) as a result of carcinogenesis, not cell proliferation or fetal gene activation. Finally, comparisons have been done of both cytosolic proteins and nuclear proteins, particularly the nonhistones. Nuclear proteins are of interest because they are presumed to contain gene regulatory proteins whose amounts might be changed during carcinogenesis. Of course, as noted above, it is just as likely that their functions will be changed, and this would go undetected.

1 . Transplantable Hepatomas A great deal of work has compared the protein maps of liver and transplantable hepatomas. Differences detected here could then be looked for in preneoplastic liver. The early work in this field is reviewed by Allfrey and Boffa (1979), Stein et al. (1978), and Gronow

(1980). Rodriguez et al. (1979) used one-dimensional SDS gels to compare the chromosomal proteins of liver with the hepatomas 9618A, 7800, 7777,253, 311C, and 252. The tumors showed an increased number (at least 10) of nonhistone chromosomal proteins (NHCPs) in the 55,000-220,000 MW range and were not lacking in any proteins observed for normal liver. It was not possible to determine if the new NHCPs were the same among all the hepatomas. Busch and colleagues have extensively catalogued the proteins produced by adult, fetal, and regenerating rat liver, as well as numerous transplantable hepatomas. Hirsch et al. (1978) used two-dimensional gels and Coomassie Blue staining to detect and compare the abundant cytosolic proteins from liver and Novikoff hepatoma and the Morris hepatomas 9618A, 8999, and 3924A. Out of about 100 proteins visualized, 1 (79/6.7) was common to all tumors, but not detectable in the liver. Variations were found in about 10% of the proteins from one hepatoma to the next, illustrating tumor heterogeneity. Much more work has been done with nuclear proteins. Takami and Busch (1979) salt-fractionated nuclear proteins and then ran two-dimensional gels

HEPATOCARCINOGENESIS AND PREMALIGNANCY

99

(“3,” analysis) comparing liver with Novikoff ascites hepatoma. With Coomassie Blue staining, over 500 proteins could be resolved. Of these, 18 were detectable in Novikoff but not liver, and 12 showed the reverse pattern. To further sort these out, Takami et al. (1979) compared liver with regenerating liver, fetal liver, and Morris hepatomas 9618A and 3924A. Proteins 79/6.4 and 6U7.2 were detectable only in the tumors (including NovikofQ, and one protein (37/6.3)was overproduced in the tumors compared with regenerating and fetal liver. These results demonstrate that, given the limits of the analysis, hepatomas do not differ greatly from normal liver. Further, hepatomas are heterogenous: Only a very small number of proteins are shared by hepatomas that are not detectable in adult, fetal, and regenerating liver. The small number of differences observed suggests that it may be possible to identify specific changes in preneoplastic liver during carcinogenesis. 2 . Preneoplastic Liver Attempts have been made to demonstrate changes in the nonhistone chromosomal proteins extracted from whole carcinogen-treated liver. Tsanev and Hadjiolov (1978), using one-dimensional SDS gels and Coomassie Blue staining, observed no differences between normal liver and liver from rats fed nitrosomorpholine for 30 or 90 days. In primary hepatomas induced by this carcinogen, slight increases in the amounts of two proteins of 43 and 63 kDa were observed. Martinez-Sales et al. (1981) saw slight increases in similar protein fractions after feeding rats for 4 or 10 weeks with DEN. These changes were not observed by Pentecost and Craddock (1983) using dimethylnitrosamine as the carcinogen. Rather, they saw a decrease in the amount of a 65 kDa protein and an increase in a 170 kDa protein. These analyses all suffered the limitation that important changes may be occurring in only a small fraction of the total liver and thus would be missed. Other investigators have looked closely at hyperplastic nodules to see whether they have proteins in common with carcinomas that are not present in normal liver. Eriksson et al. (1983) generated nodules by several different protocols and analyzed cytosolic proteins by onedimensional SDS gels. Nodules contained a 10-fold elevation in a polypeptide of 21,000 MW. Sugioka et al. (1985a) later demonstrated this protein to be a form of glutathione S-transferase normally found in kidney and placenta (see below). These same authors, using two-dimensional gels and Coomassie Blue staining to examine cytosolic proteins, also observed increases in proteins of 35 kDa in nodules and

100

S. SELL ET AL.

carcinomas, the latter generated by AAF, DEN, or 3‘-Me-DAB. Alkaliresistant phosphoproteins were also examined, and one, of molecular weight 57,000 (distinct from the previously discussed 57,000), increased about 5-fold in amount in nodules and in carcinomas compared with normal liver. The placental glutathione S-transferase (GST-P), so called because it is the most abundant GST isoenzyme in placenta, is the subject of increasing interest because its overproduction (50-1OOX over normal liver) is the most consistent phenotype so far described for rat hepatomas. Elevated GST-P protein has been observed in the transplantable hepatoma 5123D and in a variety of primary hepatomas induced by several carcinogens (Satoh et al., 1985). Increased GST-P mRNA is observed in the hepatomas 5123C, 7777, and 9098 (Knoll et al., 1986). The degree of elevation ranges from 20- to 100-fold; small amounts of the enzyme are detectable in normal liver and several other tissues. In contrast, elevation of AFP mRNA is observed in only of few of these tumors (Knoll, Longley, and Sell, unpublished). GST-P is abundant in hyperplastic nodules generated by several regimens (Eriksson et al., 1983; Sugioka et al., 1985) and in GGT-positive foci of preneoplastive livers (Sato et al., 1984). It has been proposed that the elevation of GST-P confers resistance to the cytotoxic action of carcinogens, thereby allowing proliferation of nodules into carcinomas (Satoh et al., 1985).This interesting hypothesis awaits experimental test. A further interesting point is that GST-P is only one of a large number of GST isoenzymes present in the liver, yet it is the only one found to be elevated in hepatomas (Bhargava et al., 1982; Carruthers et al., 1979; Sat0 et al., 1983). The recent isolation of cDNA clones encoding GST-P will aid in the exploration of this phenomenon (Sugioka et al., 1985b; Knoll et al., 1986). Nuclear proteins were studied by Ramagli et al. (1985) using twodimensional gels and silver staining along with computer-aided image analysis to examine more than 500 proteins. A wide spectrum of changes was observed, including four proteins of 19.3, 30.7,46.6 and 53.5 kDa, which were observed in AAF nodules and in carcinomas generated by AAF or DEN, but not in normal liver. Fourteen other proteins appeared “new” during carcinogenesis, but were present only in nodules or only in carcinomas. Sudhakar et al. (1984) examined nuclear phosphoproteins and detected one (36 kDa) in carcinomas induced by AAF or DEN and in livers of rats fed DEN. Several others were detected in carcinomas induced by one carcinogen, but not the other. To summarize, comparisons of total cytosolic or nuclear proteins

HEPATOCARCINOGENESIS AND PREMALIGNANCY

101

from normal, preneoplastic, and neoplastic liver reveal a small number of changes compared to the large number of common proteins. Some of these changes are common to both nodules and to carcinomas, although each tissue type is unique (Ramagli et al., 1985). If it is true that nodules and hepatomas are clonal in origin, then heterogeneity among nodules and carcinomas might be expected. This has been demonstrated for transplantable carcinomas (Takami et al., 1979),and some variation among nodules has been mentioned by Sugioka et al. (1985a). The evidence thus far does not prove that nodules are precursors to carcinomas, nor does the evidence disprove it. No study to date has examined the protein map of an oval cell population, nor has anyone looked at nonnodular portions of a nodular liver. No doubt future studies using detailed protein maps will be able to trace the development of normal into neoplastic cells.

VII. Summary

The cellular, biochemical, and genetic changes that occur in the liver of rats exposed to chemical hepatocarcinogens are reviewed. Multiple new cell types appear in the liver of carcinogen-treated rats including foci, nodules, ducts, oval cells, and atypical hyperplastic areas. The application of phenotypic markers for these cell types suggests that hepatocellular carcinomas may arise from more than one cell type, including a putative liver stem cell that proliferates following carcinogen exposure. Study of DNA, RNA, and proteins produced by hepatocellular carcinomas and putative premalignant cells has so far failed to identify a gene or gene product clearly associated with the malignant or premalignant phenotype. Understanding the cellular lineage from normal cell through putative premalignant cell to cancer is critical to understanding the process of carcinogenesis. Application of new immunological (monoclonal antibody, transplantation) and molecular biological (gene cloning, oncogene identification) approaches to this problem holds promise that the process of hepatocarcinogenesis will be better known in the near future. ACKNOWLEDGMENTS We thank Jackie Sanders Fagan and Annie Rose for excellent word processing assistance. The editorial suggestions of Drs. H. Shinozuka and H. Leffert are acknowledged with thanks. The authors’ research was supported in part by PHS Grants CA37150, CA34635, and CA39792, awarded by the National Cancer Institute, DHHS.

102

S. SELL ET AL.

REFERENCES Abelev, G. I. (1971).Adu. Cancer Res. 14, 295-358. Abelev, G. I., Perova, S . D., Khramkova, N. I., Postnikova, Z. A., and Irlin, I. S. (1963). Transplantation 1, 174-186. Akao, M., and Kuroda, K. (1981). Cancer Res. 41,735-740. Allfrey, V. G., and Boffa, L. C. (1979).In “The Cell Nucleus” (H. Busch, ed., Vol. 7, pp. 521-562. Academic Press, New York. Altmannsberger, M., Weber, K., Droste, R., and Osborn, M. (1985).Am. J. Pathol. 118, 85-95. Andervont, H. B., and Dunn, T. B. (1952).J.Natl. Cancer Znst. 13,455-503. Andervont, H. B., and Dunn, T. B. (1955).J . Natl. Cancer Znst. Suppl. 15, 1513-1524. Atryzek, V., Tamaoki, T., and Fausto, N. (1980). Cancer Res. 40,3713-3718. Austin, G . E., Russo, R. J., and Moyer, G. H. (1982). Carcinogenesis 3, 609-613. Baldwin, R. W. (1973).Adu. Cancer Res. 18, 1-75. Balmain, A., and Pragnell, I. B. (1983). Nature (London)303, 72-74. Balmain, A., Ramsden, M., Bowden, G. T., and Smith, J. (1984). Nature (London) 307, 658-660. Beard, J. W. (1980). In “Viral Oncology” (G. Klein, ed.), pp. 55-87 Raven, New York. Becker, F. F. (1981).A m . J. Pathol. 105,3-9. Becker, F. F. (1982). Cancer Res. 42,3918-3923. Becker, F. F., and Sell, S. (1974). Cancer Res. 34,2489-2494. Becker, F. F., and Sell, S. (1979). Cancer Res. 39, 1437-1442. Becker, F. F., Klein, K. M., Wolman, S. M., Asofsky, R., and Sell, S. 1973).Cancer Res. 33,3330-3338. Beckei, F. F., Stillman, D., and Sell, S. (1977). Cancer Res. 37,870-872. Behrens, U. J., and Paronetto, F. (1978). Zmmunology 35,289-298. Berenblum, I., and Shubik, P. (1947). Br. J. Cancer 1, 379-391. Berenblum, I., and Shubik, P. (1949). Br. J. Cancer 3, 109-118. Bernhard, M. I., Foon, K. A., Oeltmann, T. N., Key, M. E., Huang, K. M., Clarke, G. C., Christensen, W. L., Hoyer, L. C., Hanna, M. G., Jr., and Oldham, R. K. (1983). Cancer Res. 43,4420-4428. Bhargava, M., Ohmi, N., Arias, I. and Becker, F. F. (1982). Oncology 39, 378-381. Billett, E. E., Gunn, B., and Mayer, R. J. (1984). Biochem. J. 221, 765-776. Blumberg, B. S., and London, W. T. (1982). Cancer 50,2657-2665. Bone, S. N., 111, Michalopoulos, G., and Jirtle, R. L. (1985). Cancer Res. 45, 12221228. Borenfreund, E., Higgins, P. J., Steinglass, M., and Bendich, A. (1975).J. Natl Cancer Znst. 55, 375-384. Boutwell, R. K. (1978). In “Carcinogenesis” (T. Sloga, A. Sivak, and R. Boutwell, eds.), Vol. 2, pp. 49-58. Raven, New York. Bowen, J. G., and Baldwin, R. W. (1979). Znt.1. Cancer 23,826-832. Brown, J. D., Wilson, M. J., and Poirier, L. A,, (1983). Carcinogenesis 4, 173-177. Cameron, R. G., Eriksson, L. C., and Lee, G. (1984).Acta Cytol. 28, 614-620. Campisi, J., Gray, H. E., Pardee, A. B., Dean, M., and Sonenshein, G. E. (1984).Cell 36, 241-247. Capetanaki, Y., and Alonso, A. (1980). Nucleic Acids Res. 8, 3193-3214. Carruthers, C., and Baumler, A. (1979). Oncology 36, 265-270. Chiarugi, V. P. (1969). Biochim. Biophys. Acta 179, 129-135. Chiu, J. F., Huang, D. P., Burkhardt, A. L., Cote, G., and Schwartz, C. E. (1982).Arch. Biochem. Biophys. 222,310-320. Chou, J. Y., and Schlegel-Haueter, S. E. (1981).J. Cell Biol. 89, 216-222.

HEPATOCARCINOGENESIS AND PREMALIGNANCY

103

Chu, F. F., and Doyle, D. (1985).J.Biol. Chem. 260, 3097-3107. Churchill, W. H., Jr., Rapp, H . J., Kronman, B. S., and Borsos, T. (1968).J.Natl. Cancer Znst. 41, 13-19. Condamine, H., Custer, R. P., and Mintz, B. (1971).Proc. N a t l . Acad. Sci. U.S.A. 68, 2032-2036. Cook, J., Hou, E., Hou, Y., Cairo, A., and Doyle, D. (1983). Cell Biol. 97, 1823-1833. Corcos, D., Defer, N., Raymondjean, M., Paris, B., Corral, M., Tichonicky, L., Kruh, J., Glaise, D., Saulnier, A., and Guguen-Guillouzo, C. (1984). Biochem. Biophys. Res. Commun. 122,259-264. Craddock, V. M. (1972).J . Natl. Cancer Znst. 47,889-907. Craddock, V. M. (1974). Br. J . Cancer 30,503-511. Dempo, K. N., Chisaka, W., Yoshida, Y., Kaneko, A., and Onoe, T. (1975). Cancer Res. 35, 1282-1287. DeOme, K. B., Faulkin, L. J., Jr., Bern, H. A., and Blair, P. B. (1959). Cancer Res. 19, 515-520. Deschatrette, J. (1980).Cell 22, 501-511. Diamond, L. (1969). Prog. E x p . Tumor Res. 11,364-383. Drews, J., Brawerman, G., and Morris, H. P. (1968). Eur. J. Biochem. 3, 284-292. Druckrey, H., Preussmann, R., Ivankovic, S., and Schmahl, D. (1967). Z. Krebsforsch. 69, 103-201. Duesberg, P. H. (1985).Science 228,669-677. Dunsford, H. A., and Sell, S. (1987). In preparation. Dunsford, H. A., Maset, R., Salman, J., and Sell, S. (1985).A m . J .Pathol. 118,218-224. Dvorak, H. F., Harvey, V. S., and McDonagh, J. (1984). Cancer Res. 44,3348-3354. Enat, R., Jefferson, D. M., Ruiz-Opazo, N., Gatmaitan, Z., Leinwand, L. A., and Reid, L. (1984). Proc. Natl. Acad. Sci. U.S.A.81, 1411-1415. Enomoto, K., and Farber, E. (1982). Cancer Res. 42, 2330-2335. Epstein, S., Ito, N., Merkow, L., and Farber, E. (1967).Cancer Res. 27, 1702-1711. Eriksson, L. C., Shanna, R. N., Roomi, M. W., Ho, R. K., Farber, E., and Murray, R. K. (1983). Biochem. Biophys. Res. Commun. 117, 740-745. Eva, A., and Aaronson, S. (1983).Science 220, 955-956. Evarts, R. P., Marsden, E., and Thorgeirsson, S. S. (1984). Proc. Am. Assoc. Cancer Res. 25, 81. Everett, R. (1984). CRC Crit. Reu. Toxicol. 13, 235-251. Farber, E. (1956).Cancer Res. 16, 142-148. Farber, E. (1963).Ado. Cancer Res. 7, 383-474. Farber, E. (1973).Cancer Res. 33,2537-2550. Farber, E. (1980).Biochim. Biophys. Acta 605, 149-166. Farber, E. (1981).Acta Pathol.Jpn. 3, 1-11. Farber, E. (1982a).Am. J. Pathol. 106,271-296. Farber, E. (1982b). A m . . / . Pathol. 108,270-275. Farber, E. (1984a). Cancer Res. 44,4217-4223. Farber, E. (1984b). Cancer Res. 44, 5463-5474. Farber, E. ( 1 9 8 4 ~ )C. a n J . Biochem. Cell Biol. 62, 486-494. Farber, E., and Cameron, R. (1980).Ado. Cancer Res. 31, 125-226. Farber, E., Cameron, R. G., Laishes, B., Lin, J. C., Medline, A., Ogawa, K., and Solt, D. B. (1979). In “Carcinogens Identification and Mechanisms” (A. C. Griffin and C. R. Shaw, eds.), pp. 319-335. Raven, New York. Faris, R., Allison, J. P., and Hixson, D. C. (1985). Proc. Am. Assoc. Cancer Res. 25, 135 (Abstr.). Fausto, N. (1984). M o l . Cell. Biochem. 59, 131-147. Fausto, N., and Shank, P. R. (1983).Hepatology 3, 1016-1023.

104

S . SELL ET AL.

Fausto, N., Schultz-Ellison, G., Atryzek, V., and Goyette, M. (1982).J.Biol. Chem. 257, 2200-2206. Finkelstein, S. D., Lee, G., Medline, A., Tatematsu, M., Makowka, L., and Farber, E. (1983). Am. J . Pathol. 110, 119-126. Firminger, H. (1955).J . Natl. Cancer Znst. 15, 1427-1422. Fisher, B., and Fisher, E. R. (1962). Proc. SOC.E x p . Biol. Med. 109,62-64. Franke, W. W., Mayer, D., Schmid, E., Denk, H., and Borenfreund, E. (1981).E x p . Cell Res. 134, 345-365. Friedman, F. K., Robinson, R. C., Park, S. S., and Gelboin, H. V. (1983). Biochem. Biophys. Res. Commun. 116,859-865. Fujita, S., Ishizuka, H., Kamimura, N., Kaneda, H., and Ariga, K. (1975).Ann. N.Y.Acad. Sci. 259, 217-220. Fukumoto, T., Kimura, H., Naito, M., Miyamoto, M., Yamashita, A., and Sugiyama, H. (1984). Mol. Zmmunol. 21,285-291. Fukushima, S., Shibata, M., Hibino, T., Yoshimura, T., Hirose, M., and Ito, N. (1979). Toxicol. Appl. Pharmacol. 48, 145-155. Germain, L., Goyette, R., and Marceau, N. (1985). Cancer Res. 45, 673-681. Giedlin, M. A., Martin, W. J., and Callahan, G. N. (1983).]. Natl. Cancer Znst. 71,825834. Glenney, J. R., Jr., Kaulfus, P. J., Mcintyre, B. W., and Walborg, E. F., Jr. (1980).Cancer Res. 40,2853-2859. Goldfarb, S., and Pitot, H. C. (1976). Front. Castrointest. Res. 2, 194-242. Goldfarb, S., Singer, E. J., and Popper, H. (1962).Am. J. Pathol. 40, 685-698. Goustin, A. S., Leof, E. B., Shipley, G. O., and Moses, H. L. (1986). Cancer Res. 46, 1015-1029. Goyette, M. C., Petropoulos, P. R., Shank, P. R., and Fausto, N. (1983). Science 219, 510-512. Goyette, M., Petropoulos, C. J., Shank, P. R.,and Fausto, N. (1984). Mol. Cell. Biol. 4, 1493-1498. Grisham, J. W. (1983). Mol. Cell. Biochem. 53/54, 23-33. Grisham, J. W., and Porta, E. A. (1964). E x p . Mol. Pathol. 3,242-261. Gronow, M. (1980). Chem. Biol. Interact. 29, 1-30. Groopman, J. D., Haugen, A., Goodrich, G. R.,Wogan, G. N., and Harris, C. C. (1982). Cancer Res. 42,3120-3124. Groopman, J. D., Trudel, L. J., Donahue, P. R., Marshak-Rothstein, A,, and Wogan, G. N. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 7728-7731. Groudine, M., and Weintraub, H. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 5351-5354. Guerrero, I., Calzada, P., Mayer, A., and Pellicer, A. (1984a). Proc. Natl. Acad. Sci. U.S.A. 81,202-205. Guerrero, I., Villasante, A., Corces, V., and Pellicer, A. (1984b). Science 225, 11591162. Guguen-Guillouzo, C., and Guillouzo, A. (1983). Mol. Cell. Biochem. 53/54,35-56. Gunn, B., Embleton, M. J., Middle, J. G., and Baldwin, R. W. (1980). Int. J. Cancer 26, 325-330. Haddow, A. (1938). Acta Un. Znt. Cancr. 3,342-353. Hanigan, M. H., and Pitot, H. C. (1985a). Cancer Res. 45,6063-6070. Hanigan, M. H., and Pitot, H. C. (1985b). Carcinogenesis 6, 165-172. Hayashi, K., Makino, R., and Sugimura, T. (1984). Cann 75,475-478. Hayes, M. A,, Lee, G., and Farber, E. (1985). Proc. Am. Assoc. Cancer Res. 26, 133. Heidelberger, C. (1975). Annu. Reo. Biochem. 45, 79-121.

HEPATOCARCINOGENESIS AND PREMALIGNANCY

105

Heine, U. I., Wilson, M. J., and Munoz, E. F. (1984).In Vitro 20, 291-301. Heintz, N., Little, B., Bresnick, E., and Schaeffer, W. I. (1980). Cancer Res. 40, 12811285. Hellstrom, I., Rollins, N., Settle, S., Chapman, P., Chapman, W. H., and Hellstrom, K. E. (1982). Znt. J . Cancer. 29, 175-180. Hilpert, D., Romen, W., and Neumann, H.-G. (1983).Carcinogenesis 4, 1519-1525. Hill, J. (1761). Cautions Against the Immoderate Use of Snuff. Baldwin and Jackson, London. Hertzog, P. J., Shaw, A., Smith Lindsey, J. R., and Garner, R. C. (1983).J . Immunol. Methods 62,49-58. Hirsch, F. W., Nall, K., Busch, F., Morris, H. P., and Busch, H. (1978). Cancer Res. 38, 1514-1522. Hixson, D. C., Ponce, M. D., Allison, J. P., and Walborg, Jr. E. F. (1984).Exp. Cell Res. 152,402-414. Holmes, C. H., Austin, E. B., Fisk, A., Gunn, B., and Baldwin, R. W. (1984). Cancer Res. 44, 1611-1624. Holmes, E. H., and Hakomori, S. (1982).J . Biol. Chem. 257, 7698-7703. Hornberger, M., Hanser, G., and Ruhenstroth-Bauer, G. (1981). Z. Versuchstierkd. 23, 317-321. Huberman, E., Montesano, R., Drevon, C., Kuroki, T., St. Vincent, L., Pugh, T. D., and Goldfarb, S. (1979).Cancer Res. 39, 269-272. Hunt, J. M., Buckley, M. T., Onnink, P. A,, Rolfe, P. B., and Laishes, B. A. (1982). Cancer Res. 42,227-236. Hunt, J. M., Buckley, M. T., Laishes, B. A., and Dunsford, H. A. (1985). Cancer Res. 45, 2226-2233. Ishikawa, T., Takayama, S., and Kitagawa, T. (1980). Cancer Res. 40,4261-4264. Isom, H. C., Tevethia, M. J., and Kreider, J. W. (1981). Cancer Res. 41,2126-2134. Jacobs, H., and Birnie, G. D. (1980).Nucleic Acids Res. 8, 3087-3103. Jirtle, R. L., and Michalopoulos, G. (1985). Proc. A m . Assoc. Cancer Res. 26, 134. Jirtle, R. L., Michalopoulos, G., McLain, J. R., and Crowley, J. (1981). Cancer Res. 41, 3512-3518. Judah, D. J., Legg, R. F., and Neal, G. E. (1977). Nature (London)265,343-345. Kaplan, A. E., Yamaguchi, M. Y., Tralka, T. S., and Hanna, C. H. (1982). Exp. Cell Res. 138,251-260. Kaufmann, W. K., Tsao, M.-S., and Novicki, D. L. (1986). Carcinogensis 7, 669-671. Kelly, K., Cochran, B. A., Stiles, C. D., and Leder, P. (1983). Cell 35, 603-610. Kinosita, R. (1937). Truns. Soc. Pathol. Japn. 27, 665-725. Kinosita, R. (1940).Yale J . B i d . Med. 80, 231-246. Kitagawa, T. (1976).Cancer Res. 36,2534-2539. Kitagawa, T., Watanabe, R., Kayano, T., and Sugano, H. (1980). Conn 71,747-754. Klein, P. A. (1981). Transplant. Proc. 13, 4. Knochel, W., Patel, N. T., and Houloubeck, V. (1980). Biochim. Biophys. Acta 606,6775. Knoll, B. J., Longley, M. A., and Sell, S. (1986). Tumour Biol. 7, 123-125. Knook, D. L., Seffelaar, A. M., and deleeuw, A. M. (1982). Erp. Cell Res. 139,468-471. Knox, W. E. (1976). In “Fetal, Adult and Neoplastic Rat Tissues”, 2nd Ed. Karger, Basel. Koen, H., Pugh, T. D., and Goldfarb, S. (1983).A m . J . Pathol. 112,89-100. Kohler, G., Howe, S. C., and Milstein, C. (1975). Eur. J . Zmmunol. 6, 292-299. Kripke, M. L. (1981). Ado. Cancer Res. 34,69-106.

106

S. SELL ET AL.

Kuhlmann, W. D. (1978). Znt. J. Cancer 21,363-380. Kusano, M., and Mito, M. (1982). Gastroenterology 82, 616-628. Lafarge-Frayssinet, C., Morel-Chany, E., Trincal, G., and Fraysinnet, C. (1981). Cell. Mol. Biol. 27, 77-82. Lafarge-Frayssinet, C., Estrade, S., Rosa-Loridon, B., Frayssinet, C., and Cassingena, R. (1984). Cancer Lett. 22, 31-39. Laishes, B. A., and Farber, E. (1978).J. Natl. Cancer Znst. 61,507-512. Laishes, B. A., and Rolfe, P. B. (1980). Cancer Res. 40,4133-4143. Laishes, B. A., Roberts, E., and Farber, E. (1978) Znt. J. Cancer 21, 186-193. Laishes, B. A., Fink, L., and Cam, B. I. (1980). Ann. N.Y. Acad. Sci. 349, 373-382. Lando, P., Berzins, K., and Perlmann, P. (1982). Scund. J. Zmmunol. 15, 187-193. Leduc, E. H. (1959). Cancer Res. 19,1091-1095. Leduc, E. H., and Wilson, J. W. (1963).J. Natl. Cancer Znst. 30,85-99. Lee, G., Makowka, L., Kaku, T., Tatematsu, M., Finkelstein, S., and Medline, A. (1982). Proc. Am. Assoc. Cancer Res. 23,96. Lee, G., Tatematsu, M., and Makowka, L. (1983). Proc. Am. Assoc. Cancer Res. 24,106. Lee, G., Eriksson, L. C., and Cameron, R. (1985). Proc. Am. Assoc. Cancer Res. 26,74. Leffert, H. L., Moran, T., Boorstein, R., and Koch, K. S. (1977). Nature (London) 267, 58-61. Leffert, H. L., Koch, K. S., Sell, S., Skelly, H., and Shier, W. T. (1983).In “Application of Biological Markers to Carcinogen Testing” (H. A. Milman and S. Sell, eds.), pp. 119-133. Plenum, New York. Leffert, H. L., Schenk, D. B., Hubert, J. J., Skelly, H., Schumacher, M., Ariyasu, R., Ellisman, M., Koch, K. S., and Keller, G . A. (1985). Hepatology 5, 501-507. Lennox, E. S. (1980). Prog. Zmmunol. 4, 659-657. Lopez, M., and Mazzanti, L. (1955).J. Pathol. Bacteriol. 69, 243-250. Lotlikar, P. D., Enomoto, M., Miller, E. C., and Miller, J. A. (1964). Cancer Res. 24, 1835-1842. McMahon, J. B., Farrelly, J. G., and Iype, P. T. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 456-460. Magee, P. N., and Barnes, J. M. (1967). Ado. Cancer Res. 10, 163-246. Makino, R., Hayashi, K., and Sugimura, T. (1984a). Nature (London)310,697-698. Makino, R., Hayashi, K., Sato, S., and Sugimura, T. (1984b). Biochem. Biophys. Res. Commun. 119,1096-1102. Manson, M. M., Legg, R. F., Watson, J. V., Green, J. A., and Neal, G. E. (1981). Curcinogenesis 2, 661-670. Marshall, C. J., Vousden, K. H., and Phillips, D. H. (1984). Nature (London)310,586589. Martinez-Sales, V., Gabaldon, M., and Baguena, J. (1981). Cancer Res. 41, 1187-1192. Mendecki, J., Minc, B., and Chorazy, M. (1969). Biochem. Biophys. Res. Commun. 36, 494-501. Miller, E. C. (1970). Cancer Res. 30, 559-576. Miller, E. C. (1978). Cancer Res. 38, 1479-1496. Miller, S. B., Pretlow, T. P., Scott, J. A., and Pretlow, T. G. I1 (1982).J. Natl. Cancer Znst. 68, 851-857. Mito, M., Ebata, H., Kusano, M., Onishi, T., Hiratsuka, M., and Saito, T. (1979a). Trunsplant. Proc. 11, 585-591. Mito, M., Ebata, H., Kusano, M., Onishi, T. Saito, T., and Sakamoto, S. (197913).Transplantation 28, 499-505. Montesano, R., St. Vincent, L., Drevon, C., and Tomatis, L. (1975). Znt:J. Cancer 16, 550-558.

HEPATOCARCINOGENESIS AND PREMALIGNANCY

107

Montesano, R., Drevon, C., Kuroki, T., St. Vincent, L., Handleman, S., Sanford, K. K., Defeo, D., and Weinstein, I. B. (1977).J.Natl. Cancer Inst. 59, 1651-1658. Morel-Chany, E., LaFarge-Frayssinet, C., and Trincal, G. (1985). Cell Biol. Toxicol. 1, 11-22. Morris, H. P., and Meranze, D. R. (1974).Recent Results Cancer Res. 44, 103-114. Mottram, J. C. (1944).J.Pathol. Bacteriol. 56, 181-187. Nagelkerke, J. F., Barto, K. P., and van Berkel, T. J. C. (1983).J.Biol. Chem. 258,1222112227. Newberne, P. M. (1982). In “Toxicology of the Liver” (G. L. Plaa and W. R. Hewitt, eds.), pp. 243-290. Raven, New York. Novikoff, A. B. (1957). Cancer Res. 17, 1010-1017. Nowell, D. C., Morris, H. P., and Potter, V. R. (1967). Cancer Res. 27, 1565-1579. Odashima, S., and Morris, H. P. (1966). Gann Monogr. 1, 55-64. O’Farrell, P. H. (1975).J. Biol. Chem. 250, 4007-4021. Ohmori, T., Watanabe, K., and Williams, G. M. (1980).J.Natl. Cancer Inst. 65,485-490. Okita, K., Gruenstein, M., Klaiber, M., and Farber, M. (1974). Cancer Res. 34, 27582763. Okret, S., Wikstrom, A. C., Wrange, O., Anderson, B., and Gustafsson, J. A. (1984).Proc. Natl. Acad. Sci. U.S.A.81, 1609-1613. Onda, H. (1976). Gann 67,253-262. Onoe, T., Kaneko, A., Dempo, K., Ogawa, K., and Minase, T. (1975).Ann. N . Y. Acad. Sci. 259, 168-180. Opie, E. L. (1944).J. E x p . Med. 80, 231-246. Owens, R. A., and Hartman, P. E. (1984). Biochem. Biophys. Res. Commun. 122,740747. Pentecost, B. T., and Craddock, V. M. (1983). Carcinogenesis 4, 1089-1096. Peraino, C., Fry, F. J. M., Staffeldt, E., and Kisieliski, W. E. (1973). Cancer Res. 33, 2701-2708. Peraino, C., Staffeldt, E. F., Carnes, B. A., Ludeman, V. A., Blomquist, J. A., and Vesselinovitch, S. D. (1984).Cancer Res. 44, 3340-3347. Petropoulos, C., Andrews, G., Tamaoki, T., and Fausto, N. (1983).J. Biol. Chem. 258, 4901-4906. Pitot, H. C., and Sirica, A. E. (1980). Biochim. Biophys. Acta 605, 191-215. Pitot, H. C., Barsness, L., Goldsworthy, T., and Kitagawa, T. (1978). Nature (London) 271,456-458. Popper, H., dela Huerga, J., and Yesinick, C. (1953). Science 118, 80-82. Popper, H., Kent, G., and Stein, R. (1957).J. Mt. Sinai. Hosp. 24, 551-556. 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 Harbor Laboratory, Cold Spring Harbor, New York. Poralla, T., Dippold, W., Dienes, H. P., Manns, M., and Buschenfelde, K.-H. (1984).J. Immuno. Methods 68,341-348. Pott, P. (1775). In “Chirurgical Observations,” pp. 48-63. Hawkes, Clarke & Collins, London. Potter, V. R. (1961).Cancer Res. 21, 1331-1333. Potter, V. R. (1984).Cancer Res. 44,2733-2736. Pragnell, I. B., and Balmain, A. (1983). Nature (London) 303,72-74. Rabes, H. M. (1983).J. Cancer Res. Clin. Oncol. 106, 85-92. Rabes, H. M., Scholze, P., and Jantsch, B. (1972).Cancer Res. 32,2577-2586. Rabes, H. M., Buecher, Th., Hartmann, A., Linke, I., and Duennwald, M. (1982).Cancer Res. 42,3220-3227.

108

S. SELL E T AL.

Ramagli, L. S., Capetillo, S., Becker, F. F. and Rodriguez, L. V. (1985). Cancinogenesis 6,367-375. Reddy, J. K., and Lalwani, N. D. (1984). CRC Crit. Reo. Toxicol. 12, 1-58. Reddy, J. K., and Rao, M. S. (1978). Br. J. Cancer 38,537-543. Reddy, J. K., Rao, M. S., Azarnoff, D. L., and Sell, S. (1979). Cancer Res. 39, 152-161. Reddy, E. P., Reynolds, R.K., Santos, E., and Barbacid, M. (1982).Nature (London)300, 149-152. Reddy, J. K., Jirtle, R. L., Watanabe, T. K., Reddy, N. K., Michalopoulos, G., and Qureshi, S. A. (1984). Cancer Res. 44,2582-2589. Reisfeld, R. A., and Sell, S. (1985). “Monoclonal Antibodies and Cancer Therapy.” Liss, New York, NY. Reuber, M. D. (1985).J. Natl. Cancer Znst. 34,697-724. Reuber, M. D. (1966). Gann Monogr. 1,43-54. Reuber, M. D. (1971). Br. J. Cancer 25, 538-543. Reuber, M. D. (1975). Gann Monogr. 17,301-342. Reuber, M. D. (1976). Eur.J. Cancer 12, 137-141. Reuber, M. D., and Firminger, H. I. (1963).J. Natl. Cancer Inst. 31,1407-1429. Reuber, M. D., and Odashima, S. (1967). Gann 58,513-520. Reuber, M. D., Stromberg, K., and Glover, E. L. (1972).]. Natl. Cancer Znst. 48,675683. Revesz, L. (1958).]. Natl. Cancer Znst. 20, 1157-1186. Rodriguez, L. V., Klein, K. K., Amoruso, M., and Becker, F. F. (1979). Znt. J . Cancer 24, 490-497. Rogers, A. E. (1975). Cancer Res. 35,2469-2574. Rusch, H. P., and Baumann, C. A. (1939). Am. J. Cancer 35,55-62. Sala-Trepat, J. M., Dever, J., Sargent, T. D., Thomas, K., Sell, S., and Bonner, J. (1979). Biochemistry 18,2167-2178. San, R. H. C., Laspia, M. F., Soiefer, A. I., Maslansky, C. J., Rice, J. M., and Williams, G. M. (1979). Cancer Res. 39, 1026-1034. Santella, R. M., Lin, C. D., Cleveland, W. L., and Weinstein, I. B. (1984). Carcinogenesis 5, 373-377. Sasaki, T., and Yoshida, T. (1935). Virchows Arch. Pathol. Anat. 295, 175-200. Sato, K., Kitahara, A., Yin, Z., Ebina, K., Satoh, K., Tsuda, H., Ito, N., and Dempo, K. (1983). Ann. N.Y. Acad. Sci. 417, 213-223. Sato, K., Kitahara, A., Satoh, K., Ishikawa, T., Tatematsu, M., and Ito, N. (1984). Gann 75, 199-202. Satoh, K., Kitahara, A., Soma, Y., Inaba, Y., Hatayama, I., and Sato, K. (1985). Proc. Natl. Acad. Sci. U S A . 82,3964-3968. Schaeffer, W. I., and Heintz, N. H. (1978). In Vitro 14,418-427. Schenk, D. B., Hubert, J. J., and Leffert, H. L. (1984).J.Biol. Chem. 259,14941-14951. Schmidt, W. N., McKusick, K. B., Schmidt, C. A., Hofhan, L. H., and Hnilica, L. S. (1984a). Cancer Res. 44,5291-5304. Schmidt, W. N., Page, D. L., Schmidt, C. A,, McKusick, K. B., Ward, W. S., and Hnilica, E. S. (1984b). Cancer Res. 44,5867-5879. Schmidt, W. N., Page, D. L., McKusick, K., and Hnilica, L. S. (1985).Carcinogenesis, in press. Schulte-Hermann, R.,Schuppler, J., Ohde, C., and Timmermann-Trosiener, I. (1982). In “Carcinogenesis” (E. Hecker et al., eds.), Vol 7, pp. 99-104. Raven, New York. Schutzbank, T., Robinson, R., Oren, M., and Levine, A. J. (1982). Cell 30,481-490. Scott, M. R. D., Westphal, K. -H., and Rigby, P. W. J. (1983). Cell 34,557-567.

HEPATOCARCINOGENESIS AND PFWMALIGNANCY

109

Seeburg, P. H., Colby, W. W., Capon, D. J., Goeddel, D. V., and Levinson, A. D. (1984). Nature (London)312,71-75. Seglen, P. 0. (1976). Methods Cell Biol. 13,29-83. Sell, S. (1978). Cancer Res. 38,3107-3113. Sell, S. (1980). In “Cancer Markers” (S. Sell, ed.), Vol. I, pp. 249-293. Humana Press, Clifton, New Jersey. Sell, S. (1983). Cancer Res. 43, 1761-1767. Sell, S., andBecker, F. F. (1978).J. Natl. Cancer Inst. 60, 19-26. Sell, S., and Leffert, H. L. (1982). Hepatology 2, 77-86. Sell, S., and Morris, H. P. (1974). Cancer Res. 34, 1413-1417. Sell, S., and Reisfeld, R. (1985). “Monoclonal Antibodies in Cancer.” Humana Press, Clifton, New Jersey. Sell, S., and Ruoslahti, E. (1982).J. Natl. Cancer Inst. 69, 1105-1114. Sell, S., and Salman, J. (1984).A m . J. Pathol. 114, 287-300. Sell, S., Nichols, M., Becker, F. F., and Leffert, H. L. (1974). Cancer Res. 34,865-871. Sell, S., Becker, F., Lombardi, B., Shinozuka, H., and Reddy, J. (1979a).In “Carcinoembryonic Proteins” (F. G. Lehmann, ed.), Vol. I, pp. 129-136. Elsevier, Amsterdam. Sell, S., Thomas, K., Michalson, M., Sala-Trepat, J., and Bonner, J. (1979b). Biochim. Biophys. Acta 564, 173-178. In “CarcinoSell, S., Thomas, K., Michaelson, M., Scott, J., and Sala-Trepat, J. (1979~). Embryonic Proteins” F. -G. Lehman, ed.), Vol I. Elsevier, Amsterdam. Sell, S., Osborn, K., and Leffert, H. (1981a). Carcinogenesis 2,7-14. Sell, S., Leffert, H. L., Shinozuka, H., Lombardi, B., and Gochman, N. (1981b).Gann 72, 479-487. Sell, S., Becker, F., Leffert, H. L., Osborn, K., Salman, J., Lombardi, B., Shinozuka, H., Reddy, J., Ruoslahti, E. and Sala-Trepat, J. (1983).Enuiron. Sci. Res. 29,271-293. Sevier, E. D., David, G. S., Martinis, J., Desmond, W. J., Bartholomew, R. M., and Wang R. (1981). Clin. Chem. 27, 1797-1806. Shapira, F., Hatzfield, A., Weber, A., and Guillouzo, A. (1979). In “Carcinoembryonic Proteins” (F. G. Lehmann, ed.), Vol I, pp. 411-420. Elsevier, Amsterdam. Shearer, R., and Smuckler, E. (1971). Cancer Res. 31,2104-2109. Shilo, B. -Z., and Weinberg, R. A. (1981). Nature (London)289,607-609. Shimada, T., Furukawa, K., Kreiser, D. M., Cawein, A., and Williams, G. M. (1983). Cancer Res. 43, 5087-5092. Shinozuka, H., Lombardi, B., Sell, S., and Iammarino, R. M. (1978a). Cancer Res. 38, 1092-1098. Shinozuka, H., Lombardi, B., Sell. S., and Iammarino, R. M. (1978b).J . Natl. Cancer Inst. 61,813-817. Shinozuka, H., Sells, M. A., Katyal, S. L., Sell, S., and Lombardi, B. (1979).Cancer Res. 39,2515-2521. Shu, S., Fonseca, L. S., Kato, H., and Zbar, B. (1983). Cancer Res. 43,2637-2643. Singer, B., and Kusmierek, J. T. (1982). Annu. Rev. Biochem. 52,655-693. Slifkin, M., Merkow, L. P., Pardo, M., Epstein, S. M., Leighton, J., and Farber, E. (1970). Science 167, 285-287. Smuckler, E. A. (1983a). West.J. Med. 139, 115-134. Smuckler, E. A. (1983b). West.J. Med. 139, 55-74. Smuckler, E. A., Koplite, R. M., and Sell, S. (1976a). Cancer Res. 36,4558-4561. Smuckler, E. A., Koplite, R. M., and Sell, S. (1976b) I n “Oncodevelopmental Gene Expression” (W. H. Fishman, S. Sell, and R. Stewart, eds.),pp. 701-706. Academic Press, New York.

110

S. SELL ET AL.

Solt, D., and Farber, E. (1976). Nature (London) 263,701-703. Solt, D. B., Medline, A., and Farber, E. (1977). Am../. Pathol. 83,595-618. Song, B. J., Fujino, T., Park, S. S., Friedman, F. K., and Gelboin, H. V. (1983).J. Biol. Chem. 259, 1394-1392. Stein, G. S., Stein, J. L., and Thomson, J. A. (1978). Cancer Res. 38, 1181-1201. Sudhakar, S., Johnston, D. A., Becker, F. F., and Rodriguez, L. V. (1984).Cell. Mol. Biol. 30,275-289. Sugioka, Y., Fujii-Kurimaya, Y., Kitagawa, T., and Muramatsu, M. (1985a). Cancer Res. 45,365-378. Sugioka, Y., Kano, T., Okuda, A., Sakai, M., Kitagawa, T. and Muramatsu, M. (1985b). Nucleic Acids Res. 13,6049-6057. Sukumar, S., Notario, V., Martin-Zanca, D., and Barbacid, M. (1983). Nature (London) 306,658-661. Tabin, C. J., Bradley, S. M., Bargmann, C. I., Weinberg, R. A., Papageorge, A. G., Scolnick, E. M., Dhar, R., Lowy, D., and Chang, E. H. (1982).Nature (London) 300, 143-149. Takami, H., and Busch, H. (1979). Cancer Res. 39,507-518. Takami, H., Busch, F. N., Morris, H. P., and Busch, H. (1979). Cancer Res. 39, 20962105. Tarone, R. E., Chu, K. C., and Ward, J. M. (1981).J.Natl. Cancer Inst. 66, 1175-1181. Tatematsu, M., Kaku, T., Medline, A., Eriksson, L., Roomi, W., Sharma, R. N., Murray, R. K., and Farber, E. (1983a). Enuiron. Sci. Res. 29, 25-42. Tatematsu, M., Nagamine, Y., and Farber, E. (1983b).Cancer Res. 43, 5049-5058. Teebor, G. W., and Becker, F. F. (1971). Cancer Res. 31, 1-3. Thomas, P. E., Reidy, J., Reik, L. M., Ryan, D. E., Koop, D. R., and Levin, W. (1984). Arch. Biochem. Biophys. 235,239-253. Thorgeirsson, S. S., Sanderson, N., Park, S. S., and Gelboin, H. V. (1983). Carcinogenesis 4,639-641. Tsanev, R., and Hadjiolov, D. (1978). Z. Krebsforsch. 91,237-247. Tsao, M.-S., Nelson, K. G., and Grisham, J. G. (1984a).J. Cell. Physiol. 121, 1-6. Tsao, M.-S., Smith, J. D., Nelson, K. G., and Grisham, J. W. (1984b).Exp. Cell Res. 154, 38-52. Tschipysheva, T. A., Guelstein, V. I., and Bannikov, G. A. (1977).1nt.J.Cancer 20, 388393. Van Duuren, B. L. (1969). Prog. Erp. Tumor Res. 11, 31-68. Varmus, H. (1984).Annu. Reo. Genet. 18, 553-612. Vischer, P., and Reutter, W. (1978). Eur. J. Biochem. 84,363-368. Wahid, S. (1983). Acta Med. Okayama 37, 31-34. Wani, A. A., Gibson-D’Ambrosio, R. E., and D’Ambrosio, S. M. (1984). Carcinogenesis 5, 1145-1150. Weinarwin, I. B., Gatonni-Celli, S., Kirschmeier, P., Hsiao, W., Horovitz, A., and Jeffery, A. (1984). Fed. Proc., Fed. Am. SOC. E x p . Biol. 43,2287-2294. Weinberg, W. C., Howard, J. C., and Iannaccone, P. M. (1985). Science 227, 524-527. Weisburger, J. H., Pai, S. R., and Yamamoto, R. S. (1964).J. Natl. Cancer Inst. 32,881904. Wiebel, F. J., Park, S. S., Kiefer, F., and Gelboin, H. V. (1984). Eur. J . Biochem. 145, 455-462. Williams, G . M. (1976). Am. J . Pathol. 85, 739-754. Williams, G. M. (1980).Biochim. Biophys. Acta 605, 167-189. Williams, G. M., Elliott, J. M., and Weisburger, J. H. (1973). Cancer Res. 33, 606-612.

HEPATOCARCINOCENESIS AND PREMALIGNANCY

111

Williams, C. M., Klaiber, M., and Farber, E. (1977).Am. 1.Pathol. 89, 379-390. Williams, G. M., Ohmori, T., and Watanabe, K. (1980). Am. J . Pathol. 99, 1-12. Williams, J. C. (1981). In “Genetic Engineering” (R. Williamson, ed.), Vol. 1, pp. 1-59. Academic Press, New York. Wolman, S. R., Phillips, T. F., and Becker, F. F. (1972).Science 175, 1267-1269. Woodworth, C. D., Kreider, J. W., and Isom, H. C. (1984).Proc. Am. Assoc. Cancer Res. 25, 391. Yamamoto, M., Maehara, K., Takahashi, K., and Endo, H. (1983). Proc. Natl. Acad. Sci. U.S.A.80, 7524-7527. Yaswen, P., Goyette, M., Shank, P. R., and Fausto, N. (1985).Mol. Cell. Biol. 5,780-786. Yoshimura, H., Harris, R., Yokoyama, S., Takahashi, S., Sells, M. A., Pan, S. F., and Lombardi, B. (1983). A m . ] . Pathol. 110,322-332. Yuasa, Y., Srivastava, S. K., Dunn, C. Y., Rhim, J. S., Reddy, E. P., and Aaronson, S . (1983).Nature (London)303, 775-779. Zarbl, H., Sukumar, S., Arthur, A. V., Martin-Zanca, D., and Barbacid, M. (1985).Nature (London)315,382-385. Zbar, B., Wepsic, H. T., Rapp, H. J., Borsos, T., Kronman, B. S., and Churchill, W. H., Jr. (1969).J . Natl. Cancer Znst. 43, 833-841. Zurlo, J., and Yager, D. (1985). Fed. Proc., Fed. Am. SOC. E x p . Biol. 44, 1493.

This Page Intentionally Left Blank

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER Herbert Pfister lnstitut fur Klinische Virologie. Universitat Erlangen-Nurnberg, Erlangen, Federal Republic of Germany

I. Introduction

The epidemiology of human genital cancer clearly shows a correlation between this disease and sexual activity (Rotkin, 1973; Kessler, 1977). A low age at first intercourse and promiscuity are well-established risk factors for cervical cancer. Cervical and penile cancer incidence was shown to correlate for a number of countries (Waterhouse et al., 1982), suggesting that both carcinoma types are induced by the same factors. Males with multiple sexual partners and early age at first intercourse appeared to increase their wives’ risk of cervical cancer in cases where women claimed to have had no partner other than their husbands (Buckley e t al., 1981). This may be explained by the transmission of a persisting, oncogenic infectious agent. The epidemiology of genital cancer naturally parallels the epidemiology of any sexually transmitted disease. Consequently, a number of infectious agents have been incriminated as etiologic factors (Alexander, 1973), but without obtaining conclusive evidence. A possible role of papillomaviruses in the induction of cervical cancer was suggested about 10 years ago by zur Hausen (1975, 1977). This virus genus was rather neglected for many years because papillomarviruses could not (and cannot) b e propagated in cell culture. Experiments were based on material freshly isolated from wart biopsies and frequently failed because of low quantity. Molecular cloning of viral DNAs has partially overcome that problem. Techniques for in uitro labeling and sequencing of DNA helped to unravel the plurality of papillomaviruses, their prevalence in various tumors, and some aspects of their molecular biology (for review, see Pfister, 1984). Newly characterized papillomavirus DNAs were identified in benign and malignant tumors at the cervix uteri and at external genital sites in females and males (Green et al., 1982; Gissmann et al., 1983; Durst et al., 1983; Boshart et al., 1984). These data considerably substantiated 113 ADVANCES IN CANCER RESEARCH, VOL 48

Copyright 0 1987 by Academic Press, Inc All rights of reproduction in any form reserved

114

HERBERT PFISTER

speculations regarding a role of papillomaviruses in the etiology of genital cancer. It is the aim of this review to summarize our present knowledge about transforming functions of papillomaviruses and their association with genital tumors. The data will be discussed with regard to a possible etiologic role and to prospects for diagnosis and vaccination. II. Biology of Papillomaviruses

A. VIRIONSAND CLASSIFICATION Papillomaviruses are members of the Papovaviridae family. The cubic capsids are composed of one major and probably one minor protein component and harbor double-stranded, circularly closed DNA of about 8 kb (Matthews, 1982). The genus members share common antigenic determinants, which can be disclosed by a group-specific antiserum raised against detergent-disrupted particles (Jenson et al., 1980). Immunization of animals with native virions led to typespecific sera (Gissmann et d.,1977; Orth et d.,1977,1978; Pfister and zur Hausen, 1978), indicating that group-specific antigens are partially masked in assembled virions. Group-specific antisera were extensively used to screen tumor tissues for papillomavirus structural antigens of unknown specificity (Shah et aZ., 1980; Kurman et al., 1982, 1983). The DNAs of papillomaviruses cross-hybridize within subgenomic regions under conditions of low stringency, which allow base pairing in spite of a 30% mismatch (Law et al., 1979). This relationship was also exploited for a genus-specific screening of tumors (Durst et al., 1983; Lancaster et al., 1983; Boshart et al., 1984). The nucleotide sequences of DNAs from human and animal papillomaviruses revealed a strikingly similar colinear genome organization (Fig. 1).A 0.5-1 kb noncoding region harbors the origin of replication and transcription control signals. All major open reading frames are on one strand of comparable size and in similar positions (Pettersson et al., 1987). The sequences are usually highly homologous within reading frames E l , E2, and L1 and diverge most in reading frames E4 and in part of L2. A characterization of gene functions was achieved by genetic studies of bovine papillomavirus 1 (BPV 1)(Fig. 1; Section, 11,C). In contrast, our knowledge of human papillomavirus (HPV) gene functions is still very poor due to the lack of appropriate in vitro systems. The so-

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

-

transformation

115

transactivation of transcription

'

high copy maintenance

eDisomal persistence

1

transformation H

I

maior caosid .pro t e'in

minor capsid protein

HPV6

I

I

HPV16

I

I

FIG.1. Genome organization of BPV 1 (Pettersson et al., 1987), HPV 6 (Schwarz et al., 1983), and HPV 16 (Seedorf et al., 1985).The bars represent open reading frames for each potential translation frame and are labeled following current nomenclature. The position of the first ATG start codon for translation is indicated by dotted lines. Functions assigned to BPV 1 reading frames by genetic analysis are depicted below the BPV 1 genome.

called early part of the BPV 1genome (open reading frames El-E8) is expressed in transformed cells and codes for proteins, which are involved in episomal replication of viral DNA (Lusky and Botchan, 1985), high copy maintenance of viral plasmids (Lusky and Botchan, 1985), transactivation of viral transcription (Spalholz et al., 1985), and last, but not least, transformation (Schiller et al., 1984, 1986). Except for E6, the proteins are not yet identified, but are only predicted from the DNA sequence and from genetic experiments. The so-called late region covers two open reading frames, L1 and L2, coding for structural proteins (Pilacinski et al., 1984). Classification of papillomaviruses is based entirely on the host range and the relatedness of the DNAs, which are cloned in bacteria (Coggin and zur Hausen, 1979). A serological analysis of HPV 1 to HPV 5 detected no cross-reaction between different types using antigen preparations from biopsy material (Gissmann et al., 1977; Orth et al., 1977, 1978; Pfister and zur Hausen, 1978). For most types, however, it was impossible to prepare sufficient amounts of antigen for

116

HERBERT PFISTER

immunization and serology. This can now be made up by producing viral antigens in bacteria after cloning the respective genes into expression vectors (Pilacinski et ul., 1984). Papillomavirus DNA clones from one species represent independent types in the case of less than 50% cross-hybridization, which should be determined by reassociation of heterologous DNAs in liquid phase (Coggin and zur Hausen, 1979). At least 38 HPV types can be differentiated on the basis of this criterion. A number of them do not hybridize to other HPVs when tested under stringent conditions. Others form groups, members of which cross-hybridize from less than 1 to 40%. HPV 5, HPV 9, and HPV 24 are representatives of a large group of 17 more or less closely related viruses (Pfister et al., 1986; Orth et al. and H. zur Hausen et al., personal communications). It should be noted that the actual nucleotide homology is significantly higher than suggested by cross-hybridization in liquid phase (see Section 111). DNA sequence analysis revealed a markedly uneven distribution of homologies over the genomes (Pfister et al., 1986). The overall sequence homology is therefore certainly not sufficient for a clinically meaningful evaluation: Important biological differences may result from minor sequence divergence in one genome region, whereas major changes in other areas can be irrelevant in terms of pathogenic properties. As long as we do not know which genes are important in pathogenesis, we will have to deal with the present plurality. Eventually, it will be possible to focus on specific genes to simplify the systematics. OF PAPILLOMAVIRUS INFECTION B. BIOLOGY

Human papillomaviruses induce epithelial proliferations of skin or mucosa, which show a limited growth and often regress spontaneously. The incubation period varies considerably from a few weeks to several months (Rowson and Mahy, 1967). Although no direct evidence exists, it is generally believed that the virus primarily infects basal cells of the epidermis. Infection presumably depends on microlesions or local abrasion of the skin. At the cervix uteri, proliferating cells are exposed at the squamocolumnar border, and it is interesting to note that 90% of cervical HPV infections occur at this site. The persisting viral genome either increases the rate of cell proliferation or prolongs the normal life span of the keratinocytes. Both events lead to hyperplasia. A normal mitotic rate in situ and a normal growth rate in vitro after explantation were measured with laryngeal

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

117

papilloma cells (B. Steinberg, personal communication), suggesting that the benign tumor depends on a failure to differentiate properly. Epidermal cells are not permissive for papillomavirus replication in the beginning of their differentiation process. As differentiation proceeds, they become more and more permissive. Viral DNA replication can be demonstrated by in situ hybridization in suprabasal layers, and structural proteins and mature virus particles appear in the upper epidermal layers. Virus-specific cytopathogenic effects are most prominent in the stratum granulosum. HPV infection of the genital mucosa frequently induces koilocytotic atypia as defined originally by Koss and Durfee (1956).Cytoplasmic changes appear first in cells of intermediate layers and extend to the surface of the epithelium. The koilocyte is characterized by a large clear perinuclear zone, and binucleation is frequently observed. Viral capsid antigen and particles appear in some of the koilocytes, while others are negative (Morin et al., 1981; Casas-Corder0 et al., 1981). In other words, the koilocyte is no indicator of maturation of virus particles, but more likely is the result of preceding events in virus replication. Genital warts are of multicellular origin (Friedmann and Fialkow, 1976).This was shown by analysis of the glucose-6-phosphate dehydrogenase phenotype of warts from heterozygous women. They may be due to infection of several cells during initial transmission or to repeated infections by virus, which is shed from the tumor itself.

FUNCTIONS C. TRANSFORMING Cellular transformation by papillomaviruses was studied with BPV

1. The virus is distinguished from HPV types by the induction of fibropapillomas in its natural host. Within a few days after infection, fibroblasts start to proliferate, which leads to massive fibroplasia. Only after a few weeks does the epithelium become involved, showing acanthosis and hyperkeratosis (Olson et al., 1969). Oncogenic stimulation of fibroblast can be reproduced in vitro with cells from various species and was extensively analyzed with NIH 3T3 and C 127 mouse fibroblasts. Infection or transfection with viral DNA induces morphological transformation and focus formation in the monolayer cultures. The transformed cells grow anchorage independent and form colonies in soft agar and tumors in nude mice (Dvoretzky et al., 1980). BPV 1 contains two genes, corresponding to open reading frames E6 and E5, that can independently transform C 127 cells. This was shown by the following approach: BPV 1 DNA fragments were activated by ligation to promoter and enhancer elements of a retroviral

118

HERBERT PFISTER

large terminal repeat to circumvent viral transcription control mechanisms, and the transforming genes were identified genetically by deletion and linker insertion mutagenesis (Schiller et al., 1984, 1986). The transforming activity of E6 is rather low when tested under control of the BPV 1promoter (Sarver et al., 1984; Schiller et d.,1984) or as cDNA clone in an Okayama-Berg expression vector (Yang et d., 1985).In comparison to wild type, the foci appear later and grow more slowly. This may be related to the fact that open reading frame E6 is only poorly transcribed in BPV 1-transformed cells (Stenlund et ul., 1985). When promoted by the large terminal repeat, E6 induced foci as efficiently as full-length BPV 1 DNA. Cells transformed by mutants lacking E6 did not form colonies in soft agar, and tumors in nude mice appeared later and were smaller (Sarver et al., 1984). This points to a synergistic effect between E6 and E 5 functions (Yang et ul., 1985). The open reading frame E6 codes for a 15.5 kDa protein, which appears equally distributed between the nucleus and the nonnuclear membranes (Androphy et al., 1985). The most abundant virus-specific RNA in BPV 1-induced tumors (Freese et al., 1982) and transformed cells (Stenlund et al., 1985) covers open reading frame E5, and this gene seems to play a major role in oncogenesis (Sarver et al., 1984). The predicted E 5 gene product is a small (44 amino acids) hydrophobic protein which has not yet been identified. Multiple copies of BPV 1 DNA persist as plasmids in transformed cells (Law et al., 1981), indicating that transformation does not depend on integration. The establishment of the transformed state depends strongly on the function of the E2 gene, however. This gene was recently shown to be important for trans-activation of viral transcription (Spalholz et al., 1985) and is likely to play an indirect role by up-regulation of the expression of the E6 and E 5 genes. Once transformed by E2 mutants, the cells reveal the characteristic phenotype of wild-type transformed cells in spite of extremely low amounts of virus-specific transcripts (Kleiner et al., 1986). One should take care when transferring these data to other papillomaviruses and other cell systems. Expression clones of E6, for example, did not induce transformation in NIH 3T3 cells, whereas E 5 expression clones were able to transform these cells similar to C 127 cells. The target cell of human papillomaviruses is the keratinocyte and not the fibroblast, studied with BPV 1,and both cell types are not necessarily affected by the same functions. The E6 genes of HPV types reveal some homology at the amino acid level to E6 of BPV 1 (Fig. 2), suggesting common activities. A cysteine-X-X-cysteine motif

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

BPV 1 HPV 6b HPV16

119

M M E S A N A S T S A T T I D M H Q K R T A M F Q D P Q E R P R K L P

A

E ViDiA F R C M V

..*..

FIG.2. Comparison of the amino acid sequences from open reading frame E6 of BPV

1, HPV 6, and HPV 16 (Chen et al., 1982; Schwarz et al., 1983; Seedorf et al., 1985). The sequences start with the first methionine and are given in the one-letter code. Conserved amino acids are boxed and chemically similar amino acids are framed by dotted lines.

appears four times with identical spacing. This sequence is reminiscent of the small T antigens of polyomaviruses and of several early proteins of adenoviruses (Schwarz et al., 1983).The E6 gene is selectively expressed in skin carcinomas of rabbits induced by the cottontail rabbit papillomavirus (CRPV) (Nasseri and Wettstein, 1984; Danos et al., 1985)and in human cervical carcinomas (Schwarz et al., 1985; see also Section V,B). This may indicate that E6 also plays a role in oncogenesis by epidermotropic papillomaviruses. A limited amino acid sequence homology was noted between E5 open reading frames of BPV 1 and HPV 6 (Schiller et al., 1986).The homology to HPV 16 is

120

HERBERT PFISTER

less and there is no ATG codon in E 5 of HPV 16 where translation could be initiated (Seedorf et al., 1985). In seeking additional potentially transforming functions, the predicted proteins of papillomaviruses were compared with sequences from data banks. In the case of CRPV, a homology was observed between the carboxy terminus of open reading frame E2 and the mos oncogene (Giri et al., 1985), and a comparable relationship exists between open reading frame E4 of HPV 8 and the EBNA 2 protein of Epstein-Barr virus (Pfister et al., 1985). The functional significance of these homologies remains to be established. D. MALIGNANT CONVERSION-GENERAL ASPECTS Some papillomaviruses induce tumors that may progress to carcinomas, Skin warts of cottontail rabbits were first shown to convert into carcinomas in 25% of the infected animals (Rous and Beard, 1935; Kidd and ROUS, 1940; Syverton, 1952). Malignant tumors develop on the basis of long-persisting papillomas, usually not before 1 year after infection. The “latency period” and the conversion rate can be influenced by application of chemical carcinogens (Rous and Friedewald, 1944). Even without any experimental enhancement, however, papillomas rarely continue as benign growths for more than 18 months (Syverton, 1952). The rabbit system reveals basic characteristics of papillomaviral oncogenesis. The viruses are only weakly oncogenic by themselves, and carcinogenesis seems to depend on additional exposure to physical or chemical carcinogens such as UV light, X rays, or special diet components (for review, see Pfister, 1984). However, from the experimental analysis of CRPV, there can be little doubt that papillomavirus infection represents the initial and one of the major risk factors in the papilloma to carcinoma sequence. Viral DNA persists in skin cancers of rabbits (Stevens and Wettstein, 1979) and is continuously expressed (Nasseri et al., 1982). A skin carcinoma, which was induced by CRPV infection, was propagated by in vitro transplantation for more than 30 years. The viral DNA now appears highly methylated except for the early promoter region from where transcripts are still initiated (McVay et al., 1982; Wettstein and Stevens, 1983). This points to a selective pressure to keep the early promoter active and provides a strong argument that continuous viral gene expression is important to maintain the malignant phenotype. In contrast, no viral DNA was detectable in cancers

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

121

of the alimentary canal in cattle, which grow in close association and even on the basis of BPV 4-induced lesions (Campo et al., 1985). These two extremes may reflect different roles of papillomaviruses in the maintenance of the malignant phenotype. The disease epidermodysplasia verruciformis represents an excellent model in studies on malignant conversion of papillomavirus-induced tumors in man (Jablonska et al., 1972). Due to a genetic predisposition, the patients develop skin lesions during childhood, which are induced by papillomaviruses not readily observed in the normal population (Orth et al., 1979). These lesions do not regress, but persist for life, gradually spreading over the entire body. HPV types characteristic for that disease are extremely heterogeneous, comprising at least 15 types (Kremsdorf et al., 1984; Gassenmaier et al., 1984; M. Favre et al., personal communication). Some of them show extensive sequence homology in varying regions of their genomes, and nearly all cross-hybridize in Southern blot experiments. One-quarter to one-third of the patients develop cancer on average after 25 years of persisting disease. The preferential location on sunexposed skin suggests that one is looking at another example of synergism between papillomavirus infection and extrinsic factors. This is in line with the observation that the disease has a relatively good prognosis in Africans, as compared with Caucasians (Jacyk and Subbuswamy, 1979), which could be explained by the protective effect of skin pigmentation. A detailed restriction enzyme analysis of viral DNAs revealed that more than 90% of skin carcinomas are persistently infected with HPV 5 or HPV 8 (Orth et al., 1980; Ostrow et al., 1982; Pfister et al., 1983a). In a few exceptional cases, other types were detected (Yutsudo et al., 1985; G. Orth, personal communication). These data may be interpreted in terms of a special oncogenic potential of HPV 5 and HPV 8. The biological difference is noteworthy in view of the close DNA relationship with other epidermodysplasia-associated viruses, which do not occur in carcinomas. One individual patient was infected by at least six different HPV types, but only HPV 5 DNA persisted in the malignant tumor (Pfister et al., 1983a). An impaired cell-mediated immunity is a consistent feature of patients with epidermodysplasia verruciformis (Glinski et d.,1981). Malignant conversion does not seem to depend on immune status, however, because immunity parameters were similarly impaired in patients infected only by HPV 3 who developed no neoplasms. This underlines the role of specific virus types in carcinogenesis.

122

HERBERT PFISTER

Ill. Human Papillomaviruses from Genital Tumors

Individual papillomavirus types show a clear preference for specific tissues. The DNAs of 10 types were cloned from lesions on the mucosa. HPV 13, HPV 30, and HPV 32 so far seem to be confined to the oral cavity and the larynx (Pfister et al., 1983b; Kahn et al., 1986; Beaudenon et al., 1986). Viruses that prevail in genital tumors are listed in Table I. HPV 6 and HPV 11 represent closely related viruses. They show 25%cross-hybridization when tested for reassociation in liquid phase under conditions of high stringency (Gissmann et al., 1982a). The overall nucleotide homology amounts to 82% (Schwarz et al., 1983; Dartmann et al., 1986). Homologies are not evenly distributed over the genomes, however, and a considerable divergence within the open reading frame E5b allows for the construction of type-specific DNA probes (S. Wolinsky, T. Broker, D. Arvan, and L. Chow, personal communication). HPV 16 and HPV 31 are even more closely related. They crosshybridize by about 35% (Lorincz et al., 1985), which suggests a nucleotide homology around 90%, but also in this case it is possible to identify type-specific sequences (S. Wolinsky et al., personal communication). HPV 33 is only distantly related to HPV 16 (Beaudenon et al., 1986), thus posing no problem for differentiation. HPV 18 and HPV 35 have no close relatives among genital papillomaviruses up to now. However, 10% cross-hybridization was noted between HPV 18 and HPV 32, which is frequently detected in lesions of focal epithelial hyperplasia Heck of the oral cavity (Beaudenon e t al., 1986). In contrast, HPV 13, which is also prevalent in Heck’s disease, cross-hybridizes with HPV 6 and HPV 11 by about 5%(Pfister et al., 1983b) and reveals no homology with HPV 18 under stringent conditions (Boshart et al., 1984). Genital papillomaviruses and skin wart isolates may be related within small genome regions, resulting in weak cross-reactivity in Southern blot hybridization experiments (Table I). It will be interesting to map these areas and assign homology to individual viral genes. This may help elucidate common biological activities and will be essential to defining more specific probes. Viruses originally isolated from skin warts may occur in genital tumors. HPV 1 and 2 were repeatedly detected in condylomata acuminata (Krzyzek et al., 1980), as was HPV 10, which was also disclosed in 2 of 31 cervical carcinomas tested (Green et al., 1982). Using molecularly cloned DNA of HPV 1 to 5 as probe, Okagaki et al. (1983)

TABLE I HUMANPAPILLOMAVIRUSES DETECTED IN GENITAL TUMORS

Source for DNA cloning

Reference

Nucleotide homology

De Villiers et al. (1981)

HPV 11: 82%

Gissmann et al. (1982a)

HPV 6: 82%

HPV 16

Condyloma acuminatum Laryngeal papilloma Cervical carcinoma

HPV HPV HPV HPV

Cervical Cervical Cervical Cervical

Boshart et al. (1984) Lorincz et al. (1985) Beaudenon et al. (1986) Lorincz et al. (personal communication) Green et al. (1982)

HPV type HPV 6 HPV 11

18 31 33 35

carcinoma dysplasia carcinoma carcinoma

HPV 10

Skin wart

HPV 34

Bowen’s disease of the skin

Durst et al. (1983)

Kawashima et al. (1986)

Crosshybridization in liquid phase (%)

HPV HPV HPV HPV HPV

11: 25 13: 4 6: 25 13: 3 31: 35

HPV32: 10 HPV 16: 35

Cross-reactivity in Southern blot hybridization under stringent conditions

HPV 26, HPV 27

HPV 7, 10, 14, HPV 15,26, 33, 34 HPV 2,26 HPV 16

HPV 3: 36 HPV2: 6 HPV 16

124

HERBERT PFISTER

demonstrated hybridization with DNA from cervical and vaginal dysplasias. Usually more than one probe was positive in addition to HPV 6 even under high-stringency conditions. From the data it is not possible to decide if the hybridization patterns are due to multiple infections or to HPV 6 variants showing patchy homologies with skin wart papillomaviruses. HPV 34 DNA was cloned from Bowen’s disease of the skin, and a first screening revealed HPV 34 in one case of genital bowenoid papulosis out of 45 cases tested (Kawashima e t al., 1986).

IV. Characteristics of HPV-Induced Genital Lesions

A. QUESTION OF ETIOLOGY

The viruses previously discussed appear associated with various lesions in the lower genital tract of females and males. An etiologic role is suggested in each case by the more or less regular presence of viral footprints. Genital warts contain only a few mature virus capsids, but persistent electron microscopy of sections or tissue homogenates usually succeeds in demonstrating typical 50-nm particles (Oriel and Almeida, 1970; della Torre e t al., 1978; Laverty et al., 1978). The group-specific antigen of papillomaviruses can be demonstrated within the nuclei of the upper epidermal layers (Shah et al., 1980; Woodruff et al., 1980), and virus-specific DNA can be disclosed by hybridization of labeled probes to DNA extracts from tumor biopsies (Gissmann et al., 1982b; Kurman et al., 1982). For condylomata, viral etiology was clearly proved by experimental transmission from person to person using cell-free filtrates of genital warts (Waelsch, 1918; Serra, 1924; Goldschmidt and Kligman, 1958). Recently, an interesting experimental system was introduced which allows infection of human tissue free of ethical constraint (Kreider et al., 1985). Cervical tissue from the squamocolumnar junction was obtained from patients undergoing hysterectomy and was infected with extracts of condylomata acuminata. When grafted beneath the renal capsules of athymic mice, the tissues formed cysts, which differed from uninfected controls by a broader epithelium showing typical features of condylomata acuminata such as koilocytosis, nuclear hyperchromasia, and binucleation. The group-specific antigen of papillomaviruses was detected in cell nuclei, and HPV 11 DNA was demonstrated in extracts from the grafts. The system thus proves the transformation of human tissue by papillomaviruses under controlled experimental conditions. Natural transmission by sexual contact can be followed up for many

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

125

lesions. In fact, genital warts seem to be rather contagious, since 60% of sexual partners of infected individuals developed condyloma (Oriel, 1971a). The incubation period is in the range of 4-6 weeks (Barrett et al., 1954). The peak incidence of the lesions, to be discussed, generally coincides with maximum sexual activity, thus clearly suggesting venereal transmission. About 50% of male sexual partners of women with cervical condyloma or cervical intraepithelial neoplasia suffered from condyloma or penile intraepithelial neoplasia (Levine et al., 1984). Both penile bowenoid papulosis of a young male and carcinoma in situ of the cervix of his partner were shown to harbor HPV 16 DNA (Hauser et al., 1985). B. EXOPHYTIC TUMORS Until recently, exophytic condylomata acuminata were regarded as the only manifestation of papillomavirus infection in the genital area. They occur on penis and vulva, in the perianal region, in the urethra, in the vagina, and at the uterine cervix (Marsh, 1952; Oriel, 1971a,b; Murphy et al., 1983). Histologically, they are characterized by papillomatosis, elongation and thickening of the rete pegs, acanthosis, parakeratosis, and cytoplasmic vacuolization (Woodruff and Peterson, 1958). The vast majority of exophytic condylomas appear to be induced b y HPV 6 or 11 (Gissmann et al., 1983) and HPV 6 (65%) seems to be more prevalent than HPV 11(21%).Double infections with HPV 6 and 11 (Gissmann et al., 1983), 6 and 16, or 11 and 16 (Durst et al., 1983) were noted. Progression of condylomata acuminata into cancer is documented in a large number of case reports (reviewed by zur Hausen, 1977), although the relative risk appears to be very low. However, condylomata acuminata may take on greater dimensions (Buschke and Lowenstein, 1931). Still showing typical cytological features of condylomas, they nevertheless reveal invasive growth properties, but metastasize very rarely. This variant is mainly induced by HPV 6 (M. Boshart, personal communication). C. FLAT AND INVERTED CONDYLOMAS OF THE CERVIX

1 . Pathology and Prevalence Condylomata acuminata in vagina and cervix often reveal a flat growth pattern, which was previously diagnosed as mild dysplasia. They may not be visible with the naked eye, but are seen when mag-

126

HERBERT PFISTER

nified by colposcopy. The appearance of koilocytes in cervical smears is clearly indicative of papillomavirus infection, and the lesions are therefore currently referred to as flat condylomas (Meisels et al., 1976; Purola and Savia, 1977). Some authors use alternative terms such as noncondylomatous cervical wart virus infection (Laverty et al., 1978) or subclinical papillomavirus infection (Reid et al., 1982). The histology of flat condylomas shows acanthotic epithelium with mildly accentuated rete pegs, dyskeratosis, and koilocytotic atypia in superficial cells (Meisels et al., 1977). In so-called spiked condyloma, blood vessels surrounded by scanty stroma push upward through the epithelium, giving rise to an uneven surface texture (Meisels et al., 1982). Inverted endophytic condylomata are basically identical to flat ones, but are characterized by pseudo-invasive penetration into underlying stroma. A subgroup of flat condylomas displays marked nuclear atypia (see below) and has been referred to as atypical condylomata (Meisels et al., 1981). The flat condyloma is the most common type in the cervix, representing about 70% of the cases, followed by the papillary and the inverted type (Meisels et al., 1977; Syrjanen, 1979). The absolute incidence is rather high. Morphological evidence of papillomavirus infection was obtained in 1.3-1.7% of routine cervical smears (Reid e t al., 1980; Meisels and Morin, 1981). 2 . Virus Type-Specijic Effects HPV 6 and/or HPV 11 occur in about 40% of flat condylomas (Gissmann et al., 1984). They give rise to proliferations often characterized by extensive koilocytosis (Crum et al., 1985; Gross et al., 1985a; Schneider et al., 1985). The lesions histologically still resemble normal squamous epithelia, and nuclear atypia (koilocytotic atypia) is confined to superficial cells. HPV 16 was detected in about 17% of flat condylomas (Gissmann et al., 1984). Nuclear atypia was observed in all layers of the epithelium of HPV 16-infected lesions (Crum et al., 1985). Koilocytotic cells were usually low in number or even entirely absent. The presence of HPV 16 correlated with the presence of abnormal mitoses (Crum et al., 1984), which in turn are known to indicate an aneuploid karyotype (Fujii et al., 1984). HPV 16-induced genital warts thus combine features of condylomas and cervical intraepithelial neoplasia (CIN), showing the characteristics of “atypical condylomata” described by Meisels et al. (1981).Abnormal mitoses were occasionally noted in well-differentiated koilocytotic lesions (Fujii et al., 1984), unfortunately without typing the papillomavirus. It would be interesting to know if these were early HPV 16 infections

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

127

progressing to a more severe clinical picture of HPV 6/11-induced lesions, which may show similar phenomena. In the Washington, D.C. area, HPV 31 prevails in mild dysplasias (20%) (A. T. Lorincz, personal communication). It is worth investigating if the close genetic relationship between HPV 31 and HPV 16 is reflected by a similar histology. A distinct histological entity was recently described as atypical immature metaplasia (AIM) (Crum et al., 1983). It was always found within the transformation zone or within endocervical glands and could be distinguished from conventional metaplasia by an increased cellularity, variable nuclear hyperchromatism, and mild pleomorphism. AIM lacks prominent koilocytosis. Papillomavirus group-specific antigen was detected in 16% of the cases, but the HPV type has not yet been classified. It seems possible that the unusual histology reflects HPV replication in metaplastic epithelium (which does not provide fully permissive cells) rather than the presence of a specific HPV type. D. NATURAL HISTORYOF CERVICAL HPV LESIONS Follow-up studies of cervical HPV lesions are only in their beginnings and in most cases are not yet correlated with the HPV type. The frequent association of condylomata with dysplastic and neoplastic processes, however, was noted very early (Syrjanen, 1979; Meisels et al., 1982; Reid et al., 1982; Dyson et al., 1984). Both types of lesions coexist in close proximity or are even intermingled. Meisels and associates (1981)observed progression to dysplasia or even carcinoma in situ in 10% of patients with atypical condylomata ( N = 110)within 18 months. In contrast, only 5% of ordinary condylomata progressed to intraepithelial neoplasia. About 68% of the condylomata regressed and 27% remained unchanged (Meisels et al., 1982). During a 2-year prospective follow-up of 343 women, 14% of cervical HPV lesions progressed into a more severe degree of CIN and 5.5% to carcinoma in situ (Syrjanen and Syrjanen, 1985). In a retrospective study, progression was seen in 20% of 764 lesions (de Brux et al.,

1983). Condylomas and cervical cancer are linked by a spectrum of continuous morphological and biological changes (Reid et al., 1984). At any stage of cervical intraepithelial neoplasia the lesion may regress or persist unchanged, although progression is the more likely the more advanced the process is (Fu et al., 1981). A close correlation exists between DNA content and progression. About 80% of aneuploid le-

128

HERBERT PFISTER

sions persist and 12% progress whereas 91% of diploid or polyploid lesions regress (Fu et al., 1981). From this one would assume that HPV 16-induced condylomas of the stage discussed above (Section IV,C,2) are more likely to progress than HPV 6 or 11 infected warts. A rapid progression in less than 3 years was reported recently (Syrjanen et al., 1985a). The first examination of the patient revealed cytological changes consistent with HPV infection and mild dyskaryosis, but both colposcopy result and punch biopsy histology were normal. After 8 months, a typical flat condyloma was observed. When the patient returned 2 years later, an invasive squamous cell carcinoma was diagnosed, which was positive for HPV 16 and HPV 18 DNA. Peak incidence rates of cervical HPV infection precede those of cervical cancer by 20-30 years. This indicates that the average latency period between primary infection and cancer development is significantly longer than in this single case. Papillomavirus footprints can be frequently detected in premalignant and frankly malignant tumors. The group-specific antigen of papillomaviruses was disclosed in 3-25% of moderate and severe dysplasias, the percentage of positive lesions decreasing with increasing severity of CIN (Shah et al., 1980; Guillet et ul., 1983; Kurman et al., 1983; Syrjanen, 1983;Walker et al., 1983).An inverse relationship was noted between the presence of mitotic abnormalities and the expression of HPV antigen (Winkler et al., 1984).These data are not surprising because the complete replication cycle of papillomaviruses depends on differentiated epithelial cells. Less differentiating malignant cells obviously do not support capsid protein synthesis. However, HPV-specific DNA could be detected in 50-80% of CIN 3 lesions and carcinoma in situ. (McCance et al., 1983; Wagner et al., 1984; Fukushima et al., 1985; Prakash et al., 1985; Schneider et al., 1985; Scholl et al., 1985; Pfister et al., unpublished). Both HPV 6/11 and HPV 16 were observed. HPV 10 was detected in 10% of cervical swabs with cytological diagnosis Pap 11,111, or IV (H. J. Eggers and R. Neumann, personal communication). The absolute number of tested samples is still rather low, and therefore it is difficult to evaluate the prevalence of individual types. Regional geographic differences cannot be excluded. HPV 16, for example, varied between 25 and 95% in biopsies from Panama and the Detroit area of the United States, respectively (Prakash et al., 1985; A. Lorincz, personal communication) and HPV 6 likewise between 16% (3/19)and 67% (4/6) when tested in Austria and England (McCance et al., 1983; Pfister and Girardi, unpublished). Because of obvious ethical constraints, the natural history of flat

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

129

condylomas cannot be followed beyond the stage of carcinoma in situ. Looking at fully advanced invasive squamous cell carcinomas, however, it is still possible to detect persisting HPV DNA. This will be discussed in detail in Section V. E. FLATLESIONSIN

THE

ANOGENITALSKIN

Flat condyloma-like lesions also occur at external genital sites of both sexes (Gross et al., 1985a). HPV 6 and 11 induce proliferations of low epidermal atypia. HPV 16 appeared in cases of severe atypia consistent with the clinical diagnosis of bowenoid papulosis, or Bowen’s disease (Ikenberg et d . ,1983; Gross et d . , 1985a,b). Bowen’s disease is regarded as carcinoma in situ. Bowenoid papulosis shows the same histological features, but differs clinically by the age distribution of the patients (20-35 years versus more than 40) and the multicentric origin of the lesions (Lloyd, 1970; Wade et al., 1978).The clinical picture may be very inconspicuous and easily overlooked (Fig. 3 ) . Erythematous macules, reddish to violaceous, or lichenoid papules and leukoplakia-like lesions were described (Wade et al., 1978; Gross et al., 1985b). Virus particles were disclosed (Kimura et ul., 1978) in keratinocytes beneath the horny layer, which makes bowenoid papulosis an important candidate for transmission of HPV 16. It is interesting to note that many cases in females occur and regress in association with pregnancy and delivery, respectively (Kimura et ul., 1978). This suggests a hormonal activation of HPV 16.

FIG.3. Bowenoid papulosis of the penis induced by HPV 16. The clinically inconspicuous lesions (A) histologically show characteristics of carcinoma in situ (B). Photograph by A. Gassenmaier.

130

HERBERT PFISTER

The clinical course of bowenoid papulosis is usually benign and ends by spontaneous regression in spite of the severe histological picture (Kimura et al., 1978).This contrasts strikingly with the natural history of HPV 16 infections in the cervix uteri. As far as vulvar intraepithelial neoplasia (VIN) is concerned, papillomavirus footprints can be demonstrated. Structural antigen can be detected, but only infrequently within aneuploid lesions, as would be expected (Crum et al., 1982). HPV 6-related DNA was found in one carcinoma in situ of the vulva (Zachow et al., 1982), HPV 10 and HPV 16 DNA in two vuival carcinomas each, HPV 16 DNA in one penile cancer, and HPV 16 or 18 in three out of five anal carcinomas. (Green et al., 1982; Durst et al., 1983; Beckmann et al., 1985).A screenng of 18 penile carcinomas from Brazil disclosed HPV 18 DNA in 6 tumors and HPV 11 in 1 (L. Villa, personal communication).

F. INAPPARENT INFECTIONS There is evidence of a substantial percentage of subclinical infections with genital papillomaviruses. Schneider et al. (1985)obtained hybridization of an HPV 16 and 18 DNA cocktail to four cervical swabs of 229 women (2%) who were cytologically and clinically inconspicuous. HPV 6 DNA was detected in 2 of 19 cervical scrapings from women with normal colposcopic and cytological examination who were attending a sexually transmitted diseases clinic (Wickenden et al., 1985). Viral antigen was found in 8% (2 of 25) of histologically normal epithelia (Walker et al., 1983), and even virus particles were disclosed in 14 out of 22 cervical biopsies without evidence for HPVinduced lesions (Syrjanen et al., 198513).The latter result suggests that papillomaviruses can be replicated without any clinical symptoms and possibly transmitted by asymptomatic carriers. A screening of 2169 females attending the clinic for routine cancer prevention was carried out by D. Wagner and E. M . de Villiers (personal communication). Swabs of 221 out of 1997 women who were negative in colposcopy and cytology were positive for HPV DNA (i.e., 11%). The ratio of clinically apparent to subclinical HPV infection is shown in Fig. 4. A maximum of clinically detectable HPV infections between 21 and 30 years of age is likely to correspond to primary infections, whereas the following drop could reflect inapparent persistence. The rise at higher age may not be statistically significant due to small case numbers, but could indicate later recurrences. Latent papillomavirus infections are likely to be responsible for frequent recurrences after surgical removal of condylomas or in-

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER 10

,16

78

a2

22

12

a

131

N

501

620 21-30 31-10 Ll-50 51-60 61-70 a 7 1 age group

FIG.4. Relative frequency of pathological results from cytology or colposcopy among women with HPV DNA-positive cervical swabs. The absolute number of women within each age group is given on top line (D. Wagner and E. M. de Villiers, unpublished).

traepithelial neoplasms. HPV DNA was recently identified in normal skin adjacent to lesions at a distance of 15 mm (Ferenczy et al., 1985). After laser treatment, lesions recurred in 6 of 9 patients with inapparently infected skin margins, but in only 1 of 11 patients without detectable HPV DNA in the surrounding tissue.

V. Human Papillornaviruses in Cervical Cancer

A. PERSISTENCE OF VIRALDNA

1 . Prevalence of Zndividual HPV Types More than 200 squamous cell carcinomas of the cervix were screened for HPV DNA by hybridization with different viral DNAs. The prevalence of individual HPV types is summarized in Table 11. HPV 16 incidence clearly stands out in Europe and Panama, where it persists in about 60%of the carcinomas. The difference to data from Africa and South America (35%positive) is statistically significant ( p < 0.05) and will reflect differences in the prevalence of the virus in the population. The latter may also hold true for HPV 18, where the incidence difference between Europe and Africa and South America is close to significance (0.05 < p < 0.1). The incidence rate of HPV 10, HPV 6/11, HPV 31, HPV 33, and HPV 35 is very much alike and close to 5%.

132

HERBERT PFISTER

TABLE I1 PREVALENCE OF DIFFERENT HPV DNAs IN SQUAMOUS CELLCARCINOMAS OF THE CERVIXUTERI Positive tumors N

(%)

95% confidence interval

United States Austria NS"

31 18 22

6 17 5

0.8-21.4 3.6-4 1.4 0.1-22.8

United States

6

17

0.4-64.1

United States

9

0

0.0-33.6

44

0

0.0- 8.0

4

50

6.8-93.2

18 103 18

6 5 61

0.1-27.3 1.6-1 1.3 35.8-82.7

Pfister (unpublished)

18 44

28 66

9.7-53.5 50.1-79.5

United States

9

11

0.3-48.3

Panama AfricdBrasil Federal Republic of Germany Austria France

20 23 13

60 35 15

36.1-80.9 16.4-57.3 1.9-45.5

Pfister (unpublished) Orth (personal communication) Fukushima et al. (1985) Prakash et al. (1985) Durst et al. (1983) Boshart et a1. (1984)

18 44

17 7

3.6-41.4 1.4-18.7

HPV 31 HPV 33

AfricdBrazil United States France

36 39 52

25 5 4

12.1-42.4 0.6-17.3 0.5-13.2

HPV 35

United States

2

-

Virus HPV 10 HPV6/11

Country

France

HPV 16

HPV 18

Federal Republic of Germany Austria Average Federal Republic of Germany Austria France

* Not specified.

Reference Green et al. (1982) Pfister (unpublished) Gissmann et al. (1983) Lanca ter et al. (1983) Fukushima et al. (1985) Orth (personal communication) Schneider et al. (1985)

Durst et al. (1983)

Pfister (unpublished) Orth (personal communication) Boshart et al. (1984) Lorincz et al. (1985) Beaudenon et al. (1986) Lorincz (personal communication)

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

133

That viral DNA persists in the malignant cells was convincingly shown by the demonstration of HPV DNA in cervical cancer-derived human cell lines (Table 111). Seven out of nine cell lines tested contained DNA of either HPV 18 or HPV 16. HPV 16-related sequences from the CaSki line hybridized to HPV 18 under conditions of slightly reduced stringency (Yee et al., 1985), which is not true for the cloned HPV 16 prototype DNA. It was therefore concluded that CaSki cells harbor a new HPV, related closely to HPV 16 and more distantly to HPV 18. HPV 16 DNA was detected in two out of six adenocarcinomas of the cervix (E. M . de Villiers et al., personal communication). The HeLa cell line, which contains HPV 18 DNA, is also derived from an adenocarcinoma (Boshart et al., 1984). The relative prevalence of HPV DNA in carcinomas and carcinomaderived cell lines may depend mainly on three parameters: (1) the general prevalence of the virus types in a given population, (2) the ability to persist in frankly malignant cells, and/or (3)the cancerogenic potential of the virus. Viruses frequently associated with carcinomas in one part of the world (such as HPV 16 and 18) certainly deserve interest as potentially carcinogenic viruses even if they appear only rarely at another location (HPV 18 in France). A possible role in carcinogenesis is not excluded by a low prevalence rate in carcinomas, however, because this may be due to low incidence of infection or loss of viral genomes during tumor progression. A comparison of HPV 16 and HPV 31 is of special importance. The DNAs of both types are closely related (see Section 111).In contrast to HPV 16, however, HPV 31 was rarely detected in carcinomas, although it was prevalent in TABLE 111 PRESENCE AND EXPRESSION OF HUMAN PAPILLOMAVIRUSES IN CERVICAL CANCER-DERIVED CELLLINES Cell line

HPV DNA

HPV RNA

Reference

HeLa SW756 C4-I C4-I1 MS751 ME 180 SiHa CaSki C33A HT-3

18 18 18 18 18 18 16

18 18 18 18 18

Boshart et al. (1984) Schwarz et 01. (1985)

0

Pater and Pater (1985) Yee et al. (1985)

0 0

0 0

16

+ 18

16 16

134

HERBERT PFISTER

mild dysplasias (A. T. Lorincz et al., personal communication). This offers a chance to pinpoint those genes relevant to this biological difference.

2. Physical State of the Viral DNA Papillomavirus DNA is well known to persist extrachromosomally in high copy number (for review, see Pfister, 1984). This general pattern was also observed with HPV 10, HPV 11, and HPV 33 in cervical carcinomas (Green et al., 1982; Lancaster et al., 1983; G . Orth et al., personal communication). HPV 16 and 18 offer interesting exceptions to this rule (Boshart et al., 1984; Durst et al., 1985; Lehn et al., 1985). In benign tumors, the viral DNA persists in plasmid form as usual. In carcinomas, however, the majority or all of the viral DNA appears to be integrated into the host genome. Some tumors contain only one copy of the viral genome per cell, others multiple copies as head-to-tail tandem repeats and at more than one integration site. Direct evidence for integration was obtained by cloning virus-cell DNA junction fragments (Durst et al., 1985). In three carcinomas with one viral DNA copy each, the originally circular HPV DNA was opened within reading frames E 1, E2, or L2, respectively (Lehn et al., 1985).The same area of the viral genome is afflicted in the HPV DNA-positive lines HeLa, C4-I, SW756, and SiHa (Schwarz et al., 1985; Pater and Pater, 1985).Extensive deletions comprising open reading frames E2 and L2 were observed for HeLa, C4-I, C4-11, ME180, and MS751 (Schwarz et al., 1985; Pater and Pater, 1985). The rather consistent inactivation of the 3' moiety of the early region by integration and deletion may result from a selective pressure during tumor progression. It should be noted that disruption of the early transcription unit of HPV 16 and 18 uncouples the possibly transforming gene E5 from the early viral promoter. In contrast, open reading frame E6 remained intact in all tumors tested up to now. So far there is no evidence for specific integration sites within cellular DNA. Integration of HPV 16 and HPV 18 DNA may be brought about by carcinogens inducing recombinatory events or by intrinsic properties of the viruses. The open reading frame E l , which is supposed to be essential for extrachromosomal maintenance (Lusky and Botchan, 1985), appeared interrupted in the case of an HPV 16 DNA clone from a cervical carcinoma (Seedorf et al., 1985). It is of considerable interest to sequence HPV 16 DNA from benign tumors to see if this is a general characteristic of HPV 16, making it perhaps more prone to integration. Similarly interesting will be a comparison with HPV 31.

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

135

An increased tendency to integration could account for a more efficient persistence of HPV 16 and HPV 18. Integration also allows special mechanisms of carcinogenesis such as activation of viral or cellular genes by heterologous transcription control elements. Fusion transcripts and/or proteins could theoretically gain increased or decreased stability and/or new oncogenic properties. B. VIRALGENEEXPRESSION HPV 16-specific transcripts were disclosed in cervical carcinoma biopsies (Schwarz e t al., 1985; Lehn et al., 1985). This is readily achieved in the case of high DNA copy number, but may fail where HPV DNA persists only in low amounts. HPV 18 RNA could not be detected in carcinoma-derived ME 180 cells, which harbor about one copy of HPV 18 DNA (Yee et al., 1985). In most cervical cancer cell lines, however, the viral DNA is actively transcribed, as indicated in Table I11 (Schwarz e t al., 1985; Yee et al., 1985).This permits a more detailed analysis. In HeLa, C4-I, and SW 756 cells, transcripts cover open reading frames E6 and E 7 of HPV 18 and are spliced to cellular sequences using a splice donor site at the 5' terminus of E l (E. Schwarz et al., 1985, personal communication). Many of the cDNA sequences analyzed revealed an internal splice within open reading frame E6, leading to a shorter E6 protein with a different carboxy terminus. The transcription pattern differs markedly from that of HPV 6 in genital warts where the majority of RNAs is transcribed from the 3' end of the early region covering open reading frames E2, E4, and E 5 (Lehn et al., 1984). In BPV l-transformed cells, the amount of viral RNA is controlled by at least one labile protein, which is reflected by a 5- to 10-fold increase of viral transcripts 1 hr after addition of cycloheximide. In contrast, no cycloheximide effect was observed with HeLa, C4-I, and SiHa cells (Kleiner et al., 1986). It will be interesting to see if the absence of a comparable regulation mechanism is a consistent feature of all cervical carcinoma cell lines and if it depends on the cell or on the virus. VI. Speculations on an Etiologic Role in Carcinogenesis

In making an interim analysis, one can state that papillomaviruses are distinguished from all other venereally transmitted agents incriminated in the etiology of genital cancer. They were shown to cause primarily benign proliferations, which may progress through a contin-

136

HERBERT PFISTER

uous spectrum of morphological and biological changes to invasive cancer. Specific papillomavirus DNA sequences persist in the vast majority of premalignant and malignant tumors and are transcribed in a number of cervical carcinomas and carcinoma-derived cell lines. These data strongly suggest that HPV infection involves a risk to develop cancer and may indicate that papillomaviruses play a part in the maintenance of the malignant state.

A. RISK OF INDIVIDUAL HPV INFECTIONS

HPV 16 and 18 stand out for their frequent persistence in cervical carcinomas. The histology of HPV 16-induced lesions often reveals a high degree of nuclear atypia and abnormal mitotic figures (see Section IV, C and D). However, HPV 16 and/or HPV 18 reside in 20% of Pap I or Pap I1 smears (H. J. Eggers and R. Neumann, personal communication), there are many inapparent HPV 16/18 infections (D. Wagner and E. M . de Villiers, personal communication), and regression was observed in 3 out of 9 patients when controlled after an interval from 1 week to 3 months (Schneider et al., 1985). The risk of HPV 16 infections can be roughly estimated from the prevalence of HPV 16 (up to 10%; Section IV, F), and the incidence of cervical cancer (about 30 HPV-positive carcinomas per 100,000 population) can be estimated to be 1malignant tumor in about 300 infected females. An analogous calculation for HPV 6/11 (3-4 HPV 6/11-positive cervical carcinomas per 100,000 population) indicates that the risk of an infected female to develop a carcinoma is about 1per 20003000. This would be 7- to 10-fold lower than for HPV 16, but still in the same range as, for example, the risk of an HTLVI-infected Japanese to acquire adult T cell leukemia (Hunsmann and Hinuma, 1985). The calculation is subject, of course, to many imponderables. First, there is no fully randomized study thus far of the prevalence of individual virus types in the normal population. Second, one should be aware of the lesson from epidennodysplasia verruciformis. In that disease, there exist at least 15 closely related viruses, only 2 of which (HPV 5 and 8) persist in more than 90% of skin carcinomas. Premalignant cervical lesions and healthy controls were usually screened just for HPV 16-related sequences, thus picking up subtypes and crosshybridizing DNAs of other HPV types such as HPV 31. This isolate may only be the first representative of a series of HPV 16-related types. When testing premalignant cervical lesions with an HPV 16 probe, Crum et al. (1985)observed aberrant restriction enzyme cleavage patterns in 2 out of 12 cases, which is indicative of new subtypes

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

137

or types. The viral DNA from CaSki cells provides another example for an HPV 16 variant (Yee et al., 1985).A more refined classification of these HPV types is obviously necessary to differentiate between viruses of higher and lower risk. An HPV 6 variant was recently cloned from an unusually aggressive vulvar verrucous carcinoma (Rando et al., 1986). The partial nucleotide sequence revealed almost an identity with the HPV 6 prototype except for three small insertions within the noncoding region. This may indicate that minor modifications within the control region can strongly affect pathogenic properties. The cancer risk of certain subtypes may then be significantly higher than calculated above. It was already pointed out (Section IV,E) that HPV infections of the vulva and the penis have a much better prognosis than those of the cervix. Bowenoid papulosis is usually characterized by spontaneous regression in spite of a histological picture reminiscent of carcinoma in situ. The incidence of penile cancer correlates with the incidence of cervical cancer, but is usually 20-fold lower (Waterhouse et al., 1982), and penile cancer occurs about 10-15 years later. This suggests the action of the same etiologic factors, but with a different efficiency and latency period. Vulvar cancer may occur slightly more frequently than penile cancer (Waterhouse et al., 1982).

B. SYNERGISM OF OTHERRISKFACTORS

1. Carcinogens Malignant conversion of papillomavirus-induced tumors may be facilitated and accelerated by physical and chemical carcinogens (for review, see Pfister, 1984). Heavy and prolonged smoking turned out to be a significant risk factor in the development of cervical cancer (Winkelstein, 1977; Clarke et al., 1982). Nicotine, which may give rise to powerful carcinogens (N-nitrosamines), was demonstrated in vaginal fluids (Hoffmann et al., 1985),indicating that active chemicals do reach the cervical cells. Herpes simplex virus was recently shown to act like a chemical initiator in infected cells (zur Hausen, 1983).A major role of herpes simplex virus in the etiology of cervical cancer must be questioned, however, in view of a recent extensive prospective study (Vonka et al., 1984a,b). Carcinogens are known to induce mutations, recombination, and selective DNA amplification (Miller and Miller, 1977; Schimke, 1982). Persisting papillomavirus genomes may be directly subject to

138

HERBERT PFISTER

these effects. In addition, carcinogens may act on cellular genes involved in the control of papillomavirus genomes or which are themselves subject to papillomavirus functions. Finally, carcinogens may activate genes that complement or supplement viral functions. A 3- to 30-fold amplification of cellular myc and/or Ha-ras genes was noted in nine advanced (stages 3 and 4), HPV DNA-positive cervical carcinomas (Riou et al., 1984).The myc and Ha-ras genes were shown to be mutated, translocated, or amplified in a number of human lymphomas, leukemias, and carcinomas (for review, see Cooper and Lane, 1984). The genetic changes seem to activate these cellular genes in terms of oncogenic activity. In the case of cervical carcinoma progression, a possible role of myc and rus genes appears to b e confined to late stages because only one of three stage 1 tumors revealed low amplification of c-Ha-ras (Riou et al., 1984).

2 . Immunosuppression The prevalence of cervical condyloma was 8.3% in a study of 132 female transplant recipients (Schneider et al., 1983), which represents a roughly 5-fold increase if compared to nonimmunosuppressed patients. Of l l patients from this study, 6 developed cervical neoplasia within 3 years after transplantation. A 14-fold increase in the risk of developing carcinoma in situ was calculated for transplant recipients (Porreco et ul., 1975), and a higher risk was also discussed for patients who were immunosuppressed for other conditions (Norfleet and Sampson, 1978; Sillman et al., 1984). The data are highly suggestive of an increased and accelerated progression of HPV-induced tumors under conditions of impaired host immunity. They point to a role of immune surveillance in the control of the premalignant lesions.

C. MAINTENANCE OF THE MALIGNANT STATE HPV DNA cannot be demonstrated in all cervical carcinomas or in all carcinoma-derived cell lines. Although it was never definitively excluded that unrelated HPV DNA persists at low copy number, thus escaping detection by DNA hybridization, the negative data may point to an HPV-independent etiology of these tumors or to the final loss of previously present HPV DNA. The latter would imply that the continuous presence of HPV is not necessary for the maintenance of the malignant state. The same conclusion was drawn from transcript analysis. Tumors with exceptionally low amounts of viral DNA do not reveal viral RNA (Lehn et al., 1985; G . Orth et al., personal communication). In addi-

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

139

tion, an HPV 18-specific RNA was not detected in the HPV 18 DNApositive cell line ME180 (Yee et al., 1985).Of course, it is impossible to exclude a brief pulse of transcription of an RNA with a short halflife, but there exist at least considerable quantitative differences between individual cervical carcinomas. At the moment, one cannot exclude the notion that the frequent presence of HPV DNA in advanced carcinomas reflects the efficiency of the viruses to persist extrachromosomally and/or integrated into the host genome, and not necessarily an essential function in the maintenance of the malignant state. D. PROSPECTS FOR DIAGNOSIS A prognostic grading of cervical dysplasias is certainly of utmost interest. The inverse relationship between productive HPV infection and degree of dysplasia provided a first diagnostic parameter. The probability of detecting the group-specific antigen of papillomaviruses by a peroxidase-antiperoxidase test drops from 50% in lowgrade dysplasias to 3% in CIN 3 (Shah et al., 1980; Guillet et al., 1983; Kurman e t al., 1983; Walker et al., 1983).This indicates that the presence of structural antigens is suggestive of, but not proof for a lowgrade dysplasia or a flat condyloma. Eventually, a much better prognosis may arise from an HPV typespecific diagnosis. To this end, we need more information on the risk factor of an individual HPV infection. This may be obtained from data on the prevalence of an HPV type in a population and its incidence in cervical cancer (see Section V1,A). However, the best way to determine the risk will be with prospective follow-up studies, which are just beginning and will be evaluated in a few years. Summarizing our present knowledge some HPV types, namely, HPV 16 and 18, may be endowed with an increased cancerogenic potential when compared to viruses such as HPV 6, HPV 11, or HPV 31 (see Section V1,A). Differences may become clearer when classification becomes more refined, but individual types will not become black or white. Many HPV types appear to be extremely prevalent and the risk of developing malignant tumors seems to be in the range of 1 carcinoma per 3000-300 infections. It is therefore obvious that the demonstration of an HPV infection alone cannot be sufficient for a prognostic evaluation. There may be good reason for more frequent control of a patient if dysplastic lesions harbor a virus, implying a higher risk, but there is no reason for rash, radical therapy. This situation reminds one of other ubiquitous human tumor viruses such as Epstein-Barr virus, where the presence of IgG antibod-

140

HERBERT PFISTER

ies against the viral capsid antigen (VCA) or the persistence of EBV DNA in B lymphocytes is trivial. Epstein-Barr virus, however, also reveals a possible solution. A clear rise of the IgA antibody titer against VCA, apart from acute infection, is highly suggestive of an early or recurrent nasopharyngeal carcinoma (Henle and Henle, 1976). This means that we have to understand the parameters of HPV infection, which are more specifically associated with malignant conversion of HPV-induced tumors. At the moment, integration of HPV 16 and HPV 18 DNA in carcinomas seems to be the most promising starting point. It happens somewhere during tumor progression. Whether causative or a side effect, it could be used as an indicator of an early malignancy. Viruses persisting extrachromosomally do not offer any parameters to test for malignant conversion so far. More has to be learned about the mechanism of tumor progression in order to design an appropriate routine test for these infections. E. PROSPECTS FOR VACCINATION

The regular association of papillomaviruses with premalignant and malignant cervical tumors may provide an opportunity for immunotherapy by vaccination. Furthermore, decrease in the incidence of cervical cancer following successful vaccination against HPV infection would most convincingly demonstrate an essential role of papillomaviruses in tumor development. Natural papillomavirus infection in humans is followed by humoral and cellular immune response against virus particles and tumor tissue (for review, see Pfister, 1984). The special importance of cell-mediated immunity in HPV control is well established both directly by histological examination of regressing warts and indirectly by increased wart incidence in patients with cellmediated immune deficiencies. The immune response is usually very tedious and the lesions may consequently persist for months or years. This is probably at least partially due to low levels of viral antigen(s), which allow the virus to sneak through immune control. A stimulation of the immune system by vaccination with appropriate antigen preparations could circumvent this problem. Interest should not focus on neutralizing antibodies against viral capsid proteins, since the virus has a good chance to reach its target cell and establish a latent infection before any contact with antibodies. Cytotoxic T lymphocytes are likely to recognize membrane-bound virus- or tumor cell-specific antigens. The E 6 protein of bovine papillomavirus 1 was quite recently shown to be associated with nonnu-

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

141

clear membranes (Androphy et al., 1985), which is of special interest in view of E6-specific transcripts in cervical carcinoma-derived cell lines (Schwarz et al., 1985). If E6 appears to be exposed at the cell surface, it would be an important candidate for a future vaccine, which could be of both preventive and therapeutic value. VII. Concluding Remarks

Papillomaviruses are clearly proven to be the etiologic agents of anogenital lesions, with the potential to progress to squamous cell carcinomas. Viral particles, DNA, and/or capsid antigens can be demonstrated in benign precursors, and the proliferations could be transmitted from person to person by cell-free extracts (Section IV,A). Ten HPV types (1, 2, 6, 10, 11, 16, 18, 31, 33, 35) were identified infecting the anogenital mucosa. At least some of them seem to be extremely prevalent in the human population. The complete nucleotide sequences of HPV 6, HPV 11, and HPV 16 revealed the familiar genome organization of papillomaviruses and homologous sequences to those open reading frames which were shown to code for transforming functions in the case of BPV 1 (Section 11,C).A notable difference was observed between primary lesions induced by HPV 6/11 and HPV 16, respectively. HPV 6 and 11 prevail in condylomata acuminata and plana, which are characterized by a rather regular differentiation pattern and extensive koilocytosis, whereas HPV 16 infection seems to imply a spectrum of precancerous changes such as nuclear atypia and an aneuploid karyotype (Section IV,C,2). Malignant conversion of papillomavirus-induced tumors is well established for a number of animal systems and for the human disease epidennodysplasia verruciformis. The cottontail rabbit papillomavirus appears to be a high-risk virus, with 75% of infected domestic rabbits developing skin cancer (Section 11,D). HPV DNA persists in genital carcinomas either integrated into the host genome or extrachromosomally and is transcribed at least in some tumors. The worldwide prevalence of HPV 16 is noteworthy and may possibly indicate an increased cancerogenic potential of this virus (Section V,A and B). From these data there can be little doubt that papillomaviruses play an important role in the development of genital cancer and were deservedly brought into focus of intensive research. A tentative calculation indicates that the cancer risk after HPV infection equals or exceeds the risk attended with infections by other human tumor viruses such as HTLVI, Epstein-Barr virus, or hepatitis B virus. Tumor progression is certainly subject to additional factors such as chemical or

142

HERBERT PFISTER

physical carcinogens, hormones, and systemic or local immune deficiencies. A diminished exposure to these factors could account for the better prognosis of HPV infections at external genital sites when compared with cervical infections. A role of HPV in the maintenance of the malignant state is not proved at the moment. One could imagine that late activation of cellular oncogenes partially renders HPV functions superfluous (Section V1,B). It is tempting to exploit the close association between papillomaviruses and genital cancer for early cancer diagnosis and for preventive or therapeutic vaccination. It is evident, however, that we still need extensive research in order to develop routine practicing protocols. First, prospective, large-scale follow-up studies must be designed to determine the risk of malignant conversion implied by infections with well-defined HPV types. Second, the collection and classification of HPV types is probably far from complete. The example of HPV 16 and HPV 31 shows that even closely related viruses can differ exactly in their association with neoplasms, thus demonstrating the importance of type-specific probes disclosing differences in biologically significant genome regions. Finally, the molecular biology of HPV infection has to be further examined in order to learn more about the viral role in keratinocyte transformation, which will be essential to defining relevant diagnostic probes and possible candidates for therapeutic intervention (Section VI,D and E). These are obviously longterm programs, but the possible benefit in the end warrants continuous efforts.

ACKNOWLEDGMENTS I am indebted to Drs. E. Androphy, H. Eggers, A. Lorincz, G. Orth, D. Wagner, and H. zur Hausen who provided preprints and information on unpublished work. I thank Dr. P. Fuchs for his critical reading of this manuscript. Original work cited in this article was supported by the Deutsche Forschungsgemeinschaft and the Wilhelm-SanderS tiftung.

REFERENCES Alexander, E. R. (1973). Cancer Res. 33, 1485-1496. Androphy, E. J., Schiller, J. T., and Lowy, D. R. (1985). Science 230, 442-445. Barrett, T. J., Silbar, J. D., and Mc Ginley, J. P. (1954).J.Am. Med. Assoc. 154,333-334. Beaudenon, S . , Kremsdorf, D., Croissant, O., Jablonska, S., Wain-Hobson, S., and Orth, G. (1986). Nature 321,246-249. Beckmann, A. M., Daling, J. R., and Mc Dougall, J. K. (1985).]. Cell. Biochem. S u p p l . 9c, 68. Boshart, M., Gissmann, L., Ikenberg, H., Kleinheinz, A., Scheurlen, W., and zur Hausen, H. (1984).E M B O J . 3, 1151-1157.

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

143

Buckley, J. D., Harris, R. W. C., Doll, R., Vessey, M. P., and Williams, P. T. (1981). Lancet 2, 1010-1014. Buschke, A., and Lowenstein, L. (1931).Arch. Dermatol. Syph. 163, 30-46. Campo, M. S., Moar, M. H., Sartirana, M. L., Kennedy, I. M., and Jarrett, W. F. H. (1985). EMBOJ. 4, 1819-1825. Casas-Cordero, M., Morin, C., Roy, M., Fortier, M., and Meisels, A. (1981).Acta Cytol. 25,383-392. Chen, E. Y., Howley, P. M., Levinson, A. D., and Seeburg, P. H. (1982). Nature (London) 299,529-534. Clarke, E. A., Morgan, R. W., and Newman, A. M. (1982).A m . J .Epidemiol. 115,59-66. Coggin, J. R., and zur Hausen, H. (1979). Cancer Res. 39,545-546. Cooper, G. M., and Lane, M. A. (1984).Biochim. Biophys. Acta 738,9-20. Crum, C. P., Braun, L. A., Shah, K. V., Fu, Y. S., Levine, R. U., Fenoglio, C. M., Richart, R. M., and Townsend, D. E. (1982).Cancer 49,468-471. Crum, C. P., Egawa, K., Fu, Y. S., Lancaster, W. D., Barron, B., Levine, R. U., Fenoglio, C. M., and Richart, R. M. (1983). Cancer 51,2214-2219. Crum, C. P., Ikenberg, H., Richart, R. M., and Gissmann, L. (1984).N . Eng1.J.Med. 310, 880-883. Crum, C. P., Mitao, M., Levine, R. U., and Silverstein, S. (1985).J.Virol. 54, 675-681. Danos, O., Georges, E., Orth, G., and Yaniv, M. (1985).J . Virol. 53, 735-741. Dartmann, K., Schwarz, E., Gissmann, L., and zur Hausen, H. (1986). Virology 151, 124- 130. De Brux, J., Orth, G., Croissant, O., Cochard, B., and Ionesco, M. (1983).Bull. Cancer 70,410-422. Della Torre, G., Pilotti, S., De Palo, G., and Rilke, F. (1978). Tumori 64, 549-553. De Villiers, E. M., Gissmann, L., and zur Hausen, H. (1981).J.Virol. 40, 932-935. Durst, M., Gissmann, L., Ikenberg, H., and zur Hausen, H. (1983).Proc. Natl. Acad. Sci. U.S.A. 80, 3812-3815. Durst, M., Kleinheinz, A., Hotz, M., and Gissmann, L. (1985).J . Gen. Virol. 66, 15151522. Dvoretzky, I., Shober, R., Chattopadhyay, S. K., and Lowy, D. R. (1980).Virology 103, 369-375. Dyson, J. L., Walker, P. G., and Singer, A. (1984).J . Clin. Pathol. 37, 126-130. Ferenczy, A., Mitao, M., Nagai, N., Siverstein, S. J., and Crum, C. P. (1985). N . Eng1.J. Med. 313,784-788. Freese, U. K., Schulte, P., and Pfister, H. (1982). Virology 117, 257-261. Friedmann, J. M., and Fialkow, P. J. (1976). Znt. /. Cancer 17, 57-61. Fu, Y. S., Reagan, J. W., and Richart, R . M. (1981). Gynecol. Oncol. 12, S220-S231. Fujii, T., Crum, C. P., Winkler, B., Fu, Y. S., and Richart, R. M. (1984).Obstet. Gynecol. 63,99-104. Fukushima, M., Okagaki, T., Twiggs, L. B., Clark, B. A., Zachow, K. R., Ostrow, R. S., and Faras, A. J. (1985).Cancer Res. 45, 3252-3255. Gassenmaier, A., Lammel, M., and Pfister, H. (1984).J . Virol. 52, 1019-1023. Giri, I., Danos, O., and Yaniv, M. (1985).Proc. Natl. Acad. Sci. U.S.A.82, 1580-1584. Gissmann, L., Pfister, H., and zur Hausen, H. (1977). Virology 76, 569-580. Gissmann, L., Diehl, V., Schultz-Coulon, H. J., and zur Hausen, H. (1982a).J.Virol. 44, 393-400. Gissmann, L., De Villiers, E. M., and zur Hausen, H. (1982b). Znt. J . Cancer 29, 143146. Gissmann, L., Wolnik, L., Ikenberg, H., Koldovsky, U., Schnurch, H. G., and zur Hausen, H. (1983).Proc. Natl. Acad. Sci. U.S.A.80, 560-563.

144

HERBERT PFISTER

Gissmann, L., Boshart, M., Durst, M., Ikenberg, H., Wagner, D., and zur Hausen, H. (1984).J . Znuest. Dermatol. 83, 26s-28s. Glinski, W., Obalek, S., Jablonska, S., and Orth, G. (1981).Dermatologica 162,141-147. Goldschmidt, H., and Kligman, A. M. (1958).J . Znuest. Dermatol. 31, 175-182. Green, M., Brackmann, K. H., Sanders, P. R., Loewenstein, P. M., Freel, J. H., Eisinger, M., and Switlyk, S. A. (1982). Proc. Natl. Acad. Sci. U.S.A.79, 4437-4441. Gross, G., Ikenberg, H., Gissmann, L., and Hagedorn, M. (1985a).J . Znuest. Dermatol. 85, 147-152. Gross, G., Hagedorn, M., Ikenberg, H., Rufli, T., Dahlet, C., Grosshans, E., and Gissmann, L. (1985b). Arch. Dermatol. 121,858-863. Guillet, G., Braun, L., Shah, K., and Ferenczy, A. (1983).J . Znuest. Dermatol. 81,513516. Hauser, B., Gross, G., Schneider, A., De Villiers, E. M., Gissmann, L., and Wagner, D. (1985). Lancet 2, 106. Henle, G., and Henle, W. (1976). Znt. J. Cancer 17, 1-7. Hofhann, D., Hecht, S. S., Haley, N. J., Brunnemann, K. D., Adams, J. D., and Wynder, E. L. (1985).J . Cell. Biochem., Suppl. 9c, 33. Hunsmann, G., and Hinuma, Y. (1985). Ado. Viral Oncol. 5, 147-172. Ikenberg, H., Gissmann, L., Gross, G., Grussendorf-Conen, E. I., and zur Hausen, H. (1983). Znt. J . Cancer 32,563-565. Jablonska, S., Dabrowski, J., and Jakubowicz, K. (1972). Cancer Res. 32,583-589. Jacyk, W. K., and Subbuswamy, S. G. (1979). Dermatologica 159,256-265. Jenson, A. B., Rosenthal, J. R., Olson, C., Pass, F., Lancaster, W. D., and Shah, K. (1980). J . Natl. Cancer Znst. 64,495-500. Kahn, T., Schwarz, E., and zur Hausen, H. (1986). Znt. J . Cancer 37,61-65. Kawashima, M., Favre, M., Jablonska, S., Obalek, S., Croissant, O., and Orth, G. (1986). J . Virol. 57,688-692. Kessler, I. (1977). Cancer 39, 1912-1919. Kidd, J. G., and Rous, P. (1940).J . Exp. Med. 71,469-485. Kimura, S . , Hirai, A.,Harada, R., and Nagashima, M. (1978). Dermatologica 157, 229237. Kleiner, E., Dietrich, W., and Pfister, H. (1986). EMBO J . 5, 1945-1950. Koss, L. G., and Durfee, G. R. (1956). Ann. N.Y. Acad. Sci. 63, 1245-1261. Kreider, J. W., Howett, M. K., Wolfe, S. A., Bartlett, G. L., Zaino, R. J., Sedlacek, T. V., and Mortel, R. (1985).Nature (London) 317,639-641. Kremsdorf, D., Favre, M., Jablonska, S., Obalek, S., Rueda, L. A., Lutzner, M. A,, Blanchet-Bardon, C., van Voorst Vader, P. C., and Orth, G. (1984). J . Virol. 52, 1013-1018. Krzyzek, R. A., Watts, S. L., Anderson, D. L., Faras, A. J., and Pass, F. (198O).J.Virol. 36, 236-244. Kurman, R. J., Sanz, L. E., Jenson, A. B., Perry, S., and Lancaster, W. D. (1982). Znt. J . Gynecol. Pathol. 1, 17-28. Kurman, R. J., Jenson, A. B., and Lancaster, W. D. (1983).Am. J. Surg. Pathol. 7,39-52. Lancaster, W. D., Kurrnan, R. J., Sanz, L. E., Perry, S., and Jenson, A. B. (1983). Znteruirology 20,202-212. Laverty, C. R., Russell, P., Hills, E., and Booth, N. (1978). Acta Cytol. 22, 195-201. Law, M. F., Lancaster, W. D., and Howley, P. M. (1979).J . Virol. 32, 199-207. Law, M. F., Lowy, D. R., Dvoretzky, I., and Howley, P. M. (1981).Proc. Natl. Acad. Sci. U.S.A. 78,2727-2731. Lehn, H., Ernst, T. M., and Sauer, G. (1984).J . Gen. Virol. 65, 2003-2010.

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

145

Lehn, H., Krieg, P., and Sauer, G. (1985).Proc. Natl. Acad. Sci. U S A . 82,5540-5544. Levine, R. U.,Crum, C. P., Herman, E., Silvers, D., Ferenczy, A,, and Richart, R. M. (1984).Obstet. Gynecol. 64, 16-20. Lloyd, K. M. (1970).Arch. Dennatol. 101,48-51. Lorincz, A. T., Lancaster, W. D., and Temple, G. F. (1985).J. Cell. Biochem. Suppl. 9c,

75.

Lusky, M., and Botchan, M. R. (1985). J. Virol. 53, 955-965. McCance, D. J., Walker, P. G., Dyson, J. L., Coleman, D. V., and Singer, A. (1983).Br. Med. J. 287,784-788. McVay, P., Fretz, M., Wettstein, F., Stevens, J., and Ito, Y. (1982)J.Gen. Virol. 60,271-

278.

Marsh, M. R. (1952).A m . J . Obstet. Gynecol. 64,281-291. Matthews, R. E. F. (1982).Interuirology 17, 1-199. Meisels, A., and Morin, C. (1981).Gynecol. Oncol. 12, Slll-S112. Meisels, A,, Fortin, R., and Roy, M. (1976).Acta Cytol. 20, 505-509. Meisels, A., Fortin, R., and Roy, M. (1977).Acta Cytol. 21,379-390. Meisels, A., Roy, M., Fortier, M., Morin, C., Casas-Cordero, M., Shah, K. V., and Turgeon, H. (1981).Acta Cytol. 25, 7-16. Meisels, A., Morin, C., and Casas-Cordero, M. (1982).1nt.J. Gynecol. Pathol. 1,75-94. Miller, J. A., and Miller, E. C. (1977).Cold Spring Harbor Conf. Cell Prolif. 4, 605-

627. Morin, C., Braun, L., Casas-Cordero, M., Shah, K. V . ,Roy, M., Fortier, M., and Meisels, A. (1981).J. Natl. Cancer Znst. 66, 831-834. Urol. 130,84-85. Murphy, W. M., Fu, Y. S., Lancaster, W. D., and Jenson, A. B. (1983).J. Nasseri, M., and Wettstein, F. 0. (1984). J. Virol. 51, 706-712. Nasseri, M., Wettstein, F. O., and Stevens, J. G . (1982). J. Virol. 44, 263-268. Norfleet, R. G., and Sampson, C. E. (1978). Am. J. Gastroenterol. 70,383-384. Okagaki, T., Twiggs, L. B., Zachow, K. R., Clark, B. A., Ostrow, R. S., and Faras, A. J. (1983).1nt.J. Gynecol. Pathol. 2, 153-159. Olson, C., Gordon, D. E., Robl, M. G., and Lee, K. P. (1969). Arch. Enoiron. Health 19,

827-837.

Oriel, J. D. (1971a).Br. J. Vener. Dis. 47, 1-8. Oriel, J. D.(1971b).B r . J . Vener. Dis.47, 373-376. Oriel, J. D., and Almeida, J. D. (1970).Br. J. Vener. Dis. 46, 37-42. Orth, G . , Favre, M., and Croissant, 0. (1977).J. Virol. 24, 108-120. Orth, G., Jablonska, S., Favre, M., Croissant, O., Jarzabek-Chorzelska, M., and Rzesa, G. (1978).Proc. Natl. Acad. Sci. U.S.A. 75, 1537-1541. Orth, G., Jablonska, S., Jarzabek-Chorzelska, M., Obalek, S., Rzesa, G., Favre, M., and Croissant, 0. (1979).Cancer Res. 39, 1074-1082. Orth, G., Favre, M., Breitburd, F., Croissant, O., Jablonska, S., Obalek, S., JarzabekChorzelska, M, and Rzesa, G. (1980). Cold Spring Harbor Conf. Cell Prolif. 7,259-

282.

Ostrow, R. S., Bender, M., Niimura, M., Seki, T., Kawashima, M., Pass, F., and Faras, A. J. (1982).Proc. Natl. Acad. Sci. U.S.A.79, 1634-1638. Pater, M. M., and Pater, A. (1985).Virology 145,313-318. Pettersson, U.,Ahola, H., Stenlund, A., and Moreno-Lopez, J. (1987).In “The Papovaviridae: The Papillomaviruses” (N. P. Salzmann and P. Howley, eds.). Plenum, New York, in press. Pfister, H. (1984).Reu. Physiol. Biochem. Pharmacol. 99, 111-181. Pfister, H., and zur Hausen, H. (1978).Int. J. Cancer 21, 161-165.

146

HERBERT PFISTER

Pfister, H., Gassenmaier, A., Nurnberger, F., and Stuttgen, G . (1983a). Cancer Res. 43, 1436- 1441. Pfister, H., Hettich, I., Runne, U., Gissmann, L., and Chilf, G. N. (1983b).J . Virol. 47, 363-366. Pfister, H., Iftner, Th., and Fuchs, P. G. (1985). In “Papillomaviruses; Molecular and Clinical Aspects” (P. M. Howley and T. Broker, eds.), pp. 85-100. Alan R. Liss, New York. Pfister, H., Krubke, J., Dietrich, W., Iftner, Th., and Fuchs, P. G. (1986). In “Papillomaviruses” (D. Evered and S. Clark, eds.), pp. 3-22. Wiley, Chichester. Pilacinski, W. P., Glassman, D. L., Krzyzek, R. A., Sadowski, P. L., and Robbins, A. K. (1984). BiolTechnology 1, 356-360. Porreco, R. Penn, I., Droegemueller, W., Greer, B., and Makowski, E. (1975). Obstet. Gynecol. 45,359-364. Prakash, S. S., Reeves, W. C., Sisson, G . R., Brenes, M., Godoy, J., Bacchetti, S., De Britton, R. C., and Rawls, W. E. (1985). Znt.J. Cancer 35,51-57. Purola, E., and Savia, E. (1977). Acta Cytol. 21, 26-31. Rando, R. F., Groff, D. E., Chirikjian, J. G., and Lancaster, W. D. (1986).J . Virol. 57, 353-356. Reid, R., Laverty, C. R., Coppleson, M., Isarangkul, W., and Hills, E. (1980). Obstet. Gynecol. 55,476-483. Reid, R., Stanhope, C. R., Herschman, B. R., Booth, E., Phibbs, G. D., and Smith, J. P. (1982). Cancer 50,377-387. Reid, R., Crum, C. P., Herschman, B. R., Fu, Y. S., Braun, L., Shah, K. V., Agronow, S. J., and Stanhope, C. R. (1984). Cancer 53,943-953. Riou, G., Barrois, M., Tordjman, I., Dutronquay, V., and Orth, G. (1984).C . R . Acad. Sci. Paris 299,575-580. Rotkin, I . D. (1973). Cancer Res. 33, 1353-1367. Rous, P., and Beard, J. W. (1935).J . E x p . Med. 65, 523-548. Rous, P., and Friedewald W. F. (1944).J . E x p . Med. 79, 511-537. Rowson, K. E. K., and Mahy, B. W. J. (1967). Bacteriol. Reo. 31, 110-131. Sarver, N., Rabson, M. S., Yang, A. C., Byrne, J. C., and Howley, P. M. (1984).J.Virol. 52,377-388. Schiller, J. T., Vass, W. C., and Lowy, D. R. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 7880-7884. Schiller, J. T., Vass, W. C., Vousden, K. H., and Lowy, D. R. (1986).J . Virol. 57, 1-6. Schimke, R. (1982). In “Gene Amplification.” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Schneider, A., Kraus, H., Schuhmann, R., and Gissmann, L. (1985). Znt. J . Cancer 35, 443-448. Schneider, V., Kay, S., and Lee, H. M. (1983).Acta Cytol27, 220-224. Scholl, S. M., Kingsley Pillers, E. M., Robinson, R. E., and Farrell, P. J. (1985). Znt. J . Cancer 35,215-218. Schwarz, E., Durst, M., Demankowski, C., Lattermann, O., Zech, R., Wolfsperger, E., Suhai, S., and zur Hausen, H. (1983). E M B O J . 2,2341-2348. Schwarz, E., Freese, U. K., Gissmann, L., Mayer, W., Roggenbuck, B., Stremlau, A., and zur Hausen, H. (1985).Nature (London)314, 111-114. Seedorf, K., Krammer, G., Durst, M., Suhai, S., and Rowekamp, W. G. (1985).Virology 145, 181-185. Serra, A. (1924).Giorn. Ztol. Mal. Venereol. 65, 1808-1814. Shah, K. H., Lewis, M. G., Jenson, A. B., Kurman, R. J.. and Lancaster, W. D. (1980). Lancet 2, 1190.

HUMAN PAPILLOMAVIRUSES AND GENITAL CANCER

147

Sillman, F., Stanek, A., Sedlis., A., Rosenthal, J., Lanks, K. W., Buchhagen, D., Nicastri, A., and Boyce, J . (1984).Obstet. Gynecol. 150, 300-308. Spalholz, B. A., Yang, Y. C., and Howley, P. M. (1985). Cell 42, 183-191. Stenlund, A,, Zabielski, J., Ahola, H., Moreno-Lopez, J., and Pettersson, U. (1985). J. Mol. Biol. 182, 541-554. Stevens, J. G., and Wettstein, F. 0. (1979).J. Virol. 30, 891-898. Syrjanen, K. J. (1979). Arch. Gynecol. 227, 153-161. Syrjanen, K. J. (1983). Obstet. Gynecol. 62,617-624. Syjanen, K. J., and Syrjanen, S. M. (1985). Ann. Clin. Res. 17, 45-56. Syjanen, K. J., D e Villiers, E. M., Saarikoski, S., Castren, O., Vayrynen, M., Mantyjarvi, R., and Parkkinen, S. (1985a). Lancet 1, 510-511. Syrjanen, K. J., Vayrynen, M., Hippelainen, M., CastrCn, O., Saarikoski, S., and Mantyjarvi (1985b). Arch. Geschwiilstforsch. 55, 131-138. Syverton, J. T. (1952).Ann. N.Y. Acad. Sci. 54, 1126-1140. Vonka, V., Kanka, J., Jelinek, J., Subrt, I., Sucharek, A., Havrankova, A., Vachal, M., Hirsch, I., Domorazkova, E., Zavadova, H., Richterova, V., Naprstkova, J., Dvorakova, V., and Svoboda, B. (1984a). Znt. J. Cancer 33,49-60. Vonka, V., Kanka, J., Hirsch, I., Zavadova, H., Krcmar, M., Suchankova, A., Rezakova, D., Broucek, J., Press, M., Domorazkova, E., Svoboda, B., Havrankova, A., and Jelinek, J. (198413). Int. J. Cancer 33, 61-66. Wade, T. R., Kopf, A. W., and Ackennan, A. B. (1978).Cancer 42, 1890-1903. Waelsch, L. (1918).Arch. Dermatol. Syph. 124, 625-646. Wagner, D., Ikenberg, H., Boehm, N., and Gissmann, L. (1984). Obstet. Gynecol. 64, 767-772. Walker, P. G., Singer, A., Dyson, J. L., Shah, K. V., To, A., and Coleman, D. V. (1983). Br. J . Cancer 48,99-101. Waterhouse, J., Muir, C., Shanmugaratnam, K., and Powell, J. (1982). “Cancer Incidence in Five Continents,” Vol. 4. IARC, Lyon. Wettstein, F. O., and Stevens, J. G. (1983). Virology 126, 493-504. Wickenden, C., Steele, A., Malcolm, A. D. B., and Coleman, D. V. (1985).Lancet 1,6567. Winkelstein, W. Jr. (1977). Am. J . Epidemiol. 106, 257-259. Winkler, B., Crum, C. P., Fujii, T., Ferenczy, A., Boon, M., Braun, L., Lancaster, W. D., and Richart, R. M. (1984). Cancer 53, 1081-1087. Woodruff, J. D., and Peterson, W. F. (1958). Am. J. Obstet. Gynecol. 75, 1354-1362. Woodruff, J. D., Braun, L., Cavalieri, R., Gupta, P., Pass, F., and Shah, K. V. (1980). Obstet. Gynecol. 56,727-732. Yang, Y. C., Okayama, H., and Howley, P. M. (1985). Proc. Natl. Acad. Sci. U.S.A.82, 1030- 1034. Yee, C., Krishnan-Hewlett, I., Baker, C. C., Schlegel, R., and Howley, P. M. (1985).Am. J . Pathol. 119,361-366. Yutsudo, M., Shimakage, T., and Hakura, A. (1985). Virology 144, 295-298. Zachow, K. R., Ostrow, R. S., Bender, M., Watts, S., Okagaki, T., Pass, F., and Faras, A. J. (1982). Nature (London) 300, 771-773. Zur Hausen, H. (1975). Biochim. Biophy. Acta 417, 25-53. Zur Hausen, H. (1977).Curr. Top. Microbiol. Zmmunol. 78, 1-30. Zur Hausen, H. (1983). Int. Reo. E x p . Pathol. 25, 307-326.

This Page Intentionally Left Blank

HERPES SIMPLEX TYPE 2 VIRUS AND CERVICAL NEOPLASIA Vladimir Vonka,’Jiii Karika,t and Zdengk RothS

t Department of Gynaecology and Obstetrics, Faculty of Medical Hygiene, Charles University, Prague, Czechoslovakia, $ Department of Statistics, Institute of Hygiene and Epidemiology. Prague, Czechoslovakia

* Department of Experimental Virology, Institute of Sera and Vaccines, Prague. Czechoslovakia.

I. Introduction

Cervical cancer is one of the most common of cancers. On a global scale, it ranks as the second most frequent female cancer, although in most developed countries it occupies fourth or fifth place. Its incidence (age adjusted) per 100,000 per year varies from 4.2 in Israel (Parkin et al., 1984) to 79.0 in Herrera Province, Panama (Reeves et al., 1984). In the past decades there has been a continuous decrease in cervical cancer morbidity. This decrease has most probably been conditioned by a variety of factors, including changing life-styles with, most importantly, better personal hygiene and the introduction of cytological screening programs (Boyes et al., 1977). These programs have been based on the discovery by Papanicolaou that the examination of a smear of cells exfoliated from the cervix can monitor pathological changes in that area. This and the more recently introduced colposcopy have helped in the recognition of cervical intraepithelial neoplastic lesions, viz., dysplasia and carcinoma in situ. It is believed (Richart, 1967) that the natural history of cervical cancer starts with the development of a mild dysplasia, referred to as cervical intraepithelial neoplasia grade I (CIN I), and passes through the stage of moderate and severe dysplasia, referred to as CIN 11, to carcinoma in situ, referred to as CIN 111. To this stage the changes are considered reversible, with the degree of reversion decreasing with increasing severity of the condition. Further progression to invasive carcinoma (INCA) is irreversible. The concept can be schematically depicted as follows: CIN I

eCIN I1 C---- CIN I11

-

INCA

149 ADVANCES IN CANCER RESEARCH, VOL. 48

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

150

V L A D I M ~ RVONKA ET AL.

As a consequence of this reasoning, it is assumed that these different pathological conditions share the same key etiological factor and that they should have similar though not necessarily identical epidemiological characteristics. Cervical neoplasia is generally considered to be caused by a sexually transmitted carcinogen, most probably of an infectious nature. This suspicion is based on the results of epidemiological studies, which have recognized a link between the disease and sexual behavior, The first report on this dates back to the last century (Rigoni-Stern, 1842); however, most of the evidence has been obtained over the past 25 years. Gradually a number of sexual and reproductive-associated factors were identified as risk factors for cervical cancer. These include early age at first intercourse, marriage and pregnancy, marital breakdown, a high number of live births, multiplicity of sexual partners, and venereal disease (Rotkin, 1967,1973; Martin, 1967; Thomas, 1973; Kessler, 1976, 1977). Evidence for the key role of sexual factors in the development of cervical neoplasia has further been supported by the findings of an extremely rare occurrence of cervical neoplasia among nuns (Fraumeni et al., 1969) and in social groups with stable one-partner relationships and exceptional contacts with individuals from other communities (Gardner and Lyon, 1977; Sumithran, 1976). The recognition of the importance of the male factor in the development of the disease (see the review by Skegg et al., 1982) is additional evidence for its infectious nature. To mention only two instances, Kessler (1976) has demonstrated an increased risk for developing cervical cancer in the second wives of husbands whose first spouses suffered from the same disease, and Buckley et al. (1981) have documented that the sexual behavior of husbands of women who developed cervical neoplasia differs from that of husbands of control women. The latter study has made unlikely the hypothesis assuming a noninfectious role for high risk males (Singer et al., 1976). With the increasing number of epidemiological studies on cervical neoplasia, some nonsexual risk factors have also been revealed. Among these are the use of oral contraceptives (Stern et al., 1970; Meisels et al., 1977), cigarette smoking (see the review by Winkelstein et al., 1984), and low consumption of retinoids (Romney et al., 1981) and @carotene (La Vecchia et al., 1984). The recognition of these factors does not argue against the putative infectious origin of cervical neoplasia. Some factors may be covariables of sexual behavior, and others, because of the multifactorial nature of cancer etiology, may function as cocarcinogens or promoters. Since the late 1960s, herpes simplex type 2 virus (HSV-2) has most

HERPES SIMPLEX TYPE

2 AND

CERVICAL NEOPLASIA

151

often been suspected of being the etiological factor of crucial importance in cervical neoplasia. It is the purpose of this article to review the evidence for the involvement of HSV-2 in cervical neoplasia in light of recent prospective studies on this association (Vonka et al., 1984a,b; Adam et al.; 1985). Papillomaviruses will not be discussed, since they are covered in the article by Pfister. II. Criteria for a Causal Relationship between a Particular Virus and a Particular Cancer

Although Koch’s postulates have been fulfilled in the case of viruses involved in malignant diseases in animals, they are not applicable to human cancer. Before reviewing the evidence for the HSV-2 cervical neoplasia association, we believe it would be appropriate to present some methodological considerations concerning the establishment of a causal relation between a horizontally transmitted virus and a cancer, because we shall discuss the topic from this standpoint. Our concept is primarily based on the knowledge gained in studies on oncogenic DNA viruses of animals, on methodology developed in 1950s when the need for epidemiological criteria for establishing a causal relationship between newly isolated viruses and some undifferentiated clinical syndromes was recognized, as well as on the recent critical evaluations of the problem (Evans, 1976; Rawls et al., 1977; Lilienfeld, 1983). It is our belief that only a synthesis of sets of data derived from different scientific disciplines can lead to the identification of a cause. We therefore propose to base the conclusions as to the etiological involvement of a given virus in a given cancer on a collation of evidence derived from different sources. As indicated in Table I, we divide the various elements of proof for causation into the more important direct evidence and the less important indirect evidence, i.e., more or less supportive items of information, and believe that satisfying the direct criteria alone should provide evidence sufficient for proving the involvement of a given virus in the pathogenesis of a given cancer.

111. Nature of Association of HSV-2 with Cervical Neoplasia

A. FINDINGS IN PATIENTS Although the first indication of possible HSV-2 involvement in cervical neoplasia came from cytological findings (Naib et al., 1966), the

152

VLADIM~RVONKA ET AL.

bulk of present evidence has been obtained from seroepidemiological studies. Irrespective of the study design, the population investigated, or the serological techniques used, nearly all of the studies demonstrated a higher prevalence of HSV-2 antibody in patients than in control subjects (see the reviews by Melnick and Adam, 1978; Nahmias and Savanobori, 1978; Rawls et al., 1977; Aurelian, 1983; zur Hausen, 1983). In some of these studies, including our own (Janda et al., 1973), HSV-2 antibody was detected with the same frequency in dysplasia, carcinoma in situ, and invasive carcinoma patients, while in others the prevalence of HSV-2 antibody increased with the severity of the pathological condition. The interpretation of the serological studies as suggestive of a causal relationship of HSV-2 to cervical neoplasia was further supported by similarities of the epidemiological characteristics of the disease and virus spread. Venereal transmission is the usual mode of spread of HSV-2, and the prevalence of infections correlates with sexual activity and promiscuity. Both the prevalence of the disease and HSV-2 infection are also inversely related to socioeconomic status. Although properly executed studies have not been carried out until recently, there also has been some circumstantial evidence that HSV-2 infection preceded the development of cervical cancer (Naib et al., 1969; Thomas and Rawls, 1978; Catalan0 and Johnson, 1971). In addition, the link of HSV-2 to cervical cancer seemed to be specific, since similar association with other cancers was not reported. The association has also been biologically coherent. The propensity of the virus to persist in infected subjects, implying that the virus can reside in the infected cell without killing it, and the possibility that the cervical tissue can be exposed repeatedly or continually to the agent lent further credence to the etiological hypothesis. Moreover, it was demonstrated that the cervix is a common site of HSV-2 infection and that this infection involves the squamocollumnar junction, where cervical neoplasia usually originate (Naib et al., 1973). The resulting euphoria (also shared by the authors of this review) was not fully supported by molecular biological and immunological findings, however. On the basis of experience with animal DNA viruses, it was expected that if HSV-2 was indeed involved, then (1)the tumor cells would contain viral DNA and virus-coded transformation proteins and that (2) antibody to the anticipated virus-specific tumor antigens would be present in the patients’ sera. These expectations were further boosted by the rapidly expanding findings of an association of another herpesvirus, the Epstein-Barr virus (EBV), with two human neoplasms, which seemed to fit the classical model estab-

HERPES SIMPLEX TYPE

2 AND

CERVICAL NEOPLASIA

153

lished with papova- and adenoviruses in animals. Studies on the HSV2 cervical cancer relationship initially furnished a number of findings which appeared to satisfy that concept. Viral-specific DNA and/or RNA were repeatedly reported to be present in tumor biopsies (Frenkel et al., 1972; Jones et al., 1978; Eglin et al., 1981; Maitland et al., 1981; McDougall et al., 1982; Park et al., 1983; Prakash et al., 1985). However, in these reports the positive findings were limited to only a portion of the tumors and were considerably inhomogeneous. They were only exceptionally obtained by Southern blot hybridization, and to our knowledge the specificity of the DNNRNA hybridization has never been confirmed by the Northern blotting technique. Several laboratories failed to find a single case of HSV-2 DNA in cervical cancer (zur Hausen et al., 1974; Pagano, 1975; Cassai et al., 1981). We also examined for HSV-2 DNA several aneuploid cell lines derived from CIN and INCA lesions in our laboratory; these results were negative (Hirsch et al., unpublished data). As for immunological criteria, a number of papers in the early 1970s reported the presence of viral-specific antigens in tumor cells and the presence of antibody against these antigens or other virus-associated antigens in the patients’ sera. These antibodies were absent from or present at much lower frequency in sera of control subjects (Nahmias et al., 1975; Holinshead et al., 1973; Aurelian et al., 1973; Tarro, 1975; Anzai et al., 1975). With few exceptions, the procedures used for isolating these particular antigens were quite laborious and the reactive substances were not well defined. In most instances, the results have rarely been reproduced outside the laboratories that developed the tests, and the nature of the respective reactions has remained unclear. The greatest amount of systematic work has been done with the AG4 antigen (see the review by Aurelian, 1983). This antigen is extracted from HSV-2-infected cells early after infection. It is an immediate early phosphoprotein of MW 160K, and may be identical to the HSV%induced ribonucleotide reductase (Flanders et al., 1985). In the infected cell it is localized in the cytoplasm and cytoplasmic membrane and can be found in the virus envelope. IgM antibodies reactive with this antigen in the complement-fixation reaction have been reported to be present in sera of cervical cancer patients, to disappear in successfully treated patients, and to reappear in a recurrence of the disease. The antibody is reportedly absent or only rarely encountered in sera of control subjects. Should these results be confirmed and the respective antigen identified as virus coded, the observed distribution of the antibody and its fluctuation, depending on the clinical state of the disease, would be strong arguments for a causal relationship be-

154

VLADIM~R VONKA ET AL.

tween the virus and clinical cancer. The results were reportedly fully reproducible in the author’s laboratory, but only rarely outside of it. In several other laboratories (including our own), extensive efforts to reproduce these results have failed. The reasons for this discrepancy are poorly understood. They may be associated with the low reactivities of the positive sera or with the low percentage of complement fixation chosen as the threshold of positive reaction. Thus, the efforts to supplement epidemiological evidence favoring a causal association of HSV-2 with cervical neoplasia with clear-cut molecular biological and immunological data have not been particularly rewarding so far.

B. ANIMALEXPERIMENTS Attempts to induce malignant disease in animals by direct administration of the virus have also not been very successful. Experiments with live viruses were generally hampered by high mortality (Rapp and Falk, 1964; Kalter e t al., 1972). A higher frequency of cytologically determined cervical dysplasia has been revealed in intravaginally infected cebus monkeys than control animals, but no cancers were reported (Palmer et al., 1976). Neoplastic changes have been observed in a fraction of infected and honnone-treated mice (Muinoz, 1973),and cervical atypia has also been detected in rats treated simultaneously with HSV-2 and human sperm (Fish et al., 1982). The only report describing development of cervical cancer with high frequency in mice after intravaginal administration of inactivated HSV (Wentz et al., 1975) has remained unconfirmed. Sarcomas in a small percentage of hamsters inoculated in nongenital sites with UV-inactivated HSV have been observed (Nahmias et al., 1970b).The major weaknesses of these studies have been the low incidence of tumors and the lack of convincing evidence of the specificity of these effects. However, the potential of HSV-2 and also of HSV-1 to transform in vitro cultivated cells into cells with malignant properties has been demonstrated. This has provided strong, though indirect, support for the hypothesis that HSV-2 is involved in cervical cancer. Pioneering work in this field has been done by Rapp and his associates (Duff and Rapp, 1971, 1973, 1975). To transform hamster cells in culture, they used UV-irradiated virus. This method made possible partial expression of the viral genome in the infected cells without causing cell destruction, which would follow viral replication. The abortive infection resulted in low-efficiency cell transformation. Cell lines derived from individual colonies either retained the morphology of the origi-

HERPES SIMPLEX TYPE

2 AND

CERVICAL NEOPLASIA

155

nal cells or became epitheloid. A portion of the cells contained HSV antigens demonstrable by immunofluorescence. When inoculated into newborn hamsters, some of these lines were found to be oncogenic. The tumors induced resembled either fibrosarcomas or adenocarcinomas. Most of these animals possessed HSV-neutralizing antibody which further revealed the presence of HSV genetic information and its continuous expression in these cells. These results were soon confirmed in our laboratory (KutinovB et al., 1973), and several other laboratories developed similar systems mimicking Rapp’s model. In addition to hamster cells, rat cells (Macnab, 1974) and mouse cells (Boyd and Orme, 1975) were also transformed by HSV. Gradually, other techniques allowing the transfer of viral genetic information into the cell under conditions preventing viral replication were put to use for transformation purposes. These included the use of photodynamically inactivated HSV (Rapp and Li, 1974), thermosensitive mutants (Kimura et d . , 1975), and transfection with sheared viral DNA (Wilkie et al., 1974). In the transformed cells the presence of viral RNA corresponding to 13%of the genome was demonstrated (Cellard et al., 1973), and viral DNA sequences corresponding to less than onethird of the genome were detected (Frankel et al., 1976). The presence of functional viral products in transformed hamster cells was also revealed by complementation of thermosensitive mutants at nonpermissive temperatures (Benyesh-Melnick et al., 1974). Superficially, these findings were strongly reminiscent of the situation with oncogenic papova- and adenoviruses. However, there were several notable differences. In HSV-transformed cells there was no evidence of a nuclear antigen resembling the T antigens of these viruses, and also attempts to detect virus-coded TSTA failed. It also became apparent that in some cell lines viral DNA sequences were occasionally lost after multiple cell passages; this was associated with the disappearance of viral antigens, but not with the loss of oncogenic potential (Minson et al., 1976; Skinner, 1976; Kutinov6 et al., unpublished data). These observations were initially attributed to the low sensitivity of the techniques available to demonstrate the small amounts of viral genetic material or viral proteins which, however, were still sufficient to maintain the transformed phenotype. With the advent of DNA recombinant technology in vitro, these new techniques were utilized in attempts to solve the problem. Fragments of HSV DNA capable of inducing cell transformation were readily identified, virus transcripts were isolated and hybridized to viral DNA, and also virus-coded proteins were obtained from transformed cells (see the review by Galloway and McDougall, 1983). Several surpris-

156

VLADIM~RVONKA ET AL.

ing observations were derived from these studies. They can be summarized as follows: (1) The transformation regions of the virus genome capable of transforming the cells need not persist in the transformed cells; (2) the transformation regions of HSV-1 and HSV-2 are not colinear; (3) no proteins coded for by these regions have been identified; (4) although some viral antigens were present in most of the cell lines, no protein was consistently expressed in any of them; this raised the possibility that their expression is fortuitous. Evaluation of these results in context with data obtained from humans-notably the discrepancy between epidemiological evidence favoring a causal relationship and molecular biological and immunological findings not supporting it-led to the suggestion of a “hit-andrun” effect (zur Hausen, 1983; Galloway and McDougall, 1983). According to this concept, HSV would act similar to a chemical carcinogen, being responsible for the initiation of the malignant process, but not for the maintenance of the transformed phenotype. This was not just an ad hoc reconciliation hypothesis. A considerable body of evidence for its support had been accumulated. It had been known for a long time that HSV can induce chromosomal aberrations (Hampar and Ellison, 1963; Stich et al., 1964) and that in spite of shutoff of the cell macromolecular synthesis, cell DNA repair synthesis which might be error prone is enhanced in HSV-infected cells (Lorentz et al., 1977; Kucera and Edwards, 1979; Nishiyama and Rapp, 1981). More recently, zur Hausen and his co-workers demonstrated the capability of UV-inactivated HSV to induce cell mutations (Schlehofer and zur Hausen, 1982) and to amplify selectively SV40 genes in SV40transformed cells (Schlehofer et al., 1983), a phenomenon associated with the action of some known physical and chemical carcinogens (Lavi, 1981). Subsequent studies have identified the virus function involved in this process (Matz et al., 1984). Thus, a concept developed which explained the discrepancies among the various findings in humans and in animal systems. If considered seriously, it would have a tremendous impact on the methodology being developed to establish the causal role of the virus in the cancer. Of the three categories of criteria listed in Table I, only the epidemiological one could be applied, and epidemiology alone could provide data confirming that HSV-2 was etiologically involved. IV. Weaknesses of the Seroepidemiological Studies

Although nearly all seroepidemiological studies indicated a markedly higher prevalence of HSV-2 antibody in cervical cancer patients

HERPES SIMPLEX TYPE

2 AND CERVICAL NEOPLASIA

157

TABLE I EVIDENCE NEEDEDFOR ESTABLISHING A CAUSAL RELATIONSHIP BETWEEN HORIZONTALLY AND CANCER SPREAD VIRUSES

A. Direct Epidemiology Patients infected more frequently than matched control subjects; this association should be strong, consistent, specific, and biologically coherent Cancer occurrence and virus spread have the same epidemiological characteristics Infection must precede tumor development Intervention against virus results in suppression of cancer Immunology Immune reactions in patients with viral antigens, especially those expressed in tumor cells Relationship between nature and/or strength of these reactions and patients’ clinical state Molecular biology Regular presence of virus-specific macromolecules in tumor cells

B. Indirect Virus induces tumors in experimental animals Virus transforms cells in oitro Related viruses have been recognized as oncogenic agents under natural conditions Virus induces metabolic changes characteristic for known oncogenic viruses in infected cells Virus tends to persist in infected organism, etc.

than in control subjects, there were several weak points in the results. First, because of the retrospective nature of these studies, evidence was missing that HSV-2 infection preceded the development of the disease. The few studies quoted above in which groups of women were followed prospectively dealt with nonrandom population samples (Naib et al., 1969; Nahmias and Sawanabori, 1978), and in some no attempt was made to match for sex-related attributes (Catalan0 and Johnson, 1971). The second shortcoming concerns the design of the studies. In general, not enough care was given to matching controls with patients. In most of the studies, sexual and other life-style factors known as risk factors for cervical neoplasia were not taken into consideration at all or were given only superficial attention. Thus, only limited evidence was available suggesting that the different distribution of HSV-2 antibody among patients and controls could not be solely accounted for by differences in sexual behavior (Adam et al., 1973). Moreover, sera were usually obtained not immediately after the pathological cervical lesions had been detected, but in patients who had been treated for cancer for prolonged periods of time; consequently, what was being monitored was not the antibody status of the patient at

158

VLADIM~HVONKA ET AL.

diagnosis of disease, but during or after its treatment. Third, the size of the difference in HSV-2 antibody prevalence among patients and controls varied markedly in various studies, and the frequency of antibodies in controls from some geographic areas was higher than the frequency of antibodies in cancer patients elsewhere. There was only one early report which claimed HSV-2 antibody presence in all patients (Royston and Aurelian, 1970). In the other studies, a certain proportion of patients (up to 70%) was reported to be free of the antibody. This could indicate that HSV-2, if indeed involved in the pathogenesis of the disease, was not the etiological agent in all cases of cervical neoplasia (Rawls et al., 1980), or that the tests used were incapable of detecting type-specific antibody sensitively enough and thus led to an underestimation of the actual rate of experience with the agent. The latter possibility is closely related to the difficulty of demonstrating type-specific antibody in HSV infections and deserves special comment. HSV-1 and HSV-2 DNAs display about 50% homology, and the viruses share many antigenic determinants. Hence, it is nearly impossible to demonstrate antibody to one and not to another virus type b y conventional serological techniques. Antibodies to type-common and type-specific antigens are necessarily monitored simultaneously in these tests, and type-specific reactions could easily be overshadowed by the responses to type-common antigens. Moreover, a great majority of subjects are infected with HSV-1 prior to experiencing HSV-2 infection; this further complicates the outcome of the tests and their interpretation (Smith et al., 1972). The conclusion as to the presence or absence of HSV-2 antibody has usually been based on the ratio between HSV-2 and HSV-1 neutralizing antibody titers, expressed as the II/I index (Rawls et al., 1970)or the pN value (Nahmias et al., 1970b). In most of the studies, sera with the II/I ratio exceeding 85 were considered HSV-2 antibody positive. The use of this threshold was substantiated by the observation that subjects shedding HSV2 were usually within this category of reactants. Since HSV-2 infection most frequently follows HSV-1 infection, it is rather likely that the main (though not the only one) underlying immunological event is the preponderant antibody response to the type-common antigens, this antibody response is then reflected by the nearly equal neutralizing efficiency of the sera for viruses of both types. Results of crossabsorption studies suggested that this was really the case. McClung et al. (1976) reduced the chromium-releasing activity of sera for infected cells by absorbing them with heterotypically infected cells, and Snejdarova and Vonka (unpublished data) blocked the antibody-mediated release of 51Crby solubilized extracts from such cells.

HERPES SIMPLEX TYPE

2 AND CERVICAL

NEOPLASIA

159

There are several pitfalls in using the II/I ratio. First, the antibody response after HSV-1 infection may already be predominantly directed against either the type-specific or the type-common antigens, depending on the individual’s sensitivity; the marked variation of the II/I ratio observed in children presumably experiencing only HSV-1 infection reflects this. Furthermore, an antigenic variation among viruses belonging to each virus type exists, and the degree of similarity between the viruses used in the test and those responsible for the natural infection in the respective populations necessarily influences the 114 ratio. Thus, some false positives must be expected. Third, the II/I ratio is a very sensitive variable. Even small variation in the amount of one virus used in the neutralization test may influence one type of antibody titer and consequently the II/I ratio. It is therefore of crucial importance that the sera from the patient and the corresponding control be investigated in parallel, that the same virus stocks be used in successive experiments with the whole collection of sera, and that the same control sera of known specificity be included in each test. Evidence that these principles were followed in all the reported studies is missing. In general, some variation among test conditions is quite common. This made some investigators introduce a “movable” threshold that would be most capable of differentiating between patients’ sera and control sera (Adam et d.,1973). Moreover, knowledge of the fluctuation of antibodies to the type-specific and type-common antigens dependent on time, recurrences, and reinfection is insufficient. Shortcomings of the neutralization test (as well as other conventional serological techniques) were apparent and stimulated efforts to develop new assays that might better distinguish between HSV-1 and HSV-2 body. New, more sophisticated techniques were gradually introduced. They included absorption of type-common and heterotypic antibody by infected cell extracts (Forghani et al., 1975), blocking cross-reactive antigens by heterotypic antibody (Vestergaard et al., 1979), the use of type-specific antigens isolated by electrophoresis (Dreesman et al., 1979),and analysis of radiolabeled viral glycoproteins precipitated by the sera under investigation (Eberle and Courtney, 1981). The subsequent examination of these techniques was rather disappointing, however. They were either not more reliable in discriminating between type 1and type 2 antibody than the conventional tests or, because of their intricacy, were cumbersome and expensive and thus unfit for investigation of large collections of sera. Only recently have tests more adequate to their purpose been developed (SuchBnkovB et al., 1984; Adam et al., 1985; Lee et al., 1985).

160

VLADIM~RVONKA ET AL.

Taken together, a critical evaluation of the results of the case-control studies suggests that they cannot be interpreted unambiguously. Theoretically, several explanations could be offered for the higher prevalence of HSV-2 antibody in patients than in controls (1)HSV-2 is an etiological agent in cervical neoplasia; (2)HSV-2 and cervical neoplasia are two mutually independent covariables of sexual promiscuity; (3)the neoplastic changes or the immunosuppressive effects of the therapy and of psychological stress associated with the disease result in the activation of latent HSV-2 infection, which is reflected in a serological pattern characteristic of past HSV-2 infection; (4) HSV-2 infection is picked up after the disease has developed because of increased susceptibility to the infection due to the neoplastic process, type of therapy, and other events associated with the disease and its treatment. These various explanations are not mutually exclusive. V. Need for a Prospective Study

Problems involved in the interpretation of seroepidemiological studies are not new and in the past several years have repeatedly been stressed by those involved in research on the role of HSV-2 in cervical neoplasia. More than 10 years ago, it became clear that an evaluation of the various explanations offered would be greatly facilitated by a prospective study (Melnick et aZ., 1974).In the course of such a study, large numbers of healthy women with and without HSV-2 antibody would be followed and the findings on the incidence of cervical neoplasia would be correlated with various life-style factors, especially those that are sex-related and those recognized as factors increasing or decreasing the risk of the disease. With the widening discrepancies between the various findings described in the preceding sections and the birth of the hit-and-run hypothesis, such a study became indispensable and, in fact, the only means for evaluating the role of HSV-2 in cervical neoplasia. At the same time, it was becoming ever more apparent that a new test capable of more reliable discrimination between HSV-2 and HSV-1 antibody than the neutralization test had to be developed and utilized in examining the sera from such a study. VI. Prague Prospective Study

During the period from December, 1975 to May, 1983, the authors of this review and their colleagues carried out a prospective study on cervical neoplasia in one Prague district. The next section will summarize the already published data (Vonka et aZ., 1984a,b; Suchdnkov6

HERPES SIMPLEX TYPE

2 AND

CERVICAL NEOPLASIA

161

et al., 1984), include some new results from the study, and present a recently performed additional statistical analysis of the correlations among the key risk factors.

A. AIMS The main scientific aims of the study were to determine the risk of developing cervical neoplasia associated with past HSV-2 infection, and, if such a risk were found, to evaluate the diagnostic and prognostic value of antibodies against various HSV antigens. At the same time, we were aware that such a study would provide an unprecedented possibility of collecting information permitting a reevaluation of the epidemiological characteristics of cervical neoplasia patients free of biases unavoidable in case-control studies, and to utilize this information in constructing a model of women at highest risk of developing the disease. We also hoped to gain some new information on the development and kinetics of the cervical pathological changes that might be helpful toward understanding the natural history of the disease and that could be utilized in its diagnosis, therapy, and prevention as well as in future etiological studies.

B. DESIGN 1 . Enrollment Women aged 25-45 years of age living in one district of Prague, selected at random from the alphabetical register of electors, were invited to participate in the study. A total of 25,000 women received a letter of invitation in which the preventive aims of the undertaking were explained. A total of 10,683 (i.e., 42.6%)accepted the invitation and were enrolled. Two gynecological offices were established for the study, were staffed by gynecologists specially trained in colposcopy, and were used on a full-time basis. The procedure at enrollment included pelvic and a colposcopical examination, collection of smears from the exo- and endocervix for cytological investigations, blood sampling for future antibody tests, and completion of two questionnaires. One questionnaire concerned general and gynecological anamnesis and the other, taken by professional psychologists and specially trained physicians, covered a wide spectrum of information about personal life, including a number of sex-related factors. Full sets of data were obtained for 10,389 women.

162

V L A D I M ~ RVONKA ET AL.

2 . Colposcopy, Cytology, and Histology Colposcopical findings were classified according to our own system (Kafika, 1978), modified to satisfy the Proposals of the International Federation for Cervical Pathology and Colposcopy (Kolstad, 1981). The nomenclature and classification adopted are shown in Table 11. Cytological smears were evaluated according to the Papanicolaou scheme, but class I11 was subdivided into III+ and 111- to differentiate abnormal cells (+) (i.e., uniform cells from superficial and intermediate layers with enlarged nuclei, unchanged chromatin pattern, and low dyskaryotic index) from atypical cells (-) (i.e., cells from parabasal layers, with hyperchromatin nuclei and high dyskaryotic index). All cytological smears were read in one central laboratory. Biopsy specimens for histological examinations were taken by conization. All histological tests were performed in one histological laboratory.

3. Evaluation at Enrollment and Subsequent Follow-up To ensure standard evaluation, the colposcopical and cytological findings were collected on a running basis in the reference Centre for Uterine Cervix Cancer Prevention (CUCCP) at the Faculty of Medical TABLE I1 COLPOSCOPICAL GRADING SYSTEM USED Grade K1 K2

K3a K3b K3c K4

Findings“ 0, present; E, present; SCJ, visible; TZ, pink; GO, simple; VP, normal and uniform 0, present; E, present; SCJ, visible; TZ, slightly aceto- “whittish”; GO, with slight “whittish” halo; VP, normal and uniform; L, slight keratization; INZ, silent, sharp border SCJ, not visible, TZ, aceto-“whittish”; GO, acetowhite halo; VP, abnorma1 but uniform; M, fine; P, fine, small intercapillary distances; L, low SCJ, not visible; TZ, acetowhite; GO, white (L-like) halo; VP, pathological types, not uniform, disorder in branching; M, coarse; P, coarse, large intercapillary distances; L, high-(with red zones) SCJ, not visible; TZ, acetowhite, with red zone areas; GO, as in 3b, glands filled up; M, coarse, several types present; P, as in 3b, several types present; L, high, with red zones Carcinomatous tissue with or without excrescences

0, original epithelium; E, ectopy; SCJ, squamocellular junction; TZ, transformation zone; GO, gland openings; VP, vascular pattern, L, leukoplakia; INZ, iodine-negative zone; M, mosaic; P, punctuation.

HERPES SIMPLEX TYPE

WOMEN ENROLLED

/ \

> -,

NEGATIVE

SLIGHTLY

susPIcIous

2 AND

163

CERVICAL NEOPLASIA

FOLLOW-UP IN 2 YEAR INTERVAL! ~

FOLLOW-UP IN 3-18

SUSPICIOUS or POSITIVE CONE - BIOPSY (329 WOMEN)

FIG. 1. Scheme of the prospective study.

Hygiene, Charles University, headed by one of us (J.K.), where they were all evaluated by a single person (J.K.). On principle, the condition of every woman was evaluated on the basis of both colposcopy and cytology. The further follow-up of the enrolled women is shown in Fig. 1.Women with normal findings were invited for further colposcopical and cytological examination after 2 years and, if normal, once again after another 2-year period. The return in the second and third run was 9142 and 7288 women, respectively. All women with suspicious or positive findings (either at enrollment or in the course of the subsequent follow-ups) were invited to CUCCP where they were reexamined colposcopically, and additional smears were taken for repeat of the Papanicolaou test. Women with findings (after reexamination at the CUCCP) corresponding to CIN I1 or a more serious condition (see Table 111) were examined histologically. Women with suspicious findings were examined at 3- to 18-month intervals. Any worsening of the colposcopical or cytological findings in these subjects was followed by histological examination. Positive histological findings were classified as benign, CIN I (mild dysplasia), CIN I1 (moderate to severe dysplasia), CIN I11 (carcinoma in situ),and INCA (invasive carcinoma, including microinvasion). The forecast reliability of the scheme shown in Table I11 proved high. In 83% of the patients in whom histological examinations were performed, a diagnosis of CIN I1 or a more serious condition was made. The rate of underestimation (risk error) did not exceed 2%. Patients with precancerous and cancerous lesions were divided into three groups, A, B, and C, according to the time of detection of the

164

VLADIM~RVONKA ET AL.

TABLE 111 HISTOLOGICAL FINDINGS BY COLPOSCOPY AND CYTOLOGY colposcopy K3

K1

K2

PAP I

Benign

Benign

PAP I1

Benign

Benign

PAP III+ PAP III-

Benign or CIN I CIN 1-11"

PAP IV or V

INCA"

Cytology

K3a

K3b

K3c

K4

CIN 1-11

CIN 11-111

INCA

CIN 1-11

CIN 11-111

INCA

Benign or CIN I CIN 1-11"

Benign or CIN I Benign or CIN I CIN I

CIN 1-11

INCA

CIN I1

CIN 11-111

INCA"

INCA

INCA

CIN 111 or MICA* CIN I11 or MICA* INCA

INCA INCA

Most probably in cervical canal.

* Microinvasive cancer.

lesions and the nature of the findings at enrollment. Group A comprised subjects with pathological findings at enrollment. Patients with slightly suspicious findings at enrollment who subsequently developed disease were included in group B. Group C included patients with originally normal findings who subsequently developed pathological lesions. 4 . Serological Tests T o determine HSV-2 antibody presence, we used two techniques, the microneutralization test (MNT) and a type-specific solid-phase radioimmunoassay (SPRIA) recently developed in our laboratory (Suc h h k o v i et al., 1984). In MNT, all sera were tested using single stocks of HSV-1 and HSV-2, and in SPRIA, a single antigen lot was used. Sera from patients and matched controls were always examined in parallel. In MNT, the II/I ratio introduced by Rawls et al. (1970) was used as an indicator of HSV-2 antibody presence. For the SPRIA, we employed glycoprotein G of HSV-2 (gG-2), i.e., gC-2 by the older nomenclature. Antigen was isolated by elution with 0.01 M N-acetylD-galactosamine from infected cell lysates applied to Helix pomatia lectin/Sepharose B columns. The reactive antigen was identified as gG-2, and its type specificity was verified in SPRIA and in the radioimmunoprecipitation test using immune rabbit sera and monoclonal antibodies to HSV-2 glycoproteins (kindly supplied by W. E.

HERPES SIMPLEX TYPE

2 AND

Rawls). Serum reactivity was expressed in terms of the calculated from the following formula: RZz =

165

CERVICAL NEOPLASIA RZ2

index

cpm test serum with gC-2 - cpm test serum with control antigen cpm control negative serum with gC-2

An RZ2 value of 22.1 was considered evidence of anti-gG-2 antibody presence. This cutoff point was determined on the basis of testing a large set of sera collected from children possessing HSV-1 antibodies and II/I ratio lower than 85. The mean RZ2 in these sera was 0.86 0.41. Thus, the 2.1 value just exceeded the 2.09 value of the mean plus 3 SDs. Before applying SPRIA with gG-2 to the sera from those in the study, we tried to obtain confirmation of the type specificity of the assay with human sera of different origin. These data, supplemented with some recently obtained findings in patients suffering from recurrent HSV-1 or HSV-2 infection, are summarized in Table IV. These results, especially the nonreactivity of child sera, the high reactivity of sera of patients with genital herpes, and the positive correlation between number of sexual partners and percentage of reactants, made us

*

TABLE IV ANTI-gc-2 ANTIBODYPRESENCE IN HUMAN SERA AS DETERMINED BY SPRIA

Croup

Characteristic

Number

Healthy children

Aged 4-10 years, HSV-1 infected Recurrent herpes genitalis, no virus isolatedb Recurrent herpes, HSV-1 isolated Recurrent herpes, HSV-2 isolated Number of sex partners: 0" Number of sex partners: 1 Number of sex partners: 2-10 Number of sex partners: 11-50

49

Adults Adults Adults Healthy women Healthy women Healthy women Healthy women

(1

b c

16 20 24 4 88

Reactivity with gc-2" (%)

14 (87.5)

0 (-)

19 (79.2) 0 (-)

78

9 (10.2) 21 (26.9)

60

20 (33.3)

All sera examined at 1:10 dilution. No attempts to isolate virus were made. Based on interviews with women at enrollment into the study.

166

VLADIM~RVONKA ET AL.

confident that serum reactivity in SPRIA was a reliable indicator of past HSV-2 infection. At the same time, it became clear that not all HSV-2-infected subjects developed antibody against gG-2.

5. Statistical Evaluation Data were uniformly administered, coded, and committed to a computer in the form of a data base allowing retrieval and transfer of individual records, and their completion, correction, and selection according to the desired parameters. Based on the data collected at enrollment four files were set up: (1)a file of healthy women (Kl/PAP I); (2)an auxiliary file of control women (K2/PAP I, Kl/PAP 11, K2/PAP I) (not used in the present statistical analysis of risk factors); (3) a file of subjects with cytological and colposcopical findings corresponding to CIN I (see Table 111);these subjects were denoted “CIN I,” the quotation marks indicating that (with a few exceptions) no attempts were made to confirm the diagnosis histologically; (4)a file of subjects with histologically confirmed CIN I1 or CIN I11 or INCA lesions; women who developed such a condition in the course of the study were transferred from their original file to this one. All data were analyzed with the help of a contingency table program. In some instances, the different groups of patients were individually compared with the group of healthy subjects. The standard x2 method was used for the evaluation. In the contingency table program, only the association of one factor with the disease could be tested. For analyzing combined effects of more factors, the logistic regression analysis was used (Truett et al., 1967; Walker and Duncan, 1967)

C. RESULTS

1 . Prevalence of Pathological, Cytological, and Colposcopical Findings

A summary of the findings as recorded at enrollment is shown in Fig. 2. All 629 subjects with suspicious or positive findings were sent to the CUCCP for reexamination. After one or more repeated colposcopical and cytological checkups, histological examination was performed in 197 subjects (thus forming group A). In the other women, the findings were not confirmed or the condition markedly improved in the course of subsequent follow-up and treatment.

HERPES SIMPLEX TYPE

2 AND

CERVICAL NEOPLASIA

167

K 2/PAP 1-11

8 215 ( 77.4 % )

K 3b,c-U!PAP 1-11

629

( 6 . 0 %)

K 3(d,b,c)/PAP Ill+-toV 164 (

1.CW

FIG.2. Prevalence of slightly suspicious, suspicious, and positive colposcopical and cytological findings.

2 . Prevalence and lncidence Rates of Precancerous and Cancerous Lesions and Age Distribution of Disease Status In addition to the 197 subjects with pathological findings at enrollment (group A), histological investigation in the course of the study was performed in 68 women with originally slightly suspicious findings (group B) and in 64 women with initially normal findings (group C). The histological results in these 329 patients are summarized in Table V. Pathological findings were obtained in 312 women; of these, 254 were suffering from CIN 11, CIN 111, or INCA. From these data, the prevalence and incidence rates could be calculated. The prevalence rates (per 1000 women) for CIN 11, CIN 111, and INCA were 8.8,5.1,and 1.5, respectively. The incidence rates (per 1000 per year) were 1.7, 0.9, and 0.2, respectively. Definitely of greatest interest are the subjects who developed the disease after being classified normal originally. Their subdivision according to colposcopical findings at enrollment is shown in Table VI. For this particular purpose, we supplemented the group C patients with an additional 7 patients whose questionnaires were missing and who were not included in the subsequent analyses. It can be seen that

168

VLADIM~RVONKA ET AL.

TABLE V RESULTSOF HISTOLOGICAL FINDINGS of subjects

CIN I

CIN I1

CIN I11

INCA

Total cases (CIN 11-INCA)

197 68 64 329

25 18 15 58

93 27 30 150

54 17 12 83

15 3 3 21

162 47 45 254

Number Group

A B C Total

in most of the subjects, the initial colposcopical finding was classified as K2, thus indicating an increased risk of developing cervical neoplastic changes ( p < 0.01). Interestingly, however, the development of lesions with time was nearly the same in both the K 1 and K2 groups. The average interval between the first examination and the first warning signals (in 50% by both colposcopy and cytology, in 35%by cytology only, and in 15% by colposcopy only) was 29 months in both groups. These women were thereafter examined at short intervals. The average interval between detection of the first suspicious changes and the decision to perform conization and histological investigation was 9.9 months for the K 1 and 8.2 months for the K2 group. The distribution of disease status within the age groups is indicated in Table VII. Both CIN I1 and CIN I11 were significantly less frequent in the oldest group. INCA was detected less frequently in the youngest than in the other age groups. CIN I1 was most frequent in the 31- to 35-year age group, while CIN I11 peaked in the youngest age group.

TABLE VI DEVELOPMENT OF CIN 11, CIN 111, AND INCA IN ORIGINALLY HEALTHY SUBJECTS~ Histological diagnosis Original findings

Number

CIN I1

CIN 111

INCA

Total

Kl/PAP 1-11 WPAP 1-11 Total

3609 4606 8205

6 25 31

2 16 18

0

8 44 52

3 3

In addition to 45 group C patients, 7 patients were included for whom the questionnaire data were missing and who were not included in other analyses.

HERPES SIMPLEX TYPE

2 AND CERVICAL NEOPLASIA

169

TABLE VII DISEASE STATUSIN DIFFERENT AGECROUPS Condition Age groupn (years)

25-30 31-35 36-40 41-45

Healthy subjectsb

Healthy subjects‘

“CIN I”

CIN I1

CIN 111

INCA

30.3 31.2 37.0 40.1

52.9 51.8 46.6 44.0

13.7 13.6 13.3 14.2

1.9 2.1 1.8 1.0d

1.2 0.9 1.0 0.5”

0.1 0.3 0.4 0.3

Years at enrollment, figures indicate percentage distribution within the group.

* KIP1 at enrollment. d

Ancillary group of healthy subjects (K1 PAP 11, K2 PAP I, K2 PAP 11). Significantly lower ( p < 0.001). Significantly lower (P < 0.05).

3. Analysis of Answers Concerning Sexual Behavior In the past, objections were repeatedly raised that the replies concerning various aspects of sexual behavior were biased by the greater willingness of patients to be honest because of feelings of guilt and self-examination and possibly also because of the belief that telling the truth would be helpful in the cure of the disease. It has also been argued that another bias has been subconsciously introduced by the interviewer recording the data in case-control studies. Prior to determining the risks associated with promiscuity and age at first intercourse (which had been recognized as key factors in earlier studies), we attempted to estimate the validity of the answers concerning these aspects of sexual behavior. This was done by examining other variables that could or should be related to sexual behavior. To our knowledge, such an analysis had not been done in previous studies. The results are summarized in Figs. 3 and 4. They demonstrate a clear association between sexual behavior and a number of other variables. Probably the most fascinating aspect of these findings is a nearly continuous increase or decrease of the corresponding variable with both the number of sexual partners or age at first intercourse. We therefore concluded that the answers concerning these and probably other aspects of sexual behavior were true, that the bias caused by misreporting was low, and that the answers obtained provided a reliable basis for further analysis.

170

1

VLADIM~R VONKA ET AL.

FIRS19

R

I517

1 d I0

I

A 3 -20

2 -10 -20

1

3 -2,020 2 -10

d 3 -20

2 -10 -20

NO.Of SEXUAL PARTNERS

FIG.3. Association of sexual promiscuity with various factors (HG, herpes genitalis; CHL, childlessness; IR, interruption of pregnancy; FIR 5 19, first interruption at the age of 19 years or less; FI 5 17, first intercourse at the age of 17 years or less; CVD, chronic vaginal discharge; 535, age at enrollment 35 years or less; 836, age at enrollment 36 years or more; GO, gonorrhea; IMP, intercourse in menstrual period; P, using pill as contraceptive; SM > 10, smoking more than 10 cigarettedday). Note: The percentage distribution of the enrolled women in the groups was as follows: 1,30.4;2,21.1; 3, 18.1; 4-10, 27.0; 11-20, 2.3; > 20, 1.1.

4 . Epidemiological Profile of the Cervical Neoplasia Patients Only that portion of the data we consider most relevant to the topic of this review and which might be of interest to those involved in prevention of cervical cancer will be presented. An association of sexual factors with precancerous and cancerous lesions is shown in Table VIII. Of these factors early intercourse was the most consistent risk factor. On the other hand, sexual promiscuity was associated with CIN I1 by all three markers used, but such an association was not found for CIN 111. This has been further confirmed by an additional analysis (see Fig. 5). The risk of developing CIN I1 steadily increased with number of sexual partners, but such an association was not observed for CIN 111. No significant association of any type of cervical neoplasia with clinically manifest genital herpes was observed (1.0-1.4% in the various groups). Gonorrhea was reported twice as frequently in CIN I1

HERPES SIMPLEX TYPE

:[

Vt

80

dl

20

:36

SO 60

2 AND

171

CERVICAL NEOPLASIA

:II ;nkL ID

iM-10

1-2

llMP

30

20

15 -19 -25 -17 -21 -30

i -19 -25 -17 -21 -30

15 -19 -25 -17 -21 -30

15 -I9 -25 -17 -21 -30

-19 -25 17 -21 -30

15 -19 -25 -17 -21 -30

L

L -19 -25

-17 -21 -30

AGE OF 1’~INIERCOURSE IN YEARS

FIG.4. Association of age at first intercourse with various factors (CSE, completed secondary education; CVE, completed university education; D, divorced; NM, never married; AB, rather frequent consumption of alcoholic beverages; > 2 SP, two or more sex partners at any one time; M-2, married twice; the other abbreviations are the same as in Fig. 3. Note: The percentage distribution of the enrolled women in the groups was as follows: 15, 1.1; 16-17, 24.2; 18-19, 48.3; 20-21, 18.5; 22-25, 7.0; 26-30, 0.8.

patients than in healthy control subjects; however, this difference fell short of statistical significance. Also, no significant association with either personal hygiene or with hygiene and intensity of sexual life was found (data not shown; partially presented in Vonka et al., 1984a). Association with reproduction and some other factors is shown in Table IX. It may be of interest that an increased risk of CIN I11 was associated with early menarche, some menstruation problems, early pregnancy, and abortion, while none of these factors was associated with CIN 11. However, smoking represented not only the strongest risk factor, but the only one operative in all four pathological conditions studied. Moreover, the strength of this association increased nearly steadily with the severity of the pathological condition. In addition to variables increasing the risk of cervical neoplasia, several others were identified as decreasing the risk of development of the disease. They are listed in Table X. While most could be determined from previous data and their confounding nature seems appar-

172

VLADIM~RVONKA ET AL.

FIG.5. Association ofCIN I1 and CIN 111 with number of sexual partners. (A)Women with indicated number of partners compared with women with one partner only. (B) Women with indicated number of partners compared with women with one or two partners. Open columns: CIN 11. Dotted columns: CIN 111. Reprinted with the permission of International Journal of Cancer (Vonka et al., 1984a).

ent, diathermoelectrocoagulation of the ectopic epithelium and transformation zone (DKG) was clearly identified as the strongest and most consistent protective factor. Smoking is generally considered to be representative of a cluster of behaviors. As indicated in Fig. 4 and 5, smoking was associated with both sexual promiscuity and early sex. To discover whether smoking was merely a covariable of sexual behavior or an independent risk factor, an analysis of its interdependence with both of these sex-re-

TABLE VIII ASSOCIATIONOF PRECANCEROUS AND CANCEROUS CERVICAL LESIONS WITH SEX-RELATED VARIABLES Controlsn

“CIN I”b

CIN I1

CIN I11

INCA

Variable

(% of 2788)

(% of 1159)

(% of 147)

(% of 80)

(% of 21)

Age at first intercourse 518 years Age at first marriage 517 years Age of husband first marriage 518 years Divorcee Married twice Chronic vaginal discharge Sexual promiscuity Number of sex partners: 1 25 Two sex partners at one time Five or more sex partners on one occasion only

51.1

56.5‘

60.5“

73.7d

66.6

1.4

1.9

2.8

5.P

4.8

0.5

0.4

0

3.8d

0

7.0 9.0 6.4

11.0d 10.7 8.5

13.6“ 13.1 9.5

11.2 8.9 15.0d

4.8 23.g 4.8

32.6 20.2 11.8

25.2d 24.6“ 13.5

25.4 31.3“ 23.0d

31.2 23.7 10.3

14.3 33.3 5.3

1.6

3.2‘

7.6d

2.4

0

Women with K1 PAP I findings at enrollment. “CIN I”: Quotation marks indicate that the diagnosis of CIN I was expected on the basis of colposcopical and/or cytological findings (see Table 111). p < 0.05. d P < 0.01. TABLE IX ASSOCIATIONOF PRECANCEROUS AND CANCEROUS CERVICAL LESIONS WITH REPRODUCTION AND OTHERFACTORS Variable Age at menarche 511 years 215 years Duration of menstruation >7 days 5 2 days Age of first live birth, 517 years Stillbirth 1 2 Primary education onlyC Smoking >10 cigarettedday ~

Controls

“CIN I”

CIN I1

CIN I11

INCA

1.1 5.1

1.2 7.9

1.4 5.4

3.7” 7.5

0 9.5

1.3 0.4 0.5

2.3 0.6 1.o

0.7 0 2.0

6.4b 0 3.7b

0 23.8“ 4.P

7.1 0.5 30.7 15.4

6.6 1.1 33.5 23.6I,

5.4 3.4“ 33.6 31.3b

8.7 0 53.1“ 37.6b

19.0

~~~~~

p < 0.05. * p < 0.01. c Obligatory 9-year primary education.

0

36.8 33.4“

174

VLADIM~R VONKA ET AL.

TABLE X FACTORS ASSOCIATEDWITH DECREASED RISKOF PRECANCEROUS AND CANCEROUS CERVICAL LESIONS Variable

Controls

“CIN I”

CIN I1

CIN I11

INCA

Nulligravity Completed secondary education Born before 1935 Performance of D K G

183 69.3

13.5 66.5

15.6 66.4

7.5 46.9“

9.5 63.2

21.7 24.6

19.1 13.4b

10.2b 6.gb

10.0“ 8.7“

19.0 9.5“

“ p < 0.05. p < 0.01. Electrodiathermocoagulation of ectopic epithelium and transformation zone.

lated factors was performed. For this purpose, CIN I11 and INCA patients were combined in one group. As indicated in Table XI, the association between smoking and early intercourse was very strong in healthy subjects; however, the strength of this association decreased with increasing severity of the disease, and in the CIN 111-INCA group, the association was no longer significant. Also, grouping the patients according to age at first intercourse and smoking indicated that the relative risk (RR) was higher when considering both factors than only one of them (Table XII). This again indicated a degree of mutual independence of the two risk factors. Similar results were obtained when analyzing the relationship between smoking and promiscuity (data not shown).

TABLE XI ASSOCIATIONOF SMOKING ( > l o Cl(;AIIE.T.rES/l~AY) W l Tl l E A l I L Y S E X (518 YEARS) I N HEALTHY A N D IN WOMEN WITH DIFFERENT FORMSOF CERVICAL NEOPLASIA WOMEN Percentage distribution of indicated epidemiological characteristics Group

Number

S+E+‘

S+E-

S-E+

Healthy subjects “CIN I” CIN I1 CIN 111-INCA

2867 1159 147 101

9.6 16.5 23.1 27.7

5.6 7.2 8.2 8.9

41.5 40.0 37.4 44.6

S-E-

43.1 36.3 31.3 18.8

Significance (r2. a )

26.6, < 0.001 25.4, < 0.001 5.01, < 0.05 0.33, > 0.5

S + , Smoking more than 10 cigarettedday; E + , first intercourse at the age of 118 years.

HERPES SIMPLEX TYPE

2 AND

CERVICAL NEOPLASIA

175

TABLE XI1 RELATIVERISK (RR) FOR DEVELOPING DIFFERENT FORMS OF CERVICAL NEOPLASIA ASSOCIATEDWITH SMOKING (>lo CIGARETTE~DAY) AND EARLY SEX(518 YEARS)

RR associated with indicated epidemiological characteristics Group

CIN I CIN I1 CIN I11 INCA

+

S-E+'

S+Epa

S+E+'

(P)

(P)

(PI

1.1 (>0.05) 1.2 (>0.05) 2.5 (<0.001)

1.5 (<0.01) 1.9 (<0.05) 3.5 (<0.005)

2.0 (<0.0001) 3.3 (<0.001) 6.6 (<0.0001)

a Each group compared with the S-Egroup. For abbreviations, see Table XI.

Finally, a logistic regression analysis was used to examine the relationship between smoking and both sex-related factors. We tested the effect of three dichotomized factors, i.e., smoking more than 10 cigarettes per day ( S + ) , first sexual intercourse at the age of 5 1 8 (E+),and the number of sexual partners 24 ( P + ) . Each factor was scored as present or absent. The combinations of the three factors divided the subjects into eight groups. All women enrolled were included in this analysis, only CIN I1 and CIN 111-INCA patients forming separate groups. For each group of subjects, i.e., controls, CIN I1 patients and CIN 111-INCA patients, the absolute and relative frequencies were computed. These data are summarized in Table XIII. In the case of CIN 11, there is a marked increase of frequency with increased number of sexual partners; only when the two other risk factors are present does the P+ factor not increase the frequency. On the other hand, there is a marked association of CIN 111-INCA with early intercourse, while the increased number of sexual partners follows no systematic pattern. Smoking is strongly associated with the increase of frequency in both groups of patients. To study these data further, logistic regression analysis was performed for each group of patients separately. The results for CIN I1 patients are shown in Table XIV. From step A calculations, it can be seen that CIN I1 was highly significantly associated with smoking and significantly with an increased number of sexual partners, but not with early intercourse. The risk calculated from the predicted fre-

176

VLADIM~RVONKA ET AL.

TABLE XI11 ABSOLUTE AND RELATIVE FREQUENCIES OF SELECTED RISKFACTORS IN CONTROL WOMENAND WOMENSUFFERING FROM CIN I1 AND CIN I11 OR INCA CIN I11

Risk factors

S+

+ + + +

E+

+ + + + Total

0

Controls"

+ INCA

CIN I1

Total

P+

Number

%

Number

%

Number

%

(N)

+ + +

3239 947 2824 1419 344 207 587 596 10146

98.54 98.21 97.92 97.26 96.90 95.39 95.14 94.90 97.62

33 13 29 26 5 7 17 17 147

1.00 1.37 1.01 1.78 1.47 3.23 2.75 2.71 1.41

15 4 31 14 6 3 13 15 101

0.46 0.42 1.07 0.96 1.69 1.38 2.11 2.39 0.97

3287 947 2884 1459 355 217 617 628 10394

All but those who suffered from CIN 11, CIN 111, or INCA.

quencies indicates that both S + and P+ factors act almost independently. If both are present, the frequency is less than that for full independency. The results concerning the CIN 111-INCA group of patients are shown in Table XV. Step A calculations indicate that CIN 111-INCA is highly significantly associated with both smoking and early sex, but not with increased number of sexual partners. Risks calculated from the predicted frequencies indicate that both factors act nearly independently. For both risk factors present, the estimated frequency is slightly higher than that for full independency. The independence of the smoking habit implies that it operates through pathways outside the sphere of sexual behavior. 5. Serological Investigations a. HSV-2 Antibody in Sera Taken at Enrollment. The purpose of the serological investigations (and, as already stated, the main objective of the whole study) was to find out whether past infection with HSV-2 was associated with an increased risk of cervical neoplasia. To clarify the point, we examined sera from patients selected from the collection assembled at enrollment. Patients found affected at enrollment (group A, see above) and those who developed the disease in the course of the study (groups B and C) were included. In agreement with epidemiological characteristics, each patient was matched with an average of two controls (range 1-4) according to age (+1 year), age

TABLE XIV LOGISTIC ANALYSISOF SELECTEDRISKFACTORS FOR CIN 11 Computational step"

X2

Constant term

Risk factod'

Regression

Agreement

A1

-4.6232

0.1479

S+ 0.7009 2 0.1840, p < 0.001

P+ 0.3707 0.1707, p < 0.05

E+ 0.1400 2 0.1743, NSC

24.88 (4 df),d p < 0.005

2.58 (4 df),NS

A2

-4.5904 +- 0.1276

S+ 0.8694 -+ 0.2507, p < 0.001

P+ 0.4923 f 0.2058, p < 0.05

( S + ) x (P+) -0.3007 +- 0.3631, NS

24.92 (4 df), p < 0.005

2.54 (4 dfl, NS

B

S+ E+ P+ Frequency (%)

C

4

-

-

-

-

-

1.00

+

1.63

Po

=

ps+

= 1.37%

PP+

=

P ( s + )+ ( P + )

*

+

1.00

-

+

1.63

2.36

+ +

-

+ + 2.85

i-

+

2.36

+ + + 2.85

1.00%

0.64% = 1.87%

ps+/p+ = 1.24% pp+/s+ = 0.50%

a Calculation of logistic regression. In the first step (A,), all three individual factors were included. In the second step (Az), the nonsignificant factor was deleted and the interaction term of both remaining factors was determined. (B) Predicted frequencies for 8 subgroups from A2 analysis. (C) Computed risks. S+,Smoking >10 cigarettedday; P+, 2 4 sexual partners; E + , first intercourse at the age 518 years. Not significant. Four degrees of freedom.

TABLE XV LOGISTIC ANALYSIS OF SELECTEDRISK FACTORS FOR CIN 111-INCAo Computational step

X2 Risk factor

Constant term

AI

-5.3037

* 0.2032

A2

-5.4015

* 0.2299

B

C

S+ E+ P+ Frequency (%)

S+ 0.8993 2 0.2145, p < 0,001 S+ 1.2700 2 0.4072, p < 0.005

2

*

0.2144,

P+ 0.8436 t 0.2744, p < 0.005

*

-

-

-

-

0.45

-

+

+ +

0.45% 1.14% PE+ 0.59% P ( s + ) + ( E + ) = 1.81% Po

=

Ps+

= =

For explanations, see Table XIV.

E+ 0.7115 0.2284, p < 0.005 (S+) x (E+) -0.4821 0.4741, NS

P+

-0.0353 NS

+ 0.45

-

1.04

1.04

+

Regression

Agreement

32.20 (4 df), p < 0.005

1.20 (4 df),NS

31.18 (4 df), p < 0.005

0.34 (4 df), NS

+ +

-

1.58

+ +

1.58 Ps+Ip+

=

PE+,S+

=

1.22% 0.68%

2.25

+ + + 2.25

HERPES SIMPLEX TYPE

2

AND CERVICAL NEOPLASIA

179

TABLE XVI HSV-2 ANTIBODY PRESENCE AS DETERMINED BY MICRONEUTRALIZATION TESTI N SERA AND CONTROLS AT ENROLLMENT TAKENFROM PATIENTS

II/I ratio

CIN I1

Controls

Controls

(% of 147)

(% of 274)

CIN 111 (% of 79)

INCA

used

(% of 21)

(% of 206)

280 290

23.8 8.2

29.2 12.0

27.8 11.4

14.2 9.5

31.6 11.6

of first intercourse (no tolerance), number of sexual partners (1, 2-3, 4-5, ?6), smoking habits, and performance or nonperformance of DKG. Because of the somewhat different epidemiological characteristics of CIN I1 and CIN 111-INCA patients, two control groups were formed. On principle, only those subjects were selected as controls who had been free of any pathological colposcopical and cytological changes not only at enrollment, but throughout the entire observation period. This was done to reduce the possibility that a number of healthy subjects already infected with the putative oncogenic agent would develop the disease in the near future. The essence of the results is summarized in Tables XVI-XVIII and Figs. 6 and 7 . Since there was no significant difference in HSV-2 antibody prevalence among the patient groups A, B, and C, all three groups were combined into one for the sake of simplicity. (More detailed serological data have been presented elsewhere; see Vonka et al., 198413.) From the microneutralization test (MNT) results presented in Table XVI and Figs. 6 and 7 , it is clear that at any level of the II/I ratio there was no marked difference in the prevalence of HSV-2 antibody in patients and control subjects. The same is true for any of the three pathological conditions monitored. The results of examination of the same collection of sera in the type-specific SPRIA are shown in Table XVII. Again, none of the differences attained statistical significance. TABLE XVII HSV-2 ANTIBODY PRESENCE AS DETERMINED BY TYPE-SPECI~~C SPRIA I N SERATAKEN FROM PATIENTS AND CONTROLS AT ENROLLMENT CIN I1

Controls

Controls

(% of 145)

(% of 267)

CIN 111 (% of 18.4)

INCA

RZz

(% of 21)

(% of 205)

22.1

13.1

11.6

18.4

9.5

14.6

180

V L A D I M ~ R VONKA ET AL.

TABLE XVIII HSV-2 ANTIBODYPRESENCE AS DETERMINED BY EITHER THE MICRONEUTRALIZATION TESTOR SPRIA IN SERAFROM PATIENTS AND CONTROLS AT ENROLLMENT" II/I ratio

CIN IIb

Controls

CIN I11

INCA

Controls

275 285 295

39.2 18.9 14.9

38.9 26.2 15.6

40.5 24.1 19.0

19.0 14.3 14.3

42.9 26.9 17.8

A few serum specimens were tested in only one of the tests; they were also included in this table. b Percentage of sera with the indicated II/I ratio, or RZ 2 2.1, or both.

In a further analysis, we combined the results of M N T and SPRIA. There were some good reasons for doing so. The premises for which the two tests monitor past HSV-2 infection differ. Because the postinfection antibody patterns are dependent on the reactivity of the individual, reinfections, recurrences, etc., one would expect that sometimes a past HSV-2 infection might be detected by both tests, but sometimes only by one of them. In this analysis, we deliberately considered sera to be HSV-2 antibody positive at II/I ratios 275, 285,and 295 or at an RZz value 22.1. As indicated in Table XVIII, there was little or no difference between patients and control subjects.

1111 RATIO V i i ; . 6. 1)istril)iition olII/I ratios in sera from CIN I1 patients (0-0) and matched c ~ i r i t r o lstil)jc:cts (0--0). 45 represents sera without detectable antibody to both viriisi:s o r to typc: 2 only. Reprinted with the permission of International Journal of ( , ' w w r (Voiikii i * t d , 19841)).

HERPES SIMPLEX TYPE

2 AND

CERVICAL NEOPLASIA

181

RATIO FIG.7. Distribution of II/I ratios in sera from CIN 111 (0-0) and INCA (A...A) patients and matched control subjects (0-0). Reprinted with the permission of International Journal of Cancer (Vonka et al., 1984b). ll/l

In summary, the testing of sera taken at enrollment failed to provide any evidence of a higher prevalence of HSV-2 antibody in patients than in matched control subjects. b. HSV-2 Antibody in Sera Taken at Diagnosis from Women Who Developed the Disease in the Course of the Study. A majority of the subjects who developed the disease in the course of the study (i,e., groups B and C of patients) had additional blood samples taken at the time of diagnosis, i.e., usually 2 or 4 years after enrollment. A study of these sera yielded the opportunity to ascertain whether the development of the disease was associated with a change of HSV-2 antibody pattern. This information could help explain the high prevalence of HSV-2 antibody in cervical neoplasia patients found in previous casecontrol studies. In both SPRIA and MNT, sera taken at enrollment were examined in parallel with those taken at diagnosis. Sera taken at enrollment with a II/I ratio of less than 85 were considered type 2 antibody negative in MNT, and sera with RZ2 less than 2.1 were considered seronegative in SPRIA. To determine the seroconversion from HSV-2 negativity to positivity, we decided not to use an increase from less than 85 to 285 because such a switch is quite frequently a one- or two-tube phenomenon. As an indicator of seroconversion, we arbitrarily chose an increase in II/I ratio by 210. In SPRIA, similar evidence was an increase of RZz over the 2.1 threshold. The results are summarized in Table XIX. It can be seen that a seroconversion was

182

VLADIM~RVONKA ET AL.

TABLE XIX SEROCONVERSION FROM HSV-2 NECATIVITE TO POSITIVITE IN PATIENTS WHODEVELOPED THE DISEASE IN THE COURSE OF THE STUDY Test

Diagnosis

Number

Seroconversion"

MNT

CIN I1 CIN 111 All

+ INCA

26 20 46

2 (7.7) 2 (10.0) 4 (8.7)

SPRIA

CIN I1 CIN 111 + INCA All

39 24 63

l(2.6) 2 (8.3) 3 (4.8)

a In MNT, increase of II/I ratio by 210; in SPRIA, increase from <2.1 to >2.1.

quite rare by either test, and a great majority of the originally seronegative subjects remained free of detectable HSV-2 antibody at diagnosis. As will be shown elsewhere, the seroconversion rate in these patients did not differ markedly from that of healthy subjects as monitored by a longitudinal follow-up (KrEmAi. et al., 1986). These results make it seem unlikely that the development of the disease can activate latent HSV-2 infection. VII. Houston Prospective Study

The group at the Baylor College of Medicine in Houston has recently reported on a study carried out in women treated with diethylstilbestrol (DES) in utero. In the course of a 7-year observation period, 12 CIN I, 5 CIN 11, and 6 CIN I11 were detected (Adam et al., 1985). The sera taken at enrollment in subjects who developed the disease later on and matched control subjects were tested for the presence of HSV antibodies. Utilizing a combination of two tests ( M N T and SPRIA), the authors failed to demonstrate a difference in rate or level of HSV-2 antibodies between the patient and the control group. They observed a somewhat higher prevalence of HSV-1 antibodies in the patients than in the controls. However, by either test, a large proportion of the sera in each group was free of any detectable HSV antibody. Similar results were obtained when sera taken at the time of diagnosis were examined. Although during the period between the two samplings an increase in HSV-2 antibody was quite frequent, it was again nearly the same in patients (from 9% to 30%)as in the controls (from 9% to 35%). There was no comparable interval-

HERPES SIMPLEX TYPE

2

AND CERVICAL NEOPLASIA

183

related increase in HSV-1 antibody, which suggested that HSV-1 did not play a major role in genital herpes infections in the study group. VIII. General Discussion

Since the shortcomings and contradictions of the molecular biological and immunological evidence for the HSV-2-cervical cancer relationship have been discussed in other sections of this review, we will now comment on the implications of recent prospective studies on the role of HSV-2 in cervical cancer. We shall also make a few comments on future preventative measures which seem to be substantiated by the results of these studies.

A. REASONS FOR THE DISCREPANCY BETWEEN RETROSPECTIVE AND PROSPECTIVE STUDIES Both of the recent prospective studies on two different populations on two different continents disclosed no evidence for a higher prevalence of HSV-2 antibody in cervical neoplasia patients than in the matched controls. Thus, these results do not lend any support to the hypothesis that HSV-2 is involved in the pathogenesis of cervical neoplasia. In fact, they argue against the etiological hypothesis, whichever of the pathogenetic mechanisms, hit-and-stay or hit-and-run, is considered. The results of these prospective studies are at variance with retrospective studies, which nearly without exception were suggestive of a causal link between HSV-2 and cervical neoplasia. It is not easy to explain this discrepancy fully. The key to unraveling the problem may be in the matching procedure. The outcome of epidemiological studies is greatly influenced by the criteria used to select and match patient and control groups. It should be recalled that in many of the older studies on the HSV-2cervical cancer relationship, healthy women were matched with patients by age or by age and race only. Sex-related factors were only rarely used and, in most instances, rather superficially. On the other hand, in both of the prospective studies, sex-related factors were taken into consideration. In addition, these studies, in general, permit a high degree of matching unachievable in any case-control study. The reasons for this are obvious. In these studies, the follow-up interviews are with subjects who are either healthy or unaware of their disease, and thus, the information obtained is free of the biases which are unavoidable in the case-control studies that view their subjects in a reverse temporal sequence. In the Prague study, the reliability of the

184

VLADIM~RVONKA ET AL.

answers concerning sexual behavior was further validated by a demonstration of internal consistency among selected factors. The advantages of the prospective study concerning matching were further strengthened in both undertakings by the socioeconomic and ethnic homogeneity of each of the populations followed, as well as by the choice of the control group, which consisted of women who were without detectable pathological lesions not only at enrollment, but at the end of the observation period. Moreover, in the Prague study, the controls were also matched with patients by smoking habit, which was recognized as an important risk factor, and by the history of DKG, which had apparently very efficiently counteracted the exposure to the oncogenic factor(s).Thus, in the prospective studies, the epidemiological characteristics of patients and control subjects were most probably mutually closer than in the previous case-control studies. It is therefore likely that a significant portion of the evidence linking HSV-2 to cervical neoplasia gained from the case-control studies was associated with improper matching. The conclusion that HSV-2 infection is merely a covariable of sexual promiscuity has also been recently drawn by Rawls et al. (1986)from analyzing the results of casecontrol studies carried out around the world. Another distortive factor involved in some of the older studies could be the use of unsuitable serological techniques. The recent two studies differed from most of the previous undertakings not only by their prospective character, but also by the use of two different techniques of HSV-2 antibody determination, which were employed in parallel. This very probably brought the evidence of past HSV-2 infections to a higher level of precision. Still, we feel that the high prevalence of HSV-2 antibody in patients, as revealed by the case-control studies, cannot be solely accounted for by liberal matching and the inaccuracy of the serological tests used. Even after introducing the appropriate adjustments, at least some of the studies might still prove to display a higher prevalence of HSV-2 antibody in patients than in controls, although the difference might be diminished considerably. One is tempted to conclude that in these instances, HSV-2 infection and/or its manifestation by the antibody pattern characteristic for it follows rather than precedes the development of the disease. How can this occur? This is not clear at this time. Both recent studies failed to provide any evidence that the seroconversion rate during the interval between enrollment and diagnosis of the disease was higher in patients than in controls, thus indicating that the activation of latent infection by the disease, if it ever occurs, is

HERPES SIMPLEX TYPE

2

AND CERVICAL NEOPLASIA

185

not an early event. Other explanations should therefore be considered. The factors which might be suspected include those associated with the treatment of the disease and some specific features of the lifestyle of women suffering from cervical cancer. In spite of the results of the prospective studies, which strongly suggest that the previously found high prevalence of HSV-2 antibody in patients is merely a covariable of sexual behavior and possibly of the treatment of the disease, a certain degree of caution is necessary in interpreting the data. After highlighting the advantages, it is appropriate to mention the limitations of prospective studies. It is an inherent weakness of all such studies that because of frequent and generally very careful and qualified examinations of the enrolled subjects, the frequency of the most serious clinical condition is low. In the Prague study, only 21 cases of INCA were detected, and in the Houston study there was not a single case. This necessarily weakens the validity of any conclusions concerning this particular condition based on data from these two studies. It should also be noted that elderly women were not included in either study. Several previous studies had suggested the bimodality of age-specific incidence rates for both CIN I11 and INCA (Aitken-Swan and Baird, 1966; Ashley, 1966a,b; Kashgarian and Dunn, 1970; Hakama and Pertinnen, 1980). It is not impossible that the situation for women over 50 or 60 may be different. Finally, one must not neglect the capability of HSV to transform cells and induce cell mutations under certain conditions. Although HSV-2 does not seem to be the major etiological agent in cervical neoplasia, at this moment it is impossible to rule out its involvement in a minority of cases. B. RISKFACTORS IN CERVICAL NEOPLASIA In addition to investigating the role of HSV-2 in cervical neoplasia, our study provided an opportunity to test on a large and homogeneous population the validity of the risk factors cited in previous studies. In spite of the size and homogeneity of the population studied and in spite of avoiding some of the biases associated with case-control studies, our data are possibly only slightly more definitive than those from the other studies. There are two major weak points in our study. First, only 43% of the invited women participated, and of these, only 70% finished the study. This definitely meant a selection of those more interested in health and social problems and more responsive to health information. Thus, the findings are not fully representative of

186

VLADIM~RVONKA ET AL.

the population as a whole. Second, the evaluation of the male factor was not instituted in the design of the study. Most of the risk factors determined in our undertaking fit those revealed in previous epidemiological studies on cervical cancer. The most notable exception was a clear demarcation between CIN 11, on the one hand, and CIN 111, on the other, in regard to the association with sex-related factors. CIN I1 was strongly linked with promiscuity and CIN I11 with early sex. This does not necessarily speak against the single-spectrum diseases insofar as the histologically defined condition is concerned. It seems fully compatible with the findings from the prospective study that CIN I11 (and INCA) commonly passes through the CIN I1 stage, but that only a certain proportion of CIN I1 is predetermined for further progression. The strength of CIN I11 association with early sex would fit this concept. Following the leads provided by the analysis of the key risk factors, we consider it possible that the other species of CIN I1 is associated with venereal infection picked up at a later age, the probability of which grows, of course, with the number of sexual partners. This type of reasoning leads to a tentative conclusion that at least some CIN I1 are etiologically distinct from CIN I11 and INCA. Without respect to the possible inhomogeneity of the condition denoted here as CIN 11, the lack of association of CIN I11 (and INCA) with promiscuity does not provide a valid argument against the infectious etiology of cervical cancer, but may merely indicate that the infection, to be of high risk, has to occur at a young age when the metaplastic epithelium is sensitive to carcinogenic effects (Coppleson, 1970).The problem will be discussed more extensively and in relation to findings concerning papillomaviruses after the evidence collected is fully analyzed. The other gains from the prospective study are the recognition of smoking as a strong risk factor independent of sexual behavior, which supplements the recently collected and analyzed evidence (Winkelstein et al., 1984),and the recognition of the destruction of the ectopic collumnar epithelium and transformation zone as a strong protective factor. This treatment most probably results in curtailing the course of slowly developing metaplastic changes or simply in removing the initiated cells. C. IMPLICATIONS OF THE PROSPECTIVE STUDY FOR OF CERVICAL CANCER

THE

PREVENTION

Although HSV-2 infection was not recognized by the prospective studies as a risk factor in cervical neoplasia, on the basis of their results, it has been possible to construct a model of the female at

HERPES SIMPLEX TYPE (PSTEP~-

4

2 AND

4-

CERVICAL NEOPLASIA

187

2" STEP)

I I

W OB/CVY

L@ I

I

I

I

L------- CVIOLOGICAL

SERVICE

-------.I

DISTRICT O F 200 000

I

- 250 0

D . C . = District Gyn.rologimt

FIG.8. Scheme of the two-step system of uterine cervix prevention.

highest risk of developing cervical neoplasia. Preventive measures should be primarily aimed at such women. This group includes women who have had their first intercourse at an early age, have had multiple sex partners, smoke and have not undergone destruction of the ectopic epithelium and transformation zone by DKG, cryosurgery, or laser treatment. Another by-product of the study is some extension of the knowledge regarding the kinetics of pathological changes in the cervix. This and the experience gained from the organization of the study have helped to improve the system of treatment and prevention of cervical cancer on a nationwide basis. The basic elements of the new system are a two-step approach (Fig. 8) similar to that used in the Prague study (i.e., screening in district gynecological offices, treatment in regional reference centers) and a wide use of DKG or similar therapy for removing any anomaly of the cervix (Kaiika et al., 1985). Another point of interest is the observation of an increased occurrence of CIN I11 in women aged 25-30 years. In the previous studies carried out in Czechoslovakia (Kaiika, 1971) and elsewhere (Ashley, 1966a; Kashgarian and Dunn, 1970), this condition was more frequently observed in older women. The shift should be considered in preventive actions and also in future studies on cervical neoplasia. It may predict an increase in cervical cancer in the future, a tendency already reported in some countries (McGregor and Teper, 1978; Green, 1978).

188

VLADIM~RVONKA ET AL.

IX. Conclusions

The integrated knowledge from molecular biological, immunological, and epidemiological studies does not favor the concept that HSV2 is a major cause of cervical neoplasia. Although the recent epidemiological studies have strengthened the association of the disease with sexual behavior and thus the hypothesis of its infectious origin, they have at the same time identified smoking as a strong risk factor independent of sexual factors. It is likely, but not certain, that the carcinogens and promoters present in cigarette smoke (and similar substances from other sources) function in concert with the infectious agent involved in the pathogenesis of the disease. Accordingly, future etiological studies on cervical neoplasia should be structured in such a way as to permit testing for both multiple and separate involvement of different factors in the origin of this disease. REFERENCES Adam, E., Kaufman, R. H., Melnick, J. L. Levy, A. H., and Rawls, W. E. (1973).Am. J . Epidemiol. 98, 77-78. Adam, E., Kaufman, R. H., Storthz-Adler, K., Melnick, J. L., and Dreesman, G. (1985). Znt. J . Cancer 35, 19-26. Aitken-Swan, J., and Baird, D. (1966). Br. J . Cancer 20,642-659. Anzai, T., Dreesman, G. R., Courtney, R. J., Adam, E., Rawls, W. E., and BenyeshMelnick, M. (1975).J.Natl. Cancer Znst. 45, 1051-1059. Ashley, D. J. B. (1966).J. Obstet. Gynaecol. Br. Commonw. 73,372-381. Ashley, D. J. B. (1966b).J.Obstet. Cynaecol. Br. Commonw. 73, 382-389. Aurelian, L. (1983). In “Viruses Associated with Human Cancer” (L. A. Phillips, ed.), pp. 79-123. Dekker, New York. Aurelian, L., Schumann, B., and Marcus, R. L. (1973). Science 181, 161-164. Benyesh-Melnick, M., Schaffer, P. A., Courtney, R. J., Esparza, J., and Kimura, S. (1974). Cold Spring Harbor Symp. Quant. Biol. 39,731-746. Boyd, A,, and Orme, T. W. (1975). Znt. J . Cancer 16,526-531. Boyes, D. A., Nichols, T. M., Millner, A. M., and Worth, A. J. (1977). Am. J . Obstet. Gynecol. 128,692-693. Buckley, J. D., Harris, R. W. C., Doll, R., Vessey, M. P., and Williams, P. T. (1981). Lancet 2, 1010-1014. Cassai, E., Rotola, A., Meneguzzi, G., Milanesi, G., Garsia, S., Remotti, G., and Rizzi, G. (1981). Eur. J . Cancer 17,685-691. Catalano, L. W., Jr., and Johnson, L. H. (1971).J.Am. Med. Assoc. 217,447-451. Cellard, W., Thornton, H., and Green, M. (1973).Nature (London)New B i d . 243,264266. Coppleson, M. (1970). Znt. J . Cynecol. Obstet. 8,539-550. Dreesman, G. R., Matson, D. O., Courtney, R. J., Adam, E., and Melnick, J. L. (1979). Interuirology 12, 115-119. Duff, R., and Rapp, F. (1971).J . Virol. 8,469-473. Duff, R., and Rapp, F. (1973).J . Virol. 12, 209-217.

HERPES SIMPLEX TYPE

2 AND

CERVICAL NEOPLASIA

189

Duff, R., and Rapp, F. (1975).J. Virol. 15, 490-496. Eberle, R., and Courtney, R. J. (1981). Infect. Immun. 31, 1062-1970. Eglin, R. P., Sharp, F., MacLean, A. B., Macnab, J. C. M., Clements, J. B., and Wilkie, N. M. (1981). Cancer Res. 41,3597-3603. Evans, A. S. (1976). Yale J . Biol. Med. 49, 175-195. Fish, E. N., Tobin, S. M., Cooter, N. B. E., and Papsin, F. R. (1982).Obstet. Gynecol. 59, 220-224. Flanders, R. T., Kucera, L. S., Raben, M., and Ricardo, M. J., Jr. (1985). Virus Res. 2, 245-260. Forghani, B., Schmidt, N. J., and Lennette, E. H. (1975).J.Clin. Microbiol. 2,410-418. Fraumeni, J. F., Lloyd, J. W., Smith, E. M., and Wagoner, J. K. (1969).J.Natl. Cancer Inst. 42,455-468. Frenkel, N., Roizman, B., Cassai, E., and Nahmias, A. (1972). Proc. Natl. Acad. Sci. U.S.A.69, 3784-3789. Frenkel, N., Locker, H., Cox, B., Roizman, B., and Rapp, F. (1976).J. Virol. 18,885-893. Galloway, D. A., and McDougall, J. K. (1983). Nature (London) 302, 21-24. Gardner, J. W., and Lyon, J. L. (1977). Gynecol. Oncol. 5, 68-80. Green, G. H. (1978). Br. J . Obstet. Gynaecol. 85, 881-886. Hakama, M., and Pentinnen, J, (1980). Br. J . Obstet. Gynaecol. 88, 209-214. Hampar, B., and Ellison, S. A. (1963).Proc. Natl. Acad. Sci. U.S.A. 49, 474-480. Hollinshead, A. C., Lee, O., Chretien, P. B., and Rawls, W. (1973). Science 182, 713715. Janda, Z., Kaiika, J., Vonka, V., and Svoboda, B. (1973). Int. J . Cancer 12,626-630. Jones, K. W., Fenoglio, M., Shevchuk-Chaban, M., Maitland, N. J., and McDougall, Y. K. (1978). In “Oncogenesis and Herpesviruses I11 (G. de Th6, W. Henle, and, F. Rapp, eds.), pp. 917-925. IARC, Lyon. Kalter, S. S., Felsberg, P. J., Heberling, R. L., Nahmias, A. J., and Brack, M. (1972). Proc. SOC.Erp. Biol. Med. 139, 964-968. Kaiika, J. (1971). In “Complex Prevention of Uterine Cervical Carcinoma,” pp. 113119. Avicenum, Prague (in Czech). Kaiika, J. (1978). In “Complex Prevention of Uterine Cervical Cancer.” 2nd Ed., pp. 22-41. Avicenum, Prague (in Czech). Kaiika, J., Svoboda, B., Bekovia, A., and HavrAnkovA, A. (1985). Czech. Cynekol. 50, 554-557. Kashgaran, M. F., and Dunn, J. H. (1970). Am. J. Epidemiol. 92, 211-222. Kessler, I. (1976).Cancer Res. 36, 783-791. Kessler, I. (1977).Cancer 39, 1912-1919. Kimura, S., Flannery, V. L., Levy, B., and Schaffer, P. (1975). 1nt.J.Cancer 15,786-798. Kolstad, P. (1981).Proc. Study Group R. College Obstet. Gynaecol., 9th, London. pp. 303-304. KrEmAi, M., SuchinkovA, A., Kaxika, J., and Vonka, V. (1986).Int.1. Cancer 38,161-165. Kucera, L. S., and Edwards, I. (1979).J . Vrol. 29, 83-90. KutinovA, L., Vonka, V., and Broucek, J. (1973).J . Natl. Cancer Inst. 50, 759-766. LaVecchia, C., Franceschi, S., DeCarli, A., Gentile, A., Fasoli, M., Pampallona, T., and Tognoni, G. (1984).Int. J. Cancer 34,319-322. Lavi, S. (1981).Proc. Natl. Acad. Sci. U.S.A.78, 6144-6148. Lee, F. K., Coleman, R. L., Pereira, L., Bailey, P. D., Tatsuno, M., and Nahmias, A. J. (1985).J . Clin. Microbiol. 22, 641-644. Lilienfeld, A. M. (1983)./. Chronic. Dis. 36, 837-843. Lorentz, A. K., Munk, K., and Darai, G. (1977). Virology 82, 401-408.

190

VLADIM~RVONKA ET AL.

McClung, H., Sth, P., and Rawls, W. E. (1976).Am. J. Epidemiol. 104, 192-201. MeDougall, J. K., Cruzin, C. P., Fenoglio, C. M., Goldstein, L. C., and Galloway, D. A. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 3853-3857. MacGregor, J. E., and Teper, S. (1978). Lancet 2,774-776. Macnab, J. C. M. (1974).J. Gen. Virol. 24, 143-153. Maitland, N. J., Kinross, J. H., Luttusil, A., Lutgate, S. M., Smart, G. E., and Jones, K. W. (1981).J . Gen. Virol. 55, 123-137. Martin, C. E. (1967).A m . J. Publ. Health 57,803-814. Matz, B., Schlehofer, J. R., and zur Hausen, H. (1984). Virology 134,328-337. Meisels, A., Begin, R., and Schneider, V. (1977). Cancer 40,3076-3081. Melnick, J. L., and Adam, E. (1978). Prog. Erp. Tumor Res. 21,49-69. Melnick, J. L., Adam, E., and Rawls, W. E. (1974). Cancer 34, 1375-1385. Minson, A. C., Thouless, M. E., Eglin, R. P., and Darby, G . (1976). Znt. J. Cancer 17, 493-500. Munoz, N. (1973). Cancer Res. 33, 1504-1508. Nahmias, A. J., and Sawanabori, S. (1978).Prog. Erp. Tumor Res. 21, 117-139. Nahmias, A. J.. Josey, W. E., Naib, Z. M., Luce, C. F., and Duffey, A. (1970a).Am. J. Epidemiol. 91,539-546. Nahmias, A. J., Naib, Z. M., Josey, W. E., Morphy, F. A., and Luce, C. F. (1970b). Proc. SOC.E x p . Biol. Med. 134, 1065-1069. Nahmias, A. J., Naib, Z. M., Josey, W. E., Franklin, E., and Jenkins, R. (1973). Cancer Res. 33,1491-1497. Nahmias, A. J., Ibrahim, I., and Buono, I. Del. (1975). In “Oncogenesis and Herpesviruses” (G. de ThB, M. A. Epstein, and H. zur Hausen, eds.), pp. 309-313. IARC, Lyon. Naib, Z. M., Nahmias, A. J., and Josey, W. E. (1966). Cancer 19, 1026-1031. Naib, Z. M., Nahmias, A. J., Josey, W. E., and Gamer, J. H. (1969).Cancer Res. 23,940945. Naib, Z. M., Nahmias, A. J., Josey, W. E., and Zaki, M. (1973). Cancer Res. 33, 14521463. Nishiyama, Y., and Rapp, F. (1981). Virology 110,466-475. Pagano, J. S. (1975).J. Infect. Dis. 132, 209-223. Palmer, A. E., London, W. T., Nahmias, A. J., Naib, Z. M., Tunca, J., Fyccillo, D. A., Ellenberg, J. H., and Sever, J. L. (1976). Cancer Res. 36, 807-809. Park, M., Kitchner, H. C., and Macnab, J. C. M. (1983). Eur. Mol. Biol. 0 r g . J .2, 10291034. Parkin, D. M., Stjernsward, J., and Muir, C. S. (1984). Bull. W.H.O.62, 163-182. Prakash, S. S., Reeves, W. C., Sisson, G. R., Brenes, M., Godoy, J., Bacchetti, S., De Britton, R. C., and Rawls, W. E. (1985).Znt.J. Cancer 35,51-57. Rapp, F., and Falke, L. A., (1964). Proc. SOC. Erp. Biol. Med. 116, 361-365. Rapp, F., and Li., J. L. H. (1974). Cold Spring Harbor Symp. Quant. Biol. 39,747-763. Rawls, W. E., Iwamoto, K., Adam, E., and Melnick, J. L. (1970).J.Zmmunol. 104,599606. Rawls, W. E., Bacchetti, S., and Graham, F. L. (1977). Curr. Top. Microbiol. Zmmunol. 77,71-94. Rawls, W. E., Clarke, A., Smith, K. O., Docherty, J. J., Gilkman, S. C., and Graham, S. (1980), Cold Spring Harbor Conf. Cell ProliJ 7, 117-133. Rawls, W. E., Mawett, L. D., and Reeves, W. C. (1986). In “Viral Etiology of Cervical Cancer” (R. Pet0 and H. zur Hausen, eds.), Banbury Report No. 21, pp. 187-198. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

HERPES SIMPLEX TYPE

2

AND CERVICAL NEOPLASIA

191

Reeves, W. C., Brenes, M. M., D e Britton, R. C., Valdes, P. F., and Joplin, C. F. B. (1984). Am. J. Epidemiol. 119, 714-724. Richart, N. M. (1967). Clin. Obstet. Gynecol. 10, 748-784. Rigoni-Stern, D. (1842).Giorn. Seruice Prog. Pathol. Ther. 2, 507-517. Romney, S. L., Palan, P. R., Duttagulpa, C., Wassertheil-Smoller, S., Wylie, J., Miller, G . , Slagle, N. S., and Lucido, D. (1981). Am. J. Obstet. Gynecol. 141, 890-894. Rotkin, L. D. (1967).A m . J. Publ. Health 57, 815-829. Rotkin, L. D. (1973).Cancer Res. 31, 1353-1357. Royston, I., and Aurelian, L. (1970).Am. J . Epidemiol. 91, 531-538. Schlehofer, J. R., and zur Hausen, H. (1982). Virology 122,471-475. Schlehofer, J. R., Gissman, L., Matz B., and zur Hausen, H. (1983).Int.J.Cancer 32,99103. Singer, A., Reid, B. L., and Coppleson, M. (1976).Am.J. Obstet. Gynecol. 126,110-1 15. Skegg, D. C. G., Convin, P. A., Paul, Ch., and Doll, R. (1982). Lancet 2, 581-583. Skinner, G. R. (1976).Br. J. E z p . Pathol. 57, 361-370. Smith, J. W., Adam, E., Melnick, J. L., and Rawls, W. E. (1972).J. Virol. 109, 554-564. Stern, E., Clark, V. A., and Coffelt, C. F. (1970). Science 169, 497-498. Stich, H. F., Hsu, T. C., and Rapp, F. (1964). Virology 22, 439-446. Suchinkovi, A., Hirsch, I., Krfmii, M., and Vonka, V. (1984).J. Infect. Dis. 149, 964972. Sumithran, E. (1976). Cancer 39, 1570-1572. Tarro, G. (1975).In “Oncogenesis and Herpesviruses” (G. de ThC, M. A. Epstein, and zur Hausen, H., eds.), pp. 291-297. IARC, Lyon. Thomas, B. D., and Rawls, W. E. (1978).Cancer 42,2716-2725. Thomas, D. B. (1973). Am. J. Epidemiol. 93, 10-28. Truett, J., Cornfield, J., and Kannel, W. (1967).J. Chronic. Dis. 20, 511-524. Vestergaard, B. F., and Grauballe, P. C. (1979). Acta Pathol. Microbiol. Zmmunol. Scand. 87,261-263. Vonka, V., Karika, J., Jelinek, J., Subrt, I., Suchinek, A., Havrinkovi, A., Vichal, M., Hirsch, I., Domorizkovi, E., Zivadovi, H., Richterovi, V., Niprstkovi, J., Dvoiakova, V., and Svoboda, B. (1984a). Int.1. Cancer 33,4960. Vonka, V., Kaiika, J., Hirsch, I., Zivadovli, H., KrEmBi, M., Suchinkovi, A,, fiezifovi, D., Broufek, J., Press, M., Domorizkovi, E., Svoboda, B., Havrinkovi, A., and Jelinek, J. (1984b). Int. J. Cancer 33, 61-66. Walker, S. H., and Duncan, D. B. (1967). Biometrika 54, 167-179. Wentz, W. B., Reagan, J. W., and Heggie, A. D. (1975). Obstet. Gynecol. 46, 117-121. Wilkie, N. M., Clements, J. B., Macnab, J. C. M., and Subak-Sharpe, J. H. (1974). Cold Spring Harbor Symp. Quant. Biol. 39,657-667. Winkelstein, W., Shillitoe, E. J., Brand, R., and Johnson, K. K. (1984).Am. J. Epidemiol. 119, 1-8. Zur Hausen, H. (1982). Lancet 2, 1370-1372. Zur Hausen, H. (1983).Int. Reo. Erp. Pathol. 25,307-326. Zur Hausen, H., Schulte-Holthausen, H., Wolf, H., Dorries, K., and Egger, H. (1974). Int. /. Cancer 13,657-664:

This Page Intentionally Left Blank

TRANSFORMING GENES AND TARGET CELLS OF MURINE SPLEEN FOCUS-FORMING VIRUSES Wolfram Ostertag,' Carol Stocking,' Gregory R. Johnson,t Norbert Kluge,* Regine Kollek,' Thomas Franz,*.' and Norbert Hess* 'Heinrich-Pette-lnstitut fur Experimentelle Virologie and lmmunologie an der Universitat Hamburg, 2000 Hamburg 20, Federal Republic of Germany, and +Walter and Eliza Hall Institute of Medical Research, Cancer Research Unit. Melbourne, Australia

I. Introduction

In this review, we will attempt to outline the factors that determine leukemogenesis induced by acutely transforming retroviruses by interaction with either myeloid stem or progenitor cells of the mammalian system. The acutely transforming retroviruses described here will be limited to the spleen focus-forming viruses defined as those retroviruses that induce a proliferative hematopoietic disease upon intravenous injection of adult animals. We will not discuss the viruses known to interact primarily with lymphoid target cells (e.g., Abelson murine leukemia virus), nor will we review the extensive literature on the interaction of slowly transforming retroviruses with hematopoietic cells, except when relevant to the understanding of the action of spleen focus-forming viruses. The short latency of the disease induced by these acute oncogenic retroviruses implies that they interact directly with their relevant target cells, thus simplifying the analysis of the interactions that lead to leukemogenesis. However, as of yet, such direct cell interactions have not been studied in well-defined systems. This was probably due, in part, to the insufficient knowledge of normal hematopoiesis and the unavailability of selectable oncogenic retrovirus vectors, but was probably also a consequence of the lack of communication and cooperation between competent scientists involved in the study of normal hematopoiesis and those involved in analyzing the relevant retroviruses at the molecular level. Since knowledge of factors that normally determine myeloid cell differentiation and proliferation is nec1 Present address: Department of Genetics, Cambridge University, Cambridge, England.

193 ADVANCES IN CANCER RESEARCH, VOL. 48

Copyright 8 1987 by Academic Press, Inc. All rights of reproduction i n any form reserved.

194

WOLFRAM OSTERTAG

essary to understand retrovirus-cell interaction, this review will first outline normal hematopoiesis. In order to assess the relevance of the study of spleen focus-forming viruses and the retroviral-related protooncogenes to human leukemogenesis of the myeloid cell system, we will try in Section I11 to critically review the accumulating data of known factors which may determine or influence the course of leukemogenesis and/or oncogenesis. In the main section, we will discuss the molecular features of the three known groups of acutely transforming murine viruses: viruses with recombinant env genes, viruses with the mos oncogene, and finally, viruses with the rus oncogene. Data pertaining to the relevant protooncogenes and their products will also be presented. We will try to outline the critical factors of the viral genome or transforming gene, based on structure-function analysis, which determine the oncogenicity of the retroviruses and its target cell specificity. We will then attempt to discuss retrovirus-target cell interaction, an area which on a causal level is still largely unexplored. In the final part, experiments which are still necessary to fully understand the oncogenesis induced by spleen focus-forming viruses in the myeloid system will be outlined. II. Normal Hematopoiesis

Mature hemopoietic cells perform a variety of important functions, yet are relatively short-lived and therefore require constant replacement. This requirement for constant replenishment has resulted in the hemopoietic system being subdivided into three main compartments: the mature end cells, a transit population of progenitor and differentiating cells, and a multipotential stem cell compartment that produces cells for the transit population. Because of the large cell turnover required, there is considerable amplification, particularly within the transit population, in order to produce the required numbers of mature e n d cells. The rapid changes in mature hemopoietic cell numbers observed when particular situations (e.g., changes in oxygen tension, infections) arise, compared to the relatively constant numbers of such cells during steady-state hemopoiesis, suggest constant and complex regulatory systems. The unraveling of the controlling mechanisms involved in hemopoietic regulation has relied heavily upon the development of both in vivo and in vitro clonal assays for cells in the stem and transit (progenitor) cell compartments. The methods for performing these assays have been reviewed extensively (Till and McCulloch, 1980; Metcalf,

195

MURINE SPLEEN FOCUS-FORMING VIRUSES

1984) and will not be covered here. In this review, the properties of murine cells in the stem and progenitor cell compartments and the role the various known murine hemopoietic regulators play in the differentiation and proliferation of these cells will be discussed. A. HEMOPOIETIC STEMCELLS

The stem cell compartment will be defined here as the population of cells capable of producing an entire functioning hemopoietic system which itself can produce further stem cells (Fig. 1). Although it has been shown that stem cells are themselves able to produce further stem cells, controversy continues as to how long such a process may Stem

Cell

CFU-S 1

Pre-T Cell

0 BFU-E

GM-CFC

Eo-CFC

Meg-CFC

Most-CFC

Megakaryocytes

I I \\

0

Eosinophils

0

0

0

Pre-B

A 0

0

TL-CFC

BL-CFC

T Cell

B Cell

an

Macrophages

E r y i, h r o c y t e s

FIG.1. The pattern of differentiation and proliferation in the murine lymphohemopoietic system. Symbols: *, cell type can only be assayed in oiuo; curved arrow denotes that cell type has self-renewal capacity.

196

WOLFRAM OSTERTAG

occur (Wu et al., 1968; Harrison, 1980; Boggs et al., 1982). The fact that single stem cells are able to repopulate the entire hemopoietic system is well established, and it is useful to distinguish between pluripotential stem cells and multipotential stem cells. The former can repopulate both the myeloid (i.e., nonlymphoid) and lymphoid systems, while multipotential cells appear to be restricted to myeloid differentiation. The in vivo and in vitro clonal assays appear at this stage to be able primarily to detect multipotential cells, although there is some evidence that there are cells capable of producing both myeloid and B lymphoid cells (Abramson et al., 1977; Lala and Johnson, 1978; Hodgson and Bradley, 1979; Fialkow and Singer, 1985; Keller et al., 1985). Nevertheless, these assays have demonstrated considerable heterogeneity within the stem cell compartment. This heterogeneity appears to reflect a hierarchy of cells and can be demonstrated following treatment with drugs (Rosendaal et al., 1976; Hodgson and Bradley, 1979), differential in vitro growth of stem cells (Mauch et al., 1980; Bartelmez and Stanley, 1985),different kinetics of growth (Johnson et al., 1982; Magli et al., 1982; Suda et al., 1983a), or by physical separation of cells within the stem cell compartment (Worton et al., 1969a,b; Johnson and Nicola, 1984; Harris et al., 1984, 1985). These latter procedures, relying upon physical differences (Worton et al., 1969a) or differential expression of membrane antigens and glycoproteins (Johnson and Nicola, 1984; Harris et al., 1984, 1985), have resulted in partial purification of murine multipotential cells. Multipotential cells are therefore known to be of intermediate size, having a high cytoplasmic to nuclear ratio and displaying very little cytoplasmic differentiation (Worton et al., 1969a,b; Johnson and Nicola, 1984). Most studies have indicated that cells within the stem cell compartment are noncycling during normal steady-state hemopoiesis (Becker et al., 1965; van Bekkum et al., 1971; Vassort et al., 1973). That the stem cell compartment contains cells capable of multiple differentiation has been demonstrated using either karyotypic or retrovirus markers in vivo (Wu et al., 1967; Dick et al., 1985; Keller et al., 1985; Lemischka et al., 1986) or examination of the differentiated progeny of single cells in vitro (Johnson and Metcalf, 1977, 1979; Suda et al., 1983b). The ability to clone multipotential stem cells in vitro has enabled studies to be directed at the mechanisms controlling stem cell proliferation and differentiation. The regulatory mechanisms controlling these functions in vivo have proved elusive, although most studies would suggest that the controlling factors are local in nature. The fact that hemopoiesis during the normal steady state is restricted to certain

MURINE SPLEEN FOCUS-FORMING VIRUSES

197

tissues has suggested this microenvironmental concept of stem cell regulation. Numerous in vivo studies have documented the presence of both stimulatory and inhibitory substances in hemopoietic tissues which are capable of altering stem cell proliferation (Frindel et al., 1976; Lord et al., 1976, 1977; Frindel and Guigon, 1977). It is not clear whether these “factors” in altering proliferation rate also affect differentiation versus self-renewal. Thus, it has been suggested by Frindel and co-workers (Frindel, 1979; Guigon et al., 1980) that some of these factors may directly induce stem cell commitment to a particular hemopoietic lineage. This view is difficult to support due to the indirect nature of the assays. Additional evidence for the microenvironmental concept of stem cell regulation has been obtained using mutant mice Sl/Sld and W/Wv. Although the former contain cells capable of repopulating the hemopoietic system of lethally irradiated recipients, they cannot themselves act as recipients that display stem cell-initiated spleen colony formation (McCulloch et al., 1965; Russell, 1979). The W N v mice can produce spleen colonies when transplanted with normal syngeneic cells but are unable to act as donors (McCulloch et al., 1964). Additionally, S1/Sldmice have a defect in germ cell migration, and it is believed that the Sl/Sld locus leads to defective local regulation of both germ cells and hemopoietic stem cells (Russell,

1979). The development of methods for the long-term cultivation of multipotential cells has produced further insights into the requirements for stem cell maintenance and differentiation (Dexter et al., 1977; Greenberger et aZ., 1980a-c, 1983a). These long-term marrow cultures appear to require the formation of an adherent cell layer consisting of fibroblasts and epithelial and adipocyte cells, the latter appearing to be the most essential component. The establishment of the adherent layer enables the maintenance and differentiation of multipotential cells over many months. Although granulopoietic differentiation occurs under normal culture conditions, erythroid differentiation can only occur if anemic mouse plasma is added to the culture (Dexter et al., 1981a). Progenitor cells of all myeloid lineages are continuously produced in these cultures, but lymphoid differentiation is minimal (Schrader and Schrader, 1978; Jones-Villeneuve and Phillips, 1980). The duplication of the Sl/Sld and W N ”phenotype can also be demonstrated with these cultures (Dexter and Moore, 1977) as can the presence of stem cell proliferation inhibitors and stimulators (Dexter et al., 1980a,b). These similarities with in vivo studies further suggest the importance of “microenvironmental” regulation of hemopoiesis, but even with its apparent duplication in vitro, the molecular nature of this concept remains to be determined.

198

WOLFRAM OSTERTAG

The development of cloned stromal cell lines may help to elucidate some of these problems, but as yet there are few reports showing that lines developed from long-term bone marrow adherent cells are able to maintain long-term multipotential cell proliferation and differentiation (Spooncer et al., 1986). I n vitro clonal assays for multipotential cells have enabled the recognition of hemopoietic regulatory factors capable of stimulating these cells. In addition, these assays allow direct cell manipulation experiments in which cellular commitment to differentiation can b e studied (Johnson and Metcalf, 1979; Suda et al., 1983a,b). As with the in vivo assays for multipotential cells, the in vitro assays detect a hierarchy of stem cell types. These can be distinguished by physical characteristics (Johnson et al., 1982), proliferative potential, and time of onset of production of differentiated progeny (Suda et al., 1983a). The most mature multipotential cells capable of clonal differentiation in vitro are recognizable at earlier time periods of culture (e.g., after 7 days) and have a limited ability to produce additional multipotential cells (Johnson et al., 1982). By comparison, those multipotential cells that produce clones of differentiated progeny after longer periods of culture, e.g., 14 days, tend to produce more differentiated cells as well as multipotential cells (Humphries et al., 1981; Johnson et al., 1982; Suda et al., 1983a), the secondary multipotential cells being readily detected when primary colony cells are recultured. Due to the common association between erythroid cells and variable numbers of other differentiated progeny, these colonies have been referred to as mixed erythroid colonies. The multipotential cells producing these in vitro colonies have been called Mixed-colony-forming cells (MixCFC). Mixed erythroid colonies occur at a higher frequency than a third type of in vitro multipotential cell colony, referred to as blast cell colonies (Nakahata and Ogawa, 1982b). This latter colony type most probably arises from the most primitive multipotential cell able to b e detected in vitro and is characterized by slow initial proliferation without morphologically recognizable proliferation, followed by increased proliferation and production of mixed erythroid cells and subsequent differentiation (Suda et al., 1983a; Eaves and Eaves, 1984). As might be expected, a higher proportion of cells [colonyforming unit-spleen (CFU-S)] within blast cell colonies is able to generate spleen colonies in lethally irradiated mice than is found in mixed-erythroid multipotential colonies (Vassort et al., 1973). Also, the proportion of blast CFC to mixed-erythroid CFC is higher in spleen than in bone marrow or in hemopoietic tissues immediately after treatment with cytotoxic agents, e.g., 5-fluorouracil (5-FU). This

MURINE SPLEEN FOCUS-FORMING VlHUSES

199

might be expected due to the parent-progeny relationship of these cells and the different extent of hemopoietic differentiation in spleen and bone marrow in normal steady-state animals. Differentiation of multipotential cells is stimulated in vitro by at least three well-defined hemopoietic regulators: granulocyte colonystimulating factor (G-CSF) (Nicola et al., 1983), granulocyte-macrophage colony-stimulating factor (GM-CSF) (Burgess et al., 1977), and Multi-CSF (or IL-3) (Ihle et al., 1982; Cutler et al., 1985) (see Figs. 26). These regulators are able to directly stimulate single multipotential cells. These experiments with purified regulators have been performed with Mix-CFC, but experiments with single blast cell CFC have yet to be performed with purified regulators. Thus, the possibility exists that these latter cells may not respond to the known regulators. However, since blast cell colony formation is dependent upon the same conditioned media (supernatant of WEHI-3B-stimulated T cells) that have been shown to produce Multi-CSF, this molecule would be the prime candidate for having the ability to stimulate blast cell colony formation. More recently, an activity (termed hemopoietin 1)has been purified from a human carcinoma cell line which, although unable to directly stimulate colony formation, is able to stimulate apparently immature cells present in bone marrow (after treatment with 5-FU) to differentiate into cells bearing the macrophage colony-stimulating factor (MCSF) receptor (Bartelmez and Stanley, 1985; Jubinsky and Stanley, 1985). A similar type of assay has been performed by others in which not only are macrophages produced, but also multipotential cells and hemopoietic progenitor cells (Iscove et al., 1985).The activity producing this effect has been termed “factor X” and appears to preferentially stimulate primitive multipotential cells in the post-5-FU bone marrow population. Factor X is a more potent stimulator of these primitive cells than Multi-CSF (from which it is biochemically distinct), and because of the similarity of biological activities of factor X and hemopoietin 1, it seems likely that these two activities may reside in the same molecule. The relationship of hemopoietin 1 to the growth factor interleukin 4,which is capable of stimulating a series of lymphoid, mainly B, cells and, in addition, a subset of mast cells in the presence of multi-CSF (Lee et al., 1986; Mosmann et al., 1986; Noma et al., 1986), is at present uncertain. Hemopoietin 1 or factor X may also be required for maintenance of very immature hematopoietic stem cells capable of repopulating the bone marrow of irradiated mice (CFU-SI, Fig. 1)(Stanley et al., 1986).The role of Multi-CSF may thus be less important in control of hematopoietic stem cell proliferation.

200

WOLFRAM OSTERTAG

Maximum numbers of in vitro multipotential colonies are stimulated by Multi-CSF, whereas GM-CSF and G-CSF are only able to stimulate 30% and 10% of these numbers (Metcalf et al., 1980; Metcalf and Nicola, 1983) (Figs. 2-6). Additionally, GM-CSF and G-CSF, although able to stimulate multipotential cells, are not able to stimulate the continued proliferation of any hemopoietic lineages other than neutrophil and macrophage once lineage commitment has occurred within a multipotential clone. Since the addition of either GM-CSF or G-CSF to cultures maximally stimulated with Multi-CSF does not lead to increased multipotential colony numbers, this suggests that the GM- and G-CSF-responsive multipotential cells are a subset of those stimulated by Multi-CSF. Multipotential clones initiated by GM-CSF and transferred to Multi-CSF express the same patterns of hemopoietic differentiation as those stimulated by Multi-CSF (Johnson, 1981). A similar inability to alter differentiation patterns has been observed with multipotential colonies stimulated with Multi-CSF in the presence of excess concentrations of erythropoietin (Metcalf and Johnson, 1979). In this situation, increased numbers of erythroid cells were produced in each clone without altering the numbers of nonmyeloid cells. Extensive experiments with multipotential colonies derived from fetal liver cells have documented the presence of erythroid differentiation with variable proportions of other hemopoietic lineages (Johnson and Metcalf, 1977, 1979; Johnson, 1981).Additionally, liquid cultures of purified multipotential cells (in which the majority of progenitor cell production was multipotential cell derived) stimulated with Multi-CSF resulted in the ordered appearance of cells of different lineages, but always including erythroid cells (Nicola and Johnson, 1982). This apparent constant commitment to erythroid differentiation by multipotential cells associated with the progressive loss of other hemopoietic lineages suggested a hemopoietic differentiation sequence preprogrammed within the multipotential cell (Johnson, 1981; Nicola and Johnson, 1982). Based upon observations with adult multipotential cells, a stochastic model for hemopoietic commitment and differentiation has also been proposed (Nakahata et al., 1982a). Whether the differences in the two sets of Observations reflect a fetal-adult difference has yet to be determined. In addition, a genetically determined difference in the ability of erythropoietin to stimulate erythroid differentiation may have caused the different results (Johnson et al., 1982). The mouse strains used for the stochastic model require much higher levels of erythropoietin to stimulate erythroid differentiation than the mouse

MURINE SPLEEN FOCUS-FORMING VIRUSES

201

strain used in the preprogrammed model. However, in both models commitment to different hemopoietic lineages appeared to be the result of asymmetrical cell divisions. Both commitment to differentiation and self-renewal by multipotential cells appear to require cell division; however, little is known about what determines whether any given cell division will be selfgenerative or differentiative. From the preceding, it might be suggested that differentiative cell divisions result from asymmetrical cell divisions, whereas self-generative divisions result in symmetrical cell divisions. There is some evidence that the concentration of MultiCSF may be able to alter self-generative versus differentiative division (Johnson, 1983). Multipotential colonies stimulated supramaximally by Multi-CSF do not contain many secondary multipotential cells, whereas those colonies stimulated by submaximal concentrations of Multi-CSF do contain secondary multipotential cells. In both situations the mean numbers of cells contained within the primary colonies were identical (Johnson, 1983). Due to the similar physical kinetic and differentiation properties demonstrated for in uivo and in vitro multipotential cells and the fact that in uitro multipotential colonies contain CFU-S, it is certain that the cells detected in viuo and in vitro represent at least overlapping if not identical populations (Vassort et al., 1973; Nakahata and Ogawa, 198213). Although far more is known about the stimulatory factors required for in uitro proliferation and differentiation, the role of these factors in viuo has yet to be determined. B. HEMOPOIETIC PROGENITOR CELLS Hemopoietic progenitor cells are the immediate progeny of multipotential cells and are primarily characterized by being committed to one or at most two hemopoietic lineages. Additionally, progenitor cells can be distinguished from multipotential cells by their limited proliferative potential (reculture of colony cells derived from progenitor cells does not generally result in secondary colony formation), different size (Worton et al., 1969a,b; Stephenson and Axelrad, 1971) and density characteristics (Haskill et al., 1970; Moore and Williams, 1974), surface antigens (van den Engh et al., 1978; Nicola et al., 1981), and by their relatively high rate of proliferation and hence susceptibility to killing by radiation or cytotoxic drugs in viuo (Lahiri and van Putten, 1969; Iscove et al., 1970). The majority of progenitor cells for the erythroid, eosinophil, and megakaryocyte lineages appear to exist as unipotential cells, although some evidence has been presented to

202

WOLFRAM OSTERTAC

suggest that there may be minor subpopulations of bipotentialhripotential erythroid and megakaryocyte progenitors (McLeod et al., 1980; Johnson, 1981) and neutrophil/macrophage-megakaryocyte progenitors (Nakahata and Ogawa, 1982a).

PROGENITOR CELLS(GM-CFC) C. NEUTROPHIL-MACROPHAGE The best characterized bipotential progenitor, which is also the most frequent, is that which differentiates into neutrophils and macrophages (Fig. 1). This cell has been given the operational name of granulocyte-macrophage colony-forming cell (GM-CFC) due to its ability upon appropriate stimulation to differentiate in vitro and produce colonies containing neutrophils and macrophages. Fractionation studies have shown that GM-CFC are a heterogeneous population with the morphology of blast cells (Nicola et al., 1981). Although it would seem likely that this heterogeneity reflects a hierarchy of GMCFC, it has not been ruled out that some of the differences in proliferative potential observed (colony size ranging from 50 to 20,000 cells) arise stochastically as GM-CFC differentiate from multipotential precursors. To a certain extent proliferative potential (as measured by the number of progeny produced b y a single GM-CFC) of GM-CFC is dependent upon the concentration of stimulus added to cultures (Metcalf, 1970), but generally those colonies that are more responsive to stimulation form smaller colonies, whereas those GM-CFC that form larger colonies require higher concentrations of stimulus (Metcalf and MacDonald, 1975; Metcalf, 1980). This correlation between responsiveness to stimulation and colony size most probably reflects a parent-progeny relationship, with the least responsive GM-CFC being the more ancestral. Although the majority of neutrophils and monocyte-macrophages that differentiate in vitro develop from the bipotential GM-CFC, minor subsets of unipotential neutrophil and macrophage progenitors do exist in hemopoietic tissues (Metcalf, 1980). Whether these unipotential cells arise directly from multipotential cells or are the progeny of GM-CFC has yet to be determined. Differentiation of GM-CFC to neutrophils results, in the latter stages, in the development of nondividing metamyelocytes. The situation in the monocyte-macrophage pathway is different in that under certain circumstances these cells can be activated to exhibit a sustained capacity for proliferation (Lin and Stewart, 1973, 1974). Granulocyte and macrophage progenitor cells can be stimulated to differentiate by at least four distinct CSF, these being Multi-CSF, GM-CSF, G-CSF, and M-CSF (Metcalf, 1982, 1984) (Figs. 2-5). G-

MURINE SPLEEN FOCUS-FORMING VIRUSES

203

CSF appears to stimulate mainly the subset of unipotential G-CFC, whereas the three other stimuli appear to stimulate the same GM, G, and M progenitors. As a result of stimulation by GM-CSF, GM-CFC differentiate to produce approximately equal numbers of neutrophil, neutrophil-macrophage, or macrophage colonies (the actual numbers being in part dependent upon the mouse strain used, implying that there is a genetic component). In contrast, stimulation of GM-CFC by M-CSF results in over 90% of the colonies being composed only of macrophages (Johnson and Burgess, 1978; Metcalf and Burgess, 1982). Clone transfer experiments and micromanipulation of single cells within developing GM clones have indicated that GM-CSF and M-CSF can irreversibly commit the progeny of GM-CFC, respectively, to granulocyte or macrophage production (Johnson and Burgess, 1978; Metcalf and Burgess, 1982). While for some GM-CFC this occurs within 24 hr and one cell division, for the majority of cells the commitment process is slower and requires an incubation period of up to 48 hr and several cell divisions. It has not yet proved possible to determine whether the commitment event occurs due to the committing molecule acting as an instructive or selective molecule.

D. ERYTHROID PROGENITOR CELLS(BFU-E, CFU-E) As with GM-CFC, cells capable of clonal differentiation to erythroid cells exist as heterogeneous populations (Fig. 1). This heterogeneity is evident from the incubation period required for hemoglobinized progeny to appear within a clone and by the number of cell divisions occurring prior to hemoglobinization. Thus, relatively mature erythroid progenitor cells, termed CFU-E (colony-forming uniterythroid), form colonies of hemoglobinized cells (from 8 to 200) after 48 hr of incubation (Stephenson et al., 1971). With increasing periods of incubation (up to 21 days), larger multicentric hemoglobinized colonies appear in cultures, and these colonies are produced by erythroid progenitor cells, termed BFU-E (burst-forming unit-erythroid) (Axelrad et al., 1974; Gregory, 1976). BFU-E are further subdivided according to the day of incubation at which they produce recognizable hemoglobinized progeny (Gregory and Eaves, 1978). Those BFU-E which produce hemoglobinized progeny after longer periods of incubation are thought to be the most primitive, a concept that agrees with the fact that these cells also produce the most progeny (up to 20,000 cells). The different subsets of erythroid progeny can be physically separated according to size (Heath et al., 1976; Johnson and Metcalf, 1978) or surface membrane properties (van den Engh et al., 1978;

204

WOLFRAM OSTERTAG

Nicola et al., 1981).All of the available evidence would suggest that the most primitive BFU-E are the immediate progeny of multipotential cells and that the most mature BFU-E give rise to CFU-E. Fractionation studies which have produced highly enriched populations of CFU-E have shown that these cells are basophilic blast cells and their immediate progeny are most probably proerythroblasts (Nicola et al., 1981). Manipulation of the circulating levels of erythrocytes and subsequent studies of BFU-E and CFU-E numbers in spleen and bone marrow suggested that these two populations of cells were differentially regulated (Iscove, 1977). Subsequent in vitro studies have confirmed this. The most mature erythroid progenitors (CFU-E) are maximally stimulated by erythropoietin, whereas this molecule has no effect upon primitive BFU-E differentiation, with minor effects on mature BFU-E. Insulin-like growth factor I (IGF I) can replace erythropoietin for maximum stimulation of CFU-E cells (Kurtz et al., 1982; 1985). It appears to act on the same cells as erythropoietin and is most likely the erythropoietin-like activity present in many batches of fetal calf serum. In addition, a subset of CFU-E is also able to be stimulated by a nonerythropoietin molecule (Iscove, 1978; Fagg, 1981),and it is unlikely that this is Multi-CSF as supported by some experiments (Li and Johnson, 1985; Hapel et al., 1985).BFU-E proliferation and differentiation is maximally stimulated by Multi-CSF [this molecule has been termed burst-promoting activity (BPA) by others; Iscove, 19781, with smaller subsets of Multi-CSF-responsive BFU-E being also responsive to GM-CSF and G-CSF (Iscove, 1978; Metcalf et al., 1980; Metcalf and Nicola, 1983). This two-tiered model of erythropoietic stimulation observed in vitro agrees with the in vivo data, since in those situations where circulating erythropoietin levels are reduced (e.g., following hypertransfusion), CFU-E numbers decline, whereas BFU-E numbers remain unaffected (Iscove, 1978) (Figs. 1-6). Most experiments performed with committed erythroid progenitor cells suggest that these cells exist in a parent-progeny relationship (this being primitive BFU-E + mature BFU-E + CFU-E) and that the number of cell divisions required for terminal differentiation is relatively fixed. Current estimates of this number are at least 8-10, based on counts of the number of erythroblasts produced in the largest pure erythroid colonies detected (Eaves and Eaves, 1984). In normal bone marrow, the majority of CFU-E are cycling and the proportion of erythroid progenitor cells in cycle decreases progressively with immaturity (Eaves et al., 1979). There is no evidence that the number of

MURINE SPLEEN FOCUS-FORMING VIRUSES

205

division required to produce mature erythroblasts can be altered by erythropoietin or Multi-CSF concentration. Studies of this nature merely stimulate different subsets of erythroid progenitor cells that have different sensitivities to these factors. E. EOSINOPHIL AND MEGAKARYOCYTE PROGENITOR CELLS(Eo-CFC AND Meg-CFC) Colonies containing pure populations of either megakaryocytes (Metcalf et al., 1975) or eosinophils (Johnson and Metcalf, 1980) have been detected in cultures of murine bone marrow cells and provide the main evidence for the existence of committed progenitor cells for these lineages (Fig. 1).As with GM-CFC and BFU-E, Meg-CFC and Eo-CFC seem capable of only limited proliferation, since reculture of colony cells derived from these cells does not produce secondary colonies (Johnson and Metcalf, 1980). Eo-CFC and Meg-CFC can be partially separated from GM-CFC on the basis of size (Metcalf et al., 1975; Johnson and Metcalf, B980), and a recent study has succeeded in separating Meg-CFC from BFU-E and GM-CFC using anti-Qa-m2 monoclonal antisera (Harris et al., 1983). Both Meg-CFC and Eo-CFC are optimally stimulated to differentiate by Multi-CSF, and thus far, no discrete molecules capable of stimulating only megakaryocyte or eosinophil colony formation in vitro have been detected. Purified GM-CSF can stimulate fetal liver EoCFC and a subset of adult bone marrow Eo-CFC (Johnson and Metcalf, 1980; Metcalf, 1982). Although unable to stimulate colony formation directly, a megakaryocyte colony-enhancing activity has been reported which both increases the number of cells and the ploidy of cells in megakaryocyte colonies (Williams et al., 1981,1982). Whether this enhancing activity is similar to a previously reported in vivo thrombopoietic stimulating factor, thrombopoietin (Williams et al., 1979; Levin, 1983), has yet to be reported. Artifically induced changes in circulating levels of platelets (e.g., by hypertransfusion or treatment with antiplatelet antisera) result in changes in megakaryocyte ploidy and numbers of bone marrow megakaryocytes (McDonald et al., 1975; Levin et al., 1982), and it is thought that these immediate in vivo reponses are due to thrombopoietin (Williams et al., 1979; Levin, 1983). Since this putative regulator has no effect as a stimulator of Meg-CFC, it may act only on relatively mature megakaryocytes, and hence the regulation of megakaryocytopoiesis may be two-tiered, as is the case for erythropoiesis. In addition to Multi-CSF, subsets of MegCFC are able to b e stimulated by GM-CSF and G-CSF, although

206

WOLFRAM OSTERTAG

subsequent addition of Multi-CSF is required for complete terminal differentiation (Metcalf et al., 1980; Metcalf and Nicola, 1983). A. similar two-tiered mode of regulation may occur with eosinophil differentiation, as a recent report suggests the presence of a distinct molecule capable of increasing eosinophil numbers but which by itself is unable to stimulate Eo-CFC in uitro (Sanderson et al., 1985).

F. MASTCELLPROGENITOR CELLS(Mast-CFC) In uitro colonies containing pure populations of mast cells have been grown from a number of murine hemopoietic tissues (Nakahata et al., 1982b; Li and Johnson, 1984; Pharr et al., 1984) and strongly suggest the presence of a cell type committed to the mast cell lineage (Fig. 1). Experiments in uivo (Kitamura et al., 1981; Sonoda et al., 1983)and in uitro (Li and Johnson, 1984; Pharr et al., 1984) strongly suggest that committed mast cell progenitors are derived directly from multipotential cells. Unlike cells from other hemopoietic lineages, mast cell precursors (and mast cells themselves) have an unusually high proliferative capacity (Li and Johnson, 1984; Pharr et al., 1984), and this property has probably contributed to the relative ease with which mast cell lines may be produced (Hasthorpe, 1980; Nagao et al., 1981; Razin et al., 1981; Schrader, 1981; Schrader et al., 1981; Tertian et al., 1981; Galli et al., 1982). The growth of mast cell colonies and maintenance of mast cell lines occur in the presence of lectin-stimulated spleen cell conditioned media or media conditioned by the myelomonocytic cell line WEHI-3B. It appears most likely that the Multi-CSF present in these media is responsible for mast cell differentiation and proliferation (Figs. 2-5). Similarly, it appears that MultiCSF concentration may inversely modulate mast cell proliferation or differentiation (Li and Johnson, 1984). More recently, two groups have isolated and characterized a cDNA clone from a mouse helper T cell library. This clone expresses an interleukin, unique from multiCSF, which synergistically stimulates mast cell proliferation in addition to T cell proliferation. It also activates B cells to secrete immunoglobins. Due to its activity in so many biological assays, it has been named interleukin 4 (Lee et al., 1986; Mosmann et al., 1986; Noma et al., 1986) but may possibly be related to, or identical with, the synergistic growth factor, hemopoietin I (see above).

G . HEMOPOIETIC REGULATORY FACTORS Although a large and diverse range of conditioned media, cell types, and chemicals has been reported to affect hemopoiesis in uiuo and in

MURINE SPLEEN FOCUS-FORMING VIRUSES

207

vitro (for review, see Metcidf, 1984), most of these remain ill-defined at both the cellular and molecular level. This is particularly true of studies involving in vivo assays (e.g., for CFU-S) with the exception of erythropoietin. However, in vitro clonal assays have enabled the characterization (both cellular and molecular) of a number of molecules capable of interacting directly with hemopoietic multipotential and committed progenitor cells to stimulate their proliferation and differentiation. These factors have been termed colony-stimulating factors (CSFs). Based on molecular, physicochemical, and biological properties, five separate murine hemopoietic regulators have been identified. These are summarized in Table I along with their synonyms and the colony types each stimulates. Each of these molecules has been purified to homogeneity (Burgess et al., 1977; Miyake et al., 1977; Stanley and Heard, 1977; Ihle et al., 1982; Nicola et al., 1983; Clark-Lewis et al., 1984; Cutler et al., 198ij) and five (multi-CSF, GM-CSF, M-CSF, interleukin 4, and erythropoietin) have been molecularly cloned (Fung et al., 1984; Gough et al., 1984; Kawasaki et al., 1985; LeeHuang, 1984). Several growth factor genes and their respective receptors are clustered together on the same chromosome. In the human, GM-CSF, M-Cs, ,and the putative receptor for M-CSF (fms) are TABLE I PURIFIED MURINE REGULATORS Regulator

Synonym

Hemopoietin 1 Interleukin 4

M ulti-CS F

BSF-1 MCGFIU TCGFII IL-3 BPA PSF HCGF Multi-CSF

GM-CSF

MGI-1

M-CSF G-CSF

CSF-1, MGI-lh4 MGI-2

Erythropoietin

Source

Molecular weight

Urinary bladder carcinomaconditioned medium Activated T cells

20,000

WEHI-3B-conditioned medium WEHI-3B-conditioned medium Con A-stimulated spleen cellconditioned medium WEHI-3B-conditioned medium Pokeweed mitogen-stimulated spleen cell-conditioned medium Mouse lung-conditioned medium L cell-conditioned medium Mouse lung-conditioned medium Human urine

28,000 25,000 24,000

14,000

25,000 23,000

23,000 70,OOC 25,000 70,000

208

WOLFRAM OSTERTAG

all located on chromosome 5q, and their involvement in hematopoietic disorders associated with loss of or deletion of 5q has been inferred (M. F. Roussel et al., 1984; Nienhuis et al., 1985; Le Beau et al., 1986); in the murine genome both GM-CSF and Multi-CSF are closely linked (N. Gough, personal communication). Such close genetic linkage may be an indication of either shared regulatory properties or joint evolutionary descent. All are neuraminic acid-containing glycoproteins and are active at very low concentrations ( 10-11-10-13 M ) (Burgess et al., 1977; Stanley and Heard, 1977; Ihle et al., 1982; Nicola et al., 1983; Clark-Lewis et al., 1984; Cutler et al., 1985).The carbohydrate portion of these molecules does not appear to be necessary for biological action.

H. GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) AND ITS RECEPTOR GM-CSF has been purified to homogeneity from mouse lung conditioned medium (MLCM) and is a glycoprotein of MW 23,000 (Burgess et al., 1977). The loss of biological activity upon treatment with 2mercaptoethanol, without evidence for subunits, suggests that there is at least one internal disulfide bond necessary for biological activity. GM-CSF has been molecularly cloned (Gough et al., 1984) and the GM-CSF gene specifies an mRNA of 1200 nucleotides (Gough et al., 1984, 1985b). The complete predicted sequence for GM-CSF has no significant homology to any other known growth factor or oncogene. Two types of cDNA corresponding to two different mRNAs have been detected in a T lymphocyte cDNA library (Gough et al., 198513). The gene for GM-CSF exists as a single copy on chromosome 11 (Gough et al., 1984) and has recently been sequenced indicating that the two mRNAs are probably produced from different promoter regions with differential splicing (Stanley et al., 1985). GM-CSF has been found in medium conditioned by antigen and lectin-stimulated spleen cells, T lymphocytes, and T lymphocyte hybridomas (Burgess et al., 1980, 1981; Cutler et al., 1985; Kelso and Metcalf, 1985).Although the cell of origin is unknown within individual organs, GM-CSF has been extracted from or produced by almost all mouse organs (Nicola et al., 1979).Although derived from a variety of sources, in all cases each GM-CSF has been found to have similar molecular properties (Nicola et al., 1979; Burgess et al., 1981), which agrees with the hybridization data suggesting a single gene (Gough et al., 1984). Purified GM-CSF has been shown to stimulate colonies composed

MURINE SPLEEN FOCUS-FORMING VIRUSES

BFU-E GM-CFC Ea-CFC Meg-CFC --

209

Mast-CFC

ells

Neutrophils / I ----

Erythrocytes

FIG.2. Cell types stimulated in ljitro by GM-CSF are indicated by black shading. Symbols: *, cell type can only be assayed in uicjo; curved arrow denotes that cell type has self-renewal capacity.

of neutrophils, macrophages, and mixtures of neutrophils and macrophages (Burgess and Metcalf, 1977) as well as a minor population of eosinophil colonies (Johnson apd Metcalf, 1980) (Fig. 2).Additionally, purified GM-CSF can also stimulate a limited number of subdivisions of a subset of multipotential cells, BFU-E, and Meg-CFC (Metcalf et al., 1980; Johnson, 1981). In addition to colony-stimulating activity, GM-CSF has been shown to induce RNA synthesis and protein synthesis in bone marrow cells (Burgess and Metcalf, 1977), functional activation of macrophages, including plasminogen activator secretion (Lin and Gordon, 1979; Hamilton et al., 1980), and phagocytic and cytocidal activities (Handman and Burgess, 1979). Nothing is known about the site of action of GM-CSF in molecular terms. GM-CSF interacts with a cellular membrane receptor protein

2 10

WOLFRAM OSTERTAG

which is found on cells of the granulocyte-macrophage pathway and possibly some other hematopoieic cells. The reasons for specificity of GM-CSF could be on two levels: (1) interaction with its (specific) receptor, or (2) it may specifically interact with another cellular target. Both a high and a low affinity GM-CSF binding site (Walker and Burgess, 1985)have been described. Normal bone marrow cells have predominantly the high affinity receptor, tissue culture lines usually the low affinity receptor. Both types of receptors appear to be functional. The number of GM-CSF receptors is generally low (Walker and Burgess, 1985), but varies widely. It is uncertain whether the receptor/GM-CSF complex interaction occurs at a cytoplasmic or a nuclear site to induce its effects on cell proliferation and differentiation. It also is open to debate whether GM-CSF is only an active signal when interaction with its receptor takes place. Recent work utilizing a retrovirus vector with the GMCSF gene as part of the genome were interpreted so as to predict that the GM-CSF factor could act with a target (receptor?) within the cell (Lang et al., 1985).After a more detailed analysis, our group has been able to show that the first step in GM-CSF induced stimulation is autocrine in nature (i.e., both receptor and factor are expressed by the same cell, and the secreted factor is required for growth stimulation), which is then followed by a second, as yet molecularly undefined, step leading to true autonomy of cell growth (tumorigenicity) (Laker et al., submitted).

I. MACROPHAGECOLONY-STIMULATING FACTOR (M-CSF) AND ITS RECEPTOR M-CSF has been purified from media conditioned by a fibroblast cell line (L cells) and shown to be a glycoprotein of MW 70,000, consisting of two identical subunits of MW 35,000 linked by disulfide bonds (Stanley and Heard, 1977). Individual subunits are biologically inactive, and each consists of an MW 14,000 polypeptide (Das and Stanley, 1982; Stanley et al., 1983). M-CSF has also been purified from mouse yolk sac conditioned media (Johnson and Burgess, 1978) and human urine (Das et al., 1981). The human M-CSF cDNA has been cloned and has coding capacity of MW 26,000. This is much larger than the reported 15 kDa for the deglycosylated subunits of the 66 kDa mouse or 45 kDa human dimeric M-CSF (Kawasaki et al., 1985).The mature protein may be formed by processing at the NHZterminal and the COOH-terminal ends. The putative precursor protein has features reminiscent of many membrane proteins, with a transmembrane hydrophobic domain followed by three positively

MURINE SPLEEN FOCUS-FORMING VIRUSES

211

charged residues located on the cytoplasmic side. Membrane and secretory forms of M-CSF may thus exist. M-CSF stimulates predominantly macrophage colonies in vitro and the proliferation of cells of the mononuclear phagocyte lineage (Stanley, 1979; Metcalf, 1984) (Fig. 3). M-CSF, particularly at high concentrations, can stimulate GM-CFC to produce limited numbers of neutrophils. Antibodies raised against M-CSF neutralize the proliferative effects of M-CSF on colony formation in vitro, but do not cross-react with GM-CSF, G-CSF, or Multi-CSF (Shadduck and Metcalf, 1975; Burgess and Metcalf, 1980). The specificity of action of M-CSF on macrophages and immediate macrophage precursor cells (Byrne et al., 1981) is possibly a consequence of its direct interaction with a specific receptor which is found in different numbers (up to lo4 cell), depending on the degree of macrophage differentiation and only on macrophage and macrophage precursor cells. The receptor for M-CSF has been purified and shown to be a MW 160,000 protein kinase, which autophosphorylates in tyrosine on binding M-CSF (for review, see Stanley and Jubinsky, 1984). Recent data show that the felinefms oncogene is derived from the M-CSF receptor gene. Thefms oncogene product is thus closely related to the M-CSF receptor and binds M-CSF (Sherr et al., 1985; Sacca et al., 1986).This is of great interest with reference to questions of how MCSF may interact with cells. The oncogenic retrovirus of the cat has been characterized by its transforming activity on fibroblasts, but is not noted for macrophage transforming effects (McDonough et al., 1971; Roussell et al., 1984), although an fms retrovirus vector can transform macrophages or their precursors (Wheeler et al., 1986). There is some indirect evidence that links mutations in the human fms locus to macrophage-specific diseases (Sariban et al., 1985) (see Section 111). Specificity of interaction of M-CSF with cells is unlikely to be a property of only the M-CSF molecule, since its receptor appears to alter proliferation of diverse cells directly. If the receptor also induces proliferative effects on macrophages (on binding to M-CSF?), then specificity may be a function of specific synthesis of receptors in macrophage lineage cells. Nothing is known, as of yet, on the direct site of interaction of M-CSF with its receptor or the M-CSFheceptor complex with a subcellular target. J . GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF) AND ITS

RECEPTOR At low concentrations, G-CSF stimulates the formation of exclusively granulocytic colonies. At higher concentrations, it also stimu-

212

WOLFRAM OSTERTAG

Pluripotential

CFU-s

Cell

n

Pre-B Cell

Pre-T Cell

,.., BFU-E

Stem

G M - C F C E o - C F C M e g - C F C Mast-CFC

I t

::

b TL-CFC

BL-CFC

P

1 I 1 L--J

<:

0

I I

Platelets Eosinophils

I

,'

I

L-!

'

I 1

,

I

\ \

I

L---l

L-1

T Cell

B Cell

Macrophages

,! Neutrophils

L-_r Erythrocytes

FIG.3. Cell types stimulated in oitro by M-CSF are indicated by black shading. Symbols: *, cell type can only be assayed in oioo; curved arrow denotes that cell type has self-renewal capacity.

lates the formation of granulocyte-macrophage and macrophage colonies and has a weak action in initiating proliferation in other hemopoietic cell ineages (Metcalf and Nicola, 1983), but is unable to stimulate the full subset of progenitor cells seen with GM-CSF or Multi-CSF (Fig. 4).G-CSF is also unique in its strong differentiationinducing activity on WEHI-3B D+ cells. The only cell type so far identified as producing G-CSF is the macrophage, with increased levels after exposure to endotoxin (Metcalf and Nicola, 1985), although it can be detected in serum and a number of different organs, especially those of animals previously injected with endotoxin (Burgess and Metcalf, 1980; Lotem et al., 1980). GCSF has been purified from endotoxin-induced lung conditioned me-

MURINE SPLEEN FOCUS-FORMING VIRUSES

213

dium and is an MW 25,000 glycoprotein with internal disulfide bonds (Nicola et al., 1983). The core protein MW is 14,000. The human GCSF gene (Nagata et al., 1986; Souza et al., 1986), but not the murine gene, has been molecularly cloned. G-CSF receptor has been found on granulocyte and granulocyte precursor cells (Nicola and Metcalf, 1984). Mouse and human G-CSF receptors appear to be related. This may be the main reason why mouse G-CSF also affects human cells. The number of receptor sites varies within a narrow range (Nicola and Metcalf, 1985).The receptor affinity is similar to that found for the Multi-CSF receptor (see below) and the high affinity GM-CSF receptor (see above) (Nicola and Metca!f, 1984).

BFU-E GM-CFC Eo-CFC --

I

;I

Meg-CFC

Mast-CFC

Neutrophils

L-1

Erythrccytes

FIG.4. Cell types stimulated in oitro by G-CSF are indicated by black shading. Symbols: *, cell type can only be assayed in oioo; curved arrow denotes that cell type has self-renewal capacity.

214

WOLFRAM OSTERTAG

K. MULTIPOTENTIAL COLONY-STIMULATING FACTOR (Multi-CSF) Due to its multiple actions on hemopoietic cells of different lineages, Multi-CSF has acquired a number of synonyms, including interleukin-3 (IL-3) (Ihle et al., 1982), burst-promoting activity (BPA) (Iscove et al., 1982), mast cell growth factor (Yung et al., 1981), P cell stimulating factor (Clark-Lewis et al., 1984), and hemopoietic cell growth factor (HCGF) (Bazill et al., 1983). In cultures of hemopoietic cells, Multi-CSF is able to stimulate the formation of neutrophil and/ or macrophage, eosinophil, erythroid, megakaryocyte, mast cell, and probably natural cytotoxic cell colonies. Multi-CSF can also stimulate the formation of mixed-cell colonies containing all of the above cell types (Johnson and Metcalf, 1977; Metcalf et al., 1986), promote the survival and possibly proliferation of multipotential cells (CFU-S) (Clark-Lewis et al., 1984), and stimulate the production of committed progenitor cells from multipotential precursor cells (Nicola and Johnson, 1982) (Fig. 5). All the factor-dependent immortal cell lines derived from long-term cultures of murine bone marrow have been derived from Multi-CSFstimulated cultures and have been maintained in conditioned media containing Multi-CSF (Moore, 1978; Dexter et al., 1980a,b; Greenberger et al., 1983a,b). Multi-CSF has been obtained from lectin- or antigen-activated T lymphocytes and the murine myeloid leukemic cell line WEHI-3B. Despite the indication that Multi-CSF may be produced in immune reactions, it has proved extremely difficult to detect Multi-CSF in uiuo, and it may be that the action of this CSF is short range and/or that mechanisms exist to prevent accumulation of the molecule in uiuo. Two molecular species with equivalent activities have been purified from lectin-activated spleen cell conditioned medium (Cutler et al., 1985) and the lectin-activated T cell line LB-3 (N. A. Nicola, R. L. Cutler, L. Peterson, M. A. Kelso, and D. Metcalf, unpublished observations). These are both single subunit glycoproteins of MW 18,000 or 23,000. Multi-CSF purified from WEHI-3B-conditioned medium shows a broader distribution of apparent molecular weight from MW 23,000 to 30,000 (Ihle et al., 1982; Clark-Lewis et al., 1984). cDNAs for Multi-CSF from both WEHI-3B and lectin-activated T cells have been cloned and differ by only a single amino acid (Fung et al., 1984; Yokota et aZ., 1984). In normal cells, the gene for Multi-CSF exists as a single copy, and its nucleotide sequence has been determined (Gough et aZ., 1985a; Miyatake et al., 1985)and has no homology with other known growth factors. Cloned and biologically expressed Multi-

2 15

MURINE SPLEEN FOCUS-FORMING VIRUSES

i.1

0

: :

TL-CFC

BL-CFC

1 Cell

B Cell

ells]

IE r y t h r o c y t es] FIG.5. Cell types stimulated in uitro by Multi-CSF are indicated by black shading. Symbols: *, cell type can only be assayed in uiuo; curved arrow denotes that cell type has self-renewal capacity.

CSF has all of the biological properties attributed to the purified protein (Greenberger e t d., 1985; Hapel et d., 1985; Metcalf et d., 1986). Binding studies with purified iodinated multi-CSF indicate that 100-200 copies of the receptor are present on normal bone marrow cells but 700-1300 copies on diverse established cell lines (Palaszynski and Ihle, 1984; Nicola and Metcalf, 1986).Multi-CSF binding has been detected on granulocytes, eosinophils, and monocytic cells, as well as on blast cells, promyelocytes and myelocytes, but not on lymphoid cells.

216

WOLFRAM OSTERTAG

L. ERYTHROPOIETIN (Epo) The liver and the kidney are the primary source of erythropoietin synthesis (Hammond and Winnick, 1974; Sherwood and Goldwasser, 1978; Beru et al., 1986; Bondurant and Koury, 1986; Shoemaker and Mitsock, 1986). It can be produced by adult and fetal liver (Lucarelli et al., 1964; Fried, 1972). The cell type(s) responsible for Epo synthesis remains unknown. Erythropoietin appears to act only on the later stages of the erythroid differentiation lineage both in vitro and in vivo (Fig. 6). Due to the ability of Epo to act across species, this material has been purified from a number of sources (Goldwasser and Kung, 1971; Miyake et al., 1977). Based upon partial amino acid sequence (Sue and Sytowdski, 1983; Yanagawa et al., 1984a,b),human genomic and cDNA clones (Lee-Huang, 1984; Jacobs et al., 1985), as well as murine (McDonald et al., 1986; Shoemaker and Mitsock, 1986), capable of expressing biologically active Epo have recently been produced. The E p o gene appears to exist as a single copy in normal cells and comprises at least five exons and spans about 4 kilobases (kb). Comparison of sequence data has suggested that E p o has no significant homology with any published sequence of other genes (Jacobs et al., 1985).

M. GENERAL COMMENTS ON CSF RECEPTORS Binding studies have been initiated with G-CSF, M-CSF, MultiCSF, and GM-CSF to determine some aspects of the cellular specificity and the mechanisms of CSF action (Nicola and Metcalf, 1984, 1985; Palaszynski and Ihle, 1984; Stanley and Jubinsky, 1984; Walker and Burgess, 1985). With each of the CSFs, the binding to appropriate cells is rapid and dissociation is extremely slow, even when the binding is performed at 0°C. Receptors for each of the CSFs show complete specificity and no other CSF competes directly for binding by another CSF. Autoradiographic analysis using M-CSF and G-CSF has shown that the receptors for these molecules are almost entirely restricted to the macrophage or granulocytic cell lineages, respectively. The M- and G-CSF receptors appear to be present on all cells in the lineage M or G, respectively, with the frequency increasing with differentiation (Byrne et al., 1981; Nicola and Metcalf, 1984). For most of the CSFs, there are relatively small numbers of receptors on responding cells ( 102-103), although some macrophages may contain higher numbers of M-CSF receptors. Although individual CSFs show no direct cross-reactivity with

MURINE SPLEEN FOCUS-FORMING VIRUSES

CFU-SII

BFU-E

GM-CFt

Eo-CFC

217

/

ties-CFC

Pre-T Cell

Pre-B Cell

T Cell

B Cell

Mast-CFC

0 ,z.cI.,

t-I'

- i

Mac r o p h a g e s

Neutrophils

FIG.6. Cell types stimulated in oitro by erythropoietin are indicated by black shading. Symbols: *, cell type can only be assayed in uioo; curved arrow denotes that cell type has self-renewal capacity.

other CSF receptors when binding experiments are performed at WC, experiments performed at 37°C and preincubation experiments have revealed a hierarchical ability of CSF receptor complexes to downmodulate other unoccupied CSF receptors on murine bone marrow cells (Walker et al., 1985). Thus, Multi-CSF down-modulates GMCSF and M-CSF receptors over the same concentration range at which it occupies its own receptor and at somewhat higher concentrations down-modulates G-CSF receptors. GM-CSF down-modulates M-CSF receptors at low concentrations and G-CSF receptors at high concentrations. At high concentrations G-CSF down-modulates MCSF receptors and M-CSF down-modulates GM-CSF receptors.

218

WOLFRAM OSTERTAG

This pattern of down-modulation suggests that the spectrum of biological activities displayed on hemopoietic cells by CSFs may be dependent upon their ability to down-modulate or occupy lineagespecific receptors. This scheme may explain how different CSF concentrations (e.g., GM-CSF) produce different types of differentiated cells. Thus, at low concentrations of GM-CSF mainly macrophage colonies are stimulated, while at higher concentrations neutrophil colonies are also produced (Burgess and Metcalf, 1977), and this is matched by the concentration dependence of the ability of GM-CSF to down-modulate M-CSF or G-CSF receptors, respectively. The model makes specific predictions, including the existence of lineagespecific factors and receptors for other hemopoietic cell types and the ability of Multi-CSF, but not other CSFs (with the exception of GMCSF and eosinophil receptors), to down-modulate such receptors. Lineage-specific factors have been described for erythroid cells (erythropoietin), eosinophils (eosinophilopoietin), and megakaryocytes (thrombopoietin), but detailed binding studies of these factors have not yet been performed. One could thus envisage that GM-CSF and multi-CSF are proteins that interact with cells bearing receptors to permit proliferation and to down-regulate receptors for lineage-specific factors. The differentiation induced by GM-CSF or multi-CSF could be a consequence of the down-modulation and thus activation of late lineage factor receptors by interaction with cellular targets (Walker et al., 1985; Nicola et al., 1986).

Ill. Etiology of Nonviral Myeloid Neoplasms

A. INTRODUCTION Acute oncogenic retroviruses do not play a significant role, if any at all, in generating leukemia in mammals in nature. Although a few exceptions have been reported, including a retrovirus bea$ng the myc oncogene that induces a feline lymphatic leukemia (Neil et al., 1984), the etiologic agents that have been implicated in leukemogenesis are restricted to nonacute, replication-competent retroviruses (as found in mice, cats, cattle, and primates) (see review in Weiss et al., 1982; Yamamoto and Hinuma, 1985) or chemical carcinogens. Why then are these acutely transforming viruses used to study leukemogenesis? What advantages have they to other model systems? What shortcomings exist in their utilization? The induction of leukemia by nononcogenic viruses requires a long

MURINE SPLEEN FOCUS-FORMING VIRUSES

219

latency. Indeed, the onset of the disease may first require the generation of a recombinant viral intermediate (e.g., the MCF virus in the mouse) (Rowe e t al., 1980; Chattopadhyay et al., 1981, 1982; Blatt et al., 1983). The complexity of these processes and the necessity to use whole animals for study make a causal step-by-step analysis of these leukemias difficult. The study of carcinogen-induced leukemogenesis, which may reflect more closely the “normal” progression of malignancy leading to leukemia, is also encumbered by the necessity of in uivo analysis. Oncogenic retroviruses, however, act directly on a specific target cell and are thus more amenable to direct molecular analysis. The ability to genetically engineer these viruses (see Sections IV and VI). to eliminate the use of helper virus (Mann et al., 1983), and to identify and purify potential target cells (see Section V) facilitates the analysis of their action and hence an understanding of the etiology of leukemia. The specificity of the action of the oncogenic viruses simplifies their analysis; however, this may result in the oversimplification of a leukemogenic model. Most chemical carcinogens act nonspecifically, although it has been shown that the local and temporally controlled application of carcinogens such as di- or trimethylbenzanthracene (DMBA or TMBA, respectively) in rats almost always induces a specific type of erythroid leukemia (Huggins and Oka, 1972; Huggins et al., 1982; Huggins and Ueda, 1984). The transformed erythroid cells grow permanently (Kluge et al., 1976; Yamaguchi et al., 1981)and are very similar to transplantable erythroblasts transformed by Friend spleen focus-forming viruses (F-SFFV) (Friend et al., 1971; Steinheider et al., 1971; Ostertag et al., 1972). Nevertheless, caution must be taken in the interpretation of results obtained from studies utilizing oncogenic viruses for the study of leukemogenicity whose action may not duly reflect the complex interaction between mutated cellular protooncogenes, “antioncogenes,” and other somatic mutational events that may occur in viuo during malignant progression. In this section, an attempt will be made to summarize some of the reported observations of malignant progression in uiuo, so that results obtained with oncogenic viral vectors can be interpreted with these in mind. Knowledge of the complexities in uivo may also help us to understand the complex virus-cell interactions in retroviral-induced neoplasms in uitro. First, general models of leukemogenesis will be discussed. Second, we will try to outline the complexity of the interactions which lead to carcinogenesis, including the possible roles of negative-regulating factors (e.g., antioncogenes) and positive-regulating factors (e.g., mutated protooncogenes). Finally, cooperative

220

WOLFRAM OSTERTAG

models of oncogenesis and clonal progression of neoplasms will be discussed using chronic myeloid leukemia (CML) in man as a model.

B. GENERAL MODELSOF LEUKEMOGENESIS Leukemias in mammals, including man, are generally considered to be caused by one or several genomic alterations of the cell resulting in its abnormal proliferation. There are two assumptions in this simple, but generally accepted statement that may not be correct: 1. It is agreed that the malignant behavior of cells is caused by one or several inherited somatic alterations of individuals; however, the inherited stability of somatic cells may not be simply determined at the genomic DNA level. There is suggestive evidence that the cortex of developing eggs or of some protozoan species can also confer heritable stability to cells. More important is the evidence concerning the inherited stability of the differentiation pathway “chosen” and taken by different cells (review DiBerardino et al., 1984). Changes in the phenotype from one stage of differentiation to another occur at a set and very low rate and with a predictable frequency in specific directions. The mechanisms involved in this inherited stability of differentiation may be manifold. Many studies have implicated the importance of methylation in determining the set differentiation pathways (Jones and Taylor, 1980; Groudine et al., 1981; Harbers et al., 1981; Harland, 1982; Jahner et al., 1982; Ott et al., 1982; Stein et al., 1982; Asche et al., 1984). Studies utilizing cell hybrids (including heterokaryons) and involving transfer of cytoplasm from one differentiating cell to another of an alternative differentiation lineage indicate that proteins are involved in the fixation of a “metastable” state of differentiation (Kahn et al., 1981). Alterations leading to malignancy could conceivably be based on misprogramming of such normally utilized somatic mechanisms to fix a certain type of differentiation. Moreover, it has been shown by several authors that malignant cells (embryonal carcinoma cells) (Kleinsmith and Pierce, 1964; Mintz and Illmensee, 1975; Stewart and Mintz, 1982) or nuclei of malignant cells (frog renal carcinoma cells) (review DiBerardino et al., 1984) can be reprogrammed for normal differentiation. This may indicate that metastable alterations are important in the development of malignancy. It would be of great fundamental interest to establish whether stable genetic alterations are detectable in these tumors and how the cells behave on “normalization.” Present evidence indicates that there is not an increase in the incidence of cancers in these normalized cells, which argues against a stable genetic

MURINE SPLEEN FOCUS-FORMING VIRUSES

22 1

alteration in these malignant cells. This is, however, not in accord with our present knowledge of malignant transformation with other tumors. Such malignant alterations, with no apparent alteration in the DNA, may normally escape detection for two reasons: (1) Almost all cancer research is directed to detect irreversible genetic changes, and (2) analysis of alterations has been, until now, restricted to a descriptive-correlative level. 2. The second general assumption that has been called into question by several authors is the designation of the proliferating cell, as such, as abnormal. They argue the obvious: The symptoms of a malignant disorder are not necessarily directly related to the malignancy. DeLarco and Todaro (1978) found that abnormal growth factor release of transformed fibroblasts could lead to secondary alterations in the proliferation of other fibroblasts in such a way that the truly transformed state is mimicked. The inability to find viral genomes of HTLV I or HTLV I1 in all cells of leukemias induced (?) by these viruses was used as an argument for transformation of just a few cells which might secrete hormones required for proliferation of untransformed cells constituting the bulk of the abnormally proliferating cells. No concrete evidence, however, supports such a mechanism of leukemogenesis in this particular leukemia. Recent results of Allen and Dexter (1984) are suggestive that an altered Rous sarcoma virus may, in fact, modify cells of the murine hematopoietic microenvironment and thus permit hematopoietic stem cells of type I (Fig. l),which do not contain the virus or its oncogene ( v - s ~ )to, survive in culture (Boettiger et al., 1984; Spooncer e t al., 1984; Dexter et al., 1985; Wyke et al., 1986) (see also Section 11). A similar model has been proposed for the induction of myeloid leukemia by the myeloproliferative sarcoma virus (MPSV): The virus causes transformation of its target cell which is not itself grossly altered in proliferative capacity, but more in the ability to secrete hormones, the secretion of which induces myeloproliferation (Jasmin e t al., 1980; this review). Despite these objections and possible exceptions to the abovestated theory of leukemogenesis, it still appears that mutations in the DNA of somatic cells are usually the basis for malignancy. These somatic alterations in the cellular genome could conceivably affect the proper balance of proliferation and differentiation (see below) or could evoke extensive rearrangements in the temporal differentiation pattern (lineage infidelity) (McCulloch, 1983; Adler et al., 1984; McCulloch et al., 1984). Support for the latter model has been obtained from studies of blast cells of acute myeloblastic leukemia (AML) in culture. Cells from the AML cell line K562 and other cultured transformed cells were found to simultaneously express markers of erythro-

222

WOLFRAM OSTERTAG

poiesis, of granulopoiesis, and possibly of megakaryocyte/platelet differentiation. Whether this abnormal gene expression is caused by transdetermination or reversal of differentiation (see above) of a few cells in culture, thereby generating abnormal cellular differentiation heterogeneity (McCulloch et al., 1984); whether the abnormality is apparent only because of inappropriate use of markers normally expressed in several tissues; or whether secondary alterations occurred during tissue culture adaptation of leukemic cells has not been determined rigorously. Temporal reversal of differentiation or lineage infidelity within one transformed cell could thus contribute to unpredictable alterations in proliferation behavior of cells or lead to defects in cell function. Lineage infidelity, as discussed here, appears not to be the predominant mode of leukemogenesis, but it should be kept in mind that there are many ways malignancies can arise and that model studies of transformation using oncogenic retroviruses may not reveal such occurrences as lineage infidelity which may act as priming events in malignant progression. Assuming that most leukemias are, in fact, caused by cellular genetic mutations leading to abnormal proliferation of these cells and that these cells still represent, as judged by differentiation markers, faithful phenocopies of normal cells, we still have the choice of at least three alternative models of how leukemogenesis may occur: (1) by a general increase in proliferative potential of the transformed cells without alteration in the differentiation capacity; ( 2 )by both a block in differentiation and the acquisition of continued proliferative potential of the same cell; and (3)by a change in balance of cell differentiation (leading to loss of proliferative capcity) and cell renewal (leading to an increase in cell numbers) (Metcalf, 1984). A general increase in proliferative myeloid potential has been found in the myeloproliferative diseases in man, including polycythemia Vera, essential thrombocythemia, and chronic myeloid leukemia (CML) (Dameshek, 1951; Eaves and Eaves, 1978; Fialkow et al., 1978, 1981a,b; Gerwitz et al., 1983; Fialkow and Singer, 1985). FSFFV (polycythemia variant) and Rauscher SFFV (R-SFFV) cause similar myeloproliferative or erythroproliferative diseases (see Section V), but the disease is definitely not caused by the clonal expansion of precursor stem cells, as has been shown for the myeloproliferative diseases in humans (see below). Thus, the use of SFFVs as models for human myeloproliferative diseases is restrictive (SmadjaJoffe et al., 1975; Jasmin et al., 1976). A block in differentiation has been discussed as a model for some normally occurring or chemically induced human and animal leuke-

MURINE SPLEEN FOCUS-FORMING VIRUSES

223

mias (Kluge et al., 1976; Greaves, 1982; Huggins et al., 1982). The blocked cells acquire the new capacity of continued self-renewal. A block in differentiation could be considered the ultimate extreme shift in balance of self-renewal and differentiation as discussed by Metcalf (1984).Blocks in differentiation are, however, only found in rare cases of leukemias, and it is uncertain whether they are not actually a final consequence of the multiple steps of clonal progression (see below). Some acute leukemogenic retroviruses, such as the avian erythroblastosis virus (AEV) and the murine malignant histiocytosis sarcoma virus (MHSV) (see Sections IV,D and V), may also shift the balance of differentiation and self-renewal capacity to such an extreme that it mimics a complete block of differentiation (Royer-Pokora et al., 1978; Beug et al., 1980; Gazzolo et al., 1980; Moscovici and Moscovici, 1983; Boettiger and Durban, 1984; Franz et al., 1985; review Graf, 1985; and below). The problems inherent in analyzing the progeny of many cells after initial infection with an acutely transforming retrovirus and the use of mass cultures for in vitro assays do not permit the dissection of the steps leading to a block in differentiation. However, tentative evidence of one group (Boettiger and Durban, 1984) suggests that the events leading to a block in differentiation and to an increase in proliferative potential occur at different times. It is thus not excluded that a block in differentiation is just one of several steps in malignant progression, even when induced by oncogenic viruses. The disease caused by Friend or Rauscher virus (FV and RV, respectively) was also thought to be primarily the result of a block of differentiation due to the accumulation of cells in the proerythroblast stage in the spleen of infected mice. However, kinetic analysis of cell behavior in mice has revealed that the block of differentiation is not real, since preferential removal and cell death of FV- and RV-infected, excessively proliferating erythropoietic precursor cells occur following the proerythroblast stage. The proerythroblasts, if tested for differentiation capacity, appear normal except for their erythropoietin independence (Smadja-Joffe et al., 1976; Tambourin, 1979). Only a very few of the FV- or RV-infected cells are truly transformed, differentially blocked, or extremely altered in their balance of self-renewal and differentiation (review Ostertag and Pragnell, 1981; Wendling et

al., 1981). Most naturally occurring leukemias probably involve a shift in the balance of self-renewal and differentiation favoring self-renewal (Metcalf, 1984). It is still uncertain whether this shift in balance is a secondary event in the progression of the disease since clonal expansion of one stem cell with normal proliferative and differentiation

224

WOLFRAM OSTERTAG

capacity may precede changes in the balance of self-renewal and differentiation of progeny cells (see below). In conclusion, there may be multiple mechanisms for generating leukemia reflected in the diverse ways in which oncogenic retroviruses interact with target cells to generate leukemia. A unitary model of leukemogenesis cannot be maintained. OF LEUKEMOGENESIS C. COMPLEXITY

1 . Antioncogenes and Tumor Suppressors We would like to emphasize that genes other than oncogenes may be important in the development of naturally occurring leukemia. A series of genes have been implicated in some neoplasms, primarily nonleukemogenic, the functional impairment of which may be necessary before a cancer arises (Knudson, 1983). The antioncogenes appear to act epistatic with respect to the suppression of oncogenesis. Both alleles may have to be altered to allow progression of malignancy (retinoblastoma: Murphree and Benedict, 1984; Wilms’ tumor and related diseases: Koufos et al., 1984, 1985; Orkin et al., 1984; review in Klein and Klein, 1985a,b; Scott et al., 1985). Similar antioncogenes may be present in other species, such as Drosophila (Gateff, 1982; Mechler et al., 1985). The tissue-specific manifestation of tumors after mutation of these gene loci is intriguing. In cases where an inherited deficiency of these antioncogenes exists, evidence suggests that a generalized proliferation of specific cells may provide an increased chance for new somatic mutations, thereby leading to oncogenesis. The evidence that the normal alleles of these genes actually counteract oncogenesis, as the name antioncogenes would suggest, is at present slim. The close proximity of the protooncogene c-Ha-ras (see below) to the gene loci responsible for Wilms’ tumor may suggest some degree of functional or genetic relatedness to protooncogenes (Reeve et al., 1984). The recessiveness of the mutational alteration of these antioncogenes predisposing to malignancy appears in conflict with data on the dominant action of retroviral oncogenes, but is not in conflict with recent data that the normal c-Ki-ras locus may be dominant to the activated Ki-ras protooncogene (Santos et aE., 1984). The malignant cells very often not only show presence of an altered and activated protooncogene, but also deletion of the normal allele. Of equal interest is the evidence obtained by in vitro analysis of carcinogenesis for a set of genes which can be called tumor suppressors (Craig and Sager, 1985; review in Klein and Klein, 1985) and are

MURINE SPLEEN FOCUS-FORMING VIRUSES

225

identified by suppression of malignancy in cell hybrids (Wiener et ul., 1974; Stanbridge et ul., 1982). These antitumor genes may be similar to those genes which regulate expression of true oncogenes, such as an activated ras oncogene (Craig and Sager, 1985). The suppression has a genetic basis: reemergence of tumorigenicity is associated with specific chromosome loss. Moreover, the transforming effect of retroviral oncogenes can be counteracted by suppressor genes. Cellular revertants of Ki-MuSVtransformed fibroblasts have been isolated. These cells suppress rus, fes, and src action of transformation of other fibroblasts when cell hybrids with transformed cells are formed, but not that of mos, fms, and sis (Noda e t al., 1983). Whether these differences reflect functional differences of oncogene expression or the “potency” of a particular oncogene is at present uncertain, more so since study with cell hybrids has also shown that differences in gene dosage (one versus two) play a significant role in determining malignancy and nonmalignancy in cell hybrids. Other factors and genes can negatively modulate expression of tumor genes (Klein and Klein, 1985 a,b). One particular case, however, seems especially intriguing: transplantable myelocytic and erythroid chemically induced leukemias in the rat show regression if the hypophysis is removed! (Huggins and Oka, 1972; Huggins and Ueda,

1984.) 2 . Protooncogenes in Carcinogenesis Oncogenes are defined here as genes which by themselves can cause malignant proliferation when introduced into proper target cells. By this definition, the only true oncogenes are those encoded in the genomes of acutely transforming retroviruses and which are indispensable for causing malignant viral-induced cell proliferation in the animal or in vitro (Weiss et ul., 1982). Most of these viral oncogenes are closely related, but usually not identical to cellular genes called protooncogenes (Bister and Jansen, 1985; Duesberg, 1985). Some of the cellular protooncogenes are somatically altered in a series of tumors and unaltered in normal cells of the same patient or the same animal. In particular, a recent study in which over 100 fresh tumors from patients were probed with 9 protooncogenes showed frequent alterations in c-myc, c-Ha-ras, c-myb, and, to a lesser amount, c-Ki-rus. No alterations of the protooncogene in the respective normal homologous tissue of the same patient (when available) were found (Yokota et al., 1986). These data thus strongly suggest, but do not prove, that

226

WOLFRAM OSTERTAG

altered (activated) protooncogenes are generally important in cancer. Altered protooncogenes were identified (1) by a series of diverse transformation assays which usually do not reflect the original transformation specificity of the protooncogene (Shih et al., 1981; Blair et al., 1982; Cooper, 1982; Land et al., 1983; Fasano et al., 1984b; Spandidos and Wilkie, 1984; Thorgeirsson et al., 1985)or (2) by presence of amplified oncogene-related DNA sequences in some neoplasms (Collins and Groudine, 1982; Dalla Favera et al., 1982; Collins et al., 1984; Pelicci et al., 1984; Shibuya et al., 1985; Schwab et al., 1985). However, no consistent correlation to a particular type of malignancy has been found. Protooncogene expression was found altered in many cancer cells and these cases proved highly consistent. Alteration of expression could be attributed to one of two modes: (1)insertion of a retroviral or retroviral-like long terminal repeat (LTR) next to the protooncogenes myc (see Hayward, 1985),c-erbB (Nilsen et al., 1985), c-raf(Mo1ders et al., 1985), c-myb (Mushinski et al., 1983; Weiristein et al., 1985), cmos (Rechavi et al., 1982; see also Section IV,C), or to presumptive protooncogenes, such as pim-1 (Selten et al., 1985), Mlvi-2 (EconomuPachnis et al., 1985), int-1 and int-2 (Nusse and Varmus, 1982; Dickson et al., 1984), or next to growth factor genes as in the WEHI-3B myeloid cell line (Ymer et al., 1985) and two factor independent promyelocytic cell lines (Kawai and Stocking, unpublished results); or (2) specific chromosome aberrations, mostly translocations, that are diagnostic for the altered tumor cells, in some instances in close to 100% of the patients with a particular tumor or leukemia (see Rowley, 1985). The case is especially convincing in Burkitt’s lymphoma (Klein and Klein, 1985a,b), in some cases of adult acute promyelocyte leukemia with involvement of the p53 gene locus (Le Beau et al., 1985), in some cases of adult lymphocytic leukemia, and especially in chronic myeloid leukemia where the abl protooncogene is altered in a specific manner by translocation next to the bcr locus (Bartram et al., 1985; Heisterkamp et al., 1985; Shtivelman et al., 1985) (see also below). Difficulties in equating specific chromosome breakpoints or specific LTR insertions with oncogenesis arise in that (1) the approach to identify these protooncogenes or presumptive cancer genes relies entirely on correlations of specific DNA alterations with specific types of cancer; (2) these alterations may b e one of many changes necessary in malignant progression (Klein and Klein, 1985a,b, and below); and (3) the relevant genes may not be at the breakpoint or site of LTR insertion, but are located more distal and not yet identified.

MURINE SPLEEN FOCUS-FORMING VIRUSES

227

3. Cooperation of Disreguluted Protooncogenes Not only one, but several genetic changes are usually associated with the progression of malignant growth. Only acute oncogenic retroviruses appear capable of transforming cells in a single step or a series of closely related steps. A second cellular element, in addition to the oncogene, is also detected in some oncogenic retroviruses, such as erbA in the avian erythroblastosis virus in addition to erbB, ets in combination with myb in the E 26 virus, or myc in combination with ruf (or mil) in the MH2 virus (Bister and Jansen, 1985). Several of these “supplementary oncogenes” probably confer a selective growth advantage to already transformed cells. Potentially immortal cells may thus obtain factor independency for growth after introduction of a “supplementary oncogene” (review in Graf, 1985). The realization that the interaction of oncogenes with supplementary oncogenes is often necessary for carcinogenesis and the observation that standard transformation assays utilizing an immortal fibroblast cell line preferentially detected one group of altered protooncogenes (ras) made it necessary to develop new test systems which would detect the action of other oncogenes and the cooperative interaction of oncogenes. This led Weinberg’s group to use primary fibroblast cell lines (Land et al., 1983).The transformation of primary fibroblasts by previously characterized or putative oncogenes seems to require two steps involving two different potential oncogenes. One group of oncogenes supposedly acts on the nucleus to immortalize cells, and a second group of oncogenes acts on the cell membrane (or cytoplasma?) to morphologically transform cells, an event which is easily detectable in fibroblasts by the focus-forming assay. The interaction of two oncogenes from each of the two groups seems to be required for transformation. However, the subdivision of oncogenes into these two groups and the necessity of their interaction may not be completely strict. Spandidos and Wilkie (1984) showed that the same oncogene (rus) could fulfill both functions, immortalization and morphological alterations typical for transformation, if the dosage of the oncogene was adjusted properly. More recent work by Land et ul. (1986) suggests that, in addition to high expression, the absence of adjacent normal cells is also necessary to enable a singly acting ras oncogene to create a tumorigenic cell line. Such a condition would be met in vitro by infection using high-titered stocks of ras-containing retrovirus or in zjivo by recruitment, via virus spread, of large numbers of transformed cells in localized areas of an organ (Ponten, 1964; Land

228

WOLFRAM OSTERTAG

et al., 1986). The presence of normal cells may inhibit the clonal expansion of cells expressing the ras oncogene, a potentially necessary step for the full tumorigenic phenotype. Thus, a second oncogene activation may be necessary for nonviral tumorigenicity. Several other explanations for the requirement of interactions between oncogenes can be envisaged: Oncogene 1 (e.g., mos) may be toxic to a particular cell beyond a certain threshold of expression (a level which theoretically would be required for transformation). Interaction with and expression of oncogene 2 may thus permit transformation before this threshold is reached without inducing toxicity specific for oncogene 1. Recent work by Smith et al. (1986) utilizing ras antibody shows the requirement of c-ras proteins for the transformation of fibroblasts with receptor-like oncogenes (fms, src, and fes), but not some cytoplasmic oncogenes (mos and raf). This suggests that the cras proteins are necessary for transfer of signals from receptor molecules at the cell surface to cytoplasmic effectors. Such interactions or shared metabolic pathways have also been suggested by Noda et al. (1983) (see above), although discrepancies exist. Much work needs to be done to experimentally define the necessary interactions of oncogenes for transformation. How oncogenes may act in causing human leukemia or how interaction with other genetic alterations may be envisaged is best illustrated in patients developing chronic myeloid leukemia (Fig. 7)(Dube et al., 1984; Bartram et al., 1985; Fialkow and Singer, 1985; Heisterkamp et al., 1985; Shtivelman et al., 1985). Almost all cases of CML show specific chromosomal translocations resulting in two abnormal chromosomes. One of the two is easily recognized cytologically: the Philadelphia chromosome. One of the breakpoints of chromosome 22 is always within 5 kb distance of the break cluster region (bcr) and is joined to a piece of chromosome 9 with the abl protooncogene 10-100 kb downstream. This leads to a composite gene which utilizes the regulatory and part of the coding sequences of the bcr locus and coding sequences of the abl locus. A new hybrid mRNA species is formed which leads to the synthesis of an abnormally regulated fusion protein of about MW 210,000 (in K562 cells). This protein has tyrosine kinase activity contrary to the normal product of the cell locus. Tyrosine kinase activity is supposed to be critical for the oncogenic action of the abl oncogene in the Abelson virus. This protooncogene rearrangement leading to an abnormal abl product, similar to that found in cells infected by the oncogenic virus, may not, however, be sufficient for generating CML. Fialkow and collaborators (Fialkow and Singer, 1985) find that hematopoietic stem

229

MURINE SPLEEN FOCUS-FORMING VIRUSES Stem

0

cells

A

AAA

A

00.0000

'

1

L

All

B

00000 t

stem

cells e x p a n d

M u t a t i o n in one

equally

A

I

I

well

stem cell

...@... ' /

0 0 0 0 0 0 0

N o r m a l situation: t. t

1st

stage

of C M L :

\

9/2 2 rearrangement bcr/abl

Expansion of some

stem

clone ( o n l y t y p e A

G-6PD

cell

found ). N o symptoms

activation

2 n d stage

C

of CML:

clone A ( 9 / 2 2 ) , t y p i c a l

for

CML

'

I t b l o c k in differentiation

@ A(9/22D-) D

A

@WWW

I 3 r d stage of Acute

blast

CML:

I

crisis: N e w

clone

develops with a b n o r m a l differentiation

p a t t e r n ( block

of differentiation)

b b b b b b *

FIG.7. A proposed model of the progression of chronic myeloid leukemia (CML).

230

WOLFRAM OSTERTAG

cell proliferation may already be abnormal prior to the generation of a clone with the abl-bcr translocation. The nature of the first event (clonal expansion of one stem cell) is not at all understood; the protooncogene rearrangement may be secondary (at least in some cases) and only part of a whole series of events that may be necessary to obtain the full leukemia (Fig. 7). The finding that rearrangements of the bcr sequence and the abl protooncogene are also found in Philadelphia-negative patients (Bartram et al., 1985)necessitates further molecular work in combination with studies utilizing clonal markers (Fialkow and Singer, 1985) to rigorously exclude the possibility that different subsets of cells of CML are actually minimally altered or differently altered prior to the appearance of the Philadelphia chromosome which is clonally expanded in Ph’-negative patients. It is also not clear whether clonal expansion of abnormal cells eliminates all normal stem cells (Dube et al., 1984).

4 . Function of Oncogenes-Znteraction with Growth Factors Protooncogenes and oncogenes can possibly be divided into two classes, depending on the presumptive site of action (see above) (Land et al., 1983).Not much is known about the normal function of the protooncogenes except for inferences made for a group of protooncogenes whose products are related to growth factor receptors, such as the epithelial growth factor receptor [erbB-1(Downward et al., 1984); possibly neu (c-erbB-2),which is related to erbB but genetically unlinked (Schechter et al., 1985; Semba et al., 1985)l and the macrophage CSF receptor [fms (Sherr et al., 1985; Nienhuis et al., 1985)], or for growth factor-related polypeptides, such as the p chain of the platelet-derived growth factor (PDGF) [c-sis (Devare et al., 1983; Doolittle et al., 1983; Waterfield et al., 1983; Johnsson et al., 1984; Rao et al., 1986)l. The function of the ras protooncogenes and the putative influence of the ras gene product on adenyl cyclase regulation or on growth factor receptor expression will be discussed in Section IV, D. a. Oncogene Products as Autocrine or Paracrine Stimulators. Autocrine growth stimulation has been supported as at least the priming event for cell transformation by two experimental systems: the transduction of either PDGF or GM-CSF into target cells with the respective growth factor receptors by use of naturally occurring or genetically engineered retrovirus vectors (Heldin and Westermark, 1984; Johnsson et al., 1986; Laker et al., submitted). Except for three amino acid substitutions, the predicted sequence of the transforming protein

MURINE SPLEEN FOCUS-FORMING VIRUSES

23 1

p28sis of the simian sarcoma virus (SiSV) is closely related to the Nterminal amino acid residues of the p chain of platelet-derived growth factor (PDGF) (Johnsson et al., 1985a). SiSV-transformed cells secrete a product, possibly coded by v-sis, which interacts similarly with cells containing a PDGF receptor as PDGF itself (Huang et al., 1984; Johnsson e t al., 198513, 1986); treatment of SiSV-transformed cells with antiserum against PDGF inhibits cell proliferation. The human homologue (c-sis) also displays transforming activity when placed under control of a retrovirus LTR (Clarke et al., 1984; Gazit et al., 1984). SiSV-infected cells are not immortalized and require a second oncogenic event to acquire truly transformed properties (Johnsson et al., 1986). However, the first event, autocrine stimulation by introduction of growth factor genes, may precondition the cell to acquire, in a second step, true autonomy of cell growth as is indicated by recent work of our group with a retrovirus vector coding for GM-CSF (Laker et al., submitted) (see above). The second step occurs with a much higher frequency if the target cells bear the GM-CSF receptor and release GM-CSF as compared to cells which do not produce the growth factor. We have also characterized two promyelocytic tumorigenic cell lines that release GM-CSF in an autocrine fashion (M. Kawai, submitted). We have observed that, when held in culture, these altered cells mutate readily, progressing toward true autonomy (C. Stocking and C. Loliger, unpublished). PDGF is the major mitogen in serum for connective tissue-derived cells in culture (Heldin and Westermark, 1984). PDGF exhibits proliferative effects via interaction with high-affinity cell surface receptors which were identified on fibroblasts and glial cells. The proliferative signal is transduced via activation of a tyrosine-specific kinase activity associated with the PDGF receptor (Daniel et al., 1985). PDGF can also induce a migratory (chemotactic) response to cells with the PDGF receptor (Grotendorst, 1984).Whether cell migration and mitogenic response are mediated by interaction of the PDGF/PDGF receptor complex with the same intracellular target site is not known. Treatment of 3T3 fibroblasts with PDGF results in changes which are associated with the transformed state of a cell transformed by SiSV (Heldin and Westermark, 1984). But not all of the changes which are found on transformation of cells with SiSV are found on treatment with PDGF. The v-sis coded protein stimulates only those cells which have the PDGF receptor and which can be transformed by the SiSV (Leal et al., 1985).In apparent conflict to a simple hypothesis of autocrine stimulation being responsible for transformation are results of Robbins et al. (1985) showing that most of the v-sis transforming gene

232

WOLFRAM OSTERTAG

product remains associated with the cell and is not secreted, as is known for PDGF. Further circumstantial evidence that the v-sis oncogene of SiSV virus transforms cells by autocrine stimulation can be gained by a study of a series of transformed cell lines which are not transformed by SiSV, but coexpress PDGF-like growth factors and PDGF receptors [some human osteosarcoma cells (Betsholtz et al., 1984); glioma and sarcoma cells (see Heldin and Westermark, 1984)l. Less convincing are data where an abnormal secretion of PDGF-like activity or csis transcription has been found in tumors which do not normally have the PDGF receptor [neuroblastoma cells (van Zoelen et al., 1985a,b); T cells infected with T cell leukemia virus (Westin et al., 1982)l. Treatment of these neuroblastoma cells with anti-PDGF does not inhibit cell proliferation. It would be of great interest to know whether these neuroblastoma cells do express PDGF receptors. Suggestive data that autocrine stimulation of cells may lead to transformation has also been published for several unrelated systems: Schrader’s group (Schrader et al., 1983) has collected evidence that growth factor-independent P cell lines which are tumorigenic in the nude mouse assay can be isolated at a low frequency from other mast cell precursor cells (P cells) requiring Multi-CSF for growth and are not tumorigenic in the nude mouse assay. Unlike factor-dependent P cells, the factor-independent P cells release Multi-CSF. However, the nude mouse assay may not be a true indicator for tumorigenicity: Schrader’s group was also able to show that factor-dependent P cells could grow in the nude mouse if a source of Multi-CSF was provided. Adkins et al. (1984) have provided additional evidence that growth factor dependence of v-myc- or v-myb-transformed myelomonocytic cell lines can be abrogated by infection of retroviruses carrying the src oncogene. These doubly infected and transformed cells now release a growth factor which initially had to be supplied to the singly infected and transformed myeloid cells. The necessity of this factor for growth of these doubly infected cells was shown by partial inhibition of cell growth by treatment with antiserum against the growth factor. Duprey et al. (1985) have also reported the isolation of a human T cell lymphoma that constitutively expresses high-affinity cell surface receptors for IL-2 and, in addition, synthesizes IL-2, which is bound to cell surface receptors. More recently, myeloid progenitor cells isolated from acute myeloblastic leukemic (AML) patients were shown to release GM-CSF and, thus, suggested a model of autocrine stimulation for some leukemic cells (Young and Griffin, 1986). In apparent contrast to those results are data published by Rapp e t al. (1985b, 1986): A murine virus with the avian v-myc is capable of

MURINE SPLEEN FOCUS-FORMING VIRUSES

233

abrogating the requirement for Multi-CSF or interleukin-2 (IL-2) of some hematopoietic cells. None of these factor-independent cell lines produced a mitogenic activity for the parental factor-dependent cell line. No alteration in the number of Multi-CSF receptors of IL-2 receptors was found. That transformation of fibroblasts by oncogenic retroviruses carrying the mos, ras,fes, or other oncogenes can lead to the induction of growth factor release has been shown repeatedly. This correlates transformation of an oncogenic virus with release of growth factors and has its analogy in the transcriptional activation of PDGF-like activity and the c-sis protooncogene in SV40-transformed fibroblasts (Stroobant et al., 1985). Two different types of growth factors have been identified in mammalian fibroblasts transformed by mammalian sarcoma viruses: transforming growth factors of group I (TGF-I), which includes TGF-a, and TGFs of group 11, which include TGF-P. These polypeptides are able to confer a transient transformed phenotype to normal cells as long as factor is added, in contrast to truly transformed cells, which release TGFs, but stay transformed even if treated with antisera against the TGFs. TGF-I competes with the epithelial growth factor (EGF) for the E G F receptor and thus indicates a functional link between the transforming action of two groups of oncogenes (fibroblast transforming oncogenes and erbB, which is related to the E G F receptor protein). Not unexpectedly, TGF-a shows structural homology to EGF, but not to TGF-0, which binds to a different receptor. TGF-a induces phosphorylation of the E G F receptor, similarly as EGF. TGF-P controls receptor levels for E G F in normal rat kidney (NRK) fibroblasts. Both factors contribute to the conversion of normal fibroblasts to pseudo-transformed fibroblasts, and TGF-P alone can also induce fibroblasts to form colonies in soft agar (De Larco and Todaro, 1978; Reynolds et al., 1981; Anzano et al., 1983; Carpenter et al., 1983; Marquardt et al., 1983, 1984; Twardzik et al., 1983; Assoian et al., 1984; Derynck et al., 1985; Lee et al., 1985). Leof et al. (1986)were able to show that treatment of primary murine fibroblasts with TGF-P leads to an early induction of c-sis mRNA, followed by induction of c-fos, c-myc, and other PDGF-inducible genes. They suggest that the pseudotransformation of fibroblasts by release of TGF-P is mediated via PDGF release, which thus mimics the action of SiSV. Other transformed cell lines may release other factors which may phenotypically transform fibroblasts and not interact with the E G F or PDGF receptor (van Zoelen et al., 1985a). The ectopic growth factor (TGF-P) released by transformed melanoma cells may possibly be involved in the progression of malignancy (De Larco et al., 1985).

234

WOLFRAM OSTERTAG

Growth factor release can also be found in some, but not all, cell lines simply infected by retrovirus [general (Koury and Pragnell, 1982); HTLV I (Salahuddin et al., 1984)l. Such a mechanism may also explain the disease caused by MPSV (see Section V). The mechanism by which a retrovirus (with a minimum requirement of functional LTRs) can induce or enhance growth factor is at present obscure. However, the fact that growth factor release is found in almost all retrovirus-infected target cells (Koury and Pragnell, 1982; own data) and that retroviruses integrate into the cellular genome randomly (Weiss et al., 1982) argues against insertional mutagenesis. The induced release may be a consequence of unspecific interference of retroviral RNA (double stranded?) with cellular control mechanisms. Such a model would favor the enhanced release of factors constitutively expressed at low levels in the cells. This is, at present, in agreement with published data: Fibroblast infection leads to enhanced expression of GM-CSF and M-CSF, factors which are released by some fibroblasts; T cell infection leads to release of GM-CSF, Multi-CSF, y-interferon (macrophage-activating factor), IL-2, and B cell growth factor (BCGF), all factors normally inducible in T cells. If the cells are responsive to the induced factor (autocrine stimulation), transformation of the cell could occur, independent of the presence or action of an oncogene. Results by Arya et al. (1984), however, seem to rule out a specific role of IL-2 in transformation by HTLV, although levels of IL-2 receptors in HTLV-infected human B cells (Sugamura et al., 1984) or T cells (Kronke et al., 1985) may be increased. b. Oncogene Products Related to Growth Factor Receptors. Oncogene-coded products may interact with the cell not only by mimicking a growth factor, inducing a growth factor, or replacing the requirement of a growth factor as outlined above, but by acting similarly as receptors for growth factors. Indeed, two oncogenes, erbB and fms,and possibly a third, neu (erbB-2)(Schechter et al., 1985), are structurally related to growth factor receptors. erbB is the essential oncogene found within a series of acute oncogenic chicken viruses having transformation specificity mainly for erythroid precursor cells. Interestingly, erbB has sequence homology with the gene for the E G F receptor not normally expressed in erythroid precursor cells. Similarly, oncogenic sarcoma viruses carrying fms, an oncogene related to the macrophage growth factor receptor, transforms fibroblasts while the receptor is usually active only in the macrophage differentiation pathway (Stanley et al., 1983; Rettenmier et al., 1985a,b; Sariban et al., 1985).It is therefore assumed that these growth factor receptors most likely do not interact with the cell in a

MURINE SPLEEN FOCUS-FORMING VIRUSES

235

differentiation-specific manner, but their synthesis is regulated in a differentiation-specific manner. That specific expression of the c-fms is required in myeloid cells and less or not at all in other cells is supported by an acquired disease, the so-called 5s- syndrome, a deletion in genes located on chromosome 5. One of the genes lost is the c-fms protooncogene, the M-CSF receptor gene. The bone marrow of patients with the 5s- disease is characterized by a deficiency in the number of erythroblasts and an increase in immature macrophage/granulocyte precursor cells and in megakaryocytes. The defect in erythroid cells is most likely an indirect consequence of alteration of the macrophage precursor cells, since culture of bone marrow in vitro of 5q- patients leads to fonnation of erythroid colonies in normal numbers (Nienhuis et al., 1985). Interactions of macrophage factors, as implied here, could also modulate cell proliferation in other diseases involving macrophages, as during transformation of myeloid cells by the malignant histiocytosis sarcoma virus (MHSV, see following sections). 5s- defects are often acquired during progression in polycythemia Vera or in acute myeloid leukemia and are not the primary lesion in these diseases, but may be a key event in the progression to acute leukemia (block in differentiation?). Two related c-erbB protooncogenes have been identified in the cellular genome: c-erbB-1, which is the gene coding for the E G F receptor (Downward et al., 1984), and c-erbB-2, whose normal function is yet to b e defined (Schechter et al., 1985; Semba et al., 1985). The AEV has acquired part of the c-erbB-1 gene, thus coding a truncated protein consisting of the internal domain and transmembrane part of the receptor, but not the external domain containing the E G F binding region. The loss of the E G F binding domain of the receptor protein may be a prerequisite for constitutive activation of the effector and thus imparting transformation potential of the virus (Downward et al., 1984). The fact that newly generated c-erbB-1transducing viruses, even when originating from the same precursors, can evolve into one of two types of oncogenic viruses (one type transforming erythroid cells, the other inducing angiosarcoma) strongly suggests that the apparent specificity of the erythroleukemia- and the angiosarcoma-inducing viruses is not a direct property of the erbB oncogene, but could be due to acquisition of other tissue-specific signal sequences within the retrovirus genome (Tracy et al., 1985). Indeed, AEV contains a second cellular gene, v-erb-A, that while in itself is not oncogenic, may act by increasing the oncogenic potential of v-erb-B. Recent nucleotide analyses have shown that it has structural homology to the estrogen receptor (Green et al., 1986) and the

236

WOLFRAM OSTERTAG

human glucocorticoid receptor (Weinberger et al., 1985). Binding studies have led to the conclusion,that the e-erb-A gene may encode a gene related to the thyroxin receptor in chickens and humans (Sap et al., 1986; Weinberger et al., 1986). Another evolutionary divergence of oncogenic retroviruses with respect to the target cell range and the molecular basis for the alteration will be described later for the myeloproliferative sarcoma virus (see Section IV,C). A series of transformed cell lines and tumors show rearrangements, mostly amplification, of the c-erbB-1 gene [A431 epidermal carcinoma cells (Ullrich et al., 1984; Merlino et al., 1984; Xu et al., 1984a); other tumors (Xu et al., 198413; Libermann et al., 1985)l.Extensive work on the molecular nature of the transcription of the activated c-erbB-1 locus has shown the presence of extensively amplified c-erbB-1 sequences and the synthesis of mRNA coding for a truncated c-erbB-1 protein different to that observed for AEV. The aberrant RNA is generated by a gene rearrangement associated with a chromosome translocation. The altered protein can be secreted: It does not contain the transmembrane spanning region (unlike the v-erbB product), but instead presumably is homologous to the external domain of EGF receptor protein which is not present in the v-erbB product. The functions of the secreted protein are unknown, although it is possible that overproduction of this protein is linked to the transformed phenotype of the cell (Merlino et al., 1985). The c-erbB-2 protooncogene (c-neu) was first identified in transformed 3T3 fibroblasts (Shih et al., 1981). Reversion of the transformed phenotype is obtained if neu (c-erbB-2)-transformed3T3 fibroblasts are treated with antibodies against the neu product p185. The antibody is not cytotoxic, but most likely down-modulates the expression of p185 in treated cells, thus suggesting a direct link between surface expression of p185 and transformation (Drebin et al., 1985). This is similar to EGF down-regulation of the EGF receptor (Lyall et al., 1985) or down-regulation found for a series of hematopoietic growth factor receptors (see Section 11).Oncogenic retroviruses which carry a v-onc gene directly related to c-erbB-2 have yet to be detected; however, the gene locus has been found altered in several tumors (Drebin et al., 1985; King et al., 1985; Schechter et al., 1985; Semba et al., 1985). Recent data based on the isolation of a retrovirus with sequences closely homologous to the antigen receptor of T cells suggests that the altered T cell receptor protein can also act as an oncogene in T cells (Neil et al., submitted). The gene products of a family of retroviral oncogenes and the receptors for growth factors, such as the M-CSF receptor and the EGF

MURINE SPLEEN FOCUS-FORMING VIRUSES

237

receptor, display a tyrosine-specific protein kinase activity (Gilmore

et al., 1985; Rettenmier et al., 1985a,b; Sherr et al., 1985). This tyrosine kinase activity of growth factor receptors appears to be an integral part of the receptor molecules (Heldin and Westermark, 1984). Their activation leads to autophosphorylation as well as tyrosine phosphorylation of other cellular substrates. The ligand binding site is on the outside of the cell membrane and the kinase domain on the inside. The kinase activity is presumably activated by a conformational change in the receptor molecule induced by factor binding. The E G F receptor can also be activated by DMSO, a substance which is long known to induce differentiation in some cells. A well-studied example is the DMSO induction of erythroid differentiation of F-SFFV-transformed erythroleukemia cells that have lost functional interaction with the factor erythropoietin (Kluge et al., 1974) required for differentiation of normal but not erythroleukemic cells (see previous section). DMSO induces conformational changes in receptor proteins, e.g., the E G F receptor (Rubin and Earp, 1983) and also induces an increase in erythropoietin receptors in Friend cells. Oncogene-derived tyrosine kinases as well as the receptors for EGF, TGF-I, PDGF, and insulin contain phosphate bound to serine and in some cases to threonine. Interestingly, some other oncogene products (mos, raf) are possibly also associated with serinekhreonine kinase activity (Arlinghaus, 1985; Rapp et al., 1985a). None of the target proteins or lipids (?) of any of the oncogenic tyrosine or threoninetserine kinases is known. Tyrosine kinases and certain serinehhreonine kinases might thus normally function in a regulatory network with feedback and feedforward functions in the control of the cell cycle. Some retroviruses have acquired such genes for kinase functions or other kinases involved in growth control. These virally encoded kinases may perturb the regulatory network and cause uncontrolled activation of the cell cycle (Heldin and Westermark, 1984). c. Oncogene Products Znvolved in Other Cell Processes. Oncogenes have also been implicated in the chain of events triggered by the ligandheceptor interaction which leads to transformation (see Rapp et al., 1985a). Stimulation of fibroblasts with serum or purified growth factors leads to induction of the protooncogene expression of c-fos followed by c-myc (Cochran et al., 1984; Greenberg and Ziff, 1984; Kruijer et al., 1984; Muller et al., 1984). Exposure of myeloid precursor cells to growth factors or agents that induce myeloid differentiation activate the fos protooncogene specifically in the macrophage lineage (Gonda and Metcalf, 1984). Expression of the p53 gene has also been frequently detected in SV40-transformed or cell lines of spontaneous tumors (Crawford et al., 1981). Several unidentified

238

WOLFRAM OSTERTAG

TABLE I1 INDUCTION OF FACTOR INDEPENDENCE OF MURINE HEMATOPOIETIC PRECURSOR CELLSBY SPLEENFOCUS-FORMING VIRUSES

THE

Factor requirement oP Virus

Cell type

Normal cells

Transformed cells None for proliferation, but Epo for terminal differentiation None Apparently none

FVa, Cas-SFFV

Proerythroblasts

Erythropoietin (Epo) or Multi-CSF

FVP MPSV

Proerythroblasts Mix-CFU, BFUE, CFU-GM Proerythroblasts, BFU-E

Epo or Multi-CSF Multi-CSF or other factors Epo or Multi-CSF

CFU-GM, CFU-M CFU-M

GM-CSF, M-CSF, or Multi-CSF GM-CSF, M-CSF, or Multi-CSF

RV

MHSV HaSV

None for proliferation, but Epo for terminal differentiation reduced None Subfraction of CFU-M may be independent

a Factor requirement is defined here as the necessity to add factors in colony assays that are carried out in the presence ofmany other cells which may also supply other required hormone(s).

mRNA proteins have been described which are induced in cells stimulated by growth factors or expressing oncogenes. One mRNA species described by Matrisian et al. (1985) can be induced by transformation with polyoma middle T, src and Ha-ras oncogenes, as well as by EGF. PDGF-stimulated and transformed or tumor-promoting activity (TPA)-treated fibroblasts lead to coordinate expression of several identical proteins (Frick et al., 1985). The function and necessity for protooncogene expression are as yet uncertain, but the complexity of the interactions, only briefly here described, is apparent. Although the mechanism of action of some oncogenes that interfere with proliferation response is here only cursorily described, it provides us with some working models for the action of SFFVs on target cells. These acute oncogenic viruses also interfere with growth factor interactions of the cell (Table 11), some by modulating growth factor receptor levels and other by inducing release of growth factors.

5. Other Positive Factors Involved in Malignant Progression There are a whole series of other specific and nonspecific genetic alterations which may influence progression of malignant diseases. We have already briefly discussed possible cellular mechanisms that

MURINE SPLEEN FOCUS-FORMING VIRUSES

239

inhibit the oncogenic expression of cellular protooncogenes (see above). Yet there also seems to be factors which favor oncogenic activation and expression: DNA sequences closely linked to the protooncogene may be responsible for its activation (Krontiris et al., 1985); the regions where protooncogenes are located may be especially susceptible to chromosome breakage and thus predisposed for rearrangements that result in activation (Yunis and Soreng, 1984); and chromosomal changes, such as trisomies, probably play a key role in certain neoplasias (Klein, 1981a; Hayata et al., 1983; Yunis, 1983). Gene amplification, whether as a consequence of chromosomal imbalance or as a result of the duplication of genes within one chromosome [which may be favored in tumor cells (Sager et al., 1985)], may enhance tumor proliferation. Observations made by analysis of karyotype alterations can thus give us some insight into the genes involved in the progression of malignancy which cannot be obtained from studies using oncogenic retroviruses. Involvement of chromosome 15, on which c-myc is located (Cory et al., 1983, 1985), is observed in leukemias, lymphomas, and other neoplasms in mice, whether they emerge spontaneously (Dofuku et al., 1975) or after radiation (Wiener et al., 1978a,b), chemical carcinogenesis (Wiener et al., 1978b),or retroviral infection (Miller et al.,1979). In one study (Jonasson et al., 1977), only mouse somatic cell hybrids of melanoma with diploid cells which had duplications of chromosome 15 were able to grow as tumors. Joint application of the two oncogenes, v-myc and v-Ha-ras, to primary fibroblasts results in transformed foci; the presence of both viral oncogenes is supposedly sufficient for transformation (Land et al., 1983). A study of the karyotype of cell lines derived from such foci of transformed Syrian hamster embryo cells, however, revealed that these cells, as opposed to normal parental cells, were no longer fully diploid, but had lost chromosome 15. These results not only indicate that neoplastic progression of normal diploid cells may involve more than t w o steps, but also that the normal chromosome 15 either has genes which interfere with malignant progression or has recessive “tumor” genes which only contribute to transformation if the presumptive normal allele is lost (Oshimura et al., 1985). However, some trisomy 15 cases may have evolved secondarily to a primary alteration in the myc protooncogene expression to increase dosage of the altered myc protooncogene (Klein, 1981b, 1983): All three chromosomes in one T lymphoma cell line, TIKAUT, had an identical rearrangement. This suggests an amplification process by

240

WOLFRAM OSTERTAG

double nondisjunction, concurrently with the loss of the nonrearranged normal chromosome. These findings are thus compatible with a general hypothesis on semispecific chromosomal rearrangements by dosage increase of genes (including protooncogenes) directly involved in generating the malignancy.

6. Stem Cells in Leukemia Normal myelopoiesis (see Section 11) proceeds from an ancestral stem cell compartment of CFU-SI and CFU-SII to progenitor and terminally differentiating cells (macrophages, granulocytes, eosinophils, erythrocytes, platelets, and mast cells). The terminally differentiating cells have lost the option for self-renewal. Leukemic myelopoietic cells could be pictured as cells with extensive self-renewal capacity and loss of response to normal differentiation signals (Fig. 26). Only hemato- or myelopoietic stem cells have self-renewal capacity and can thus be considered the first likely target cells for leukemic progression; cells in the progenitor compartment, however, if committed to a defined and lower number of cell divisions until cell death by terminal differentiation, would have to gain a much larger degree of self-renewal capacity. Self-renewal capacity of a hierarchy of stem cells is inversely related to their capacity to enter cell cycle and to contribute to the differentiation of hematopoietic progenitor cells. A mutation in early stem cells that increased the probability of entry into the cell cycle would lead to preferential utilization of this stem cell for myeloid differentiation. Normal cells would then be expected to stay dormant, since the progeny of one abnormal stem cell would supply the need of differentiating progenitors. Stem cells having an increased probability of entering cell cycle would also generate a larger pool of the same stem cells (clonal), and the probability of a further mutation within this compartment (perhaps later in the pathway of the stem cell), leading to further proliferative distortion, would be much increased. Such a proliferative distortion (e.g., lack of response to normal differentiation signals, altered distribution of self-renewal and differentiation, see above) would-in a normal stem cell-not lead to overt leukemia, since the leukemic stem cell would still have to compete with the normal stem cells for entering cycle. A further proliferative distortion within an already existing stem cell clone (see above), resulting in extended utilization of its progeny for differentiation, would, however, lead to an extensive distortion of the proliferative capacity of all the cells: A second clone with increased malignant potential would emerge (see Fig. 7). Many, perhaps the majority of

MURINE SPLEEN FOCUS-FORMING VIRUSES

24 1

leukemias may thus be caused initially by defects in stem cell utilization (Nowell, 1976; Fialkow and Singer, 1985). Questions of stem cell clonality can be investigated in subjects with at least two genetically distinct types of cells, cellular mosaicisms. The most useful probe to investigate mammalian myeloproliferative disorders is the natural mosaicism in females due to inactivation of one X chromosome in each somatic cell, which is a random process occurring early in development and which generates a limited subset of hematopoietic stem cells of each type (see Section 11).The X-linked polymorphism of the glucose-&phosphate dehydrogenase gene in man (Fialkow, 1976; Fialkow et al., 1978; Fialkow and Singer, 1985) and of the phosphoglycerate kinase gene in mice (Nielsen and Chapman, 1977; Bucher et al., 1980; Rabes et al., 1982) has been used most extensively to study clonal evolution of malignant tumors. Insertion of a retrovirus with a resistance marker into a pluripotent stem cell and transfer of thus marked stem cells to congenic mice would be another feasible alternative to establish usable chimerism (Dick et al., 1985; Keller et al., 1985). Karyotype rearrangements, if already present in hematopoietic stem cells such as the Philadelphia chromosome (Fig. 7),can also be useful to trace cell lineage and progression in leukemia (Nowell and Hungerford, 1960). Using such markers, it has been established that the large majority of myeloid leukemia in patients, including CML (Fialkow et al., 1978, 1981b; Fialkow and Singer, 1985), with polycythemia Vera (PV) (Fialkow et al., 1978), with essential thrombocytopenia (Fialkow et al., 1981a), with myelodysplasia (Fialkow and Singer, 1985),and some cases of acute nonlymphocytic leukemias (Fialkow et al., 1981c; Fialkow and Singer, 1985), are of monoclonal origin. A few cases of acute nonlymphocytic leukemia, however, show clonality of the disease in cells with restricted differentiative capacity (granulocyte/macrophage differentiation). Two mechanisms could be responsible for this lineage restriction: The progenitors for the leukemia were cells analogous to committed precursor cells (GM-CFU) or they were multipotent stem cells incapable of erythroid and megakaryocyte differentiation. Not all of the cells derived from an abnormal stem cell clone need be abnormal in differentiation capacity. An interesting case is PV. In this disorder, altered erythropoietin responsiveness of the erythropoietic cells is a well-established feature. Most other hematopoietic cells, although derived from the same stem cell clone, are unaltered in differentiation and proliferation. In in vitro assays, a proportion of the total number of erythroid precursor cells yields well-hemoglobinized

242

WOLFRAM OSTERTAG

CFU-E and BFU-E under low Epo conditions. These Epo-“independent” cells appear to represent a second population (Eaves and Eaves, 1978) to the normal Epo-requiring population. However, data obtained by dividing 7- to 8-day BFU-E and resuspending in Epo-containing and Epo-depleted methylcellulose imply mixed phenotypes from single BFU-E, i.e., production of both Epo-dependent and Epoindependent progeny from the same precursor cell (Cashman et al., 1983). The interpretation of these results suggests that the mechanism responsible for Epo independence may be variably expressed in clonal derivatives, not only between the two daughter cells of a single division, but also from one cell generation to the next throughout the period of differentiation when continuous exposure to Epo is normally required. Since PV is a clonal disease of the pluripotent stem cell, it thus follows that the defect becomes effective to a variable extent in the normally Epo-sensitive phase of differentiation. Philadelphia chromosome-positive and -negative CML, PV, myeloid metaplasia, essential thrombocytopenia, and most cases of acute nonlymphocytic leukemia in adults involve multipotent stem cells. However, there are marked variations in the manifestations of the diseases. One possibility to explain these differences is that the progression of these diseases involves a series of induced alterations in the genomes of somatic cells. These changes may be different in each disorder and determine which cell type will predominate: granulocytic cells in CML, erythroid cells in PV, and platelet precursor cells in essential thrombocytopenia (Fialkow et al., 1981a). These data collected from many patients with myeloid proliferative diseases showing that most of those leukemias have a stem cell origin raise serious questions as to the applicability of studies gained by infecting and transforming myeloid precursor cells (and not stem cells!) with oncogenic retroviruses (see below). None of the acutely transforming oncogenic retroviruses discussed later in this review (myeloproliferative sarcoma virus, viruses with the ras oncogene, Friend and Rauscher SFFV) has been shown to cause a clonal disease of the hematopoietic stem cell. The major target cells for the ras oncogenic viruses, for Friend and Rauscher virus, and perhaps MPSV (see below), are cells already committed to one differentiation pathway. The primary diseases induced by these viruses are not monoclonal but polyclonal, with the possible exception of the disease induced by MHSV. Clonal tumor cells appear only late in the diseases caused by these viruses (Miller et al., 1979; Tambourin, 1979; Ostertag and Pragnell, 1981;Wendling et al., 1981; Yamamoto et al., 1981; MoreauGachelin et al., 1985).These transplantable erythroblastic tumor cells represent clones of committed precursor cells and not of stem cells,

MURINE SPLEEN FOCUS-FORMING VIRUSES

243

but whether they originate by transformation of committed cells (as generally assumed) or of stem cells with subsequent shift to the committed precursor levels remains to be solved. The above-mentioned data on naturally occurring or chemically induced myeloid leukemias set limits for the use of oncogenic retroviruses as models. At present they can only be used to study induction of leukemia in later phases of progression of the disease. Our knowledge of human myeloid proliferative disorders also indicates the need to study interaction of oncogenic agents with truly pluripotent hematopoietic stem cells and putative microenvironmental cells required to control the cycling of very early hematopoietic stem cells (see Section 11). Several acutely transforming retroviruses may also alter stem cell proliferative behavior: Extended self-renewal capacity of pluripotent hemopoietic or an increase of hematopoietic stem cells was found associated upon infection with F-SFFV (Eckner et al., 1982), with RSFFV (Mol et al., 1982; Ostertag et al., 1982; Merchav et al., 1985), and with MPSV (Klein et al., 1981; Ostertag et al., 1981). The data published by Dexter’s group (Spooncer et al., 1984; Wyke et al., 1986) are also of great potential interest: Multipotential stem cells survive in vitro if a recombinant src ontogenic virus is used to infect stem cell cultures. The hematopoietic stem cell itself does not contain the src virus. The authors therefore infer that the src oncogenic virus alters (transforms?) cells of the hematopoietic microenvironment to provide signals permitting survival of uninfected (?) hematopoietic stem cells in culture. IV. Molecular Biology of Murine Spleen Focus-Forming Viruses

A. INTRODUCTION The acute leukemogenic viruses discussed in this review all induce focal cellular proliferation in spleens of adult mice (spleen foci) and hence have all been named spleen focus-forming virus. The foci can be seen as early as 6-8 days after infection, and it is assumed that the first transformation event occurs immediately on infection of relevant target cells. However, the nature of the leukemia each virus causes is unique. This uniqueness is also reflected in the molecular structure of the viral genome. The genetic information embedded in a particular molecular structure is responsible for the function these viruses express in a specific environment. Detailed analysis of the genetic structure of a virus and its variants can therefore be helpful to elucidate this structure-function relationship as well as to obtain information about their origin and evolution.

244

WOLFRAM OSTERTAG

Since the establishment of molecular methods, many retroviruses of avian, murine, and other mammalian origin have been analyzed by these means. Data revealed a surprising uniformity of the overall genomic structure of these microorganisms which consist mostly of four genetic elements: (1)the gag region, coding for the structural proteins of the nucleocapsid, (2) the gene (pol) for RNA-directed DNA polymerase (reverse transcriptase), which in many cases contains deletions rendering the respective virus replication defective and therefore dependent upon the equivalent gene product of a helper virus, (3) the envelope (enu)gene, in many cases substituted by or fused to an oncogene (onc), either one of which is often involved and plays an essential role in the pathogenicity of the virus, and (4) the LTR sequences which are indispensable elements for the regulation of replication and gene expression. In this section, we will try to define the features of the genomic structure of the acutely transforming viruses that account for their leukemogenicity and that influence the specificity of the virus and thus its unique pathology. The transforming genes will be discussed, and where relevant, the differences that distinguish them from their cellular protooncogenic equivalents. Although the action of the oncogene-coded proteins is not clearly understood, we will try to summarize the relevant data obtained from studies of these gene products.

B. ENV-RECOMBINANT SFFV Env-recombinant spleen focus-forming viruses (SFFV) were the first acute oncogenic retroviruses described to cause a short-latency disease in adult mice (Friend, 1957; Rauscher, 1962). Although FSFFV is one of the first molecularly cloned acutely oncogenic viruses (Linemeyer et al., 1980), the relationship of viral sequences to the rapid disease induced by the virus is nevertheless one of the least understood in comparison to other oncogenic retroviruses. One of the main reasons for this deficiency in knowledge is the lack of a cellularly related oncogene in SFFV (see below). In this subsection, we will try to outline molecular features of the different SFFV isolates which may permit us to ask the pertinent question of how this unique group of viruses can transform erythroid precursor cells by virtue of a viral recombinant oncogenic sequence.

1 . Origin of SFFVs and Dual Nature of the Virus Complex Three independent isolates of erythroleukemia-inducing SFFV have been studied (Friend-, Rauscher- and Cas-SFFV). All of these are complexes composed of at least two components: a replication-

MURINE SPLEEN FOCUS-FORMING VIRUSES

245

competent murine leukemia virus (MuLV) and a defective SFFV. The SFFV component is responsible for spleen focus formation and rapid induction of leukemogenesis. Although the different viruses have been isolated independently, they are all to some extent similar in their pathogenicity (see also Section V). All of the SFFV complexes induce a proliferative expansion of the late erythroid precursor population, the late BFU-E or the CFU-E cells, on infection of adult F V - ~ ~ mice (Tambourin, 1979; Ostertag and Pragnell, 1981; Seidel, 1982). An expansion, even though at a reduced level, can also be found if helper-free SFFV of the Friend or Rauscher virus complex is used to infect mice (Berger et al., 1985; Bestwick et al., 1985; Wolff and Ruscetti, 1985).This interaction of SFFV with erythroid precursor cells interferes with the natural erythropoietin response of erythroid cells (Tambourin, 1979; Ostertag and Pragnell, 1981). However, there are important differences in pathogenicity with different SFFV isolates (see Section V; Tables I1 and VI). The original isolate of Friend virus complex was obtained 2 weeks after injection of extracts of Ehrlich ascites mouse sarcoma cells into a Swiss mouse b y Charlotte Friend (Friend, 1957). Further passages as cell-free extracts through Swiss mice yielded a virus that induced an erythroleukemia, splenomegaly, and hepatomegaly, but did not transform fibroblasts in uitro. The virus was passaged independently in several laboratories as uncloned Friend virus preparations (Axelrad and Steeves, 1964; Mirand et al., 1968; Lilly and Steeves, 1973; Ikawa et al., 1976). The first biologically active SFFV in cloned cells became available later (Ostertag et al., 1972, 1974). During passage in mice, virus stocks were isolated which had altered biological properties: In contrast to the mild anemia induced by the original isolate (FVa), mice inoculated with these virus stocks (FVp) developed a rapid polycythemia. Reflecting their distinct pathology, the different viral complexes were named FVa and FVp for anemia- and polycythemia-inducing Friend virus, respectively (Fig. 8). The anemia-inducing F-SFFVs are characterized by their property to cause a mild anemia when injected into adult mice. Infection of erythroid cells in uiuo with R-SFFV or F-SFFVa replaces the erythropoietin requirement for proliferation of erythroid cells up to the late BFU-E stage (Steinheider et al., 1979; Tambourin, 1979; Hankins and Troxler, 1980). The terminal differentiation of erythroid cells is still dependent on the presence of erythropoietin (Steinheider et al., 1979; Hankins and Troxler, 1980). SFFVa (Friend, 1957; Troxler and Scolnick, 1978; Steinheider et al., 1979; Fagg et al., 1980; MacDonald et al., 1980b; Mager et al., 1981; Ostertag et al., 1982) induces a similar anemia as R-SFFV (Brommer and Bentvelzen, 1974; de Both et al.,

246 HELPER

WOLFRAM OSTERTAG F-MuLV

VIRUSES

F-MULV V I R U S COMPLEXES

ANEMIA V I R U S E S

I I

t

R-RuLV

F-HCF

R-MULV

1 t

R-NCF

GAS-MuLV

CAS-NULV

+

CAS-nCF

FV-A (F-MuLV + F-SFFVA)

RV (R-HULV + R-SFFV)

CAS-V (GAS-MULV + CAS-SFFV)

I

R-SFFV

CAS-SFFV

F-SFNA

I

.1

POLYCYTHEMIA VIRUSES

F - S F F W AX

F-SFFVP SL

F-SFFVP M I

F-SFFVP IK

FIG.8. Origin of the SFFVs. Recombination events that led to the SFFV were probably similar for Friend-, Rauscher-, and Cas-SFFVs. Helper virus recombined with cellular “MCF-like” sequences, generating MCFV. Further recombination events may have resulted in defective anemia-inducing viruses. The polycythemia-inducing F-SFFVs evolved by additional alterations of the F-SFFVa genome.

1978; Hasthorpe, 1978; Mol et al., 1982; Sytkowski et al., 1983) and the Cas-SFFV (Langdon et al., 1983a,b). The polycythemia-inducing F-SFFV contains four related strains of F-SFFVp: the Axelrad (AX) strain (Axelrad and Steeves, 1964), the Mirand (MI) strain (Mirand, 1968), and the Steeves-Lilly (SL) strain (Lilly and Steeves, 1973),and the Ikawa (IK) strain (Ikawa et al., 1976) (Fig. 8).The three FVp strains differ remarkably in the genome constitution with respect to the gag and the pol gene as shown by immunoprecipitation of viral proteins, by oligonucleotide fingerprints, and by nucleic acid hybridization experiments (Bilello et al., 1980; Mol et al., 1982; Langdon et d., 198313; Ruscetti and Wolff, 1984). Molecular cloning and nucleotide sequencing confirmed the heterogeneity of SFFVp substrains. The four strains can be characterized by their gag products (for review, see Troxler et al., 1980b). The genetic heterogeneity of SFFVp is most likely a consequence of two processes: new recombinations of the SFFV genome with replication-competent mink cell focus-forming virus (MCFV) or MuLV and accumulation of mutations in those parts of the genome which are not required for biological activity. New recombination between SFFV and helper virus sequences can be inferred from data of Mol et al. (1981)and Obata et al. (1984). Whether the genetic heterogeneity of SFFVp is also of biological consequence is at present uncertain. An interesting indication of biological divergence of SFFVp variants is still unexplained: Most SFFVp variants induce a large increase in CFU-E density in the spleen and only a very small increase, if at all, in the bone marrow (Liao and Axelrad, 1975; Peschle et ul., 1980).

MURINE SPLEEN FOCUS-FORMING VIRUSES

247

This is different for an SFFVp complex released by some Friend cells and cloned in fibroblasts (Fagg et al., 1980; Ostertag and Pragnell, 1981; Fagg and Ostertag, 1982). The Rauscher virus complex was isolated after serial transplantation of an ascites tumor derived from a virus-induced leukemia. These tumor cells that had been passaged many times through Swiss and BALB/c mice were found to release virus capable of inducing an erythroleukemia in susceptible adult mice (Rauscher, 1962). The virus complex was characterized by the induction of an acute disease with enlarged spleen and liver as well as the appearance of nucleated red cells in the peripheral blood. Depending on the dose of virus injected, most of the animals die within 40 days. Animals surviving the first mortality peak develop a lymphatic leukemia (Tambourin, 1979; Ostertag and Pragnell, 1981). A new SFFV (Cas-SFFV) was isolated by Langdon et ul. (1983a,b). Cas-SFFV is a virus found on inoculation of newborn NFS/N mice with a cell-free extract from a splenic lymphoma induced by the wild mouse ecotropic MuLV Cas-Br-M. Serial passages in NFS/N mice and cloning in tissue culture followed. Cas-SFFV injection in adult mice results in an erythroleukemia similar to that induced by F-SFFVa. The original virus complex contains also MCF virus. However, this MCF virus complex does not induce erythroleukemia in adult animals. The erythroproliferative disease induced by this group of viruses is caused by a viral complex which is composed of at least one helper virus, MuLV, and the defective SFFV. First evidence of this viral complex came from the separation of the viral components by physical methods and isolation of nonproducer cell lines on end-point dilution (Fieldsteel et al., 1969; Steeves and Mirand, 1969; Bentvelzen et al., 1972; Steeves, 1975; Troxler et al., 1977a; Pragnell et al., 1978; Bilello et al., 1980; Ruta and Kabat, 1980; Troxler et ul., 1980). The two-component nature of the FV complex was also established by analysis of the viral RNA (Maisel et al., 1973). The RNA molecules coding for helper virus (longer RNA) and for SFFV functions (shorter RNA) associate as homodimers in the viral particles of the FV complex (Dube et al., 1976). Rescue of the defective SFFV component in fibroblast nonproducer cells by different helper viruses was carried out. The resulting FV complexes (with different helper viruses) all induce erythroleukemia in adult mice, whereas the helper virus by itself does not cause erythroleukemia (Troxler et al., 1977d; Bilello et al., 1980; Ruta and Kabat, 1980). Pseudo-types of SFFVp with the Moloney-MuLV, the Friend-MuLV, and the Gross-MuLV transfer erythropoietin indepen-

248

WOLFRAM OSTERTAG

dence to infected CFU-E (Fagg et al., 1980; Fagg and Ostertag, 1982). Reciprocal exchanges of F-MuLV of FVa and of FVp complexes were done and only FV complexes with SFFV from FVp origin confer erythropoietin independence to infected erythroid precursor cells (MacDonald et al., 1980b). All of these experiments as well as the generation of a transient erythroleukemia by helper-free SFFV (see above) show that transformation specificity is a property of the SFFV component and not of F-MuLV. 2. Role of the Helper Virus F-MuLV or R-MuLV may also have their own pathogenic specificity (Fieldsteel et al., 1969; Tambourin, 1979; Troxler et al., 1980a; Ostertag and Pragnell, 1981; Habara et al., 1982; Balachandran et al., 1984). This could be of potential relevance to the evolution of the recombinant derivative virus. Rauscher-MuLV may have some degree of B cell specificity of transformation (Balachandran et al., 1984). An independent clonal isolate of R-MuLV was shown to induce T cell lymphoma (Vogt, 1982). Steeves and Lilly (1971) and others (for review, see Tambourin, 1979; Troxler et al., 1980a; Ostertag and Pragnell, 1981) have shown that the ecotropic F-MuLV is able to induce a splenic leukemia when injected into newborn susceptible mice (Troxler and Scolnick, 1978; MacDonald et al., 1980a; Oliff et al., 1980; Troxler et al., 1980b; Ishimot0 et al., 1981; Ruscetti et al., 1981a).Two types of F-MuLV helper virus have been cellularly cloned and biologically characterized. One type, termed Friend lymphatic leukemia virus (F-LLV) (Fieldsteel et al., 1969), induces a lymphatic leukemia 2-4 months after injection into newborn sensitive animals. The other type, F-MuLV, induces erythroid hyperplasia (Troxler et al., 1980a) with a somewhat shorter latency after injection of the virus into newborn animals. The disease specificity of the helper viruses appears to be determined by the viral LTR (Bosze et al., 1986). Recombinants between F-MuLV and Mo-MuLV, as well as between F-MuLV and amphotropic MuLV (and other MuLVs) constructed by exchanging fragments containing the 3’ end of the genomes, demonstrate that the LTR is responsible for the tropism of the viruses (Chatis et al., 1983, 1984; Des Groseillers and Jolicoeur, 1984; Des Groseillers et al., 1983; Oliff et al., 1984; Holland et al., 1985). F-MuLV-induced disease in newborn mice progresses relatively fast (6-9 weeks) compared to that induced by other ecotropic helper viruses, such as Mo-MuLV (3-6 months). The long latency in inducing helper virus-related prolifera-

MURINE SPLEEN FOCUS-FORMING VIRUSES

249

tive alterations of hematopoietic cells makes it likely that F-MuLV does not act directly. In accordance with the slower onset of disease as compared to that induced by SFFV (5-9 days), it was observed that infection of newborn mice with MuLV generates a new recombinant virus, the mink cell focus-inducing virus (MCFV). This new virus is characterized by its dual tropism (Hartley et al., 1977), the ability to infect not only cells of murine or rat origin, but also cells of other species. Friend-MCFV (F-MCFV) causes an accelerated erythroid hyperplasia (compared to F-MuLV) when injected into newborn mice (Troxler et al., 1978). The activation of MCFV in induction of erythroblastosis was analyzed in detail using different mouse strains inoculated with F-MuLV. The action of MCFV virus appears to interfere with helper virusinduced disease, since mice, e.g., C57BL/6 and DBN2, which express endogenous MCFV enu proteins are remarkably resistant to F-MuLVinduced disease (Ruscetti et al., 1981a, 1985). Expression of MCF-like envelope protein, as has been reported in DBN2 (Bassin et al., 1982), could possibly lead to blockage of the specific MCF cell surface receptors leading to resistance to MCF viral infections. Indeed, a strong correlation between cellular resistance F-MuLV infection and expression of a glycoprotein has been reported (Kai et al., 1986).Observed resistance of certain mouse strains to F-MuLV which express MCFlike glycoproteins would support the hypothesis that recombination of F-MuLV with endogenous xenotropic sequences to create a MCFV is necessary for the pathology of F-MuLV. Resistance could also be correlated to the presence of additional MCFV-specific DNA fragments in mice resistant to F-MuLV-induced erythroleukemia (Silver, 1984). The acceleration of leukemogenicity by MCFV as compared to MuLV was suggested to be partly a consequence of synergistic effects of both helper viruses. The formation of MuLV envelope pseudotyped particles of MCFV may allow the reinfection of susceptible cells, even if they are already infected with F-MuLV (Rein, 1982; Chesebro et ul., 1984). Pseudo-types of F-MuLV and F-MCFV may also have specific pathogenic effects not generated by either F-MuLV or F-MCFV alone (Sitbon et al., 1985). Similar to Friend helper viruses, it seems likely that the original Rauscher virus (RV) complex also contains two distinct replicationcompetent helper viruses: R-MuLV and R-MCFV (Mol et al., 1982). The analysis of one RV complex, however, showed the presence of at least four distinct viruses, all of which have been cellulary cloned (van Griensven and Vogt, 1980). In addition to the previously described ecotropic Rauscher helper virus (R-MuLV) and the SFFV, an MCFV-

250

WOLFRAM OSTERTAG

type and an XC-negative replication-competent virus could be isolated. Results on the detailed analysis of the genomes, the gene products and nucleotide sequences of helper viruses, and helper virus-associated recombinant viruses suggested that SFFVs arise by recombinational events involving at least ecotropic MuLV and MCFV (see below). Analysis of the genomic structure of F-SFFV by hybridization experiments reveals homologies not only to the ecotropic F-MuLV, but also to mouse xenotropic viruses (Troxler et al., 1977b,c). Hybridization experiments show that F-SFFVp also has sequences homolo. led to the hypothesis that gous to MCFV (Troxler et al., 1 9 7 7 ~ )This MCFV is the precursor of SFFV. In experiments designed to isolate SFFV-related viruses from MuLV-infected animals, F-SFFV cDNA was hybridized to the RNA from spleens of leukemic mice. Suprisingly high amounts of RNA with xenotropic-related sequences could be detected in the spleens of the infected mice (Troxler and Scolnick, 1978). Replication-competent F-MCFV from ecotropic helper-infected mice can be isolated easily (Troxler et al., 1978). Attempts to directly observe conversion of F-MCFV to an SFFV with properties of F-SFFV or R-SFFV, however, were not successful (Troxler et al., 1978). 3. Genomic Structure The finding that all acute oncogenic retroviruses except SFFV show transformation-specific sequences related to cellular protooncogenes led some groups to question the oncogenesis of SFFV (Duesberg, 1985). SFFV possibly could induce a disease solely by virus reinfection and short-term proliferative response of hematopoietic target cells. Infection with a helper-free virus has shown that the defective SFFV subunit by itself induces erythroleukemia, which, however, is transient (Berger et al., 1985; Bestwick et al., 1985; Wolff and Ruscetti, 1985), although infection of a few cells with SFFV alone may be sufficient to generate a truly malignant erythroleukemia (Wolff et al., 1986). The uniqueness of F-SFFV or R-SFFV in generating a fast proliferative disease and lacking sequences related to cellular oncogenes raised speculations whether endogenous MCFV en0 sequences actually represent normal regulatory signals for some hematopoietic cells and are thus possibly counterparts of protooncogenes (Mak et al., 1980; Risser et al., 1980). Molecular analysis should give us a hint as to which part of the SFFV genome is required for its unique functions. With the exception of the recently isolated Cas-SFFV, all strains of SFFV have been

25 1

MURINE SPLEEN FOCUS-FORMING VIRUSES

molecularly cloned (Linemeyer et al., 1980; Yamamoto et al., 1981; Kaminchik et al., 1982; Bestwick et al., 1984; Hess et al., 1984) and sequence information of the enu and LTR region of most SFFVs is available (Amanuma et al., 1983; Clark and Mak, 1983; Bestwick et al., 1984; Wolff et al., 1985). In the following, we will try to summarize some of these data. a. General Structural Features of SFFVs. The genome size of both the anemia and the polycythemia-inducing SFFVs is remarkably variable. This probably reflects their “long” evolutionary history in different laboratories and also indicates that large parts of the genome are not essential for the functions for which SFFVs have been tested, namely, rapid induction of erythroleukemia in adult animals. The genome of F-SFFVa is only 5.2 kb in length (Kaminchik et al., 1982); one R-SFFV isolate has a genome size of 8.3 kb (Bestwick et al., 1984; Hess et al., 1984) (Fig. 9), another is similar to that of SFFVp (Mol et F - SFFV, 5 s

B P

BB

5SP

5 5

P

5 5

P

B9 1 0

5 5

5 5

B

S

B

I

l

B

B

S

R

5 5

B

l

1 P

P

F - SFFV

I S

P P

S P

; t

B

P

5 s

Psti

P

P Ik

F - SFFVp Ls

I ’kbP I FIG.9. Comparison of the restriction maps of the env-recombinant SFFV. Key: S, SstI; K, KpnI; B, BumHI; Bg, BglII; Bs, BstEII; H, HindIII; P, PouII; Ps, PstI; R, EcoRI. The restriction maps are taken from Kaininchik et al., 1982; Wolff et ul., 1984: FSFFV, Amanuma et ul., 1983: F-SFFVp Ik; Yamamoto et ul., 1981; Clark and Mak, 1983: F-SFFVp Ax; Linemeyer et al., 1980; Wolff et al., 1985: F-SFFVp LS.

252

WOLFRAM OSTERTAG

al., 1982). The genomes of the F-SFFVp group of virus passaged in animals vary between 6.0 and 6.3 kb (Linemeyer et al., 1980; Yamamot0 et al., 1981; Amanuma et al., 1983). An intriguing size heterogeneity of SFFVp in erythroid-transformed Friend cell lines has been reported (Ostertag and Pragnell, 1978; Housman et al., 1980). SFFV genomes released by Friend cells induced to differentiate show a larger genome size than those found in the supernatant of uninduced cells (Ostertag and Pragnell, 1978). Whether this change is related to the fact that SFFVp released by uninduced Friend cells versus Friend virus released by induced Friend cells has a different target cell pattern (T. Franz e t al., unpublished; see also above) still remains an open question. The heterogeneity in genome size of different SFFVp is mainly due to deletions in the gag-pol region of the genome. This variability is paralleled by a comparable heterogeneity in the overall genome: The restriction maps of the SFFVs show remarkable diversities (Fig. 9). Although common restriction sites are usually found within the LTRs, many restriction sites are different. It is therefore not possible to determine the location of the gag-pol deletion in the genome of the SFFVs on the basis of restriction site analysis alone. More information about the general mechanism for the evolution of SFFVp variability can be aquired by direct comparison of the nucleotide sequence. Only one of the SFFVp genomes is completely sequenced (Clark and Mak, 1983). Comparative analysis of this sequence suggests that SFFVp is generated by a complex sequence of recombinations involving most likely a minimum of three retroviruses, namely, an endogenous retrovirus related to AKV, FMuLV, and a virus with xenotropic sequences. The recombination point of xenotropic/ecotropic SFFV-enu sequences is similar to that of the F-MCF virus. The region 3’ from the 5’ LTR (U5) to the 3’ half of the gag gene shows more than 95% nucleotide sequence homology between SFFVp and AKV. The remainder of the gag region and the nondeleted part of pol of SFFVp has 75-95% homology to both AKV and F-MuLV: More than 90%homology to xenotropic and xenotropicderived MCFV sequences is found from the 3’ end of pol to the middle of the env gene. The 3’ e n d of env is 90-100% homologous to FMuLV. The 3’ LTR (see below) is similar to that of MCFV. Comparison of nucleotide sequence homologies is helpful to determine the putative recombination points between different viruses, even if homology is found on the level of the amino acid sequence (e.g., for the SFFV-env gene; see below). b. LTR-US Region a n d the gag Gene of SFFV. The most 5’ part of

MURINE SPLEEN FOCUS-FORMING VIRUSES

253

the retroviral RNA genome, the US of the LTR, is similar to that of an endogenous retrovirus (see above). In replication-competent retroviruses, the gag and the pol gene code for a polyprotein Prl80gag-pol which is cleaved to Pr65gag and pol. The gag polyprotein is further processed to p15, p12, p30, and p10, which contribute to the formation of the viral capsid (for a review, see Weiss et al., 1982). Most of the SFFVs have an altered gag structure as a consequence of deletions in the gag-pol region. The variation in expression of gag proteins in the different SFFV strains may reflect this feature (Barbacid et al., 1978; Bilello et al., 1980; Ruscetti et al., 1980; Ruta and Kabat, 1980). The molecularly cloned R-SFFV has a larger genome size and codes for normal gag and pol proteins (Bestwick et al., 1983). Since no correlation could be found between the expression of gag protein and the pathogenicity of various SFFV strains, it appears unlikely that gag is important for the induction of the erythroleukemia. Studies on recombinant and mutated viruses also appear to exclude the gag region as an important structural region specifying the leukemogenic action of SFFV (Linemeyer et al., 1982; Ruta et al., 1983).This is in contrast to data on leukemia induced by the Friend helper virus F-MuLV, where gag sequences may contribute to leukemogenesis (Oliff et al., 1985). c. The pol Gene. The pol gene of replication-competent viruses codes for the reverse transcriptase, an RNA-dependent DNA polymerase, which also has RNase H activity (Weiss et al., 1982). Other functions are in addition encoded by the pol gene: An endonuclease activity was found in Rauscher virus preparation (Kopchick et a2., 1981).The int sequences of pol code for a trans-acting factor which is involved in the integration of the proviral genome. Other sequences at the 3' end of pol determine the efficiency of enu RNA splicing in a cisacting way (Hwang et al., 1984). Most SFFVs have deletions in the po2 gene. Only the R-SFFV with the larger genome size expresses a functional reverse transcriptase (Bestwick et al., 1983). This finding is remarkable, but the fact that a second R-SFFV most likely does not code for a functional polymerase (Mol et al., 1982) makes it unlikely that reverse transcriptase is important for the acute pathogenic effect of Rauscher virus. 3' pol sequences of various lengths are still retained in different SFFVs as a conquence of different deletions in pol. Whether the remaining sequences of pol are still functional is uncertain. The reading frame of the po2 and enu gene overlaps by 58 nucleotides as reported for F-MuLV and FMCFV (Koch et al., 1984) and for R-SFFV (Bestwick et al., 1984). Only three amino acid exchanges within the frame of po2 have been

+3 AA (F-MuLV)

1.

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

.....

+ 3 4 AA ( F - M u L V )

. ...-GDRE.V..GNHPLWTWW.

. / . W . P...M.ALS.C..PLT----SLTPRCNTAW

ttt

ttt

.

F-HuLV pF-HuLV57 1 .ACSTLP.SP D.RD LFLS.KG.RSAAQGS E. .V F-MCFV o F M 5 4 B 2 REGPAFSKPLKDKlNPWGPLlVLGlLIRAGYSYPHDSPH~VFNVTWRVTNLHTG~TANATSLLGTRTDAFPKL FDLCDLIGDDWDETGL----GCRTPGGRKR F-RCFV ~ F H ~ / ~ F H 3 Z .ACST .......................... i...........Q....................................... V Y..OPEPDIGD........ R. 4 .ACST I...........O. K..... R-RCFV-1 F-HCFV F r N x 5 Q M. .. F-SFFVa 6 0 1 1 1 3 6 1 Q....................................... .. R-SFFV 7 1 q....................................... M F-SFFVp L S 5 2 - 3 6 8 5 T.......I...........QL......S.........................E..... F-SFFVp I k K-1 9 .K N ................. Q....................................... .. F-SFFVp A x 205 10 .K E..... D.................. R. PI-RCFV 11 I Q V

....

____

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

...... .. . . . . . . . . . . . . . . . ._ ._ ._ ._ . . . ................................. ..................................... . . . . . . .- . . . . . . . . . . . ..................... ........... _--_ ...................... ..................... ........... ...... ................... ..... ....... ........ ............................... ............................... ................... . . . . . . . . . . . . . . . ._- ---_- _ ..................... ........... ........................ ..................................... +lo

AA ( F - R u L V )

. -. .- - ...... 1...... .... - - -

.- .......- ............. - ..- ..... -

- ..- ..- ..- ......

.... ..^ ~ . .

-..

I-,-

2

. I

............ .............................. ........ .. .................................................................. ................................. ................................................. ............. ...... ................... ...................................................................... ........................... ........................................................ ...-.... ...... ............ ..... ........................................................ ...... ............ ........................................................ .......-.... ...... ...... ...........

3 4

5 6 7 8 9 10 11

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

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

+13 A A ( F - H U L V )

3 4

5 6 7

8

9 11 1 0

tt.

f ......... ....... ....................... ....... ............ I ............................ G . . . . . . . . . . R . . . . . . P P P . . . . . R V P G T A P P S Q . . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . ........ . P . . .................. . V . . ...... I . . ..... . R . . ........ P . P . . .. . V P E T T P P S P . . E . . .. .Q-----........ . S . l r ] .............................. . V . . ......................... . P . P . . .. . V P E T A P P S Q . . .............o. .......................... D ....V . . . S . . . T . . . . . L . . . . . T . P . . . L . . P . P . . . . . Y P E T A P P P q . . . A . . . . . . . . . . . . . . ............................... V ........S..S................P.P.....VPETAPPSO.........H........ ... .Q......... N . . . . . . . . . ......... P . . I . . ......................... I G . . .......V R . . .... . P . . ... . R V P G T A P P S Q . . .=. ............ I .................. V S ........................... P . P .....V P E T A P P S Q ................ R...............

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

q k......... HV V...R GL1.GIRLRVQ.L Y.......LA...SLP..NP....K.AK....PTQPP.........PA......... K ~ W G L R L V R S T G T D P V T R F S L T R Q V L N l G P R l P l G P N P V ~ T D ~ l P P S R P V Q l M L P R P P Q P S P T G A A S ~ - - - - - - - - - Q P G T G D R L L N LD G A V Q A L N L T S P D K T Q E C W L R Y........I...................P.P.....VPETAPPSQ........... R V........I...................P.P.....VPETAPPSO...........

t195 AA (F-RuLV,F-MCFVs)

1 2 3

t*t *** 1 . ..............................................................................

C L V S G P P V Y E G V A V L G T Y S N H T S A L K E E C C F Y A D H T G L V R D S ~ A K L R E R L T Q R ~ K L F E S S ~ ~ W F E G L F N R S P ~ FSTTTILM G P L I I L L - - L I L L F G P C I L AE.

...

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

.............I . ...................................

N

........ T . . . . . . . . . . . . . . . . . .

300

+ 3 3 AA (F-HuLV.F-MCFV) I 399

_.....................

__ ..................... ... 1 . S UTLHS ..I . . . . . . . .....WTLHS ... . . . . . F ..... .... ..... L . . . . . . . . . ........ W . . - -M. ........

F .A 11

200

R.LKL ~n H . K ~ uS~..AS KV ~.IVUNNL.I--------.U UVLKUNKW---HI N . ..yu..i I B ARTFDFVYCPGHTVPTGCGGPREGVCGKWGCETTGQAVUKPSSSUDLISLKRGNTPRNQGPCVDSSAVSSGIQGATPGGRCNPLWLEFTDAGKKASWDGP A L............Q.............D.K........................... T.LV T l.....G..................................KD........-................................. Y A. T...... KO VP 0.K R A. KO..... VL R ....... KDR VQ K 5. KDR VQ N.K

1

1 2

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

100

MURINE SPLEEN FOCUS-FORMING VIRUSES

255

found if F-MCFV and F-MuLV are compared. In contrast, 12 amino acids differ in the frame of enu. Thus, the 3' pol region is conserved to a higher degree than the 5' end of the enu, which may suggest that these sequences are of importance. An area not longer than 165 bp located 3' to the splice junction of the enu mRNA in Mo-MuLV has been ascribed to b e important for efficient splicing of env mRNA (Hwang et al., 1984). This sequence should also be conserved in all SFFVs. d . The enu Gene. The recombinant enu genes of all SFFVs are highly related, which in itself provides strong correlative evidence that this region is essential for leukemogenicity. The 5' part of the gene is related to MCFV enu, whereas the 3' end is homologous to the ecotropic F-MuLV enu gene. Studies on enu gene mutants and recombinants of Friend and Rauscher virus generated in viuo and in vitro prove that this part of the genome is required for the rapid erythroleukemia (Linemeyer et al., 1982; Ruta et al., 1983; Machida et al., 1984, 1985). Most enu genes of the cloned SFFV have been sequenced to identify common sequences which may be of importance for leukemogenicity. Comparison of these sequences with those of F-MCFV, FMuLV, and M-MCFV reveals the following distinguishing and shared features for all SFFVs: (1)All SFFVs have a 585 bp deletion which removes the junction of gp70 and p15E, and (2) a single base pair insertion 3' in the p15E region that leads to a shift in reading frame and premature termination in the p15E region of the eno gene (Amanuma et al., 1983; Clark and Mak, 1983, 1984; Wolf et al., 1983, 1985; Bestwick et al., 1984). These two features, in addition to the recombinant nature of the enu gene (see above), may thus be important to generate a protein which interacts specifically and directly with erythroid cells to induce a proliferative response (see below) (Fig. 10). FIG. 10. The recombinant proteins of SFFV and related viruses. The enu amino acid sequences of all viruses are deduced from the nucleotide sequences and compared to the enu amino acid sequence of F-MCFV using the standard single letter code. Symbols: -,missing residues; ?, possible common glycosylation sites. For best alignment, part of the sequences were omitted at position indicated by /. Sites of recombination between xenotropic and ecotropic sequences, as determined by nucleotide sequence data, are enclosed within horizontal boxes (amino acid 270-300). The sequence data were taken from Koch et al., 1984: F-MuLV pF-MuLV57; Koch et al., 1984: F-MCFV pFM54B and pFMlIpFM2; Adachi et al., 1984: F-MCFV FrNx; Wolff et al., 1985: FSFFVa; Bestwick et al., 1984: R-SFFV; Wolff et al., 1983: F-SFFVp LS; Amanuma et al., 1983: F-SFFVp Ik K-1; Clark and Mak, 1983: F-SFFV Ax; Vogt et al., 1985: RMCFV-1; Bosselmann e t al., 1982: M-MCFV c116.

256

WOLFRAM OSTERTAC

All SFFVps have a 6 bp duplication at the 3' end of the enu gene (Fig. 10). This feature is not shared by R-SFFV and F-SFFVa, both of which induce anemia and do not confer complete independence to erythropoietin interaction of infected erythroid precursor cells as compared to normal erythroid precursors (see below). There are a series of other shared features of the env region of the SFFVps which makes them distinct from the anemia-inducing virus and which may have functional consequences. (1)All SFFVps have the same mutations in positions 328 and 348 which lead to an altered charge (glutamine replaced by lysine residues) in the enu SFFVp. (2)The abovementioned duplication of two codons in SFFVp leads to an insertion of two leucine residues in positions 388/389 in the hydrophobic domain of the enu protein of SFFVp. (3)A serine residue in position 367 replaces a leucine residue in SFFVp. This replacement could alter the attachment of fatty acids, which may alter the membrane attachment of the env protein of SFFVp as compared to that of SFFVa or R-SFFV (Fig. 10). Construction of a recombinant virus between SFFVp and SFFVa has clearly demonstrated that the 3' end of the env gp52 of SFFVp determines the biological difference (Ruscetti and Wolf, 1985b). However, further recombinants, as well as site-specific mutagenesis, are necessary to define the contributing lesion. Compared to the putative, ancestral MCF recombinant virus, there are no specific changes shared by SFFVa and R-SFFV which are not also found in SFFVp. A missing glycine in position 366 of SFFVa (not of R-SFFV) could potentially alter the enu protein coded for by SFFVa. Most of the interesting amino acid changes (see above) are located in the 3' part of the enu gene (Bestwick et al., 1984; Wolff et al., 1985) (Fig. 10).This region is possibly responsible for the altered erythropoietin requirement for erythroid differentiation of SFFVpinfected mice (Kaminchik et al.,1982). Three major conclusions can be drawn from a pure sequence comparison of the enu regions of the SFFVs and the putative ancestral recombinant virus(es): (1)A large and identical deletion joining the gp70 and p15E domain (see below) and a frame shift in the p15E domain is found in all SFFVs. This must be significant for the specific function of the recombinant env oncogene if we assume that R-SFFV and F-SFFVs are products of two independent ancestral recombinant viruses. (2) There are a whole series of coordinate changes in the 3' half of the env oncogene of all SFFVps, which sets SFFVp apart from the R-SFFV and the F-SFFVa. All of these changes which result in amino acid alterations of the enu oncogene could be of significance to the specific feature of SFFVp, i.e., its unique property to completely

MURINE SPLEEN FOCUS-FORMING VIRUSES

257

replace the requirement for erythropoietin in infected erythroid precursor cells. These common changes in all SFFVs may also imply that all SFFVs (including R-SFFV) have the same origin. The coordinate changes of SFFV in the enu region could reflect the recombination with an endogenous virus-like sequence already having these SFFV-specific alterations. It appears less likely that several coordinate changes (as outlined above) have occurred in sequence. The progressive change from an MCFV-type enu region to an SFFVdR-SFFV-like enu region to an SFFVp-like env oncogene would suggest that SFFVa is a precursor of SFFVp and thus the ancestral FVa. This has been pointed out repeatedly by C. Friend (Brown et al., 1985). Further alterations of SFFVa (smaller genome size and other changes) must have followed in the divergent evolution of SFFVs. ( 3 )Lastly, it appears that secondary recombinations were important for the evolution of SFFVs as indicated by different points of crossover of SFFVp and SFFVa. The different crossover point in R-SFFV could either indicate its independent evolution or secondary recombination as has been shown to occur for SFFVp with related viral sequences (Mol et al.,

1982). Rearrangements that generated the ancestral R- or F-SFFV may have evolved stepwise by independent env recombination events. First, recombination of an ecotropic helper virus with endogenous MCFV-like sequences generating an MCFV-type env sequence should have occurred. Deletion duplications and insertions characteristic for SFFV should then have occurred. An alternative model of how the cellular MCFV-enu-related sequences could have generated SFFV-like structures independently is that the MCFV-like SFFV env gene is present in the cellular genome of mice and possibly fulfills some functions analogous to protooncogenes. The fact that Mo-MuLV and F-MuLV recombine with different, but specific endogenous retroviral sequences (Evans and Cloyd, 1984, 1985) could favor a specific process of incorporation of preexisting SFFV-like MCFV enu genes to generate SFFV. e. The LTR-U3 Region. The integrated provirus is flanked by identical sequences termed long terminal repeat (LTR). The U3 region of the LTR not only plays an important role in replication and integration of the virus, but also contains sequences responsible for the regulated expression of the viral genes. Most elements of the LTRs found to be functional in other retroviruses are also present in the LTRs of the SFFVs: (1) inverted repeats at each end of the LTR, (2) an enhancer sequence located in the single direct repeat, ( 3 ) a CAAT box

258

WOLFRAM OSTERTAG

which acts as a polymerase binding site, (4) a TATA box which presumably is necessary for the proper initiation of transcription, and (5) a polyadenylation site which is functional only in the 3’ LTR (Fig. 11). The most striking difference in the U3 region of the LTR of SFFV compared to ecotropic MuLV is the deletion of one copy of the direct tandem repeat containing an enhancer sequence in the U3 region (Bestwick et al., 1984; Clark and Mak, 1984; Wolff et al., 1985). Several of the MCFVs have identical, some different deletions (Adachi et al., 1984; Koch et al., 1984; Vogt et al., 1985). The MCFVs with identical deletions to SFFVs are most likely related to the putative precursor viruses of SFFV. The observations that all SFFVs have lost one part of the direct repeat led to the assumption that this change in the structure of the LTR is significant for the pathogenicity induced by the virus (see below). However, it could also reflect recombination with a specific MCFV LTR (see above). Comparison of several SFFV LTR sequences reveals only very few other variations. In fact, the differences between the LTRs of all SFFVs (about 95%homology) (Bestwick et d., 1984; Wolff et al., 1985) are most likely random. Only the sequence GCG (nucleotide no. 104-106) is common to the F-SFFVps and FMCF 54B, but is different in F-SFFVa and R-SFFV. It is unlikely that this change is of relevance to the biology of SFFVp. It is more likely a change which occurred in the ancestor of all SFFVps prior to evolutionary divergence. A comparison of the U3 LTR sequences of several retroviruses reveals a region of dishomology in the presumptive glucocorticoid receptor binding region flanking the direct repeats between those viruses which alter erythroid proliferation (e.g., SFFV/F-MuLV) and those which presumably interact with lymphoid target cells (e.g., Mo-MuLV/MPSV/Abelson virus family) (see Section IV,C) (Figs. 11 and 16). Reciprocal exchange of the LTRs between M-MuLV and F-MuLV suggests that the LTR determines the lymphoid or erythroid target cell specificity of these MuLVs (Chatis et al., 1983; Des Groseillers and Jolicoeur, 1984; Vogt et al., 1985). This assumed specificity may be a function of the enhancer region of F-MuLV and of F-MCFV (Koch et al., 1984). Indeed, Bosze et al. (1986)have recently shown by using transient expression assays with heterogenous promotors that the enhancer within the F-MuLV LTR has erythroid specificity. The transition of T and A of F-MuLV to C and G (nucleotide no. 149 and 153, respectively) and of three SFFV and one MCFV isolates in the enhancer consensus sequence most likely is not of primary impor-

259

MURINE SPLEEN FOCUS-FORMING VIRUSES

L S 52-16

8

MPSY 0 1 8 - b b l

10

M-MulY PMLVI

11

5 b

n 9

LO 11

1.

.......... . . . . . . . . . . . . . . . . . . . . ...G. ..... : . . . . . .A , . .... . . . . . . . . . . ...G.G. . . . . . . . . . . . . .... . . . G.G. . . . . .l . . . .. I ................ . . . . . . .. A . . . G A l G G .. ....G . A l A ....... ....... . A . . . G A G . - .. . . G U G . A l A . . .... ....... . A . . . G A l G G .. . . . .G . A l A .......

t

enhancer

I

2 3 I

AALlGGAlAiC

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

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

... .C.l.. .c 1 .C.l.... -d.,.

................... .................. ................. . . . . . 1 . A . . . . . . . . ... 1 . A ............ . . . .1 . A . . . . . . . . . . . .. . .

A.GAGAA1l 1GAAlA

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

-01 '0°

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

5

61

8 9 10 I 1 CAAl-box

1

.

4"O

2 4J 5 bI

a 9

10 I1 500

I 2 I I 5

bi

n 9 10

I1

I 2 I

P

i bj 8 9

I0 I1

FIG.11. Comparison of LTR nucleotide sequences of SFFV and related viruses with the LTR of F-MuLV. Missing nucleotides are indicated by -. Possibly significant nucleotide changes are indicated as small boxes. Regulatory sequences are enclosed within large boxes and labeled. The putative glucocorticoid receptor binding sites are indicated by cross-hatched bars. Inverted repeat sequences are labeled i.r.; direct repeats are labeled d.r. The sequences are taken from Koch et ul., 1984: F-MuLV c157, FMCFV pFM54B; Adachi et al., 1984: F-MCFV FrNx; Wolff et al., 1985: F-SFFVa, FSFFVp LS; Bestwick et al., 1984: R-SFFV; Clark and Mak, 1982: SFFVp AX; Bosselmann et al., 1982: M-MCFV c116; Stacey et ul., 1984: MPSV; Shinnick et al., 1981: M-MuLV pMLV1; Vogt et al., 1985: R-MCFV-1.

260

WOLFRAM OSTERTAG

tance, since these changes are not consistent in all erythroid-specific SFFVs (Fig. 11). However, they could, in concert with env-specific regions, be of secondary importance. Experiments with recombinants between F-MCFV/F-MuLV and M-MuLV confirm that the F-MCFV with F-MuLV LTRs contains the sequence responsible for induction of progressive erythroleukemia by F-MCFV (Chatis et al., 1983,1984; Ishimoto et al., 1985).Virus genomes were also generated with independently exchanged env genes and LTRs of Mo-MuLV and FMCFV. Infection of NFS mice with these recombinants led to various leukemias with different latencies. These data suggest that the disease specificity of F-MCFV is not only determined by genetic elements within the LTR, but also by the env gene. Similar results were obtained with R-MCFV/Mo-MuLV recombinants (Vogt et al., 1985). The target cell specificity of slowly transforming MuLV/MCF viruses for erythroid and lymphoid leukemia may thus reside in the LTR region and in the env gene [or the pol-env gene region (Ishimoto et al., 1985)l. A specific interaction of gp70 coded by Mo-MuLV and lymphoid target cells leading directly to altered proliferation of a subset of lymphoid cells has been proposed by Weissmann et al. (1985). We may thus conclude from data on specificity of long latency leukemia viruses on inducing a particular leukemia that both env and LTR (U3) regions are involved. What is presently known about specificity of the acutely transforming SFFVs? We already have pointed out that the env region of SFFV can be considered analogous to a retroviral oncogene (see above). On the other hand, it appears likely that the F-MuLV/F-MCFV and R-MCFV LTRs may confer erythroid specificity in the long latency disease which is only indirectly a consequence of infection by these viruses; however, the direct cause of inducing a transplantable leukemia by these viruses is most likely related to MCFV LTR insertion in the vicinity of an oncogene (see Section 111,C). Surprisingly, the LTR of SFFV seems to play a minor role for generating disease specificity. Viruses with recombinant genomes constructed by the substitution of the SFFVa LTR with LTRs of M-MuLV, M-MCFV, F-MuLV, and F-MCFV showed no pronounced differences in their biological properties (Wolff and Ruscetti, 1986). In the following subsections, we will try to reconcile these seemingly contradictory results to generate a model of the interaction of the LTRs and of the oncogenic regions of MPSV and of SFFV (see below). 4 . Expression and Function of SFFV-Coded env Proteins a. Structure of Recombinant env Proteins. The function of the env protein of SFFVs, which act like oncogene-coded proteins of conven-

MURINE SPLEEN FOCUS-FORMING VIRUSES

261

tional, acutely transforming retroviruses, is obviously of central interest for the understanding of transformation and specificity of transformation by SFFV. Furthermore, knowledge of the cell interaction of the gp70 of the nononcogenic precursor viruses of SFFV appears crucial to causally follow the process of transformation by SFFV. Except for the work of Kabat’s group (see below), gp70 or gp55 (SFFV env protein) interactions with erythroid cells have not received enough attention. All retroviral env proteins are glycoproteins. The unglycosylated form of glycoproteins is synthesized at the rough endoplasmic reticulum, where the first steps of glycosylation occur. Further glycosylation follows during transport to the cell membrane via the Golgi apparatus. The envelope gene of F-MuLV and R-MuLV codes for a glycoprotein of 70,000 Da (gp70), which shares common features with the gp70 of many other MuLVs. The envelope protein precursor pr85 is a polyprotein that is processed by peptide cleavage to gp70 and prl5E, which are connected by at least one disulfide bond (Pinter and Honnen, 1983).The prl5E protein is processed further to p15E by removal of 18 amino acids from the carboxyl terminus by proteolytic cleavage. An additional R peptide predicted from the 3’ nucleotide sequence of MMuLV and precipitated as a 10,000 Da protein with an antibody generated against a synthetic oligopeptide is probably that portion of prl5E which is cleaved to generate p15E (Weiss et al., 1982).A hydrophobic stretch of amino acids in p15E makes p15E a transmembrane protein. The gp70 is located at the outer side of the membrane and is relatively easy to dissociate from the membrane (Marcus et al., 1978; Pinter and Honnen, 1983). The membrane-bound complex of gp70/ p15E in the membrane of the infected cell is used for the final virus assembly of released virions in the process of budding from the membrane and is then utilized as external virus envelope. To determine regions of the env proteins which are possibly responsible for different biological actions, the structure of these env proteins was compared on the basis of amino acid sequences deduced from nucleotide sequence data. Comparison of MCFVs with the parental MuLVs could elucidate the role of env sequences in pathogenesis. The structure of the F-MCFVs differs mainly from F-MuLV in several common deletions (3 amino acids at position 18, 34 amino acids deleted at position 83, 10 amino acids at position 105, and 13 amino acids at position 259) (Fig. 10). How these changes alter the secondary structure and which of them affect the biological properties is still unknown. Construction of MCFVs with deletions in the env genes should help to determine which changes in the env gene alter the biological property of the virus.

262

WOLFRAM OSTERTAG

Comparison of MCFV env sequences to those of SFFV should reveal differences which alter oncogenic properties so drastically: All SFFVs have, in addition to the deletions in the env protein of the MCFVs as compared to MuLV, an additional deletion of 195 amino acids, which removes the junction region of gp70/pr15E (Fig. 10).The altered gp55f52 protein coded by the env region of SFFV was first described by Racevskis and Koch (1977, 1978). gp55 possesses 5 of 8 potential glycosylation sites of F-MuLV and MCFVs. The insertion of one nucleotide leads to a frame shift and results in a unique C-terminus of gp55 in all SFFVs. Premature termination as a consequence of this frame shift shortens the 3' end of gp55 by 33 amino acids. All SFFVps have a duplication of 6 nucleotides in the 3' end of gp55, which results in 2 additional amino acids. Whether this change is responsible for the altered erythropoietin requirement for differentiation of SFFVp-infected erythroid precursor cells remains to be shown. Although the intracellular level of gp55 is similar, or even higher, to that of gp70, only small amounts of gp55 are expressed on the surface of infected, nontransformed fibroblasts or transformed erythroid cells (Ruta and Kabat, 1980; Wolff et al., 1985).This is presumably due to an altered processing and intracellular transport of the gp55 (Ruta and Kabat, 1980; Ruscetti et al., 1981b; Srinivas and Compans, 1983a,b). ,Only about 10%of the env gene protein of SFFVp and less of R-SFFV is expressed on the cell surface of infected cells, whereas the amount of membrane-associated env protein of SFFVa is below detectable levels (Ruscetti et al., 1981b; Wolff et al., 1985). This difference in expression on the cell surface membrane may reflect the putative interaction of gp55 with a growth factor receptor (for erythropoietin?) located in the cell membrane. Recent results of Krantz's laboratory indicate that erythroid cells infected by FVa display one to two orders of magnitude higher levels of erythropoietin receptors (Krantz et al., 1987). Whether this is a direct consequence of SFFVa interaction with target cells or whether this reflects a normal property of the target cells is at present uncertain. It has been recently observed that the mature gp55/52 product of both SFFVp and SFFVa, gp65 and gp60, respectively, are efficiently secreted from cells. The truncated SFFV env protein has lost the hydrophilic carboxyl-terminus normally observed in MuLV env, believed to be important in anchoring the protein to the cell membrane. It has been proposed that loss of these sequences allows the secretion of the protein and perhaps plays an important role in the leukemogenicity of the virus (Pinter and Honnen, 1985). b . Function of Recombinant env Genes. Infection of cells by re-

MURINE SPLEEN FOCUS-FORMING VIRUSES

263

troviruses depends on the interaction of cellular membrane proteins (receptors) with a surface protein (env gp70) of the virus particles (for review, see Andersen and Nexo, 1983; Handelin and Kabat, 1985). Murine retroviruses are divided into four classes based upon their host range, neutralization, and interference properties: ecotropic, xenotropic, amphotropic, and dualtropic (MCF) (Chesebro and Wehrly, 1985; Cloyd et al., 1985).These classes differ in their viral env genes. Little homology is found between the ecotropic, xenotropic, and amphotropic env genes based on analysis of the amino acid and nucleotide sequence. The env genes of dualtropic viruses, on the other hand, share large homology regions with ecotropic and xenotropic env genes (see above). The four groups of murine retroviruses utilize different membrane receptors (Rein, 1982; Rein and Schultz, 1984). The interaction of gp70 with cellular specific receptors is assumed to be the basis for interference with infection within each group of retroviruses. The gp55 which is found in the membrane of SFFV nonproducer cells does not interfere with infection by helper virus of any group. Very little, if any, SFFV-coded gp55 is found in the viral particles (Troxler et al., 1980b; Ruscetti and Wolff, 1984). No data exist as to whether the SFFV-coded gp55 with MCF-like properties actually binds to the MCF receptor or any other specific site of the membrane of, e.g., fibroblast nontarget or, more importantly, erythroid target cells. The SFFV env recombinant “oncogene” codes for gp55 which has been found on the cell surface and accumulated within the cell (see above). Much more gp55 is made in transformed erythroid cells than in nonproducer fibroblasts (Bilello et al., 1980; Ruta and Kabat, 1980). This could reflect differential transcription either of the subgenomic 21s mRNA for gp55 or of all SFFV transcription products (Bilello et al., 1980) and possibly is a property of the LTR which may interact specifically with a product of erythroid cells. Unpublished experiments (Nobis and Ostertag) show that gp55 is found specifically in spleen and bone marrow, but not other tissues, in FV-infected sensimice ~ ) when Mo-MuLV is used as a helper virus, whereas tive ( F V - ~ gp70, coded by Mo-MuLV, can be found in many other tissues. Mice resistant (Fv-2‘) to the erythroleukemia-inducing action of SFFV (see Section V) express somewhat lower levels of gp55 on infection with FVp but show similar tissue distribution as in infected F V -animals. ~~ gp55 or related membrane-active products coded by the env gene of SFFV could either interact in a specific manner with the pathway leading to terminal erythroid differentiation (qualitative model of in-

264

WOLFRAM OSTERTAG

teraction) or could interact with the cell membrane of any cell which permits high levels of gp55 synthesis (quantitative model). What is the location of interaction of the env gene product with regulatory elements of the relevant target cell? Is it the membranebound species which may act as a transmembrane protein, the secreted protein, or the protein which is found within the cytoplasm or even within the nucleus? Kabat and his group isolated a series of mutant SFFVs (Kabat et al., 1980; Ruta et aZ., 1983; Machida et al., l984,1985a,b) by selectively screening SFFV nonproducer fibroblasts deficient in inserting gp55 (or a related glycoprotein) into the membrane. Analysis of these mutants shows that some viral mutants thus isolated are also deficient in causing an erythroid hyperplasia or erythroleukemia. The molecularly cloned R-SFFV mutant 4-3 lacks the carboxy-terminal membrane anchor of its envelope glycoprotein (ecotropic domain) and is only a weakly erythroproliferative virus in vivo (Machida et al., 1985a,b). These data strengthen the hypothesis that the transmembrane location of gp55 is essential for its biological activity. They are in accordance with correlative data that SFFVp, with its strong interference with erythropoietin responsiveness of infected erythroid cells, codes for a gp55 found proportionately more in membrane as the gp55 of SFFVa, which shows less interference with erythropoietin responsiveness (Ruscetti et aZ., 1981b; Machida et al., 1985). Machida et al. suggested as a model for interaction of gp55 with erythroid cells that the two domains, the xenotropic-related aminoterminal region and the carboxy-terminal ecotropic-related regions of gp55, are folded independently and connected by a proline-rich flexible linker. The carboxy-terminal part anchors gp55 to the cell membrane, and the amino-terminal xenotropic-related domain is responsible for inducing erythroblast proliferation. The mutational changes in SFFV which lead to altered membrane insertion of the gp55 in the outer cell membrane could, however, also have unpredictable consequences on the interaction of the major intracellular subfraction of gp55 and its possible interaction with other internal subcellular compartments. A direct approach to study interaction of the SFFV-coded gp55 with the erythropoietin response is obviously necessary to understand the interference of SFFVs with erythroid cell proliferation. C. THE ~OS-ONCOGENIC VIRUSES The mos protooncogene c-mos is a conserved single gene and is located on chromosome 4 of the mouse (Swan et al., 1982) and 8 (band

265

MURINE SPLEEN FOCUS-FORMING VIRUSES

q l l ) in humans (Caubet et al., 1985).The c-mos protooncogenes of the mouse (Oskarsson et al., 1980), of the rat (van der Hoorn et al., 1982), and of man (Blair et al., 1984) have been molecularly cloned. When the coding region of the mouse or rat c-mos gene (but not human) is coupled to viral LTRs, it becomes a transforming gene analogous to the viral mos (v-mos) gene (Blair et al., 1981, 1984; van der Hoorn et al., 1982, 1985). Although the function of the c-mos gene is not well understood, the fact that its transforming activity is indistinguishable from that of v-mos allows us to infer its cellular interactions from studies with the mos-oncogenic viruses. Two independent replication-defective retroviruses bearing the mos oncogene have arisen presumably by recombination between a competent murine leukemia virus and mouse protooncogenic sequences (c-mos) (Fig. 12). Of the rnos-oncogenic viruses investigated to date, including the numerous variants of the Moloney murine sarcoma virus (Mo-MuSV) and the Gazdar murine sarcoma virus (GzMuSV), only the MPSV induces a short-latency, myeloproliferative disease in adult mice. Transforming activity in the animal can be assayed by spleen focus formation. In this section, the viral structure and its relation to the unique function of MPSV will be discussed.

1. Origin of the Myeloproliferatiue Sarcoma Virus Moloney murine sarcoma virus (Mo-MuSV) was first isolated from a sarcoma that appeared following injection of a BALB/c mouse with Moloney murine leukemia virus (Mo-MuLV) (Moloney, 1966). When LTR

LTR

env

gag

Crnos 1

A M o M uS V [MPSV

'1 I1

;

I;

-

]

; I I

Pol

1

7

I f (

I

"mas

1 I

1 I

1

1

I I

I

I

M TRANSLATION

FIG.12. Schematic representation of the recombination of Mo-MuLV with the c-mos protooncogene resulting in acute transforming retroviruses, Mo-MuSV and MPSV. Arrows represent the two translation patterns that would generate the p37 enu-mos protein of Mo-MuSV and the p34 mos protein of MPSV.

266

WOLFRAM OSTERTAG

injected into newborn mice, uncloned Mo-MuSV causes solid tumors, presumably rhabdomyosarcomas. Such a virus-positive solid tumor of the regressing type (Weiland et al., 1979) was transplanted serially in adult BALB/c mice (Chirigos et al., 1968). The virus released by one of the resulting tumors induces “undifferentiated” sarcomas and splenomegaly, and was hence named myeloproliferative sarcoma virus (MPSV) (Jasmin et al., 1980; Ostertag et al., 1980). Symptoms similar to that induced by MPSV may also be the consequence of infection of adult mice with a new acutely transforming retrovirus isolate, the myeloproliferative leukemia virus (MPLV), which in the absence of any known oncogene neither transforms fibroblasts nor elicits tumor nodule formation in animals but generates a myeloproliferative disease (Wendling et al., 1986). Infection of adult mice with MPSV results in a myeloproliferative disease characterized by changes in hematopoietic precursor number and density, initially in the spleen and later in other hematopoietic and nonhematopoietic organs. Myelofibrotic symptoms become dominant in the bone marrow and later, to a lesser degree, also in the spleen. Stem cells and progenitors of all subcompartments of the myeloid system (macrophage, granulocytic, erythroid, and megakaryocytic) have altered kinetic properties (Fagg et al., 1980; Jasmin et al., 1980; Le Bousse-Kerdiles et al., 1980, 1981, 1986; Ostertag et al., 1980; Klein et al., 1981, 1982; Smadja-Joffe et al., 1981). A decline in myeloid cells in bone marrow and later in the spleen is a late symptom of the MPSV disease and also characteristic of the myeloproliferative disease found in some patients (see Section V). Until the virus was biolQgically cloned in 1980 (Ostertag et al., 1980),it was unclear whether the sarcoma-inducing and spleen focusforming properties were coded for by a single viral genome. Endpoint dilution of virus on rat fibroblasts revealed the virus consists of two separable biological entities: a helper virus component, Mo-MuLV, and a replication-defective, fibroblast-transforming virus. The helper virus alone does not induce the myeloproliferative disease. The transforming viral component, however, when propagated with a leukemia helper virus, caused characteristic spleen foci after infection in adult mice. Furthermore, the phenotypical appearance of the MPSV-induced disease was independent of the nature of the helper virus (Fagg et al., 1980,1983; Kollek et al., 1984; Ostertag et al., 1984). The defective component was initially referred to as MPV-SFFV, but is since commonly called MPSV. Cell lines carrying diverse temperature-sensitive (ts) MPSV mutants were also established (Ostertag et d., 1984). These ts viruses

MURINE SPLEEN FOCUS-FORMING VIRUSES

267

transform fibroblasts at permissive (32°C) but not at nonpermissive (39"C), temperatures and are inefficient in causing leukemic transformation in mice at normal body temperatures. These results supported the hypothesis that the fibroblast and hematopoietic transforming properties were related.

2. Genomic Structure a. Relationship to Mo-MuSV. At the time the MPSV complex was biologically characterized, it was still unclear if MPSV with its unique pathogenicity was a variant of the Mo-MuSVlMo-MuLV complex or a new isolate of independent origin. The molecular relationship of MPSV to its progenitors was ascertained by liquid hybridization to specific cDNA probes of Mo-MuSV and Mo-MuLV as well as to probes of other SFFVs (Pragnell et ul., 1981). It was shown that MPSV consisted only of sequences related to the Mo-MuSVlMo-MuLV complex. It had been demonstrated earlier that Mo-MuSV contained sequences homologous to cellular sequences, first called src (Frankel and Fischinger, 1976) and subsequently mos for mouse oncogenic sequences. Southern blotting techniques confirmed the presence of these sequences in MPSV (Kollek et al., 1984). Thus, MPSV, as MoMuSV, is derived entirely of Mo-MuLV and c-mos sequences. No other cellular sequences or sequences unique to other SFFVs had been acquired by MPSV to explain its altered pathogenicity. Whether MPSV w a G result of a new recombination of helper virus and the cmos gene or a result of mutations in the Mo-MuSV genome could not be ascertained. I n order to localize the structural elements responsible for the unique pathogenicity of MPSV and to establish the relationship of Mo-MuSV and MPSV, the integrated proviral DNA was molecularly cloned from NRK nonproducer cells. Concurrently, the proviral DNA of two independently isolated ts mutants was also cloned, ts124 and ts159 (Kollek et al., 1984). Transfection of cloned MPSV DNA in rat fibroblasts and virus rescue upon infection with either F-MuLV or Mo-MuLV yielded an MPSV/MuLV complex, which transformed fibroblasts in vitro and also induced spleen foci in adult mice, thus proving unequivocally that both properties are coded by the same viral genome (Kollek et al., 1984). Transfection using cloned ts mutant DNA yielded cell lines which express a transformed morphology at permissive but not at restrictive temperatures (Ostertag et al., 1984; J. Friel et al., 1987). These results demonstrated that the lesion leading to the ts phenotype

268

WOLFRAM OSTERTAC

is intrinsic to the ts mutant genome and not due to changes in the host cell. Restriction enzyme, heteroduplex, and partial nucleotide sequencing analysis of the cloned genomes enabled a closer examination of heterogeneity between MPSV and Mo-MuSV. b. Deletions in gag and pol Genes. By comparing restriction enzyme cleavage patterns (Fig. 13), two main regions of fragment heterogeneity were observed: (1) in the defective pol gene, where MPSV and the two cloned ts mutants are different from Mo-MuSV and from each other, and (2) in the area 3’ to the mos gene, which was identical in MPSV and its t s mutants, but different from other Mo-MuSV variants (see below) (Kollek et al., 1984). Deletions of varying degrees in the pol gene have occurred in all the cloned Mo-MuSV strains analyzed to date (Tronick et al., 1979; Vande Woude et al., 1979; McClements et al., 1980; Reddy et al., 1981; van Beveren et al., 1981a,b). Except for the newly described variant, 78A-1 (Devaux et al., 1985; Le Bousse-Kerdiles et al., 1985a), MPSV contains the smallest deletion. Restriction analysis of MPSV ts124 indicated a rearrangement in the pol gene, but no deletion as compared to MPSV. Clone ts159, on the other hand, contained a substantial deletion of the pol gene (Fig. 13). The structural features of the molecularly cloned MPSV strains were also analyzed by heteroduplex mapping (Stacey et al., 1984). The results essentially confirmed the findings described above. MPSV wild-type, compared to Mo-MuLV, has a 1 kbp deletion of the pol gene, and most of the Mo-MuLV env gene is substituted by about 1.3 kbp of mos-related sequences. Heteroduplexes formed by MPSV and ts124 are identical. The changes leading to the alterations detected by restriction enzyme analysis or to the ts phenotype are obviously too small to be detected by this method. Heteroduplexes of ts159 to MPSV showed that in addition to the 1 kbp deletion present in MPSV, ts159 contained a further deletion of 1.5 kbp in the pol gene. The deletions or rearrangements within the ts mutant genomes, compared to wild-type MPSV, are of secondary origin, however, since the uncloned precursor cell lines ts124 and ts159 do not show these size alterations. Both exhibit the same biological and ts properties as the biologically and molecularly cloned progenies ts124/1 and ts159/ 511. Furthermore, recombinant analysis of the ts mutant and wild-type MPSV proved that the gross sequence alterations in the pol gene were not necessary for the acquisition of temperature sensitivity (J. Friel et al., submitted).

269

MURINE SPLEEN FOCUS-FORMING VIRUSES

MO-MulV

p18-663

p19-124

p22-143

p20-159

MSV-124

HT I

MI FIG. 13. Comparison of MPSV (p18-663) and its temperature-sensitive mutants (p19-124, p20-159) to Mo-MuLV and the Mo-MuSV variants (MSV-124; HT-1, and m l ) (Kollek et al., 1984). Only the restriction enzyme cleavage sites which are significant for the comparative presentation of the genomes are shown. Heavy lines indicate LTR; cross-hatched bar, v-rnos; dashed lines, deletions.

No deletions or rearrangements were observed in the gag gene by restriction enzyme analysis (Kollek et d., 1984); however, partial nucleotide sequence indicates a small deletion at the gag-pol junction in MPSV (N. Walther, unpublished) which would consequently disrupt p10 expression. A p65 gag-precursor protein has been detected in

270

WOLFRAM OSTERTAG

MPSV-infected fibroblasts with Moloney anti-p30 and anti-pl2 monoclonal antibodies (Ostertag et al., 1982, and unpublished). c . mos and env Gene Junctions. Nucleotide sequencing was necessary to establish if the site of integration of the MPSV v-mos gene in the env region of Mo-MuLV differed from that of v-mos of Mo-MuSV. Sequence data covering the mos helper junctions of the Mo-MuSV variants MSV-124, HT-1, m-1, and Gazdar-MuSV were used as the basis for a proposed interrelationship among Mo-MuSV variants and to the Mo-MuLV helper virus (Donoghue and Hunter, 1983; Seth and Vande Woude, 1985): The 3’ mos-env junction is quite variable between these strains, whereas the 5’ env-mos junction has the same position throughout. The later junction creates an open reading frame for an env-mos fusion protein using the AUG start codon of the MoMuLV env gene. MPSV, however, has one less cytosine residue at this junction point, which may be regarded either as a different junction or as a subsequent deletion (Stacey et al., 1984). This frame-shift mutation establishes the twentieth codon of the hypothetical fusion protein as an amber terminator. The next AUG, located 92 bp downstream of the first AUG (env coded) and within the acquired mos sequences, reopens the main reading frame (Fig. 14) (Stacey et al., 1984). The precise position of the 3’ mos-env junction of MPSV is ambiguous from the sequence data. Perfect homology with c-mos stops at exactly the same junction point as MSV-124,50 bp downstream of the mos protein stop codon; homology to Mo-MuLV commences 12 nucleotides further downstream (Fig. 14). Approximately 200 bp of the 3’ env coding sequences of Mo-MuLV are still present, and due to a frame shift in this region, a novel peptide designated pl5ER’ could be synthesized. d . Comparison of the mos Genes. Is the mos gene identical to either c-mos or to the Mo-MuSV v-mos gene, or does it contain sequence alterations leading to the unique pathogeneity of MPSV? The first AUG in the mos open reading frame in the MPSV genome corresponds to the predicted start codon of c-mos (van Beveren et al., FIG.14. Nucleotide s6quence of MPSV from 895 bp upstream of the mos gene to the 5’ terminus of the LTRs (Stacey et al., 1984). Sequence includes the entire mos gene and indicates the integration sites of the oncogene within the eno-coding region of MoMuLV. Symbols: 0,nucleotide is the same as c-mos; 0, nucleotide is the same as MSV124; W, nucleotide is the same as Mo-MuLV; A, nucleotide differs from c-mos; A, nucleotide differs from MSV-124; 0,nucleotide differs from Mo-MuLV; A, deletion , from MPSV as compared with another sequence (indicated by the number); V, region present in MPSV, but absent in another sequence (indicated by the number). 1, c-mos, 2, MSV-124; 3, Mo-MuLV.

271

MUFUNE SPLEEN FOCUS-FORMING VIRUSES "l"'lll M D M V l V

.P~~~CTTGTGCACAAGTCAACGCCAGCAAGTCTGCCGTTAAACAGGGAACTAGGGTCCGCCG~CACCGGCCCGGCACTCA~TGGGAGA~C~ATTTCACCGA~A~AAAGCC~GCAT~~,~AT~ 0 T

-Pel

Ill.

GCCGACACCAG~~TAAGAACCTAGAACClCGCTGGAAAGGACCTTACACAGlCCTGCTGACCACCClCACCGCCClCAAAGlAGACGGCAlCGCAGClTGGAlACACGCCGCCCACGTGA 0 840 1

xbgl

MoMulV-mas

----BTGGACCATCCTCTIGI\ClGACATGGCGCGllCAACGCATGClCCCAAACTTCCClGGClGllCCTA A ilCATTlCTCCCTAGTGlCTCAlGlGAClGlCCCA960

VXCTGCCGACCCCGGGG

I

.

m

v

f t :

TClGAGGGGGTAATGCClTCGCClCTAAGCClGlGTCGC~ACClCCClCGlGAGCTGlCGCCATCGGl~GAClCGCGGTCCTGCAGCAT~CClllGGlGGCCCCGAGGAAGGCAGGGAAG

f

S

G

A

l

1080

A

T

ClCTlCClGGGGACCACTCCTCCTCGGGClCCCGGAClGCCACGCCGGCTGGCClGGllClCCAlAGACTGGGAACAGGTAlGlCTGAlGCAlAGGCTGGGCTClGGAGGGl

TTGGCTCG 1200

GlGTATAAAGCCAClTACCACGGTGTTCClGTGGCCATCAAGCAAGlAAACAAGTGCACCAAGGACClACGCGCATCCCAGCGGAGlTTCTGGGCTGAACTGAACATTGCAAGAClACGC 1320

Ic"1

t

CACGACAACATAGTTCGGGTTGTGGCTGCCAGCACGCGCACGCCCGAAGACTCCAACAGCCTAGG~ACCATAA~CATGGAGT~~GGGGGCAACG~GAC~CTACACCAAGTCATC~ACGGT 1440

GCCACCCGCTCACCGGAGCCTCTCAGCTGCAGAGAACAAC~AAG~TTGGGGAAGTGCC~CAAGTA~TCCCTAGATGT~GTTAACGG~TGC~T~TTC~CCACTCACAAAGCAT~TTGCAC 1560

t

e

1.1

? 4

GGCACGTACACGCACCAAGCTCCGGAGATCCTGAAAGGAGAGAT~GCCACGCCCAAAGCTGACA~CTACTCTTTTGGAATCACCCTGTGGCAGATGACCACCAG~AGGTGCC~TACTCC 1800

H

GGCGAACC~CAGT ACGT GCAGT ATG CAG ~G C~AG CCTACAATCTG CCCCCCTCAC~G ACAG G G G CG GTGTTC A C C GC C TC C C ~ GA C TGGA A A GA C A C TGC A GA A C A TC A ~ C C A GA A C ~ GC ttszo

G

H

4

l

X CI.1

1.3

A

c

...,

I I

272

WOLFRAM OSTERTAG

1981b; Stacey et al., 1984). Based on sequence analysis of MPSV, one would predict a 34,000 Da mos protein. This is in contrast to the 37,000 Da enu-mos protein detected in MSV-124 transformed NIH 3T3 cells (Papkoff et al., 1982, 1983) and strongly implicated as the transforming protein. However, overlapping series of proteins of MW 37,000, 33,000, 13,000, and 18,000 have been found in in uitro translates of MSV-124 and Gazdar genomic and subgenomic RNA, presumably due to initiation at the different AUG codons (Papkoff et al., 1982). The predicted amino acid sequence of the MPSV mos protein differs from that of c-mos by only three residues. In contrast, when the second AUG is employed, the MSV-124 mos varies from c-mos by 10 amino acids and 12 residues from MPSV mos (van Beveren et al., 1981b) and HT-1 codes for the identical predicted amino acid sequence as c-mos (Seth and Vande Woude, 1985) (Fig. 15). The significance of these novel attributes of the MPSV mos protein and their relationship to the unique biological properties of the virus is difficult to assess without knowledge of the role of these domains in the mos protein function. e. Comparison of the LTRs. Differences in the LTR region of MPSV as compared to Mo-MuLV and Mo-MuSV were not initially detected by restriction enzyme analysis. However, sequence analysis revealed a number of nucleotide alterations in all of the Mo-MuLV derivatives (Dhar et al., 1980; Shinnick et al., 1981; van Beveren et al., 1981b; Stacey et al., 1984). MPSV shows 19 bases unlike MoMuLV, 27 bases unlike m-1, and 38 bases unlike MSV-124. Although point mutations are shared by some of the Mo-MuSV variants, no significant pattern is discernible (Fig. 16). The Mo-MuSV LTR direct repeats have been shown to act as enhancers (Levinson et d.,1982), which are involved in regulation and species specificity of gene expression (de Villiers et al., 1982; Khoury and Gruss, 1983). The position of these repeats within the LTR is identical to the 75 bp repeats of Mo-MuLV, although the length of MPSV repeats is reduced to 74 bp by a matched pair of single base deletions. 3. Structure-Function Relationship mos expression under LTR control has been previously shown to lead to transformation of fibroblasts (Oskarsson et al., 1980; Blair et al., 1981). Work with MPSV ts mutants suggested that the mos gene was also involved in the hematopoietic transformation (Ostertag et al.,

273

MURINE SPLEEN FOCUS-FORMING VIRUSES

1

HT- 1 MSV-124

MARSTPCSQTSLAVPTHFSLVSHVTVPSQGV N

c-mos MPSV HT-1 MSV- 1 24

MPSPLSLCRYLPRELSPSVDSRSCSIPLVAPRKAGKLFLGTTPPRPGLPRRLAWFSIDW

c-mos MPSV HT- 1 MSV-124

EQVCLMHRLGSGGFGSVYKATYHGVPVAIKQVNKCTKDLRASQRSFWAELNIARLRHDNI' O'

c-mos MPSV HT- 1 MSV-124

VRWAASTRTPEDSNSLGTI IMEFGGNVTLHQVIYGATRSPEPLSCREQLSLGKCLKY SL'"

-

6o

A

G

E

D

K

c-mos MPSV HT- 1 MSV-124

G

P

c-mos MPSV HT- 1 MSV-124

THQAFEILKGEIATPKADIY SFGITLWQMTTREVPYSGEPQYVQYAWAYNLRPSLAGAV3m T

c-mos MPSV HT- 1 MSV- 1 2 4

€TASLTGATLQNIIQSCWEARALQRPGAELLQRDLKAFRGALG % N A

G

S

T

FIG.15. Comparison of the predicted amino acid sequence of the mouse c-mos (Van Beveren et al., 1981a) with that of v-mos found in MPSV (Stacey et ul., 1984) and two Mo-MuSV variants, HT-1 (Seth and Vande Woude, 1985) and MSV-124 (Van Beveren et ul., 1981b). The standard single letter code for amino acid residues was used. The first methionine in the v-mos reading frame in HT-1 and MSV-124 is coded for by the enu gene of Mo-MuLV. The arrow represents the start of the acquired mos sequences. Position 1 represents the first methionine in-frame for c-mos and v-mos in MPSV. Differences between c-mos sequences and v-mos sequences are indicated. A dash (-) denotes a deletion. The asterisk (*) denotes the single base change found in the coding region of the ts159 mutant of MPSV (G + R). The putative ATP binding is marked as indicated: overbar, glycine-rich sequence; A, lysine. The conserved amino acid sequence APE that forms part of the sequence KWTAPEA, thought to confer tyrosine protein kinase activity to pp6OSrc,is marked with a dashed line (Van der Hoorn and Firzlaff, 1984).

WOLFRAM OSTERTAG

274 MPSV WMULV m-MuSv Ab-MULV

T T

G C

G

d.r. Mpsv

R

M-WV m-MuSv AB-MULV

-

-

T

A

T G G

CT C

T

A

T

a G

T

CT

G

t

d.r. d.r.

MPSV W-MULV

m-Musv Ab-MULV

drl_

P T A - T A m - R A T A T G G G C C A A A C A G W \ T A A A

CT C

cr

G T

-

C

G

G

MPSV b-MULV WMUSV

Ab-MULV Mpsv

WMULV

m-Musv

Ab-MULV MPSV W-MULV

m-Musv Ab-MULV

MPSV WdlULV

m-Musv

Ab-MULV

G G

G

FIG. 16. Nucleotide sequence comparison of the LTR of MPSV, Mo-MuLV, MoMuSV ( m l variant), and Ab-MuLV. Nucleotide variances between MPSV and that of the three other viruses are noted. Hyphens (-) indicate a deletion and the asterisk (*) represents an insertion of a G residue after the indicated base. The U3, R, and U 5 regions are labeled. IR indicates inverted repeat; heavy solid line denotes homology to glucocorticoid receptor binding site; d.r. designates direct re_peats;hyphenated superscores indicate enhancer core consensus sequence. Promoter sequences are enclosed in a box and labeled. Sequence data were obtained from Stacey et al. (1984), Shinnick et al. (1981), Dhar et al. (1982), and Reddy et al. (1983).

1984). These results were confirmed by recombinant ts viruses and nucleotide sequencing which showed that a point mutation in the mos coding sequence imparted temperature sensitivity to both hematopoietic and fibroblast transforming capabilities of MPSV (J. Friel et d.,1987, and see below). The necessity of the mos gene in the leukemogenic pathology was also demonstrated by an MPSV deletion mutant (Stocking et d., 1985). The mos coding sequences were deleted from an MPSV recombinant encoding the gene for neomycin resis-

275

MURINE SPLEEN FOCUS-FORMING VIRUSES

tance (Ostertag et al., 1985).In the absence of the transformed phenotype, the resistance gene enabled easy selection of transduced cell lines. Although the mos- MPSV variant transfers resistance to fibroblasts, it has lost its ability to transform fibroblasts in vitro and transform cells of the hematopoietic compartment in vivo (Fig. 17; Table

111). These results, however, did not explain why MPSV and not any of the other Mo-MuSV variants causes the myeloproliferative syndrome. An obvious way to elucidate the molecular basis of the unique pathology of MPSV is to construct recombinants of the genetic components of MPSV with structural and functional analogs of related viruses of different biological activity. The following hypotheses were formulated, and consequent recombinants were tested for induction of spleen foci in mice: a. Function of the Altered mos Protein. Due to a single nucleotide deletion at the 5' junction of the helper and oncogene sequences, MPSV encodes for an amino-terminal truncated mos protein (p34) versus the env-mos fusion protein (p37) of Mo-MuSV that has been detected and implicated as the transforming protein in fibroblasts (Papkoff et al., 1982,1983).This and other changes induced by single transformation

fibroblasts S'LTR

J

mos

deleted

1

temperature sensitive mor mutant

gas- deleted

pl5E

deleted

A

J

A

L T R 3'

mor

Poi

909

MPSV

I

+++

of

hematopoietir cells

+++

-rlA1#7 3Z0 + + +

A A

380

A

!

J

A

I

not

done

+++

++.I

+++

+++

FIG.17. Leukemogenicity of MPSV mutants that contain deletions in protein-coding regions or that are temperature sensitive. The ability of the virus to induce in oitro transformation of fibroblasts and to induce spleen foci in infected adult mice (hematopoietic transformation)was determined. The gene for neomycin resistance was inserted in the mos- construct to faciIitate selection (see Fig. 21).

276

WOLFRAM OSTERTAC

TABLE I11 SPLEENFOCUS-FOFIMING ACTIVITY OF MPSV RECOMBINANTS IN MICE ~

Virus Wild types MPSV Mo-MuSV MuLV mos deletion and exchange pC6M- neo pCB61 pCH61

Clone

Genome structure

pC663 ml HT-1 Moloney Friend

MPSV Mo-MuSV Mo-MuSV MuLV MuLV

11 11/3 2 3 12 1 21 28

Deletion of mos

pCH62

5 22 27

pCLS3

5 6 9

Deletion mutants PC6 pC615

~~

4

4 5

c-mos replaces vmos in MPSV genome v-mos of HT-1 replaces v-mos in the MPSV genome v-mos of MPSV replaces v-mos in the HT-1 genome c-mos replaces vmos in MoMuSV genome Deletion of gag Deletion of pl5ER'

6 LTR exchange pCM61 pCL61

12 13 3

MPSV 3' end combined with m l 5 ' end LTR of Mo-MuLV replaces LTR of MPSV

FFU tested in mice

MPSV activity

0.07-0.25 0.00002 0

+

105)

-

106)

0.05 0.02

0 0

5 x lo4 108 3 x 104 (>10R) (>lOS) (2 x (3 x 1x 1.4 x 4.2 x 1x 6.2 x 7x

SFFU/FFU

102 105 10' 105 104 103

-

0.006 0.15 0.15

0 0 0 0

+ + + + + +

3 x 103 5 x 103 2 x 104

0 0 0

0 0 0

2 x 105 4.2 x 105 5 x 105

0 0 0

0 0 0

0.04 0.05 0.06 0.08

+ + +

9 x 104 6 X lo4

0.01 0.013

+ +

4 x 105

0.00009

0

1.4 x 5x 7.0 x 5.0 x

104 104 104 104

t

277

MURINE SPLEEN FOCUS-FORMING VIRUSES

base mutations could have altered the structure of the mos protein sufficiently to gain an increased transforming capacity, and thus the unique pathology. Recombinants carrying the mos gene of different origin were constructed (Stocking et al., 1985).Using these recombinant virus preparations, it was shown that the origin of the mos gene was irrelevant with respect to the action of the virus upon the murine hematopoietic system. Virus particles carrying the mos gene derived from either MPSV, Mo-MuSV, or cellular origin introduced into the MPSV genome were still able to induce spleen foci in mice. However, introduction of these genes into the Mo-MuSV background did not result in a virus with spleen focus-forming activity (Fig. 18; Table 111). tronrformotion 5’LTR

9-0

Llll

POI

l a

InOS

4

J.

fibroblorts

of

hemotopoietir cells

+ ++

+++

+++

-

pC061

+++

+++

pcn.51

+++

+++

pcn6i

+ ++

MPSV

MoMuSV

‘rnos A

c

pCLS3

c++

FIG.18. Leukemogenicity of MPSV mutants that encode either mouse c-mos or vmos derived from Mo-MuSV variant m l and mutants of Mo-MuSV and Mo-MuLV that carry the v-mos coding region of MPSV. The ability of the virus to induce in oitro transformation of fibroblasts and to induce spleen foci in infected adult mice (hematopoietic transformation) was determined. The box labeled sup in the schematic of c-mos represents the UMS sequences described in the text which suppress the transformation capacity of the protooncogene.

278

WOLFRAM OSTERTAG

It could be concluded that mos genes of different origin (e.g., either v-mos or c-mos)are functionally identical and that the increased target cell range of MPSV was not due to an altered mos protein. Furthermore, these results were comparable with those of Anderson and Scolnick (1983), who demonstrated that a change in the cell type specificity and pathological effects of the src oncogene could be brought about by inserting the gene into mouse amphotropic viral sequences, including the LTR. This suggested that some other viral structure may play an important role in determining the target cell specificity of the oncogene. b. Interaction of Other Protein(s) with the Transforming Protein. It could be envisaged that a second protein encoded within the MPSV genome could interact either directly with the mos gene product or with the expression of the mos gene. Two candidates were considered: protein(s) coded by the gag gene or the hypothetical pl5ER’ peptide coded by 3’ enu sequences (see above). A series of MPSV mutants and recombinants was constructed to determine the role of these MPSV-encoded genes in inducing myeloproliferation. Mutants containing deletions of the residual enu sequences, which could code for a truncated, enu-related peptide, were tested and shown to be unaltered in their ability to induce a disease of wild-type phenotype (Fig. 17; Table 111) (Stocking et al., 1985). A gag deletion of MPSV was also generated to exclude an essential role of the gag region of MPSV which can still code for most of the gag proteins (p15, p12; and p30), but possibly not for p10 (N. Walther, unpublished). An essential role of gag was implied for a series of results on acutely oncogenic chick retroviruses by Duesberg and others (review Duesberg, 1980; Bister and Jansen, 1985) and for the Abelson virus (Prywes et al., 1983).The necessity for gag in the gagabl oncogenic protein of the Abelson virus is a consequence of instability of the gag-abl protein in lymphoid cells if parts of gag are deleted (Prywes et al., 1985).The deleted gag- MPSV virus, however, is equally as efficient in generating a myeloproliferative disease and fibroblast transformation as the wild-type virus (Fig. 17; Table 111) (Stocking et aZ., 1986). In contrast to the results with mos- mutants, these results indicate that neither the gag region nor the truncated enu peptide is necessary for the oncogenic potential of MPSV, as their deletion does not abolish the unique pathogenicity of this virus compared to Mo-MuSV. c. The Role of the Long Terminal Repeats. It could be envisaged that the type of disease induced by the oncogene is not a function of the oncogene itself, but rather of the different cell type(s) most com-

MURINE SPLEEN FOCUS-FORMING VIRUSES

279

patible with the particular enhancer structure of the infecting virus. Analysis of differences in the tandem repeats of nonacute MuLVs suggested that these enhancers are important for different tissue tropisms of various virus strains (Chatis et d.,1983, 1984; Des Groseillers et al., 1983, 1984; Koch et al., 1984; Lenz et al., 1984; Holland et al., 1985; Yoshimura et al., 1985). However, the studies on retrovirus LTR specificity have been limited to transformation induced by helper virus (MuLV). The mechanism of the long-latency disease that develops after infection with these viruses is not fully understood, but may require the insertion of an LTR next to an oncogene (see Section 111) and also (at least for the murine system and for true malignant transformation) an MCF intermediate recombinant virus. MPSV, however, is an acute oncogenic virus that most likely interacts directly with the target cell (see Section V). The question of involvement of the LTR in targeting acutely transforming retroviruses is part of a long-lasting controversy on the specificity of oncogenes. Several groups, especially that of Graf and Beug (1978), have proposed specific interactions of oncogenes with specific differentiated cells, whereas Duesberg and others (Bister and Jansen, 1985; Duesberg, 1985) have questioned the evidence in support of such an interpretation. Tissue specificity of oncogenes interacting with specific target cells has never been demonstrated conclusively, with the possible exception of the Friend and Rauscher SFFVs (this review; reviewed by Ostertag and Pragnell, 1981; Ruscetti and Wolff, 1984). On the other hand, many studies have demonstrated that the LTR contains signals which allow tissue-specific transcription (Laimins et al., 1982; Khoury et al., 1983; Oliff et al., 1984; Ishimoto et al., 1985; Rosen et al., 1985). To determine if the LTR was responsible for the unique target specificity of MPSV, recombinants of MPSV were constructed with either part or all of the Mo-MuSV LTR (Table 111; Fig. 19). The results indicated that recombinants carrying all or only the U3 region of the Mo-MuSV LTR still transform fibroblasts in zjitro, but do not induce hematopoietic changes in mice (Stocking et al., 1985).To test whether the MPSV LTR was functionally different from that of Mo-MuLV from whence it was derived, further recombinants were constructed replacing the MPSV LTR with that of Mo-MuLV. The resulting recombinant was at least two orders of magnitude less active than wild-type MPSV (Fig. 19; Table 111) (Stocking et al., 1986). Thus, this is the first reported example demonstrating that the altered target cell range of an acutely transforming retrovirus was due to properties of the LTR and not the oncogene itself.

280

WOLFRAM OSTERTAG t r a n s t o r m o t l o n of flbvoblasts

hematopmlmtlr

rell. MPSV

MoMuSV

+++

+++

+++

-

+ ++

++

env Mo MulV

UJUI pCM6l

A

pCL6I

+++

FIG.19. Leukemogenicity of recombinants between Mo-MuLV and MPSV and between Mo-MuSV and MPSV. The ability of the virus to induce in uitro transformation of fibroblasts and to induce spleen foci in infected adult mice (hematopoietic transformation) was determined.

On the basis of these results, the function of the MPSV LTR can be directly tested in putative target cells. Furthermore, these results have implications for studies on target cells for other acutely transforming retroviruses. The extensive molecular data on the transforming activity of SFFV/F-MuLV and the limited data on the MHV (see below) indicate that MPSV is not the only acutely transforming retrovirus whose target cell range is determined in part by its LTRs. However, it should be kept in mind that alterations of the LTR U3 region of MPSV have not limited the viral specificity, but have extended the target cell range of MPSV expression from fibroblasts to hematopoietic cells. Recent reports indicate that MPSV contains regulatory sequences which are highly active not only in fibroblasts, as is Mo-MuSV, and target cells involved in producing myeloproliferation, but also in F9 embryonal carcinoma cells (Franz et al., 1986a) and epithelial secretory cells (Fusco et al., 1985) and hepatoma cells (unpublished). What are the crucial regions of the MPSV LTR that permit increased transcriptional activity in a wide range of target cells? The tandemly repeated sequences of the U3 region, identified as a transcriptional enhancer element (Blair et al., 1980; Laimins et al., 1982; Levinson et al., 1982), may be the most likely candidates. There are three coordinate changes and one singular change in the direct repeat of MPSV as compared to Mo-MuLV. Mo-MuSV (and MPSV) LTRs also possess

MURINE SPLEEN FOCUS-FORMING VIRUSES

28 1

recognition sequences for glucocorticoid receptor proteins which in part overlap with the enhancer region (see Fig. 16). There are also several alterations in these sequences between Mo-MuLV and MPSV LTRs. This region is altered similarly in the Abelson virus and much more drastically in the Friend SFFV/MuLV (with erythroid target cell specificity) as compared to Mo-MuLV/MPSV/Abelson virus (with lymphoid target cell specificity?). We will readdress this question in connection with our discussion on the target cells of SFFVs and will propose a hypothesis that can easily be tested to account for the target cell specificity of SFFVs (see Section V and above).

4 . Expression and Function of the mos Gene Product a. Transcription of the mos Oncogene. Our knowledge of the transcription of the c-mos protooncogene (Propst and Vande Woude, 1985) and the v-mos oncogene is equally as sparse as our information on mos protein function (see below). Transcriptional analysis has been impeded by the extremely low levels of mos-specific mRNA expressed. The number of protein molecules coded by the mos gene required for transformation is extremely low (Papkoff et al., 1982), and hence 1-10 copies of mos mRNA appear to be sufficient for transformation (Seth and Vande Woude, 1985). Furthermore, the high levels detected immediately after infection with Mo-MuSV appear to be toxic to fibroblasts (Papkoff et al., 1982; Papkoff and Ringold, 1984). The implicit toxicity of the rnos oncogene could lead to selective cell death. Only cells expressing low levels of mos, altered by a cellular control mechanism or with a mutated viral genome, survive. Expression of mos mRNA is regulated by transcriptional control signals encoded in the proviral genome (Dhar et al., 1980; Shinnick et al., 1981). The LTR sequences of Mo-MuSV were found to enhance the transforming activity of the v-mos gene, and it was demonstrated that the essential region within the LTR responsible for this enhancement is a region within the U3 region of the LTR containing the 74 b p tandem repeat sequence (Wood et al., 1983). In a more recent study, recombinants lacking CAAT, TATA, and poly(A) signals within the LTR were shown to be able to transform with an efficiency comparable to that of the wild-type MSV-124 genome. Deletion of two of the tandem repeat units, but not of one, abolished v-mos gene function (Narayaman et al., 1984). The addition of a fragment containing one tandem repeat 3’ to v-mos resulted in efficient activation of the transforming function. The results suggest that the transcript for the MSV124 transforming gene is not normally initiated within the 5’ LTR, but instead uses promoter signals in close proximity to v-mos and enhancer elements localized in the 3’ LTR (Fig. 20). A putative promoter

282

WOLFRAM OSTERTAG

5' LTR

gag

A pol

I

+U31IU5.

3' LT R

mos

--I=*

NPSV

I

An

by using t h e e n v splicing

I

Provirus

An

1

possi b l e mos-coding mRNAs

bl

by u s i n g a c r y p t i c p r o m o t e r

I enhancement

by

U3')

FIG.20. Transcription patterns of MPSV. From the integrated provirus, a full-length mRNA encoding the gag and neoRproteins is transcribed. The mos protein could be translated from an mRNA that is spliced from the full-length mRNA or, alternatively, an mRNA subgenomic species promoted from putative transcription signals directly upstream of mos, but enhanced by sequences in the 3' LTR.

sequence proximal to v-mos has been postulated for MSV-124 by

Reddy et al, (1981). Although this interpretation is not in conflict with that of previous studies which have indicated that the LTR of the HT1 or m-1 strains of Mo-MuSV is equally efficient in potentiating MoMuSV transforming activity when positioned in either 5' or 3' locations (Blair et al., 1980), it is noteworthy that the LTR promoter-like structures may not be necessary for the activity of the mos gene. Recent results of Graves et al. (1985a,b) suggest, moreover, that the Mo-MuSV LTR may contain several signals in addition to the direct repeat/glucocorticoid receptor binding sequence and the CAT and TATA box which may modify transcription selectively. Transcription of Mo-MuSV in frog oocytes does not require the direct repeat, but a distal signal between the TATA box and the enhancer region, whereas the enhancer appears necessary for fibroblast transcription. The second signal acts only weakly in fibroblasts, even if the enhancer is deleted, but is necessary for the 100- to 200-fold trans-induction found after herpes simplex virus infection. This sequence, however, appears to be unimportant for the specific action of MPSV, since it is unaltered as compared to Mo-MuSV/Mo-MuLV. Insertion of the neoRgene in the deleted pol region of MPSV does not disrupt expression of the mos gene (Ostertag et al., 1986). Although this would be consistent with the two-promoter theory, it

MURINE SPLEEN FOCUS-FORMING VIRUSES

283

would not exclude the alternative model in which the gag-pol (and, in this case, neoR)region would be acting as a mos intron. Studies with the chicken leukosis virus have shown that insertion or deletions in the enu intron do not disrupt expression of the env gene (Cullen et al., 1982). Furthermore, putative splicing sequences, necessary for the efficient splicing of the env gene in Mo-MuLV (Weiss et al., 1982; Hwang et al., 1984; Keller and Noon, 1984), are all present in MoMuSV and MPSV (Fig. 21). Whether mos mRNA is transcribed from the 5' LTR or from its own promoter, a subgenomic species should be detectable in Mo-MuSVinfected cell lines. Due to its low copy number, however, it has been difficult to detect the subgenomic message. It is thus uncertain whether the subgenomic mos RNA detected by some authors in virusinfected cells is actually real or an artifact due to degradation of the genomic-length RNA (Dina and Nadal-Ginard, 1980). A discrete, but faint, 2.6 kb mos subgenomic RNA has been observed in MPSV-infected fibroblasts and embryonal carcinoma cells (Seliger et al., 1986). Work with MPSV t s or deletion mutants, where mos expression would not be toxic to infected cells, may resolve these questions and perhaps give some insight on how quantitative differences in mos expression are regulated. In summary, the mechanism by which the transforming gene of the Mo-MuSV system is normally expressed is not well understood. Until now, analysis of RNA transcripts of the different Mo-MuSV variants has not produced conclusive data to establish a convincing theory as to how these viruses express their oncogenic potential. Transcription of the c-mos gene is also not well understood. The murine c-mos locus has only one exon which may code for the complete c-mos product, yet upstream sequences in position -331-29 from the first ATG are identical to consensus splice acceptor signals (Blair et al., 1984).The mouse and rat, but not the human c-mos locus, contain additional sequences (UMS) 0.8-1.8 kb upstream of the first ATG which prevents c-mos activation by a 3' LTR (Fig. 12). Insertion of the UMS region 3' or 5' to v-mos prevents 3' LTR enhancement of transforming activity of v-mos. This inhibition is dependent on position and functions only when inserted between v-mos and its promoter. These data may indicate that the c-mos promoter is located further upstream and does not function in either normal fibroblasts or a series of other normal murine cells (Wood et al., 1984). The question of whether RNA accumulation of c-mos is blocked directly by interference with transcription or by another mechanism has been tested for the rat c-mos locus. In uitro transcription of a c-mos LTR vector with

284

WOLFRAM OSTERTAG

H

c

S

5 pC663 tk L

S

5

pc159 n e o c

z

5

r

f

'td

0

splice a c c e p t o r

pmor-'

MURINE SPLEEN FOCUS-FORMING VIRUSES

285

the inhibitory rat sequences is not inhibited in either rodent or human cells, but RNA accumulation is inhibited in rodent but not human cells. The upstream mouse and rat inhibitory sequences thus act in a species-specific manner (van der Hoorn et al., 1985). The transcriptional start region of the c-mos locus has not been identified, but must be upstream of the inhibitory region (Wood et al., 1984). In addition to containing upstream sequences which prevent expression, the mouse c-mos gene may also be transcriptionally inactive due to hypermethylation of c-mos in many tissues (Gattoni et al., 1982). However, treatment with azacytidine of 3T3 cells, which leads to extensive loss of methylation of the c-mos locus, did not lead to fibroblast transformation (Hsiao et al., 1984). This may be the consequence of a negative, cis-acting control exerted by the UMS sequences. However, these negative controls (inhibitory upstream regions and possibly methylation) can be overcome in some mouse tissues during normal development (Propst and Vande Woude, 1985). c-mos-related transcripts have been found in mouse embryos, testes, and ovaries. The mos transcripts in testis RNA are 1.7 kb long, and the entire mos open reading frame is present. Different transcripts of 1.4 kb in ovaries and of 2.3 and 1.3 kb are found in embryos. This suggests differential controls for transcription of the c-mos gene. The mos transcripts in testis start 160 and 310 bp upstream of the conserved mos initiation codon. This region is just distal to a region conserved to 75% between mouse and human (MuH; see Fig. 12). The embryonic mos transcripts extend further upstream and thus may either be promoted and/or spliced differently in testes, ovaries, and embryos. The presence of an additional 6 kb mos transcript in embryos and epididymis may suggest even further complexity of transcription (and function?) of the c-mos locus (Propst and Vande Woude,

1985). Activation of c-mos expression has been reported in two mouse myeloma cell lines, but not in any other tumor cells (Cohen e t al., 1983; Gattoni-Celli et al., 1983; Horowitz et al., 1984). The activation of c-mos resulted from the integration of an endogenous retroviruslike DNA element (intracisternal A-type particle genome, IAP) within FIG.21. Insertion of the gene coding for neomycin resistance or thymidine kinase activity into the wild-type MPSV genome, the MPSV temperature-sensitive genome (p20-159) (Kollek et al., 1984), and several mos--MPSV mutants, in which the coding region of rnos has been disrupted by an approximate 1-2 kb deletion (Ostertag et al., 1986; Seliger et nl., 1986). The neon or t k genes were inserted in the defective pol coding region. Interference of mos expression in MPSV neo has not been observed.

286

WOLFRAM OSTERTAG

or upstream to the coding region of c-mos. Each IAP LTR insertion occurred between the inhibitory UMS region and the c-mos coding region. In one cell line (XRPC 24), the insertion is in a head-to-head orientation and splits the c-mos into the 5’ rc-mos and 3’ rc-mos. The 3’ rc-mos is actively transcribed into 1.2 kb mRNA and transforms NIH 3T3 cells. Two main mRNA start sites were identified in IAP LTR 3‘ rc-mos constructs, one mapping to the junction of the 3’ rc-mos and the 5’ LTR and the other located 10 nucleotides upstream within the 5’ LTR (Horowitz et al., 1984). The 3’ rc-mos thus is activated by insertion of a new promoter provided by the LTR of an IAP genome. The 5’ LTR thus appears to have promoter activity in both directions. The lack of major transforming activity (of mouse fibroblasts) of the human c-mos locus (Blair et al., 1984) may explain why no human tumors have been found involving activation of the c-mos locus. Speculations that c-mos is involved in generating AML in cases of chromosome (8;21) translocations (Diaz et al., 1985) were discredited, as the c-mos locus lies an appreciable distance from the breakpoint (Caubet et al., 1985). b. mos Protein and Its Functional Domains. Identification of the genomic regions of MPSV which are involved in the transformation of the target cell (see above) is not sufficient to understand the mechanism of transformation. It is also necessary to study the interaction of the mos protein with its cellular “receptors.” Although data on the MPSV mos protein itself are still preliminary, conclusions can probably be drawn from results obtained with the Mo-MuSV system and the protooncogene c-mos. Unlike the human cmos gene, the murine c-mos gene is unique in its capacity to transform fibroblasts in conjunction with an Mo-MuSV LTR (Blair et al., 1984; Wood et al., 1984) or to enhance proliferation of hematopoietic precursor cells in conjunction with the MPSV LTR (Stocking et al., 1985). One of the Mo-MuSV variants (HT-1) actually codes for mos protein identical to the c-mos protooncogene, except for the addition of amino-terminal residues (Seth and Vande Woude, 1985). This results in a p37 env-mos fusion protein in Mo-MuSV-infected fibroblasts (Papkoff et al., 1982) and a p34 mos protein in MPSV-infected fibroblasts (Stacey et al., 1984) (Fig. 15). A mos-specific protein has never been detected in normal cells, and in transformed cells the protein is present at very low levels (Papkoff et al., 1983). It is only in acutely infected cells that the p37 protein is produced at high enough levels to be detected. A temperature-sensitive variant of MSV-124, however, codes for a gag-mos fusion protein that is easily detected in chronically infected cells by antisera against

MURINE SPLEEN FOCUS-FORMING VIRUSES

287

the gag as well as the mos component. The 85,000 Da protein contains p15, p12, and about 25% of p30 fused to a shortened form of the v-mos gene product. Revertants of this ts virus code for pl00 gag-mos protein, which contains p15, p12, and an extended portion of p30 compared to the t s mutant in addition to the mos component. The ts defect in the mutant affects the splicing of the viral mRNA and enhances the turnover of the fusion protein. The work on the tsllO Mo-MuSV system was recently reviewed by Arlinghaus (1985). p37 of MSV-124 was characterized as a soluble cytoplasmic protein (Papkoff et al., 1983). Data were reported on fatty acid addition to the gag-mos protein of the tsllO Mo-MuSV mutant virus, and indirect evidence supports that fatty acids may also be attached to the mos portion of the gag-mos fusion protein (Gallick and Arlinghaus, 1984). These data suggest that mos may also interact with the cell membrane. Although no data exist concerning the function of the mos protein, it was found that the gag-mos fusion protein of the tsllO variant exhibits serinekhreonine protein kinase activity shown to be correlated with morphological changes of cells infected with the t s mutant in temperature shift experiments (Kloetzer et al., 1983, 1984; Maxwell and Arlinghaus, 1985). Analysis of a p40 mos protein encoded by a bacterial expression vector failed to detect phosphokinase activity, but demonstrated mos-related ATPase activity and ATP binding (Seth and Vande Woude, 1985).These data are consistent with protein sequence data showing that the v-mos gene encodes both a lysine residue (position 90 in c-mos; Fig. 15) in a position equivalent to the pp60"" lysine residue that interacts with an ATP analog, and an adjacent cluster of glycines (G.X*G*X.X*G; G = glycine and X = other; Fig. 15) thought to be involved in ATP binding (Kamps et al., 1984). Indeed, site-specific mutation of the lysine residue abolishes the ability of the mos gene to transform cells (Hannick and Donoghue, 1985). These residues are also found in the bovine CAMP-dependent kinase, related to v-mos (Barker and Dayhoff, 1982) and also demonstrating predominantly protein-serine kinase, but also protein-threonine kinase activity (Cohen et al., 1977). Homology of the c-mos protein to the epidermal growth factor precursor protein composed of 1200 amino acids, but not to E G F proper (53 amino acids), may suggest a functional tie to growth factors (Baldwin, 1985).The role of pro-EGF, however, is obscure, and functions in cell-cell interactions are only implied (Baldwin, 1985). The correlation between serine-threonine kinase activity and transforming capacity of the ts gag-mos fusion protein suggests a causative mechanism, as predicted for the pp60"" kinase activity and neoplastic

288

WOLFRAM OSTERTAC

transformation (Bishop, 1985). However, this work is not conclusive. An in-frame deletion of codons 83-119 (Fig. 15)containing the ATPbinding consensus sequence and within domain 1, previously shown to be important for the activation of the human mos gene (Blair et al., 1984), results in loss of the transforming capacity of the mos protein (Seth and Vande Woude, 1985). However, this loss of activity may be due to alterations in steric configuration and not loss of putative binding sites; interaction of the three postulated domains of the mos protein are critical for transformation, as shown by human/mouse mos hybrids (Blair et al., 1984). A characterized ts mos protein with a base substitution coding for an arginine instead of a glycine at position 338 (Fig. 15) and rendering the protein inactive at 39°C also indicates the importance of C-terminal sequences (J. Friel et al., 1987). Deletion mutants have also shown that some portion of the 23 amino acids at the C-terminus of p37 mos is necessary for biological activity (Bold and Donoghue, 1985). Whether these sequences are necessary for the kinase activity of mos or are involved in other postulated functions of mos, i.e., DNA binding (G. Vande Woude, personal communication), remains to be seen. Expression of v-mos has been correlated with the inhibition of transcription of both type 1collagen and fibronectin genes via some cellular trans-activating factor(s) (Schmidt et al., 1985; Setoyama et al., 1985). More recently, a reduction in steady-state levels of a MW 55,000protein in a rat myoblast cell line has been observed (Singh et al., 1986). How mos transforms cells is unclear; however, the specificity of the oncogenic effect (and thus the disease) is probably not determined by the mos gene itself, but by transcription signals regulating its expression and factors interacting with the protein in the target cells (see Section V). D. THE US-ONCOCENIC VIRUSES The ras protooncogenes are a family of cellular genes that may have related, but different, functions in normal cells. Much data have been gathered recently about the ras genes, both in the normal and activated (oncogenic) state. Thus, we will first review what is known about the ras loci and its association with malignant diseases, in particular with respect to myeloid leukemias, and potential target cell restriction of ras action. We will then briefly outline the origin and genomic structure of four acute oncogenic retroviruses that carry an activated ras gene and thus have contributed greatly to the analysis of the function of the rus oncogene in malignancy. In the next part, we

MURINE SPLEEN FOCUS-FORMING VIRUSES

289

will discuss the rus protein: its expression and localization, its proposed function in both normal and malignant growth, and the significant structural domains based on work with the cellular and viral rus, as well as the equivalent genes found in yeast ( R A S 1 and RAS 2 ). In the last part, the interaction of the rus protein with growth factor responses will be discussed.

1 . The rus Protooncogenes: Normal Function and Involvement in Malignancy

The rus protooncogenes are a family of at least three, most likely four, cellular genes: The first is related to the Harvey sarcoma virus oncogene, c-Ha-rus; the second to the Kirsten sarcoma virus oncogene, c-Ki-rus; the third is related to both and occurs as an activated protooncogene in some tumors, c-N-rus (review Shih and Weeks, 1984); and the last, the rho genes, which have not yet been shown to be activated in any malignant disease, but also code for a related p21 protein as do the other genes of the rus gene family (Madaule and Axel, 1985). The mammalian-active c-rus genes are located on different chromosomes: c-Ki-rus on chromosome 1 (humans)or chromosome 6 (mouse), c-Ha-rus on chromosome 11 (human) or chromosome 7 (mouse), and N-rus on chromosome 1 (human) (Sakaguchi et ul., 1984; Rowley, 1985). The c-Ha-rus and c-Ki-rus genes have presumably maintained linkage to adjacent marker genes during evolution in mammals (Sakaguchi et ul., 1984). Nonfunctional pseudo-genes that are derived from spliced rus genes have been found in variable numbers in mammals. The rus pseudo-genes are, as far as is known, unlinked to the active genes and thus may have originated by transpositional events. Part of the c-rus genes of mouse, rat, and humans have been molecularly cloned. The active c-Ha-rus-1 locus is a gene with three introns interspersed among four exons coding for the p21 protein and is in total about 2 kb in length (see Fig. 22). The c-Ki-rus-2 locus spans more than 45 kbp, the coding region of which is also separated into four exons. The c-Ki-rus-2 gene has two alternative fourth coding exons, exon 4A and 4B, which can be used in differential splicing of the mRNA to encode two different p21 protein molecules with alternate C-terminal sequences (see Fig. 23). The p21 encoded by the alternate exon 4B is 188 residues long while all other p21 proteins contain 189 residues. Both mRNAs are expressed in vivo. The significance of the presence of two mRNAs is unclear (Change et ul., 1982b; Capon et ul., 198313; McGrath et ul., 1983; Shimizu et UZ., 1983; review in Shih and Weeks, 1984).

290

WOLFRAM OSTERTAG

Each subgroup of the ras gene family is conserved evolutionarily within the mammalian species and thus may have specific normal functions. The ras genes of mammalians are closely related to invertebrate, yeast, and slime mold ras genes (Shilo and Weinberg, 1981a,b; Ellis et al., 1982a,b; yeast: Gallwitz et al., 1983; Powers et al., 1984; Tatchell et al., 1984; Fukui and Kaziro, 1985; Dictyostelium: Reymond et al., 1984; Drosophila: Neumann-Silberberg et al., 1984; Mozer et d., 1985). The study of the function of the ras and ras-related genes has been facilitated by a series of mutants in yeast of the two closely related RAS genes. Genetic experiments indicate that function of either wildtype gene is necessary for haploid cell viability (Kataoka et al., 1984; Tatchell et al., 1984). Yeast cells carrying the mutated RAS-2 gene coding for a protein altered in position 19 (valine), a mutant analogous to the c-Ha-ras (Val 12) mutant of the human bladder carcinoma line (see below), have a defective response to nutritional deprivation (Kataoka et al., 1984; Toda et al., 1985).They are phenotypically similar to yeast strains with mutations in the cyclic AMP effector pathway and are also similarly functionally defective (Broek et al., 1985; Toda et al., 1985). RAS proteins are required for and regulate adenylate cyclase activity: Levels are low in ras 1- ras 2- yeast, whereas they are elevated in RAS-2”a119 yeast. Addition of purified yeast R A S 1 or RAS 2 and human c-Ha-ras proteins activates yeast adenylate cyclase in the presence of GTP (Broek et al., 1985).The human c-Ha-ras coded p21 or the viral Ha-ras p21, if introduced into yeast cells, allows vegetative growth of otherwise nonviable adenylate cyclase-deficient ras 1- ras 2- yeast strains (Clark et al., 1985; DeFeo-Jones et al., 1985; Kataoka et al., 1985). In addition, yeast-mammalian hybrid genes and a deletion mutant yeast R A S - 1 gene induce morphological transformation of mouse NIH 3T3 cells when the genes have a point mutation analogous to one that increases the transforming activity of mammalian ras genes (DeFeo-Jones et al., 1985). The high levels of ras protein in prestalk cells of slime molds (Dictyostelium) may suggest a role in differentiation (Reymond et al., 1984). The ras gene encodes two mRNA species, one of which is found in vegetative cells and disappears rapidly upon initiation of development. The protooncogene can be induced with cyclic AMP and thus may be similar to the yeast analog in respect of adenylate cyclase interaction. Examination of the role of the ras gene of Drosophila in development yielded no pronounced correlations to developmental changes (Lev et al., 1985; Mozer et al., 1985).Both c-Ha-ras and c-Ki-ras genes are expressed in many, perhaps all, mammalian

MURINE SPLEEN FOCUS-FORMING VIRUSES

291

adult dividing tissues. Nearly constant levels can be found in development of the mouse fetus and of extraembryonal structures (Muller et ul., 1982; review Muller and Verma, 1984).A 1.4 kb and less of a 4.3 kb transcript of c-Ha-rus and 2.0 kb as well as 4.4 kb transcripts of c-Ki-rus have been observed. The level of v-rus transcripts is usually much higher in virus-transformed cells as compared to c-rus related transcripts in normal cells. Studies of normal c-rus expression need to be extended to specific types of normal cells during differentiation to establish meaningful correlations which would allow speculations about the rus function in differentiation and proliferation of cells. Ramsden et ul. (1985)suggest that c-Ha-rus may have a specific role in cells of epithelial origin; the c-Ha-rus locus is undermethylated in normal epidermis as compared to NIH 3T3 fibroblasts. Intermediate levels of methylation are observed in normal brain, neuroblastoma, or erythroleukemia cell DNAs. Tumors with activation of c-Ha-rus gene generally seem to occur in epithelial cell types. c-Ha-rus activation has been demonstrated in bladder, lung, and renal pelvis carcinoma in humans (Cooper, 1982; Der et ul., 1982; Parada et ul., 1982; Santos et ul., 1982; Yuasa et ul., 1983), mouse epidermal papilloma and carcinoma (Balmain and Pragnell, 1983; Balmain et d . ,1984), and in mammary epithelial tumors (Zarbl et ul., 1985). The only reported exceptions to this restriction of c-Ha-rus activation to epithelial cell tumors are melanomas (Albino et ul., 1984; Sekiya et ul., 1984) and one (long latency) virus-induced myeloid leukemia (Vousden and Marshall, 1984). This is in contrast to the lack of specificity of most v-Ha-rus oncogenic viruses and for the v-Ha-rus oncogenic MHSV with a preference for macrophage precursor transformation (see below). A much larger number of human tumors with activated c-Ki-rus-2 has been identified. These tumors are mainly lung and colon carcinoma (Der et ul., 1982; Pulciani et ul., 1982; McCoy et ul., 1983), but the activated gene has also been correlated with a series of other solid tumors (Pulciani et ul., 1982), with chemically induced mouse fibrosarcomas (Eva and Aaronson, 1983), and with lymphomas (Eva et ul., 1983; Guerrero et ul., 1984). c-Ki-rus activation thus seems to lack specificity with respect to the type of target cell. N-rus activation is most frequently found in hematopoietic tumors (Eva et ul., 1983; Gambke et ul., 1984, 1985; Guerrero et ul., 1984; Vousden and Marshall, 1984; Bos et ul., 1985), including AML in humans (Gambke et ul., 1984, 1985; Bos et ul., 1985), but also, with a lower frequency, in other neoplasms such as a neuroblastoma, a lung carcinoma, and teratocarcinoma cells, if adopted to prolonged tissue

292

WOLFRAM OSTERTAG

culture growth (Tainsky et al., 1984; Vousden and Marshall, 1984; Yuasa et al., 1984). Activated ras oncogenes are, however, not found in the majority of cases of human tumors. They constitute a small fraction of the total number of tumors of a particular type and thus cannot be considered the primary cause of these tumors. Activation of different protooncogenes resulting in the same cancers may imply similar action of these putative and nonspecific protooncogenes in normal cell physiology. Activated ras oncogenes, however, are involved in a major fraction of tumors induced in animals by carcinogens or irradiation (Balmain et al., 1984; Guerrero et al., 1984). Not much is known about whether the expression of an activated ras is required for the initiation of a tumor or whether its continuous expression is needed for the maintenance and the progression of the tumoral tissue. ras activation was found in the preneoplastic benign stage of chemically induced skin papillomas in mice (Balmain et al., 1984). Yet, in spontaneous human colon carcinomas, elevated expression of p21 was detected only in late malignant stages (Thor et al., 1984). ras alterations are quantitative, as with c-Kiras in one mouse adrenocortical tumor (Schwab et al., 1983) and one human lung carcinoma (Pulciani et al., 1985),but are usually single point mutations (Table IV) in similar positions as found for viral ras oncogenes (see below).

2 . Origin of Retroviruses with the ras Oncogene Five independent retrovirus isolates, Harvey murine sarcoma virus (Ha-MuSV) (Harvey, 1964), Kirsten murine sarcoma virus (Ki-MuSV) (Kirsten and Mayer, 1967), Rasheed murine sarcoma virus (Ra-MuSV) (Rasheed et al., 1978), BALB murine sarcoma virus (BALB-MuSV) (Peters et al., 1974; Andersen et al., 1981a,b), and MHSV, previously named AF-1 virus (Ostertag et al., 1982; Franz et al., 1985; Lohler et al., 1987), have been described which contain either c-Ha-ras (HaMuSV, BALB-MuSV, Ra-MuSV, MHSV) or c-Ki-ras (Ki-MuSV) related oncogenic sequences. No oncogenic retrovirus with N-ras related sequences has yet been found. Three of the ras oncogenic retrovirus isolates (Ha-MuSV, Ki-MuSV, Ra-MuSV) are of rat origin, two of mouse origin (BALB-MuSV, MHSV). a. c-Ha-ras-Transducing Viruses. Harvey (1964) isolated the HaMuSV from tumors induced b y Mo-MuLV in rats. The virus was identified as a murine type C retrovirus that had recombined with rat cellular sequences (Scolnick and Parks, 1974) and induces sarcomas and erythroblastosis in newborn mice (Harvey, 1964). Ha-MuSV also

MURINE SPLEEN FOCUS-FORMING VIRUSES

293

induces a proliferative disease of hematopoietic cells in adult animals (see below). Very little is known about the biology of the Ra-MuSV (Rasheed et al., 1978). Rasheed e t al. isolated the Ra-MuSV by in vitro cocultivation of a rat cell line releasing retrovirus with another rat cell line derived from tumors induced by a chemical carcinogen. Ra-MuSV also induces erythroblastosis and sarcomas in newborn mice; nothing is known about its effect on adult mice. The transforming gene of RaMuSV codes for a gag-ras fusion protein of 29K (p29) (Young et al., 1979, 1981). The BALB-MuSV, in contrast to Ha-MuSV, Ki-MuSV, and RaMuSV, is a recombinant product of an endogenous murine retrovirus with the mouse protooncogene (Peters et al., 1974; Andersen et al., 1981a,b). Balb-MuSV causes erythroblastosis and sarcomas in adult and newborn mice (Peters et al., 1974). The last murine retrovirus carrying a c-Ha-ras-related oncogene that will be discussed in this review, MHSV or AF-1 (Ostertag et al., 1982; Franz et al., 1985; Lohler et al., 1987), was isolated after passage of a cloned Friend helper virus, 643/22F (Ostertag et al., 1981), through newborn BALB/c mice. A virus was isolated from sarcomas induced during passage that causes splenomegaly and solid tumors in vivo and transforms fibroblasts in vitro. MHSV also transforms macrophage precursor cells in vivo and in vitro and induces a malignant histiocytosis in adult mice (Franz et al., 1985; Lohler et al., 1987). b. c-Ki-ras-Transducing Viruses. The Kirsten murine sarcoma virus (Ki-MuSV) was isolated after passage of Kirsten murine leukemia virus in Wistar-Furth rats (Kirsten and Mayer, 1967). The Ki-MuSV is a replication-defective retrovirus that causes multiple sarcomas, osteolytic lesions, and erythroblastic splenomegaly in rats when pseudo-typed with Ki-MuLV or Mo-MuLV (Scher et al., 1975). The defective subunit of the viral complex contains rat cellular sequences (Scolnick et al., 1973) and a c-Ki-ras-related oncogene. 3. Genomic Structure a. Ha-MuSV and Ki-MuSV. Although independent isolates, HaMuSV and Ki-MuSV are closely related (Figs. 22 and 23). Both have been molecularly cloned and parts of their genomes have been sequenced (H. w. Chang et al., 1980; Tsuchida and Uesugi, 1981; Weiss et al., 1982). The replication-defective transforming genomes of HaMuSV and Ki-MuSV were compared to each other and to their presumptive ancestors by heteroduplex mapping (Chien et al., 1979; Young et al., 1981). These studies revealed that only about 1.0 kb at

294

WOLFRAM OSTERTAG

30s R N A + c-

Ha- r a s

30 S c-Ha- ras

ras

HaMuSV

FIG.22. Putative steps of recombination which led to Ha-MuSV (Ellis et ul., 1980). Recombination of 30 S RNA with c-Ha-ras generated a 30 S c-Ha-rus intermediate. A second recombination step with Mo-MuLV resulted in Ha-MuSV (Ellis et ul., 1980).30 S RNA homologous sequences are retained 5' and 3' of the v-Hams gene (Ellis et ul., 1981).

30s R N A +

TT2 3

1

c-Ki-ros

LA

48

30 S c-Ki-ras +

Ki MuLV

I #

H

P

Ki MuSV

FIG.23. Putative steps of recombination involved in generating Ki-MuSV. Recombination of c-Ki-rus (shown as human c-Ki-rus-2; McGrath et ul., 1983) with 30 S RNA generated a 30 S c-Ki-rus intermediate. In a second step, Ki-MuLV recombines with the 30 S c-Ki-ras, resulting in Ki-MuSV (Tsuchida et ul., 1982). Sequences homologous to 30 S RNA are retained on both ends of the v-Ki-rus gene.

MURINE SPLEEN FOCUS-FORMING VIRUSES

295

the 3' end and less than 0.2 kb at the 5' end were related to the respective helper viruses, Mo-MuLV and Ki-MuLV. Large portions of the defective genomes contain sequences that align with a rat 30 S RNA found in multiple copies in the rat genome and having no identified product or function (Ellis et al., 1980). The irrelevance of the 30 S RNA-derived sequences for the action of Ha-MuSV and Ki-MuSV was later demonstrated by the retention of the transforming capacity of subgenomic fragments lacking 30 S RNA homology (Goldfarb and Weinberg, 1981). To localize the transforming region, insertion-deletion mutants of cloned Ha-MuSV were used in transfection experiments (E. Chang et al., 1980; Wei et al., 1980). The transformation-specific sequences, termed rus, were mapped within the first 2.0 kb at the 5' end. This region was shown to be that coding for a phosphoprotein of 21,000 Da (p21) which had previously been associated with transformation induced by both Ha-MuSV and Ki-MuSV (Shih et al., 1979a).A temperature-sensitive mutant of Ki-MuSV coding for a thermolabile p21 proved that the maintenance of the transformed state in fibroblasts depends on the presence of a functional p21 (Shih et al., 1979b). In spite of the close relationship between the p21 of Ki-MuSV and Ha-MuSV, as judged by function and antigenicity, heteroduplex analysis showed little homology between the two genes (Chien et al., 1979). The 1.75 kb ras sequence of Ki-MuSV, nonhomologous to 30 S RNA sequences, showed only a short region of homology with the 0.9 kb ras sequences of Ha-MuSV and hybridized to it only under nonstringent conditions in Southern analysis. Protein analysis, however, showed that two-thirds of the tryptic peptides of the two p21 proteins are identical (Ellis et al., 1981). The p21 coding regions of both Ha-MuSV and Ki-MuSV are derived from unique rat cellular sequences (Ellis et al., 1980). The v-Ki-ras oncogene is derived from the c-Ki-ras-2 locus of the rat: It lacks the introns of c-Ki-ras-2 and utilizes exon 4A and not the alternative exon 4B (McGrath et al., 1983; Shimizu et al., 1983; Iritani et al., 1986). Using a specific probe for the Ha-MuSV ras gene, two different c-Haras alleles were identified in the rat and human genome (DeFeo et al., 1981;Chang et al., 1982a,b; Ruba et al., 1986). One, termed c-Ha-ras-1 of about 2.8 kb length, is colinear with the viral ras. The v-ras oncogene incorporates all four exons of c-Ha-ras-1, but lacks its introns (Fig. 22, 23). b. M H S V and Ra-MuSV. The genomic structures of these two viruses are at this point not well understood. Both carry sequences that hybridize to the c-Ha-ras allele; however, the Ra-MuSV genomes en-

296

WOLFRAM OSTERTAG

code a gag-ras fusion protein of 29,000 Da (p29) (Young et al., 1979, 1981). Ra-MuSV contains sequences of a rat endogenous virus (SD-1) and 30 S RNA (Rasheed et al., 1978,1983). MHSV has the same length and many common restriction sites as Ha-MuSV (Franz et al., 1985). Partial nucleotide analysis has shown that although MHSV seems to have the same 5’junction between MuLV and 30 S RNA sequences as Ha-MuSV, the LTR is derived from F-MuLV instead of Mo-MuLV (Padua et al., submitted). 3’junction sites have not been determined, nor have differences that might exist in the junctions between ras and 30 S sequence and within the ras coding region. Of special note is the deletion of a region within the direct repeats in the LTR, and a triplication of a single motif within the LTR. The significance of this rearrangement has not been determined but is postulated to alter the specificity of MHSV and action of the rus oncogene with respect to Ha-MuSV (Padua et al., submitted). c. BALB-MuSV. The BALB-MuSV genome has been molecularly cloned (Andersen et al., 1981a) (Fig. 24). The v-ras oncogene is intergrated between the pol and the env region of an endogenous helper virus which is closely related to the Rauscher MuLV (Reddy et al., 1985). Although termed bas, this sequence is similar, if not identical, to the Ha-ras sequences present in the mouse genome. Transcription of the mRNA coding for the p21 protein of BALB-MuSV is not yet understood. 4. The ras Protein

a. Localization and Expression. All of the v-ras genes of the aforementioned spleen focus-forming viruses, except Ra-MuSV, encode one protein of 21,000 Da (p21). A precursor of MW 22,000 (p22) is synthesized on non-membrane-bound ribosomes and then moves to the plasma membrane after processing of the C-terminus (Shih et al., 1982). Immunohistochemical studies have localized 95% of the viral

mouse c-Ha-ras

-gag MuSV I C I FIG.24. Putative evolvement of Balb-MuSV by recombination of c-Ha-ras (DeFeo e t al., 1981) with endogenous MuLV to generate Balb-MuSV (Reddy et al., 1985). Balb

297

MURINE SPLEEN FOCUS-FORMING VIRUSES

p21 at the inner surface of the plasma membrane (Willingham et al., 1980; Furth et al., 1983). The different p21 proteins are closely reIated and are related to the endogenous p21 of many vertebrate and invertebrate species (Shih et al., 1979a,b; Langbeheim et al., 1980;Temeles et al., 1985).In spite of this close relationship, transfection of c-ras alleles does not transform fibroblasts in vitro (DeFeo et al., 1981; Shilo and Weinberg, 1981a,b). “ Activated” c-ras genes, however, are often found associated with tumors and leukemias (see above). Activation of cellular genes and subsequent transforming activity may be the result of either point mutations (Der et al., 1982; Santos et al., 1982; Tabin et al., 1982; Taparovsky et al., 1982; Capon et al., 1983a)b; Der and Cooper, 1983; Reddy, 1983; Nakano et al., 1984; Santos et al., 1984) or the elevated expression of the normal protooncogene (DeFeo et al., 1981; Pulciani et al., 1982) (Table IV). TABLE IV AMINOACID DIFFERENCES IN ACTIVATEDrus PROTEINS Amino acid Protein c-Ha-rus Normal EJ/T24 bladder carcinoma NIH 3T3 spontaneous activation Hs242 lung carcinoma v-Ha-rus Harvey sarcoma virus Balb sarcoma virus Rasheed sarcoma virus c-Ki-rus Normal Calu-1 lung carcinoma SW480 colon carcinoma A1698 bladder carcinoma v-Ki-rus Kirsten sarcoma virus c-N-rus Normal SK-N.SH neuroblastoma SW1271 lung carcinoma Acute myeloid leukemia Acute myeloid leukemia Acute myeloid leukemia Human teratocarcinoma

12 Glycine Valine Aspartic acid -

13 Glycine

-

Arginine Lysine Arginine

-

Glycine C ysteine VaIine Arginine

Glycine -

Serine Glycine

-

Aspartic acid Aspartic acid

-

61 Glutamine -

Leucine

Glutamine -

-

-

-

-

-

-

G 1y ci n e Valine Aspartic acid -

Glutamine Lysine Arginine -

-

-

-

298

WOLFRAM OSTERTAG

DeFeo and colleagues (1981) demonstrated that enhanced expression of the two c-Ha-rus alleles under control of viral LTRs results in transformed 3T3 cells. Unusually high copy numbers of c-Ha-ras, if introduced into these cells, can also induce fibroblast transformation (Pulciani et ul., 1985).Work with primary fibroblasts, however, led to different biological effects. Enhanced expression of either c-rus or the T24 bladder carcinoma rus in an early passage of rodent fibroblasts causes rescue from senescence, but only the T24 rus induces malignant transformation (Spandidos and Wilkie, 1984) (see below). What constitutes an activated rus protein? Comparison of the biological activities and structural alterations of the normal protooncogene with that of the activated gene, e.g., the viral rus gene or the ras gene associated in human bladder carcinomas, has given us some insight into the transforming domains of the ras protein. b. Function of the rus Protein. Although the exact function of the normal rus protein in mammalian cells is not known, it has been suggested that it may be required to initiate DNA synthesis and entry into S phase in fibroblasts (Mulcahy et UZ., 1985; see also above). Utilizing a transformation-competent mutant, v-Ha-rus, whose protein products are not immunoprecipitated by a monoclonal antibody that has been shown to react to other rus proteins, Papageorge et ul. (1986) have shown that a transforming rus gene can provide an essential function normally supplied by c-rus which allows entry into S phase. A temperature-sensitive mutant of Ki-MuSV has facilitated the study of the requirement of the ras protein for cells to transit G and to start replicating DNA in the absence of serum (Durkin and Whitfield,

1986). Hurley et al. (1984) showed that guanine nucleotide-binding proteins (G-proteins), which transduce signals elicited by ligand binding to membrane receptors into intracellular changes in metabolism, share sequence homology in limited but presumably vital domains with the rus gene product. The a-subunit of transducins (G-protein analogs) bind GTP similarly as known for the protein coded by the rus protooncogene (Gilman, 1984; Lochrie et ul., 1985; Medynski et ul., 1985; Tanabe et ul., 1985). Since one function of G-proteins is to regulate adenylate cyclase in mammalian cells, it was speculated that rus proteins may have a similar function. An interaction with adenyl cyclase, similar to that found for the yeast RAS protein, has been suggested based on the finding that fibroblasts and epithelial cells transformed by Ha-MuSV and Ki-MuSV have a reduced adenyl cyclase activity. However, this reduction appears not to be directly related to p21 M S protein interaction with adenyl cyclase (Beckner et

MURINE SPLEEN FOCUS-FORMING VIRUSES

299

al., 1985).A recent study shows reduced hormone-stimulated adenylate cyclase activity in fibroblasts expressing an activated ras oncogene and, to a lesser extent, with c-ras (Tarpley et al., 1986). The mechanism of inhibition is not understood, but does seem to be a simple replacement of normal cellular G-proteins with activated ras. The association of the ras protein with the EGF, one of the proteohormones required for divisions of fibroblasts was also suggested (see Section 111). E G F was shown to augment GTP binding of p21 after serum starvation (Kamata and Feramisco, 1984), and coprecipitation of the E G F receptor and p21 had been reported (Finkel and Cooper, 1984). However, since elevated GTP binding levels of p21 were also demonstrated with insulin, it is unclear whether this effect is a consequence of the reprisal of cell growth or EGF directly. Furthermore, coprecipitation results have been attributed to artifacts (Harford, 1984). The binding of p21 with GTP and GDP, but not with other nucleotides, such as ATP, UTP, or CTP, had been shown (Scolnick et al., 1979; Shih et al., 1980; Papageorge et al., 1982). Recently, it was reported that p21 has a 5- to 10-fold higher GTPase activity in cells than the activated or viral p21, although GTP binding was essentially the same (Gibbs et al., 1984b; McGrath et al., 1984; Sweet et al., 1984). GTPase, evolutionarily highly conserved, is a key enzyme in normal cells and may be required to initiate S phase in 3T3 cells (Mulcahy et al., 1985). GTPase activity has also been attributed to the RAS gene product of yeast (Temeles et al., 1985) (see above). It is thus possible that the ras protein is a regulatory molecule controlling cell division or differentiation similar to that implied for other GTP binding proteins (transducins) (Hurley et al., 1984). The active signal could thus be a complex of p21 and GTP that dissipates as soon as the GTP molecule is hydrolyzed. Inhibition of hydrolysis, perhaps by mutation of p21, could result in a persistent activation of the system, producing uncontrolled cell division and transformation. c. Structure-Function Relationship. The structural difference between the normal cellular rm gene and the activated gene, e.g., the viral ras gene or the ras gene found in human bladder carcinomas, was shown to be very subtle (Table IV). Codons corresponding to amino acids 12, 13, 59, and 61 are frequently altered, suggesting that these codons are crucial for the transforming activity of activated ras protooncogenes or viral oncogenes (Table IV). Analysis of induced mutations with the c-Ha-ras-1 gene confirmed this notion (Fasano et al., 1984a; Chipperfield et al., 1985): Mutated Ha-ras DNA was tested for fibroblast-transforming activity. Mutations in positions 12, 13, 59,

300

WOLFRAM OSTERTAG

61, or 63 could confer transformation potential to the c-Hams gene. How are these alterations related to ras protein functions? Position 12 is most frequently affected. All viral ras oncogenes are altered in position 12 (Table IV): Replacement of glycine by arginine (Ha-MuSV, Dhar et al., 1982; Ra-MuSV, Rasheed et al., 1983), serine (Ki-MuSV, Tsuchida et al., 1982), or lysine (BALB-MuSV, Reddy et al., 1985) has been detected. Several activated ras protooncogenes are altered in either position 12 or 13(Bos et al., 1985)(Table IV). That an alteration in position 12 is necessary for transformation was shown by the inhibition of transformation with an antibody that interacts only with oncogenic p21 altered in position 12 (Clark et al., 1985; Feramisco et aZ., 1985). Although this antibody inhibits binding of GTP to p21, it was demonstrated that positions 12 and 13 of p21 are not only involved in GTP binding, but also in conferring GTPase activity to the p21 protein (Clark et al., 1985). Furthermore, studies using insertion mutagenesis showed that alterations at position 12 do not alter the nucleotide binding capacity of mutated p21 (McGrath et al., 1984), which is in agreement with previous studies of GTP binding of normal and activated p21 (Finkel et al., 1984). The GTPase activity, however, is drastically affected if oncogenic ras (mutated in position 12) is compared to the normal cellular p21 protein (Gibbs et al., 1984b; McGrath et al., 1984; Manne et aZ., 1985). These data establish a strong correlation between a structural alteration in a functional domain resulting in an activated, transforming gene. The viral proteins of Ha-MuSV and Ki-MuSV, but not of Balb-MuSV and Ra-MuSV, were shown to contain a threonine instead of an alanine at position 59. Although the normal p21 at position 59 is not extensively phosphorylated (Papageorge et al., 1982; Der and Cooper, 1983),the viral protein is. This phosphorylation increases the stability of the p21 protein (Ulsh and Shih, 1984); however, its importance for the transforming function is unclear. Phosphorylation of a threonine residue at position 59 was independent of the amino acid at position 12 in hybrid p21s encoded by recombinants of viral and cellular ras genes (McGrath et al., 1984). Mutations at position 59 in activated p21 proteins also decrease GTP binding, but do not alter specificity of binding (Finkel et al., 1984). It could be envisaged that the region around position 12 and 13 cooperates with the region proximal to residues 59-63 for binding and hydrolyzing GTP (McCormick et al., 1985). Changes in both positions, as found in the Ha-MuSV or KiMuSV coded p21, may provide an even stronger signal for the disruption of the GTP binding and hydrolyzing function of p21 ras, resulting

MURINE SPLEEN FOCUS-FORMING VIRUSES

301

in transformation. This would be in agreement with other studies indicating that alterations in position 12 alone may not lead to efficient transformation (Nakano et ul., 1984). An alteration at amino acid 143 in the p21 rus protein of Balb-MuSV as compared to the rat c-Ha-rus-1 protein is also found. However, this may be an alteration that is present in the mouse protooncogene (which is not cloned) and then is not involved in activation (Reddy et al., 1985). Comparison of the amino acid sequence of the v-Ki-rus and v-Ha-rus proteins showed that 110 of 120 residues at the N-terminal half are identical, but only 3 of 22 at the C-terminal half (Dhar et ul., 1982; Tsuchida et ul., 1982). The preservation of the amino acid sequence in spite of an abundance of mutational events in the gene suggested the importance of the structural integrity of the N-terminal half for the function of p21. However, the C-terminal domain of p21 was recently also implicated in the transformation process. Deletion of amino acids 184-189 of Ha-rus-encoded p21 abolished fibroblast transformation (Willumsen et ul., 1984a,b). Although autophosphorylation and GTP binding activity were not impaired, mutations or deletions in position 186 cysteine and 197 serine led to defects in lipid binding and membrane anchorage. Inhibition of processing of the p22 precursor to the p21 protein results in localization in the cytoplasm and the subsequent loss of transforming capacity (Shih and Weeks, 1984). Lipid binding of p21 has also been implicated in the process of membrane anchorage and is necessary for the transformation capability of HaMuSV and Ki-MuSV (Sefton et ul., 1982). Thus, residues 186 and 197 are probably necessary for the proper processing of p21 for membrane association, which in turn is apparently essential for the rus protein transforming function (Willumsen et ul., 1984a,b). The sequence divergence near the C-terminus of the three mammalian rus genes (Ki-rus, Ha-rus, N-rus) may have functional significance. These specific features are thus retained and conserved during evolution of the Ha-rus genes; however, they may not be essential for transformation, as was shown by the functional exchange of the 3' terminal region of the Drosophilu rus and the 5' part of an activated rus (Schejter and Shilo, 1985). Thus, it could be summarized that the catalytic domain encompasses the first 160 amino acids of the mammalian protein. Residues 164-183 are divergent among rus genes and are not necessary for transformation (Lacal et ul., 1986),and residues 186189 are required for membrane localization of the protein and transformation. Mutational analysis in a limited region of the catalytic do-

302

WOLFRAM OSTERTAG

main indicated that mutations at positions 32-42 and at 78 disrupt the adenylate cyclase stimulation in yeast and transformation in mammalian cells (Sigal et al., 1986). A more systematic mutational analysis of the ras catalytic domain was reported by Willumsen et al. (1986). They defined three nonessential segments which span amino acids 64-76, 93-108, and 120138 and are all hydrophilic regions, implying location at the molecule surface. Six segments were identified as being essential for ras activity. Loss of GDP binding activity in three of these segments was correlated with loss of efficient cell transformation as has been reported previously (Lacal et al., 1986). Lesions in one segment did not impair GDP binding or membrane localization, but interfered with transformation. It was speculated that this region (residues centered around position 30) may interact with the putative target. Comparison of the sequence homology between rus and other necleotide-binding proteins, such as EF-Tu, shed some light on the catalytic domain. Major homology has been noted in three segments: residues 7-21, 57-61, and 109-121; the first two segments are implicated in phosphoryl binding, the latter in guanosyl binding (Sigal et al., 1986). This led Walter et al. (1986) to analyze residue 116, which from structural grounds predicted a contact site with the guanine residue. As expected, GTP binding and hydrolysis were reduced, both in the normal c-Ha-rus allele and activated v-Ha-ras; however, transformation of v-Ha-ras was not altered and, indeed, c-Ha-ras was activated. By analogy to other signal-transducing proteins whose activity is mediated by the binding of guanine nucleotides, p21 is thought to be activated as a result of an interaction with a cell surface receptor that promotes the exchange of GTP for GDP (Gibbs et al., 1985). Impairment of deactivation (i.e., GTP + GDP) is believed to account for the increased transformation potential of activated ras. It is postulated by Walter et al., (1986) that activation of c-Ha-rus by reducing the affinity of p21 for guanine nucleotides also acts by impairing the deactivation step, in this case by enhancing the dissociation rate of GDP by uncoupling the normal mechanisms of control. 5. Interaction of the ras Protein with Growth Factor Responses There are as yet no consistent responses which distinguish transformation by ras oncogenes. A reduced requirement for growth factor supplements has been shown on transformation of epithelial cells with the v-ras oncogene (Yoakum et al., 1985). Introduction of v-Haras into the MCF7 breast cancer cells removes the dependence for

MURINE SPLEEN FOCUS-FORMING VIRUSES

303

these cells for estrogen in tumorigenesis (Kasid et al., 1985). The Multi-CSF (IL-3)requirement for mast cells is not altered by infection with Harvey sarcoma virus, but the probability of immortalization is increased significantly (Rein et al., 1985). Data on transformation of macropahge precursor cells by MHSV appear to be of special interest (Franz et al., 1985; Johnson et al., 1985;Klingler et al., submitted). Macrophage precursor cells normally require either of three growth factors for proliferation and differentiation: Multi-CSF, GM-CSF, or M-CSF (see Section 11).MHSV has the unique property that infected macrophage precursor cells acquire growth factor independence immediately after infection. About 50% of all potential macrophage precursor cells respond to factor-independent colony growth upon infection with MHSV. These cells are not immortal but possibly have a somewhat extended proliferation capacity. Supernatants of such colonies do not contain detectable amounts of CSFs, as judged by biological response assays (Johnson et al., 1985, and unpublished). Permanent macrophage cell lines can be derived at a low frequency from factor-independent macrophage colonies or from spleen of MHSV-infected mice (see below). These cell lines are growth factor independent but do not release detectable growth factor. They also clone linearly, independent of the cell number plated, thus excluding an autocrine mechanism of stimulation (Klingler et al., submitted). The number of growth factor receptors (GM-CSF, multiCSF, M-CSF) is, however, markedly increased in all of these transformed cell lines. The number of GM-CSF receptors in newly infected cells increases consistently 5- to 10-fold above the level of normal macrophages and remains high in established cell lines. This finding suggests that one early event in transformation of macrophages by MHSV is the increase in growth factor receptor levels. One could speculate that the ras oncogene of MHSV interacts with growth factor receptors, such as the GM-CSF or M-CSF receptors, in a regulatory manner. The M-CSF receptor status in these cells is particularly intriguing since recent data show activation of the M-CSF receptor gene in several cases of human malignant histiocytosis, a disease which is duplicated in mice infected by MHSV. Interaction of the ras oncogene p21 product with growth factor receptors or peripherally acting oncogenes has been invoked by a recent report which shows that fms,erbB, and other peripheral oncogenes cannot transform cells if the function of the cellular p21 ras is inhibited by antibody injection into single cells (Smith et al., 1986) (also, see above). The specificity of the ras oncogenic viruses also needs to be addressed in this review. Why are Ha-MuSV, Ki-MuSV, and Balb-MuSV

304

WOLFRAM OSTERTAG

less efficient oncogenic viruses than MHSV in generating leukemia? What determines that the MHSV specifically transforms macrophage precursor cells in the animal and in vitro (see Section V), whereas the other rus-containing viruses cause a general proliferative disorder in the adult animal? It should be pointed out that the transformation specificity of MHSV is not absolute or unique. Other rus-oncogenic viruses also transform macrophage precursors, but at a lower efficiency (Greenberger, 1979; Pierce and Aaronson, 1985; Franz et ul., 1985; Lohler et ul., 1987). The rus oncogene itself can interact with and transform any of a series of cell types, indicating that other parts of the MHSV genome must be responsible for its affinity for macrophage transformation. A study similar to that done for the myeloproliferative sarcoma virus (see above) is necessary to define the structure-function relationships for MHSV and to exclude the possibility that MHSV contains not only one, but two oncogenes. V. Target Cells for Transformation

A. INTRODUCTION An understanding of the complex interaction between viral genes and cellular components that leads to a malignant disease requires a comprehensive knowledge of the contributing factors of both the transforming virus and its target cell. In the previous section, we have attempted to summarize the available data on the viral genes and their products which have been implicated in transformation and to identify other viral features which may regulate the expression of these genes in specific target cells. In this section, we will try to summarize and relate the available data on the hemopoietic target cells of these transforming genes. The course of viral-induced leukemogenesis is determined by many factors: the availability of cycling hematopoietic target cells for infection and their distribution in specific organs; the availability of hematopoietic stimulating factors and receptors and their interaction with each other and with newly introduced or activated gene products; and the efficacy of the immune system or other cellular factors to remove transformed or excessive differentiating cells. A series of host genes regulate the proliferation and differentiation of hematopoietic cells, the complexity of which is slowly being unraveled (see Section 11). No doubt more than several alternative regulatory pathways for hematopoietic maintenance which allow the organ-

MURINE SPLEEN FOCUS-FORMING VIRUSES

305

ism to cope with changes in the internal environment, i.e., infections and other stress situations, will be unveiled. These regulatory genes are also subject to variations of the genetic pool within a defined population which allow a proper response to diverse external stress situations and subsequent survival of the species. Several genetic polymorphisms influencing hematopoietic cell proliferation have been detected due to their variant phenotypic expression either under normal conditions or when subjected to adverse conditions. Although such mutant gene loci have been characterized in respect to their influence on normal hematopoiesis, only a few of these have been tested in respect to their influence on the action of the acute leukemogenic viruses. Such an analysis has important implications in the identification of presumptive target cells of these viruses as the availability and distribution of hemopoietic cells may be governed by these host genes. Thus, in the first part of this section, we would like to outline briefly some of the polymorphic gene loci involved in the regulation of hemopoiesis and virus restriction and to use these data for the classification of the spleen focus-forming viruses. In the main part, we would like to summarize the data obtained from in vivo and in vitro studies on the effects of these viruses on hematopoietic CFC. These results, taken together with our knowledge of polymorphisms and infectivity, can be used to draw some conclusions on their presumptive target cells and to discuss their possible interaction. Finally, we would like to suggest experiments necessary to study these interactions at the molecular and cellular level. B. HOSTRANGEFOR TRANSFORMATION BY SPLEENFOCUS-FORMING VIRUSES Several polymorphic gene loci have been described that affect the infectivity and pathogenicity of the leukemogenic viruses: those that determine resistance to helper virus infection and/or replication, such as the Fv-l locus (Lilly and Pincus, 1973; Odaka, 1973,1974), the Fv-4 locus (Odaka et aZ., 1980, 1981; Yoshikura and Odaka, 1982; O’Brien et d.,1983; Ikeda et al., 1985), and possibly the Rmcy locus (Ruscetti et al., 1985);those that determine resistance to the spleen focus-forming viruses, such as the W and S l loci (review by Russell, 1979), the Fv-2 locus (Lilly and Pincus, 1973; Odaka, 1973), the Mpsv resistance locus (Ostertag et aZ., 1981), and the putative Fv-5 locus determining specificity of the Friend virus complex (Shibuya and Mak, 1982,a,b; Kitagawa et aZ., 1983); and those that indirectly influence viral-induced pathogenicity by affecting cell removal, such as the H - 2 linked

306

WOLFRAM OSTERTAG

loci and others (Lilly and Pincus, 1973; Kitagawa et al., 1983; Meruelo and Bach, 1983). For the purpose of this review, we will primarily discuss the group of polymorphic loci which influence directly the action of the spleen focus-forming viruses. Loci controlling helper virus replication also play an important role in the infectivity of the transforming viruses, as all virus genomes are carried to the target cell within a helper virus envelope; however, unique pathology of the various spleen focusforming viruses seems to be specified to a greater degree by their own genome. Although not too much is known about the Mpsv and Fv-5 loci, especially in regard to their effect on normal hematopoietic differentiation, it is known that the W, S2, and Fv-2 loci alter hematopoietic cell production. Functionally defined stem cells are found at a very low concentration in WNV' mice; the number of splenic 8-day BFU-E cells is greatly reduced. Conversely, Sl/Sld mice have normal numbers of hematopoietic stem cells (CFU-S) and CFU-C; the defect in these mice appears to occur in the erythroid precursor cells at the late BFUE stage (Russell, 1979). One study implies that a second BFU-E-like cell, the TE-CFU, is grossly deficient or entirely lacking in Sl/Sld and W/Wv mice (Wiktor-Jedrzejczak et a2., 1979, 1981), and the lack of this TE-CFU cell is responsible for the much increased radiosensitivity of these mutant anemic mice (Gregory et al., 1975; Wiktor-Jedrzejczak et TABLE V MOUSEHOSTRANGEOF SPLEEN FOCUS-FORMING VIRUSES" ____

~~

Fu-2, Mpsu loci Virus

FVP RV MPSV HaMuSV MHV

rlrb

slsb

rlrb

slsb

rlrc

rlrc

s/sc

SISC

-

+++

-

+++ +++

+(+)

+(+)

-

+

+

-

+++ +++ ++ +++

+++ +++ +++ +++ +++

w locus w w W+" iz

+++ +++ +++ +++

+++ +++ +++ +++ +++

s1 locus SIISld

SI+/SI+

-

+++ +++ +++ +++ +++

+++ +++ +++ +++ ~~

The Mpsu'resistance gene could be one locus or several independent loci in C57BL mice. It is not certain whether the same C57B1 genes control resistance to MPSV, RV, a

HV, and HaMuSV. * FV-2 locus. c Mpsu locus.

MURINE SPLEEN FOCUS-FORMING VIRUSES

307

al., 1979, 1981). The Fu-2 locus seems to affect the BFU-E cycling (Suzuku and Axelrad, 1980). Analysis of the host range of the spleen focus-forming viruses in respect to these genetic mutations reveals three classifications of virus (Table V): (1)viruses restricted in F~-2~/Fv-2', in Sl/Sld, and in W/Wv mice, but not influenced by the state of the M p s u locus, e.g., the Friend-SFFVdMuLV complex (FVp) (Bennett et al., 1968; Steeves et al., 1968; Odaka, 1969; Lilly and Pincus, 1973; Ostertag et al., 1981); (2) viruses not restricted in S1/Sld,in WNV", or in Fv-2'/Fv-2' mice, e.g., the ras oncogenic viruses, Ha-MuSV, and MHSV (Ostertag et al., 1982; Franz et al., 1985, 1986a); and (3) viruses not restricted in S1/Sld or W/Wv, but restrictive to Fv-2'/Fv-2' when one or several other resistant loci (Mpsu'?) are present, as in C57BL animals, e.g., MPSV and the R-SFFV/MuLV complex (RV) (Ostertag et al., 1981, 1982; Mol et al., 1982; Hess et al., 1984). The diverse host range pattern with respect to leukemia induction allows us not only to classify the acutely oncogenic murine retroviruses, but also is an indication that the different oncogenic viruses have different hematopoietic target cells. Some of the possible reasons determining host range restriction of spleen focus-forming virus-mediated transformation will be discussed below. C. EFFECTSOF THE SPLEENFOCUS-FORMING VIRUSES ON HEMATOPOIETIC CELLS

1. F-SFFV The Friend virus complex consists of the Friend helper virus (FMuLV) and a defective SFFV component (see Section IV,B). F-MuLV is a leukemogenic agent when injected into newborn mice of certain mouse strains (Ruscetti et al., 1981; 1985; Shibuya and Mak, 1982a,b). After a long latency, F-MuLV induces erythroleukemia associated with a rapid increase in BFU-E (Niho et al., 1982). Permanent erythroid cell lines isolated from such mice can be induced to differentiate in uitro (Oliff et al., 1981), some of which may be blocked earlier in differentiation than F-SFFV-transformed cell lines (Shibuya and Mak, 1983a). Precursor cells within a colony still have migratory capacity unlike differentiating Friend cells infected and transformed (?) by SFFV. The cells, however, do not respond noticeably to Multi-CSF as would be expected for BFU-E or early erythroid precursor cells. These cells probably do not represent the normal target cells of FMuLV and how they are transformed remains unclear. Other erythroid cell lines have been isolated from F-MuLV-infected mice.

308

WOLFRAM OSTERTAG

These cell lines secrete erythropoietin and arise as a rare artifact of the F-MuLV-induced disease (Tambourin et al., 1983; Choppin et al., 1984; Hankins et al., 1986).Established Epo-releasing cell lines are a minor subfraction of the erythroid cell lines isolated following longer exposure of newborn mice to F-MuLV. These cell lines not only release Epo but also have Epo receptors, thus suggesting a model of autocrine stimulation due to rearrangement of the Epo gene. However, none of the established Epo-producing cells requires Epo for growth and thus appear to have undergone a second mutation as discussed for the myeloid cell lines in which the GM-CSF gene has been activated (see above). In other mouse strains, F-MuLV causes a myelomonocytic leukemia with a dramatic increase of GM-CFU in the spleen, 40% of which differentiate in the absence of added stimulating factor (Shibuya and Mak, 1982b, 1983b). It i s not understood why F-MuLV does not cause comparable alterations in the adult mouse. Several mouse strains have been investigated which, after infection with F-MuLV as newborns, develop first erythroid hypoplasia and later erythroleukemia, and much later myeloid leukemia or lymphoid leukemia (Chesebro et al., 1983). MCFV generation is frequent and appears almost obligatory in some mouse strains (Ruscetti et al., 1981,1985; Chesebro et al., 1983). The disease induced by F-MuLV alone, in contrast to that elicited by the SFFV/MuLV virus complex (FV), can be cured by hypertransfusion, i.e., infected cells still react to normal erythropoietic control mechanisms, and truly transformed cells arise only very late in the disease (Tambourin, 1979; Wendling et al., 1981). The disease induced by FV (SFFV) is an acute disease which is noticeable soon after infection of sensitive adult or newborn mice; tumorigenic cells arise within the first 1 to 2 weeks after infection (Tambourin, 1979; Ostertag and Pragnell, 1981;Wendling et al., 1981) (Table VI). After a short latency, the anemia-inducing strain and the polycythemia-inducing strain of Friend virus (FVa and FVp, respectively) induce an erythroleukemia in susceptible adult mice, which is characterized by a massive increase of CFU-E in bone marrow and spleen (Horoszewicz et al., 1975; Liao and Axelrad, 1975). Most, if not all, of the CFU-E in FVp-infected mice are erythropoietin independent, while those infected by FVa remain erythropoietin dependent (Ostertag and Pragnell, 1981; Steinheider et al., 1979). As reported for in vivo infections, FV infection of long-term marrow culture induces a rapid increase of CFU-E (Dexter et al., 1981a,b). The different biological properties of the two isolates seem to be caused by different env

MURINE SPLEEN FOCUS-FORMING VIRUSES

309

TABLE VI FORMATION OF HEMATOPOIETIC CELLSON INFECTION WITH SPLEEN COLONY FOCUS-FORMING VIRUSES OF THE ADULT MOUSE Virus

Type of disease

FVP

Erythroleukemia and pol ycythemia

FVa

Erythroleukemia and anemia

RV

Erythroleukemia and anemia

MPSV

Myeloproliferative syndrome, anemia, erythroleukemia, myelofibrosis

HaMuSV

Erythroblastosis and benign histiocytoma

MHSV (AF-1) Malignant histiocytosis

Alteration of hematopoietic colony-forming cells Increase of spleen CFU-E with all SFFVp strains Increase of bone marrow CFU-E on infection with some, but not all SFFVp strains All CFU-E acquire complete independence of erythropoietin (Epo) V-BFU-E formation on in uitro infection independent of Epo Increased self-renewal capacity of CFU-S Increase of spleen, but not bone marrow CFU-E All CFU-E independent of Epo in proliferation, but not terminal differentiation Increase of spleen CFU-E All CFU-E independent of Epo in proliferation, but not terminal differentiation Reduced dependence of BFU-E on multiCSF Increase in proliferative capacity of CFU-S Increase in macrophage-granulocyte colony-forming cells Increase of CFU-E, BFU-E, CFU-GM, megakaryocyte precursor cells, Mix-CFU, and of CFU-S in spleen Apparent independence of precursor cells and Mix-CFU for Multi-CSF Increase of colony-forming cells in longterm bone marrow cultures Transient increase of CFU-E in bone marrow and spleen Decrease of BFU-E in bone marrow and spleen Increase of CFU-M in spleen and later on in bone marrow Factor independence of CFU-M and of part of the CFU-GM Decrease in CFU-E, BFU-E, CFU-G, and CFU-GM in spleen and bone marrow

310

WOLFRAM OSTERTAG

gene products, since a subgenomic fragment of FVa comprising the enu region and the two LTRs are sufficient to cause all the characteristics of the FVa-induced disease (see Section IV,B; Kaminchik et al., 1982). The spleen focus formation by the Friend virus complex was shown to be caused by the SFFVp component, since pseudo-types with varying helper virus isolates induced similar erythroleukemias when infected (Fagg et al., 1980; MacDonald et al., 1981a,b; Fagg and Ostertag, 1982). Wolff and Ruscetti (1985)demonstrated that F-SFFV alone is sufficient to cause erythroid expansion by using a helper-free SFFV to infect NIH Swiss mice, a mouse strain that is unlikely to provide endogenous helper virus for replicating the SFFVp genome (see above). While most of the above-mentioned work suggested that CFU-E cells may be the target for FV, since it was the most obvious affected cell type, other work also found significant changes in BFU-E levels of FVp or FVa-infected mice: an increase in the peripheral blood and spleen, and a decrease in the bone marrow (Peschle et al., 1980). The increase of BFU-E cells in cycle after infection with either strain of Friend virus suggested that it is one of the main target cells of FV. However, BFU-E expansion has not been observed in FVp-infected long-term marrow cultures (Dexter et al., 1981a,b) nor in vivo using molecularly cloned SFFV pseudo-typed with F-MuLV (Yamamoto et al., 1981).The altered in vivo levels of BFU-E after FV infection is therefore a matter of controversy. Different results may be ascribed to the cell culture conditions and the source of virus used. Amplification of both the CFU-E and the mature BFU-E compartment by FV was also found in the Fv-2”derived fraction of spleen cells in chimeras of mice congenic at the Fv-2 locus (Behringer and Dewey, 1985). This supports the notion that FV induces the expansion of two different mature erythroid CFC. No residual erythropoietin dependence of erythroid precursor cells (CFU-E) was detectable after FV infection in viuo, even in serum-free conditions (MacDonald et al., 1981a,b). The defective subunit of Friend virus also seems to alter proliferation of other hematopoietic precursor cells. Although infection of Rauscher-MuLV alone increases production of GM-CFU and CFU-S in bone marrow cultures, these cultures are sustained for a much longer period after infection with F-SFFVp pseudo-types. The increase of CFU-S is not due to an altered self-renewal capacity of these progenitors (Greenberger et al., 1983a). Similarly, factor-dependent permanent cell lines, characterized as either multipotential cells or committed granulocytic and mast cells, were isolated at an increased frequency from F-SFFV-infected cultures as compared to those in-

MURINE SPLEEN FOCUS-FORMING VIRUSES

311

fected by F-MuLV alone (Greenberger et al., 1980a,b, 1983a). The multipotential cell lines are dependent on WEHI-3-conditioned medium and are not tumorigenic. They are not pluripotent stem cells, since they do not form splenic colonies in the CFU-S assay (Greenberger et al., 198313).The role of SFFV in the immortalization of such cell lines is not clear, since neither its transforming gene product gp55 nor its genome has been detected within these cells; only F-MuLV expression has been observed (Kluge et al., 1985). Eckner and Hettrick (1982) described SFFVp-associated effects on CFU-S in B6/B6 isologous chimeras, infected shortly after bone marrow transplantation. SFFVp, but not helper virus, appeared to increase the serial transplantability of CFU-S more than 5-fold. This indicates that SFFVp can alter the self-renewal capacity of CFU-S and that this effect is not controlled by the Ft1-2~locus. Previous studies had shown that 12% of FV-infected F~-2'/Fv-2~ chimeras developed erythroleukemia and that most of them contained dormant leukemic cells which rapidly induce leukemia of donor origin when trans~ ~ (Eckner and Hettrick, 1982). planted into congenic F V -mice Friend virus infection of C57BL/6-DBA/2 allophenic mice caused erythroleukemia, if at least 15% of the blood cells were of DBN2 origin. The erythroleukemic effects were almost exclusively noted in ~~ a the susceptible DBN2-derived cells. This indicates that F V -exerts permissive effect on the expression of FV-induced erythroleukemia and that Fv-2' cells cannot confer their restrictive potential on F V - ~ ~ cells. Thus, Fv-2' and F V -do ~ not ~ release soluble factors that can be transmitted to the other cell types in a chimeric mouse (Dewey and E ldridge, 1982). The erythroproliferative phase of the Friend disease depends on an intact microenvironment, since microenvironment-defective Sl/Sld mice do not develop splenomegaly on infection with Friend virus, but permit the proliferation of transformed Friend cells (Mager et al., 1980). It was reported that the Fu-5 gene of the mouse determines the induction of early anemia or polycythemia even if FVp is used to infect certain substrains of CBA or C3H/He mice (Shibuya and Mak, 1982a,b; Kitagawa et al., 1983). These findings cannot at present be interpreted, since normal hematopoiesis and interaction of FV with hematopoietic target cells in these mice are as yet undefined. The results cited above were obtained by the analysis of hematopoietic cells of infected mice. Such studies, however, need to be augmented by an analytical approach in uitro to obtain a better understanding of the interaction of virus with target cells. Hankins and others (Kost et al., 1979,1981; Hankins and Krantz, 1980; Hankins and

312

WOLFRAM OSTERTAC

Troxler, 1980; Koury et al., 1982; Bondurant et al., 1983) have attempted to define conditions for testing the effect6 of FV infection in uitro. Erythroid bursts are induced by FVa and FVp on in vitro infection of bone marrow cells. FVp-induced bursts require no erythropoietin for hemoglobinization, whereas FVa-induced bursts do. These results confirmed earlier in uivo work that indicated that FVa also induces a proliferative response that normally requires erythropoietin (Tambourin, 1979). The terminal maturation of FVa-infected cells within the virus-induced burst, however, still requires erythropoietin. The FVa-infected cells, if not exposed to erythropoietin, will divide for up to 5 days in culture. If erythropoietin is then added, they maintain the potential for cell differentiation. These cells are thus ideal to study the erythropoietin response on erythroblasts (Bondurant et al., 1983, 1985). The erythropoietin-independent burst formation after in vitro infection with FVp is impaired when erythropoietin is not present in the growth medium (serum-free conditions) prior to infection. The addition of erythropoietin prior to infection reverses these results, suggesting that cells responsive to FVp require this factor (Hankins, 1983) and pass to the factor-independent state after infection. This again is in agreement with in vivo results of Tambourin (1979): The number of FVp target cells in the animal is decreased markedly if mice are manipulated in a way that reduces erythropoietin production (e.g., by hypertransfusion). What conclusions can be drawn on the identification of the target cells of FV action? The type of colony which is obtained on in vitro infection with FV is morphologically a large, compact-appearing, burst-type colony. It does not require Multi-CSF, but does require erythropoietin; thus, it is probably not the relative early erythrocyte progenitor, BFU-E, although its number is increased after in uivo infection. Initial velocity sedimentation studies showed that cells that give rise to large erythroid colonies after in vitro infection with FV (vBFU-E) coelute with the CFU-E, but not the BFU-E fraction (Kost e t al., 1979). Further studies, however, showed that V-BFU-E coelute with day 3 BFU-E and that both cell types are increased in plethoric mice (Kost et al., 1981). It is as yet not well understood why FVp should be restricted by S1/ Sld or W/Wvmice. W/Wvmice appear to generate almost normal numbers of early BFU-E cells in the bone marrow and depressed BFU-E levels in the spleen, whereas a much less pronounced decrease is found for Sl/Sld mice. This moderate depression of BFU-E in these mice does not agree with the assumption that the BFU-E is actually the target cell for FVp action (Hankins and Troxler, 1980). Lack of cycling of BFU-E cells in W/Wv mice is an unlikely explanation for the

MURINE SPLEEN FOCUS-FORMING VIRUSES

313

resistance, since cycling of the BFU-E must be high to account for the compensatory increase of proliferation of erythroid precursor cells in W/Wv mice. One study implies that a second BFU-E-like cell, the TECFU, is grossly deficient or entirely lacking in Sl/Sld and W/Wv mice (Wiktor-Jedrzejcak et al., 1979, 1981).The lack of a marked reduction of TE-CFU in mice heterozygous for the W locus [(W/+)or Wv/+] and the much reduced sensitivity of these mice to FVp as published by others (Steeves et al., 1968) appeared to exclude the TE-CFU as a target cell for FVp. Our results, which show normal sensitivity of these heterozygous mice (Ostertag et al., 1982), would suggest that TE-CFU cells may, in fact, be the target cell for FVp. This is not in contrast to the interpretation of the extensive data of Hankins' group, who did not distinguish between TE-CFU and BFU-E (Hankins and Troxler, 1980). However, the inability of other groups, as well as our own, to reproduce or extend the in vitro results of Hankins' group, using purified erythroid precursor cells from fetal or adult F v - mice, ~ ~ puts the interpretation of these results into question. There are several critical points in the in vitro protocol of Hankins' group: (1)The mice used to isolate cells are treated with phenylhydrazine, which induces a hyperproliferative state of erythroid precursor cells. Thus, these precursor cells may not only represent normal erythroid precursors, but also cells which are possibly utilized only during stress-induced erythropoiesis (see Section 11).Such cells may be similar to those deficient in W and S1 mice, and thus would be consistent with the findings that these mutant mice are deficient in TE-CFU cells and are relatively resistant to FV-induced erythroleukemia. (2) The virus preparations used are of a very high titer, much more than necessary for observing direct in uiuo effects. Best results are usually obtained with virus preparations from infected mice and not from tissue culture. The sera used to provide optimal conditions are usually rich in erythropoietinlike activity, perhaps IGF-1 (Kurtz et al., 1982, 1985), resulting in a high background of normal erythroid burst formation. This may imply that other undefined factors are additionally required to obtain target cells for FV action. (3) The most serious objection, however, is that at least lo4 cells must be plated per milliliter to obtain any effect of FV in in vitro infection. Such a protocol does not exclude effects on the target cell due to cell interaction, even if purified precursor cells are used. Thus, previous interpretations of these data were perhaps too simplified. Infection with SFFV preparations of single erythroid precursor cells, colonies, or progeny thereof is necessary to obtain more definitive data. Analysis at the single colony or single cell level, however, requires

314

WOLFRAM OSTERTAG

a selecting procedure to confirm infection of the cell/colony. Screening for erythropoietin-independent growth transferred by FVp infection would be one possible approach; however, this would preclude analysis of all infected cells, as those cells which are infected, but not yielding factor-independent growth would escape detection. Joyner and Bernstein (1983), as well as our group (Hess, 1985), have attempted to modify molecularly cloned SFFV by the introduction of a selectable marker gene. These attempts encountered unexpected difficulties: The introduction of a gene between the 5’ LTR and the enu gene interferes with proper expression of the env mRNA coding for gp55, required for transformation (see Section IV,B). However, we have recently been successful in constructing a chimeric virus between the neoR-MPSV (Ostertag et d., 1986) and F-SFFVp-SL genomes. This virus contains the entire env coding region, splice acceptor sequences, and U 3 LTR from SFFV and the gag-neoR sequences and U5 LTR from MPSV. It efficiently transfers G418 resistance to infected cells and induces the typical course of Friend erythroleukemia upon infection of sensitive mice (N. Hunt et aZ., 1987). Such a vector may prove invaluable in ascertaining the SFFV target cell. Promising results were obtained from binding purified erythropoietin to FVa-infected spleen cells, demonstrating the presence of Epo receptors in these cells (Krantz and Goldwasser, 1984) and thus the feasibility of studying directly the interaction of SFFV-coded gp55 protein with erythropoietin receptors when infected target cells are isolated by drug resistance. In summary, not only the Friend viral complex, but also F-SFFV ~ ~ The alone induces an erythroproliferative disease in adult F V -mice. expanded erythroid precursor pool is composed mainly of late erythroid precursors (CFU-E). In vitro studies (which are certainly not conclusive) indicate that the target cells of FV are erythropoietinresponsive late BFU-E cells. Upon infection with either FVa or FVp, these cells acquire proliferative potential for up to 5 days in culture without added erythropoietin. After this time, FVp-infected colonies differentiate terminally without added factor, but FVa-infected colonies require erythropoietin. F-SFFV appears to interact specifically with (some) erythroid precursor cells, since myeloid (Colletta et aZ., 1983a,b) or fibroblast cells (Ostertag and Pragnell, 1981) infected with SFFV do not show obvious alterations in their growth requirement. 2 . Rauscher- and Cas-SFFV The Rauscher virus is molecularly and biologically more closely related to FVa than to FVp (see Sections IV,B and V,B,l). The RV

MURINE SPLEEN FOCUS-FORMING VIRUSES

315

complex causes an erythroproliferative syndrome in adult mice (Opitz et at., 1977; Hasthorpe, 1978; Hasthorpe and Bol, 1979). One isolate of RV consisted of two forms of helper virus (R-MuLV), R-SFFV, and an associated MCFV (Vogt, 1982); however, R-SFFV, in conjunction with any helper virus, is sufficient to induce the typical RV-induced leukemogenesis (Mol et al., 1982). Molecularly cloned R-SFFV pseudo-typed with F-MuLV also causes the amplification of the CFUE compartment in the spleen and shows the pattern of host range restriction typical of RV (Hess et al., 1984). RV induces in vitro formation of poorly hemoglobinized V-BFU-E, similar to those observed after infection with FVa (Hankins and Troxler, 1980) (Table VI). These V-BFU-E are highly sensitive to erythropoietin. Exceeding the extent of compartment amplification of FV, RV also induces proliferation of BFU-E, GM-CFU, and CFU-S and increases their sensitivity to stimulating factor (Opitz et al., 1977; Hasthorpe, 1978; Mol et al., 1982; Ostertag et al., 1982) (Table 11).The fact that a subset of BFU-E of RV-infected mice apparently does not require Multi-CSF for proliferation (Ostertag and Pragnell, 1981; Mol et al., 1982), unlike normal BFU-E and mixed bursts (Table 11), raises the question whether RV transforms early precursor cells, including perhaps stem cells, or whether it infects (and transforms?) cells which secrete hormones related to or identical with Multi-CSF in biological activity (see discussion on MPSV). The Cas-SFFV is another enu-recombinant isolate which causes erythroblastosis in adult mice (Langdon et al., 1983a,b). Spleen focus formation by this virus is absolutely restricted by the Fv-2' locus in DDD mice, as it is with FV, but in contrast to RV. Cas-SFFV induces erythroid bursts after infection of bone marrow cells in vitro. These bursts are not fully hemoglobinized in the absence of erythropoietin, similar to those induced by FVa and RV (Langdon et al., 1983b). The hematology of cells infected by RV- and Cas-SFFV is even less understood than that of FV-infected cells. The action of RV is especially interesting, since it differs markedly from that of FV even though molecular data on genome structure would suggest great similarity (see Section IV,B). RV, in contrast to FV, induces a disease in homozygous Fv-2' DDD mice; only mice carrying at least one other resistance gene (Mpsu?), in addition to the homozygote Fv-2' locus, are resistant to RV-induced leukemogenesis. RV also induces leukemia in W and S1 mice (Ostertag et al., 1981; Mol et al., 1982; Hess et al., 1984). These data and the fact that some of the virus-infected BFUE and mixed colonies do not require growth factors may indicate that RV also transforms earlier precursor or stem cells or affects cells that

316

WOLFRAM OSTERTAG

secrete hormones, thereby indirectly affecting BFU-Es (see MPSV discussion). The analysis of R-SFFV or Cas-SFFV interaction with putative target cells requires the development of better colony assays, purified progenitors, and a selectable virus construct (see previous discussion). 3. Myeloproliferative Sarcoma Virus a. In Vivo Effects of MPSV. MPSV transforms fibroblasts in vitro and induces a variety of hematological alterations in adult mice (Table VI). MPSV is not restricted by the Fv-2 resistance locus in DDD mice, yet it cannot cause splenomegaly in C57BL/6 mice if these are Fv-2' (Ostertag et al., 1981).The putative Mpsv resistance locus which is present in C57BL/6 mice is active in hematopoietic cells, but not in fibroblasts. In susceptible F V - ~Mpsvs ~ , mice, MPSV causes splenomegaly and anemia, with a marked elevation of CFU-S (Jasmin et al., 1980), an increase of GM-CFU (LeBousse-Kerdiles et al., 1980; Klein et al., 1982) of megakaryocyte precursor cells (LeBousse-Kerdiles et al., unpublished), and a pronounced increase of CFU-E in the spleen (Ostertag et al., 1982). Some of the GM-CFU and BFU-E of MPSVinfected mice mature without the addition of stimulating factor (Klein et al., 1982, 1983; Ostertag et al., 1982; Fagg and Ostertag, 1983). The pronounced increase of CFU-E is mainly responsible for the enlargement of the spleen. The effect of MPSV on erythroid precursor cells was thought to be a direct one, since in vitro infection of bone marrow cells with MPSV caused erythroid burst formation (Hankins et al., 1982). The increased number and factor independence of GM-CFU and CFU-S could be reproduced in vitro in long-term marrow cultures (Mori et al., 1983; Le Bousse-Kerdiles et al., 198513). Infected cultures give rise to transformed fibroblastic cell lines, presumably derived from the microenvironment formed in such cultures. Only a single permanently growing myeloid (monocyte!) cell line and no erythroid cell line has been so far reported after infection of myeloid cells with MPSV (Le Bousse-Kerdiles et al., 1985b). The lack of erythroid transformation, despite induction of erythroid proliferation of these cells by MPSV, would support the conclusion that the helper virus in the FV complex does not cause erythroid transformation during progression of the FV-induced disease (block in differentiation, as in Friend cells). The variety of hematopoietic alterations caused by MPSV suggested either a direct influence on the hematopoietic stem cell or the induced secretion of stimulating factors by cells infected with MPSV. Indeed, GM-CSF activity was found in conditioned medium of MPSV-trans-

MURINE SPLEEN FOCUS-FORMING VIRUSES

317

formed fibroblasts (Koury and Pragnell, 1982); however, the presence of GM-CSF-like activity in the supernatant of transformed fibroblasts is not specific for MPSV-transformed cells, since other fibroblasts (even untransformed) release a similar factor when infected with a murine retrovirus. More relevant are data by Le Bousse-Kerdiles et al. (1983, 1985b): MPSV-infected, but not uninfected, spleen cells release a Multi-CSF-like activity. These results were repeated and confirmed with molecularly cloned MPSV and MPSV ts mutants (C. Laker, 1987). Cells which have been reported to release CSFs necessary for hematopoietic cell differentiation are either T lymphocytes or, for stem-cell-level stimulation, more likely stroma cells or, possibly, also macrophages (see below). b. Znteraction of MPSV with Potential Target Cells in Vitro. Direct interaction with the potential target cell of any acutely transforming virus can be studied at a molecular level only if the target cells can be isolated free of other cells and the infected target cell can be separated from the noninfected cell. The separation of infected and noninfected cells requires introduction of a dominant selectable marker into the retrovirus genome to allow removal of noninfected cells by growth in selection medium. We were successful in introducing the geneticin resistance gene (neoR)into MPSV without impairing its transforming potential (Ostertag et al., 1986; Seliger et al., 1986) (Fig. 21). The use of retroviruses bearing mutants of the transforming gene enables investigation of whether some effects are a consequence of viral infection or if the (continued?) presence of the oncogene is required. Selectable mutants of MPSV were therefore generated: neoRts mos mutants of MPSV (Kollek et al., 1984; Ostertag et al., 1984) and neoR-mos deletion mutants of MPSV (neoR-mos-') (Stocking et al., 1985; Seliger et al., 1986) (Fig. 21). The use of psi-2 helper cell lines as described by Mann et al. (1983) makes it possible to generate virus particles which only contain the genome of replication-defective neoR-MPSV or its mutants. This not only precludes reinfection of other cells, but also eliminates the possibility that (continued) helper virus functions may contribute to the altered phenotype of an infected cell, Thus, such helper-free neoR-MPSV viral isolates were used in in uitro target cell experiments described below (Johnson, Nicola and Ostertag, unpublished). Two types of potential target cells were initially utilized for infection with neoR-MPSV: (1) a prepurified cell population of murine hematopoietic precursor cells (CFC) (see Section 11) or enriched stem cells (CFU-S), or (2) bone marrow cells of mice treated with 5-FU to obtain enrichment of CFCs by killing cycling progenitor cells. Infec-

318

WOLFRAM OSTERTAG

tion with neoR-MPSV or neoR-mos-' MPSV (Fig. 21) was done at different times after initiation of in vitro culture and was followed by selection for infected cells with geneticin. The results were essentially the same for all infections regardless of the type of virus or of target cells tested: (1)The infected potential target cells and the noninfected cells show the same distribution of CFC, i.e., the proportion of Mix-CFC, of BFU-E, of GM-CFU, or of later colonies was not grossly altered. (2) The infected cells still require, as do normal cells, Multi-CSF for colony formation, similarly as known for normal CFC. It is still uncertain whether the Multi-CSF requirement is altered quantitatively or whether other factor requirements are impaired. Growth potential of stem cell cultures may possibly be altered by introduction of neoR-MPSV in stem cell cultures. The above cited results show that some of the potential target cells (BFU-E, Mix-CFC, or GM-CFU) which acquire apparent factor independence on infection of mice with MPSV (Ostertag et al., 1980; Klein et al., 1981, 1982; Fagg et al., 1983) are not truly factor independent when neoR-MPSV is introduced. Two models for obtaining factor independence have been discussed above (see also Jasmin et al., 1980; Le Bousse-Kerdiles et al., 1981,1983, 1983; Laker, 1987):factor independence acquired as a consequence of direct infection with MPSV (which now seems unlikely) or as a consequence of factor production by an alternative target cell. This other presumptive target cell cannot be present in long-term stem cell cultures which are maintained in the presence of Multi-CSF. T lymphocytes which are known to secrete growth factors on exposure to either antigens or lectins, including Multi-CSF, do not grow in stem cell cultures unless IL-2 is supplied (see also Section 11). neoRMPSV and neoR-mos-l were therefore used to infect established T cell line LB3, as described by Kelso and Metcalf (1985),and to isolate neoR-LB3 cells (Graning, Kelso, Laker, and Ostertag, unpublished). LB3 cells infected by either of the neoR-MPSV viruses do not acquire factor independence with respect to IL-2, but a large proportion of cells infected with neoR-MPSV and by neoR-mos-' acquire independence of antigen stimulation for continued growth in uitro. Noninfected LB3 cells do not secrete growth factors such as MultiCSF or GM-CSF (see Section 11); they can, however, be induced to release high levels of GM-CSF and of Multi-CSF when exposed to concanavalin A (Con A) (Kelso and Metcalf, 1985). Cells infected with neoR-MPSV or neoR-mos- were tested for release of these growth factors in the absence of any normal stimulator of factor release. Cells

MURINE SPLEEN FOCUS-FORMING VIRUSES

319

infected by either of the two viruses (regardless of the mos oncogene!) release GM-CSF but no or only small amounts of Multi-CSF, however, at lower levels than released by conA-induced LB3 cells. The fact that GM-CSF and not Multi-CSF is released by MPSV-infected LB3 cells is in contrast to previously cited results that MPSV-infected spleen cells release factors similar in biological properties to MultiCSF but not to GM-CSF (Le Bousse-Kerdiles et al. (1983). Our results would therefore not explain the myeloproliferative disease. We have shown that the U3 region of the LTR of MPSV is essential for the disease induced by MPSV in mice (Stocking et al., 1985; 1986). Based on these data, we have attempted to discriminate between potential target cells by infection with neoR-MPSV constructs with either the MPSV or the Mo-MuLV U3 region. T cells and all other hematopoietic cells which have been tested so far can be infected and express virally controlled genes equally efficiently as in fibroblasts, regardless of the U3 region. The U3 region of MPSV, however, appears to be necessary for expression in a series of diverse epithelial cell lines, such as the embryonal carcinoma cell line F9 (Franz et al., 1986) and hepatoma cells (unpublished). It will be interesting to test the effect of MPSV on stroma cells. 4 . ras Oncogenic Viruses

The ras oncogenic viruses induce myeloproliferative diseases, with a bias of erythroproliferation (Ki-MuSV, Ha-MuSV, BALB-MuSV) or histiocytosis (MHSV) on infection of sensitive mice (Table VI). When the ratio of fibroblast transformation potential to spleen focus formation is compared, the first three aforementioned viruses are much less efficient than MHSV in inducing spleen foci of proliferating cells in adult mice (Franz et al., 1985; Lohler et al., 1987).Very little information is available concerning the alterations of CFC or on virus-induced pathology of the ras oncogenic viruses in adult mice, except for a recent comparative study of MHSV and Ha-MuSV (Franz et al., 1985, Lohler et al., 1987). The earliest characterization of the HaMuSV and Ki-MuSV showed that these two viruses cause erythroblastosis and multiple sarcomas in newborn mice (Harvey, 1964; Kirsten and Mayer, 1967).Using biologically cloned Ha-MuSV and Ki-MuSV and ts mutants, it was shown that the pathogenicity was a property of the defective subunit of those viral complexes (Scher et al., 1975; for a review see Weiss et al., 1982). Ha-MuSV also induces a monocytic tumor in adult DDD, but not in C57BL/6 mice. The development of

320

WOLFRAM OSTERTAG

the tumor begins in the red pulp of the spleen and is associated with a monophasic erythroproliferative response, but not by erythroleukemia (Lohler et al., 1987). The tumor is characterized by its slow growth and subsequent regression. Attempts to isolate permanent cell lines from the tumor have failed so far. The transient erythroproliferative response is associated with a marked increase of CFU-E, a fraction of which differentiates in the absence of added erythropoietin. A minor fraction of the M-CFU and GM-CFU does not require growth factor addition. Whether these myeloid factor-independent colonies are truly factor independent remains to be shown. A similar study with hematopoietic cells of newborn mice infected by either HaMuSV or Balb-MuSV by Pierce and Aaronson (1985)showed evidence for myeloid perturbations induced by these viruses. Whether these abnormal proliferating myeloid colonies actually grow without addition of growth factor is unclear. The effect of Ha-MuSV and BalbMuSV does not depend on the type of helper virus used. The recently isolated malignant histiocytosis sarcoma virus (MHSV or AF-1) rapidly induces splenomegaly and multiple solid tumors not only in adult DDD, but also C57BU6 mice, thereby exceeding the host range of Ha-MuSV (Franz et al., 1985; Lohler et al., 1987). MHSV causes transformation of monocytic cells which are clustered in the marginal zone of the spleen. Transformed macrophages appear as early as 3-5 days following infection with MHSV. These cells are easily transplantable and form solid histiocytic tumors. The “fibroblastic” cells express macrophage-specific markers, such as unspecific esterase, acid phosphatase, F4/80 antigen, and lysozyme (Franz et al., 1985; Klinger et al., submitted). The disease induced by MHSV is similar to the malignant histiocytosis found in humans (Huhn and Meister, 1978), where it is also associated with a transient erythroproliferative response. The MHSV-induced transient expansion of the erythroid lineage is limited to the CFU-E compartment of the spleen, whereas the BFU-E begins to decrease rapidly 4 days postinfection. GM-CFU, G-CFU, and Mix-CFC also decrease markedly and in parallel with the decrease in BFU-E. A few days later, the number of late CFCs also declines. This synchronous decay of CFCs (except for macrophage precursor cells) suggests the depletion of stem cells is either directly caused by infection with MHSV or is an indirect consequence of the virus that is hormone mediated. The fact that the erythroid precursor pool does not mature properly in uiuo in the 5s- disease in which the M-CSF receptor (fms) locus has been deleted (see Section 111), but that these cells are nevertheless present in in vitro studies, suggests that macrophage precursors may interact with erythroid

MURINE SPLEEN FOCUS-FORMING VIRUSES

32 1

precursors in an inhibitory fashion (Nienhuis et al., 1985). This fact may also explain the deficiency in CFCs in mice infected with MHSV. The splenomegaly caused by MHSV is, thus, not a consequence of erythroid expansion, as found after infection with any of the other spleen focus-forming viruses (SFFV, MPSV, Ha-MuSV), but due to the proliferation of macrophage-type tumor cells. This is also reflected in the increase of M-CFU, the majority of which proliferate in the absence of added growth factors (Franz et al., 1985). A few growth factor-independent colonies are also of the GM-CFU type; however, these colonies differentiate along the macrophage pathway on successive colony transfer (Johnson et al., 1984). Almost all of these M-CFU or GM-CFU colonies retain their factor-independent growth after serial dilution in microtiter wells, indicating that they are truly factor independent and do not require factors produced by other cells. Of factor-independent M-CFU and GM-CFU from MHSV-infected mice, 25%can be grown in culture as permanent cell lines, even as early as 4 days after infection of mice (Franz et al., 1985; Klingler et al., unpublished). This suggests that the macrophage-transforming action of MHSV is direct (Table 11). Acute transforming retroviruses containing rus oncogenes can alter the growth property of a series of different cell types. Epithelial cells can be transformed in vitro in addition to fibroblasts and hematopoietic cells (Fusco et al., 1981; Colletta et al., 1983b; Rhim et al., 1985), and it appears likely that ras oncogenic viruses, as discussed for the mos oncogenic viruses, can transform a wide range of differentiated cells. The specific effects of the rus oncogenic viruses on hematopoiesis upon intravenous injection of adult mice are at present unexplained. The effects of ras oncogenic viruses on hematopoietic cells were also tested in in vitro systems (Greenberger, 1979; Hankins and Scolnick, 1981; Johnson et al., 1985; Pierce and Aaronson, 1985; and unpublished results). Greenberger (1979) infected stem cell cultures with Ki-MuSV and obtained a reduction of the proliferation of granulocytic precursors and of hematopoietic stem cells. The effect on erythroid precursor cells was not tested. Macrophage precursors were morphologically altered and possibly transformed. They grow in agar to form small clusters of 4-49 cells in the absence of growth factors. The limited growth precluded analysis by colony transfer experiments. Effects of helper virus and cell-cell interactions could not be excluded. Two groups studied the effect of rus-containing viruses on erythroid and myeloid precursor cells in vitro. Ha-MuSV and Ki-

322

WOLFRAM OSTERTAG

MuSV induce a proliferative response in erythroid precursor cells in the presence of erythropoietin and independent of the type of helper virus used. Arrested differentiation of erythroid cells was not observed (Hankins and Troxler, 1981).Pierce and Aaronson (1985)have shown evidence for a growth-promoting effect on myeloid precursor cells with Balb-MuSV- or Ha-MuSV-infected murine bone marrow cells. Both groups utilized a high cell number and essentially unsorted hematopoietic cells for infection. More defined experiments have been done with the malignant histiocytosis sarcoma virus (Johnson et al., 1985; Klingler et al., submitted). MHSV, if added to prepurified CFC or to single CFC, induces in up to 50% of the granulocytic/macrophage precursor cells factor-independent colony formation. Factor-independent growth of colonies without a macrophage phenotype was not obtained. MHSV is thus the first acute oncogenic retrovirus where transformation of single-cell hematopoietic cultures has been observed. Direct studies of virus-cell interactions and their causal effect on transformation thus appear feasible. The macrophage or macrophage precursor cell is one definite target cell of MHSV. What determines the target cell specificity of MHSV is not understood. All of the myeloid colonies which grow autonomously after in vitro infection of single cells with MHSV have a limited life span (Johnson et al., 1985). With a frequency of 1 in lo3, we also obtained large myeloid colonies after infection with MHSV which did not require tissue culture adaptation for permanent growth (Klingler et al., submitted). In Section 111, we have summarized other data regarding the phenotype of macrophage precursors transformed by MHSV and have concluded that the most likely model to explain the transformation process induced by MHSV is the increase of growth factor receptors, mainly for M-CSF and GM-CSF, as a consequence of an altered interaction of the oncogenic p21 ras protein encoded by MHSV with these receptors. Many lines of evidence suggest that the hematopoietic target cells for ras oncogenic viruses are primarily found within the macrophage precursor lineage. The malignant histiocytosis sarcoma virus is the most extreme example of this virus group in its almost exclusive macrophage specificity of transformation. Associated erythroproliferative alterations which are also induced by these viruses (Ha-MuSV, Ki-MuSV, Balb-MuSV, but much less MHSV) are not truly leukemic. The ubiquity of macrophages in parenchymal organs and connective tissue culture cells makes it likely that macrophage tumors can appear at many sites, not only the spleen.

MURINE SPLEEN FOCUS-FORMING VIRUSES

323

VI. Perspectives

The purpose of this review was 3-fold: (1)to summarize current data on the molecular structure, biological function, and target cells of the acutely transforming murine retroviruses that induce alterations in myeloid proliferation; (2)to contrast the proposed process of transformation by these oncogenic viruses to the process of leukemogenesis observed in chemically induced or naturally occurring neoplasms and to identify viral factors or experimental artifacts that may account for discrepancies or similarities between the proposed models; and ( 3 )to define experiments, based on our current knowledge, utilizing already existing spleen focus-forming viruses or newly generated recombinant viruses that will further our understanding of leukemogenesis. Molecular analysis of the viral genome and in uiuo and in uitro analysis of hematopoietic alterations occurring after infection of the animal or of bone marrow cultures has not yet revealed a general mechanism of how spleen focus formation occurs in the course of the viral-induced myeloproliferative diseases. Three groups of spleen focus-forming viruses were compared: Each group contains a unique transforming gene (enu-recombinant, mos, or rus), and virus of each group alters the proliferative capacity of more than one cell type and thus appears to have a number of different target cells. The transforming gene itself does not appear to be restrictive to one particular target cell. Retroviruses transducing a rus oncogene may alter erythroid, granulocyte/macrophage, and potentially other hematopoietic cell proliferation both in viuo and in in uitro mass cultures. This lack of specificity of a defined target cell is also reflected in the diversity of tumors associated with the activation of the cellular rus protooncogene. Although a bias for one neoplasm or another may exist, this is most likely not a feature of the protooncogene itself. Similarly, the transduction and efficient expression of mos within a retroviral genome (e.g., MPSV) is capable of not only inducing a proliferative disregulation of myeloid cells, but also of transforming fibroblasts, epithelial cells, and embryonal carcinoma cells. Only the env-recombinant product, at least when expressed within the retroviral genome, seems to interact specifically with erythroid precursor and erythroid cells, and possibly also with stem cells (Friend virus group). This specificity is not strict, however, as evidenced by the broader effect on hematopoietic cell proliferation induced by the Rauscher virus and the peculiar host range pattern of this enu-recombinant virus which has marked similarity to the mos-oncogenic MPSV.

324

WOLFRAM OSTERTAG

The permissive nature of the transforming genes is consistent with the association of a number of different oncogenes with a variety of neoplasms, including leukemias, observed in patients, but does not explain the diverse pathological effects of the different spleen focusforming viruses. Two main questions remain unclear: What other feature of the virus determines its target cell specificity and thus its pathology? What are the real target cells of the virus? To address the first question, two viruses should be examined: MPSV, which has a broader host range than the closely related MoMuSV (see Section IV,C), and MHSV, which induces a more specific effect than the related Ha-MuSV (see Section IV,D). We have summarized results showing that the alterations in the U3 region of the LTR, which carries tissue-specific sequences, have resulted in the expansion of the target cell range of MPSV in comparison to Mo-MuSV. MPSV transforms fibroblasts, as Mo-MuSV, but also hematopoietic (?), epithelial, and embryonal carcinoma cells. One could speculate that the inverse applies to MHSV (and perhaps F-SFFV): Specific sequences within the LTR may permit high expression of the transforming genes in only specific tissues. Further analysis of the specificity of transformation by the enu-recombinant product itself, as well as the genomic makeup of MHSV, is necessary if other factors are involved. However, we feel that it may be more than coincidence that alterations within the LTR U3 region of MPSV as compared to Mo-MuSV are clustered around the glucocorticoid receptor binding regions, and even more striking dyshomology occurs in this same region when the Mo-MuLV-derived LTRs (e.g., MPSV, Abelson virus, Mo-MuLV) are compared with the F-MuLV-derived LTRs (e.g., F-SFFV, F-MCFV, F-MuLV) (Fig. 11). One corollary to these predictions would be the emergence of regulatory proteins which specifically bind to these sequences within the LTR and are perhaps also of major importance to cell differentiation. Characterization of such proteins may help us to understand basic mechanisms of differentiation and proliferation. Analysis of the action of the transforming gene specifically expressed in a target, as well as regulatory proteins that determine the expression, demands identification of the target cell. Analysis of potential target cells, however, was until recently limited to the study of these viruses in conditions where cell interactions could severely bias the conclusions. Unlike retroviral-induced transformation of fibroblasts, infection and transformation of hematopoietic cells by retroviruses are much more complicated owing to the complex interactions of the cells. It is therefore necessary to study only a subset of

MURINE SPLEEN FOCUS-FORMING VIRUSES

325

infected cells within a population and to study only the progeny of infected cells by eliminating noninfected cells selectively. Initial studies of potential target cells using single cell cultures are now feasible and have been carried out for MHSV (Johnson et al., 1985). Such studies can be facilitated by using retrovirus vectors carrying dominant selectable genes. Such vectors are available for MPSV (Ostertag et al., 1986; Seliger et al., 1986), but not for any of the ras oncogenic retroviruses (Ki-MuSV, Ha-MuSV, Balb-MuSV, RasheedMuSV, or MHSV), and only for one env-recombinant SFFV (F-SFFVp). Without single cell culture systems or selectable markers within the oncogenic virus, analysis of the direct interaction of the virus and its transforming gene with the target cell is severely constrained. The action of the oncogene transduced by the acute leukemia virus in the cell has become less esoteric, although by no means understood, owing to the increasing, mostly correlative evidence on protooncogene activation in various neoplasms. All currently fashionable models imply that oncogenes interfere with cell proliferation by diverse mechanisms and at various sites: as a growth factor analog outside of the membrane (sis), as membrane-spanning altered receptor molecules fms,erbB, neu, erbA), as gene regulators (myc,myb?), as components of the membrane interacting with receptors or signal transduction (possibly ras, SFFV enu), or, even more obscure, as intracellular signal transducers (possibly mos). Study of the function of oncogenes with respect to cell proliferation will require more direct data on the presumptive site of action. The alteration and recombination of oncogenes with cellular target signals, e.g., signals for the nucleus [SV40 T antigen sequences (Kalderon et al., 1984); influenza virus sequences (Davey et al., 1985)l or signals for membrane structures of extranuclear cellular components (Kreil, 1981; Walter et al., 1984), may give us more information on their site of action. Also of importance are studies where genetic mutations are employed to define those genes which directly determine the proliferative behavior of higher eukaryotic cells, similar to what is being done with yeast cells and other lower eukaryotes. The availability of a series of well-analyzed, cell cycle mutants would certainly help to define the function of protooncogenes and the putative misregulation by homologous oncogenes. The most striking feature of normal mammalian hematopoietic cells which makes these so attractive for studying malignant diseases is the vast and rapidly advancing knowledge of hematopoietic growth fac-

326

WOLFRAM OSTERTAG

tors and their receptors (see Section 11).This knowledge can be used to determine the “anatomy” of a disease with respect to alterations in growth factor requirements induced by acute oncogenic murine viruses. Indeed, an astonishing feature of the disease induced by most oncogenic retroviruses is the apparent altered growth factor requirement of abnormally proliferating cells (Table 11). Factor-independent growth has been observed in cells infected by viruses of the env-recombinant group and for the mos-oncogenic MPSV, although the latter may induce factor independence indirectly. Of the rus oncogenic viruses, only MHSV clearly alters growth factor requirements, conferring growth factor independence to macrophage and macrophage/granulocyte precursor cells. Alteration of factor requirements of cells infected by Ki-MuSV, Ha-MuSV, and BalbMuSV remains obscure despite intensive studies. The mechanisms by which each virus induces the alteration of growth factor requirements in the hematopoietic system appear to be different: The products encoded by R-SFFV and perhaps F-SFFV appear to interact directly or indirectly with the erythropoietin receptor and/or receptors related to Multi-CSF or functionally related growth factors. The TUS product of MHSV and, presumably, the other rus-oncogenic viruses appear to interact with a larger group of growth factors, including the hematopoietic receptors in MHSV-infected macrophage precursor cells. MPSV, bearing the mos oncogene, most likely interacts in two ways with hematopoietic cells. First, it may alter growth properties in all hematopoietic cells, similar to that of infected fibroblasts or epithelial cells, by a mechanism that is not well understood, but involves changes in the cytoskeleton and other cellular structures. In addition, some aspect of the expression of MPSV (and not necessarily the mos product itself) may induce factor release by possibly mimicking antigen stimulation of T cells or by inducing factor release in cells of the hematopoietic microenvironment (stroma cells?). Released growth factors (e.g., Multi-CSF, GM-CSF) may then cause the generalized myeloproliferative disease. The different mechanisms involved in the malignant progression appear just not to be a feature of viral-induced leukemias, but also of naturally occurring leukemias (see Section 111). We have already stated that most leukemias in mammals appear to arise as a consequence of multiple genetic alterations of a cell, each change increasing the malignant potential of the cell; yet, many oncogenic retroviruses appear to cause malignant conversion in one step or a series of temporally closely linked steps. This peculiar feature of oncogenic retroviruses implies a dominant action of its oncogene, in

MURINE SPLEEN FOCUS-FORMING VIRUSES

327

contrast to data on activated c-ras and other activated protooncogenes which may require homozygosity or hemizygosity (see Sections I11 and IV,D). The dominant action of the retorviral oncogene, however, may be attributed to an elevated level of LTR-regulated transcription, mimicking the effect of gene amplification or increased gene dosage. Other alterations which appear necessary for malignant progression, such as karyotype changes, interactions with antioncogenes, and, most importantly, stem cell alterations (which appear to be the primary step in many leukemias), cannot, at present, be studied using retrovirusmediated transformation. Oncogenic retroviruses have been used as tools to study alterations in the microenvironment which may be necessary to permit growth of pluripotent hematopoietic stem cells in culture. An src-oncogenic recombinant virus and MPSV either alter T lymphocyte proliferation, and thereby induce abnormal growth factor release, or transform other interacting cells which permit growth of stem cells [src virus (Boettiger et al., 1984; Pierce et al., 1984; Spooncer et al., 1984,1986);MPSV (see Section IV)]. These stem cells can be used for studies ofpotential targets for the SFFVs reviewed here or other retrovirus vectors, and, perhaps, a system could be developed where the progression of malignancy from a stem cell can be studied. Alternatively, one could envisage that oncogenic viruses, such as the R-SFFV and F-SFFV, known to alter not only erythroid precursor proliferation, but also hematopoietic stem cell proliferation, could be used as vectors to infect stem cells in vitro in order to permit permanent growth (and study) of such stem cell lines with respect to malignant progression. To further understand the interaction of oncogene products with normal cellular functions, it will be useful to develop selectable vectors to transfer oncogenes (and other genes) to mammalian hematopoietic cells. Although these vectors need not be limited to retroviruses, their efficiency of transfer into the cell and their ability to integrate, without incurring rearrangements, into the cellular genome make them ideally suited as transducing vectors (Wei et al., 1981; review Weiss et al., 1982; Hwang and Gilboa, 1984). Unforeseen difficulties, such as integration mutagenesis or LTR-mediated transcription of neighboring genes, may render episomal viruses (e.g., papovavirus, parvovirus) more useful for vector construction. An incomplete understanding of retroviral functions, such as splicing, transcriptional controls, and mechanisms of transcriptional enhancement, also contributes to difficulties in constructing retrovirus vectors. Introduction of selectable markers may lead to alterations in other genome functions by an epigenetic mechanism (Emerman and

328

WOLFRAM OSTERTAG

Temin, 1984) or disruption of correct splicing patterns (Joyner and Bernstein, 1983). The use of internal transcriptional signals for a newly introduced gene allows independent regulation. Such a construct is especially useful when retroviral LTR transcriptional signals are inefficient, as in embryonal carcinoma cells (Rubenstein et al., 1984; Wagner et al., 1985). Vectors which allow undisrupted expression of a marker gene and a controlled level expression of a second gene product, either by use of conditional mutants of genes (Fig. 21) [ts mos (Friel et al., 1987)SV40based ts vectors (Rio et al., 1985)l or by introducing elements that permit modulation at the transcriptional level [hormone inducibility (Miller et al., 1984); MMTV glucocorticoid control regions (Ponta et al., 1985; Overhauser and Fan, 1985); cadmium and hormone inducibility (Karin et al., 1983); utilization of glucose-regulating proteins (Attenello and Lee, 1984)], would also be useful for oncogene analysis. Tissue-specific activation signals, as described for MMTV (Ross and Solter, 1985) and MPSV (Stocking et al., 1985, 1986) LTRs, should also be analyzed more thoroughly and incorporated in the design of new vectors. Initial results of several groups indicate that selectable retrovirus vectors cannot only be transferred to pluripotent hematopoietic cells, but also to pluripotent stem cells that are capable of repopulating the hematopoietic system of stem cell-deficient mice (Cepko et al., 1984; Williams et al., 1984; Dick et al., 1985; Keller et al., 1985; Lemischka et al., 1986). The marker gene transduced by the retrovirus facilitates analysis of cell lineage (e.g., for studying clonality of leukemic cells) and permits selection of infected cells. The dhfr marker gene appears to be especially useful for such studies, since methotrexate can be used for both in vivo and in vitro selection (Miller et al., 1985). Such positive results indicate that direct studies of the possible effects of oncogenes that cause hematopoietic proliferative diseases on stem cells and their progenitors can ensue. It is, however, still uncertain whether hematopoietic stem cells permit expression of retroviral LTRs . Finally, introduction of selectable retrovirus vectors into embryonal cells may permit us to study effects of oncogenes throughout the development, in particular, hematopoiesis. controlled studies in tissue culture require utilization of embryonal cell lines (Bradley et al., 1984) and a retrovirus vector which utilizes either a nonretroviral promoter (Van der Putten et al., 1985; Wagner et al., 1985) or a retroviral promoter which is expressed in embryonal cells (Franz et al., 1986).

MURINE SPLEEN FOCUS-FORMING VIRUSES

329

ACKNOWLEDGMENTS This review would not have been possible without the dedicated and extremely efficient help of E. Danckers in typing and editing this manuscript. Figures were drawn professionally by G. Arman. The unpublished work cited by our group was supported by funds of the Deutsche Forschungsgemeinschaft ( 0 s 31/12), by the Deutsche Krebshilfe (design of some recent retrovirus-based vectors), and by the Stiftung Volkswagenwerk. The Heinrich-Pette-Institut is financially supported by Freie und Hansestadt Hamburg and Bundesministerium fur Jugend, Familie und Gesundheit.

REFERENCES Abramson, S., Miller, R. G., and Phillips, R. A. (1977).J . Exp. Med. 145, 1567-1579. Adachi, A., Sakai, K., Kitamura, N., Nakanishi, S., Niwa, O., Matsuyama, M., and Ishimoto, A. (1984).J . Virol. 50, 813-821. Adkins, B., Leutz, A., and Graf, T. (1984). Cell 39, 439-445. Albino, A. P., LeStrange, R., Oliff, A. J., Furth, M. E., and Old, L. J. (1984). Nature (London) 308,69-72. Alder, S., Ciampi, C., and McCulloch, E. A. (1984).J.Cell. Physiol. 118, 186-192. Allen, T. D., and Dexter, T. M. (1984). E x p . Hematol. 12,517-521. Amanuma, H., Katori, A., Obata, M., Sabata, N., and Ikawa, Y. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 3913-3917. Andersen, K. B., and Nexo, B. A. (1983). Virology 125, 85-98. Andersen, P. R., Devare, S. K., Tronick, S. R., Ellis, R. W., Aaronson, S. A., and Scolnick, E. M. (1981a). Cell 26, 129-134. Andersen, P. R., Tronick, S. R., and Aaronson, S. A. (1981b).J . Virol. 40, 431-439. Anderson, S. M., and Scolnick, E. (1983).J . Virol. 46, 594-605. Anzano, M. A., Roberts, A. B., Smith, J. M., Sporn, M. B., and DeLarco, J. (1983).Proc. Natl. Acad. Sci. U.S.A. 80, 6264-6268. Arlinghaus, R. B. (1985).J. Gen. Virol. 66, 1845-1853. Arya, S. K., Wong-Staal, F., and Gallo, R. (1984).Science.223, 1086-1087. Asche, W., Colletta, G., Warnecke, G . , Nobis, P., Pennie, S., King, R. M., and Ostertag, W. (1984).Mol. Cell. Biol. 4, 923-930. Assoian, R. K., Frolik, C. A,, Roberts, A. B., Miller, D. M., and Sporn, M. B. (1984). Cell 36,36-41. Attenello, J. W., and Lee, A. S. (1984). Science 226, 187-190. Axelrad, A. A., and Steeves, R. A. (1964). Virology 24,513-518. Axelrad, A. A., McLeod, D. L., Shreeve, M. M., and Heath, D. S. (1974). In “Hemopoiesis in Culture” (W. A. Robinson, ed.), pp. 226-234. DHEW Publication No (NIH) 74-250, Washington, D.C. Balachandran, R., Reddy, E. P., Dunn, C. Y.,Aaronson, S. A., and Swan, D. C. (1984). EMBO. J . 3,3199-3207. Baldwin, G . S. (1985).Proc. Natl. Acad. Sci. U.S.A.82, 1921-1925. Balmain, A., and Pragnell, I. B. (1983).Nature (London) 303,72-74. Balmain, A,, Ramsden, M., Bowden, G. T., and Smith, J. (1984). Nature (London) 307, 658-660. Barbacid, M., Troxler, D. H., Scolnick, E. M., and Aaronson, S. A. (1978).J . Virol. 27, 826-830. Barker, W. C., and Dayhoff, M. 0. (1982). Proc. Natl. Acad. Sci. U.S.A. 79,2836-2839.

330

WOLFRAM OSTERTAG

Bartelmez, S. H., and Stanley, E. R. (1985). J. Cell.Physiol. 122,370-378. Bartram, C. R., Kleihauer, E., de Klein, A,, Grosveld, G., Teyssier, J. R., Heisterkamp, N., and Groffen, J. (1985).EMBOJ.4,683-686. Bassin, R. H., Ruscetti, S., Ah, I., Haapala, D., and Rein, A. (1982).Virology 123, 139151. Bazill, G. W., Haynes, M., Garland, J., and Dexter, T. M. (1983).Biochern.J.210,747759. Becker, A. J., McCulloch, E. A., Siminovitch, L., and Till, J. E. (1965).Blood 26,296308. Beckner, S. K., Hattori, S., and Shih, T. Y. (1985).Nature (London)317, 71-72. Behringer, R. R., and Dewey, M. J. (1985).Cell 40,441-447. Bennett, M., Steeves, R. A., Cudkowicz, G., Mirand, E. A., and Russell, L. B. (1968). Science 162,564-565. Bentvelzen, P., Aarssen, A. M., and Brinkhof, J. (1972).Nature (London)New Biol. 239, 122-123. Berger, S. A., Sanderson, N., Bernstein, A., and Hankins, W. D. (1985). Proc. Natl. Acad. Sci. U S A . 82, 6913-6917. Beru, N., McDonald, J., Lacombe, C., and Goldwasser, E. (1986).Mol. Cell. B i d . 6, 2571-2575. Bestwick, R.,Ruta, M., Kiessling, A., Faust, C., Linemeyer, D., Scolnick, E., and Kabat, D. (1983).J. Virol. 45, 1217-1222. Bestwick, R. K., Boswell, B. A., and Kabat, D. (1984).J. Virol. 51,695-705. Bestwick, R. K., Hankins, W. D., and Kabat, D. (1985). J. Virol. 56, 660-664. Betsholtz, C., Westermark, B., Ek, B., and Heldin, C. H. (1984).Cell 39,447-457. Beug, H., Kitchener, G., Doederlein, G., Graf, T., and Hayman, M. J. (1980).Proc. Natl. Acad. Sci. U.S.A. 77, 6683-6686. Bilello, J. A., Colletta, G., Warnecke, G., Koch, G., Frisby, D., Pragnell, I. B., and Ostertag, W. (1980).Virology 107, 331-344. Bishop, J. M. (1985).Cell 42, 23-38. Bister, K., and Jansen, H. W. (1986).Ado. Cancer Res. 47, in press. Blair, D. G., McClements, W. L., Oskarsson, M. K., Fischinger, P. J., and Vande Woude, G. F. (1980).Proc. Natl. Acad. Sci. U.S.A. 77, 3504-3508. Blair, D.G., Oskarsson, M., Wood, T. G., McClements, P., Fischinger, J., and Vande Woude, G. (1981).Science 212,941-943. Blair, D. C., Cooper, C. S., Oskarsson, M. K., Eader, L. A., and Vande Woude, G. F. (1982).Science 218, 1122-1125. Blair, D. G., Wood, T. G., Woodworth, A. M., McGeady, M. L., Oskarsson, M. K., Propst, F., Rainsky, M. A., Cooper, C. S., Watson, R., Baroudy, B. M., and Vande Woude, G. F. (1984).In “Cancer Cells,” Vol. 2,pp. 281-284.Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Blatt, C., Mileham, K., Haas, M., Nesbitt, M. N., Harper, M. E., and Simon, M. I. (1983). Proc. Natl. Acad. Sci. U.S.A. 80,6298-6302. Boettiger, D., and Durban, E. (1984).J. Virol. 49,841-847. Boettiger, D., Anderson, S., and Dexter, T. M. (1984).Cell 36, 763-773. Boggs, D. R., Boggs, S. S., Saxe, D. F., Cress, L. A., and Canfield, D. R. (1982). J. Clin. Invest. 70, 242-253. Bold, R. J., and Donoghue, D. J. (1985).Mol. Cell. Biol. 5, 3131-3138. Bondurant, M., Koury, M., Krantz, S. B., Blevins, T., and Duncan, D. T. (1983).Blood 61,751-758. Bondurant, M. C., Lind, R. N., Koury, M. J., and Ferguson, M. E. (1985). Mol. Cell. Biol. 5,675-683.

MURINE SPLEEN FOCUS-FORMING VIRUSES

33 1

Bondurant, M. C., and Koury, M. J. (1986). Mol. Cell B i d . 6, 2731-2733. Bos, J. L., Toksoz, D., Marshall, C. J., Verlaan-de Vries, M., Veeneman, G. H., van der Eb, A. J., van Boom, J. H., Janssen, J. W. G., and Steenvoorden, A. C. M. (1985). Nature (London) 315,726-730. Bosselmann, R. A., van Straaten, F., van Beveren, C., Verma, I. M., and Vogt, M. (1982). J. Virol. 44, 19-41. Bosze, Z., Thiesen, J.-J., and Charnay, P. (1986). EMBO J. 5, 1615-1623. Bradley, A., Evans, M., Kaufman, M. H., and Robertson, E. (1984).Nature (London)309, 255-256. Broek, D., Samiy, N., Fasano, O., Fujiyama, A., Tamanoi, F., Northup, J., and Wigler, M. (1985). Cell 41, 763-769. Brommer, E. J. P., and Bentvelzen, P. (1974).Eur. J. Cancer 10, 827-833. Brown, E. C., Zajac-Kaye, M., Pogo, B. G.-T., and Friend, C. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 5925-5929. Bucher, T., Bender, W., Fundele, R., Hofner, H., and Linke, I. (1980). FEBS Lett. 115, 319-324. Burgess, A. W., and Metcalf, D. (1977).J. Cell. Physiol. 90,471-484. Burgess, A. W., and Metcalf, D. (1980).Int. J. Cancer 26,647-654. Burgess, A. W., Camakaris, J., and Metcalf, D. (1977).J. Biol. Chem. 252, 1998-2003. Burgess, A. W., Metcalf, D., Russell, S. H. M., and Nicola, N. A. (1980). Biochem. J. 185, 301-304. Burgess, A. W., Bartlett, P. F., Metcalf, D., Nicola, N. A., Clark-Lewis, I., and Schrader, J. W. (1981). E x p . Hematol. 9, 893-903. Byrne, P. V., Guilbert, L. J., and Stanley, E. R. (1981).J. Cell B i d . 91, 848-853. Canaani, E., Dreazen, O., Klar, A., Rechavi, G., Ram, D., Cohen, J. B., and Givol, D. (1983). Proc. Natl. Acad. Sci. U.S.A.80, 7118-7122. Capon, D. J., Chen, E. Y., Levinson, A. D., Seeburg, P. H., and Goeddel, D. V. (1983a). Nature (London)302,33-37. Capon, D. J., Seeburg, P. H., McGrath, J. P., Hayflick, J. S., Edman, U., Levinson, A. D., and Goeddel, D. V. (198313). Nature (London)304,507-513. Carpenter, G., Stoscheck, C. M., Preston, Y. A., and DeLarco, J. E. (1983).Proc. Natl. Acad. Sci. U.S.A. 80, 5627-5630. Cashman, J., Henkelman, D., Humphries, K., Eaves, C., and Eaves, A. (1983). Blood 61, 876-884. Caubet, J.-F., Mathieu-Mahul, D., Bernheim, A., Larsen, C.-J., and Berger, R. (1985). EMBO J. 4,2245-2248. Cepko, C. L., Roberts, B. E., and Mulligan, R. C. (1984). Cell 37, 1053-1062. Chang, E. H., Maryak, J. M., Wei, C.-M., Shih, T. Y., Shober, R., Cheung, H. L., Ellis, R. W., Hager, G. L., Scolnick, E. M., and Lowy, D. R. (1980).J. Virol. 35, 76-92. Chang, E. H., Furth, M. E., Scolnick, E. M., and Lowy, D. R. (1982a).Nature (London) 297,479-483. Chang, E. H., Gonda, M. A., Ellis, R. W., Scolnick, E. M.,and Lowy, D. R. (1982b).Proc. Natl. Acad. Sci. U.S.A.79, 4848-4852. Chang, H. W., Garon, C. F., Chang, E. H., Lowy, D. R., Hager, G. L., Scolnick, E. M., Repaske, R., and Martin, M. A. (1980).J. Virol. 33, 845-855. Chatis, P. A., Holland, C. A., Hartley, J. W., Rowe, W. P., and Hopkins, N. (1983).Proc. Natl. Acad. Sci. U.S.A. 80, 4408-4411. Chatis, P. A., Holland, C. A., Silver, J. E., Fredrickson, T. N., Hopkins, N., and Hartley, J. W. (1984).J. Virol. 52, 248-254. Chattopadhyay, S. K., Lander, M. R., Gupta, S., Rands, E., and Lowy, D. R. (1981). Virology 113, 465-483.

332

WOLFRAM OSTERTAG

Chattopadhyay, S. K., Cloyd, M. W., Linemeyer, D. L., Lander, M. R., Rands, E., and Lowy, D. R. (1982).Nature (London)295,25-30. Chesebro, B., and Wehrly, K. (1985). Virology 141, 119-129. Chesebro, B., Portis, J. L., Wehrly, K., and Nishio, J. (1983). Virology 128,221-233. Chesebro, B., Wehrly, K., Nishio, J., and Evans, L. (1984).J. Virol. 51, 63-70. Chien, Y. H., Lai, M., Shih, T. Y., Verma, I. M., Scolnick, E. M., Roy-Burman, P., and Davidson, N. (1979).J. Virol. 31, 752-760. Chipperfield, R. L. G., Jones, S. S., Lo, K. M., and Weinberg, R. A. (1985). Mol. Cell. B i d . 5, 1809-1813. Chirigos, M. A., Scott, W., Turner, W., and Perk, K. (1968). Znt. J. Cancer 3,223-237. Choppin, J., Lacombe, C., Casadevall, N., Muller, O., Tambourin, P., and Varet, B. (1984). Blood 64,341-347. Cichutek, K., and Duesberg, P. H. (1986). Proc. Natl. Acad. Sci. U.S.A.83,2340-2344. Clark, G., Wong, G., Arnheim, N., Nitecki, D., and McCormick, F. (1985). Proc. Natl. Acad. Sci. U.S.A.82, 5280-5284. Clark, S. G., McGrath, J. P., and Levinson, A. D. (1985). Mol. Cell. Biol. 5,2746-2752. Clark, S. P., and Mak, T. W. (1983). Proc. Natl. Acad. Sci. U.S.A.80, 4408-4411. Clark, S. P., and Mak, T. W. (1984).J. Virol. 50, 759-765. Clarke, M. F., Westin, E., Schmidt, D., Josephs, S. F., Ratner, L., Wong-Staal, F., Gallo, R. C., and Reib, M. S. (1984). Nature (London) 308,464-467. Clark-Lewis, I., Kent, S. B. H., and Schrader, J. W. (1984).J. Biol. Chem. 259, 74887494. Cloyd, M. W., Thompson, M. M., and Hartley, J. W. (1985). Virology 140,239-248. Cochran, B. H., Zullo, J., Verma, I. M., and Stiles, C. D. (1984).Science 226,1080-1082. Cohen, J. B., Unger, T., Rechavi, G., Canaani, E., and Givol, D. (1983).Nature (London) 306,797-799. Cohen, P., Rylatt, D. B., and Nimmo, G. A. (1977). FEES Lett. 76, 182-186. Colletta, G., DiFiore, P. P., Fusco, A,, Lettieri, F., Covelli, A., and Peschle, C. (1983a). Cancer Res. 43,598-603. Colletta, G., Pinto, A., Di Fiore, P. P., Fusco, A., Ferrentino, M., Avvedimento, V. E., Tsuchida, N., and Vecchio, G. (1983b). Mol. Cell. B i d . 3,2099-2109. Collins, S. J., and Groudine, M. (1982). Nature (London)298,679-681. Collins, S. J., Kubonishi, I., Miyoshi, I., and Groudine, M. T. (1984).Science 225,72-74. Cooper, G. M. (1982).Science 217,801-806. Cory, S . , Gerondakis, S., and Adams, J. (1983). EMBOJ. 2,697-703. Cory, S., Graham, M., Webb, E., Corcoran, L., and Adams, J. M. (1985).EMBOJ. 4,675681. Craig, R. W., and Sager, R. (1985). Proc. Natl. Acad. Sci. U.S.A.82, 2062-2066. Cullen, B. R., Kopchick, J. J., and Stacey, D. W. (1982). Nucleic Acids Res. 10, 61776190. Cutler, R. L., Metcalf, D., Nicola, N. A., and Johnson, G. R. (1985).J.Biol. Chem. 260, 6579-6587. Dalla Favera, R., Wong-Staal, F., and Gallo, R. B. (1982). Nature (London)299,61-63. Dameshek, W. (1951). Blood 6,372-375. Daniel, T. O., Tremble, P. M., Frackelton, A. R., and Williams, L. T. (1985). Proc. Natl. Acad. Sci. U.S.A.82, 2684-2687. Das, S. K., and Stanley, E. R. (1982).J. B i d . Chem. 257, 13679-13684. Das, S. K., Stanley, E. R., Guilbert, L. J., and Forman, L. W. (1981).Blood 58,630-641. Davey, J., Dimmock, N. J., and Colman, A. (1985). Cell 40,667-675. DeBoth, N. J., Vermey, M., van’t Hull, E., Klootwyk-van Dyke, E., van Griensven, L. J. L. D., Mol, J. N. M., and Stoof, T. J. (1978). Nature (London)272, 626-628.

MURINE SPLEEN FOCUS-FORMING VIRUSES

333

DeFeo, D., Gonda, M. A., Young, H. A., Chang, E. H., Lowy, D. R., Scolnick, E. M., and Ellis, R. W. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 3328-3332. DeFeo-Jones, D., Tatchell, K., Robinson, L. C., Sigal, I. S., Vass, W. C., Lowy, D. R., and Scolnick, E. M. (1985). Science 228, 179-184. DeLarco, J. E., and Todaro, G. (1978). Proc. Natl. Acad. Sci. U.S.A. 75,4001-4005. DeLarco, J. E., Pigott, D. A., and Lazarus, J. A. (1985). Proc. Natl. Acad. Sci. U.S.A.82, 50 15-50 19. Der, C. J., and Cooper, G. M. (1983). Cell 12,201-208. Der, C. J., Krontiris, T. G., and Cooper, G. M. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 3637-3640. Derynck, R., Jarrett, J. A., Chen, E. Y., Eaton, D. H., Bell, J. R., Assoian, R. K., Roberts, A. B., Sporn, M. B., and Goeddel, D. V. (1985). Nature (London)316,701-705. Des Groseillers, L., and Jolicoeur, P. (1984).J. Virol.52,945-952. Des Groseillers, L., Rassart, E., and Jolicoeur, P. (1983).Proc. Natl.Acad. Sci. U.S.A. 80, 4203-4207. Devare, S. G., Reddy, E. P., Law, J. D., Robbins, C., and Aaronson, S. A. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 731-735. Devaux, J., Jouanneau, J., Quibriac, M., Longuet, M., Le Bousse-Kerdiles, M. C., Auger-Buendia, M. A., and Tavitian, A. (1985).J.Gen. Virol. 66, 2407-2414. De Villiers, J., Olson, L., Tyndall, C., and Schaffner, W. (1982). Nucleic Acids Res. 10, 7965-7976. Dewey, M. J., and Eldridge, P. W. (1982). E x p . Hematol. 10, 723-731. Dexter, T. M., and Moore, M. A. S. (1977). Nature (London)269,412-414. Dexter, T. M., Allen, T. D., and Lajtha, L. G. (1977).J . Cell. Physiol. 91, 335-344. Dexter, T. M., Garland, J., Scott, D., Scolnick, E., and Metcalf, D. (1980a).J.E x p . Med. 152, 1036-1047. Dexter, T. M., Spooner, E., Toksoz, D., and Lajtha, L. G. (1980b).J . Supramol. Struct. 13,513-524. Dexter, T. M., Allen, T. D., Testa, N. G., and Scolnick, E. (1981a).J. Erp. Med. 154, 594-608. Dexter, T. M., Testa, N. G., Allen, T. D., Rutherford, T., and Scolnick, E. (1981b). Blood 58,699-707. Dexter, T. M., Boettiger, D., and Spooncer, E. (1985). In “Modern Trends in Human Leukemia” (R. Neth et al., eds.), Vol. VI, pp. 363-371. Springer-Verlag, Berlin. Dhar, R., McClements, W. L., Enquist, L. W., and Vande Woude, G. F. (1980). Proc. Natl. Acad. Sci. U.S.A. 77,3937-3941. Dhar, R., Ellis, R. W., Shih, T. Y., Oroszlan, S., Shapiro, B., Maizel, J., Lowy, D., and Scolnick, E. M. (1982). Science 217, 934-937. Diaz, M. O., Le Beau, M. M., Rowley, J. D., Drabkin, H. A., and Patterson, D. (1985). Science 229,767-769. DiBeradino, M. A., Hoffner, N. J., and Etkin, L. D. (1984).Science 224, 946-952. Dick, J. E., Magli, M. C., Huszar, D., Phillips, R. A., and Bernstein, A. (1985).Cell 42, 71-79. Dickson, C., Peters, G., Smith, R., and Brookes, S. (1984). In “Cancer Cells” (G. Vande Woude, ed.), Vol. 2, pp. 195-203. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Dina, D., and Nadal-Ginard, B. (1980). Cold Spring Harbor Symp. Quant. Biol. 44, 90 1-905. Dofuku, R., Breidler, J. L., Spengler, B. A., and Old, L. J. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 1515-1517. Donoghue, D. J., and Hunter, T. (1983).J . Virol. 45, 607-617.

334

WOLFRAM OSTERTAG

Doolittle, R. F., Hunkapiller, M. W., Devare, S. G., Robbins, K. C., Aaronson, S. A., and Antoniades, H. N. (1983).Science 221,275-277. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A., Schlesinger, J., and Waterfield, M. D. (1984). Nature (London) 307, 521-527. Drebin. J. A., Link, V. C., Stern, D. F., Weinberg, A., and Greene, M. I. (1985). Cell 41, 695-706. Dube, I. D., Arlin, Z. A., Kalousek, D. K., Eaves, C. J., and Eaves, A. C. (1984). Blood 64, 1284- 1287. Dube, S . K., Kung, H. J., Bender, W., Davidson, H., and Ostertag, W. (1976).J.Virol. 20, 264-272. Duesberg, P. H. (1980). Cold Spring Harbor Symp. Quant. Biol. 44, 13-29. Duesberg, P. H. (1985).Science 228,669-677. Duprez, V., Lenoir, G., and Dautry-Varsat, A. (1985). Proc. Natl. Acad. Sci. U.S.A.82, 6932-6936. Durkin, J. P., and Whitfield, J. F. (1986). Mol. Cell. Biol. 6, 1386-1392. Eaves, A. C., and Eaves, C. J. (1984). Clin. Haematol. 13,371-391. Eaves, C . J., and Eaves, A. C. (1978). Blood 52, 1196-1203. Eaves, C. J., Humphries, R. K., and Eaves, A. C. (1979). In “Cellular and Molecular Regulation of Hemoglobin Switching” (G. Stamatoyannopoulos and A. W. Nienhuis, eds.), pp. 251-273. Grune & Stratton, New York. Eckner, R. J., and Hettrick, K. L. (1982). Virology 122, 171-185. Eckner, R., Hettrick, K. L., Greenberger, J. S., and Bennett, M. (1982).Cell 31,731-738. Economou-Pachnis, A., Lohse, M. A., Furano, A. V., and Tsichlis, P. N. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 2857-2861. Ellis, R. W., DeFeo, D., Maryak, J. M.,Young, H. A., Shih,T. Y.,Chang, E. H., Lowy, D. R., and Scolnick, E. M. (1980).J. Virol. 36,408-420. Ellis, R. W., DeFeo, D., Furth, M. E., and Scolnick, E. M. (1982a). Mol. Cell. Biol. 2, 1339-1345. Ellis, R. W., Lowy, D. R., and Scolnick, E. M. (198213).Adu. Viral Oncol. 1, 107-126. Emerman, M., and Temin, H. M. (1984).Cell 39,459-467. Eva, A., and Aaronson, S. A. (1983). Science 220, 955-956. Eva, A., Tronick, S. R., Gol, R. A., Pierce, J. H., and Aaronson, S. A. (1983). Proc. Natl. Acad. Sci. U.S.A.80, 4926-4930. Evans, L. H., and Cloyd, M. W. (1984).J.Virol. 49, 772-781. Evans, L. H., and Cloyd, M. W. (1985). Proc. Natl. Acad. Sci. U.S.A. 82,459-463. Fagg, B. (1981). Nature (London) 289, 184-186. Fagg, B., and Ostertag, W. (1982).J . Natl. Cancer Inst. 68,457-460. Fagg, B., Vehmeyer, K., Ostertag, W., Jasmin, C., and Klein, B. (1980). In “In Vivo and In Vitro Erythropoiesis: The Friend System; (G. B. Rossi, ed.), pp. 163-172. Elsevier, Amsterdam. Fagg, B., Ostertag, W., Klein, B., and LeBousse, C. (1983).J.Cell. Physiol. 116, 17-20. Famulari, N. G. (1983). Curr. Top. Microbiol. Immunol. 103, 76-108. Fasano, O., Aldrich, T., Tamanoi, F., Taparowsky, E., Furth, M., and Wigler, M. (1984a). Proc. Natl. Acad. Sci. U S A . 81,4008-4012. Fasano, O., Birnbaum, D., Edlund, D., Fogh, J., and Wigler, M. (198413).Mol. Cell. Biol. 4, 1695-1705. Feramisco, J. R., Clark, R., Wong, G., Arnheim, N., Milley, R., and McCormick, F. (1985).Nature (London) 314,639-642. Fialkow, P. J. (1976).Biochim. Biophys. Actn 458,283-321. Fialkow, P. J., and Singer, J. W. (1985). In “Leukemia” (I. L. Weissman, ed.), pp. 203222. Dahlem Konferenzen 1985. Springer-Verlag, Berlin.

MURINE SPLEEN FOCUS-FORMING VIRUSES

335

Fialkow, P. J., Denman, A. M., Singer, J., Jacobson, R. J., and Lowenthal, M. N. (1978). In “Differentiation of Normal and Neoplastic Hematopoietic Cells” (B. Clarkson, P. A. Marks, and J. E. Till, eds.), Vol. 5, pp. 131-144. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Fialkow, P. J., Faguet, G. B., Jacobson, R. J., Vaidya, K., and Murphy, S. (1981a).Blood 58,916-919. Fialkow, P. J., Martin, P. J., Najfeld, V., Penfold, G. K., Jacobson, R. J., and Hansen, J. A. (1981b). Blood 58, 158-163. Fialkow, P. J., Singer, J. W., Adamson, J. W., Vaidya, K., Dow, L. W., Ochs, J., and Moohr, J. W. ( 1 9 8 1 ~ )Blood . 57, 1068-1073. Fieldsteel, A. H., Karahara, C., and Dawson, P. J. (1969).Nature (London) 223, 12741276. Finkel, T., and Cooper, G. M. (1984). Cell 36, 1115-1121. Finkel, T., Der, C. J., and Cooper, G. M. (1984). Cell 37, 151-158. Fraenkel, A. E., and Fischinger, P. J. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 37053709. Franz, T., Lohler, J., Fusco, A., Pragnell, I. B., Padua, R., and Ostertag, W. (1985). Nature (London) 315, 149-151. Franz, T., Hilberg, J., Seliger, B., Stocking, C., and Ostertag, W. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 3292-3296. Frick, K. K., Doherty, P. J., Gottesman, M. M., and Scher, C. D. (1985). Mol. Cell. Biol. 5,2582-2589. Fried, W. (1972).Blood 40, 671-677. Friel, J., Stocking, C., Stacey, A., and Ostertag, W. (1987).J . Virol., in press. Friend, C. (1957).J . E x p . Med. 105, 307-318. Friend, C., Scher, W., Holland, J., and Sato, T. (1971).Proc. Natl. Acad. Sci. U.S.A. 68, 378-382. Frindel, E. (1979). In “Cell Lineage, Stem Cells and Cell Determination” (N. Le Douarin, ed.), INSERM Symp. 10, pp. 227-239. Elsevier, Amsterdam. Frindel, E., and Guigon, M. (1977). E x p . Hematol. 5, 74-76. Frindel, E., Croizat, H., and Vassort, F. (1976).Exp. Hematol. 4, 56-61. Fukui, Y., and Kaziro, Y. (1985). E M B O J . 4, 687-691. Fung, M. C., Hapel, A. J., Ymer, S., Cohen, D. R., Johnson, R. M., Campbell, H. D., and Young, I. G. (1984).Nature (London) 307,233-237. Furth, M. E., Davis, L. J., Fleurdelys, B., and Scolnick, E. M. (1983).J.Virol. 43,294304. Fusco, A., Pinto, A,, Ambest-Impiombato, F. S., Vecchio, G., and Tsnchida, N. (1981). Int. J . Cancer 28, 655-662. Fusco, A., Portella, G., Di Fiore, P. P., Berlingieri, M . T., Di Lauro, R., Schneider, A. B., and Vecchio, G. (1985).J . Virol. 56, 284-292. Galli, S. J., Dvorak, A. M., Marcum, J. A., Ishizaka, T., Nabel, G., Der Simonian, H., Pyne, K., Goldin, J. M., Rosenberg, R. D., Cantor, H., and Dvorak, H. F. (1982).J . Cell Biol. 95, 435-444. Gallick, G. E., and Arlinghaus, R. B. (1984).Virology 133,228-232. Gallwitz, D., Donath, C., and Sander, G. (1983). Nature (London) 306, 704-707. Gambke, C., Signer, E., and Moroni, C. (1984). Nature (London)307,476-478. Gambke, C., Hall, A., and Moroni, C. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 879882. Gateff, E. (1982). Adrj. Cancer Res. 37, 33-74. Gattoni, S., Kirschmeier, P., Weinstein, I. B., Escobedo, J., and Dina, D. (1982).Mol. Cell Biol. 2. 42-51.

336

WOLFRAM OSTERTAG

Gattoni-Celli, S., Hsiao, W.-L. W., and Weinstein, I. B. (1983). Nature (London) 306, 795-796. Gazit, A., Igarashi, H., Chiu, I.-M., Srinivasan, A., Yaniv, A., Tronick, S. R., Robbins, K. C., and Aaronson, S. A. (1984). Cell 39, 89-97. Gazzolo, L., Samarut, J., Bouabdelli, M., and Blanchet, J. P. (1980).Cell 22,683-691. Gerwitz, A. M., Bruno, E., Elwell, J., and Hoffman, R. (1983). Blood 61, 384-389. Gibbs, J. B., Ellis, R. W., and Scolnick, E. M. (1984a).Proc. Natl. Acad. Sci. U S A . 81, 2674-2678. Gibbs, J., Sigal, I. S., Poe, M., and Scolnick, E. M. (1984b).Proc. Natl. Acad. Sci. U S A . 81,5704-5708. Gilman, A. G. (1984). Cell 36,577-579. Gilmore, T., DeClue, J. E., and Martin, G. S. (1985). Cell 40,609-618. Goldfarb, M. P., and Weinberg, R. A. (1981).J . Virol. 38, 136-150. Goldwasser, E., and Kung, K-H. (1971). Proc. Natl. Acad. Sci. U.S.A. 68, 697-698. Gonda, T. J., and Metcalf, D. (1984).Nature (London) 310,249-251. Cough, N. M., Cough, J., Metcalf, D., Kelso, A., Grail, D., Nicola, N. A., Burgess, A. W., and Dunn, A. R. (1984). Nature (London) 309,763-768. Cough, N. M., Metcalf, D., Cough, J., Grail, D., and Dunn, A. R. (1985). EMBOJ. 4, 645-653. Graf, T. (1985). In “Leukemia” (I. L. Weissman, ed.), pp. 131-145. Dahlem KonferenZen. Springer-Verlag, Berlin. Graf, T., and Beug, H. (1978). Biochim. Biophys. Acta 516,269-299. Graves, B. J., Eisenman, R. N., and McKnigh, S. L. (1985a). Mol. Cell. Biol. 5, 19481958. Graves, B. J., Eisenberg, S. P., Coen, D. M., and McKnight, S. L. (198513).Mol. Cell. Biol.5, 1959-1968. Greaves, M. F. (1982).1.Cell. Physiol. Suppl. 1,23-30. Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J.-M., Argos, P., and Chambon, P. (1986). Nature (London) 320, 134-139. Greenberg, M. E., and Ziff, E. B. (1984). Nature (London) 311,433-438. Greenberger, J. G. (1979).J . Natl. Cancer Znst. 62,337-348. Greenberger, J. S., Eckner, R. J., Ostertag, W., Colletta, G., Boschetti, S., Nagasawa, M., Karpas, A., Weichselbaum, R. R., and Moloney, W. C. (1980a).Virology 105,425435. Greenberger, J. S., Newburger, P. E., Lipton, J. M., Moloney, W. C., Sakakeeny, M. A., and Jackson, P. L. (1980b).J . Natl. Cancer Znst. 64,867-878. Greenberger, J. S., Wroble, L. M., and Sakakeeny, M. A. (198Oc).J. Natl. Cancer Znst. 65,841-851. Greenberger, J. S., Hofhan, N., Liebeman, M., Botnick, L. E., Sakakeeny, M. A., and Eckner, R. J. (1983a).J . Natl. Cancer Znst. 70,323-331. Greenberger, J. S., Sakakeeny, M. A., Humphries, R. K., Eaves, C. J., and Eckner, R. J. (198313).Proc. Natl. Acad. Sci. U S A . 80,2931-2935. Greenberger, J. S., Humphries, R. K., Messner, H., Reid, D. M., and Sakakeeny, M. A. (1985).E x p . Hematol. 13,249-260. Gregory, C. J. (1976).J. Cell. Physiol. 89, 289-302. Gregory, C. J., and Eaves, A. C. (1978). Blood 51,527-537. Gregory, C. J., McCulloch, E. A., and Till, J. E. (1975).J . Cell Physiol. 86, 1-8. Grotendorst, G. R. (1984). Cell 36,279-285. Groudine, M., Eisenman, R., and Weintraub, H. (1981).Nature (London)292,311-317.

MURINE SPLEEN FOCUS-FORMING VIRUSES

337

Guerrero, I., Calzada, P., Mayer, A., and Pellicer, A. (1984).Proc. Natl. Acad. Sci. U.S.A. 81,202-205. Guigon, M., Enouf, J., and Frindel, E. (1980). Leuk. Res. 4, 385-391. Habara, A., Reddy, E. P., and Aaronson, S. A. (1982).J. Virol. 44, 731-735. Hamilton, J. A., Stanley, E. R., Burgess, A. W., and Shadduck, R. K. (1980). J . Cell Physiol. 103, 435-445. Hammond, D., and Winnick, S. (1974). Ann. N.Y. Acad. Sci. 230,219-227. Handelin, B. L., and Kabat, D. (1985).Virology 140, 183-187. Handman, E., and Burgess, A. W. (1979).J . Immunol. 122, 1134-1137. Hankins, W. D. (1983).J. Natl. Cancer Znst. 70, 725-734. Hankins, W. D., and Krantz, S. B. (1980). Proc. Natl. Acad. Sci. U.S.A.77, 5287-5291. Hankins, W. D., and Scolnick, E. M. (1981). Cell 26,91-97. Hankins, W. D., and Troxler, D. (1980). Cell 22, 693-699. Hankins, W. D., Kost, T. A., and Pragnell, I. B. (1982).Mol. Cell Biol. 2, 138-146. Hankins, W. D., Schooley, J., and Eastment, C. (1986). Blood 68,263-268. Hannink, M., and Donoghue, D. J. (1985).Proc. Natl. Acad. Sci. U.S.A.82,7894-7898. Hapel, A. J., Fung, M. C., Johnson, R. M., Young, I. G . ,Johnson, G . R.,and Metcalf, D. (1985). Blood 65, 1453-1459. Harbers, K., Schnieke, A., Stuhlmann, H., Jahner, D., and Jaenisch, R. (1981). Proc. Natl. Acad. Sci. U S A . 78, 7609-7613. Harford, J. (1984). Nature (London)311,673-675. Harland, R. M. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 2323-2327. Harris, R. A., Hogarth, P. M., McKenzie, I. F. C., and Penington, D. G . (1983). Erp. Hematol. 11, 527-541. Harris, R. A., Hogarth, P. M., Wadeson, L. J., Collins, P., McKenzie, I. F. C., and Penington, D. G. (1984). Nature (London)307, 638-639. Harris, R. A., Sandrin, M. S., Sutton, V. P., Hogarth, P. M., McKenzie, I. F. C., and Penington, D. G. (1985).J. Cell Physiol. 123, 451-458. Harrison, D. E. (1980). Blood 55, 77-81. Hartley, J. W., Wolford, N. K., Old, L. J., and Rowe, W. P. (1977).Proc. Natl. Acad. Sci. U.S.A.74, 789-792. Harvey, J. J. (1964).Nature (London) 204, 1104-1105. Haskill, J. S., McNeill, T. A., and Morre, M. A. S. (1970).J. Cell. Physiol. 75, 167-180. Hasthorpe, S. (1978). In “In Vitro Aspects of Erythropoiesis” (M. J. Murphy, ed.), pp. 172-176. Springer Verlag, New York. Hasthorpe, S. (1980).J . Cell Physiol. 105, 379-384. Hasthorpe, S., and Bol, S. (1979).J . Cell. Physiol. 100, 77-86. Hayata, I., Seki, M., Yoshida, K., Hirashima, 0. K., Sado, T., Yamagiwa, J., and Ishihard, T. (1983). Cancer Res. 43,367-373. Hayward, W. S. (1985). I n “Leukemia” (I. L. Weissman, ed.), pp. 147-162. Dahlem Konferenzen. Springer-Verlag, Berlin. Heath, D. S., Axelrad, A. A., McLead, D. L., and Shreeve, M. M. (1976). Blood 47,777792. Heisterkamp, N., Stam, K., and GroKen, J. (1985). Nature (London) 315, 758-761. Heldin, C. H., and Westermark, B. (1984). Cell 37, 9-20. Hess, N. (1985).Doctoral thesis, University of Hamburg, Hamburg, Federal Republic of Germany. Hess, N., Franz, T., Kollek, R.,and Ostertag, W. (1984).J . Gen. Virol. 65, 2225-2235. Hodgson, G. S., and Bradley, T. R. (1979). Nature (London)281,381-382.

338

WOLFRAM OSTERTAG

Holland, C. A., Wozney, J., Chatis, P. A., Hopkins, N., and Hartley, J. W. (1985).J.Virol. 52, 152-157. Horoszewicz, J. S . , Leong, S. S., and Carter, W. A. (1975).J . Natl. Cancer Inst. 54,265267. Horowitz, M., Luria, S., Rechavi, G., and Givol, D. (1984). EMBOJ. 3,2937-2941. Housman, D., Levenson, R., Volloch, V., Tsiftsoglou, A., Gusella, J., Parker, D., Kernen, J., Mitrani, A., Weeks, V., Witte, O., and Besmer, P. (1980). Cold Spring Harbor Symp. Quant. Biol. 44, 1177-1185. Hsiao, W.-L. W., Gattoni-Celli, S., Kirschmeier, P., and Weinstein, B. (1984).Mol. Cell. B i d . 4, 634-641. Huang, J. S., Huang, S. S., and Deuel, T. F. (1984). Cell 39, 79-87. Huggins, C. B., and Oka, H. (1972). Cancer Res. 32,239-242. Huggins, C. B., and Ueda, N. (1984). Proc. Natl. Acad. Sci. U.S.A.81, 598-601. Huggins, C. B., Grand, L., and Ueda, N. (1982). Proc. Natl. Acad. Sci. U.S.A.79,54115414. Huhn, D., and Meister, P. (1978). Cancer 42, 1341-1352. Humphries, R. K., Eaves, A. C., and Eaves, C. J. (1981).Proc. Nutl. Acad. Sci. U.S.A.78, 3629-3633. Hunt, N., Laker, C., Stocking, C., Hess, N., Nobis, P. Sieb, J., and Ostertag, W. (1987).In “NATO Advanced Research Workshop: Molecular and Cellular Aspects of Erythropoiesis” (I. N. Rich, ed.). In press. Hurley, J. B., Simon, M. I., Teplow, D., Robishaw, J. D., and Gilman, A. G. (1984). Science 226,860-863. Hwang, L.-H. S., and Gilboa, E. (1984).J. Virol. 50,417-424. Hwang, L.-H. S., Park, J., and Gilboa, E. (1984). Mol. Cell. Biol. 4, 2289-2297. Ihle, J. N., Keller, J., Henderson, L., Klein, F., and Palaszynski, E. (1982).J.Immunol. 129,2431-2436. Ikawa, Y., Aida, M., and Inoue, Y. (1976).Gann 67,767-770. Ikeda, H., Laigret, F., Martin, M. A., and Repaske, R. (1985).J . Virol. 55, 768-777. Iritani, A., Katayama, N., Tahira, T., Hayashi, K., and Tsuchida, N. (1986). Bull. Tokyo Med. Dent. Uniu. 33, 35-40. Iscove, N. N. (1977). Cell Tissue Kinet. 10, 323-334. Iscove, N. N. (1978). ICN-UCLA Symp. Mol. Cell. B i d . 10,37-52. Iscove, N. N., Till, J. E., and McCulloch, E. A. (1970). Proc. S O C . E x p . B i d . Med. 134, 33-36. Iscove, N. N., Roitsch, C. A., Williams, N., and Guilbert, L. J. (1982).J. Cell. Physiol. Suppl. 1,65-78. Iscove, N. N., Keller, G., and Roitsch, C. (1985). In “Stem Cell Physiology” (J. Palek, ed.). Liss, New York. Ishimoto, A., Adachi, A., Sakai, K., Yorifuji, T., and Tsuruta, S. (1981). Virology 113, 644-655. Ishimoto, A., Adachi, A., Sakai, K., and Matsuyama, M. (1985). Virology 141, 30-42. Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S. D., Kaufman, R. J., Mufson, A., Seehra, J., Jones, S. S., Hewick, R., Fritsch, E. F., Kawakita, M., Shimizu, T., and Miyake, T. (1985). Nature (London)313,806-809. Jahner, D., Stuhlmann, H., Stewart, C. L., Harbers, K., Lohler, J., Simon, I., and Jaenisch, R. (1982).Nature (London)298, 623-628. Jasmin, C., Smadja-Joffe, F., Klein, B., and Kerdiles-LeBousse, C. (1976). Cancer Res. 36,603-606. Jasmin, C., LeBousse, M. C., Klein, B., Smadja-Joffe, F., Mori, K. J., Fagg, B., Ostertag,

MURINE SPLEEN FOCUS-FORMING VIRUSES

339

W., Vehmeyer, K., and Pragnell, I. B. (1980). In “In vivo and In Vitro Erythropoiesis: The Friend System” (G. B. Rossi, ed.), pp. 183-192. Elsevier, Amsterdam. Jenkins, J. R., Rudge, K., and Currie, G. A. (1984). Nature (London) 312, 651-653. Johnson, G. R. (1981). In “Experimental Hematology Today 1981” (J. J. Baum, G. D. Ledney, and A. Khan, eds.), pp. 13-20 Karger, Basel. Johnson, G. R. (1983). Leuk. Res. 7,655-666. Johnson, G. R., and Burgess, A. W. (1978).J. Cell Biol. 77, 35-47. Johnson, G. R., and Metcalf, D. (1977).Proc. Natl. Acad. Sci. U.S.A. 74, 3879-3882. Johnson, G. R., and Metcalf, D. (1978)./. Cell Physiol. 94, 243-252. Johnson, G. R., and Metcalf, D. (1979). In “Cell Lineage, Stem Cells and Cell Determination” (N. Le Douarin, ed.), INSERM Symposium 10, pp. 199-213. Elsevier, Amsterdam. Johnson, G. R., and Metcalf, D. (1980). E x p . Hematol. 8, 549-561. Johnson, G. R., and Nicola, N. A. (1984).J. Cell Physiol. 118, 45-52. Johnson, G. R., Keller, G. M., and Nicola, N. A. (1982).J.Cell. Physiol. Suppl. 1,23-30. Johnson, G. R., Ostertag, W., and Nicola, N. A. (1985). In “Modern Trends in Human Leukemia” (Neth, Gallo, Greaves, and Janka, eds.), Vol. 6, pp. 376-379. Johnsson, A., Heldin, C.-H., Wasteson, A,, Westermark, B., Deuel, T., Huang, J., Seeburg, P., Gray, A,, Ullrich, A., Scrace, G., Stroobant, P., and Waterfield, M. (1984).E M B O J. 3,921-928. Johnsson. A., Betsholtz, C., Heldin, C.-H., and Westermark, B. (1985a).Nature (London) 317,438-440. Johnsson, A,, Betscholtz, C., von der Helm, K., Heldin, C.-H., and Westermark, B. (1985b). Proc. N Q t l . A c Q ~Sci. . U.S.A. 82, 1721-1725. Johnsson, A., Betsholtz, C., Heldin, C.-H., and Westermark, B. (1986).E M B O J . 5,15351541. Jonasson, J., Povey, S., and Harris, H. (1977).J. Cell Sci. 24,217-254. Jones, P. A., and Taylor, S. M. (1980). Cell 20,85-93. Jones-Villeneuve, E., and Phillips, R. A. (1980). E x p . Hematol. 8, 65-76. Joyner, A. L., and Bernstein, A. (1983).Mol. Cell. Biol. 3, 2180-2190. Jubinsky, P. T., and Stanley, E. R. (1985).Proc. Natl. Acad. Sci. U.S.A. 82,2764-2768. Kabat, D., Ruta, M., Murray, M. J., and Polonoff, E. (1980).Proc. Natl. A c Q ~Sci. . U.S.A. 77,5741. Kahn, C. R., Bertolotti, R., Ninio, M., and Weiss, M. C. (1981). Nature (London) 290, 7 17-720. Kai, K., Sato, H., and Odaka, T. (1986).Virology 150, 509-512. Kalderon, D., Richardson, W. D., Markham, A. F., and Smith, A. E. (1984). Nature (London) 311,33-38. Kamata, T., and Feramisco, J. R. (1984). Nature (London) 310, 147-150. Kaminchik, J., Hankins, W. D., Ruscetti, S. K., Linemeyer, D. L., and Scolnick, E. M. (1982).J. Virol. 44, 922-931. Kamps, M. P., Taylor, S. S., and Sefton, B. M. (1984). Nature (London) 310, 589-592. Karin, M., Cathala, G., and Nguyen-Huu, M. C. (1983).Proc. Natl. Acad. Sci. U.S.A. 80, 4040-4044. Kasid, A., Lippman, M. E., Papageorge, A. G., Lowy, D. R., and Gelmann, E. P. (1985). Science 228,725-728. Kataoka, T., Powers, S., McGill, C., Fasano, O., Strathern, J., Broach, J., and Wigler, M. (1984).Cell 37, 437-445. Kataoka, T., Powers, S., Cameron, S., Fasano, O., Goldfarb, M., Broach, J., and Wigler, M. (1985). Cell 40, 19-26.

340

WOLFRAM OSTERTAG

Kawasaki, E. S., Ladner, M. B., Wang, A. M., van Arsdell, J., Warren, M. K., Coyne, M. Y., Schweickart, V. L., Lee, M.-T., Wilson, K. J., Boosman, A., Stanley, E. R., Ralph, P., and Mark, D. F. (1985). Science 230, 291-296. Keller, E. B., and Noon, W. A. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 7417-7420. Keller, G., Paige, C., Gilboa, E., and Wagner, E. F. (1985). Nature (London)318, 149154. Kelso, M. A., and Metcalf, D. (1985).J . Cell. Physiol. 123, 101-110. Khoury, G., and Gruss, P. (1983). Cell 33,313-314. King, C. R., Kraus, M. H., and Aaronson, S. A. (1985). Science 229, 974-976. Kirsten, W. H., and Mayer, L. A. (1967).J . Natl. Cancer Znst. 39, 311-335. Kitagawa, M., Matsubara, O., and Kasuga, T. (1983). Bull. Tokyo Med. Dent. Univ. 30, 95-107. Kitamura, Y., Yokoyama, M., Matsuda, H., and Ohno, T. (1981). Nature (London)291, 159-160. Klein, B., Le Bousse, C., Fagg, B., Smadja-Joffe, F., Vehmeyer, K., Mori, J. K., Jasmin, C., and Ostertag, W. (1981).J . Natl. Cancer Znst. 66,935-940. Klein, B., LeBousse-Kerdiles, M. C., Smadja-Joffe, F., Pragnell, I., Ostertag, W., and Jasmin, C. (1982). E x p . Hematol. 10,373-381. Klein, B., Akouala, J. J., Le Bousee-Kerdiles, C., Smadja-Joffe, F., and Jasmin, C. (1983). Blood 62,305-307. Klein, G. (1981a). Mod. Trends Hum. Leuk. 26,71-78. Klein, G. (1981b). Nature (London)294,313-318. Klein, G . (1983). Cell 32, 311-315. Klein, G., and Klein, E. (1985a). Zmmunol. Today 6,208-215. Klein, G., and Klein, E. (1985b). Nature (London)315, 190-195. Kleinsmith, L. J., and Pierce, G . B. (1964). Cancer Res. 24, 1544-1553. Klingler, K., Johnson, G. R., Nicola, N., Walker, F., and Ostertag, W. (1987). J . Cell. Physiol., submitted. Kloetzer, W. S., Maxwell, S. A., and Arlinghaus, R. B. (1983). Proc. Natl. Acad. Sci. U.S.A.80,412-416. Kloetzer, W. S., Maxwell, S. A., and Arlinghaus, R. B. (1984). Virology 138, 143-155. Kluge, N., Gaedicke, G., Steinheider, G., Dube, S., and Ostertag, W. (1974). E x p . Cell Res. 88,257-262. Kluge, N., Ostertag, W., Sugiyama, T., Jovin-Arndt, D., Steinheider, G., Furusawa, M., and Dube, S. K. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 1237-1240. Kluge, N. K., Ostertag, W., Fusco, A., Pennie, S., and Greenberger, J. S. (1986). Leuk. Res. 10, 187-193. Knudson, A. G. (1983). Prog. Nucleic Acids Res. Mol. Biol. 29, 17-25. Koch, W., Hunsmann, G., and Friedrich, R. (1983).J . Virol. 45, 1-9. Koch, W., Zimmermann, W., Oliff, A., and Friedrich, R. (1984).J . Virol. 49, 828-840. Kollek, R., Stocking, C., Smadja-Joffe, F., and Ostertag, W. (1984).]. Virol. 50,717-724. Kopchick, J. J., Harless, J., Geisser, B. S., Killam, R., Hewitt, R. R., and Arlinghaus, R. B. (1981).J . Virol. 37,274-283. Kost, T. A., Koury, M. J., Hankins, W. D., and Krantz, S. B. (1979). Cell 18, 145-152. Kost, T. A., Koury, M. J., and Krantz, S . B. (1981). Virology 108, 309-317. Koufos, A., Hansen, M. F., Lampkin, B. C., Workman, M. L., Copeland, N. G., Jenkins, N. A., and Cavence, W. K. (1984). Nature (London) 309, 170-172. Koufos, A., Hansen, M. F., Copeland, N. G., Jenkins, N. A., Lampkin, B. C., and Cavence, W. K. (1985). Nature (London)316,330-334. Koury, M. J., and Pragnell, I. B. (1982). Nature (London)299, 638-640.

MURINE SPLEEN FOCUS-FORMING VIRUSES

34 1

Koury, M. J., Bondurant, M. C., Duncan, D. T., Krantz, S. B., and Hankins, W. D. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 635-639. Krantz, S. B., Sawyer, S. T., Koury, M. J., and Bondurant, M. C. (1987). In “NATO Advanced Research Workshop: Molecular and Cellular Aspects of Erythropoiesis” (I. N. Rich, ed.). In press. Kreil, G. (1981). Annu. Reu. Biochem. 50, 317-348. Kronke, M., Leonard, W. J., Depper, J. M., and Greene, W. C. (1985). Science 228, 1215-1217. Krontiris, T. G., Di Martino, N. A., Colb, M., and Parkinson, D. R. (1985). Nature (London)313,369-373. Kruijer, W., Cooper, J. A., Hunter, T., and Verma, I. M. (1984). Nature (London)312, 71 1-716. Kurtz, A., Jelkmann, W., and Bauer, C. (1982). F E B S Lett. 149, 105-108. Kurtz, A., Hartl, W., Jelkmann, W., Zapf, J., and Bauer, C. (1985).J. Clin. Inuest. 76, 1643- 1648. Lacal, J. C., Anderson, P. S., and Aaronson, S. A. (1986). EMBOJ. 5,679-687. Laimins, L. A., Khoury, G., Gorman, C., Howard, B., and Gruss, P. (1982). Proc. Natl. Acad. Sci. U S A . 79,6453-6457. Laker, C. (1987).Doctoral thesis, University of Hamburg, Hamburg, Federal Republic of Germany. Lala, P. K., and Johnson, G. R. (1978).J.E x p . Med. 148, 1468-1477. Land, H., Parada, L. F., and Weinberg, R. A. (1983). Nature (London) 304, 596-602. Land, H., Chen, A. C., Morgenstern, J. P., Parada, L. F., and Weinberg, R. A. (1986). Mol. Cell. Biol. 6, 1917-1925. Lang, R. A., Metcalf, D., Cough, N., Dunn, A. R., and Gonda, T. J. (1985). Cell 43,531542. Langbeheim, H., Shih, T. Y., and Scolnick, E. M. (1980).Virology 106, 292-300. Langdon, W. Y., H o h a n , P. M., Silver, J. E., Buckler, C. E., Hartley, J. W., Ruscetti, S. K., and Morse, H. C. (1983a).J . Virol. 46, 230-238. Langdon, W. Y., Ruscetti, S. K., Silvr, J. E., Hankins, W. D., Buckler, C. E., and Morse, H. C. (1983b).J. Virol. 47, 329-336. Leal, F., Robbins, K. C., and Aaronson, S. A. (1985). Science 230,327-330. Le Beau, M. M., Westbrook, C. A., Diaz, M. O., Rowley, J. D., and Oren, M. (1985). Nature (London)316,826-828. Le Beau, M. M., Westbrook, C. A., Diaz, M. O., Larson, R. A., Rowley, J. D., Gasson, J . C., Golde, D. W., and Sherr, C. J. (1986). Science 231, 984-987. Le Bousse-Kerdiles, M. C., Smadja-Joffe, F., Klein, B., Caillou, B., and Jasmin, C. (1980).Eur. J. Cancer 16,43-51. Le Bousse-Kerdiles, M. C., Jasmin, C., Smadja-Joffee, F., Klein, B., Pragnell, I. B., and Ostertag, W. (1981).Blood Cells 7, 105-123. Le Bousse-Kerdiles, M. C., Smadja-Joffe, F., Klein, B., Jasmin, C., Comisso, M., and Ostertag, W. (1983). Blood 61, 520-524. Le Bousse-Kerdiles, M. C., Dumenil, D., Smadja-Joffe, F., Bertoli, A. M., Degiorgis, V., Auger-Buendia, M. A., Tavitian, A,, and Jasmin, C. (1985a).J.Gen. Virol. 66,2415242 1. Le Bousse-Kerdiles, M. C., Smadja-Joffe, F., Bertoli, A. M., Comisso, M., Mori, K. J., and Jasmin, C. (1985b). Leuk. Res. 9, 1181-1188. Le Bousse-Kerdiles, M. C., Fernandez-Delgado, R., Smadja-Joffe, F., Massier, E., Degiorgis, V., Bertoli, A. M., Paulus, J. M., Prenant, M., and Jasmin, C. (1986). Submitted.

342

WOLFRAM OSTERTAC

Lee, D. C., Rose, T. M., Webb, N. R., and Todaro, G. J. (1985). Nature (London) 313, 489-491. Lee, F., Yokota, T., Otsuka, T., Meyerson, P., Villaret, D., Coffman, R., Mosmann, T., Rennick, D., Roehm, N., Smith, C., Zlotnik, A., and Arai, K. (1986). Proc. Natl. Acad. Sci. U.S.A.83, 2061-2065. Lee-Huang, S. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 2708-2712. Lemischka, I. R., Raulet, D. H., and Mulligan, R. C. (1986). Cell 45, 917-927. Lenz, J., Celander, D., Crowther, R. L., Patarca, R., Perkins, D. W., and Hazeltine, W. A. (1984). Nature (London)308,467-470. Leof, E . B., Proper, J. A., Goustin, A. S., Shipley, G. D., DiCorleto, P. E., and Moses, H. L. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 2453-2457. Lev, Z., Kimchie, Z., Hessel, R., and Segev, 0. (1985). Mol. Cell. Biol. 5, 1540-1542. Levin, J. (1983). Blood 61, 617-623. Levin, J., Levin, F. C., Hull, D. F., and Penington, D. G. (1982). Blood 60, 989-998. Levinson, B., Khoury, G., Vande Woude, G. F., and Gruss, P. (1982).Nature (London) 295,568-572. Li, C. L., and Johnson, G. R. (1984).J.Cell Physiol. 121, 167-173. Li, C. L., and Johnson, G. R. (1985). Nature (London) 316,633-636. Liao, S. K., and Axelrad, A. A. (1975). Int. J . Cancer 15,467-482. Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, I., Soreq, H., Whittle, N., Waterfield, M. D., Ullrich, A., and Schlesinger, J. (1985). Nature (London),313, 144-146. Lilly, F., and Pincus, T. (1973). Adu. Cancer Res. 17,231-277. Lilly, F., and Steeves, R. A. (1973). Virology 55, 363-370. Lin, H. S., and Gordon, S. (1979).J . E r p . Med. 150,231-245. Lin, H. S., and Stewart, C. L. (1973). Nature (New Biol.) 243, 176-177. Lin, H. S., and Stewart, C. L. (1974).J. Cell. Physiol. 83, 369-378. Linemeyer, D. L., Ruscetti, S. K., Menke, J. G., and Scolnick, E. M. (1980).J . Virol. 35, 7 10-72 1. Linemeyer, D. L., Menke, J. G., Ruscetti, S. K., Evans, L. H., and Scolnick, E. M. (1982). J . Virol. 43, 223-233. Lochrie, M. A., Hurley, J., and Simon, M. I. (1985). Science 228, 96-99. Lohler, J., Franz, T., Fusco, A., Pragnell, I., and Ostertag, W. (1987). Leukemia 1, in press. Lopez, A. R., Barker, J., and Deisseroth, A. B. (1986). Proc. Natl. Acad. Sci. U.S.A.83, 2042-2046. Lord, B. I., Mori, K. J., Wright, E. G., and Lajtha, L. G. (1976). Br.1. Haematol. 34,441445. Lord, B. I., Mori, K. J., and Wright, E. G. (1977). Biomedicine 27, 223-226. Lotem, J., Lipton, J. H., and Sachs, L. (1980). Int. J . Cancer 25, 763-771. Lucarelli, G. P., Howard, D., and Stohlman, F., Jr. (1964).J.Clin. Inuest 43,2195-2203. Lyall, R. M., Pastan, I., and Willingham, M. C. (1985)./. Cell. Physiol. 122, 166-170. McClements, W. L., Enquist, L. W., Oskarsson, M., Sullivan, M., and Vande Woude, G. F. (1980).J . Virol. 35,488-497. McCool, D., Mak, T. W., and Bernstein, A. (1979).J . E x p . Med. 149,837-846. McCormick, F., Clark, B. F. C., La Cour, T. F. M., Kieldgaard, M., Norskov-Lauritsen, L., and Nyborg, J. (1985). Science 230, 78-82. McCoy, M. S., Toole, J.. Cunningham, J., Chang, E., Lowy, D., and Weinberg, R. (1983). Nature (London)302,79-81. McCulloch, E. A. (1983). Blood 62, 1-13. McCulloch, E. A., Siminovitch, L., and Till, J. E. (1964). Science 144, 844-846.

MURINE SPLEEN FOCUS-FORMING VIRUSES

343

McCulloch, E. A,, Siminovitch, L., Till, J. E., Russell, E. S., and Bernstein, S . E. (1965). Blood 26,399-407. McCulloch, E. A,, Motoji, T., Smith, L. J., and Curtis, J. E. (1984). J . Cell. Physiol. S u p p l . 3, 13-20. MacDonald, M. E., Reynolds, F. H., van de Ven, W. J . M., Stephenson, J. R., Mak, T. W., and Bernstein, A. (1980a).J . E x p . Med. 151, 1477-1492. MacDonald, M. E., Mak, T. W., and Bernstein, A. (1980b).J.E x p . Med. 151,1493-1503. MacDonald, M. E., Johnson, G. R., and Bernstein, A. (1981). Virology 110, 231-236. McDonald, J. D., Lin, F.-K., and Goldwasser, E. (1966). Mol. Cell. Biol. 6, 842-848. McDonald, T. P., Clift, R., Lange, R. D., Nolan, D., Tribby, E., and Barlow, G. H. (1975). J . Lab. Clin. Med. 85, 59-66. McDonough, S. K., Larsen, S., Brodey, R. S., Stock, N. D., and Hardy, W. D., Jr. (1971). Cancer Res. 31,953-956. McGrath, J. P., Capon, D. J., Smith, D. H., Chen, E. Y., Seeburg, P. H., Goeddel, D. V., and Levinson, A. D. (1983). Nature (London)304,501-506. McGrath, J. P., Capon, D. J., Goeddel, D. V., and Levinson, A. D. (1984). Nature (London) 310,644-649. McLeod, D. L., Shreeve, M. M., and Axelrad, A. A. (1980). Blood 56,318-322. Machida, C. A., Bestwick, R. K., and Kabat, D. (1984).J. Virol. 49, 394-402. Machida, C. A., Bestwick, R. K., Boswell, B. A., and Kabat, D. (1985a). Virology 144, 158- 172. Machida, C. A., Bestwick, R. K., and Kabat, D. (198513).J . Virol. 53, 990-993. Madaule, P., and Axel, P. (1985).Cell 41, 31-40. Mager, D., Mak, T. W., and Bernstein, A. (1980).Nature (London) 288, 592-594. Mager, D. L., Mak, T. W., and Bernstein, A. (1981).Proc. Natl. Acad. Sci. U.S.A. 78, 1703- 1707. Magli, M. C., Iscove, N. N., and Odartchenko, N. (1982).Nature (London)295, 527528. Maisel, J., Klement, U., Lai, M. M.-C., Ostertag, W., and Duesberg, P. (1973).Proc. Natl. Acad. Sci. U.S.A. 70, 3536-3540. Mak, T. W., Gamble, C. L., MacDonald, M. E., and Bernstein, A. (1980). Cold Spring Harbor Symp. Quant. Biol. 44,893-899. Mann, R., Mulligan, R. C., and Baltimore, D. (1983). Cell 33, 153-159. Marcus, S. L., Smith, S. W., Racevskis, J., and Sarkar, N. H. (1978). Virology 86, 398412. Marquardt, H., Hunkapiller, M. W., Hood, L. E., Twardzik, D. R., De Larco, J. E., Stephenson, J. R., and Todaro, G. J. (1983). Proc. Natl. Acad. Sci. U.S.A.80,46844688. Marquardt, H., Hunkapiller, H. W., Hood, L. E., and Todaro, G. J. (1984). Science 223, 1079-1082. Matrisian, L. M., Glaichenhaus, N., Gesnel, M.-C., and Breathnach, R. (1985).EMBO J . 4, 1435-1440. Mauch, P., Greenberger, J. S., Botnick, L., Hannon, E., and Hellman, S. (1980).Proc. Natl. Acad. Sci. U.S.A.77, 2927-2930. Maxwell, S. A., and Arlinghaus, R. B. (1985).J. Virol. 55,874-876. Mechler, B. M., McGinnis, W., and Gehring, W. J. (1985). EMBO J . 4, 1551-1557. Medynski, D. C., Sullivan, K., Smith, D., van Dop, C., Chang, F.-H., Fung, B. K.-K., Seeburg, P. H., and Bourne, H. R. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 43114315. Merchav, S., Wagemaker, G., and van Bekkum, D. W. (1985).J. Natl. Cancer Znst. 75, 361-368.

344

WOLFRAM OSTERTAG

Merlino, G. T., Xu, Y-H., Ishii, S., Clark, A. J. L., Semba, K., Toyoshima, K.,Yamamoto, T., and Pastan, I. (1984). Science 224, 418-419. Merlino, G. T., Ishii, S., Whang-Peng, J., Knutsen, T., Xu, Y-H., Clark, A. J. L., Stratton, R. H., Wilson, R. K., Ma, D. P., Roe, B. A., Hunts, J. H., Shimizu, N., and Pastan, I. (1985). Mol. Cell. Biol. 5 , 1722-1734. Meruelo, D., and Bach, R. (1983).Adu. Cancer Res. 40, 107-188. Metcalf, D. (1970).J . Cell. Physiol. 76, 89-100. Metcalf, D. (1980). Proc. Natl. Acad. Sci. U.S.A.77, 5327-5330. Metcalf, D. (1982). Natl. Cancer Inst. Monogr. 60, 123-131. Metcalf, D. (1984). “The Hemopoietic Colony Stimulating Factors.” Elsevier, Amsterdam. Metcalf, D., and Burgess, A. W. (1982).J. Cell. Physiol. 111,275-283. Metcalf, D., and Johnson, G. R. (1979).J. Cell. Physiol. 99, 159-174. Metcalf, D., and MacDonald, H. R. (1975).J . Cell. Physiol. 85, 643-654. Metcalf, D., and Nicola, N. A. (1983).J. Cell. Physiol. 116, 198-206. Metcalf, D., and Nicola, N. A. (1985). Leuk. Res. 9, 35-50. Metcalf, D., MacDonald, H. R.,Odartchenko, N., and Sordat, B. (1975). Proc. Natl. Acad. Sci. U S A . 72, 1744-1748. Metcalf, D., Johnson, G. R.,and Burgess, A. W. (1980). Blood 55, 138-147. Metcalf, D., Begley, C. G., Johnson, G. R.,Nicola, N. A., Lopez, A. F., and Williamson, D. J. (1986). Blood 68, 46-57. Miller, A. D., Ong, E. S., Rosenfeld, M. G., Verma, I. M., and Evans, R. M. (1984). Science 225, 993-998. Miller, A. D., Law, M.-F., and Verma, I. M. (1985). MoZ. Cell. Biol. 5,431-437. Miller, D. A., Tantravahi, R., Newman, B., Dev, V. G., and Miller, 0.J. (1979). Cancer Genet. Cytogenet. 1, 103-113. Mintz, B., and Illmensee, K. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 3585-3589. Mirand, E. A. (1968).Ann. N . Y. Acad. Sci. 149,468-496. Mirand, E. A., Steeves, R.A., and Lange, R. D. (1968). Proc. Soc. E r p . Biol. Med. 128, 844-849. Miyake, T., Kung, C. K. H., and Goldwasser, E. (1977).J. Biol. Chem. 252,5558-5564. Miyatake, S., Yokota, T., Lee, F., and Arai, K-I. (1985). Proc. Natl. Acad. Sci. U.S.A.82, 3 16-320. Molders, H., Defesche, J., Muller, D., Bonner, T. I., Rapp, U. R.,and Miiller, R.(1985). E M B O J . 4,693-698. Mol, J. N. M., Vonk, W. P., Pragnell, I. B., and Stoof, T. J. (1981).J. Gen. Virol. 54,367377. Mol, J. N. M., Ostertag, W., Bilello, J., and Stoof, T. J. (1982).J.Gen. Virol. 63,45-56. Moloney, J. B. (1966). Natl. Cancer Inst. Monogr. 22, 139-142. Moore, M. A. S. (1978). In “Hematopoietic Cell Differentiation” (D. W. Golde, M.J. Cline, D. Metcalf, and C. F. Fox, eds.), pp. 445-459. Academic Press, New York. Moore, M. A. S., and Williams, N. (1974).In “Hemopoiesis in Culture” (W. A. Robinson, ed.), pp. 17-27. DHEW, Washington, D. C. Moreau-Gachelin, F., Robert-Lezenes, J., Wendling, F., Tavitian, A., and Tambourin, P. (1985).J . Virol. 53, 292-295. Mori, K. J., Smadja-Joffe, F., Barre-Sinoussi, F., Le Bousse-Kerdiles, M. C., Klein, B., Ostertag, W., and Jasmin, C. (1983). Leuk. Res. 7, 77-86. Moscovici, M. G., and Moscovici, C. (1983).Proc. Natl. Acad. Sci. U . S A . 80,1421-1425. Mosmann, T. R.,Bond, M. W., C o f h a n , R. L., Ohara, J., and Paul, W. E. (1986). Proc. Natl. Acad. Sci. U.S.A. 83,5654-5658. Mozer, B., Marlow, R.,Parkhurst, S., and Corces, V. (1985). Mol. Cell. Biol.5,885-889.

MURINE SPLEEN FOCUS-FORMING VIRUSES

345

Muller, K., Bravo, K., Burckhardt, J., and Curran, T. (1984).Nature (London)312,717721. Muller, R.,and Verma, I. M. (1984). Curr. Top. Microbiol. Immunol. 112, 73-115. Muller, R. C., Slamon, D. J., Tremblay, J. M., Cline, M. J., and Verma, I. (1982).Nature (London)299,640-644. Mulcahy, L. S., Smith, M. R., and Stacey, D. W. (1985).Nature (London)313,241-243. Murphree, L. A., and Benedict, W. F. (1984). Science 223, 1028-1033. Mushinski, J. F., Potter, M., Bauer, S. R., and Reddy, E. P. (1983). Science 220,795-798. Nagao, K., Yokoro, K., and Aaronson, S. A. (1981). Science 212, 333-335. Nagata, S., Tsuchiya, M., Asano, S., Kaziro, Y., Yamazaki, T., Yamamoto, O., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., and Ono, M. (1986). Nature (London) 319, 4 15-4 18. Nakahata, T., and Ogawa, M. (1982a).J . Cell. Physiol. 111,239-246. Nakahata, T., and Ogawa, M. (1982b). Proc. Natl. Acad. Sci. U.S.A.79,3843-3847. Nakahata, T., Gross, A. J., and Ogawa, M. (1982a).J . Cell. Physiol. 113,455-458. Nakahata, T., Spicer, S. S., Cantey, J. R.,and Ogawa, M. (1982b). Blood 60,352-361. Nakano, H., Yamamoto, F., Neville, L., Evans, D., Mizuno, T., and Perucho, M. (1984). Proc. Natl. Acad. Sci. U.S.A.81, 71-75. Narayaman, R.,Srinivasan, A., and Aaronson, S. A. (1984). Virology 137, 32-40. Neil, J., Hughes, D., McFarlane, R.,Wilkie, N. M., Onions, D., Lees, G., and Jarrett, 0. (1984).Nature (London)308,814-820. Neumann-Silberberg, F. S., Schejter, E., Hoffmann, F. M., and Shilo, B.-Z. (1984). Cell 37, 1027-1033. Nicola, N. A,, and Johnson, G. R. (1982).Blood 60, 1019-1029. Nicola, N. A., and Metcalf, D. (1984). Proc. Natl. Acad. Sci. U.S.A.81, 3765-3769. Nicola, N. A., and Metcalf, D. (1985).J . Cell. Physiol. 124, 313-321. Nicola, N. A., and Metcalf, D. (1986).J. Cell. Physiol. 128, 180-188. Nicola, N. A., Burgess, A. W., and Metcalf, D. (1979).J . Biol. Chem. 254, 5290-5299. Nicola, N. A,, Metcalf, D., von Melchner, H., and Burgess, A. W. (1981). Blood 58,376387. Nicola, N. A., Metcalf, D., Matsumoto, M., and Johnson, G . R.(1983).J.Biol. Chem. 258, 90 17-9023. Nielsen, J. T., and Chapman, V . M. (1977). Genetics, 87, 319-327. Nienhuis, A. W., Bunn, H. F., Turner, P. H., Gopal, T. V . , Nash, W. G., O’Brien, S. J., and Sherr, C. J. (1985). Cell 42,421-428. Niho, Y., Shibuya, T., and Mak, T. W. (1982).J.E x p . Med. 156, 146-158. Nilsen, T., Maroney, P. A,, Goodwin, R. G., Rottman, F. M., Crittenden, L. B., Raines, M. A., and Kung, H.-J. (1985). Cell 41, 719-726. Noda, M., Selinger, H., Scolnick, E. M., and Bassin, R. H. (1983). Proc. Natl. Acad. Sci. U.S.A.80, 5602-5606. Noma, Y., Sideras, P., Naito, T., Bergstedt-Lindquist, S., Azuma, C., Severinson, E., Tanabe, T., Kinashi, T., Matsuda, F., Yaoita, Y., and Honjo, T. (1986). Nature (London) 319,640-646. Nowell, P. C. (1976).Science 194, 23-28. Nowell, P. C., and Hungerford, D. A. (1960). Science 132, 1497-1502. Nusse, R., and Varmus, H. E. (1982). Cell 31,99-109. Obata, M., Anamura, H., Harada, Y., Sagata, N., and Ikawa, Y. (1984).Virology 136,435438. O’Brien, S. J., Berman, E. J., Estes, J. D., and Gardner, M. B. (1983).J.Virol. 47,649651. Odaka, T. (1969).J . Virol. 3, 543-548.

346

WOLFRAM OSTERTAG

Odaka, T. (1973). Int. J. Cancer 11,567-574. Odaka, T. (1974). Znt. J. Cancer 14,252-258. Odaka, T., Ikeda, H., and Akatsuka, T. (1980). Int. J. Cancer 25,757-762. Odaka, T., Ikeda, H., Yoshikura, H., Moriwaki, K., and Suzuki, S. (198U.J.Natl. Cancer Inst. 67, 1123-1 127. Oliff, A., Hager, G. L., and Chang, E. H. (1980).J. Virol. 33,475-486. Oliff, A., Ruscetti, S., Douglass, E. C., and Scolnick, E. M. (1981). Blood 58, 244-254. Oliff, A,, Signorelli, K., and Collins, L. (1984).J. Virol. 51, 788-794. Oliff, A., McKinney, M. D., and Agranovsky, 0. (1985).J. Virol. 54, 864-868. Opitz, U., Seidel, H. J., and Rich, I. S. (1977). Blut 35, 35-44. Orkin, S. H., Goldman, D. S., and Sallan, S. E. (1984). Nature (London) 309, 173-174. Oshimura, M., Gilmer, T. M., and Barrett, J. C. (1985). Nature (London) 316,636-639. Oskarsson, M., McClements, W. L., Blair, D. G., Maize], J. V., and Vande Woude, G. (1980). Science 207, 1222-1224. Ostertag, W., and Pragnell, I. B. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 3278-3282. Ostertag, W., and Pragnell, I. B. (1981).Curr. Top. Microbiol. Zmmunol. 94/95,143-208. Ostertag, W., Melderis, H., Steinheider, G., Kluge, N., and Dube, S. K. (1972). Nature (London)239,231-234. Ostertag, W., Cole, T., Crozier, T., Gaedicke, G., Kluge, N., Kind, J., Krieg, C., Roesler, G., Steinheider, G., Weimann, B., and Dube, S. K. (1974). Symp. Differ. Control Malig. Tumor Cells Tokyo, 1973 pp. 493-520. Ostertag, W., Vehmeyer, K., Fagg, B., Pragnell, I. B., Paetz, W., Le Bousse, M. C., Smadja-Joffe, F., Klein, B., Jasmin, C., and Eisen, H. (1980).J. Virol. 33,573-582. Ostertag, W., Odaka, T., Smadja-Joffe, F., and Jasmin, C. (198l).J.Virol. 37, 541-548. Ostertag, W., Pragnell, I. B., Fusco, A., Hughes, D., Freshney, M., Klein, B., Jasmin, C., Bilello, J., Warnecke, G., and Vehmeyer, K. (1982). I n “Expression of Differentiated Functions in Cancer Cells” (R.Revoltella, ed.), pp. 401-416. Raven, New York. Ostertag, W., Freshney, M., Vehmeyer, K., Jasmin, C., and Rutter, G. (1984).J.Virol.49, 253-261. Ostertag, W., Seliger, B., Kollek, R., Stocking, C., Bergholz, U., and Smadja-Joffe, F. (1986).J. Cen. Virol. 67, 1361-1371. Ott, M.-O., Sperling, L., Cassio, D., Levilliers, J., Sala-Trepat, J,, and Weiss, M. C. (1982). Cell 30, 825-833. Overhauser, J., and Fan, H. (1985).J. Virol. 54, 133-144. Padua, R.,Barrett, K. B., Hughes, D. C., Pragnell, I., Friel, J., and Stocking, C. (1986).J. Virol., submitted. Palaszynski, E. W., and Ihle, J. N. (1984).J. Zmmunol. 132, 1872-1878. Papageorge, A., Lowy, D., and Scolnick, E. M. (1982).J. Virol. 44,509-519. Papageorge, A. G., Willumsen, B. M., Johnsen, M., Kung, H.-F., Stacey, D. W., Vass, W. C., and Lowy, D. R. (1986). Mol. Cell Biol. 6, 1843-1846. Papkoff, J., and Ringold, G. M. (1984).J. Virol. 52,420-430. Papkoff, J., Verma, I., and Hunter, T. (1982). Cell 29,417-426. Papkoff, J., Nigg, E. A., and Hunter, T. (1983). Cell 33, 161-172. Parada, L., Tabin, C. J., Shih, C., and Weinberg, R. A. (1982). Nature (London) 297, 474-478. Pelicci, P.-G., Lanfrancone, L., Brathwaite, M. D., Wolman, S. R.,and Dalla-Favera, R. (1984). Science 224, 1119-1121. Peschle, C., Migliaccio, G., Lettieri, F., Migliaccio, A. R.,Ceccarelli, R.,Barba, P., Titti, F., and Rossi, G. B. (1980). Proc. Natl. Acad. Sci. U.S.A. 77,2054-2058.

MURINE SPLEEN FOCUS-FORMING VIRUSES

347

Peters, R. L., Rabstein, L. S., van Vleck, R., Kelloff, G. J., and Huebner, R. J. (1974).J. Natl. Cancer Znst. 53, 1725-1729. Pharr, P. N., Suda, T., Bergmann, K. L., Avila, L. A,, and Ogawa, M. (1984).J. Cell Physiol. 120, 1-12. Pierce, J. H., and Aaronson, S. A. (1985). Mol. Cell. B i d . 5, 667-674. Pierce, J. H., Aaronson, S. A., and Anderson, S. M. (1984). Proc. Natl. Acad. Sci. U.S.A. 81,2374-2378. Pinter, A., and Honnen, W. J. (1983).J. Virol. 46, 1056-1060. Pinter, A,, and Honnen, W. J. (1985).Virology 143, 646-650. Ponta, H., Kennedy, N., Skroch, P., Hynes, N. E., and G o n e r , B. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 1020-1024. Ponten, J. (1964).J. Natl. Cancer Znst. Monogr. 17, 131-135. Powers, S., Kataoka, T., Fasano, O., Goldfarb, M., Strathern, J., Broach, J., and Wigler, M . (1984).Cell 36, 607-612. Pragnell, I. B., McNab, A., Harrison, P. R., and Ostertag, W. (1978). Nature (London) 272,456-458. Pragnell, I. B., Fusco, A., Arbuthnott, C., Smadja-Joffee, F., Klein, B., Jasmin, C., and Ostertag, W. (1981).J.Virol. 38, 952-957. Propst, F., and Vande Woude, G. (1985). Nature (London) 315,516-518. Prywes, R., Foulkes, J. G., Rosenberg, N., and Baltimore, D. (1983). Cell 34,569-579. Prywes, R., Hoag, J., Rosenberg, N., and Baltimore, D. (1985).J. Virol. 54, 123-132. Pulciani, S., Santos, E., Lanver, A. V., Long, L. K., Aaronson, S. A,, and Barbacid, ,M. (1982).Nature (London) 300,539-542. Pulciani, S., Santos, E., Long, L. K., Sorrentino, V., and Barbacid, M. (1985). Mol. Cell. Biol. 5,2836-2841. Rabes, H. M., Bucher, T., Hartmann, A., Linke, I., and Diinnwald, M. (1982).Cancer Res. 42,3220-3227. Racevskis, J., and Koch, G. (1977).J. Virol. 21, 328-337. Racevskis, J., and Koch, G. (1978). Virology 87, 354-365. Ramsden, M., Cole, G., Smith, J., and Balmain, A. (1985). E M B O J . 4, 1449-1454. Rao, C., Igarashi, H., Chiu, I.-M., Robbins, K. C., and Aaronson, S. A. (1986).Proc. Natl. Acad. Sci. U.S.A. 83, 2392-2396. Rapp, U. R., Bonner, T. I., Moelling, K., Bister, K., and Ihle, J. (1985a). In “Recent Results in Cancer Research” (K. Havemann, G. Sorensen, and C. Gropp, eds.), in press. Rapp, U. R., Cleveland, J. R., Brightman, K., Scott, A., and Ihle, J. N. (198513).Nature (London) 317,434-438. Rasheed, S., Gardner, M.B., and Huebner, R. J. (1978).Proc. Natl. Acad. Sci. U.S.A.75, 2972-2976. Rasheed, S., Norman, G. L., and Heidecker, G. (1983).Science 221, 155-157. Rauscher, F. J. (1962).J. Natl. Cancer Znst. 29,515-532. Razin, E., Cordon-Cardo, C., and Good, R. A. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 2559-2561. Rechavi, G., Givol, D., and Canaani, E. (1982). Nature (London) 300, 607-611. Reddy, E. P. (1983). Science 220, 1061-1063. Reddy, E. P., Smith, M. J., and Aaronson, S. A. (1981). Science 214,445-450. Reddy, E. P., Lipman, D., Andersen, P. R., Tronick, S. R., and Aaronson, S. A. (1985).J. Virol. 53, 984-987. Reeve, A. E., Housiaux, P. J., Gardner, R. J. M., Chewings, W. E., Grindley, R. M., and Millow, L. J. (1984).Nature (London) 309, 174-176.

348

WOLFRAM OSTERTAG

Rein, A. (1982). Virology 120,251-257. Rein, A., and Schultz, A. (1984). Virology 136, 144-152. Rein, A., Keller, J., Schultz, A. M., Holmes, K. L., Medicus, R., and Ihle, J. N. (1985). Mol. Cell. Biol. 5, 2257-2264. Rettenmier, C. W., Chen, J. H., Roussel, M. F., and Sherr, C. J. (1985a) Science 228, 320-322. Rettenmier, C. W., Roussel, M. F., Quinn, C. O., Kitchingman, G. R., Look, A. T., and Sherr, C. J. (1985b). Cell 40,971-981. Reymond, C. D., Gomer, R. H., Mehdy, M. C., and Firtel, R. A. (1984).Cell 39,141-148. Reynolds, F. H., Todaro, G. J., Fryling, C., and Stephenson, J. R. (1981). Nature (London) 292,259-262. Rhim, J. S., Jay, G., Arnstein, P., Price, F. M., Sanford, K. K., and Aaronson, S. A. (1985). Science 227, 1250-1252. Rio, D. C., Clark, S. G., and Tjian, R. (1985).Science 227, 23-28. Risser, R., Grunwald, D., Sinaiko, C., and Jelen, P. (1980). Cold Spring Harbor Symp. Quant. Biol. 44, 1195-1204. Robbins, K. C., Leal, F., Pierce, J. H., and Aaronson, S. A. (1985). EMBO J . 4, 17831792. Rosen, C. A., Haseltine, W. A., Lenz, J., Ruprecht, R., and Cloyd, M. W. (1985).J . Virol. 55,862-866. Rosendaal, M., Hodgson, G. S., and Bradley, T. R. (1976).Nature (London)264,68-69. Ross, S. R., and Solter, D. (1985). Proc. Natl. Acad. Sci. U S A . 82, 5880-5884. Roussel, M. F., Rettenmier, C. W., Look, A. T., and Sherr, C. J. (1984).Mol. Cell. Biol. 4, 1999-2009. Rowe, W. P., Cloyd, M. W., and Hartley, J. W. (1980).Cold Spring Harbor Symp. Quant. Biol. 44, 1265-1268. Rowley, J. D. (1985). In “Leukemia” (I. L. Weissman, ed.), pp. 179-202. Dahlem Konferenzen. Springer-Verlag, Berlin. Royer-Pokora, B., Beug, H., Claviez, M., Winkhardt, H. J., Friis, R. R., and Graf, T. (1978). Cell 13, 751-760. Rubenstein, J. L. R., Nicolas, J.-F., and Jacob, F. (1984).Proc. Natl. Acad. Sci. U.S.A. 81, 7137-7140. Rubin, R. A., and Earp, H. S. (1983). Science 219,60-63. Ruff, M., and Pert, C. B. (1984). Science 225, 1034-1036. Ruscetti, S., and Wolff, L. (1984). Curr. Top. Microbiol. Zmmunol. 122,21-44. Ruscetti, S., and Wolff, L. (1985).J . Virol. 56, 717-722. Ruscetti, S. K., Troxler, D., Linemeyer, D., and Scolnick, E. (1980).J . Virol. 33, 140151. Ruscetti, S., Davis, L., Feild, J., and Oliff, A. (1981a).J . Erp. Med. 154, 907-920. Ruscetti, S. K., Feild, J. A., and Scolnick, E. M. (1981b).Nature (London)294,663-665. Ruscetti, S . , Matthai, R., and Potter, M. (1985).J . E z p . Med. 162, 1579-1587. Russell, E. S. (1979). Ado. Genet. 20, 357-459. Ruta, M., and Kabat, D. (1980).J . Virol. 35,844-853. Ruta, M., Bestwick, R., Machida, C., and Kabat, D. (1983). Proc. Natl. Acad. Sci. USA. 80,4704-4708. Ruta, M., Wolford, R., Dhar, R., Defeo-Jones, D., Ellis, R. W., and Scolnick, E. M. (1986). Mol. Cell. Biol. 6, 1706-1710. Sacca, R., Stanley, E. R., Sherr, C. J., and Rettenmier, C. W. (1986). Proc. Natl. Acad. Sci. U S A . 83, 3331-3335. Sager, R., Gadi, I. K., Stephens, L., and Grabowy, C. T. (1985). Proc. Natl. Acad. Sci. U.SA. 82,7015-7019.

MURINE SPLEEN FOCUS-FORMING VIRUSES

349

Sakaguchi, A. Y., Lalley, P. A., Zabel, B. U., Ellis, R. W., Scolnick, E. M., and Naylor, S. L. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 525-529. Salahuddin, S. Z., Markham, P. D., Lindner, S. G., Gootenberg, J., Popovic, M., Hemmi, H., Sarin, P. S., and Gallo, R. C. (1984).Science 223, 703-707. Sanderson, C. J., Warren, D. J.. and Strath, M. (1985).J . E r p . Med. 162, 60-74. Santos, E., Tronick, S. R., Aaronson, S. A., Pulciani, S., and Barbacid, M. (1982).Nature (London),298,343-347. Santos, E., Martin-Zanca, D., Reddy, E. P., Pierotti, M. A., Della Porta, G., and Barbacid, M. (1984). Science 223, 661-664. Sap, J., Mufioz, A., Damm, K., Goldberg, Y., Ghysdael, J., Leutz, A., Beug, H., and Vennstrom, B. (1986).Nature (London) 324,635-640. Sariban, E., Mitchell, T., and Kafe, D. (1985). Nature (London), 316, 64-66. Schechter, A. L., Hung, M.-C., Vaidyanathan, L., Weinberg, R. A., Yang-Feng, T. L., Francke, U., Ullrich, A., and Coussens, L. (1985).Science 229, 976-978. Schejter, E. D., and Shilo, B. Z. (1985). E M B O J . 4, 407-412. Scher, C. D., Scolnick, E. M., and Siegler, R. (1975). Nature (London) 256,225-226. Schmidt, A., Setoyama, C., and de Crombrugghe, B. (1985).Nature (London)314,286289. Schrader, J. W. (1981).J . lmmunol. 126,452-458. Schrader, J. W., and Crapper, R. M. (1983). Proc. Natl. Acad. Sci. U.S.A. 80,6892-6896. Schrader, J. W., and Schrader, S. (1978).J . E x p . Med. 148, 823-828. Schrader, J. W., Lewis, S. J., Clark-Lewis, I., and Culvenol, J. G. (1981). Proc. Natl. Acad. Sci. U S A . 78, 323-327. Schrader, J. W., Clark-Lewis, I., Crapper, R. M., and Wong, G. W. (1983).lmmunol. Reu. 76, 78-104. Schwab, M., Alitalo, K., Varmus, H. E., Bishop, J. M., and George, D. (1983).Nature (London) 303,497-501. Schwab, M., Ramsay, G., Alitalo, K., Varmus, H. E., Bishop, J. M., Martinsson, J., Levan, G., and Levan, A. (1985). Nature (London) 315, 345-347. Scolnick, E. M., and Parks, W. P. (1974).J.Virol. 13, 1211-1219. Scolnick, E. M., Rands, E., Williams, D., and Parks, W. P. (1973).J.Virol. 12,458-463. Scolnick, E. M., Papageorge, A. G., and Shih, T. Y. (1979). Proc. Natl. Acad. Sci. U.S.A. 76,5355-5359. Scott, I., Cowell, J., Robertson, M. E., Priestley, L. M., Wadey, P., Hopkins, B., Prichard, J., Bell, G. I., Rall, L. B., Graham, C. F., and Knott, T. J. (1985). Nature (London) 317,260-262. Sefton, B. M., Trowbridge, I. S., Cooper, J. A., and Scolnick, E. M. (1982). Cell 31,465474. Seidel, H. J. (1982).Cancer Res. Clin. Oncol. 104, 1-12. Sekiya, T., Fushimi, M., Hori, H., Hirohashi, S., Nishimura, S., and Sugimura, T. (1984). Proc. Natl. Acad. Sci. U.S.A.81, 4771-4775. Seliger, B., Kollek, R., Stocking, C., Franz, T., and Ostertag, W. (1986). Mol. Cell. Biol. 6,286-293. Selten, G., Cuypers, H. T., and Berns, A. (1985). EMBOJ. 4, 1793-1798. Semba, K., Kamata, N., Toyoshima, K., and Yamamoto, T. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501. Seth, A., and Vande Woude, G. F. (1985).J . Virol. 56, 144-152. Setoyama, C., Liau, G., and d e Crombrugghe, B. (1985). Cell 41, 201-209. Shadduck, R. K., and Metcalf, D. (1975).J . Cell Physiol. 86, 247-252. Sherr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F., Look, A. T., and Stanley, E. R. (1985). Cell 41, 665-676.

350

WOLFRAM OSTERTAG

Shenvood, J. B., and Goldwasser, E. (1978).Endocrinology 103,866-870. Shibuya, M., Yokota, J., and Ueyama, Y. (1985). Mol. Cell. Biol. 5,414-418. Shibuya, T., and Mak, T. W. (1982a). Nature (London) 296,517-519. Shibuya, T., and Mak, T. W. (1982b). Cell 31,483-493. Shibuya, T., and Mak, T. W. (1983a). Proc. Natl. Acad. Sci. U.S.A. 80, 3721-3725. Shibuya, T., and Mak, T. W. (1983b).J. Cell Physiol. 117,283-289. Shih, C., Padhy, L., Murray, M., and Weinberg, R. A. (1981).Nature (London)290,261266. Shih, T. Y., and Weeks, M. 0. (1984). Basic Sci. Reo. Cancer Invest. 2, 107-121. Shih, T. Y., Weeks, M. O., Young, H. A., and Scolnick, E. M. (1979a). Virology 96,6479. Shih, T. Y., Weeks, M. O., Young, H. A., and Scolnick, E. M. (197913).J. Virol. 31,546556. Shih, T. Y., Papageorge, A. G., Stokes, P. E., Weeks, M. O., and Scolnick, E. M. (1980). Nature (London) 287,686-691. Shih, T. Y., Weeks, M. O., Gruss, P., Dhar, R., Oroszlan, S., and Scolnick, E. M. (1982).J. Virol. 42, 253-261. Shilo, B., and Weinberg, R. A. (1981a). Proc. Natl. Acad. Sci. U.S.A.78,6789-6792. Shilo, B. Z., and Weinberg, R. A. (1981b). Nature (London) 289,607-609. Shimizu, K., Goldfarb, M., and Wigler, M. (1983). Proc. Natl. Acad. Sci. U.S.A.80,383387. Shinnick, T. M., Lerner, R. A., and Sutcliffe, J. G. (1981). Nature (London) 293, 543548. Shoemaker, C. B., and Mitsock, L. D. (1986). Mol. Cell. Biol. 6, 849-858. Shtivelman, E., Lifshitz, B., Gale, R. P., and Canaani, E. (1985). Nature (London)315, 550-554. Sigal, I. S., Gibbs, J. B., D’Alonzo, J, S., Temeles, G. L., Wolanski, B. S., Socher, S. H., and Scolnick, E. M. (1986a). Proc. Natl. Acad. Sci. U.S.A. 83, 952-956. Sigal, I. S., Gibbs, J. B., D’Alonzo, J. S., and Scolnick, E. M. (198613).Proc. Natl. Acad. Sci. U.S.A. 83, 4725-4729. Silver, J. (1984).J. Virol. 50, 872-877. Singh, B., Sparrow, J. T., Hedge, A.-M., and Arlinghaus, R. B. (1986). Proc. Natl. Acad. Sci. U.S.A. 83,3629-3633. Sitbon, M., Nishio, J., Wehrly, K., and Chesebro, B. (1985). Virology 140, 144-151. Smadja-Joffe, F., Jasmin, C., Kerdiles, C., and Klein, B. (1975). Eur.1. Cancer 11,831840. Smadja-Joffe, F., Klein, B., Kerdiles, C., Feinendegen, L., and Jasmin, C. (1976). Cell Tissue Kinet. 9, 131-145. Smadja-Joffe, F., LeBousse-Kerdiles, M. C., Costagliola, D., Klein, B., Malaise, E. P., and Jasmin, C. (1981).E x p . Hematol. 9, 137-148. Smith, M. R., DeGudicibus, S. J., and Stacey, D. W. (1986). Nature (London)320,540543. Sonoda, T., Kitamura, Y., Haku, Y., Hara, H., and Mori, K. J. (1983). Br.J. Haematol. 53, 611-620. Souza, L. M., Boone, T. C., Cabrilove, J., Lai, P. H., Zsebo, K. M., Murdock, D. C., Chazin, V. R., Bruszewski, J., Lu, H., Chen, K. K., Barendt, J., Platzer, E., Moore, M. A. S., Mertelsmann, R., and Welte, K. (1986). Science 232, 61-65. Spandidos, D. A., and Wilkie, N. M. (1984). Nature (London) 310,469-475. Spooncer, E., Boettiger, D., and Dexter, T. M. (1984). Nature (London) 310, 228230.

MURINE SPLEEN FOCUS-FORMING VIRUSES

351

Spooncer, E., Heyworth, C. M., Dunn, A,, and Dexter, M. (1986). Differentiation 31, 111-118. Srinivas, R. V., and Compans, R. W. (1983a). Virology 125, 274-286. Srinivas, R. V., and Compans, R. W. (1983b).J . Biol. Chem. 258, 14718-14724. Stacey, A., Arbuthnott, C., Kollek, R., Coggins, L., and Ostertag, W. (1984).J . Virol. 50, 725-732. Stanbridge, E., Der, C., Doersen, C.-J., Nishimi, R., Peehl, D., Weissman, B., and Wilkinson, J. (1982). Science 215, 252-259. Stanley, E. R. (1979).Proc. Natl. Acad. Sci. U.S.A. 76, 2969-2973. Stanley, E. R., and Heard, P. M. (1977).J . Biol. Chem. 252, 4305-4312. Stanley, E. R., and Jubinsky, P. T. (1984). Clin. Haematol. 13, 329-348. Stanley, E. R., Guilbert, L. J., Tushinski, R. J., and Bartelmez, S. H. (1983). J . Cell. Biochem. 21, 151-159. Stanley, E. R., Metcalf, D., Sobieszczuk, P., Gough, N. M., and Dunn, A. R. (1985). E M B O J . 4,2569-2573. Stanley, E. R., Bartocci, A., Patinkin, D., Rosendaal, M.,and Bradley, T. R. (1986). Cell 45,667-674. Steeves, R. A. (1975).J . Natl. Cancer Znst. 54, 289-297. Steeves, R. A,, and Mirand, E. A. (1969).Proc. A m . Assoc. Cancer Res. 10, 86-94. Steeves, R. A., Bennett, M., Mirand, E. A., and Cudkowicz, G. (1968). Nature (London) 218,372-374. Stein, R., Gruenbaum, Y., Pollack, Y., Razin, A., and Cedar, H. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 61-65. Steinheider, G., Melderis, H., and Ostertag, W. (1971).Znt. Symp. Synth. Struct. Function Hemoglobin Bad Nauheim pp. 225-235. Steinheider, G., Seidel, H. J., and Kreja, L. (1979). Experientia 35, 1173-1176. Stephenson, J. R., Axelrad, A. A. (1971). Blood 37, 417-427. Stephenson, J. R., Axelrad, A. A., McLeod, D. L., and Shreeve, M. M. (1971).Proc. Natl. Acad. Sci. U.S.A. 68, 1542-1546. Stewart, T. A., and Mintz, B. (1982).J . E x p . 2001.224, 465-496. Stocking, C., Kollek, R., Bergholz, U., and Ostertag, W. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 5746-5750. Stocking, C., Kollek, R., and Bergholz, U. (1986).Virology 153, 145-149. Stroobant, P., Gullick, W., Waterfield, M. D., and Rozengurt, E. (1985). E M B O J . 4, 1945-1949. Suda, T., Suda, J., and Ogawa, M. (1983a).J . Cell. Physiol 117, 308-318. Suda, T., Suda, J., and Ogawa, M. (1983b).Proc. Natl. Acad. Sci. U.S.A.80,6689-6693. Sue, J. M., and Sytowdski, A. J. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 3651-3655. Sugamura, K., Fujii, M., Kobayashi, N . , Sakitani, M., Hatanaka, M., and Hinuma, Y. (1984).Proc. Natl. Acad. Sci. U.S.A. 81, 7441-7445. Suzuki, S., and Axelrad, A. (1980). Cell 19, 225-236. Swan, D., Oskarsson, M., Keithley, D., Ruddle, F. H., D’Eustachio, P., and Vande Woude, G. F. (1982).J . Virol. 44, 752-754. Sweet, R. W., Yokoyama, S., Kamata, T., Feramisco, J. R., Rosenberg, M., and Gross, M. (1984). Nature (London) 311,273-276. Sytkowski, A. J., Salvado, A. J., Smith, G. M., McIntyre, C. J., and d e Both, J. (1980). Science 210,74-76. Sytkowski, A. J., Perrine, S. P., Bicknell, K. A., and Kessler, C. J. (1983). In “Globin Gene Expression and Hematopoietic Differentiation” (Stamatoyannopoulos and Neinhaus, eds.), pp. 335-343. Liss, New York.

352

WOLFRAM OSTERTAG

Tabin, C. J., Bradley, S. M., Bargmann, C. I., Weinberg, R. A., Papageorge, A. G., Scolni k, E. M., Dhar, R., Lowy, D. R., and Chang, E. H. (1982). Nature (London) 300, 1h-149. Tainsky, M. A., Cooper, C. S., Giovanella, B. C., and Vande Woude, G. F. (1984). Science 225,643-645. Tambourin, P. E. (1979). Br. SOC. Cell Biol. Symp. 2, 259-316. Tambourin, P., Casadevall, N., Choppin, J., Lacombe, C., Heard, J. M., Fichelson, S., Wendling, F., Hankins, W. D., and Varet, B. (1983).Proc. Natl. Acad. Sci. U.S.A.80, 6269-6273. Tanabe, T., Nukada, T., Nishikawa, Y., Sugimoto, K., Suzuki, H., Takahashi, H., Noda, M., Haga, T., Ichiyama, A,, Kangawa, K., Minamino, N., Matsuo, H., and Numa, S. (1985). Nature (London)315,242-245. Taparowsky, E., Suard, Y., Fasano, O., Shimizu, K., Goldfarb, M., and Wigler, M. (1982). Nature (London)300,762-765. Tarpley, W. G., Hopkins, N. K., and Gorman, R. R. (1986). Proc. Natl. Acad. Sci. U.SA. 83,3703-3707. Tatchell, K., Chaleff, D. T., de Feo-Jones, D., and Scolnick, E. M. (1984). Nature (London)309,523-527. Temeles, G. C., Gibbs, J. B., D’Alonzo, J. S., Sigal, I. S., and Scolnick, E. M. (1985). Nature (London),313,700-703. Tertian, G., Yung, Y. P., Guy-Grand, D., and Moore, M. A. S. (1981).J . Immunol. 127, 788-793. Thor, A., Horan-Hand, P., Wunderlich, D., Caruso, A., Muraro, R., and Schlom, J. (1984). Nature (London)311,562-565. Thorgeirsson, U. P., Turpeenniemi-Hujanen, T., Williams, J. E., Westin, E. H., Heilman, C. A., Talmadge, J. E., and Liotta, L. A. (1985). Mol. Cell Biol. 5, 259262. Till, J. E., and McCulloch, E. A. (1980). Biochim. Biophys. Acta 605,431-459. Toda, T., Uni, I., Ishikawa, T., Powers, S., Kataoka, T., Broek, D., Cameron, S., Broach, J., Matsumoto, K., and Wigler, M. (1985). Cell 40,27-36. Tracy, S. E., Woda, B. A., and Robinson, H. L. (1985).J. Virol. 54,304-310. Tronick, S. R., Robbins, K. C., Canaani, E., Devare, S. G., Andersen, P. R., and Aaronson, S. A. (1979). Proc. Natl. Acad. Sci. U.S.A. 76, 6314-6318. Troxler, D. H., and Scolnick, E. M. (1978). Virology 85, 17-27. Troxler, D. H., Boyars, J. K., Parks, W. P., and Scolnick, E. M. (1977a)J. Virol. 22,361372. Troxler, D. H., Lowy, D., Howk, R., Young, H., and Scolnick, E. M. (1977b). Proc. Natl. Acad. Sci. U.S.A. 74, 4671-4675. Troxler, D. H., Lowy, D., Howk, R., Young, H., and Scolnick, E. M. (1977c).J.Virol. 22, 361-372. Troxler, D. H., Parks, W. P., Voss, W. C., and Scolnick, E. M. (1977d). Virology 76,602615. Troxler, D. H., Yuan, E., Linemeyer, D., Ruscetti, S. K., and Scolnick, E. M. (1978).J . E x p . Med. 148,639-653. Troxler, D. H., Ruscetti, S. K., Linemeyer, D. L., and Scolnick, E. M. (1980a). Virology 102,28-45. Troxler, D. H., Ruscetti, S. K., and Scolnick, E. M. (1980b).Biochim. Biophys. Acta 605, 305-324. Tsuchida, N., and Uesugi, S. (1981).J . Virol. 38, 720-727. Tsuchida, N., Ryder, T., and Ohtsubo, E. (1982). Science 217,937-939.

MURINE SPLEEN FOCUS-FORMING VIRUSES

353

Twardzik, D. R., Todaro, G. J., Reynolds, T. H., and Stephenson, J. R. (1983). Virology 124,201-207. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A., Lee, J., Yarden, Y., Libermann, T., Schlesinger, J., Downward, J., Mayes, E., Whittle, N., Waterfield, M. D., and Seeburg, P. (1984). Nature (London) 309,418-425. Ulsh, L. S., and Shih, T. Y. (1984). Mol. Cell. Biol. 4, 1647-1652. Van Bekkum, D. W., van Noord, M. J., Maat, B., and Dicke, K. A. (1971). Blood 38,547558. Van Beveren, C., van Straaten, F., Galleshaw, J. A,, and Verma, I. M. (1981a). Cell 27, 97-108. Van Beveren, C., Galleshaw, J. A., Jonas, V., Berns, A. J. M., Doolittle, R. F., Donoghue, D. J., and Verma, I. M. (1981b). Nature (London) 289,258-262. Van den Engh, G., Russell, J., and de Cicco, D. (1978). In “Experimental Hematology Today” (S. S. Baum and G. D. Ledney, eds.), pp. 9-15. Springer-Verlag, Berlin and New York. Van der Hoorn, F. A., and Firzlaff, J. (1984).Nucleic Acids Res. 12, 2147-2156. Van der Hoorn, F. A., Hulsebos, E., Berns, A. J. M., and Bloemers, H. P. J. (1982). E M B O J . 1, 1313-1317. Van der Hoorn, F. A., Miiller, V., and Pizer, L. I. (1985). Mol. Cell. Biol. 5, 406-409. Van der Putten, H., Botteri, F. M., Miller, A. D., Rosenfeld, M. G., Fan, H., Evans, R. M., and Verma, I. M. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 6148-6152. Vande Woude, G. F., Oskarsson, M., Enquist, L. W., Nomura, S., Sullivan, M., and Fischinger, P. J. (1979). Proc. Natl. Acad. Sci. U.S.A. 76,4464-4468. Van Griensven, L. J. L. D., and Vogt, M. (1980). Virology 101,376-388. Van Zoelen, E. J. J., van Ooshvaard, T. M. J., van der Saag, P. T., and de Laat, S. W. (1985a).J. Cell Physiol. 123, 151-160. Van Zoelen, E. J. J., van de Ven, W. J. M., Franssen, H. J., van Oostwaard, T. M. J., van der Saag, P. T., Heldin, C.-H., and de Laat, S. W. (1985b). Mol. Cell. Biol. 5,22892297. Vassort, F., Winterholer, M., Frindel, E., and Tubiana, M. (1973). Blood 41, 789-796. Vogt, M. (1982).Virology 118, 225-228. Vogt, M., Haggblom, C., Swift, S., and Haas, M. (1985).J. Virol. 55, 184-192. Vousden, K. H., and Marshall, C. J. (1984). EMBO J. 3, 913-917. Wagner, E. F., Vanek, M., and Vennstrom, B. (1985). E M B O J . 4,663-666. Walker, F., and Burgess, A. W. (1985). E M B O J . 4,933-939. Walker, F., Nicola, N. A., Metcalf, D., and Burgess, A. W. (1985). Cell 43, 269-276. Walter, M., Clark, S. G., and Levinson, A. D. (1986). Science 233, 649-652. Walter, P., Gilmore, R., and Blobel, G. (1984). Cell 38,5-8. Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C.-H., Huang, J. S., and Deuel, T. F. (1983). Nature (London) 304,35-39. Wei, C.-M., Lowy, D. R., and Scolnick, E. M. (1980).Proc. Natl. Acad. Sci. U.S.A. 77, 4677-4678. Wei, C.-M., Gibson, M., Spear, P. G., and Scolnick, E. M. (1981).]. Virol. 39,935-944. Weiland, F., Weiland, E., and Mussgay, M. (1979). Br. J. Cancer 40, 932-942. Weinberger, C., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. (1985). Nature (London) 318,670-672. Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Gruol, D. J., and Evans, R. M. (1986). Nature (London) 324,641-645. Weinstein, Y., Ihle, J. N., Lavu, S., and Reddy, E. P. (1985). Cell, in press.

354

WOLFRAM OSTERTAG

Weiss, R., Teich, N., Varmus, H., and Coffin, J. (1982). “RNA Tumor Viruses.” Cold Spring Harbor Laboratory, Cold Spring Habor, New York. Weissman, I. L., McGrath, M. S., and Tamura, G. (1985). In “Leukemia” (I. L. Weissman, ed.), pp. 235-249. Dahlem Konferenz 1985. Springer-Verlag, Berlin and New York. Wendling, F., Moreau-Gachelin, F., and Tambourin, P. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 3614-3618. Wendling, F., Varet, P., Charon, M., and Tambourin, P. (1986). Virology 149,242-246. Westin, E. H., Wong-Staal, F., Gelmann, E. P., Dalla Favera, R. D., Papas, T. S., Lautenberger, J. A., Eva, A., Reddy, E. P., Tronick, S. R., Aaronson, S. A., and Gallo, R. C. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 2490-2494. Wheeler, E. F., Rettenmier, C. W., Look, A. T., and Sherr, J. (1986). Nature (London) 324,377-379. Wiener, F., Klein, G., and Harris, H. (1974).J. Cell Sci. 15, 177-183. Wiener, F., Ohno, S., Spira, J., Haran-Ghera, N., and Klein, G. (1978a).J.Natl. Cancer Inst. 61, 227-237. Wiener, F., Spria, J., Ohno, S., Haran-Ghera, N., and Klein, G. (1978b).1nt.J.Cancer 22, 447-453. Wiktor-Jedrzejczak, W., Ahmed, A., Sharkis, S. J., McKee, A., and Sell, K. W. (1979).J. Cell. Physiol. 99, 31-36. Wiktor-Jedrzejczak, W., Szczylik, C., Gornas, P., Sharkis, S. J., and Ahmed, A. (1981). Cell Tissue Kinet. 14, 211-217. Williams, D. A., Lemischka, I. R., Nathan, D. G., and Mulligan, R. C. (1984). Nature (London) 310,476-480. Williams, N., McDonald, T. P., and Rabellino, E. M. (1979). Blood Cells 5,43-55. Williams, N., Jackson, H. M., Eger, R. R., and Long, M. W. (1981). In “Megakaryocyte Biology and Precursors: In Vitro Cloning and Cellular Properties” (B. L. G. Evatt, R. F. Levine, and N. T. Williams, eds.), pp. 59-73. Elsevier, Amsterdam. Williams, N., Eger, R. R., Jackson, H. M., and Nelson, D. J. (1982).J.Cell Physiol. 110, 101- 104. Willingham, M. C., Pastan, I., Shih, T. Y., and Scolnick, E. M. (1980). Cell 19, 10051014. Willurnsen, B. M., Christensen, A., Hubbert, N. C., Papageorge, A. G., and Lowy, D. R. (1984a).Nature (London) 310,583-586. Willumsen, B. M., Norris, K., Papageorge, A. G., Hubbert, N. L., and Lowy, D. R. (1984b). EMBOJ. 3,2581-2585. Willumsen, B. M., Papageorge, A. G., Kung, H.-F., Bekesi, E., Robins, T., Johnsen, M., Vass, W. C., and Lowy, D. R. (1986). Mol. Cell. B i d . 6,2646-2654. Wolff, L., and Ruscetti, S. (1985). Science 228, 1549-1552. WOE, L., and Ruscetti, S. (1986). Proc. Natl. Acad. Sci. U.S.A.83, 3376-3380. Wolff, L., Scolnick, E., and Ruscetti, S. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 47184722. Wolff, L., Kaminchik, J., Hankins, D. W., and Ruscetti, S. K. (1985).J. Virol. 53, 570578. Wolff, L., Tambourin, P., and Ruscetti, S. (1986). Virology 152, 272-276. Wood, T. G., McGeady, M. L., Blair, D. G., and Vande Woude, G. F. (1983).J.Virol. 46, 726-736. Wood, T. G., McGeady, M. L., Baroudy, B. M., Blair, D. G., and Vande Woude, G . F. (1984).Proc. Natl. Acad. Sci. U.S.A. 81, 7817-7821. Worton, R. G., McCulloch, E. A., and Till, J. E. (1969a).J. Erp. Med. 130, 91-103.

MURINE SPLEEN FOCUS-FORiMING VIRUSES

355

Worton, R. G., McCulloch, E. A., and Till, J. E. (1969b).J. Cell. Physiol. 74, 171-182. Wu, A. M., Till, J. E., Siminovitch, L., and McCulloch, E. A. (1967).J.Cell. Physiol. 69, 177-184. Wu, A. M., Till, J. E., Siminovitch, L., and McCulloch, E. A. (1968).1.E x p . Med. 127, 465-471. Wyke, J. A., Stoker, A. W., Searle, S., Spooncer, E., Simmons, P., and Dexter, T. M. (1986).Mol. Cell. Biol. 6, 959-963. Xu, Y.-H., Ishii, S., Clark, A. J. L., Sullivan, M., Wilson, R. K., Ma, D. P., Roe, B. A., Merlino, C. T., and Pastan, I. (1984a). Nature (London)309, 806-810. Xu, Y.-H., Richert, N., Ito, S., Merlino, C. T., and Pastan, I. (1984b). Proc. Natl. Acad. Sci. U.S.A. 81, 7308-7312. Yamaguchi, Y., Kluge, N., Ostertag, W., and Furusawa, M. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 2325-2329. Yamamoto, K. R., Gamble, C. L., Clark, S. P., Joyner, S. P., Shibuya, T., MacDonald, M. E., Mager, D., Bernstein, A,, and Mak, T. W. (1981).Proc. Natl.Acad. Sci. U.S.A.78, 6893-6897. Yamamoto, N., and Hinuma, Y. (1985).J. Gen. Virol. 66, 1641-1660. Yanagawa, S., Hirada, K., Ohnota, H., Sasaki, R., Chiba, H., Veda, M., and Goto, M. (1984a).J . Biol. Chem. 259, 2707-2710. Yanagawa, S., Yokoyama, S., Hirada, K., Sasaki, R., Chiba, H., Ueda, M., and Goto, M. (1984b). Blood 64, 357-364. Ymer, S., Tucker, W. Q. T., Sanderson, C. J., Hapel, A. J., Campbell, H. D., and Young, I. C. (1985). Nature (London) 317,255-258. Yoakum, G. H., Lechner, J. F., Gabrielson, E. W., Korba, B. E., Malan-Shibley, L., Willey, J. C., Valerio, M. G., Shamsuddin, A. M., Trump, B. F., and Harris, C. C. (1985). Science 227, 1174-1179. Yokota, T., Lee, F., Rennick, D., and Hall, C. (1984).Proc. Natl. Acad. Sci. U.S.A.81, 1070- 1074. Yokota, J., Tsunetsugu-Yokota, Y., Battifora, H., Le Fevre, C., and Cline, M. J. (1986). Science 231,261-265. Yoshikura, H., and Odaka, T. (1982).J.Natl. Cancer Znst. 68, 1005-1009. Yoshimura, F. K., Davison, B., and Chaffin, K. (1985).Mol. Cell. Biol. 5, 2832-2835. Young, D. C., and Griffin, J. D. (1986).Blood 68, 1178-1181. Young, H. A., Shih, T. Y., Scolnick, E. M., Rasheed, S., and Gardner, M. B. (1979).Proc. Natl. Acad. Sci. U.S.A. 76, 3253-3257. Young, H. G. A., Rasheed, S., Sowder, R., Benton, C. V., and Henderson, L. E. (1981).J. Virol. 38, 286-293. Yuasa, Y., Srivastava, S. K., Dunn, C. Y., Rhim, J. S., Reddy, E. P., and Aaronson, S. A. (1983).Nature (London) 303,775-779. Yuasa, Y., Col, R. A., Chang, A., Chin, I . M., Reddy, E. P., Tronick, S. R. and Aaronson, S. A. (1984). Proc. Natl. Acad. Sci. U.S.A 81, 3670-3674. Yung, Y.-P., Egar, R., Tertian, C., and Moore, M. A. S. (198l).J.Zmmunol. 127,794-799. Yunis, J. J. (1983). Science 221, 227-236. Yunis, J. J., and Soreng, A. L. (1984).Science 226, 1199-1203. Zarbl, H., Sukumar, S., Arthur, A. V., Martin-Zanca, D., and Barbacid, M. (1985).Nature (London) 315,382-285.

This Page Intentionally Left Blank

INDEX

A Abelson virus, spleen focus-forming viruses and, 228, 278, 281 Acanthosis, human papillomaviruses and, 117, 125 N-2-Acetylaminofluorene (AAF), hepatocarcinogenesis and 42, 43 complex regimens, 45 cyclic feeding, 40, 42 gene expression, 95, 100 in oitro culture, 81 markers for cellular lineage, 47, 48, 50, 52,55 monoclonal antibodies, 64, 67, 69 promotion, 44 transplantation, 78-81 Activation, hepatocarcinogenesis and, 38, 39 Acute myeloblastic leukemia, 221, 286, 291 Acute myeloid leukemia, 255 Acute nonlymphocytic leukemia, 242 Acute promyelocyte leukemia, 226 Adenocarcinomas herpes simplex type 2 and, 155 human papillomavirus and, 133 Adenoviruses, 119 Adenyl cyclase, spleen-focus forming viruses and, 230, 298 Adenylate cyclase, spleen-focus forming viruses and, 290, 298, 299, 302 Adipocyte cells, spleen focus-forming viruses and, 197 Affinity chromatography, hepatocarcinogenesis and, 65 Aflatoxin B,, hepatocarcinogenesis and, 65,81 Alanine, spleen focus-forming viruses and, 300 357

Albumin hepatocarcinogenesis and, 46, 49-51, 69 placental alkaline phosphatase and, 14 Alimentary canal, human papillomaviruses and, 121 Alkaline phosphatase, hepatocarcinogenesis and, 61 Amino acid hepatocarcinogenesis and, 88, 90 human papillomaviruses and, 118, 119 placental alkaline phosphatase and, 5, 6, 8, 17, 21, 26 spleen focus-forming viruses and env-recombinant, 252, 255, 256, 261-263 hematopoiesis, 214 leukemogenesis, 230, 231 rnos-oncogenic viruses, 272, 287, 288 ras proteins, 297, 298, 300-302 0-Aminoazotoluene, hepatocarcinogenesis and, 37 Amnion, placental alkaline phosphatase and, 1 8 , 2 3 Anemia, spleen focus-forming viruses and env-recombinant, 245, 251, 256 transformation, 306, 308, 311, 316 Antigens hepatocarcinogenesis and monoclonal antibodies, 57, 58, 6062, 64, 65 transplantation, 75, 76 herpes simplex type 2, 153, 155, 156 Prague study, 161, 164 seroepidemiological studies, 158, 159 human papillomaviruses and biology, 114, 115, 117, 119 carcinogenesis, 139, 140 genital lesions, 124, 128, 130

358

INDEX

placental alkaline phosphatase and, 12, 14, 18, 27 spleen focus-forming viruses hematopoiesis, 196, 201, 208, 214 ras-oncogenic viruses, 295 transformation, 318, 320 Antioncogenes, 224,225 Arginine, spleen focus-forming viruses and, 288, 300 ATP binding, spleen focus-forming viruses and, 287, 288, 299 ATPase hepatocarcinogenesis and, 63 spleen focus-forming viruses and, 287 Atypical immature metaplasia, 127 Autophosphorylation hematopoiesis and, 211 spleen focus-forming viruses and, 301 Autoradiography hepatocarcinogenesis and, 50 spleen focus-forming viruses and, 216 Avian erythroblastosis virus (AEV), 223, 227,235,236

B cell growth factor, 237 BALB murine sarcoma virus (BALBMuSV), 292, 293, 296, 300, 301, 303, 320,322,326 Bile canaliculi, hepatocarcinogenesis and, 63, 67 Bile duct cells, hepatocarcinogenesis and, 42,50-52,57 monoclonal antibodies, 64, 67, 70 proliferation, 46 Binucleation, human papillomaviruses and, 124 Bladder carcinoma, spleen focus-forming viruses and, 290, 291, 298,299 Bovine papillomavirus, genital cancer and, 114, 115-119, 121, 141 carcinogenesis, 140 cervical cancer, 135 Bowenoid papilosis, 124, 125, 129, 130,

137 Brain tumors, placental alkaline phosphatase and, 13 Breast cancer, spleen focus-forming viruses and, 302

Bromelain, placental alkaline phosphatase and, 6, 8, 26 Bromodeoxyuridine, placental alkaline phosphatase and, 16 Bronchogenic cancer, placental alkaline phosphatase and, 1 Burkitt’s lymphoma, 226 Burst-forming unit-erythroid, spleen focus-forming viruses and env-recombinant, 245 hematopoiesis, 203-206, 209 leukemogenesis, 241 transformation, 306, 310, 312, 313, 315, 316, 318, 320 Burst-promoting activity (BPA), hematopoiesis and, 204, 214 Butylated hydroxytoluene, hepatocarcinogenesis and, 44 Butyrate, placental alkaline phosphatase and, 14-16, 18,25,28

C Calcitonin, placental alkaline phosphatase and, 23 CAMP placental alkaline phosphatase and, 16 spleen focus-forming viruses and, 287, 290 Carbohydrate, placental alkaline phosphatase and, 10 Carbon tetrachloride, hepatocarcinogenesis and, 44 Carcinoembryonic antigen, placental alkaline phosphatase and, 3, 23 Carcinogenesis human papillomaviruses and, 121, 133-136 diagnosis, 139, 140 individual infections, 136, 137 malignancy, 138, 139 synergism, 137, 138 vaccination, 140, 141 spleen focus-forming viruses and, 219, 224-227,239 Carginogens herpes simplex type 2 and, 156, 186 human papillomaviruses and, 120, 137, 138, 142

INDEX

spleen focus-forming viruses and, 219, 293 /%Carotene, herpes simplex type 2 and, 5 Cas-SFFV, 315, 316 cDNA hepatocarcinogenesis and, 94-96, 100 human papillomaviruses and, 118, 135 placental alkaline phosphatase and, 5, 6 spleen focus-forming viruses and env-recombinant, 250 hematopoiesis, 206, 208, 210, 214 mos-oncogenic viruses, 267 Cell-mediated immunity, human papillomaviruses and, 121, 140 Cervical intra-epithelial neoplasia (CIN) herpes simplex type 2 and, 149, 153, 185 Houston study, 182 Prague study, 163, 167, 168, 170, 171, 174-176, 179, 180 prevention, 187 risk factors, 186 human papillomaviruses and, 126-128, 139 Cervical neoplasia, herpes simplex type 2 and, see Herpes simplex type 2, cervical neoplasia and Cervix human papillomaviruses and, 113 biology, 116, 119 carcinogenesis, 136, 138, 139 genital lesions, 125-130 genital tumors, 122, 124 viral DNA, 131-135 viral gene expression, 135 placental alkaline phosphatase and, 20, 21,25 Chick embryo fibroblasts (CEF), hepatocarcinogenesis and, 93, 97 Cholangiocarcinomas, hepatocarcinogenesis and, 42, 64, 75 Choline deficiency, hepatocarcinogenesis and, 44, 45, 50, 51, 67, 69, 79, 81, 95 Choriocarcinoma, placental alkaline phosphatase and, 1, 2, 10, 16-18, 25 Chromatin, placental alkaline phosphatase and, 16, 18, 28 Chromium, herpes simplex type 2 and, 158

359

Chronic myeloid leukemia (CML), 220, 226,228-230,241,242 Cleavage, spleen focus-forming viruses and, 261,268 Clones hepatocarcinogenesis and, 44, 88, 101 human papillomaviruses and, 113, 116, 118, 122, 124, 133, 134 placental alkaline phosphatase and, 2, 5, 22, 26, 28 spleen focus-forming viruses and env-recombinant, 244-246, 248, 253, 254 hematopoiesis, 196, 198, 200, 203, 206-208,210,214 leukemogenesis, 223,228,230, 240242 mos-oncogenic viruses, 266-268 ras-oncogenic viruses, 289, 293, 296 transformation, 314, 315, 317, 319 c-Myc gene hematopoiesis and, 90, 91 leukemogenesis and, 239 growth factors, 233, 237 protooncogenes, 225 Collagen, spleen focus-forming viruses and, 288 Colon carcinoma hepatocarcinogenesis and, 61 placental alkaline phosphatase and, 15 spleen focus-forming viruses and, 291, 292 Colony-forming cells (CFC), spleen focus-forming viruses and hematopoiesis, 198, 199 transformation, 305, 318-322 Colony-forming unit-erythroid (CFU-E), spleen focus-forming viruses and env-recombinant, 246 hematopoiesis, 203-205 leukemogenesis, 241 transformation, 308, 310, 312, 315, 316, 320 Colony-forming unit-spleen (CFU-S), spleen focus-forming viruses and env recombinant, 245, 248 hematopoiesis, 198, 201 transformation, 306, 310, 311, 315, 316 Colony-stimulating factors, spleen focusforming viruses and hematopoiesis, 207

360

INDEX

receptors, 216-218 transformation, 317 colposcopy herpes simplex type 2 and, 149,161-

Dexamethasone, placental alkaline phosphatase and, 14,17 4,4’-Diaminodiphenylmethane(DDPM), hepatocarcinogenesis and, 46,51 Dichlorodiphenyltrichlorethane, hepato163,166-168,179 carcinogenesis and, 44 human papillomaviruses and, 126,128, Diethylnitrosamine (DEN), hepatocar130 cinogenesis and, 43,44 Condyloma gene expression, 91,99,100 human papillomaviruses and, 125,130 in vitro culture, 81 carcinogenesis, 139 markers for cellular lineage, 47,48,55 cervix, 125-129 monoclonal antibodies, 67 Condylomata, human papillomaviruses transplantation, 74,78-80 and, 124 Diethylstilbestrol, herpes simplex type 2 Condylomata acuminata, human papilloand, 182 maviruses and, 122,124,125 DMBA, hepatocarcinogenesis and, 43, Corticosteroids, placental alkaline phos44,88,89 phatase and, 11, 15 DMSO, leukemogenesis and, 237 Cottontail rabbit papillomavirus, genital DNA cancer and, 119,120,141 hepatocarcinogenesis and, 39,101 Cross-hybridization, human papillogene expression, 86-88,90,93,94, maviruses and 96 biology, 114,116,121 monoclonal antibodies and, 65,66,70 genital tumors, 122 herpes simplex type 2 and, 152,153, Croton oil, hepatocarcinogenesis and, 43 155,156,158 Cryosurgery, herpes simplex type 2 and, human papillomaviruses and, 113 187 Cycloheximide biology, 114-118,120,121 human papillomaviruses and, 135 carcinogenesis, 136-140 placental alkaline phosphatases and, genital lesions, 127,129-131 11,12 genital tumors, 122,124 Cysteine viral, 131-135 placental alkaline phosphatase and, 12, human papillomaviruses and, 118 16 spleen focus-forming viruses and, 301 spleen focus-forming viruses and Cytochrome P-450, hepatocarcinogenesis env-recombinant, 249 and, 64 leukemogenesis, 220,221,226,238 Cytokeratins, hepatocarcinogenesis and, mos-oncogenic, 267,285,288 69,70 ras-oncogenic, 291,298,299 C ytotoxicity DNA polymerase, spleen focus-forming hepatocarcinogenesis and, 82 viruses and, 244,253 placental alkaline phosphatase and, 27 DNA virus, herpes simplex type 2 and, spleen focus-forming viruses and 151,152 hematopoiesis, 198,201,214 Drosophila, spleen focus-forming viruses leukemogenesis, 236 and, 290,301 Dyskaryosis, human papillomaviruses and, 128 D Dyskeratosis, human papillomaviruses and, 126 Deathermoelectrocoagulation (DKG), Dysplasia herpes simplex type 2 and, 171,172, herpes simplex type 2 and, 149,152,

179,184,187

154

36 1

INDEX

human papillomaviruses and, 124, 125,

127, 128, 134

E Electron microscopy human papillomaviruses and, 124 placental alkaline phosphatase and, 6,

11 Electrophoresis hepatocarcinogenesis and, 97 herpes simplex type 2 and, 159 placental alkaline phosphatase and,

12-14, 17, 19, 21 ELISA, hepatocarcinogenesis and, 67 Endoplasmic reticulum, placental alkaline phosphatase and, 6, 11 Endotoxin, spleen focus-forming viruses and, 212 Enu gene, spleen focus-forming viruses and, 194 Env-recombinant SFFV genomic structure, 250-260 helper virus, 248-250 origin, 244-248 protein, 260-264 Eosinophil cells, spleen focus-forming viruses and, 201, 214, 215, 218 Eosinophil colony-forming cells (EoCFC), hematopoiesis and, 205, 206,

209 Epidermal growth factor, spleen focusforming viruses and, 287 Epidermis, human papillomaviruses and,

116, 117, 124, 129 Epidermodysplasia verruciformis, human papillomaviruses and, 121, 136, 141 Epithelial cells hepatocarcinogenesis and, 43, 44, 81-

85 spleen focus-forming viruses and, 323,

326 hematopoiesis, 197 mos-oncogenic, 280 ras-oncogenic, 291, 298, 302 transformation, 319, 321 Epithelial growth factor, leukemogenesis and, 230,233-237,299 Epithelium herpes simplex types 2 and, 186, 187

human papillomaviruses and biology, 116, 117 genital lesions, 124, 126, 127, 130 genital tumors, 122 Epitopes hepatocarcinogenesis and, 57, 59, 61-

63, 66, 67, 69, 70 placental alkaline phosphatase and, 8 Epstein-Barr virus herpes simplex type 2 and, 152 human papillomaviruses and, 120,

139-141 Erythroblasts, spleen focus-forming viruses and env-recombinant, 249, 264 hematopoiesis, 204 nonviral myeloid neoplasms, 219, 235,

242 ras-oncogenic, 292, 293 transformation, 312, 315, 319 Erythrocytes, hematopoiesis and, 204 Erythroid cells, spleen focus-forming viruses and, 323, 327 env recombinant, 245,252,255-258,

260-264 hematopoiesis, 198, 200-202, 214, 218 molecular biology, 244 nonviral myeloid neoplasms, 219 leukemogenesis, 225, 234, 237, 241,

242 transformation, 307, 312-314, 316, 321,

322 Erythroid hyperplasia, 248, 249, 264 Erythroid leukemia, 219, 260 Erythroid progenitor cells, hematopoiesis and, 203-205 Erythroleukemia, spleen focus-forming viruses and env-recombinant, 244-245, 248-252,

255, 260,263,264 growth factors, 237 ras-oncogenic viruses, 291 transformation, 307, 308, 310, 311, 313,

314,320 Erythropoiesis, spleen focus-forming viruses and hematopoiesis, 205 leukemogenesis, 221, 222 transformation, 313 Erythropoietin, spleen focus-forming viruses and 326

362

INDEX

env-recombinant, 247, 248, 256, 257,

ras-oncogenic viruses, 291, 295, 298,

262,264

299, 301

hematopoiesis, 200, 204, 207, 216 leukemogenesis, 233, 237, 241, 242,

245 transformation, 307, 308, 310, 312, 314,

315,320,322 Erythroproliferative diseases, spleen focus-forming viruses and, 222, 311,

314,319,320,322 Escherichia coli, placental alkaline phosphatase and, 8 Estrogen, sp1ee.i focus-forming viruses and, 303 Ethionine, hepatocarcinogenesis and, 42, 43, 50, 67, 76, 81, 95 Eukaryotes, placental alkaline phosphatase and, 11

F Factor X, spleen focus-forming viruses and, 199 a-Fetoprotein hepatocarcinogenesis and, 43 cellular, 48-50 lesions, 52, 55 liver proteins, 100 models, 40, 42 monoclonal antibodies, 69, 70 oval cell proliferation, 50, 51 protooncogene expression, 90 serum concentrations, 46-48 tumor mRNA complexity, 97 placental alkaline phosphatase and, 23 induction, 13, 14 oncotrophoblast genes, 3 Fibroblasts human papillomaviruses and, 117, 118 placental alkaline phosphatase and, 2 spleen focus-forming viruses and 323,

transformation, 314, 316, 317, 319-

32 1 Fibronectin hepatocarcinogenesis and, 56,57 spleen focus-forming viruses and, 280 Fibropapillomas, human papillomaviruses and, 117 Fibroplasia, 117 Fibrosarcomas hepatocarcinogenesis and, 89 herpes simplex type 2 and, 89 N-2-Fluorenyldiacetamide,hepatocarcinogenesis and, 75, 76 5-Fluorouracil (5-FU), spleen focusforming viruses and, 198, 199 Friend lymphatic leukemia virus (FLLV), 248 Friend-MCFV, 249,250, 253, 255, 260 Friend MuLV (F-MuLV), 324 env-recombinant, 248, 249, 252, 253,

255,257,258,260-262 mos-oncogenic, 280 transformation, 307, 308, 310, 311, 315 Friend spleen focus-forming viruses (FSFFV), 219,222,237,243,326,327 env-recombinant, 244-246, 250, 251,

256,257 mos-oncogenic, 281 transformation, 307-314 Friend virus (FV) leukemogenesis and, 223 SFFV env-recombinant, 245-248, 252, 255,

263 ras-oncogenic. 293 transformation, 305, 308, 310-312, 315,

316

ti

324,326 env-recombinant, 245-247, 262-264 hematopoiesis, 197, 210, 211 leukemogenesis, 221, 225, 227, 228,

Gastrointestinal cancer cell lines, placental alkaline phosphatase and, 17,

231,233,234,236-239 mos-oncogenic viruses, 266, 267, 270, 272, 274, 275, 279, 280, 282-286

Gazdar murine sarcoma virus (GzMuSV), 265 GDP, spleen focus-forming viruses and,

18

299,302

363

INDEX

Gene expression, hepatocarcinogenesis and liver proteins, 97-101 protooncogene expression, 90-92 protooncogene mutations, 86-90 tumor mRNA complexity, 92, 93 competitive hybridization, 94 kinetic hybridization, 94 saturation hybridization, 93, 94 sequence complexity, 95,96 specific transcripts, 96, 97 Genital cancer, human papillomaviruses and, see Human papillomaviruses Glial cells, spleen focus-forming viruses and, 231 Glucocorticoid induction of alkaline phosphatase, 13, 14, 16, 17 Glucocorticoid receptor, spleen focusforming viruses and, 258, 280, 282, 324 y-Glutamyltranspeptidase (GGT), hepatocarcinogenesis and 56, 62, 67, 69, 78,90, 100 Gluthathione S-transferase (GST-P), hepatocarcinogenesis and, 56, 99, 100 Glycine, spleen focus-forming viruses and env recombinant, 256 mos-oncogenic, 287,288 ras-oncogenic, 300 Glycoprotein herpes simplex type 2 and, 159, 164 spleen focus-forming viruses and env-recombinant, 249, 261, 264 hematopoiesis, 196, 208, 210, 213 Glycoprotein antigens, hepatocarcinogenesis and, 62, 63 Glycosylation, placental alkaline phosphatase and, 6, 10, 16 Colgi apparatus placental alkaline phosphatase and, 6, 11 spleen focus-forming viruses and, 261 G-Proteins, spleen focus-forming viruses and, 298,299 Granulocyte, spleen focus-forming viruses and, 323, 326 hematopoiesis, 203, 211, 213, 215, 216

leukemogenesis, 235, 242 transformation, 310, 322 Granulocyte colony-forming cells (GCFC), 203 Granulocyte colony-stimulating factor (G-CSF), 199,202-205,211-213, 216-218 Granulocyte-macrophage colony forming cells (GM-CFC), 203-205 Granulocyte-macrophage colony-stimulating factor (GM-CSF), spleen focus-forming viruses and, 303 hematopoiesis, 199, 200, 202, 203, 205, 207-213, 216-218 leukemogenesis, 230, 231, 234 transformation, 308, 316-319, 322 Growth factors, spleen focus-forming viruses and, 325-327 leukemogenesis, 230-238 molecular biology, 262, 302-304 transformation, 315, 319-322 GTP, spleen focus-forming viruses and, 290,298-302 Guanine, spleen focus-forming viruses and, 298,302

H Harvey murine sarcoma virus (HaMuSV), 324, 326 hematopoietic cells, 307, 319-322 ras-oncogenic viruses, 292-296, 298, 300, 301, 303 Harvey sarcoma virus, 289-293, 296, 298-303 Heck’s disease, 122 Hemangiosarcoma, 73 Hematopoiesis, spleen focus-forming viruses and, 193-195, 328 CSF receptors, 216-218 Eo-CFC, 205,206 erythroid progenitor cells, 203-205 erythropoietin, 216 G-CSF, 211-213 GM-CFC, 202,203 GM-CSF, 208-210 hemopoietic regulatory factors, 206208 Mast-CFC, 206

364

INDEX

M-CSF, 210,211 Meg-CFC, 205,206 Multi-CSF, 214, 215 progenitor cells, 201, 202 stem cells, 195-201 Hematopoietic cells, spleen focus-forming viruses and, 323-328 env-recombinant, 248, 250 leukemogenesis, 221, 228, 230, 232, 236,240,241,243 mos-oncogenic, 266, 267, 272, 274, 275,277,279,280,286,291 transformation, 304-307, 322 Cas-SFFV, 315, 316 F-SFFV, 307-314 myeloproliferative sarcoma virus, 316-319 ras-oncogenic virus, 319-322 Rauscher SFFV, 314-316 Hematopoietic stem cells, 195-201, 228, 230, 243,306,321 Hemoglobinization, spleen focus-forming viruses and hematopoiesis, 203 transformation, 312, 315 Hemopoietic cell growth factor (HCGF), 214 Hemopoietic regulatory factors, 206-208 Hemapoietin 1, 199, 206 Hepatocarcinogenesis, premalignancy and, 37, 38, 101 activation, 38, 39 complex regimens, 45, 46 gene expression liver proteins, 97-101 protooncogene expression, 90-92 protooncogene mutations, 86-90 tumor mRNA complexity, 92-97 initiation, 39 markers for cellular lineage, 55-57 cellular AFP, 48-50 lesions, 52-55 oval cell proliferation, 50-52 serum AFP concentrations, 46-48 models, 40-43 monoclonal antibodies, 57, 58, 70 carcinogen adducts, 65, 66 carcinogenic metabolizing enzymes, 64 liver cells, 61-64

murine tumors, 58-61 putative premalignant cells, 66-70 phenotype analysis, 70, 76 in oitro culture studies, 70-72, 8186 in oiuo transplantation, 70-81 transplantation, 70-72 promotion, 39, 43, 44 Hepatocytes, placental alkaline phosphatase and, 13 Hepatoma cells, spleen focus-forming viruses and, 280 Hepatomas gene expression and, 90, 93-101 transplantation and, 72-77, 81, 85, 86 Hepatomegaly, 245 Herpes simplex type 2, cervical neoplasia and, 149-151, 188 animal experiments, 154-156 discrepancy between studies, 183-185 findings in patients, 151-154 Houston study, 182, 183 Prague study, 160, 161 aims, 161 design, 161-166 results, 166-182 prevention, 186, 187 risk factors, 185, 186 seroepidemiological studies, 156-160 Herpes simplex virus, human papillomaviruses and, 137 Heteroduplex, spleen focus-forming viruses and, 268, 295 Heterogeneity hepatocarcinogenesis and gene expression 87, 98, 99 in oitro culture, 81 monoclonal antibodies, 58 transplantation, 71,77 spleen focus-forming viruses and env-recombinant, 246, 252 hematopoiesis, 196, 202, 203 mos-oncogenic, 268 ras-oncogenic, 293-295 Histaminase, placental alkaline pjosphatase and, 2 Histochemistry, placental alkaline phosphatase and, 1 L-Homoarginine, placenta1 alkaline phosphatase and, 9, 12

365

INDEX

Homology herpes simplex type 2 and, 158 human papillomaviruses and, 141 biology, 114, 116, 118-121 genital tumors, 122, 124 spleen focus-forming viruses and, 325 env-recombinant, 250, 252, 255, 263 hematopoiesis, 208, 214 leukemogenesis, 233-236 mos-oncogenic, 267,270,287 ras-oncogenic, 295, 298, 302 Human chorionic gonadotropin, placental alkaline phosphatase and, 1, 1719,23-25 Human papillomaviruses, 113, 114, 141, 142 biology infection, 116, 117 malignant conversion, 120, 121 transforming functions, 117-120 virions, classification and, 114-1 16 carcinogenesis, etiologic role in, 135, 136 diagnosis, 139, 140 individual infections, 136, 137 malignancy, 138, 139 synergism, 137, 138 vaccination, 140, 141 cervical cancer, 131-135 genital lesions anogenital skin, 129, 130 condylomas of cervix, 125 etiology, 124, 125 exophytic tumors, 125 history, 127-129 inapparent infections, 130, 131 genital tumors, 122-124 Hybridization hepatocarcinogenesis and, 93, 96 competitive, 94 kinetic, 94, 95 saturation, 93, 94 human papillomaviruses and biology, 116, 117 carcinogenesis,l38 cervical cancer, 131, 133 genital lesions, 124, 130 genital tumors, 122, 124 herpes simplex type 2 and, 153, 155

spleen focus-forming viruses and env-recombinant, 250 leukemogenesis, 220, 225, 228, 239, 246 mos-oncogenic, 267, 288 ras-oncogenic, 290, 295 H ybridoma hepatocarcinogenesis and, 57, 60, 61, 66,67 spleen focus-forming viruses and, 208 Hydrocortisone, placental alkaline phosphatase and, 14 Hydrolysis placental alkaline phosphatase and, 1 spleen focus-forming viruses and, 299, 302 Hyperacetylation, placental alkaline phosphatase and, 15 Hyperkeratosis, human papillomaviruses and, 117 Hyperosmolar induction, placental alkaline phosphatase and, 15, 16, 28 H yperplasia hepatocarcinogenesis and, 52, 101 gene expression, 98-100 in uitro culture, 81, 82, 86 transplantation, 74-77, 80 human papillomaviruses and biology, 116 genital tumors, 122 Hysterectomy, 124

I Immunoelectron microscope, placental alkaline phosphatase and, 8 Immunization, human papillomaviruses and, 114, 116 Immunofluorescence, herpes simplex type 2 and, 155 Immunoglobins, hematopoiesis and, 206 Immunohistochemistry, hepatocarcinogens and, 63 Immunolocalization, placental alkaline phosphatase and, 27 Immunosuppression, human papillomaviruses and, 138 Immunotherapy, placental alkaline phosphatase and, 27

366

INDEX

Inhibition, placental alkaline phosphatase and, 1, 10, 15, 17, 19, 21, 26, 28 Initiation, hepatocarcinogenesis and, 39,

40 Insulin, spleen focus-forming viruses and, 237,299 Insulin-like growth factor I, 204 Interleukin-2, 232-234 Interleukin-3, 199, 214, 303 Interleukin-4, 199, 200 Intestinal alkaline phosphatase (IAP), 9,

10, 13, 14, 25, 26, 28 Intestine, placental alkaline phosphatase and, 1, 24 oncotrophoblast genes, 3, 4 Invasive carcinoma, herpes simplex type 2 and, 149, 153, 185 Prague study, 163, 167, 168, 174-176,

179, 180 risk factors, 186 Iododeoxyuridine, placental alkaline phosphatase and, 16

K Karyotype, leukemogenesis and, 239, 24 1 Karyotypic markers, spleen focus-forming viruses and, 196 Kasahara isoenzyme, placental alkaline phosphatase and, 4, 8, 18 Keratinocytes, hepatocarcinogenesis and,

116, 118, 129, 142 Kirsten murine sarcoma virus (Ki-MuSV),

292-295,300,303,319, 321, 326 Kirsten sarcoma virus, 289-295, 298, 301 Koilocyte, human papillomaviruses and,

117, 124, 126, 127, 141

Leucine, spleen focus-forming viruses and, 256 L-Leucine, placental alkaline phosphatase and, 1, 19, 25, 26 Leukemias, human papillomaviruses and, 138 Leukemogenesis, spleen focus-forming viruses and, 193, 194, 218-224, 323-

326 antioncogenes, 224, 225 env-recombinant, 245, 249, 253, 255,

262 malignant progression, 238, 239 molecular biology, 244 mos-oncogenic, 274 oncogenes, growth factors and, 230-

238 protooncogenes, 225-230 ras-oncogenic, 304 stem cells, 240-243 transformation, 304, 305, 307, 308, 311,

315 Liver placental alkaline phosphatase and, 1, 24 induction, 14, 17 oncotrophoblast genes, 3, 9 Long terminal repeat (LTR), spleen focus-forming viruses and, 244, 324,

327,328 env-recombinant, 248,251-253,257-

260,263,265 leukemogenesis, 226, 231 rnos-oncogenic, 272,278-283, 286 ras-oncogenic, 296, 298 transformation, 308, 314, 319 Lung placental alkaline phosphatase and, 1, 4, 5, 17, 21, 25 spleen focus-forming viruses and, 291,

292

L Laminin, hepatocarcinogenesis and, 56,

57 Larynx, human papillomaviruses and,

122 Lectin, spleen focus-forming viruses and hematopoiesis, 214 transformation, 318

Lymphoid cells, spleen focus-forming viruses and hematopoiesis, 196, 197, 199, 215 molecular biology, 258, 260, 281 Lymphomas human papillomaviruses and, 138 spleen focus-forming viruses and env-recombinant, 248 leukemogenesis, 239 ras-oncogenic, 291

INDEX

Lysine, spleen focus-forming viruses and, 287,300

M Macrophage, spleen focus-forming viruses and, 323, 326 hematopoiesis, 199, 200, 203, 209, 211, 212,214,216, 218 leukemogenesis, 235,237 molecular biology, 293, 303, 304 transformation, 317, 320-322 Macrophage colony-stimulating factor (M-CSF), spleen focus-forming viruses and, hematopoiesis, 199, 202, 203, 207, 210, 211, 216-218 leukemogenesis, 230, 234-236 molecular biology, 303 transformation, 322 Macrophage growth factor receptor, 234 Major histocompatibility complex, hepatocarcinogenesis and, 60, 78, 79 Malignancy herpes simplex type 2 and, 154 human papillomaviruses and, 120, 121, 136, 138, 139 spleen focus-forming viruses and, 326, 327 molecular biology, 288-292, 298 nonviral myeloid neoplasms, 219222,224-226,233,238, 239,24 1 transformation, 304 Malignant histiocytosis sarcoma virus (MHSV), 235, 295,296, 303, 304, 307, 319, 320, 322, 324, 326 Mammary adenocarcinoma, 59, 60 Mammary tumors, 76 Mast cell progenitor cells, 206, 214 MCF, spleen focus-forming viruses and molecular biology, 246, 247, 256-258, 263, 279 transformation, 308 3’-Me-DAB, hepatocarcinogenesis and, 42, 47, 91, 96, 100 Medullary thyroid cancer, 23 Megakaryocyte cells, spleen focus-forming viruses and hematopoiesis, 201, 202, 214, 218 leukemogenesis, 222, 235, 241 transformation, 316

367

Megakaryocyte-colony forming cells, 205,206, 209 Megaryocytopoiesis, 205 Melanoma cells, spleen focus-forming viruses and, 233, 239, 291 Metamyelocytes, spleen focus-forming viruses and, 202 Methotrexate, placental alkaline phosphatase and, 2 3-Methylcholanthrene, hepatocarcinogenesis and, 64 Methylnitrosurea (MNU), hepatocarcinogenesis and, 81 MHV, 280, 325 Microneutralization test, herpes simplex type 2 and, 164, 179-181 Mink cell focus-inducing virus (MCFV), 249, 250, 252, 255, 257, 258, 261, 262 Mitochondria hepatocarcinogenesis and, 63 placental alkaline phosphatase and, 6 Mitogens, spleen focus-forming viruses and, 231, 233 Mitosis, human papillomaviruses and biology, 116 carcinogenesis, 136 genital lesions, 128 Mix-CFC, spleen focus-forming virus and hematopoiesis, 198, 199 transformation, 318, 320 Maloney-murine leukemia virus (MoMuLV), 255, 257, 258, 260, 263, 265-268,270,272,281,282,292, 293,295,319 Moloney-murine sarcoma virus (MoMuSV), 265-268,272,275, 275-283, 286, 287, 324 Monoclonal antibodies hepatocarcinogenesis and, 57, 58, 70 carcinogen adducts, 65, 66 carcinogenic metabolizing enzymes, 64 liver cells, 61-64 murine tumors, 58-61 putative premahgnant cells, 66-70 placental alkaline phosphatase and, 3, 8, 14, 19,25-27 spleen focus-forming viruses and, 270

368

INDEX

Morphology hepatocarcinogenesis and, 40, 42, 46, 55, 57, 62, 75, 80, 93 herpes simplex type 2 and, 154 human papillomaviruses and, 117, 126, 127, 136 placental alkaline phosphatase and, 12, 18 spleen focus-forming viruses hematopoiesis, 198, 202 leukemogenesis, 227 mos-oncogenic, 267, 287 ras-oncogenic, 290 transformation, 312 Morris hepatoma, hepatocarcinogenesis and, 67-69,90,99 Mos oncogene, spleen-focus forming viruses and, 194 Mos-oncogenic viruses, 264, 265 expression, 281-288 genomic structure, 267-272 myeloproliferative sarcoma virus, 265267 structure, 272-281 Mosaicism, leukemogenesis and, 240 mRNA hepatocarcinogenesis and, 89, 91-93 hybridization, 92-95 liver proteins, 97, 100 sequence complexity, 95, 96 specific transcripts, 96, 97 placental alkaline phosphatase and, 16 spleen focus-forming viruses env-recombinant, 255,263 hematopoiesis, 208 leukemogenesis, 228,233,236, 237 mos-oncogenic, 281,283,286,287 ras-oncogenic, 289, 290, 296 transformation, 314 Multi-CSF, spleen focus-forming viruses and, 326 hematopoiesis, 199-202, 204-206, 208, 211-218 leukemogenesis, 232-234 molecular biology, 303 transformation, 312, 315, 317-319 Murine leukemia virus (MuLV), spleenfocus forming viruses and env-recombinant, 244, 246,247, 249, 250,258,261,262

mos-oncogenic, 267, 279, 281 ras-oncogenic, 296 M urine malignant histiocytosis sarcoma virus (MHSV), 223, 242, 292, 293 Murine spleen focus-forming viruses, see Spleen focus-forming viruses, murine Mutagenesis human papillomaviruses and, 118 spleen focus-forming viruses and, 234, 300,327 Mutation hepatocarcinogenesis, 86-90 herpes simplex type 2 and, 155 human papillomaviruses and, 137 placental alkaline phosphatase and, 4, 17 spleen focus-forming viruses and, 328 env-recombinant, 246, 256 mos-oncogenic, 267, 268, 272, 274, 278,283,287 nonviral myeloid neoplasms, 219, 221,222,224, 240 ras-oncogenic, 290, 292, 297-302 transformation, 306,317 Myelodysplasia, 241 Myeloid cell, spleen focus-forming viruses and, 193, 194, 323 hematopoiesis, 196, 197 molecular biology, 266 transformation, 316, 321, 322 Myeloid leukemia, spleen focus-forming viruses and, 288, 291 leukemogenesis, 221,242 transformation, 308 Myeloid metaplasia, 242 Myeloid neoplasms, nonviral, spleen focus-forming viruses and, 218-220 leukemogenesis, 220-224 antioncogenes, 224,225 env-recombinant, 245, 249, 253, 255, 262 malignant progression, 238, 239 molecular biology, 244 mos-oncogenic, 274 oncogenes, growth factors and, 230238 protooncogenes, 225-230 ras-oncogenic, 304 stem cells, 240-243

369

INDEX

transformation, 304, 305, 307, 308,

311,315 Myeloma, hepatocarcinogenesis and, 60 Myeloma cell, spleen focus-forming viruses and, 285 Myelopoiesis, 240 Myeloproliferative diseases, 222, 265,

266,319,323,326 Myeloproliferative leukemia virus (MPLV), 266 Myeloproliferative sarcoma virus (MPSV), spleen focus-forming viruses and, 323-328 leukemogenesis, 221, 223, 224, 226,

243 mos-oncogenic, 265-270,272,274-

278,280-283,286 ras-oncogenic, 304 transformation, 307, 314, 316-319

N Nagao' isoenzyme, placental alkaline phosphatase and, 1,25 1-Napthylisocyanate (ANIT), hepatacarcinogenesis and, 46, 51 Nasopharyngeal carcinoma, human papilloma viruses and, 140 Necrosis, hepatocarcinogenesis and, 45,

48 Neomycin, spleen focus-forming viruses and, 274 Neoplasia, herpes simplex type 2 and,

121, 125, 127, 131, 138, 142 Neuraminic acid, hematopoiesis and, 208 Neuraminidase, placental alkaline phosphatase and, 1, 10, 12 Neuroblastoma, spleen focus-forming viruses and, 232, 291 Neutrophil, hematopoiesis and, 200, 202,

Nonhistone chromosomal proteins, hepatocarcinogenesis and, 98 Novikoff hepatoma, hepatocarcinogenesis and, 64, 94, 98, 99 Nuclear atypia, human papillomaviruses and, 126,141 Nuclear hyperchromasia, human papillomaviruses and, 127

0 Oncomodulin, placental alkaline phosphatase and, 2 Oncotrophoblast genes, placental alkaline phosphatase and, 3-6, 26, 28 biosynthesis, 10, 11 cervix, 20 gene product, 6-9 isozymes, 9, 10 lung, 21 mechanisms, 24, 25 oncodevelopmental biology, 21-23 ovary, 19,20 testes, 19 thymus, 20 Oral cavity, human papillomaviruses and, 122 Oval cells, hepatocarcinogenesis and,

101 cellular AFP, 49 ethionine, 42 gene expression, 92, 101 monoclonal antibodies, 64, 67, 69, 70 proliferation, 50-52 transplantation, 79 Ovary, placental alkaline phosphatase and, 1, 2, 19, 20, 25, 28

P

203, 209, 211, 214, 218 Neutrophil-macrophage progenitor cells,

202, 203 Nicotine, human papillomaviruses and,

137 NIH 3T3 cells hepatocarcinogenesis and, 86-90 human papillomaviruses and, 117, 118 spleen focus-forming viruses and, 272,

286,290, 291

P cell stimulating factor, hematopoiesis and, 214 Papillomas, hepatocarcinogenesis and,

44, 89 Papillomaviruses, see Bovine papillomavirus; Human papillomaviruses Parakeratosis, 125 Partial hepatectomy (PH), transplantation and, 78-81,91

370

INDEX

Penile bowenoid papulosis, 125 Penile cancer, human papillomaviruses and, 113-137 Peroxisome proliferators, hepatocarcinogenesis and, 43, 80 Phenobarbital, hepatocarcinogenesis and, 44,48 monoclonal antibodies and, 64 transplantation, 77, 79, 80 Phenotype hepatocarcinogenesis and, 39, 101 gene expression, 86, 87, 100 in uitro culture studies, 70-72, 81-86 in uiuo transplantation, 70, 72-81 markers, 46, 55, 56 monoclonal antibodies, 61, 62 transplantation herpes simplex type 2 and, 155, 156 human papillomaviruses and, 117, 118, 120, 121 placental alkaline phosphatase and, 5, 17 spleen focus-forming viruses and leukemogenesis, 220, 228, 233, 236, 24 1 spleen focus-forming viruses and, 266-268,275,278,290 transformation, 305, 307, 322 L-Phenylalanine, placental alkaline phosphatase and, 1, 10, 12, 17, 19, 20, 26 Phosphokinase activity, spleen focusforming viruses and, 287 Phosphoprotein hepatocarcinogenesis and, 100 herpes simplex type 2 and, 153 Phosphorylation placental alkaline phosphatase and, 11, 15 spleen focus-forming viruses and, 233, 236,300 Pituitary hormone, hepatocarcinogenesis and, 44 Placental alkaline phosphatase, 1, 2, 28 immunolocalization, 27 induction in cultured cells, 11 butyrate, 14, 15 CAMP, 16 DNA synthesis inhibitor, 16 first trimester, 17

gastrointestinal cancer cell lines, 17, 18 glucocorticoid, 13, 14 halogenated nucleoside, 16 hyperosmolar, 15 individuality, 15, 16 isoenzymes, 11, 12 induction mechanisms, 18, 19 oncotrophoblast genes, 3, 4 biosynthesis, 10, 11 cervix, 20 evolution, 4, 5 gene product, 6-9 isozymes, 9, 10 lung, 21 mechanisms, 24,25 oncodevelopmental biology, 21-23 ovary, 19, 20 PLAP gene, 5 , 6 structure, 26 testes, 19 thymus, 20 PLAP-like enzymes, 25 Placental glutathione S-transferase, (GST-P), hepatocarcinogenesis and, 100 Plasmids, human papillomaviruses and, 115, 134 Plasminogen activator, spleen focusforming viruses and, 209 Platelet-derived growth factor (PDGF), Ieukemogenesis and, 230-233,237 Pleomorphism, human papillomaviruses and, 127 Polyadenylation, spleen focus-forming viruses and, 258 Polycyclic hydrocarbons, hepatocarcinogenesis and, 37 Polycythemia, spleen focus-forming viruses and, 245, 246, 251, 308, 311 Polycythemia Vera (PV), 222, 241, 242 Polymaviruses, 119 Polymorphism placental alkaline phosphatase and, 26 spleen focus-forming virus and, 305, 306 Prednisolone, placental alkaline phosphatase and, 12-15, 28 Pregnancy herpes simplex type 2 and, 150, 171

371

INDEX

human papillomaviruses and, 129 placental alkaline phosphatase and, 3 Premalignancy, hepatocarcinogenesis and, see Hepatocarcinogenesis Proerythroblasts, spleen focus-forming viruses and hematopoiesis, 204 leukemogenesis, 223 Promotion, hepatocarcinogenesis and, 39 Promyelocytes, spleen focus-forming viruses and, 215, 226, 231 Protein kinase, spleen focus-forming viruses and, 211, 287 Protein synthesis, spleen focus-forming viruses and, 209 Proteolysis, placental alkaline phosphatase and, 5, 6, 8, 26 Protooncogenes hepatocarcinogenesis and expression, 90-92 mutations, 86-90 spleen focus-forming viruses and, 244, 323,325, 327 env-recombinant, 250, 257 leukemogenesis, 225-230,233, 235238 mos-oncogenic, 264, 265, 286 nonviral myeloid neoplasms, 219,224 ras-oncogenic, 288-293,297-301

R Radioimmunoassay, hepatocarcinogenesis and, 64 Ras-oncogenic viruses, spleen focusforming viruses and, 194, 288, 289 genomic structure, 293-296 growth factor, 302-304 leukemogenesis, 225 protein, 296-302 protooncogenes, 289-292 retroviruses, 292, 293 transformation, 319-322 Rasheed murine sarcoma virus (RaMLISV),292, 293, 295, 296, 300 Rauscher-MCF, 260 Rauscher-MCFV, 249 Rauscher-MuLV, 248, 249,261,296,310 Rauscher SFFV, 222,243, 245,250,251, 253,256

Rauscher virus, 323 env-recombinant SFFV, 247,249, 253, 255 leukemogenesis, 223 transformation, 315 Rectal cancer, placental alkaline phosphatase and, 15, 17 Regan isoenzyme, placental alkaline phosphatase and, 1, 8, 13, 17, 19, 25 Retinoids, herpes simplex type 2 and, 150 Retroviruses hepatocarcinogenesis and, 87, 90 spleen focus-forming viruses and, 193, 243,323-328 antioncogenes, 224, 225 env-recombinant, 244, 250, 252, 257, 258,261,263 growth factors, 230-236 hematopoiesis, 210 mos-oncogenic, 265,266,278-286 nonviral myeloid neoplasms, 218, 219,222-224,239 protooncogenes, 225-227 ras-oncogenic, 288, 292, 293 stem cells, 241-243 transformation, 307, 317, 321 RNA hepatocarcinogenesis and, 39, 89, 9297, 101 herpes simplex type 2 and, 153 human papillomaviruses and, 118, 135, 138, 139 spleen focus-forming viruses and, 244 env-recombinant, 247, 253 leukemogenesis, 234,236 mos-oncogenic, 272, 283, 285 ras-oncogenic, 295, 296 RNA synthesis placental alkaline phosphatase and, 11 spleen focus-forming viruses and, 209 Rous sarcoma virus, 221

S Saponin, placental alkaline phosphatase and, 6 Sarcomas herpes simplex type 2 and, 154

372

INDEX

spleen focus-forming viruses and, 292, 293 Saturation hybridization, hepatocarcinogenesis and, 93-95 Seminoma, placental alkaline phosphatase and, 2, 19, 21, 25, 28 Serine, spleen focus-forming viruses and env-recombinant, 256 leukemogenesis, 237 mos-oncogenic, 287 ras-oncogenic, 300, 301 Seroepidemiological studies, herpes simplex type 2 and, 152, 156-160 Serology, herpes simplex type 2 and, 152, 164-166, 176, 179-182, 184 Simian sarcoma virus (SiSV), 231, 233 Smoking herpes simplex type 2 and, 171, 172, 174, 175, 184, 186-188 human papillomaviruses and, 137 Solid-phase radioimmunoassay (SPIRA), herpes simplex type 2 and, 164-166, 179-181 Spleen focus-forming viruses, murine, 193, 194,243, 244, 323-328 env-recombinant genomic structure, 250-260 helper virus, 248-250 origin, 244-248 proteins, 260-264 hematopoiesis, 194, 195 CSF receptors, 216-218 Eo-CFC, 205,206 erythroid progenitor cells, 203-205 erythropoietin, 216 G-CSF, 211-213 GM-CFC, 202,203 GM-CSF, 208-210 hemopoietic regulatory factors, 206208 Mast-CFC, 206 M-CSF, 210,211 Meg-CFC, 205,206 Multi-CSF, 214, 215 progenitor cells, 201, 202 stem cells, 195-201 leukemogenesis, 220-224 antioncogenes, 224,225 malignant progression, 238, 239

oncogenes, growth factors and, 230238 protooncogenes, 225-230 stem cells, 240-243 mos-oncogenic, 264, 265 expression, 281-288 genomic structure, 267-272 myeloproliferative sarcoma virus, 265-267 structure, 272-281 nonviral myeloid neoplasms, 218220 ras-oncogenic, 288, 289 genomic structure, 293-296 growth factor, 302-304 protein, 296-302 protooncogenes, 289-292 retroviruses, 292, 293 transformation, 319-322 transformation, target cells for, 304, 305 Cas-SFFV, 315, 316 F-SFFV, 307-314 host range, 305-307 MPSV, 316-319 ras-oncogenic, 319-322 R-SFFV, 314-316 Splenomegaly, 245, 266, 293, 316, 320 Squamocolumnar border, human papillomaviruses and, 116, 124 Squamocollumnar junction, herpes simplex type 2 and, 152 Squamous cell carcinoma, 129, 131, 132, 141 Squamous epithelia, human papillomaviruses and, 126 Stem cells, spleen focus-forming viruses and, 230,240-243,266,318,323, 327,328 Steroids, hepatocarcinogenesis and, 44 Stroma cells, spleen focus-forming viruses and, 198, 317,319 Subtilism, placental alkaline phosphatase and, 6 Synergism human papillomaviruses and, 118, 121, 137, 138 placental alkaline phosphatase and, 15, 16

373

INDEX

spleen focus-forming viruses and, 206, 249

T T cell leukemia virus, 232 T cell lymphocytes, spleen focusforming viruses and, 208, 214 T cell lymphoma, 232 Target cells, spleen-focus forming viruses and, 194, 250, 264, 279-281, 288, 324, see also Transformation, spleen-focus forming viruses and Testicular teratocarcinoma, 19, 21 Testis, placental alkaline phosphatase and, 2, 19, 21, 25, 28 Testosterone, hepatocarcinogenesis and, 44 Threonine, spleen focus-forming viruses and, 237,287,300 Thrombocytepenia, 222, 241, 242 Thrombopoietin, 205 Thymidine, hepatocarcinogenesis and, 50,81 Thymus, placental alkaline phosphatase and, 20, 21, 25, 28 Transcription human papillomaviruses and biology, 114, 115, 118, 120 cervical cancer, 134, 135 spleen focus-forming viruses and, 280, 327,328 leukemogenesis, 236 mos-oncogenic, 281-286,288 ras-oncogenic, 291, 296 Transformation, spleen focus-forming viruses and, 324 leukemogenesis, 231, 232 mos-oncogenic, 272,279-282,286,288 ras-oncogenic, 293, 295, 297-299, 301, 304 target cells for, 304, 305 Cas-SFFV, 315, 316 F-SFFV, 307-314 host range, 305-307

MPSV, 316-319 ras-oncogenic, 319-322 R-SFFV, 314-316 Transforming growth factors, leukemogenesis and, 233,237 Transplantation, hepatocarcinogenesis and, 70-72, 86 enzymatically dissociated premalignant liver cells, 77-81 hepatomas, 72-74 in oitro culture, 85 putative premalignant liver lesions, 74-77 Triphenylamine hepatocarcinogenesis and, 88, 89 leukemogenesis and, 237 Trypsin, placental alkaline phosphatase and, 618, 626 Tumorigenicity hepatocarcinogenesis and, 66, 82, 85, 87, 88, 90 spleen focus-forming viruses and, 303 leukemogenesis, 227,228,231, 232 transformation, 310 Tunicamycin, placental alkaline phosphatase and, 10 Turnover, spleen focus-forming viruses and, 194,287 Tyrosine, hematopoiesis and, 211 Tyrosine kinase, leukemogenesis and, 228,231,236,237

v Vaccination, human papillomaviruses and, 114 Vagina, human papillomaviruses and, 124, 125 Venereal disease, herpes simplex type 2 and, 150 Viral capsid antigen (VCA), human papillomaviruses and, 140 Vulvar intraepithelial neoplasia (VIN), 130 Vulvar verrucous carcinoma, 137

This Page Intentionally Left Blank