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ADVANCESINCANCERRESEARCH VOLUME 48

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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

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(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


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).


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


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.


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-


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


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.


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.,


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.


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.


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


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.


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.

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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.


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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.

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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.


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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.

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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).

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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.)

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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.


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

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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?


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


(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-


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.


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-


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.


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


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-


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


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

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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


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.


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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

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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.


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

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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-


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


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-

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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


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-

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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,


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

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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-


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

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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


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 aigturn+ 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)


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).


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


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-


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-

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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


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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

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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


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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.

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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


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(“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

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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


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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.

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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.


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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

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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

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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


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

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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


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

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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


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.

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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

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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


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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-

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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


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-

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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


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.

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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%.

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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)


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

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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.


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-

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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


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

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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-


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-

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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-


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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

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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.

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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

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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

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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

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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-

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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-

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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-

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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-

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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

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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

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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.

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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


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


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


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


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


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


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


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 (

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